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Woods, Nicholas Taylor.
Regulation of bax activation and apoptosis by src and acetylated mutant p53
h [electronic resource] /
by Nicholas Taylor Woods.
[Tampa, Fla] :
b University of South Florida,
Title from PDF of title page.
Document formatted into pages; contains 183 pages.
Dissertation (Ph.D.)--University of South Florida, 2008.
Includes bibliographical references.
Text (Electronic dissertation) in PDF format.
ABSTRACT: Apoptosis is an inherent suicide mechanism that cells invoke for a variety of reasons including embryo cavitation, tissue homeostasis, excessive DNA damage and aberrant oncogene activation. Apoptosis is regulated by a diverse set of proteins including, but not limited to, the Bcl-2 family. This family set is comprised of both pro-death and pro-survival proteins whose relative expression, localization and/or modifications regulate the balance between life and death for each cell. The keystones to this system are the proapoptotic proteins Bax and Bak, which are regulated by their conformation and localization. However, the exact mechanisms by which Bax and Bak become activated remains to be resolved. Similarly, research focusing on the cancer cell's ability to deregulate apoptosis by preventing the activation of Bax or Bak will provide further insight into the development of targeted therapies for cancer that will hopefully contribute to the cure of this formidable disease.Src, the classic oncogenic kinase, has been found to deregulate Bax activation in response to the detachment of a cell from its substratum support thereby preventing anoikis, the Bax-dependent apoptotic response involved in the impairment of metastatic dissemination of cancer. Our findings indicate that Src deregulates this response by altering the relative expression of Bcl-2 family members Mcl-1 and Bim through the PI3-K/Akt and Erk1/2 pathways. However, Src retains its ability to prevent anoikis even in the setting of Akt and Erk1/2 signaling inhibition. Further evaluation of the role of Src in this process revealed that Bif-1, a protein known to associate with and activate Bax, could be directly phosphorylated by Src which prevented the association of Bax with Bif-1 and impaired the anoikis response.In addition, our studies have also found that Bax activation in response to treatment with type I and II histone deacetylase inhibitors is dependent on the expression of the tumor suppressor p53. Acetylation of p53 at carboxy-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here, we demonstrate that p53 acetylation at K320/K373/K382 is also required for its transcription-independent functions in Bax activation, ROS production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) SAHA and LAQ824. Knockout of p53 in HCT116 cells markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cancer cells to HDACi-mediated Bax-dependent apoptosis, whereas knockdown of endogenous mutant p53 inhibited HDACi-induced apoptosis. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70.The association between these two proteins was acetylation independent, but acetylation of p53 could prevent and disrupt the Ku70/Bax complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.
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Advisor: Hong-Gang Wang, Ph.D.
x Cancer Biology
t USF Electronic Theses and Dissertations.
Regulation of Bax Activation and Apoptosis by Src and Acetylated Mutant p53 By Nicholas Taylor Woods A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cancer Biology College of Graduate Studies University of South Florida Major Professor: Hong-Gang Wang, Ph.D. Alvaro Monteiro, Ph.D. Jiandong Chen, Ph.D. Jie Wu, Ph.D. Date of Approval: August 25 2008 Keywords: Anoikis, Bif-1, Cancer, Ku70, Mcl-1, Metastasis Copyright 2008, Nichol as Taylor Woods
Note to the Reader The original of this document contains colo r that is necessary for understanding the data. The original dissertation is on file with the USF library in Tampa, Florida.
Dedication I would like to dedicate this disserta tion to all my family friends who have supported me throughout the years. I owe the en tirety of my success in the pursuit of my doctorate degree to my wife Neha. She has be en a constant source of love and support, and our countless conversations on science have immeasurably enhanced my growth as a researcher. This work is also dedicated to my parents who have taught me the value of hard work and perseverance that has driven me to accomplish my goals, my friends who have provided needed laughter and distract ion, and to my grandfather Royal who has been a source of unlimited inspiration. Words cannot begin to express my gratitude to all of you.
Acknowledgments I would like to acknowle dge my mentor Dr. HongGang Wang whose diligent guidance and encouragement has been the key to my growth as a researcher. Thank you for devoting your time and resources to my development. He has given me many opportunities to enhance my education and rese arch which have allowed me to build a solid foundation onto which I hope to build a successful career. I would like to thank Dr. Hirohito Yamaguchi for the training he gave me during our time working together in the lab. He has been an excellent collaborator on each of the three projects found below, and has contribu ted equally to the data found in chapters three and four within this dissertation. I would like to thank my committee members for the time they have given to support my pursuit of the doctorate degree. Your comments and suggestions have provided valuable insight into my research projects and enhanced my education. I would like to thank Dr. St even Frisch for taking time to come to my dissertation defense and act as the outside chair. I would also like to than k the Cancer Biology Program for the opportunity to be accepted into the program and for the rese arch it has allowed me to pursue.
i Table of Contents List of Figures....................................................................................................................iv Abstract....................................................................................................................... .........x Introduction................................................................................................................... .......1 Cancer ......................................................................................................................1 Hallmarks of Cancer....................................................................................1 Oncogenes................................................................................................................2 Src................................................................................................................3 Structure and Regulation of Src...................................................................4 Src Signaling................................................................................................5 Anoikis.....................................................................................................................7 Integrin Signaling.........................................................................................8 Src Mediated Suppression of Anoikis........................................................10 Apoptosis...............................................................................................................11 Extrinsic Apoptotic Pathway.....................................................................13 Intrinsic Apoptotic Pathway......................................................................14 Bax Activation...........................................................................................16 Bcl-2 Family Regulation of Bax....................................................17 Regulation of Bax by non-Bcl-2 Family Proteins..........................19 Bim.....................................................................................20 Mcl-1..................................................................................21 Bif-1...................................................................................22 Ku70...................................................................................24 p53..........................................................................................................................25 Deregulation of p53 in Cancer...................................................................26 Transcription-Dependent p53 Apoptosis...................................................27 Transcription-Independent p53 Apoptosis.................................................29 Src Mediated Suppression of Anoikis................................................................................31 Abstract..................................................................................................................31 Results ....................................................................................................................31 Src Signaling Ablates th e Anoikis Response due to an Inhibition of Bax Activation.................................................................31 Mcl-1 and Bim are Critical Regulators of Anoikis....................................34 Mcl-1 and Bim Regulate DetachmentInduced Bax Activation.............................................................................40 Anoikis is Regulated by Prot easomal Degradation of Mcl-1....................42 Mcl-1 Degradation Elicit s a Robust Anoikis Response.............................46 Mcl-1 Degradation is Mediated by GSK-3 ..............................................49
ii Src Regulated Akt and Erk Signaling Control Mcl-1 and Bim Expression........................................................................53 Metastatic Cancers Exhibit Reduced Mcl-1 Degradation and Bim Induction......................................................................................56 Discussion..............................................................................................................61 Materials and Methods...........................................................................................64 Reagents.....................................................................................................64 Plasmids.....................................................................................................65 Cell Culture and Transfection....................................................................66 Caspase-3, Chymotrypsin, and LDH release.............................................66 Semi-Quantitative RT-PCR.......................................................................67 In Vivo Metastasis Model...........................................................................67 Src Phosphorylation of Bif-1 Disrupts th e Interaction of Bif-1 with Bax and Inhibits Anoikis.......................................................................................................68 Abstract..................................................................................................................68 Results ....................................................................................................................68 Bif-1 Phosphorylation In Vivo Is Repressed upon Apoptotic Stimuli.......................................................................................68 Src Interacts with Bif-1 in Both Yeast and Mammalian Cells...................71 Src Phosphorylation of Bif-1 at Y80.........................................................75 Src Phosphorylates Bif-1 at Y80 in Intact Cells........................................77 Bif-1Y80 Phosphorylation by Src Prevents Bif-1 Mediated Bax Activation during Anoikis..................................................80 Discussion..............................................................................................................83 Materials and Methods...........................................................................................86 Metabolic Labeling and In Vitro Kinase Assays.......................................86 Yeast Two-Hybrid Assays.........................................................................86 Expression and Purifi cation of GST-Bif-1................................................87 Immunoprecipitation and Immunoblot Assays..........................................87 Retrovirus Production................................................................................88 Cell Culture and Transfection....................................................................89 p53 Acetylation Is Crucial for Its TranscriptionIndependent Proapoptotic Functions..............................................................................90 Abstract..................................................................................................................90 Results ....................................................................................................................90 Transactivation Activity of p53 Is Dispensable for Apoptosis Induced by SAHA or LAQ824.................................................90 C-Terminal Acetylation Is Essential for Mutant p53Mediated Apoptosis in Response to SAHA or LAQ824 Treatment .................................................................................102 SAHAor LAQ824-Mediated ROS Production Is Dependent on p53 Acetylation and Bax..................................................110 Acetylated p53 Binding to Ku70 Activates Bax by
iii Disrupting the Bax/Ku70 Complex.........................................................113 Discussion............................................................................................................127 Transcription-Indepe ndent p53 Mechanism of HDACi-induced Apoptosis......................................................................129 p53 Localization and HDACi-Induced Apoptosis...................................131 p53 Acetylation and Bax/Ku70 Disruption..............................................133 Materials and Methods.........................................................................................135 Cell Culture and Transfection..................................................................135 Reagents...................................................................................................136 Tandem Affinity Purification of p53QS Binding Protein.........................136 GST-Ku70/Bax/p53 In Vitro Binding Assay...........................................137 Immunoprecipitation................................................................................137 Subcellular Fractionation.........................................................................138 Caspase Assay, ROS Measurement and Annexin V Staining.................138 Luciferase Assays....................................................................................139 Protein Purification..................................................................................140 In Vitro Disruption of Endogenous Bax/Ku70 Complex.........................141 Immunostaining.......................................................................................142 Scientific Significance & Future Directions....................................................................143 Src Regulation of Anoikis........................................................................144 Ku70 and p53 Interaction.........................................................................147 HDAC Inhibitor Regulation of Apoptosis...............................................150 References Cited..............................................................................................................1 53 About the Author...................................................................................................End Page
iv List of Figures Figure 1. Domain structur e of c-Src and v-Src.................................................................5 Figure 2. Integrin signaling networks..............................................................................10 Figure 3. Apoptotic Pathwa ys and Bax Organization.....................................................15 Figure 4. Posttranslational modifications of p53.............................................................26 Figure 5. Active Src prevents anoikis.............................................................................32 Figure 6. Active Src prevents de tachment-induced Bax activation................................33 Figure 7. Src inhibition restores Bax activation in response to detachment...................34 Figure 8. De novo protein synthesis promotes anoikis....................................................36 Figure 9. Detachment induced changes in the microarray profile of apoptosis related genes...............................................................37 Figure 10. Caspase activity is not required for detachment-induced Bax activation.............................................................37 Figure 11. Bcl-2 family protei n expression during anoikis...............................................38 Figure 12. Semi-quantitative RT-P CR analysis of Mcl-1 and Bim in response to anoikis...................................................................................40 Figure 13. Knockdown of Bim prevents Bax activation in response to detachment..............................................................................41 Figure 14. Overexpression of Mcl-1 or Bim alters the anoikis response..........................42 Figure 15. Proteasome inhibition prevents detachment-induced Bax activation............................................................43 Figure 16. MG132 mediated protea some inhibition represses detachment-induced caspase-3 activation..................................................44 Figure 17. Velcade-mediated proteasome inhibition stabilizes Mcl-1 and prevents detachment-induced Bax activation............................45
v Figure 18. Proteasome mediated suppression of anoikis is mediated by the accumulation of Mcl-1....................................................46 Figure 19. Mcl-1 degradation alone is sufficient to induce anoikis in the presence of Bim...............................................................................48 Figure 20. Anoikis in Mcl-1-IR ES-Bim cells induced by CHX can be prevented by proteasome inhibition................................................49 Figure 21. Inhibition of GSK-3 signaling stabilizes Mcl-1 and prevents Bax activation and anoikis.......................................................51 Figure 22. Knockout of GSK-3 prevents detachment-induced Mcl-1 degradation.......................................................................................52 Figure 23. Mutation of the GSK-3 phosphorylation site on Mcl-1 prevents detachment-induced degradation......................................52 Figure 24. Src signaling through Akt and Erk1/2 controls the expression profiles of Mcl-1 and Bim during detachment...........................54 Figure 25. Knockdown of Mcl-1 enha nces the anoikis response in c-Src 527F cells treated with kinase inhibitors......................................55 Figure 26. Metastatic progression in osteosarcoma is associated with deregulation of Mcl-1 and Bim...........................................................57 Figure 27. The concerted effects of decreased Mcl-1 and increased BimEL expression correlate with enhanced anoikis response.....................59 Figure 28. Mcl-1 depletion sens itizes human breast cancer cells to anoikis.............................................................................................60 Figure 29. Mcl-1 depletion re duces human breast cancer metastasis in vivo ........................................................................................61 Figure 30. Bif-1 phosphorylation......................................................................................69 Figure 31. Phosphorylation of Bif-1 is inhibited by cellular stress...................................70 Figure 32. Bif-1 is dephosphoryl ated in a time-dependent manner upon treatment with staurosporine..................................................70 Figure 33. Bif-1 interacts with c-Src in the yeast two-hybrid assay.................................71
vi Figure 34. Schematic representation of the domain structure of Bif-1 and mapping the Sr c binding domain of Bif-1...................................72 Figure 35. Bif-1 interacts w ith Src in mammalian cells....................................................74 Figure 36. Dephosphorylation of Bif1 can be achieved via two independent mechanisms in response to cellular stresses...........................74 Figure 37. Minimal Bif-1 sequence required for Src phosphorylation.............................76 Figure 38. Src phosphorylates Bif-1 at Y80 in vitro .........................................................77 Figure 39. Src phosphorylates Bif-1 in intact cells...........................................................78 Figure 40. Src phosphorylates Y 80 of Bif-1 in whole cells..............................................79 Figure 41. Endogenous phosphorylation of Bif-1 by c-Src...............................................80 Figure 42. Src phosphorylation of Bif-1 inhibits Bax conformational activation during anoikis....................................................82 Figure 43. Src phosphorylation of Bif-1 inhibits caspase-3 activation and cell death in response to cell detachment.............................83 Figure 44. Treatment with HDACi does not robustly induce p53QS transactivation....................................................................................91 Figure 45. p53 status affect s HDACi-induced cell death..................................................92 Figure 46. The transactivation-deficient p53QS mutant sensitizes HCT116 p53-/cells to HDA Ci-induced caspase-3 activation....................93 Figure 47. HDACi promotes stabiliza tion of exogenously expressed p53.......................93 Figure 48. Expression of p53 mutants in HCT116 p53-/background enhances Bax activation induced by HDACi...........................94 Figure 49. Expression of p53 muta nts in HCT116 p53-/background enhances caspase-3 activation induced by HDACi.....................................95 Figure 50. Naturally occurring p53 mutations promote Bax activation after HDACi treatment................................................................95
vii Figure 51. Expression of naturally occurring p53 mutants promotes caspase-3 activity in response to HDACi....................................................96 Figure 52. p53 expression and acetyl ation in response to HDACi and proteasome inhibition in H1299 cells...................................................97 Figure 53. HDACi but not proteasome i nhibitor induced cell death is enhanced by mutant p53 expression..........................................................98 Figure 54. Knockdown of mutant p53 in SW480 and HT-29 cell lines............................99 Figure 55. Knockdown of mutant p53 in SW480 and HT-29 cell lines prevents caspase-3 activation and cell death....................................100 Figure 56. Knockdown of mutant p53 in SW480 cells prevents HDACi-induced apoptosis........................................................................101 Figure 57. Knock-in of mutant p53 in MEFs enhances HDACiinduced apoptosis......................................................................................102 Figure 58. Substitution of K320, K373 and K382 to R attenuates the proapoptotic activity of mutant p53 in response to HDACi......................104 Figure 59. Mutation of K320, K373 and K382 to R attenuates the proapoptotic activity of p53QS initiated by HDACi in HCT116 cells............................105 Figure 60. p53-dependent HDACi-induced cell death is repressed by mutation of three C-terminal Lysine residues............................................106 Figure 61. Overexpression of SirT1 doe s not impact p53 acetylation or p53-dependent HDACi-induced apoptosis...............................................107 Figure 62. Pharmacological inhibition of SirT1 enhances p53 acetylation....................108 Figure 63. SirT1 inhibition does no t affect p53-dependent HDACiinduced apoptosis......................................................................................109 Figure 64. HDACi-induced p53-dependent ROS production promotes cell death.........110 Figure 65. K3R mutation prevents HDACi-induced p53-dependent ROS production........................................................................................111 Figure 66. Bax expression promot es HDACi-induced cell death...................................112 Figure 67. Bax expression promotes ROS generation in response to HDACi ...............113
viii Figure 68. TAP-p53QS mediated HDACi-induced cell death..........................................114 Figure 69. Identification of Ku70 in complex with TAP-p53QS.....................................115 Figure 70. Ku70/p53 binding is independent of p53 acetylation at K320/373/382...........................................................................................115 Figure 71. Increased p53 and Ku70 expression and complex formation is induced by proteasome inhibition or HDACi............................................116 Figure 72. In vitro pull-down of GST-Ku70 and purified p53........................................116 Figure 73. Ku70 associates with endogenous mutant p53 with and without HDACi treatment..........................................................117 Figure 74. Acetylation of p53QS is required for HDACi induced Bax activation without impact on Bcl-2 family member expression........................................................................118 Figure 75. Ku70/Bax complex stability after HDACi treatment is regulated by p53....................................................................119 Figure 76. Endogenous wild type p53 re tains the ability to disrupt the Bax/Ku70 complex...................................................................................119 Figure 77. Acetylation deficient p5 3 expression prevents HDACiinduced disruption of Ku70/Bax complex................................................120 Figure 78. Purified acetylated p53 disrupts Bax/Ku70 complexes in vitro.....................121 Figure 79. Ku70 knockdown preferentially en hances apoptosis in p53-/cells.............122 Figure 80. Cellular localization of p53/Ku70/Bax interactions......................................124 Figure 81. Cellular localization of p53, Ku70 and Bax in HCT116 cells.......................125 Figure 82. p53-dependent HDACi-induced Bax translocation from the nucleus to the cytoplasm............................................................................126 Figure 83. HDACi-induced ace tylated p53-dependent Bax activation and mitochondrial accumulation.............................................127 Figure 84. Proposed model for p53/Ku70/ Bax interaction disrupted by HDACi...........128
ix Figure 85. Wild type and mutant p53 interaction with Bcl-XL and Bak........................132
x Regulation of Bax Activation and Apopto sis by Src and Acetylated Mutant p53 Nicholas T. Woods ABSTRACT Apoptosis is an inherent suicide mech anism that cells invoke for a variety of reasons including embryo cavitation, tissu e homeostasis, excessive DNA damage and aberrant oncogene activation. Apoptosis is regulated by a diverse set of proteins including, but not limited to, the Bcl-2 family. This family set is comprised of both prodeath and pro-survival proteins whose relative expression, lo calization and/or modifications regulate the bala nce between life and death for each cell. The keystones to this system are the proapoptotic proteins Bax and Bak, which are regulated by their conformation and localization. However, th e exact mechanisms by which Bax and Bak become activated remains to be resolved. Si milarly, research focusing on the cancer cells ability to deregulate apoptosis by preventing the activation of Bax or Bak will provide further insight into the development of targ eted therapies for cancer that will hopefully contribute to the cure of th is formidable disease. Src, the classic oncogenic kinase, has been found to deregulate Bax activation in response to the detachment of a cell from its substratum support thereby preventing anoikis, the Bax-dependent a poptotic response involved in th e impairment of metastatic dissemination of cancer. Our findings indicate that Src deregulates this response by altering the relative expression of Bcl-2 fa mily members Mcl-1 and Bim through the PI3K/Akt and Erk1/2 pathways. However, Src retain s its ability to prevent anoikis even in
xi the setting of Akt and Erk1/2 si gnaling inhibition. Further evaluation of the role of Src in this process revealed that Bif-1, a protein known to associate with and activate Bax, could be directly phosphorylated by Src which preven ted the association of Bax with Bif-1 and impaired the anoikis response. In addition, our studies have also fo und that Bax activa tion in response to treatment with type and II histone deacetylase inhibitors is dependent on the expression of the tumor suppressor p53. Acetylation of p53 at carboxy-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here, we demonstrate that p53 acetylation at K320/K373/K382 is also required for its transcription-independent f unctions in Bax activation, ROS production, and apoptosis in response to the histone deacetylase inhib itors (HDACi) SAHA and LAQ824. Knockout of p53 in HCT116 cells markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 va riants sensitized p53null cancer cells to HDACi-mediated Bax-dependent apoptosis, whereas knockdown of endogenous mutant p53 inhibited HDACi-induced apoptosis. Evalua tion of the mechanisms controlling this response led to the discovery of a nove l interaction between p53 and Ku70. The association between these two proteins was acetylation independent, but acetylation of p53 could prevent and disrupt the Ku70/Bax complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.
1 Chapter One: Introduction Cancer Cancer is a complex group of diseases that exhibit similar cellu lar traits such as uncontrolled growth, invasiveness, and someti mes metastasis of abnormal cells. When the spread of cancer is uncontrolled the re sult is often death. The lifetime risk of developing cancer in men is 1 in 2, and for wo men it is 1 in 3. Cancer can originate at many different sites but the cancer with the hi ghest mortality rate is lung and bronchus in both men and women. Cancer associated mo rtalities remain alarmingly high accounting for 1 in every 4 deaths and averaging over ha lf a million per year in the United States alone. Although cancer is the second leading cause of death in the United States after heart disease, over the years little advancement in improving survival rates has occurred compared to that for heart disease (Jemal et al., 2007). The diversity and heterogeneity of cancer has been the largest obstacle to advanced clinical therapies. It has become clear that the path to a cure will likely be a long-term research endeavor culminating in personalized therapeutics that will incorporate the lessons learned in a variety of different fields. Hallmarks of Cancer The causes of cancer are diverse in nature and include environmental factors such as chemical carcinogens, ionizing radiation and viruses as we ll as internal factors such as inherited genetic mutations, hormones and th e immune system. Theref ore, the cause of
2 cancer is generally cryptic in nature with the exception of those caused by cigarette smoking or alcohol consumption, but even in these cancers it appears that the combination of genetic predisposition and carcinogen consumption determines cancer formation and progression (Sellers et al., 1992 ). Nonetheless, all cancer cells exhibit specific traits termed the Hallmarks of Cancer that dictate malignant progression (Hanahan and Weinberg, 2000). These hallmarks are self-sufficiency in growth signals, resistance to anti-growth si gnals, evasion of cell-death, li mitless replicative potential, angiogenesis, and tissue invasion and metastasis. The multiplicity of these hallmarks reflects the progressive nature cancer must exhi bit to overcome biologi cal barriers to their formation and dissemination. Multiple molecu lar pathways have been identified that control each one of these hallmarks and curre nt research has focused on targeting these different pathways to eliminate cancer. Oncogenes Oncogenes are protein encoding genes th at drive malignant transformation and progression when mutated, overexpressed or hyp eractive. There have been a number of oncogenes identified since the in itial discovery of Src in th e 1970s (Erikson et al., 1980). The normal cellular counterparts are termed pr oto-oncogenes to indicat e their potential to drive oncogenesis if activated. Proto-onc ogenes can be activat ed by qualitative or quantitative mechanisms. Chromosomal transloc ations can cause oncogenes to be driven by promoters that are constitutively active in the target cells, such as the well known Philadelphia chromosome translocation t( 9:22)(q34:q11) resulting in the oncogenic fusion product BCR-Abl found in chronic myelogenous leukemia and acute leukemia
3 (Kurzrock et al., 1987). Gene amplifications of oncogenes can also lead to aberrant signaling. An example of th is is the dihydrofolate reductase gene ( DHFR ) amplification found in methotrexate-resistant acute lymphoblastic leukemia (Alt et al., 1978). Qualitative alteration of BRAF through the mutation V599E results in a constitutively active protein that drives th e MAP kinase pathway (Davie s et al., 2002). Since oncogenes are well studied and their role in cancer is generally pronounced, many therapeutic agents have been derived that specifically target th em. One of the most successful inhibitors of oncogenes developed has been imatinib (Gl eevec) targeting BCR-Abl, which has been highly effective in the treatment of Philadelphia chromosome positive leukemias. Src The discovery of the first oncogene, Src, was set in motion by the discovery of the cell-free transmission of a malignant avian virus by Peyton Rous in 1910, for which he was awarded the Nobel Prize (1966). This re trovirus was later found to encode for the viral oncogene v-Src which shared seque nce homology with DNA found in normal avian genetic material (Stehelin et al., 1976). Src was determined to exhibit protein kinase activity required for its tran sformation potential (Collett et al., 1979; Levinson et al., 1978). The original v-Src was later determined to have a deletion of the c-terminal negative regulatory domain and was therefore constitutively active. The full-length mammalian homolog, c-Src, is often overexpressed, hyperactivated or mutated in a number of cancers of epithelial origin (Fra me et al., 2002; Irby et al., 1999; Jacobs and Rubsamen, 1983; Rosen et al., 1986). Infreque nt Src mutation in cancers results in a truncated and activated protei n (Irby et al., 1999; Sugimura et al., 2000). Given Srcs well
4 established role in tumorigenesis, therapeuti c disruption of its mech anisms of action has been aggressively pursued. Structure and Regulation of Src Src is a non-receptor tyrosine kinase that localizes in the cytoplasm, but can also be recruited to membranes via N-terminal myristolation. Its three key domains are the SH3 domain, SH2 domain and the kinase dom ain (Figure 1). The SH2 domain recognizes pTyr motifs and is responsible for the auto inhibition of cSrc as it binds phosphorylated Tyr530 ( Homo sapiens nomenclature; Tyr527 in chicken) in the c-termin al regulatory domain. Binding of the SH2 domain to pTyr530 also promotes the interaction of the SH3 domain with the polyproline tract located within the linker regi on between the SH2 and kinase domains of Src. These coordinated eff ects lead to structural changes in Src that inactivate the kinase activity of the pr otein (Yamaguchi and Hendrickson, 1996). Phosphorylation of Tyr530 can be accomplished by proteins such as CSK or its homolog CHK. Alternatively protein tyro sine phosphatases SHP1, SHP2, PTP or PTP1B can dephosphorylate Src resulting in an open activ e conformation of the kinase (Yeatman, 2004). Competitive binding of the SH2 do main by focal adhesion kinase (FAK), epidermal growth factor receptor (EGFR) or erbB2 can also promote the active conformation of Src (Cobb et al., 1994; Schaller et al., 1994). Li kewise, SH3 binding by the HIV associated Nef protein can promote Sr c family kinase activity (Moarefi et al., 1997). Full activation of Src is accompan ied by autophosphorylation of tyrosine 419 within the kinase domain. These multiple modes of Src regulation intricately regulate downstream signaling that control cell su rvival, proliferation, angiogenesis and
metastasis. Figure 1. Domain structure of c-Src and v-Src. (Yeatman, 2004) Src Signaling Src is involved in diverse and numerou s signaling pathways. Src family kinases (SFKs), including Lck, Lyn, Fyn, Blk, Fgr, Hc k, Yrk, and Yes, are also involved in a broad spectrum of cellular signa ling, but for the sake of brev ity this introduction will only attempt to summarize important Src signaling ca scades relevant to the research outlined below. For an excellent comprehensive revi ew on SFK signaling in different biological pathways please refer to Thomas and Brugge, 1997. One of the most studied functions of Src is its role in the signaling cascade of focal adhesions. These structures regula te cell morphology, attachment and mobility. 5
6 Many focal adhesion associated proteins are also substrates for v-Src such as focal adhesion kinase (FAK), p130CAS, paxillin, talin, vinculin, tensin, and 1 integrin subunit, which are phosphorylated on tyrosine residu es when localized to these structures (Thomas and Brugge, 1997). Specifically, Sr c directly phosphorylates FAK which controls FAK kinase activity and creates binding sites for recr uitment of proteins to FAK complexes thereby perpetuating downstream signaling that promote cell migration and survival (Calalb et al., 1996; Cobb et al., 1994; Schlaepfer and Hunter, 1996). The role of Src in cell migration was also demonstrated in src-/fibroblasts which show reduced cell motility that could be rescued by kinase-ac tive but not inactive Src (Hall et al., 1996). However, there is also evidence that the SH 2 and SH3 domains of Src act as a scaffold responsible for assembly of FAK signaling complexes independent of Srcs kinase activity (Brunton et al., 2005; Schlaepfer et al., 1997). Src is also an upstream mediator of su ch pathways as PI3-K, Erk1/2, and STAT3 (Fincham et al., 2000; Park et al., 1999; Yu et al., 1995). Like Src, these cell signaling pathways are responsible for regulating a di verse set of biological functions. The phosphatidylinositol 3-kinase (PI3-K) and ex tracellular regulated kinase (Erk1/2) pathways are often deregulated in cancer by overexpression or mutation of key regulatory elements such as Src. The PI3-K regulator y subunit p85 can interact with Lck and Abl which directly phosphorylate the subunit at tyrosine 688 allo wing the catalytic domain of p110 of PI3-K to become activated (von Wille brand et al., 1998). The PI3-K pathway is also deregulated by mutation or loss of phos phatase with tensin homology (PTEN). When PTEN is disrupted, the cell lo ses the ability to negatively regulate PI3-K dependent formation of the second messenger lipid phos phatidylinositol (3,4,5) triphosphate (PIP3),
7 which results in constitutive activation of the Akt pathwa y responsible for apoptosis inhibition and cell cycle progression (Cully et al., 2006). Overactive Src signaling can also promote aberrant signaling thro ugh the Erk1/2 pathway by promoting phosphorylation of the adaptor protein Shc which then leads to enhanced signaling through the Grb2-SOS-Ras pathway (Blake et al., 2000; Ravichandr an, 2001; Salcini et al., 1994). Ras activation can also lead to the activation of PI3-K th rough direct binding to the p110 subunit (Sjolander et al., 1991; Walker et al., 1999). These convoluted pathways are essential to the progression of cancer as they collectively promote the tumor phenotype. However, for the purposes of this re port the focus of their signaling outcomes will be their antiapoptotic potentiation. Anoikis The development of a metastatic phenotype is a late event in tumor progression. Metastases are an indicator of poor prognosis. In fact, 90% of the mortalities associated with cancer arise from metastatic dissemi nation (Weigelt et al., 2005). In order for a tumor cell to acquire the metastatic phenot ype it must first overcome or bypass the intrinsic cellular response of anoikis, detach ment induced apoptosis. When normal cells are detached from the extracellular matrix (E CM) and deprived of integrin signaling they die by a process termed anoikis (Frisch a nd Francis, 1994). Anoikis is essentially a normal apoptotic reaction in response to th e specific stimulus of improper integrin engagement. The execution of a cell by anoikis stimulation can rely on several different mechanisms of apoptosis. For instance, inhi bition of the extrinsic apoptotic cascade results in the inhibition of a poptosis (Frisch, 1999). However, regulation of the intrinsic
8 pathway of apoptosis can also effectively prevent anoikis in a variety of systems. Anoikis is essential for common biological processes involved in homeostasis, morphogenic changes, and inhibition of cancer metastasis. Integrins not only function as structural adhesive support be tween the cell and the ECM, they also serve as a scaffold for intracellular signaling proteins (Hynes, 2002). This signaling activates pathways involved in survival and proliferation which pr events anoikis. Cells of different origins express unique sets of integr ins and preferentially adhere to specific complements of ECM ligands. Therefore, when a cell comes in to contact with the improper complement of ECM ligands, signaling becomes insufficien t and anoikis soon follows (Pullan et al., 1996). However, upon mobilization some metastat ic cells can change their complement of integrin receptors to adapt to a new ECM environment to prevent anoikis (Montgomery et al., 1994). Therefore, it is es sential to understand in tegrin function and their signaling pathways to decipher the mechanisms of anoikis initiation. Integrin Signaling The ECM provides adhesive support to normal tissues and controls numerous signals that regulate divers e cellular processes such as survival, growth, and differentiation (Hynes, 1999). These signaling mechanisms are regulated by integrins. Integrins are heterodimeric pr oteins that consist of two and type-I transmembrane subunits. There are 18 and 8 subunits that can be expressed in mammals, which can combine to form 24 recognized integrins. Thes e integrins bind specific components of the ECM, but some overlapping is found between su bstrate recognition of different integrins (Guo and Giancotti, 2004). Integrin engagement of extracellular matrix substrates leads
9 to the formation of focal adhesions, as men tioned previously. These focal adhesions are membrane attachment plaques that couple th e ECM attached transmembrane integrins to the intracellular actin cytoskel eton and other cellular structural components (Jockusch et al., 1995). Integrin signaling affects many di fferent pathways such as the Erk1/2, PI3K/Akt, NFB and others through Src family kinase mediated signal propagation (Figure 2). In this way, integrins regulate biological functions such as cell su rvival, proliferation and migration. Integrin activation of the aforem entioned signaling pathways is highly reminiscent of extracellular grow th factor receptors. In fact, growth factor signaling by epidermal growth factor receptor (EGFR) can suppress the anoikis response in mammary epithelial cell line MCF-10A (Reginato et al., 2003). Detachment of this cell line leads to a downregulation of EGFR expression, repr essed Erk1/2 signaling, and enhanced Bim expression leading to apoptosis. Further crosstalk between growth factor receptors and integrins has been documented in both primary oligodendrocytes and mammary epithelial cells that enhances growth factor signaling and survival (Gilmore, 2005). Given the high interconnectedness between integrin and grow th factor receptor pathways, it will be important to determine the individual contri butions of inhibited growth factor and integrin signaling in anoikis.
Figure 2. Integrin signaling networks (Guo and Giancotti, 2004). Src Mediated Suppression of Anoikis When cells are deprived of integrin signaling due to ECM detachment these prosurvival pathways are ablated which triggers the anoikis response. Therefore, in order for a cell to suppress anoikis it must acquire the means to compensate for the lost signaling and prevent apoptosis. Aggressive breast, co lon, and lung malignancies with a propensity to metastasize have been shown to lack th e normal apoptotic response after detachment from the supporting matrix (Shanmugathasan and Jothy, 2000; Wei et al., 2001; Yawata et al., 1998). Moreover, cancer cel ls that are more apt to re sist anoikis due to genetic 10
11 mutation or overactive survival signaling ar e increasingly likely to initiate distal metastases (Douma et al., 2004). Src has been implicated in processes th at increase the metastatic potential of cancer cells including cell adhe sion (Kaplan et al., 1995), migration (Rahimi et al., 1998), and invasion (Zhang et al., 2004). Transient in creases in Src activity upon detachment in both normal and cancer cells is critical to in hibition of anoikis (L oza-Coll et al., 2005; Wei et al., 2004). Active c-Src likely promotes survival once the cell is detached from the ECM by compensating for the loss of integrin and growth factor signaling. However, the direct mechanisms that potentiate Src mediated anoikis suppression are poorly understood. Two of the projects th at have been included in this report attempt to delineate specific mechanisms of Src action in the pr evention of anoikis a nd the promotion of metastatic dissemination of cancers (Woods et al., 2007; Yamaguchi et al., 2008). The findings presented herein provi de insights into Src mediated disruption of Bax activation required for anoikis. Apoptosis Apoptosis is a multi-step process that cu lminates in the self -destruction of an individual cell. This self-killing mechanis m proceeds through a highly regulated set of events that lead to the activ ation of cellular cysteine pr oteases known as caspases (Wolf and Green, 1999). Apoptosis is characterized by specific bi ochemical and morphological characteristics. This process is essential to counteract normal proliferation resulting in relatively steady state in an organisms total cell number. The deregulation of apoptosis has been characterized in many different dis eases. Neurodegenerative disorders are often
12 the result of increased apoptosis of neuronal lineage cells. Al ternatively, inhibition of the normal apoptotic response often results in the development of cancer when combined with sustained proliferation (Thompson, 1995). Luckily, there are many built-in regulatory mechanisms that maintain apoptosis at acceptable levels. Morphological and biological features that distinguish apoptosis from other forms of cell death such as necrosis or autophagy include cell shrinkage, membrane blebbing, nuclear condensation, activation of cellula r caspases, DNA fragmentation, mitochondrial membrane disruption, and exposure of phosphatidyl serine (PS) on the ou ter leaflet of the plasma membrane (Fadok et al., 1992; Green and Evan, 2002; Reed, 1995). Unlike necrosis, apoptosis is a rather clean proce ss in that there is limited or no release of inflammatory intracellular components. The expo sure of PS results in the recruitment of macrophages that recognize the signa l and engulf the apoptotic cell. Apoptosis can be triggered by a number of different stimuli. Depending on the specific stimulus, apoptosis can be initiated through one of two independent pathways referred to as intrinsic or extrinsic based on the origin of the initiating apoptotic signal. The intrinsic pathway of apoptos is is regulated by the integr ity of the outer mitochondrial membrane (OMM) and the Bcl-2 family members that control it. Alternatively, the extrinsic pathway of apoptosis is regulated by transmembrane receptors that are activated by ligand specific binding. Whereas the intrinsic apopt otic response is determined by the integrity of the OMM, extrinsic apoptosis may or may not require OMM disruption for efficient apoptosis.
13 Extrinsic Apoptotic Pathway The extrinsic pathway of apoptosis is initiated upon binding of ligand to their cognate receptors. These receptors, ominous ly dubbed death receptors such as TRAIL, TNFR1-2, DR4, DR5 and Fas/CD95 all contain cysteine rich extracellular domains that allow specific binding of lig and resulting in trimerization and activation (Naismith and Sprang, 1998). This clustering of death receptors results in the recruitment of cytoplasmic adapter proteins FADD or TRADD to the comp lexes, which interact with the death domains (DD) of the receptor via their own DD. FADD also contains a death effecter domain (DED) which recruits initiator caspase-8 and/or caspase-10, via their own DED domain, to the receptor resulting in the formation of the death inducing signaling complex (DISC). The enhanced proximity of these initiator caspases promotes their proteolytic processing into mature proteases (Ashkenazi, 2002) (Figure 3A). Apoptosis resulting from death receptor signaling can be classified as type I or type II. Type I extrinsic apoptosis occurs when activation of initiator casp ases alone is sufficient to activate effecter caspases-3 and 7 which then promote the enzymatic destruction of the cell. However, the much more common type II extrinsic apoptosis relies on the involvement of the intrinsic apoptotic pathway and outer mitochondrial membrane permeabilization through caspase-8 mediated enzymatic cleavage of Bid into two fragments. The C-terminal 15 kDa fragment, tBid, translocates to the mitochondria and acts as a potent inducer of a poptosis through its interaction with Bcl-XL and Bax (Luo et al., 1998). This mechanism allows for the amplif ication of the initial apoptotic insult and the efficient execution of apoptosis.
14 Intrinsic Apoptotic Pathway Bcl-2 was originally identified in follicular lymphoma based on the common translocation t(14:18) in this tumor type (Tsuji moto et al., 1985). Originally classified as an oncogene, it was later found that expressi on of Bcl-2 had no effect on cell cycle progression and instead specifically prev ented cell death in response to stimuli (Thompson, 1995). Subsequent identification of th e rest of the Bcl-2 family members and several non-Bcl-2 proteins has led to our current understandi ng of the basic functions of these proteins in the regulation of apoptosis. The Bcl-2 family is composed of opposing subsets of proteins such as antiapoptotic members Bcl-2, Bcl-XL, and Mcl1 which protect the integrity of the OMM through inhibition of the multi-domain proapopto tic proteins Bax and Bak. Activation of proapoptotic molecules Bax and Bak is associat ed with the loss of OMM integrity and the release of apoptogenic proteins such as cytochrome c, AI F, endonuclease G, HtrA2/Omi and Smac/Diablo (Green, 2005). Release of cyto chrome c into the cytoplasm allows it to bind Apaf-1 culminating in the formation of the apoptosome with the recruitment of procaspase-9 (Figure 3A). The clustering of procaspase-9 results in autolytic processing and maturation of the initiator caspase-9 and subsequent activation of downstream effecter caspases and the resultant apoptos is. Release of HtrA2/Omi and Smac/Diablo prevent the inhibition of cellular caspase activity by inactivating the inhibitors of apoptosis (IAP) proteins such as cIAP and XIAP. Endonuclease G and AIF are both nucleases that once release d, translocate to the nucl eus and participate in DNA fragmentation (Daugas et al., 2000; van Loo et al., 2001).
A B Figure 3. Apoptotic Pathways and Bax Organization. (A) Extrinsinc and Intrinsic Apoptotic Pathways (Ashkenazi, 2002). (B) Native conformation of inactive Bax (Suzuki et al., 2000). 15
16 Bax Activation Bax activation is a multi-step process th at converts cytoplasmic monomers into pore forming membrane bound oligomers and is a key initiating step in the execution of intrinsic apoptosis and anoikis (Gilmore et al., 2000; Lindsten et al., 2000). Activation involves a conformational rearrangement, r ecruitment to the mitochondrial membrane, insertion into the membrane, oligomerizati on and pore formation. In aqueous solution Bax exists as a globular prot ein similar to other Bcl-2 family members (Petros et al., 2004). Bax is composed of 9 helices separated by amorphous loop regions (Figure 3B). The two core helices 5 and 6 are largely hydrophobic and are surrounded by the seven remaining amphipathic helices. These seven helices maintain th eir hydrophobic regions toward the center of the protein and their hydrophilic regions toward the exterior, which promotes the solubility of the protein and its localization to the cytoplasm. The BH1-3 domains of Bax form a hydrophobic pocket that is occupied by the C-terminal helix 9. Bax conformational activation involves the dissociation of helix 9 from the hydrophobic pocket. This C-terminal helix of Bax is responsible for the anchoring of Bax to intracellular membranes. Akt mediat ed phosphorylation of S184 in helix 9 or mutation to a charged residue prevents Bax localization to the mitochondria even under apoptotic conditions (Gardai et al., 2004). Similarly, S 184 deletion or mutation to valine results in constitutive association of Ba x with the mitochondria, but wi thout another stimulus this alone does not stimulate the apoptotic res ponse (Nechushtan et al ., 1999). Rather, the core hydrophobic helices 5 and 6, which strongly resemble the pore forming domain of diphtheria toxin, are required to be exposed in order for pore formation to occur. Exposure of these two helices is responsible fo r insertion of Bax into the OMM and as an
17 isolated region they able to insert into s ynthetic membrane lipids and form pores (Annis et al., 2005; Garcia-Saez et al., 2006). However, Bax localization to the OMM is independent of helices 5 and 6 and instead requires the exposure of the normally restricted N-terminal domain consisting of amino acids 20-37 (Cartron et al., 2003). Exposure of this N-terminal domain can be assessed using the conformation specific Bax 6A7 antibody. As evidenced by these previous findings, in order for efficient Bax mediated apoptosis this pr otein must undergo extensive rearrangement of its threedimensional structure for proper targeting, insertion and pore formation. Although much is already known about the changes in Bax required for OMM permeabilization, the exact mechanism by which Bax is persuaded to undergo such gross rearrangement remains to be resolved. Bcl-2 Family Regulation of Bax Bax activation status is re gulated by other Bcl-2 protei ns consisting of both proand antiapoptotic molecules with opposing functions. The antiapop totic Bcl-2, Bcl-XL, Mcl-1, and Bcl-w proteins seque ster proapoptotic BH3-only pr oteins as well as Bax and Bak. Currently two models have been propos ed to explain the function of BH3-only molecules in the activation of Bax and Ba k. In the displacement model, BH3-only proteins bind to antiapoptotic proteins a nd cause the release of Bax and Bak allowing them to become activated. In this model, BH 3-only proteins Bim, tB id, and Puma are the most potent inducers of Bax activation pres umably for their broad affinity for the majority of the antiapoptotic proteins. Ind eed, the different BH3only proteins exhibit drastically different affinities to antia poptotic molecules (Certo et al., 2006).
18 Contrastingly, in the direct binding model Bim, tBid and Puma are classified as activators because they can directly activate Bax (D esagher et al., 1999; Harada et al., 2004; Kuwana et al., 2005; Letai et al., 2002; Wei et al., 2000). The remaining BH3-only proteins, such as Noxa, Bmf, Hrk, Bik, Bad, Bl k, and Bnip3, are classified as sensitizers and bind to antiapoptotic proteins and preven t their inhibitio n of the BH3-only activators as well as Bax and Bak. Although both models adequately account for the regulation of Bax and Bak activation, definitive experiment al evidence is still needed to resolve discrepancies in both models. Although BH3-only proteins may affect Ba x activation through several different mechanisms as hypothesized in the direct and indirect models, problems with both models still remain to be resolved. For instance, evidence that BimEL can directly associate with Bax is lacking. Co-immunoprecipi tation of the two proteins has only been successfully tested in exogenous expression systems and this model is highly contested due to the lack of interacti on seen by others (Harada et al ., 2004; Marani et al., 2002). Interestingly, the BimS isoform is able to induce apoptosis wit hout interacting with antiapoptotic proteins (Weber et al., 2007). This accumulating evidence suggests that the multitude of different Bim isoforms can disp lay proapoptotic functions in a mutually exclusive manner. Our lab has also determin ed that Bcl-XL can protect against Bax activation and cytochrome c release independent of its direct inte raction with Bax and Bim (Yamaguchi and Wang, 2002). Also, chemi cal inhibition of Bcl-2 and Bcl-XL by ABT-737 does not promote substantial a poptosis when Mcl-1 is expressed. Concomitantly, when Mcl-1 is knocked down, a poptotic induction still requires another stimulus. Interaction between the Bcl-2 family members is highly dependent on
19 conformation. Therefore, it is essential to cri tically analyze experimental methods used to probe the molecular interactions between these proteins as detergents used to make cell lysates have a large impact on the findings. Fu ture work is needed to determine how the molecular interactions occur, where they occur and their biological significance. Regulation of Bax by nonBcl-2 Family Proteins Non-Bcl-2 proteins Ku70, Bif-1, human in peptide, p53, 14-3-3, cardiolipin, Tom22, and ASC have been identified as poten tial mediators of Bax activation and it is likely that their individual ro le is determined by several f actors such as the apoptotic insult and cell lines tested (Chipuk et al., 2004; Cohen et al., 2004a; Guo et al., 2003; Kuwana et al., 2002; Nomura et al., 2003; Ohtsuka et al., 2004; Ott et al., 2007b; Takahashi et al., 2005). Ku70, 14-3-3 and hum anin control apoptosis through their inhibition of Bax activation by sequestering inactive Bax away from the mitochondria and preventing its activation (G uo et al., 2003; Nomura et al ., 2003; Subramanian et al., 2005). These retention factors may explain how Bax is kept in the inactive state when localized to the cytoplasm w ithout direct interaction with Bcl-XL which resides on the mitochondria. Although less well characterized than Bcl-2 proteins, these retention factors add another layer of control into the scheme of Bax activation and provide additional prospective therapeutic targets to potentiate an apoptotic response. Non-Bcl-2 family membrane proteins are also required for Bax activation, as pretreatment of purified mitochondrial membra nes with protease K prevents tBid induced Bax oligomerization (Roucou et al., 2002). Similarly, tBid and Bax can efficiently release pre-loaded dextran from outer mitochondrial vesicles compared to chemically defined
20 protein-free liposomes (Kuwana et al., 2002). However, controve rsy persists as to which mitochondrial proteins are required for Ba x activation (Ott et al ., 2007b). The research and discussion found in the following secti ons will explore the mechanisms of Mcl-1, Bim and Bif-1 regulation and their impacts on Bax activation in response to detachment from adhesive support, as well as the novel mechanism of acetylated p53 regulation of Bax activity through a Ku70 dependent manner. Bim As mentioned previously, the two competing hypotheses of direct and indirect BH3-only protein regulation of Ba x activation remain to be re solved. One of the proteins at the center of this conflict is Bim. A lthough the exact mechanism is debated, the fact remains that there is a positive correlation between Bim expression and apoptosis which can be prevented by overexpression of antiapoptot ic proteins or the absence of Bax and Bak. The expression of Bim is regulated at bot h the transcriptional and posttranslational levels. Transcription is controlled by seve ral transcription factors, most notably FKHRL1, Foxo3a (Dijkers et al ., 2000; Gilley et al., 2003). Akt directly phosphorylates Foxo3a to promote its interaction with 14-3-3 and retention in the cytoplasm (Brunet et al., 1999). There have been many different is oforms of Bim identified but the most predominantly expressed are BimEL, L and S (EL, extra long; L, long; S, short) (Miao et al., 2007). At the posttranslational level, Bim can be regulated by phosphorylation events. Erk1/2 phosphorylates BimEL on Ser69 thereby targeting the protei n for ubiquitination and degradation via the proteasome, which leads to inhibition of apopt osis as a result of repressed expression (Akiyama et al., 2003; Luciano et al., 2003). Akt can also
21 phosphorylate Bim at Ser87 and mutation of th is site is associat ed with enhanced proapoptotic activity (Qi et al., 2006). Mouse mode ls have also identified phosphorylation of Bim at Thr112 to be impor tant for Bcl-2 bindi ng and repression of apoptosis (Hubner et al., 2008). Bi m is also maintained in an inactive state by binding to the dynein motor complex or by sequestration to the mitochondria by antiapoptotic Bcl-2 family members, such as Bcl-XL and Mcl1 (Puthalakath et al ., 1999; Yamaguchi and Wang, 2002). One of the reasons Bim is thought to be such an efficient activator of apoptosis is its ability to inte ract with most antiapoptotic Bcl-2 proteins. However, Mcl-1 has been shown to have a higher affinity for Bim compared to Bcl-2 and Bcl-XL (Gomez-Bougie et al., 2005). Mcl-1 Mcl-1 is a short-lived prot ein with a half-life of a pproximately 30 minutes. It sequesters BH3-only proteins such as Bim a nd tBid to the mitochondria and prevents activation of Bax and Bak (Clohessy et al., 2006; Opferman et al., 2003). Mcl-1 is regulated transcriptionally by the Src/ STAT3 pathway (Niu et al., 2002), and posttranslationally by Mule, M cl-1 u biquitinl igase E 3, which is able to ubiquitinate Mcl1 and promote its degradati on (Zhong et al., 2005). GSK-3 phosphorylation of Ser159 on Mcl-1 enhances its ubiquitination and de gradation via the proteasome (Maurer et al., 2006). The resulting decrease in Mcl-1 expressi on levels promotes apoptosis. Mcl-1 is also a substrate for cellular caspases. Truncati on of Mcl-1 may result in the acquisition of a proapoptotic function that can enhance the a poptotic response similar to that observed for caspase cleaved Bcl-2 and Bcl-XL, but ther e is still some uncertainty as to what
22 impact this truly has (Clohessy et al., 2004; Weng et al., 2005). In relation to metastasis, it was determin ed in a Lewis lung carcinoma model that the expression of Mcl-1 was dire ctly related to metastatic pot ential of the tumor as these cells were more resistant to hypoxia-indu ced apoptosis (Koshikawa et al., 2006). Therefore, the selection of tu mor cells with high levels of Mcl-1 may also give rise to anoikis resistant metastatic ce lls. Indeed, our research has demonstrated Mcl-1 to be a critical regulator of anoikis. Specifically, Src activity can preven t detachment induced Mcl-1 depletion through Akt me diated suppression of GSK-3 activity. This results in decreased phosphorylation of Mcl-1 at S 159 which promotes its stabilization and inhibition of Bax activation afte r detachment from the ECM. In this study, we have id entified the stabilization of Mcl-1 and suppression of Bim as critical events during oncogenic suppression of anoikis. Th e transition of cells to a metastatic phenotype correlates not only with increased Mcl-1 expressi on, but also to the altered regulation of its degradation profile in response to detach ment. Inhibition of survival signals mediated by active Src or downstream Akt and Erk1/2 kinases that control Mcl-1 degradation and Bim induction is able to rest ore Bax activation and anoikis susceptibility. Furthermore, Mcl-1 repressi on, but not Bcl-2 or Bcl-XL inhibition, is capable of initiating anoikis in metastatic cancer cells. This study is the first to characterize Mcl-1 degradation in anoikis and the oncogenic signaling that can disrupt this essential mechanism in human cancers. Bif-1 Bax interacting factor-1 (Bif-1), also known as SH3GLB1 or Endophilin B1, was
23 initially identified by two independent groups as a Bax interacting molecule (Cuddeback et al., 2001; Pierrat et al., 2001) even though it lacks homol ogy with the Bcl-2 family. Bif-1, like other members of the Endophilin family, contains a carboxy-terminal SH3 domain, binds to membranes through thei r N-BAR domain and promotes membrane curvature (Gallop et al., 2006; Masuda et al ., 2006; Peter et al., 2004). Bif-1 localizes on the membranes of intracellular organelles such as the Golgi and mitochondria (Karbowski et al., 2004; Takaha shi et al., 2005; Yang et al., 2006). Importantly, Bif-1 directly interacts with Bax and enhances the kinetics of apoptosis induction by promoting conformational activation of Bax and Bak in response to intrinsic apoptotic signals (Cuddeback et al., 2001; Takahashi et al., 2005). Bax pore formation in large unilamellar vesicles is accompanied by structural changes in the lipid bilayer caused by monol ayer curvature (Terrones et al., 2004), and Bax has a tendency to accumulate at sites of fission and fusion on the OMM where lipids are likely to deviate from a bilayer structure (Karbowski et al., 2002). Together, these previous findings suggest that Bax pore formation can be regulated by mitochondrial proteins and those that alter membrane curvature, such as Bif-1, are increasingly likely to affect Bax conformational activation. However, the mechanism by which Bif-1 accomplishes this regulation of Bax remains unclear. The data presented below illustrates that Src signaling can prevent the initiation of anoikis by inhibiting both Mcl-1 degradati on and Bim induction. However, depletion of Mcl-1 along with induction of Bim by blocking the Akt and Erk1/2 signaling is unable to illicit a robust anoikis response in the pres ence of Src activity. Ther efore, Src must be acting at multiple levels to prevent Bax act ivation during detachment. In this study, we
24 found that Src directly intera cts with Bif-1 and phosphorylates Bif-1 on tyrosine 80. This phosphorylation event has a direct impact on th e ability of Bif-1 to bind Bax which correlates with a repression of Bax activation during anoikis. Ku70 Ku70 was originally iden tified as an essential re gulator of nonhomologous end joining (NHEJ) DNA double strand break repa ir and is also e ssential for V(D)J recombination (Lieber et al., 2003). Ku70 associates with K u80 to form the heterodimeric protein Ku that binds to both fragment ends of DNA double strand breaks to maintain them in close proximity. Recruitment of other NHEJ proteins such as DNA-PKCS, Artemis and DNA-ligase IV results in the repair of these DNA lesions. The Ku70/Ku80 complex has also been shown to be an essential regulator of telomere maintenance (Tuteja and Tuteja, 2000). Ku70 knock-out mice are viable but smalle r than their wild type counterparts, exhibit increased apoptosis in neuronal cells during development, are immune compromised and are hypersensitive to ionizing radiati on (Gu et al., 2000; Manis et al., 1998; Ouyang et al., 1997). Ku70 is an abundant protein in the nucleus but also has been found to localize to the cytoplasm and may play a role in cell-cell or cellmatrix adhesion (Muller et al., 2005; Subramanian et al., 2005). In addition to its role in DNA damage repair, Ku70 forms an inhibitory complex with Bax that impairs apoptosis initiation. This complex is disrupted by acetylation of the carboxy-terminus of Ku70 mediated by CBP /p300, PCAF, SirT1 and HDACi treatment (Cohen et al., 2004a; Cohen et al., 2004b; Subramanian et al., 2005). The interaction between Ku70 and Bax also leads to decrease d levels of ubiquitinated Bax but does not
25 affect the total pool of unmodified Bax (A msel et al., 2008). This may lead to the enrichment of unmodified Bax that is more efficient at initia ting apoptosis. Ku70/Bax association may serve a dual purpose of bot h inhibiting Bax translocation to the mitochondria while ensuring that adequate pools of functional Bax are available when needed. Specifically, we have found that acetylated p53 can also have a profound effect on the association between Ku70 and Bax. p53 The tumor suppressor p53 is a highly regul ated transcription factor that has a fundamental role in the prevention of tu morigenesis and response to chemotherapy, making it one of the most comprehensively st udied molecules in cancer research. Normal cells exhibit low levels of p53 in resting conditions to maintain cellular homeostasis and prevent aberrant apoptosis. Cellular stress leads to rapid incr eases in p53 protein levels as a result of combined increases in tran scription, translation and posttranslational modifications that repress de gradation and enhan ce its activity. The characterization of p53 function is often accompanied by specific posttranslational modifications such as acetylation, methylation, ubiquitination, neddylation, and sumoyl ation (Figure 4) (Brooks and Gu, 2003; Toledo and Wahl, 2006). These m odifications regulate diverse aspects of p53 function ranging from cellular localization, DNA binding, protein-protein interactions, and transcriptional regulation.
Figure 4. Posttranslational modifications of p53 (Toledo and Wahl, 2006) Deregulation of p53 in Cancer A multitude of different tumo rigenic mutations of p53 have been characterized. It is estimated that approximately half of all cancers have mutated p53. The other half retains wild type p53 but exhi bit deregulated pathways of p53 control which generally leads to the constitutive repression of p53 expression thereby prohi biting its proapoptotic functions. The vast majority of p53 mutati ons occur in the central DNA-binding domain of p53. These mutations prevent p53 from binding DNA and inhibit th e transcriptional activation of its target genes. The deregulati on of wild type p53 in cancer can be caused by the overexpression of its negative regulator MDM2. Aberrant MDM2 mediated degradation of p53 can be caused by gene amplification of MDM2 or the loss of ARF, which directly binds to MDM2 in response to cellular stresses and i nhibits its function (Momand et al., 1998; Ruas and Peters, 1998). This amazing rate of deregulation across 26
27 cancers illustrates the central role of p53 as a tumor suppressor. Knock-out of mdm2 results in an embryonic lethal phenotype that can be rescued by complimentary knock-out of p53. This is presumably because in the absence of Mdm2, p53 becomes overexpressed and causes aberrant apoptosis during embryogenesis. Mutation of a single allele of p53 is thought to functionally inactivat e wild type p53 as it exists in a tetramer for proper transcriptiona l activity and the presence of a single mutant protein in this complex is inhibitory. Interestingly, p53+/R172H / mdm2-/mice fail to be born, however p53+/R172H embryos exposed to ionizi ng radiation show decreased apoptosis in the hypothalamus compared to p53+/mice (Lang et al., 2004). Furthermore, tumor development and metastatic potential in p53+/R172H mice have distinct spectrums dependent upon the strain of mice (Lang et al ., 2004; Olive et al., 2004). Therefore, the function of mutant p53 varies dependi ng upon the genetic background and cellular context in which it is assayed. Much rese arch remains to be done to determine the multitude of factors that determine the func tion of both wild type and mutant p53 in different cellular settings a nd in response to stresses. Transcription-Dependent p53 Apoptosis Expression of Mdm2, an E3-ligase respons ible for targeting p53 for proteasomal degradation, is transcriptionally activated by p53, which results in a negative feedback loop designed to maintain p53 at appropriate levels. Other E3 ligases including COP1, Pirh2 and ARF-BP1 are also capable of ubi quitinating p53 and repressing its expression (Chen et al., 2005; Dornan et al., 2004; Leng et al., 2003). Cellular stresses cause posttranslational modifications to p53 which negatively affects the ability of Mdm2 to
28 bind p53. For instance, DNA damage results in phosphorylatio n of p53 by ATM and Chk2 at serine 15 and serine 20, respectivel y (Toledo and Wahl, 2006). These two sites are within the Mdm2 binding domain of p53 and prevent the interaction between these two proteins. Phosphorylation of the N-te rminus of p53 results in the increased association with histone acetyltransferases p300/CBP which can also cause acetylation of p53 at multiple lysine residues. Interestingly, Mdm2 appears to have several contacts points with p53 as it has recently been disc overed that the C-te rminus and DNA binding domains of p53 can also bind Mdm2 (Tang et al., 2008; Yu et al., 2006). Recruitment of Mdm2-p53 complexes to p53 responsive prom oters results in th eir transcriptional repression (Ohkubo et al., 2006). Acetylation of p53 can prevent Mdm2 interaction with p53 at the p21 promoter and relieve its repr essive effects. These multiple modes of Mdm2 mediated repression of p53 has led to the development of novel small molecule inhibitors that disrupt their interact ion such as Nutlin-3 (Vassilev, 2007). Activated p53 can potentia te cell cycle arrest and apoptosis by inducing transcription of numerous cel l cycle regulators and proapoptotic genes such as p21, Bax, DR5 and Puma as well as transcriptional repr ession of antiapoptotic proteins Bcl-2 and Mcl-1 (el-Deiry, 1998; Fridman and Lowe, 2003; Miyashita et al., 1994; Pietrzak and Puzianowska-Kuznicka, 2008; Yu et al., 2001). Histone acetyltransferases (HATs) such as p300/CBP or PCAF and histone deacetylases (HDACs) such as HDAC1, 2, 3 or SIRT1 regulate the acetylation of p53 at specific lysine re sidues (Gu and Roeder, 1997; Luo et al., 2000; Sakaguchi et al., 1998; Vaziri et al., 2001) The amount and specificity of p53 acetylation is regulated by diverse mechanisms in response to various cellular stresses (Luo et al., 2004; Sakaguchi et al., 1998). Acetylation of p53 enhances its DNA
29 binding activity and stability and may be responsible for determining the specific context in which p53 will function (Gu and Roed er, 1997; Luo et al., 2004). HDACi-induced hyperacetylation of p53 results in p53 transactivation-depend ent apoptosis (Henderson et al., 2003; Roy et al., 2005; Terui et al., 2003). Transcription-Independent p53 Apoptosis In addition to its function as a tran scription factor, p53 promotes cell death through transcription-independent mechanisms as it can cause apoptosis in the presence of transcription and translation inhibitors (Caelles et al., 1994). In addition, p53 induces transcription-independent apoptosis through increasing Fas cell surface translocation and activation of caspase-8 (Bennett et al., 1998; Ding et al., 2000). Interestingly, mutant p53 lacking transcriptional activity is fully capable of inducing apoptosis (Bissonnette et al., 1997; Chipuk et al., 2003; Haupt et al., 1995; Kokontis et al., 2001; Yamaguchi et al., 2004). The best characterized transcriptionindependent apoptotic function of p53 involves the intrinsic mitoc hondrial apoptotic pathway. Although normally a cytoplasmic protein, cellular stresses promote wild t ype p53 accumulation in the cytoplasm and mitochondria where it induces mitochondria l outer membrane permeabilization through displacement of BH3-only proteins from Bcl-2 and Bcl-XL, promoting the oligomerization of Bak and the activation of Bax (Chipuk et al., 2003; Dumont et al., 2003; Erster et al., 2004; Leu et al., 2004; Pietsch et al., 2007). Furthermore, p53 may directly interact with Bax and promote it s activation (Chipuk et al., 2004). Conversely, unlike their wild type count erparts, some naturally occurring DNA-binding domain
30 mutants of p53 lack the capacity to bind Bcl-XL and promote cytochrome c release at the mitochondria (Mihara et al., 2003; Tomita et al., 2006). This leaves open the possibility that mutant p53 may promote apoptosis in a transcription-independe nt manner other than its previously described role at the mitochondri a. It is increasingly apparent that p53 has a dual role in the initiation of apoptosis through both tr anscription-dependent and independent mechanisms. Transcriptionindependent binding to Bcl-XL and displacement of Bax can combine with tran scription-dependent upr egulation of Puma which can directly activate Bax. In this report, we provide evidence that HDACi induce apoptosis through a p53 acetylation-dependent but transactivationindependent mechanism. Expression of transactivation deficient p53 mutants in p53-null HCT116, H1299, K562, and MEF cells enhances apoptosis initiated by HDACi. C onversely, knockdown of endogenous mutant p53 in HT-29 and SW480 cells abrogates SAHA or LAQ824 induced apoptosis. Purification of p53 binding prot eins and mass spectrometry analysis revealed a novel interaction between p53 and Ku70. This intera ction is independent of p53 acetylation. However, acetylation at its carboxy-terminus is required for p53 to prevent and/or displace Bax from its inhib itory interaction with Ku70, thus allowing this key proapoptotic member of the Bc l-2 family to target mitochondria, generate ROS, and initiate apoptosis in resp onse to HDACi. Furthermore, knockdown of Ku70 promotes apoptosis in p53-null but not p53 mutant ce lls treated with HDACi. These results highlight a novel mechanism by which acetylat ed p53 restrains the Bax/Ku70 interaction to potentiate HDACi-induced apoptosis.
31 Chapter Two: Src Mediated Suppression of Anoikis Abstract Anoikis, a Bax dependent apoptosis triggered by detachment from the extracellular matrix, is often dysfunctional in metastatic canc er cells. Using wild type and c-Src transformed NIH3T3 cells as a mode l we identified Mcl-1 degradation and Bim upregulation as a critical dete rminant of anoikis initiation. Detachment rapidly degraded Mcl-1 via a GSK-3 -dependent proteasomal pathway a nd transcriptionally upregulated Bim expression. Mcl-1 degradation in the presence of Bim was sufficient to induce anoikis. By analyzing non-me tastatic Saos-2 and metastatic derivative LM7 cells, we confirmed that dysregulation of Mcl-1 degr adation and Bim inducti on during detachment contributes to decreased anoikis sensitivity of metastatic cells. Furthermore, knockdown of Mcl-1 or pharmacological inhibition of the PI3-K/Akt and MAPK pathways that suppress Mcl-1 degradation a nd Bim expression could markedly sensitize metastatic breast cancer cells to anoiki s and prevent metastases in vivo Therefore, Mcl-1 degradation primes the cell for Bax activa tion and anoikis, which can be blocked by oncogenic signaling in metastatic cells. Results Src Signaling Ablates the A noikis Response due to an Inhibition of Bax Activation The anoikis response is known to be i nhibited in cells expressing the Src oncogene (Windham et al., 2002), but the mechanisms involved are poorly understood.
To determine the characteristics of the anoi kis response, parental and Src transformed NIH3T3 cells were forcibly detached on polyH EMA coated plates fo r varying lengths of time. Wild type cells were observed to rapidl y loose viability in a time dependent manner while cells with active c-Src 527F and v-Src exhibited limited cell death as determined by a trypan blue exclusion assay (Figure 5A). The loss of viability in wild type cells correlated to the activation of caspase-3 as measured by the DEVDase activity (Figure 5B). Figure 5. Active Src prevents anoikis. (A) Wild type, c-Src 527F, and v-Src NIH3T3 cells were detached on poly-HEMA coated plates and assayed for viability at the indicated timepoints by trypan blue exclusion assay. (B) Activity of caspase-3 in the aforementioned ce ll lines after 0, 8, and 12 hours of detachment. Data represented as mean + SD, n=3. The activation of Bax is known to be an initiating event in the activation of the caspase cascade during anoikis (Valentijn et al., 2003). Therefore, we assessed the activation status of Bax by i mmunoprecipitation (Figure 6) with the Bax 6A7 monoclonal antibody and immunostaining (Figure 7) with the Bax N20 polycl onal antibody. Both of 32
these antibodies specifically recognize conformationally active Bax. Figure 6 illustrates that Bax activation is completely inhibited in 527F and v-Src cells while wild type cells exhibit a time dependent increase in the conformationally changed Bax protein beginning as early as 8 hours post detachment. Sim ilarly, the immunofluorescence staining in Figure 7 demonstrates that Bax becomes active in wild type cells but not 527F expressing cells in response to cell detachment. Howeve r, treatment with the Src family kinase inhibitor dasatinib clearly restored det achment-induced Bax activ ation in 527F cells (Figure 7A & B). Figure 6. Active Src prevents detachment-induced Bax activation. Wild type, v-Src, and c-Src 527F cells were forcibly de tached on poly-HEMA coated plates for 0, 8, 12, and 24 hours. Active Bax was immunoprecipitated using the conformation specific Bax 6A7 antibody. 33
Figure 7. Src inhibition restores Bax activation in response to detachment. (A) NIH3T3 wild type and c-Src 527F cells were immunostained for active Bax in attached and 8 hours detached samples using the conforma tion specific Bax N20 antibody. c-Src 527F cells were treated with control DMSO or 50 nM dasatinib during detachment. (B) Quantif ication of the percentage of cells staining positive for active Bax; e xperiment represented as average + SD, n=3. Att, attached. Det, de tached. Das, dasatinib. Mcl-1 and Bim are Critical Regulators of Anoikis To determine the role of protein neog enesis in the activation of Bax during anoikis, cycloheximide (CHX) was used to block de novo protein synthesis in both wild type and 527F cells detached on polyHEM A. CHX markedly reduced detachment34
35 induced Bax activation and cellular caspase-3 act ivity in wild type cells as well as 527F cells treated with dasatinib (Figure 8A & B). This prompted us to examine the gene expression profiles associated with anoikis in 527F cells treated with or without dasatinib by microarray (Figure 9). There was a clear increase in the induction of Bim and Puma, both known activators of Bax. There was also a slight decrease in transcription of the antiapoptotic proteins Mcl-1 and Bcl-XL. Memb ers of the caspase family were generally unchanged; however, their invol vement in Bax activation was ruled out through the use of the pan caspase inhibitor z-VAD-fmk which was unable to inhibit Bax conformational change during detachment (Figure 10).
Figure 8. De novo protein synthesis promotes anoikis. Wild type and c-Src 527F NIH3T3 cells were cultured on polyHEMA-coated plates containing DMSO control or 50 nM dasatinib, 10 g/mL CHX or the combination of dasatinib and CHX for 8 hours and subjected to (A) immunoprecipitati on with anti-Bax 6A7 antibody and (B) caspase-3 activity assay; mean + SD, n=3. 36
Figure 9. Detachment induced change s in the microarray profile of apoptosis related genes. NIH3T3 cells expressing cSrc 527F were detached on polyHEMA-coated plates in the presen ce of DMSO or 50 nM dasatinib for 8 hours and subjected to microarray analysis. The bar graph shows the upand down-regulation of apoptosis genes by da satinib compared to DMSO control. Figure 10. Caspase activity is not required for detachment induced Bax activation. NIH3T3 wild type cells were assayed for conformationally active Bax in control attached samples and 8 hours det ached conditions in the presence of either DMSO control or 25 M z-VAD-fmk. The microarray analysis allowed us to form a short list of Bcl-2 family proteins that are differentially transcribed between anoikis responsive and unresponsive cells. However, the prevalence or absence of tran scripts does not always coincide with the 37
expression of the protein product. To this end, we examined the protein expression profiles of BimEL, Puma, Bcl-XL, Mcl-1 a nd Bax in 527F cells detached and treated with dasatinib or DMSO and compared them to wild type cells (Figure 11). Several reports have suggested that Bc l-XL is induced as the result of Src signaling and that this provides resistance to anoiki s (Coll et al., 2002; Rosen et al., 2001). However, we observed little or no decrease in Bcl-XL at the time of Bax activation in either dasatinib treated 527F or wild type cells. Similarly, ther e was no significant increase in the protein levels of Bax in either of the two cell type s. We did find that Mcl-1 and Bim were the most dynamically regulated proteins analy zed. In particular, Mcl-1 expression was substantially reduced while Bim was increase d during anoikis; this response was also found in detached 527F cells treated with dasa tinib indicating the relevance to a restored anoikis response by Src inhibition. Puma was moderately induced in dasatinib treated 527F but not in wild type cells, suggesting that Puma may not be a key regulator of anoikis. Figure 11. Bcl-2 family protei n expression during anoikis. Wild type and c-Src 527F 38
39 NIH3T3 cells were cultured in normal conditions or detached on polyHEMA-coated plates with DMSO or 50 nM dasatinib (Das) for 8 hours and subjected to western blot The results from the microarray in rela tion to Mcl-1 and Bim were validated using semi-quantitative RT-PCR in samples of wild type and 527F cells (Figure 12). Transcripts of Mcl-1 were marginally d ecreased in dasatinib treated 527F cells. Interestingly, there did not appe ar to be any decrease in the transcription of Mcl-1 in the wild type samples, indicating that post-transcriptional regu lation was responsible for the observed decrease in protein levels. Contra stingly, Bim transcri pts were increased dramatically by detachment in wild t ype and dasatinib treated 527F cells. Bim transcription is known to be positively regulated through the transcription factor Foxo3a which is negatively regulated by Akt (Gilley et al., 2003). Therefore, the ability of Akt signaling in 527F cells to i nhibit Bim expression was assessed through the use of LY294002, a PI3-K inhibitor. Indeed, this in hibitor restored Bi m induction, indicating that the Src/Akt/Foxo3a pathway is likely involved in the transcri ptional suppression of this proapoptotic protein.
Figure 12. Semi-quantitative RT-PCR analys is of Mcl-1 and Bim in response to anoikis. Wild type and c-Src 527F NIH3T3 cells were cultured in normal conditions or detached on polyHEMAcoated plates with DMSO, 50 nM dasatinib (Das), or 50 M LY294002 for 8 hours and subjected to semiquantitative RT-PCR analysis. Mcl-1 and Bim Regulate Detachme nt Induced Bax Activation The functional importance of increased Bim expression in the anoikis response was assessed by shRNA mediated targeted knockdown. Both wild type and 527F cells were infected with Bim shRNA (shBim) or cont rol retrovirus. Stable pools were detached on polyHEMA plates and their ability to init iate Bax activation and anoikis was assayed. Decreased Bim expression led to a similar decr ease in the activation of Bax (Figure 13A) as well as the caspase-3 activity (Figure 13B) in cells cultured on polyHEMA plates. These results suggest that Bim is the major ac tivator of Bax during anoikis, and the Puma induction seen in Figure 7 is of little functional relevance. 40
Figure 13. Knockdown of Bim prevents B ax activation in response to detachment. Wild type and c-Src 527F cells were in fected with control (Puro) or Bim shRNA (shBim) retroviruses and selected for 14 days on puromycin. The resulting puromycin-resistant transfectants were maintained in normal culture (Att) or detached (Det) on polyHEMA-coated plates with DMSO or 50 nM dasatinib for 8 hours and subjected to (A) immunoprecipitatio n with anti-Bax 6A7 antibody and (B) assayed for caspase-3 activity; mean + SD, n=3. To test the functional significance of Mcl-1 repression during detachment, increasing amounts of the Mcl-1 construct were transfected into wild type cells along with the pGL3 Luciferase repor ter construct. By measuring luciferase activity, we found that overexpression of Mcl-1 led to a dose dependent suppression of cell death in 41
detached as compared to attached conditions (Figure 14A). Bim alone was able to induce an apoptotic response in 527F cells as tr ansfection of increasi ng amounts of the Bim construct led to a dose dependent decrease in cell viability (Figure 14B). This demonstrates that decreased Mc l-1 expression is vital to the induction of anoikis and that overexpression of Bim can induce apoptosis in 527F expressing cells, presumably through a mechanism that activates Bax once antiapoptotic Bcl-2 me mbers are saturated by Bim. Figure 14. Overexpression of Mcl-1 or Bim alters the anoikis response. (A) Wild type (WT) and (B) Src 527F NIH3T3 cells were co-transfected with increasing amounts of Mcl-1 or Bim expr ession plasmids, respectively, along with 10 ng pGL3-actin luciferase repor ter plasmid. Luciferase expression was assayed as a measure of cell viability; mean + SD, n=3. Anoikis is Regulated by Prot easomal Degradation of Mcl-1 Mcl-1 is an ephemeral protein that is degraded in a proteasome dependent manner. This regulatory mechanism allowed us to study if stabili zed endogenous Mcl-1 42
contributes to inhibition of anoikis. As shown in Figure 15, the proteasome inhibitor MG132 was able to stabilize Mc l-1 in wild type NIH3T3 cel ls detached on polyHEMA in a dose dependent manner with maximal stab ilization occurring at 100 and 1000 nM. This stabilization of Mcl-1 correlated with a similar dose dependent decrease in Bax activation. We also observed th e stabilization of Mcl-1 and the complete inhibition of Bax activation in detached 527F cells treate d with the combination of dasatinib and MG132 (Figure 15). The addition of MG132 to the lysate of detached wild type cells had no effect on the ability to immunoprecipi tate active Bax (data not shown). Figure 15. Proteasome inhibition prev ents detachment-induced Bax activation. Wild type NIH3T3 cells were maintain ed as attached on normal plates or detached on polyHEMA-coated plates containing DMSO or increasing amounts of MG132. c-Src 527F NIH3T3 ce lls were detached and treated with DMSO or 50 nM of dasatinib with or without 100 nM of MG132 for 8 hours. Cells were then su bjected to anti-Bax 6A7 immunoprecipitation/immunoblot analysis. Samples from the wild type cells det ached and exposed to the MG132 gradient were also assayed for chymotrypsin-lik e activity and caspase-3 activity through in vitro 43
protease assays. In concordance with the above results, caspase-3 activity was decreased similar to the chymotrypsin activity inhi bited by MG132 (Figure 16). The repression of Bax activation and anoikis in wild type NI H3T3 cells was also observed in a similar manner using Velcade, a proteasome inhibitor curr ently in clinical trials (Figure 17). This study identifies the ability of proteasome inhibitors to pr event Bax activation and anoikis in response to detachment. Figure 16. MG132 mediated proteasome inhibition represses detachment-induced caspase-3 activation. Wild type NIH3T3 cells were collected in normal attached conditions (Att) or after 8 hours of forced detachment with treatment of DMSO control or increasing amounts of MG132. Chymotrypsin activity was assessed to determine the i nhibition of the proteasome mediated by MG132 and caspase-3 assay was performed to analyze apoptotic induction. 44
Figure 17. Velcade-mediated proteasome i nhibition stabilizes Mcl-1 and prevents detachment-induced Bax activation. Wild type NIH3T3 cells were assayed for Mcl-1, Bim, and Bax expression and the activation of Bax in attached (Att) and 8 hours detached cel ls that were untreated (NT) or incubated with DMSO or the indicated concentration of Velcade. To determine whether MG132 mediated in hibition of Bax activation was due to the stabilization of Mcl-1 but not anothe r unidentified protein( s), retroviral shRNA targeting Mcl-1 was infected into wild type cells to decrease its accumulation upon MG132 treatment. Three shRNA constructs we re designed, of which constructs 1 and 3 (c-1 and c-3) were the most effective at inhibiting Mcl-1 expression (Figure 18A). Treatment with MG132 led to the nearly comp lete inhibition of Bax activation in the Puro control line, but was less effective in suppressing the Bax c onformational change when there was less Mcl-1 st abilized. An inverse correl ation was found between the amount of Mcl-1 accumulate d in MG132 treated samples and the activation of Bax. Similarly, there is decrea sed repression by MG132 on caspase-3 activation in Mcl-1 knockdown cells compared to control (Figure 18B). These observations strongly suggest that anoikis is dependent on the proteasomal depletion of Mcl-1. 45
Figure 18. Proteasome mediated suppression of anoikis is mediated by the accumulation of Mcl-1. NIH3T3 cells stably ex pressing Mcl-1 shRNA (c-1 or c-3) were left in normal cultur e (control) or detached on polyHEMAcoated plates in the presence of DMSO or 100 nM MG132 for 8 hours and subjected to (A) anti-Bax 6A7 immunoprecipitation /immunoblot analysis and (B) caspase-3 assay. Mcl-1 Degradation Elicits a Robust Anoikis Response 46 Thus far it has been evident that bot h Mcl-1 degradation and Bim induction are key regulators of the normal anoikis res ponse. While overexpression of Mcl-1 can undoubtedly protect cells from anoikis (Figure 18), other groups have shown that overexpression of other antiapoptoti c Bcl-2 family proteins can also have the same effect (Rosen et al., 2000). Therefore, the vitality of Mcl-1 degradation for anoikis induction
47 was evaluated using retrovira lly transduced NIH3T3 cells co-expressing Bcl-2, Bcl-XL, or Mcl-1 with the proapoptotic BimEL count erpart via a bicistronic mRNA construct (Figure 19A). Repression of protein translation using CHX illus trates that the instability of Mcl-1 is essential for the induction of anoikis, while the co-expression of Bcl-2:Bim or Bcl-XL:Bim had no effect on cell death due to the maintenance of the antiand proapoptotic balance (Figure 19A & B). The anoikis response to Mcl-1 degradation could be completely resolved by MG132 mediated proteasome inhibition (Figure 20A & B). We confirmed that releasing Bim from th e Bcl-XL:Bim complex by ABT-737, a specific Bcl-2/Bcl-XL inhibitor, was al so capable of inducing an apoptotic response (Figure 20B). The uniquely short half-life of Mcl-1, compar ed to other antiapoptotic proteins such as Bcl-2 and Bcl-XL, makes this an esse ntial element of anoikis initiation.
Figure 19. Mcl-1 degradation alone is sufficient to induce anoikis in the presence of Bim. NIH3T3 cells were retrovirally tran sduced with constructs expressing Bcl-2-IRES-Bim, Bcl-XL-IRES-Bim, or Mcl-1-IRES-Bim. Cells were untreated or pretreated with 10 M CHX for 30 minutes then detached for the indicated times in the presence of CHX and analyzed for (A) expression of the proteins by immunoblot and (B) caspase-3 activation; mean + SD, n=3. 48
Figure 20. Anoikis in Mcl-1-I RES-Bim cells induced by CHX can be prevented by proteasome inhibition. NIH3T3 cells expressing Mcl-1-IRES-Bim or BclXL-IRES-Bim were untreated or pret reated for 30 minutes with CHX or CHX and MG132 then detached for 5 hours in the presence of their respective inhibitors and analyzed for (A) protein expression and (B) caspase-3 activation; mean + SD, n=3. Mcl-1 Degradation is Mediated by GSK-3 49 The phosphorylation of Mcl-1 by GSK-3 at Ser159 is known to promote an increased turnover of Mcl-1 (Maurer et al., 2006). To determine if this phosphorylation controls Mcl-1 degradation during anoikis, the GSK-3 inhibitor TDZD-8, a non-ATP
50 competitive inhibitor, was used to treat cells beginning immediately with detachment. As shown in Figure 21A, TDZD-8 was able to st abilize Mcl-1 in both wild type and 527F cells when detached or sensitized with dasa tinib, respectively. This stabilization was once again associated with a decr ease in Bax activation and inhibition of the apoptotic response (Figure 21A & B). To furt her validate the role of GSK-3 in the degradation of Mcl-1 induced by detachment, wild type and GSK-3 knockout MEFs were either attached or detached on polyHEMA plates for 12 hours in the presence of DMSO or LY294002. Detached wild type MEFs showed a d ecrease in the protein levels of Mcl-1 as did treatment with LY294002 (Figure 22). However, the GSK-3 null cells did not exhibit a decrease in Mcl-1 protein levels in response to detachment, and treatment with LY294002 resulted in only a slight decrease of Mcl-1; this d ecrease is likely due to the functional redundancy of GSK-3
Figure 21. Inhibition of GSK-3 signaling stabilizes Mcl-1 and prevents Bax activation and anoikis. Wild type and c-Src 527F 3T3 cells were maintained in normal culture or detached on polyHEMA-coated plates containing DMSO, 25 M TDZD-8, 50 nM dasatinib, or both inhibitors for 8 hours and subjected to (A) anti-B ax 6A7 immunoprecipitation/immunoblot and (B) caspase-3 activity assays; mean + SD, n=3. 51
Figure 22. Knockout of GSK-3 prevents detachment-induced Mcl-1 degradation. Wild type and GSK-3 null MEFs were treated with DMSO or 25 M LY294002 in normal or forced detachments conditions for 12 hours and Mcl1 expression was determined by western blot. To determine if phosphorylation of Ser 159 is required for Mcl-1 degradation during anoikis, constructs encoding the human Mcl-1 wild type and S159A mutant were transfected into wild type NIH3T3 cells. These cells were then detached and treated with CHX to observe the degradation of Mcl-1. Figure 23 shows that the half-life of wild type Mcl-1 is very short when compared to that of the S159A mutant. Th is suggests that Mcl-1 is degraded in response to a phosphorylation prim ing event that in turn targets the protein for ubiquitination and degradation. Moreover, detachment was found to decrease the levels of phospho-Akt in wild type NIH3T3 cells (data not shown), which may explain the increased degradation of Mcl-1 regulated by GSK-3 Figure 23. Mutation of the GSK-3 phosphorylation site on Mcl-1 prevents detachment-induced degradation. NIH3T3 cells transfected with wild type or S159A mutant Mcl-1 were deta ched on polyHEMA plates for the 52
53 indicated times in the presence of CHX to determine the half-life of Mcl-1 during detachment by immunoblot analysis. Src Regulated Akt and Erk Signaling Control Mcl-1 and Bim Expression Src is known to promote survival signa ling through multiple pathways. Two of the most well defined pathways controlled by Src are Akt and Erk, both of which have been implicated in preventing anoikis but debate remains over their involvement (Fukazawa et al., 2004; Martin et al., 2006). We therefore explored the consequences of Src, Akt, and Erk inhibition on Mcl-1 and Bim protein levels in 527F cells treated with dasatinib, LY294002, U0126, or the combin ation of LY294002 and U0126 during detachment (Figure 24A). Dasatinib inhibite d signaling through both Akt and Erk, while treatment with LY294002 or U0126 specifically inhibited their targets pathways. Inhibition of Akt but not Erk1/2 resulted in a significant decrease in Mcl-1 levels but did not extend to the levels observe d in dasatinib treated cells. Al so, Akt inhibition led to the appearance of multiple bands of Bim that are presumed to be Erk phosphorylated because U0126 inhibited these slower migrating bands and stabilized the Bim protein. Unlike dasatinib, however, treatment with LY294002, U0126 or the combination did not lead to the activation of Bax and caspase-3 (Figure 24A & B).
Figure 24. Src signaling through Akt and Erk1 /2 controls the expression profiles of Mcl-1 and Bim during detachment. c-Src 527F NIH3T3 cells were cultured in normal conditions (Att) or detached on polyHEMA-coated plates containing DMSO, 50 nM dasati nib, 50 M LY294002, 10 M U0126, or the combination of LY294002 and U0126 for 8 hours and subjected to (A) immunoprecipitation/immunoblot analysis with the indicated antibodies and (B) caspase-3 activity assay; mean + SD, n=3. It is possible that decreasing Mcl1 and increasing Bim expression by LY294002 and/or U0126 could not reach the threshold to shift the antiand proapoptotic balance of the cell towards anoikis. To test this possi bility, we used the c-3 shMcl-1 construct (Figure 18) to knockdown Mcl-1 expression in detached 527F cells treated with 54
dasatinib, LY294002, U0126, or the combin ation of LY294002 and U0126. Knockdown of Mcl-1 led to a more anoikis sensitive phenotype in the presence of these kinase inhibitors determined by casapse-3 activity (Figure 25A). Importan tly, the activation of Bax was also found to be enhanced in Mcl-1 knockdown cells over that of control (Figure 25B). This not only indicates that the level of Mcl-1 is critical for the initiation of Bax activation in response to Akt and Erk inhibiti on, but also illustrates the multi-faceted role of Src and implicates other unidentified Src pathways that contribute to anoikis suppression. Figure 25. Knockdown of Mcl-1 enhances the anoikis response in c-Src 527F cells treated with kinase inhibitors. c-Src 527F NIH3T3 cells stably expressing Mcl-1 shRNA (shMcl-1) or control vector (Puro) were treat ed as in Figure 24 55
56 and subjected to (A) caspase-3 activ ity assay and (B) immunoprecipitation with anti-Bax 6A7 antibody; mean + SD, n=3. Metastatic Cancers Exhibit Reduce d Mcl-1 Degradation and Bim Induction Resistance to anoikis is known to enhance the metastatic potential of cancer cells by affording them increased survival potenti al once detached from the ECM. Using the non-metastatic osteosarcoma cell line Saos-2 an d its metastatic deriva tive LM7 (Jia et al., 1999), we found that parental Saos-2 cells were sensitive to anoikis which correlated with Mcl-1 degradation and Bim induction (Figure 26A & B). However, LM7 cells exhibited increased basal levels of Mcl-1 and mainta ined this expression during detachment, as well as decreased induction of Bim. Inhibition of Src signa ling using dasatinib in both cell lines caused Bim induction and Mcl-1 degradation and increases in caspase-3 activity. The relatively slight increase in dasatinib sensitivity in LM7 cells likely indicates that oncogenic signaling other than Src is responsibl e for anoikis resistance. To determine if the stabilization of Mcl-1 in LM7 cells was required for their resistance to anoikis, we inhibited Mcl-1 expression using lentiviral delivered shRNA. Knockdown of Mcl-1 was able to restore caspase-3 activation to levels similar to parental Saos-2 cells, while non-targeting scrambled shRNA had no ef fect on anoikis compared to uninfected LM7 cells (Figure 26C).
Figure 26. Metastatic progression in osteosarcoma is associated with deregulation of Mcl-1 and Bim. Saos-2 and LM7 cells were untreated or detached on polyHEMA in the presence of DMSO or 50 nM dasatinib for 24 hours. Protein expression profiles were assayed by (A) immunoblot, and (B) apoptotic index was measured by caspase-3 activity. (C) LM7 cells were infected with lentivirus containing c ontrol shScr or shMcl-1 constructs. Stably infected cells were analyz ed for Mcl-1 expression by immunoblot (insert). The anoikis response of the transfectants was measured by the activation of caspase-3 in response to detachment and compared to the anoikis resistant parental LM7 cells; mean + SD, n=3. Similarly, the highly metastatic breas t cancer MDA-MB-231 cells were also 57
58 observed to be resistant to anoikis. This was correlated with the maintenance of Mcl-1 expression and repressed Bim induction (F igure 27, lower panel), indicating the dysregulation of the normal response of these proteins to detachment is a contributing factor to metastatic poten tial. To decipher the signaling pathways involved in the observed expression profiles of Mcl-1 and Bi m during detachment, we treated the cells with dasatinib, LY294002, U0126, or the combination of LY294002 and U0126. Inhibition of MEK or PI3-K i ndividually resulted in si milar apoptotic indices as measured by caspase-3 activation (Figure 27, upp er panel). Similar to results seen in Figure 24A, PI3-K inhibition cau sed a decrease in Mcl-1 expression, likely through the derepression of GSK-3 as well as a limited increase in Bim expression, albeit the Bim protein was highly phosphorylated and likely rapidly turned over (Figure 27, lower panel). Inhibition of MEK with U0126 had no e ffect on Mcl-1 expression but did confirm an earlier report of increase d Bim expression upon Erk1/2 in hibition (Fukazawa et al., 2004). Importantly, the combin ational treatment of LY294002 and U0126 enhanced the anoikis response which correla ted with Mcl-1 degradation and Bim induction. The failure of dasatinib to sensitize these cells to anoikis is likely the result of independent downstream oncogenic signaling mediated by pr eviously identified mutations in K-RAS and BRAF (Hollestelle et al., 2007) which activate both the PI3-K/Akt and MEK/Erk pathways (Campbell et al., 1998).
Figure 27. The concerted effects of decreased Mcl-1 and increased BimEL expression correlate with enhanced anoikis response. MDA-MB-231 breast cancer cells were untreated or detached on polyHEMA-coated plates containing DMSO, 50 nM dasati nib, 25 M LY294002, 10 M U0126, or the combination of LY294002 and U0126 for 24 hours and subjected to immunoblot analysis with the indicated antibodies (lower pa nel) and caspase3 activity assay (upper panel). To determine if Mcl-1 degradation alone was able to promote anoikis in the MDA-MB-231 cells similar to PI3-K inhibition, we agai n knocked down Mcl-1 (Figure 28A, insert) and assayed the re sponse to detachment. Afte r only 24 hours of detachment, cells with depleted Mcl-1 displayed mark edly increased caspase-3 activity and by 48 hours contained a much higher fraction of d ead cells (Figure 28A & B). To further confirm that the loss of Mcl-1 function but not other antiapoptotic Bcl-2 members is essential for initiation of anoikis in metastat ic cancer cells, we tested the ability of ABT737, which targets Bcl-2, Bcl-XL and Bcl-w but not Mcl-1 (Oltersdorf et al., 2005), to 59
induce apoptosis of detached MDA-MB-231 ce lls. As expected, treatment with ABT-737 was unable to trigger anoikis in contro l cells, although it significantly enhanced detachment induced apoptosis of Mc l-1 knockdown cells (Figure 28A & B). Furthermore, in vivo experiments demonstrated that knockdown of Mcl-1 alone can significantly reduce tumor establishment of metastatic MDA-MB-231 cells in lungs of nude mice (Figure 29A &B). These results st rongly imply that inhibition of Mcl-1 degradation during detachment is vital to the establishment of metastatic cells. Figure 28. Mcl-1 depletion sensitizes hum an breast cancer cells to anoikis. MDAMB-231 cells were infected with lentivirus encoding control shScr or shMcl1 constructs and selected by 0.5 g/mL puromycin for 14 days. The knockdown of Mcl-1 was confirmed by immunoblot (insert). The anoikis response of shScr and shMcl-1 cells cultured in the pres ence of DMSO or 100 nM ABT-737 was measured by (A) caspase-3 activation and (B) LDH release after 24 or 48 hours deta chment respectively; mean + SD, n=3. 60
Figure 29. Mcl-1 depletion reduces human breast cancer metastasis in vivo MDAMB-231 cells constitutively expressing luciferase were infected with shScr or shMcl-1, injected into nude mice and tumor establishment was measured three weeks post-injection. (A) Re presentative mice and (B) photon emissions were quantified; mean + SD, n=9. Discussion Despite recent advances in early detection and new therapeutic options for cancer patients, metastatic progression is attributed to 90% of human cancer fatalities (Weigelt et al., 2005). Anoikis is a vita l regulatory mechanism that can prevent metastases in a process that requires Bax tran slocation to mitochondria, whic h is inhibited by survival kinase signaling found in many human cancers (Gilmore et al., 2000). In spite of our current knowledge of apoptosis, how metast atic cancers escape anoikis remains poorly defined. In this report, we are the first to provide evidence of th e unique importance of Mcl-1 degradation in the anoikis response. The Mcl1 protein rapidly undergoes proteasome-dependent degradation in normal NIH3 T3 cells at very early time points after detachment, which allows the subsequently induced Bim to activate Bax. Indeed, 61
62 depletion of Mcl-1 alone is not enough to activate Bax under conditions in which the induction of Bim is blocked. Conversely, stab ilization of Mcl-1 by proteasome inhibition suppresses Bax activation during anoikis, whereas overexpression of Bim causes dose dependent apoptosis. Together, these result s suggest a model for anoikis where Mcl-1 degradation is required as a priming event to sensitize the cell towards Bax activation, which occurs in a manner that is depe ndent on the BH3-only protein Bim induction. These events are suppressed by active Src, Akt, and Erk1/2 signaling thereby conferring cancer cells with activating mutations in these pathways a survival advantage upon detachment from the ECM. The ability of cancers to inhibit anoiki s is propagated by the inability of these cells to activate Bax after the loss of a dhesion. Bim, a known activator of Bax, is suppressed in active Src expressing cells through a mechanism controlled by Akt and Erk1/2 signaling pathways. The transcriptional repression of Bim in 527F cells is likely through the Akt pathway, which is known to phosphorylate the Bim transcription factor Foxo3a and promote its retention in the cytopl asm by interacting with 14-3-3 (Brunet et al., 1999). BimEL is also regulated posttran slationally by phosphorylation of Ser69 by Erk1/2, which promotes its targeting to the proteasome and degrad ation (Luciano et al., 2003). JNK is also known to control transcri ption and sequestration of Bim (Lei and Davis, 2003; Whitfield et al., 2001), but upon treat ment of 527F cells with JNK inhibitors there are no significant changes in the Bim e xpression or apoptosis (data not shown). Although Src can directly phosphorylate STAT3 thereby promoting the expression of Mcl-1 (Bowman et al., 2001), our microarray and semi-quantitative RTPCR analyses indicate that the Mcl-1 transcri pts decrease marginally during anoikis in
63 dasatinib sensitized 527F but not wild type cells. At posttranslationa l levels, however, we found that GSK-3 mediated phosphorylation at Se r159 of Mcl-1 in response to detachment promotes its rapid proteasoma l degradation. Akt negatively regulates GSK3 by phosphorylation at Ser9 (Cross et al., 1995 ). Consistently, our studies in 527F and human metastatic cancer cells indicate that Mcl-1 expression is mainly regulated posttranslationally throu gh inhibition of GSK-3 activity by Akt. However, in 527F cells the lower expression of Mcl-1 and higher sensitivity to a noikis observed when treated with dasatinib compared to LY294002 suggest that other regula tory mechanisms mediated by Src, such as STAT3-dependent transcription, are also involved. Mcl-1 degradation is required but by itself is insufficient to induce anoikis which requires the subsequent inducti on of Bim. Once there is an excess of Bim in relation to Mcl-1 the activation of Bax is initiated. These coordinated events determine the anoikis response, which are deregulated in cells wi th active oncogenic signaling. The comparison of human osteosarcoma cell line Saos-2 and it s metastatic derivative LM7 illustrates that stabilization of Mcl-1 during detachment can afford metastatic cells considerable survival advantage. Consistent with this finding, knoc kdown of Mcl-1 enhances the sensitivity of metastatic LM7 and MDA-MB-231 cells to an oikis and can prevent establishment of metastases. Likewise, repression of the PI 3-K/Akt and MAPK signaling pathways that control Mcl-1 stability and Bim expression can resensitize metastatic breast cancer cells to anoikis. The unique role of Mcl-1 in the anoikis re sponse is likely attr ibuted to its short half-life as its depletion coincides with th e commitment of the cell to anoikis. The mechanism of Mcl-1 mediated a noikis inhibition app ears to be non-redundant with that of
64 Bcl-2 or Bcl-XL as ABT-737 is unable to in itiate anoikis whereas loss of Mcl-1 does. However, the failure of initiation of anoi kis by inhibition of Bc l-2/Bcl-XL does not exclude the role that these antiapoptotic Bc l-2 members play in suppression of anoikis. Indeed, treatment with ABT-737 enhances an oikis initiated by knockdown of Mcl-1 in MDA-MB-231 cells. This finding is consistent with others that show an increased apoptotic response when Mcl-1 inhibition is combined with ABT-737 (Chen et al., 2007). Moreover, there is no significant decrease in the protein levels of Bcl-XL at early timepoints of anoikis where Bax conformational change and caspase activation are observed. Therefore, Bcl-XL overexpression in cancer cells may promote viability by repressing the velocity of the apoptotic insult initiated by Mcl-1 degradation when detached from the ECM. The prolonged viab ility may give the cells more time to reattach to the ECM at a distal site or for th e accumulation of additi ve mutations that can disrupt anoikis execution. Due to the unique characteristics of Mcl-1, such as a short halflife, higher affinity to Bim, and verified in tricate regulation, we propose that Mcl-1 serves at the convergence point of many resultant si gnals downstream of detachment from the ECM that mediates the initiation of an anoi kis response and the prev ention of metastasis. Materials and Methods Reagents Poly(2-hydroxyethyl methacrylate) (polyHEMA), caspase-3 assay kit, cycloheximide, MG132, oligonucleotides for shRNA constructs, and monoclonal antibodies specific for Bax (clone 6A7), -tubulin and -actin were purchased from Sigma. 3-[3-cholamidopropyl)-dimethylam monio]-1-propanesulfonate (CHAPS) was
65 purchased from MP Biomedical. Anti-mous e Mcl-1 was purchased from Rockland. Monoclonal anti-Akt and polyc lonal anti-p473 Akt antibodies were purchased from R&D Systems. Anti-p202/204 Erk1/2 and Anti-Erk1/2 polyclonal antibodies and U0126 were purchased from Cell Signaling. Polyclonal Bax (N20) and Goat anti-rabbit IgG-HRP were purchased from Santa Cruz. Poly clonal anti-Bim antibody, TDZD-8, and the fluorogenic chymotrypsin substrate III were purchased from Calbiochem. The antihuman Mcl-1 antibody was purchased from BD Biosciences. LY294002 was purchased from Alexis. Plasmids The pcDNA3-BimEL vector was descri bed previously (Yamaguchi and Wang, 2002). The pcDNA3.1/V5-His-TOPO plasmids encoding wild type and mutant Mcl-1 were described previously (Maurer et al., 2006). Oligos for shRNAs targeting Bim 5GTTCTGAGTGTGACAGAGA-3 and Mcl1 5-GAGGACGACCTATACCGCC-3 (c1) and 5-GCCCTAATTAACAACGTTG-3 (c-3) we re synthesized and cloned into the Bgl II and Sal I restriction sites of LTRH 1-puro (Ken Watanabe, National Center for Geriatrics & Gerontology, Ai chi, Japan). The retroviral constructs expressing Bcl-2IRES-Bim, Bcl-XL-IRES-Bim, a nd Mcl-1-IRES-Bim were desc ribed previously (Kim et al., 2006). The pLKO.1-based lentivir al shRNA targeting human Mcl-1, TRCN0000005517 was purchased from Open Bios ystems. The pLKO.1-based scrambled control shRNA vector was purchased from Sigma.
66 Cell Culture and Transfection Wild type, v-Src, and c-Src 527F NIH3T3 cells were previously described (Yu et al., 1995). GSK-3 -/MEF cells were kindly provided by James Woodgett (Hoeflich et al., 2000). NIH3T3 and MEF cells were maintained in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Saos-2, LM7, and MDA-MB-231 cell lines were maintained in MEM medium supplemented with 10% FBS, 1% penicillin/streptomycin, 1 mM NaPyruvate, and 1x MEM non-essential amino acids. To induce anoikis, cells were maintained in the same media supplemented w ith 1% (for mouse cell lines) or 10% (for human cell lines) FBS in polyHEMA coated plates. Transfection was completed with Lipofectamine2000 (Invitrogen) according to the manufacturers recommendations. Recombinant retrovirus and lentivirus were produced in Amphotropic 293T packaging cells and 293FT cells with ViraPowerTM Packaging Mix (Invi trogen), respectively. Caspase-3, Chymotrypsin and LDH release Caspase-3 activation was assayed as DEVDase activity with the Caspase-3 fluorescence assay kit (Sigma). Briefly, cells were lysed in 1% Chaps lysis buffer and cleared by centrifugation at 13,000g for 5 minutes. The supe rnatant was collected and protein concentration was determined by BCA method. 1X reaction mixture was aliquoted into a 96-well plate and 50 g total protein lysa te was added. The reaction was incubated at room temperature in the dark for 1 hour and the fluorescence was measured by an automated plate reader. Values are repr esented as the change in fluorescent units ( FU) per g protein per hour. The fluorogenic chymotrypsin substrate III (Calbiochem)
67 was used to measure chymotrypsin-like activity using the same protocol as that used for the caspase-3 assay Cell death was measured using the LDH cytotoxicity assay (Biovision) according to the ma nufacturers recommendations. Semi-Quantitative RT-PCR Semi-quantitative RT-PCR was complete d using the Qiagen OneStep RT-PCR system according to the manuf acturers recommendations. Primers used for the reactions are: 5-TGCAGGGTTATGGAATCCTC-3 and 5-GCCCCTACCTCCCTACAGAC-3 for Bim; 5-GCAGCTTCAAGTCCACCTTC-3 and 5AGATGGCGTAACAAACTGGG-3 for Mcl-1; 5-AATGTGTCCGTCGTGGATCT-3 and 5-CCCTGTTGCTGTAGCCGTAT-3 for GAPDH. In Vivo Metastasis Model MDA-MB-231-luc-D3H2LN cells were obtained from Xenogen and infected with lentiviral Mcl-1 shRNA (shMcl-1) or contro l scrambled shRNA (shScr). After 10 days selection with 0.5 g/mL puromycin, 1 x 106 cells were injected into 10-12 week-old Harlan nude mice via the tail vein. Three w eeks post-injection mice were imaged using the IVIS200 system (Xenogen) as per the manufacturers recommendations. Statistical significance was determined using Students t-test.
68 Chapter Three: Src Phosphorylation of Bif-1 Disrupts the Interaction with Bax and Inhibits Anoikis Abstract Bif-1 interacts with Bax and enhances its conformational rearrangement resulting in apoptosis. However, the molecular mechanism governing the interaction between Bif-1 and Bax is poorly defined. Here we provide evidence that Bif-1 is phosphorylated, an event which can be repressed by apoptotic stim uli. The protein kinase c-Src binds to and directly phosphorylates Bif1 on tyrosine 80. Moreover, Src phosphorylation of Bif-1 suppresses the interaction between Bif-1 and Bax resulting in the inhibition of Bax activation during anoikis. Together, these re sults suggest that phosphorylation of Bif-1 impairs its binding to Bax and represses apoptosis, providing another mechanism by which Src oncogenic signaling can prevent cell death. Results Bif-1 Phosphorylation In Vivo is Repressed upon Apoptotic Stimuli Because Bif-1 binding to Bax is induced by apoptotic stimuli with no apparent changes in either proteins expression profile (Cuddeback et al., 2001; Takahashi et al., 2005; Yamaguchi et al., 2002), we reasoned that the ability of Bif-1 to activate Bax might be regulated by posttranslational modificat ions. To determine if Bif-1 could be phosphorylated in intact cel ls, 293T cells were metabolically labeled with 32PO4 and subjected to immunoprecipitation with anti-Bif -1 or control anti-My c antibodies (Figure
30). A specific 32P-labeled band corresponding to Bif-1 was pulled down with anti-Bif-1 but not control anti-Myc antibody. Figure 30. Bif-1 phosphorylation. 293T cells were metabolically labeled with 32PO4 for 6 hours; lysates were collected and subjected to immunoprecipitation with anti-Bif-1 or anti-Myc antibodi es followed by SDS-PAGE and autoradiography. To determine the phosphorylation status of Bif-1 in stressed versus unstressed cells, 293T cells expressing Myc-tagged Bi f-1 were metabolically labeled with 32PO4 and treated with DMSO, thapsigargin (THG), or se rum starvation (Figure 31). The levels of phosphorylated Bif-1 were found to be highest in unstressed cells. Similarly, the level of tyrosine phosphorylation of Bif-1 was repressed in a time dependent manner upon treatment with staurosporine (STS) (Figure 32). The kineti cs of Bif-1 dephosphorylation correlated with the activa tion of Bax as determined by immunoprecipitation of conformationally active Bax w ith the 6A7 monoclonal antibody (Figure 32). Together, these results clearly indicate that Bif-1 is phosphorylated in whole cells and apoptotic 69
stresses promote the dephosphorylation of Bif-1. Figure 31. Phosphorylation of Bif-1 is inhibited by cellular stress. 293T cells were transfected with Myc-Bif-1 and labeled with 32PO4 for 6 hours after serum deprivation (FBS) for 24 hours or exposure to 2 M thapsigargin (THG) or DMSO for 8 hours. Myc-Bif-1 protei n was immunoprecipitated with antiMyc antibody and subjected to SDS-PAGE/immunoblot and autoradiography. Figure 32. Bif-1 is dephosphorylated in a time-dependent manner upon treatment with staurosporine. 293T cells were transfected with Myc-Bif1 and treated with 1 M staurosporine (STS) for various times prior to immunoprecipitation/immunoblot analysis with the indicated antibodies. 70
Src Interacts with Bif-1 in Both Yeast and Mammalian Cells A search of Bif-1 interacting proteins through the use of a yeast two-hybrid assay resulted in the identification of c-Src kinase. The LexA-c-S rc fusion protein specifically interacted with the wild type Bif-1 fused to the B42 transactivation domain in yeast (Figure 33). The region of Bif-1 required for the interaction with c-Src was determined using a series of Bif-1 deletion mutants. It was found that full length Bif-1 was the most effective at binding c-Src but appeared to require the presence of the region between amino acid residues 238-285 (Figure 34). Figure 33. Bif-1 interacts with c-S rc in the yeast two-hybrid assay. LexA-c-Src and B42-Bif-1 fusion proteins interact in yeast. 71
Figure 34. Schematic representation of th e domain structure of Bif-1 and mapping the Src binding domain of Bif-1. LexA-c-Src was transfected into yeast with the indicated deletion mutants of Bif-1 fused to B42. Interactions of pairs of fusion proteins were dete rmined by visual examination of galactosidase activity and represented graphically. To determine if Bif-1 was a binding pa rtner for c-Src in mammalian cells, 293T cells were transfected with empty vector, Flag-tagged c-Src or the na turally occurring Src mutant, Src531, which results in a truncated pr otein that has been shown to be activating, transforming, and tumorigenic (Irby et al., 1999). Immunoprecipita tion with anti-Flag antibody revealed that endogenous Bif-1 coul d indeed interact with both c-Src and mutant Src531 (Figure 35A). Similarly, Bif-1 was found to interact w ith wild type c-Src and mutant c-Src (Y527F) in 3Y1 rat fibrobl asts and NIH3T3 cells, respectively (Figure 72
73 35B). Src binding to Bif-1 is therefore a co mmon event with implications in Bif-1 regulation. Since both STS and THG prevented the phosphorylation of Bif-1 (Figures 27 & 28), we sought to determine whether apoptotic stimulation affects the binding of Src to Bif-1. As shown in Figure 36, H1299 cells treated with STS did not prevent the association of Bif-1 with Src at endogenous levels as compared to DMSO control, but instead prevented the kinase activity of Src as determined by the activation associated pTyr-416 western blot. Alternatively, THG treatment could effectively prevent the association between Bif-1 and Src. Treating cells with THG did not suppress but rather enhanced the activation of Src, which is consistent with previous observations (Chung et al., 2001). Together, these results suggest th at apoptotic stresses can prevent the phosphorylation of Bif-1 through either inhibiti on of Src interacting with Bif-1 or by preventing Src activity.
Figure 35. Bif-1 interacts with Src in mammalian cells. (A) 293T cells were transiently transfected with pFlag-c-Sr c or c-Src531 or parental pFlag-CMV2 vector. After 2 days, immunoprecipitation was performed using anti-Flag antibody or anti-Myc (control) follo wed by SDS-PAGE/immunoblot analysis with anti-Bif-1 polyclonal antiseru m. (B) 3Y1 rat fibroblasts stably expressing c-Src or NIH3T3 cells stably expressing a constitutively active cSrc (Y527F) were subjected to immu noprecipitation with anti-c-Src or control antibody followed by SD S-PAGE/immunoblot analysis. Figure 36. Dephosphorylation of Bif-1 can be achieved via two independent mechanisms in response to cellular stresses. H1299 cells were treated with 74
75 1 M STS, 2 M THG, or DMSO for 8 hours. Left panel, lysates were immunoprecipitated with anti-Bif-1 antibody or control Flag antibody and then probed with anti-Src and anti-Bif -1 antibodies. Right panel, whole cell lysates were probed for Src act ivity with anti-pY416 antibody. Src phosphorylation of Bif-1 at Y80 We next sought to determine whether Src could directly phosphorylate Bif-1 using an in vitro kinase assay. Incubation of recomb inant c-Src with GST-Bif-1 fusion protein but not GST alone resulted in detectable phosphorylation determined by autoradiography (Figure 37A). The subseque nt Src phosphorylation site on Bif-1 was narrowed down through the use of deletion mutants (Figure 37B).
Figure 37. Minimal Bif-1 sequence required for Src phosphorylation. Recombinant c-Src kinase was incubated with GST, GST-Bif-1, or various GST-Bif-1 deletion mutants and [ -32P]-ATP at 30C for 15 minutes before analysis by SDS-PAGE and autoradiography. Src mediated phosphorylation of Bif-1 occu rred in the N-terminal region of Bif-1 encompassing amino acids 47-126. A closer evalua tion of this region revealed a potential Src phosphorylation consensus motif (EEFVY80EKLD), and substitution of Y80 to F abrogated Src mediated Bif1 phosphorylation (Figure 38). 76
Figure 38. Src phosphorylates Bif-1 at Y80 in vitro Recombinant c-Src kinase was incubated with GST, GST-Bif-1 wild t ype (WT), or GST-Bif-1 Y80F mutant and [ -32P]-ATP at 30C for 15 minutes be fore analysis by SDS-PAGE and autoradiography. Coomassie staining s hows the input of GST and GST-Bif-1 proteins. Src Phosphorylates Bif-1 at Y80 in Intact Cells Although the in vitro kinase assay illustrates that Sr c directly targets Y80 in Bif-1, it remained to be determined whether this ev ent could be recapitula ted in whole cells. Therefore, 293T cells were co-transfected w ith Flag-Bif-1 along with empty, c-Src, or Src531 constructs. Bif-1 was then immunopr ecipitated from lysates using anti-Flag antibody and subjected to immunoblotting with antibodies specific for phosphotyrosine or Bif-1 (Figure 39A). Co-expression of Sr c with Bif-1 led to a marked increase in tyrosine phosphorylation of Bif-1. It was also found that expression of Src531 could induce the phosphorylation of both endogenous and ectopic Bif-1 regardless of tag presence in 293T cells (Figure 39B). 77
Figure 39. Src phosphorylates Bif-1 in intact cells. (A) 293T cells were transiently cotransfected with Flag-Bif -1 and either c-Src, c-Src531 mutant or empty vector. After two days, Flag-Bif-1 was immunoprecipitate d with anti-Flag antibody, followed by SDS-PAGE/immunobl ot analysis with anti-Bif-1 rabbit antiserum or anti-phosphotyrosin e PY20 antibody. (B) 293T cells were transiently co-transfected with Src5 31 and plasmids encoding Bif-1, FlagBif-1 or parental vector Cells were lysed 2 da ys later and Bif-1 was immunoprecipitated with anti-Bif-1 monoclonal antibody and subjected to SDS-PAGE/immunoblot analysis with antibodies specific for Bif-1 or phosphotyrosine. Performing the same co-transfection e xperiments confirmed that Y80 was the major phosphorylation target of Src as the Bif-1Y80F mutant showed a significant reduction in the level of tyrosine phosphoryl ation of Bif-1 (Figure 40). However, it 78
appears as if there is some level of phosphorylation still pr esent in the Bif-1Y80F mutant. This is likely due to phosphorylation at other tyrosine residues re gulated by Src or the immunoprecipitation of endogenous Bif-1. Figure 40. Src phosphorylates Y8 0 of Bif-1 in whole cells. 293T cells were transfected with Bif-1 or Bif-1(Y80F) alone or together with c-Src expression plasmids. Bif-1 proteins were immunoprecipi tated and analyzed 2 days post transfection. Similarly, A431, 293T, and NIH3T3 cells we re assayed for the ability of EGF to stimulate endogenous Bif-1 phosphorylation (Fig ure 41A). EGF was found to promote an increase in Bif-1 phosphorylati on, which could be inhibited by co-treatment with the Src specific inhibitors PD180970 in NIH3T3 cells as well as dasatinib in A431 and 293T cells, indicating that this phosphor ylation is associated with in tracellular signals that are associated with oncogenic suppression of a poptosis. Pretreatment with pervanadate, a broad spectrum tyrosine phospha tase inhibitor, was found to enhance the amount of Bif-1 phosphorylation on tyrosine by Src (Figure 41B). This indicates that the phosphorylation 79
of Bif-1 is a tightly regulated process. B A Figure 41. Endogenous phosphorylation of Bif-1 by c-Src. (A) A431, 293T, and NIH3T3 cells were serum starved for 16 hours then treated with or without 500 nM PD180970 or 50 nM dasatinib for 2 hours, and then stimulated with 50 ng/mL recombinant EGF for 5 minutes. Cell lysates were prepared and subjected to immunoprecipitation with anti-Bif-1 antibody followed by SDSPAGE/immunoblot with anti-phosphotyr osine or anti-Bif-1 antibody. (B) A431 cells were treated as in A with and without pervanadate, which was added 10 minutes prior to the addition of EGF and maintained during the 5 minute incubation with EGF. Bif-1Y80 Phosphorylation by Src Prevents Bif-1 Mediated Bax Activation during Anoikis Based on the previous knowledge of Bif-1 activation of Bax upon apoptotic stimuli and our current findings of Src medi ated phosphorylation of Bif-1, we sought to 80
81 determine the role of Src mediated Bif1 phosphorylation in the context of anoikis initiation. Bif-1-/MEFs expr essing empty control vector (p KI) or re-established wild type or Y80F mutant Bif-1 in the presen ce of Src531 or control GFP expression were created (Figure 42A) and detached on poly-HE MA coated plates for 24 hours or left attached on normal culture dishes. Lysates were then collected and the interaction between Bif-1 and Bax was determined (Figur e 42B). Detachment caused an increase in Bif-1/Bax binding, consistent with our previo us findings that apoptotic stimuli increase this interaction (Cuddeback et al., 2001; Takahashi et al., 2005). The Bif-1Y80F mutant was able to bind Bax with the same affinity as wild type. Importa ntly, co-expression of Src531 with wild type Bif-1 repressed the abilit y of Bif-1 to interact with Bax in response to detachment. However, the non-phosphorylatable Bif-1Y80F mutant retained Bax binding potential even in the presence of Sr c expression (Figure 42B ). This interaction was specific as immunoprecipitation with nor mal rabbit serum (NRS) was unable to pull down either Bax or Bif-1 (Figure 42C).
Figure 42. Src phosphorylation of Bif-1 inhibits Bax conformational activation during anoikis. (A) Bif-1 -/MEFs were infected with retrovirus encoding empty control, Bif-1 wild type (WT) or Bif-1 mutant (Y80F) and selected on 1.0 g/mL puromycin for 10 days. These cells were then re-infected with retrovirus encoding GFP or Src531 prot eins. Expression of transgenes was confirmed by immunoblot. (B) The cells created in A were left attached or detached on poly-HEMA coated plates for 24 hours. Lysates were prepared and subjected to immunoprecipitation w ith anti-Bax polyclonal antiserum or monoclonal 6A7 antibody. The resulting immune complexes and whole cell lysates were analyzed by immunoblotting with anti-Bax or anti-Bif-1 antibody. (C) Lysates prepared from Bi f-1(Y80F) cells detached for 24 hours were subjected to immunoprecipitation with anti-Bax polyclonal antibodies or normal rabbit serum. 82
Detachment induced Bax activation wa s increased in cells expressing Bif-1. Importantly, the enhanced Bax conformational change was inhibited by co-expressing Src531 in wild type Bif-1 but not Bif-1Y80F cells (Figure 42B). Si milarly, co-expression of Src531 could partially rescue cells expressing empt y or wild type Bif-1 from caspase-3 activation, but overexpression of Src531 had no inhibitory e ffect on cells expressing the non-phosphorylatable Bif-1Y80F mutant (Figure 43A). These results were confirmed by a cell death assay measuring LDH release (Fig ure 43B). These findings clearly indicate that Src phosphorylation of Bif-1 has direct biological impacts that affect the ability of a cell to undergo apoptosis. Figure 43. Src phosphorylation of Bif-1 i nhibits caspase-3 activation and cell death in response to cell detachment. Cells were left attached or detached on poly-HEMA coated plates for 24 hours. Lysates were prepared and subjected to (A) caspase-3 assays or (B) LDH release. LDH release representation is normalized to their attached conditions; mean + SD, n=3. Discussion Bax activation is known to control the in itiation of apoptosis. However, the exact 83
84 mechanism by which the conformational rearra ngement and insertion of Bax into the OMM is controlled remains elusive. Alt hough it is generally believed that activating BH3-only molecules such as tBid, Puma, and Bi m can directly facilita te the activation of Bax, there remains debate as to whether Bax can directly interact with these molecules (Kim et al., 2006; Willis et al., 2007). Furtherm ore, it appears that activating molecules, other than BH3-only proteins, are crucial to the ability of Bax to form pores in membranes (Lutter et al., 2000; Ott et al., 2007b; Roucou et al., 2002). These observations have led to the pursuit and discovery of novel Bax interacting proteins, such as Bif-1. Identification of Bif-1 as a novel Bax inte racting factor has led to the finding that Bif-1 is able to potentiate the activation of Bax as well as Bak (Cuddeback et al., 2001; Takahashi et al., 2005). Reasons as to why Bif1 is able to enhance the activation of Bax are not entirely understood. On e possible explanation is that Bif-1 can alter the mitochondrial membrane structure such th at it becomes suitable for conformational rearrangement and/or insertion of Bax into the mitochondria. As a member of the endophilin family of proteins, Bif-1 contains the N-BAR domain which is known to promote membrane curvature (Gallop et al ., 2006; Peter et al., 2004). Given that Bax accumulates at fission and fusion sites on the mitochondrial membrane and that Bif-1 is known to regulate mitochondrial morphology (K arbowski et al., 2004; Karbowski et al., 2002), it is likely that the Bif-1/Bax interacti on observed is the consequence of changes in membrane structure mediated by Bif-1. In terestingly, the use of deletion mutants has identified the first eleven amino acids in Bi f-1 as essential for its ability to bind Bax (Pierrat et al., 2001). This regi on of Bif-1 is a part of th e amino terminal amphipathic
85 helix know as helix zero, which is esse ntial for binding and tubulating membranes (Farsad et al., 2001; Gallop et al., 2006). The overlapping Bax and membrane binding functions of this region in Bif-1 may indicate that the interaction between Bif-1 and Bax is dependent on membrane dynamics. Given the aforementioned role of Bif-1 in the activation of Bax and apoptosis (Cuddeback et al., 2001; Takahashi et al., 2005) as well as its newl y defined role in autophagy (Takahashi et al., 2007), the mechan isms that regulate Bif-1 function are of increasing importance. Yeast twohybrid analysis has identified Src as a Bif-1 interacting protein, an association that is also evident in both e xogenous and endogenous expression systems. Furthermore, we have shown that Src can directly phos phorylate Bif-1 at Y80. This residue is part of the internal am phipathic helix common to members of the endophilin family but weakly conserved ther ein. Previous deletion mutation analysis revealed that this domain is required for membrane binding and t ubulating activities of Endophilin A1 (Gallop et al., 2006). Similarly, mutating positively charged residues to negative ones in this region of Endophilin A1 also inhibits membrane binding and tubulation (Gallop et al., 2006). Phosphorylation of Y80 on Bif-1 is likely to alter membrane binding by changing the charge distribution in the internal amphipathic helix, as well as altering the helix organization with the addition of a bulky phosphate group. Mutant Bif-1Y80F would lack the ability of Src to alter the charge structure of the helix and allow binding to membranes, which could explain why this muta nt maintained Bax binding potential in cells with active Src. Fu ture studies will be needed to determine specifically how Src mediated phosphorylation at Y80 of Bif1 affects helix organization, membrane binding, and tubulati on potential of the protein.
86 Materials and Methods Metabolic Labeling and In Vitro Kinase Assays For detection of Bif-1 phosphorylation, 293T cells were incubate d for overnight in phosphate-free Dulbecco's modified Eagle's medium (DMEM) containing 5% dialyzed fetal bovine serum (FBS), and then labeled with 0.5-1.0 mCi of [32P]-orthophosphate for 6 hours in the presence of 10% dialyzed FB S. Plates were washed with ice-cold phosphate-buffered saline, and lysed with radi oimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. Lysates were pre-cleared with Protein G agarose prior to immunoprecip itation with anti-Bif-1 or an ti-Myc monoclonal antibodies. For in vitro kinase assays, a recombinant c-Src was obtained from Upstate Biotechnology. The phosphorylation reaction was carried out at 30oC for 15 min in 40 l reaction mixture (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.015% Brij 35, 0.1 mg/ml BSA, 10 M ATP, 5 Ci [ -32P]ATP (3000 Ci/mmole), 5 units Src kinase) containing 1 g of indi cated GST fusion protein. The reaction was stopped by addition of 14 l of 4 x SDS loading buffer and heat denaturation, resolved by SDS-PAGE and analyzed by autoradiography. Yeast Two-Hybrid Assays Two-hybrid assays were performed as previously described (Cuddeback et al., 2001). Briefly, c-Src fused to the LexA DNA binding domain was transfected into S. cerevisiae EGY48 cells with the full length or de letion mutants of Bif-1 fused to the B42 transactivation domain. Five independent tr ansformants were grown on either galactose
87 or glucose containing agar plat es for inducing or repressing -galactosidase activity, respectively. Expression and Purification of GST-Bif-1 The GST-Bif-1 wild type and deletion mu tants proteins were cloned into pGEX4T-1 plasmid and expressed in the DH5 strain of E. coli. Briefly, transformed cells were grown in LB medium containing ampici llin (100 g/ml) at 37C to an OD600nm of 0.8, then 1 mM IPTG was added to induce protein expression at 37C for 3 hours. Cells were lysed in PBS (pH=7.4) containing protease in hibitors by sonicati on and centrifuged at 14,000 x g for 30 min. The resulting supernat ant was incubated with glutathionesepharose 4B (Amersham Bioscience) at 4 fo r 1 hour then washed three times with PBS, the protein was eluted with 10 mM of redu ced glutathione in 50 mM Tris-HCl (pH=8.0) and dialyzed against 50 mM Tris-HCl buffer (pH=7.5). Immunoprecipitation and Immunoblot Assays Cells were lysed using 1% Chaps lysi s buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 1% Chaps) containing protease and phospha tase inhibitors. Immunocomplexes were pulled down with the indicated antibodies and washed 3 times in lysis buffer. Immunoprecipitates were resolved using SDS-PAGE and immunoblotted with the indicated antibodies. For detection of ac tive Bax, anti-Bax 6A7 monoclonal antibody (Sigma) was used for immunoprecipitation a nd anti-Bax N20 polyclonal antibody (Santa Cruz) was used for subsequent immunoblot analysis. The Bax/Bif-1 interaction was determined by immunoprecip itation of Bax using polycl onal anti-mouse/rat Bax 1696
88 antiserum (Krajewski et al., 1994) followed by immunoblotting with anti-Bif-1 monoclonal antibody (Imgenex). The Src/Bif-1 interaction was determined in H1299 cell lysate in 1% Chaps lysis buffer by imm unoprecipitation of Bif-1 with monoclonal antibody (Imgenex) and subsequent immunobl otting for Src (Cell signaling #2108). For detection of tyrosine phosphorylation of Bif-1, A431, 293T, and NIH3T3 cells were deprived of serum for 18 hours in DMEM containing 0.1% BSA. Cells were then incubated with Src inhibitors PD180970 or dasatinib for 2 hours. Cells were then stimulated by the addition of EGF to 50 ng/ mL for 5 minutes, washed once with 5 mL ice-cold PBS, and collected in 900 L Buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 2 g/ml aprotinin, 2 g/mL le upeptin, 100 g/ml phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 20 mM p-nitrophenyl phosphate, 1% Triton X-100) as previously described (Ren et al., 2004). One mg of pre-cleared cell lysate was incubated with Bif-1 rabbit polyclonal antibody for one hour on ice and th en 20 l Protein A agarose was added and rocked overnight at 4C. Beads were washed three times with Buffer A and then boiled in 20 l Laemmli sample buffer and resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane (Bio-Rad) and blocked in 3% chicken egg white albumin in TBS-T for 2 hours. The phosphorylated Bif-1 protein was detected with HRP-conjugated anti-phos pho-tyrosine (PY20) mouse monoclonal antibody (BD Biosciences). Retrovirus Production Retroviral plasmids encoding Bif-1 wild type and Y80F mutant were cloned into
89 the BglII-XhoI sites of pKI vector. For Src expression, active c-Src531 mutant was cloned into the EcoRI-XhoI sites of pBMN-IR ES-GFP vector. Retrovirus production was carried out in Amphotropic 293T cells. Briefly, cells were transfected with the indicated retroviral vector us ing the calcium phosphate method overnight in the presence of 25mM Chloroquine. Media were replaced the next morning and 36-48 hours later the viral containing media were collected and used to infect the target Bif-1-/MEFs in the presence of 8 g/mL polybrene. Cell Culture and Transfection 293T, NIH3T3, 3Y1, and Bif-1-/MEFs we re maintained in DMEM containing 10% FBS supplemented with 100 g/mL streptomycin and 100 U/mL penicillin. Transfection was performed using the calci um phosphate precipitation method. For anoikis assays, the indicated MEFs were grown to approxim ately 80-90% confluency and then forcibly detached on poly(2-hydroxyeth yl methacrylate; poly-HEMA) coated culture plates for 24 hours in 1% FBS as previously described (Woods et al., 2007). Control attached cells were also cultured for 24 hours in 1% FBS before collection.
90 Chapter Four: p53 Acetylation Is Crucial for Its Tr anscription-Independent Proapoptotic Functions Abstract Acetylation of p53 at carboxy-terminal lysi ne residues enhances its transcriptional activity associated with cell cycle arrest a nd apoptosis. Here, we demonstrate that p53 acetylation at K320/K373/K382 is also requi red for its transcription-independent functions in Bax activation, ROS production, and apoptosis in response to the histone deacetylase inhibitors (HDA Ci) SAHA and LAQ824. Knockout of p53 in HCT116 cells markedly reduced HDACi-induced apopt osis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p 53-null cancer cells to HDACimediated Bax-dependent apoptosis, whereas knockdown of endogenous mutant p53 inhibited HDACi-induced apoptosis. Evalua tion of the mechanisms controlling this response led to the discovery of a nove l interaction betw een p53 and Ku70. The association between these two proteins was acetylation independent, but acetylation of p53 could prevent and disrupt the Ku70/Bax complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis. Results Transactivation Activity of p53 Is Dispens able for Apoptosis Induced by SAHA or LAQ824
To investigate the transc ription-independent functi on of p53 in HDACi-mediated cell death, we utilized the previously established p53-/HCT116 cell line expressing the p53QS transactivation deficient mutant that retains the proapoptoti c function of p53 in which two key amino acids (Leu22 and Trp23) in the transactivation domain were replaced with Gln and Ser, respectively (Yam aguchi et al., 2004). Th e inability of this mutant to transcriptionally activate p53 target genes was confirmed by using p53 responsive luciferase reporter plasmids c ontaining the p21 or Mdm2 promoter (Figure 44). Figure 44. Treatment with HDACi does not robustly induce p53QS transactivation. HCT116 p53-/cells stably expressi ng control empty Puro, p53 wild type (WT) or p53QS mutant were transiently co-trans fected with firefly luciferase constructs driven by either p21 or md m2 promoter together with pRL-SV40 renilla luciferase vector. The cells were then treated with DMSO, 5 M SAHA or 200 nM LAQ824 and subjected to Dual-Luciferas e assay according to manufacturers instructions (Promega ). The results are represented as the mean ratio of firefly/renilla luciferase activities + SD, n=3. 91 When compared to p53+/+ HCT116 cells, p53-/HCT116 cells displayed a drastically reduced cell death (Figure 45) a nd caspase-3 activity (Figure 46) following
SAHA or LAQ824 treatment. Surprisingly, tr ansfection of not only wild type p53 but also the p53QS mutant with Puromycin (Puro) selec tion marker completely resensitized p53-/HCT116 cells to SAHA and LAQ824 (Fi gures 45-48), suggesting that HDACi require the presence of p53 but not its transc riptional activity to induce apoptosis. Similar results were obtained in three additional i ndependent transfection clones harboring p53QS with Blasticidin (Bsd) selection marker (Figures 45-48). Figure 45. p53 status affe cts HDACi-induced cell death. Wild type HCT116 (p53+/+) and HCT116 p53-/cells stably transfect ed with empty vector (Puro or Bsd), p53 WT or p53QS were treated with either 5 M SAHA or 200 nM LAQ824 for the indicated periods of time. Th e percentage of cell viability was determined by trypan blue dye exclusion assay; mean + SD, n=3. 92
Figure 46. The transactivation-deficient p53QS mutant sensitizes HCT116 p53-/cells to HDACi-induced caspase-3 activation. HCT116 cell lines expressing p53 as outlined in Figure 45 were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 28 hours a nd subjected to caspase-3 assay; mean + SD, n=3. Figure 47. HDACi promotes stabilizat ion of exogenously expressed p53. The same cell lines used in Figures 45 & 46 were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 18 hours and subjected to immunoblot analysis. Mutations in p53 have been documented in more than half of all human cancers, of which most arise in the DNA binding domain. Since there are no known naturally 93
occurring mutation at both Leu22 and Trp23 residues of p53 and the transcriptional activity of p53QS is not equal to other common mu tations (Johnson et al., 2005), the ability of tumor-associated p53 mutants, R175H, R273H, and D281G (Hinds et al., 1990), to promote apoptosis in response to HDACi treatment in cancer cells was assessed. Consistently, all of these transactivation deficient p53 mutants potentiated HDACi-induced apoptosis in p53-/HCT116 cells, with p53QS remaining the most potent inducer of apoptosis (Figures 48 & 49). Figure 48. Expression of p53 mutants in HCT116 p53-/background enhances Bax activation induced by HDACi. HCT116 p53-/cells were stably transfected with empty, Myc-p53QS, p53R175H, p53R273H or p53D281G, treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 24 hours and subjected to anti-Bax 6A7 immunoprecipitation and we stern blot analysis. 94
Figure 49. Expression of p53 mutants in HCT116 p53-/background enhances caspase-3 activation induced by HDACi. HCT116 p53-/cells were stably transfected with empty, Myc-p53QS, p53R175H, p53R273H or p53D281G, treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 24 hours and subjected to caspase-3 activity assay. Additionally, K562 and H1299 cells (both p53-null) stably transfected with p53 constructs containing R175H or D281G mutations, resp ectively, showed a marked increase in apoptotic response to LAQ824 tr eatment, as demonstrated by the ability to activate Bax and caspase-3 (Figures 50 & 51). Bax activation was determined by immunoprecipitation with anti-Bax 6A7 monoc lonal antibody that only recognizes the active conformer of Bax (H su and Youle, 1997). Figure 50. Naturally occurri ng p53 mutations promote Bax activation after HDACi 95
treatment. K562 and H1299 cells were stably transfected with p53R175H and p53D281G, respectively, treated with DM SO or LAQ824 (100 nM for K562 and 200 nM for H1299) for 24 hours and s ubjected to western blot analysis and anti-Bax 6A7 immunoprecipitation. Figure 51. Expression of naturally occu rring p53 mutants promotes caspase-3 activity in response to HDACi. K562 and H1299 cells were stably transfected with p53R175H and p53D281G, respectively, treated with DMSO or LAQ824 (100 nM for K562 and 200 nM for H1299) for 24 hours and subjected to caspase-3 assay. Notably, after exposure to SAHA or LAQ824, the exogenously expressed p53 proteins were robustly increased (Figures 47, 48, & 50). Proteasome-directed degradation of p53 is tightly regulated through specific ubi quitin ligases such as Mdm2 (Honda and Yasuda, 1999). The increase in p53 protein le vels by HDACi coincides with acetylation of p53 (Figures 48 and 50) that contributes to the inhibition of Mdm2-mediated p53 ubiquitination (Li et al., 2002). In addition, mi croarray analysis indicated that the mRNA levels of exogenous p53 were increased two to five fold by HDACi treatment (data not shown). We speculate that th e exogenously integrated p53 gene s are normally silenced by HDACs and reactivated by HDACi. This ma y explain at least in part why the 96
upregulation of endogenous p53 by HDACi was less than that of exogenous p53 (Figure 47). To evaluate the consequences of p53 stabilization after HDAC inhibition, H1299 p53-/and p53D281G cells were treated with DMSO (vehicle), MG132 (proteasome inhibitor), or LAQ824. Inhibition of the proteasome has been shown to induce p53 accumulation without specific modifications a nd activation (Sakaguchi et al., 1998). Both MG132 and LAQ824 promoted a similar am ount of p53 stabilization, but only LAQ824 treatment resulted in an increase in p53 acetylation (Figure 52). While MG132 caused a similar loss of viability in both p53-/and p53D281G cell lines, LAQ824 specifically promoted apoptosis in p53D281G expressing cells (Figure 53). Therefore, the presence of mutant p53 does not predispose cells to apoptosis under certain stresses, but HDACi preferentially promotes apopt osis in p53 expressing cells. Th is unique ability of HDACi correlates with the ace tylation of p53 but not just its accumulation. Figure 52. p53 expression and acetylation in response to HDACi and proteasome inhibition in H1299 cells. H1299 p53-/and p53D281G cells were treated with DMSO, 200 nM LAQ824, or 1 M MG132 for 24 hours and subjected to western blot analysis. 97
Figure 53. HDACi but not proteasome inhibitor induced cell death is enhanced by mutant p53 expression. H1299 p53-/and p53D281G cells were treated with DMSO, 200 nM LAQ824, or 1 M MG132 for 24 hours and subjected to Annexin V-APC/7AAD staining and anal ysis by flow cytometry. Data are represented as mean + SD, n=3. 98 To further evaluate the potential of p53 to initiate HDACi-induced apoptosis, two colon carcinoma cell lines SW480 and HT29 harboring R273H/P309S and R273H p53 mutations, respectively, were infected with lentiviral constructs expressing p53 shRNA (shp53), scrambled (shScr) shRNA or empty v ector. Infection with the shp53 lentivirus
effectively knocked down p53 expression in bo th cell lines (Figure 54). Loss of mutant p53 conferred significant resistance to HDA Ci-induced caspase-3 activation and cell death (Figure 55A & B). The viability and caspase-3 results were also confirmed by Annexin V-APC/7AAD staining of shScr and shp53 SW480 clones (Figure 56). Furthermore, knock-in of murine p53-R172H corresponding to hum an R175H mutation, results in enhanced cell deat h response of MEFs over cont rol p53 null cells when treated with LAQ824 (Figure 57). These results highlight the functional significance of endogenous p53 status in determination of apoptotic index in response to HDACi regardless of its transactivational ability. Figure 54. Knockdown of mutant p 53 in SW480 and HT-29 cell lines. SW480 and HT-29 colon carcinoma cell lines were infected with lentiviral empty, scrambled shRNA (shScr) or p53 shRNA (shp53) constructs and selected on puromycin for 10 days. Cells were tr eated with DMSO, 5 M SAHA or 200 nM LAQ824 for 36 hours and subjected to immunoblot analysis. 99
Figure 55. Knockdown of mutant p53 in SW480 and HT-29 cell lines prevents caspase-3 activation and cell death. (A) Cells described in Figure 54 were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 36 hours and subjected to caspase-3 assay. (B) Ce lls were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 24 and 48 hours and subjected to trypan blue exclusion assay. 100
Figure 56. Knockdown of mutant p53 in SW480 cells prevents HDACi-induced apoptosis. SW480 cells were infected with either scrambled control (shScr) or p53 targeting (shp53) lentivirus as described in Figure 54. Cells were treated with DMSO or 200 nM LAQ824 for 24 hours and subjected to Annexin V-APC/7AAD staining and anal ysis by flow cytometry. Data are represented as mean + SD, n=3. 101
Figure 57. Knock-in of mutant p53 in ME Fs enhances HDACi-induced apoptosis. MEFs derived from p53-/or knock-in p53R172H were treated with the indicated concentration of LAQ824 for 24 hours and subjected to Annexin V-APC/7AAD staining and analysis by flow cytometry. Graphical representation of the different cell p opulations are presented as the mean + SD, n=3. C-Terminal Acetylation Is E ssential for Mutant p53-Mediat ed Apoptosis in Response to SAHA or LAQ824 Treatment Inhibition of HDACs by SAHA or LAQ824 resulted in a significant increase in the levels of acetylated p53 (Figures 48, 50, and 52). To investigate the possible 102
103 involvement of p53 acetylation in its transc ription-independent proapoptotic functions, three lysine residues (K3 20, K373, and K382), which are known to be acetylated by HDACi treatment (Terui et al., 2003), in human p53R175H protein were mutated to arginine individually and in triplicate (K3R) to mimic the unacetylated state. These p53 constructs were cloned into retroviral vectors and infected into HCT116 p53-/cells to comparable levels (Figure 58A). The apoptot ic index was then measured by caspase-3 activity after LAQ824 treatment (Figure 58B). Single mutations at the individual lysine residues yielded marginal decreases in apoptos is. However, mutation of all three lysines completely abolished the proapoptotic activity of p53R175H in response to HDAC inhibition.
Figure 58. Substitution of K320, K373 and K382 to R attenuates the proapoptotic activity of mutant p5 3 in response to HDACi. HCT116 p53-/cells were infected with retroviral empty (pKI), p53R175H, p53R175H/K320R, p53R175H/K373R, p53R175H/K382R or p53R175H/K3R constructs. (A) p53 expression was confirmed by western blot analysis. (B) The ab ility of p53 mutants to enhance HDACiinduced apoptosis was assessed by caspase-3 activation; mean + SD, n=3. 104 To further validate the importance of these residues in transactivational deficient p53, Myc-tagged p53QS or p53QS/K3R (all three lysines, K320/373/382, were mutated to arginine) was stably expressed in HCT116 p53-/cells (Figure 59A). Immunoblot analysis with anti-acetylated K382-p53 antibody conf irmed the absence of HDACi-induced K382 acetylation in cells expressing the p53QS/K3R mutant (Figure 59A). Consistently, the ability of p53QS to induce caspase-3 activation and apoptotic cell death in response to SAHA or LAQ824 was drastically impaired by the K3R mutations (Figures 59B and 60).
Figure 59. Mutation of K320, K373 and K 382 to R attenuates the proapoptotic activity of p53QS initiated by HDACi in HCT116 cells. HCT116 p53-/cells stably transfected with empty (Puro), Myc-p53QS or Myc-p53QS/K3R were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 18 hours and subjected to (A) immunoblot analys is and (B) caspase-3 assay; mean + SD, n=3. 105
Figure 60. p53-dependent HDACi-induced cell death is repressed by mutation of three C-terminal lysine residues. HCT116 p53-/stably transfected with empty (Puro), Myc-p53QS or Myc-p53QS/K3R were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 18 hour s and subjected to Annexin VFITC/PI staining and flow cytometry analysis. SirT1 is a class III HDAC whose activity also influences p53 acetylation status, but is not inhibited by LAQ824 or SAHA. To determine if SirT1 has a functional role in LAQ824 mediated apoptosis, H1299 p53-/and p53D281G cells were transfected with empty control or SirT1 expressing plasmids Overexpression of SirT1 resulted in a minimal reduction in acetylated K382-p53 (Fi gure 61A) and did not impact caspase-3 activation in either ce ll line treated with LAQ824 (Figure 61B). 106
Figure 61. Overexpression of SirT1 does not impact p53 acetylation or p53dependent HDACi-induced apoptosis. H1299 p53-/and p53D281G cells were transfected with empty pCDNA3.1 or pCDNA3.1-SirT1 expression vectors and then treated with DM SO or 200 nM LAQ824 for 24 hours and subjected to (A) western blot and (B) caspase-3 assay. Because SirT1 can be specifically inhibited by the compound EX527 (Napper et al., 2005), we treated p53-/and p53D281G H1299 cells with EX527, LAQ824, or the combination of both compounds. Although EX527 alone had no effect on K382 acetylation, when combined with LAQ824 there was a significant increase in p53 acetylation (Figure 62). However, EX527 faile d to enhance LAQ824-meciated apoptosis (Figure 63). This could possibly be due to a saturation of the acetylated p53 needed for 107
apoptosis induction after LAQ824 treatment. Over all, it appears that SirT1 does not play a significant role in LAQ 824-mediated cytotoxicity. Figure 62. Pharmacological inhibition of SirT1 enhances p53 acetylation. H1299 p53-/and p53D281G cells were treated with DMSO, 1 M EX527, 200 nM LAQ824 or the combination of 1 M EX527 and 200 nM LAQ824 for 24 hours and subjected to western blot analysis. 108
Figure 63. SirT1 inhibition does not affect p53-dependent HDACi-induced apoptosis. H1299 p53-/and p53D281G cells were treated with DMSO, 1 M EX527, 200 nM LAQ824 or the combination of 1 M EX527 and 200 nM LAQ824 for 24 hours and subjected to Annexin V-APC/ 7AAD staining and flow cytometry analysis. 109
SAHAor LAQ824-Mediated ROS Production Is Dependent on p53 Acetylation and Bax ROS production plays a central role in HDA Ci-mediated cell death (Ruefli et al., 2001). In addition, ROS is a mediator of p53-in duced apoptosis (Polyak et al., 1997). We therefore examined the co rrelation between ROS genera tion and p53 transactivationindependent proapoptotic function after HDACi treatment. To this end, HCT116 p53-/cells stably expressing p53QS or empty control vector (Puro) were treated with SAHA or LAQ824 and the ROS levels were determined as previously described (Chandel et al., 2001). LAQ824 increased the ROS level in p53QS but not control cells (Figure 64A). The antioxidant NAC reversed this ROS generation (Figure 64A) and decreased cell death (Figure 64B) induced by SAHA or LAQ824 in p53QS cells. Figure 64. HDACi-induced p53-dependent RO S production promotes cell death. (A) HCT116 p53 -/cells stably expressing control Puro or p53QS were treated with 200 nM LAQ824 in the pres ence or absence of 10 mM NAC for 12 hours and the changes in intracellu lar ROS level were determined. (B) HCT116 p53-/cells stably expressing p53QS were treated with 5 M SAHA or 200 nM LAQ824 in the presence or absence of 10 mM NAC for 48 hours. The percentage of cell viability was determined by trypan blue dye exclusion 110
assay; mean + SD, n=3. Moreover, substitution of K320/373/382 to R (K3R) completely abolished the ability of p53QS to induce ROS generation after L AQ824 treatment (Figures 65A & B). These results suggest that SAHA and L AQ824 require p53 acetylation but not its transcriptional activity to induce ROS production that is essential for HDACi-mediated cytotoxicity. Figure 65. K3R mutation prevents HDACi-induced p53-dependent ROS production. HCT116 p53-/stably transfected with p53QS or p53QS/K3R were treated with 200 nM LAQ824 and intrace llular ROS levels were determined by analysis of DCF staining us ing (A) cell-free analysis by spectrofluorometry or (B) live ce ll analysis by flow cytometry. Mitochondria are the main s ource of cellular ROS and Bc l-2 family proteins are essential mediators of the amount of RO S production by HDACi (Ott et al., 2007a). To 111
determine the role of Bax in HDACi-indu ced ROS production and cell death, we took advantage of HCT116 Bax knockout cells (Z hang et al., 2000). Similarly to p53-/HCT116 cells, Bax-/HCT116 cells were also resist ant to SAHAor LAQ824-induced cell death regardless of norma l p53 expression (Figure 66). Figure 66. Bax expression prom otes HDACi-induced cell death. Bax +/and Bax -/HCT116 cells were treated with 5 M SAHA or 200 nM LAQ824 for the indicated periods of time and subjected to trypan blue dye exclusion assay. Furthermore, the upregulation of ROS levels induced by SAHA or LAQ824 was not observed in Bax-null HCT116 cells (Figur es 67A & B). Taken together, these data suggest that p53 exerts its tr anscription-independent functi on upstream of Bax signaling to control ROS generation and apoptos is in response to HDACi treatment. 112
Figure 67. Bax expression promotes RO S generation in response to HDACi. Bax +/and Bax -/HCT116 cells were treated with 5 M SAHA or 200 nM LAQ824 for 12 hours and the changes in intracellular ROS level were determined by DCF using (A) cell-free analysis by spectrofluorometry or (B) live cell analysis by flow cytometry. Acetylated p53 Binding to Ku70 Activates Bax by Disrupting the Bax/Ku70 Complex To gain further insight into the trans activation-independent proapoptotic function of p53 in response to HDACi, we took a proteo mics approach combined with the tandem affinity purification (TAP) (Wa ng et al., 2004) to identify p53QS binding proteins. The Nterminal TAP-tagged p53QS fusion protein was stably expressed in HCT116 p53-/cells 113
and its ability to enhance HDACi-mediated cytotoxicity was confirmed (Figure 68). Figure 68. TAP-p53QS mediated HDACi-induced cell death. HCT116 p53 -/cells stably expressing control TAP or TAP-p53QS were treated with 200 nM LAQ824 for indicated periods of time. The percentage of cell viability was determined by trypan blue dye exclusion assay. These cells were then treated with LAQ 824 and subjected to TAP purification and SDS-PAGE/mass spectrometry analysis. As a result, we identified Ku70 in the p53QS complex (Figure 69). This association was not disrupted by DNase or ethidium bromide, indicating that the p53 /Ku70 interaction is not mediated by DNA (data not shown). To determine if p53 acetylation is required for its binding to Ku70, we performed coimmunoprecipitation analysis in p53-/HCT116 cells stably transfected with empty control vector (Puro), Myc-p53QS, or Myc-p53QS/K3R constructs (Figure 70). A specific interaction between Myc-p53QS and endogenous Ku70 was observed after LAQ824 treatment. However, substitution of K320/373/382 to R (K3R) in p53QS did not affect this interaction, suggesting that ace tylation at these lysines is dispensable for p53 binding to Ku70. 114
Figure 69. Identification of Ku70 in complex with TAP-p53QS. Cell lysates prepared from HCT116 TAP and TAP-p53QS cells treated with 200 nM LAQ824 for 20 hours were subjected to TAP purif ication followed by SDS-PAGE and mass spectrometry analysis. Figure 70. Ku70/p53 binding is independe nt of p53 acetylation at K320/373/382. HCT116 p53-/cells stably transf ected with empty (Puro), Myc-p53QS or Myc-p53QS/K3R were treated with DMSO or 200 nM LAQ824 for 16 hours. Cell lysates were subjected to immun oprecipitation with anti-Myc antibody. The observed increase in p53/Ku70 intera ction after LAQ824 treatment is likely due to the increased protein levels of exogenous p53, because stabilization of p53 with MG132 also led to increased p53/Ku70 interaction (Figure 71). Moreover, in vitro GST 115
pull-down assays using GST-Ku70 and purified unacetylated or acetylated recombinant p53 revealed that the interaction between p53 and Ku70 is direct and acetylationindependent (Figure 72). Additionally, endogenous p53 could co-immunoprecipitated with endogenous Ku70 in both LAQ824 treated and untreated SW480 cells (Figure 73), providing further evidence that the p53/Ku70 interaction occu rs at physiological protein levels in an acetylation-independent manner. Figure 71. Increased p53 and Ku70 expression and complex formation is induced by proteasome inhibition or HDACi. H1299 p53-/and p53D281G cells were treated with DMSO, 1 M MG132 or 200 nM LAQ824 for 24 hours and subjected to immunoprecipitation for Ku70. Figure 72. In vitro pull-down of GST-Ku70 and purified p53. Purified recombinant GST and GST-Ku70 was immobilized on Glutathione Sepharose 4B agarose 116
and incubated with purified non-acety lated or acetylated p53 for 2 hours, washed, and subjected to western analysis to determine p53 binding. Figure 73. Ku70 associates with endogenous mutant p53 with and without HDACi treatment. SW480 cells were treated with DMSO or 200 nM LAQ824 for 24 hours and subjected to immunoprecipita tion with HA or Ku70 monoclonal antibodies and normal rabbit serum (N RS) or p53 polyclonal antibodies. The interaction between Ku70 and Bax pr events Bax conformational change and mitochondrial translocation (Cohen et al., 2004a; Li et al., 2007). Mo reover, it has been shown that Ku70 acetylation reduces the Ba x/Ku70 association and plays a role in HDACi-induced cell death (Subramanian et al., 2005). Thus, we speculated that the interaction between acetylate d p53 and Ku70 may be involved in Bax activation induced by HDACi. After LAQ824 treatment, a dras tic conformational activation of Bax measured by exposure of the 6A7 epitope was observed in p53QS cells compared to p53QS/K3R and Puro control cells that coincide d with increased p53 acetylation (Figure 74). Analysis of Bcl-2 family proteins in these cell lines revealed that LAQ824 treatment resulted in an increase in BimEL, with a lesser extent to Bak a nd Mcl-1, and a slight decrease in Bcl-2 (Figure 74). However, th ere are no clear differences in these Bcl-2 family members between p53QS, p53QS/K3R, and control Puro expressing cells, suggesting that acetylated p53 has other modes of acti on besides regulating Bcl-2 family protein 117
expression for the induction of a poptosis in response to HDACi. Figure 74. Acetylation of p53QS is required for HDACi-induced Bax activation without impact on Bcl-2 family member expression. HCT116 p53-/-, p53QS and p53K3R cells were treated with DMSO or 200 nM LAQ824 for 18 hours and subjected to Bax 6A7 IP and western blot analysis. Interestingly, the Bax/Ku70 interactio n was increased at 12 hours following LAQ824 treatment (Figures 75). However, while the enhanced Bax/Ku70 complex was maintained up to 36 hours in p53-/(Puro) cells, the Bax/Ku70 association was decreased at 24 hours in p53QS cells. Similar results were obtained in p53-/and p53+/+ HCT116 cells (Figure 76). 118
Figure 75. Ku70/Bax complex stability after HDACi treatment is regulated by p53. HCT116 p53-/cells stably expr essing empty (Puro) or Myc-p53QS were treated with 200 nM LAQ824 for the indicated times and subjected to immunoprecipitation with anti-Bax pol yclonal antibody or control NRS. Figure 76. Endogenous wild type p53 retai ns the ability to disrupt the Bax/Ku70 complex. HCT116 p53 -/and p53+/+ cells we re treated with 200 nM of LAQ824 for the indicated times and s ubjected to immunoprecipitation with anti-Bax polyclonal an tibody or control IgG. Interestingly, the interaction betw een Ku70 and Bax was increased in p53QS/K3R cells during LAQ824 treatment and the p53QS/K3R protein was detected in the Bax/Ku70 complex after 24 hours treatment (Figure 77). These findings s uggest that HDACi treatment initially promotes an increase in the interaction between Bax and Ku70, but prolonged treatment leads to the disruption of this complex probably through acetylated 119
p53. Figure 77. Acetylation deficient p53 expression prevents HDACi-induced disruption of Ku70/Bax complex. HCT116 p53 -/cells stably expressing Myc-p53QS or Myc-p53K3R were treated with 200 nM of LAQ824 for the indicated times and subjected to immunoprecipitation with anti-Bax polyclonal antibody or control NRS. Since p53 interacts with Ku70 independently of its acetylation status, the ability of acetylated p53 to disrupt the Bax/Ku70 interaction was assayed in vitro The Bax/Ku70 immune complexes were isolated from HCT116 p53-/cell lysate and incubated with purified non-acety lated or acetylated recombin ant p53 (Piluso et al., 2005) (Figure 78A). Only acetylated p53 was able to release Ku70 from the Bax/Ku70 complex when compared to control vehicle and unacetylated p53. Similar results were also obtained in a GST-Ku70 pull-down assay; a cetylated p53 could prevent Bax binding to Ku70, whereas non-acetylated p53 had no effect (Figure 78B). These results clearly indicate that p53 acetylation is impor tant for Bax release from Ku70. 120
Figure 78. Purified acetylated p53 disrupts Bax/Ku70 complexes in vitro (A) HCT116 p53-/cell lysate was immunopr ecipitated with anti-Bax polyclonal antibody and the resulting isolated imm unocomplexes were incubated with purified non-acetylated or acetylated p53 for 30 mi nutes. The supernatant was collected and the immunocomplexes were washed 3 times and subjected to western analysis. (B) Purified recombinant GST and GST-Ku70 were isolated by Glutathione Sepharose 4B agarose pull-down, after which purified Bax, non-acetylated or acetylated p53 were incubated together for 2 hours. Samples were washed and s ubjected to immunoblot analysis. To elucidate the role of Ku70 in HDACi-induced apoptosis, H1299 p53-/and p53D281G cells were infected with control sh Scr or shKu70 lentiv irus. Knockdown of Ku70 was assayed by Western bl ot (Figure 79A). These cells were then treated with LAQ824 for 24 hours and subjected to apoptos is analysis with Annexin V-APC/7AAD staining and flow cytometry (Fig ure 79B). Knockdown of Ku70 in p53-/cells promotes an enhanced apoptotic response over shScr control cells. Howeve r, knockdown of Ku70 in p53D281G cells did not significantly impact LAQ824 induced apoptosis. This result suggests that the Bax/Ku70 complex is essential to the prevention of HDACi-induced apoptosis in p53-/cells. However, when p53 is present and able to disrupt the Bax/Ku70 complex, reduction in Ku70 expression is redundant and unnecessary to promote 121
HDACi-induced apoptosis. Figure 79. Ku70 knockdown p referentially enhances HDACi-induced apoptosis in p53-/cells. H1299 p53-/and p53D281G cells were infected with shScr or shKu70 lentivirus. 24 hours after infection cells were treated with DMSO or 200 nM LAQ for an additional 24 hours and analyzed for (A) Ku70 knockdown by western blot and (B) apoptotic response measured by Annexin V-APC/7AAD staining and flow cytometry. Data represented as mean + SD, DMSO treated n=3, LAQ treated n=5. To determine the cellu lar localization of the Bax/Ku70 complex, coimmunoprecipitation experiments were performed using heavy membrane, cytosolic, and 122
123 nuclear fractions isolated from p53-/HCT116 cells transfected w ith control empty vector (Puro), or Myc-tagged p53QS or p53QS/K3R (Figure 80). The Bax/Ku70 complex was increased in both cytosolic and nuc lear fractions of Puro and p53QS/K3R cells after LAQ824 treatment. Since the complex formation was only assayed at 24 hours after treatment, the accumulation of the Bax/Ku70 complex was not observed in p53QS cells as it was at 12 hours seen in Figure 74. Furthermore, the Ku70/p53 complex was most readily detected in the nuclear fraction corre sponding to the prominent localization of p53 and Ku70 to this cellular compartment. Howe ver, p53 could also be found localized to the cytosolic and heavy membrane fractions and in a complex with Ku70 at reduced levels compared to that in the nuclear fr action after LAQ824 treatment. The localization of p53QS, Bax, and Ku70 was also confirmed by i mmunofluorescence (Figures 81A & B).
Figure 80. Cellular localization of p53/Ku70/Bax interactions. (A) HCT116 p53-/cells stably expressing Puro, Myc-p53QS or Mycp53QS/K3R were treated with DMSO or 200 nM LAQ824 for 24 hours a nd subjected to cell fractionation and immunoprecipitation with the indicated antibodies. 124
125 p53-QS p53 -/A p53-QS p53 -/B Figure 81. Cellular localization of p53, Ku70 and Bax in HCT116 cells. (A) HCT116 p53-/or p53QS cells were treated with 5 M SAHA for 18 hours and then immunostained for Bax and HSP60 w ith DAPI counter staining. (B) HCT116 p53-/or p53QS cells were treated with 5 M SAHA for 18 hours and then immunostained for Ku70 and p53 with DAPI counter staining.
To determine whether p53QS affects the intracellular redistribution of Bax after HDACi treatment, we performed subcellu lar fractionation and immunoblot analysis. Ku70 localization was not affected by p53QS after HDACi treatment, while the nuclear Bax was decreased in p53QS cells but not in p53-/cells (Figure 82). Conversely, the cytosolic Bax was increased in p53QS cells after HDACi treatment. These results support the hypothesis that Bax dissociation from K u70 and subsequent translocation to the cytoplasm after HDACi treatment is mediat ed by acetylated p53.Consistent with the retained association of Ku70 with Bax, HDA Ci-induced Bax conformational activation at the mitochondria was attenuated in p53-/and p53QS/K3R compared to p53QS cells (Figure 83A), which correlated with an apparent increase in Bax translocation to the mitochondrial fraction specifically in HDACi treated p53QS cells (Figure 83B). Figure 82. p53-dependent HDACi-induced B ax translocation from the nucleus to the cytoplasm. HCT116 p53-/cells stably expressing Puro or p53QS were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 18 hours and subjected to subcellular fracti onation and immunoblot analysis. 126
Figure 83. HDACi-induced acetylated p53-dependent Bax activation and mitochondrial accumulation. (A) Immunoprecipita tion using Bax 6A7 antibody in mitochondrial fract ions of HCT116 p53-/-, p53QS and p53QS/K3R cells treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 18 hours. (B) HCT116 p53-/cells stably tr ansfected with Puro, Myc-p53QS or Mycp53QS/K3R were treated with DMSO, 5 M SAHA or 200 nM LAQ824 for 16 hours and subjected to subcellular fractionation and immunoblot analysis. Discussion HDACi represent a new class of chemot herapeutic agents that have shown promise in pre-clinical and clinical trials a nd work by modifying the acetylation status of histone and non-histone proteins. Acetyl ation is accepted as an important posttranslational modification th at impacts protein structure and/or function but relatively few non-histone proteins have been identifie d as regulated by acetylation. Of those identified are several important apopto tic molecules such as Ku70, p53, Rb, and p73 (Chan et al., 2001; Cohen et al., 2004a; Costan zo et al., 2002). The findings presented in 127
this report indicate that HDACi can i nduce apoptosis by a p53 transactivationindependent apoptotic mechanism. Expression of various mutants of p53 in p53-/backgrounds enhances HDACi-induced apopt osis, and knockdown of endogenous mutant p53 results in an abrogated apoptotic respons e. Both mutant and wild type p53 bind Ku70 and promote Bax dissociation from Ku70 in an acetylation dependent manner. Additionally, HDACi can induce substantial apoptosis in p53-/but not mutant p53 expressing cells when Ku70 is knocked down. Therefore, we propose a model where the interaction between Ku70 and p53 is acetylation independent; however, acetylation of p53 is absolutely required for Bax dissoci ation from Ku70, conformational activation, and enhanced apoptosis upon HDACi treatment (Figure 84). Given that p53 is frequently mutated in human cancers, our findings that mutant p53 sensitizes cancer cells to HDACi-induced apoptosis via Ba x activation further explain w hy this class of inhibitors displays preferential an ti-neoplastic potential. Figure 84. Proposed model for p53/Ku70/ Bax interaction disrupted by HDACiinduced acetylation. In normal resting cells p53/Ku70/Bax can likely be found in complex. After HDACi, Bax is displaced from Ku70 by acetylated p53 and becomes activated to induce apoptosis. 128
129 Transcription-Independent p53 Mechani sm of HDACi-induced Apoptosis The transcriptional activity of p53 is able to efficien tly enhance apoptosis in response to various cellular st resses. Genotoxic stresses re sult in activation of DNA damage response pathways that activate kina ses such as ATM, ATR, CHK1 and CHK2 or methyltransferases such as Set7/9 that respectively phosphoryl ate or methylate p53 which in turn promotes the acetylation of p53 at C-terminal lysines resulting in enhanced protein stability and sequ ence specific DNA binding (Ivanov et al., 2007; Toledo and Wahl, 2006). However, through the course of our experiments into the nature of p53dependent HDACi-induced apoptosis we found that the transcript ional activity of p53 was dispensable but acetylation of C-termin al lysines was not. There are conflicting reports as to what role p53 transcription has on HDACi-induced apoptosis. Much of this could be attributed to global changes in transcription due mainly because of histone acetylation and maybe less so through acetylatio n of other factors such as p53 or other transcription factors. Indeed, our experiments have found that expr ession of wild type p53 marginally enhanced HDA Ci-induced caspase-3 activ ity over the response from expression of mutant p53QS (Figure 46), although this doe s not necessarily correlate exactly with the cell death response (Figure 45). This could indicate several possible scenarios. First, expression of wild type p53 could preferentially promote apoptosis through transcriptional regulation. However, our results have shown that wild type p53 still functions to disrupt the Bax/Ku70 complex when acetylated by HDACi treatment, suggesting that this pathway is active even in the presence of functional tr ansactivation activity. This gives favor to the alternative scenario wh ere wild type acetylated p53dependent HDACi-induced apoptosis functi ons in a predominantly transcription-
130 independent manner to illicit such a response, likely through disrupt ion of the Bax/Ku70 complex as shown above. This would explain why there was limited additive effect on HDACi-induced caspase-3 activa tion in wild type p53 over p53QS expressing cells. However, this does not explain why there is enhanced viability in wild type p53 cells compared to p53QS after HDACi treatment. This could possibly be caused by the increased stability of p53QS over wild type p53 as the Mdm2 binding site is disrupted in this mutant. Nonetheless, more experiment s will be needed to rectify the roles of acetylated wild type p53 in transcripti on-dependent and independent HDACi-induced apoptosis. We sought to confirm that mutant p53QS lacks HDACi-induced transcriptional activity by luciferase reporter gene assays (Figure 44). Expression of wild type p53 dramatically enhanced transcription from the p21 and MDM2 promoters, but p53QS also exhibited low levels of target pr omoter activation compared to p53-/cells. However, this residual transactivati on potential of p53QS likely plays a minor role in HDACi-induced apoptosis for several reasons. First, microa rray analysis revealed that there was no specific increase in p53 responsive target ge ne expression after treatment with HDACi (data not shown). The expression of p21 wa s also found to be similar between p53-/-, p53QS, and p53QS/K3R stably expressing cell lines (Figur e 74). Indeed, it has been shown that p21 expression is enhanced by HDACi in a p53-independent manner (Huang et al., 2000). Although p53QS has been reported to retain the potential for transcriptional activation of Bax and repression of Bcl-2 (Johns on et al., 2005), we we re unable to detect changes in apoptotic pr oteins controlled by p53QS such as Bax, Bcl-2, and Puma after HDACi treatment in (Figure 74). Additionall y, studies with transfection of prominent
131 naturally occurring p53 mutant s into p53 deficient HCT116, K562, and H1299 cells, and knockdown of endogenous mutant p53 in SW48 0 and HT-29 cells further support our hypothesis that p53 retains a transcription-inde pendent proapoptotic function mediated by acetylation. p53 Localization and HDACi-Induced Apoptosis Although it is widely accepted that p53 has a transcription-independent mechanism of apoptosis induction, several cont roversies remain. For instance, it has been shown that p53 translocates to mitochondria where it interacts with antior proapoptotic Bcl-2 family proteins including Bcl-XL, Bcl-2, and Bak to induce Bax and Bak activation, thereby leading to the release of cytochrome c from mitochondria to the cytoplasm (Chipuk et al., 2004; Deng et al., 2006; Leu et al., 2004; Mi hara et al., 2003). However, other reports suggest that the leve l of mitochondrial p53 does not correlate with the level of apoptosis (Essmann et al., 2005; Mahyar-Roemer et al., 2004). Upon HDACi treatment, relatively low levels of p53QS and p53QS/K3R were detected in the mitochondrial fraction (Figure 80). Furthermore, there we re no differences between wild type p53, p53QS, and p53QS/K3R for their ability to interact w ith Bcl-XL (Figure 85), even though p53QS/K3R cannot significantly enhance HDACi-i nduced apoptosis, suggesting that the mitochondrial translocation of mutant p53 may not play a major role in HDACi-mediated apoptosis. However, the small increase in HDACi-induced apoptosis in p53QS/K3R over p53-/cells may rely on its ability to bind Bcl-XL at the mitochondria. Alternatively, this observation could possibly be due to another mechanism such as residual transcriptional
activity of p53QS/K3R. Although our results suggest that p53QS and p53QS/K3R have no impact Bcl-2 family or p21 expression pr ofiles in response to HDACi treatment compared to p53-/cells, other unexamined proteins may be affected which could theoretically also lead to the slight increase in apoptosis seen in the p53QS/K3R cell line. The exact mechanism by which this occurs remains to be resolved. Figure 85. Wild type and mutant p53 interaction with Bcl-XL and Bak. p53-/HCT116 cells were transiently transfected with the indicated p53 expression plasmids. 36 hours after transfection, th e cells were treated with LAQ for additional 12 hours. The cells were then lysed and subjected to IP with myc monoclonal antibody, followed by immunoblot analysis with anti-Bcl-XL or Bak polyclonal antibodies. As a transcription factor, p53 principally localizes in the nucleus, but certain apoptotic stimuli can promote p53 accumula tion in the cytosolic and mitochondrial fractions. Ku70 is an important mainly nuclear protein involved in non-homologous end joining DNA repair and V(D)J recombination (Gu et al., 1997). Therefore, it is not surprising that the nucleus is the compartment that contains the highe st concentration of p53 associated with Ku70 (Figure 80). Alte rnatively, Bax has been classified as a predominantly cytosolic protein that loosely associates with the mitochondrial membrane in unstressed cells. Enigmatically, Bax has also been classified as a nuclear protein and 132
133 under certain stresses interacts with p53 (Nishita et al., 1998; Raffo et al., 2000). Similarly, our results indicat e that a substantial amount of Bax can be found in the nucleus of HCT116 cells (Figures 80 and 81A). It seems that HDACi-induced p53 acetylation disrupts the Ku70/Bax complex, which results in Bax conformational change and translocation from the nucleus to the cytoplasm. However, cytosolic p53 is also equally capable of disrupting Bax from K u70. The individual contri butions of Ku70/Bax dissociation in the separate cellular compartments remain to be determined. Bax has been traditionally characterized as a cytoplasmic and mitochondrial protein, however increasing ev idence has suggested that Bax can also be found in the nucleus but its function there is almost co mpletely unknown. Here we provide evidence that nuclear Bax activation can be initiat ed in response to HDACi treatment through displacement from Ku70 by acetylated p53 and translocation to the cytoplasm. Nuclear Bax localization may predispose cells to enhanced HDACi-induced apoptosis as Ku70/Bax interactions there have an increa sed probability of di ssociation due to p53 enrichment. This localization of Bax may also have impacts on other proapoptotic signals originating in the nucleus such as genotoxic stresses. However, future research is still required to fully understand this se emingly misplaced population of Bax. p53 Acetylation and Bax/Ku70 Disruption LAQ824 treatment results in a transient increase in the asso ciation between Ku70 and Bax (Figures 75-78). This phenomenon could be due to several reasons. First, this apoptotic stimulus may encourage a cellular re sponse designed to prevent Bax activation by sequestration through Ku70 binding. The incr eased association could be caused by a
134 variety of different mechanisms such as pos ttranslational modifications or intracellular distribution. Another intrigui ng possibility is that the increase in Ku70/Bax complex could serve a dual role as K u70 promotes the deubiquitination of Bax in addition to its sequestration (Amsel et al., 2008). Thus, this interaction could prime cells for apoptosis by increasing the pool of unmodified Bax able to promote apoptosis after release from Ku70. In either case, prolonged HDACi treatment that results in the disruption of the Ku70/Bax complex can enhan ce the apoptotic response. Expression of wild type and mutant p53 cap able of being acety lated promotes the dissociation of Bax from Ku70 after a pr olonged HDACi treatment (Figures 75-78). Moreover, acetylated but not unacetylated recombinant p53 is able to disrupt the Ku70/Bax complex in vitro (Figures 78A & B). However, the acetylation deficient p53QS/K3R mutant retains the ability to inte ract with Ku70 and accumulates in the Ku70/Bax complex during HDACi treatment (F igures 77 and 80). Since acetylation of p53 alters its structure (Giordano and Avantaggi ati, 1999), it is plausible that once p53 is acetylated changes in conformation lead to competition for the Bax binding site on Ku70. Acetylation and altered struct ure of p53 could also lead to conformational changes in Ku70, which may depend its own acetylation, and the release of Bax as previously proposed (Cohen et al., 2004a). Furthermore, past studies into th e acetylation of Ku70 and the regulation of apoptosis were carried out primarily in 293T cells which would preclude p53 transactiva tional activity but not necessarily its transcriptional-independent role in HDACi-induced apoptosis (Cohen et al., 2004a; Li et al., 2007). Our results suggest that the cellular status of both p53 and Ku70 determines apoptotic responses (Figure 79B) and is warrant to be further eval uated as biomarkers fo r clinical therapeutic
135 response to HDACi such as SAHA and L AQ824. Additional assays designed to determine how acetylated p53 affects the Ku70/ Bax complex at the structural level will give additional insights into this novel in terplay between proteins with hopes of developing advanced therapeutic stra tegies to benefit cancer patients. Materials and Methods Cell Culture and Transfection HCT116 cells were grown in McCoys 5A medium, K562, SW480 and HT-29 cells were grown in RPMI, and H1299 and MEFs were grown in DMEM. Medium was supplemented with 10% FBS and 1X peni cillin/streptomycin. MEFs p53-/and p53R172H/R172H were a kind gift from Guillerm ina Lozano and were described previously (Lang et al., 2004). Cell transfection using Lipofectamine 2000 (Invitrogen) was described previously (Yamaguchi et al., 2004). The plasmids expressing p53 WT, QS, R175H, R273H, and D281G mutants were de scribed previously (Hinds et al., 1990; Yamaguchi et al., 2004). The p53QS/K3R mutant was prepared by two-step PCR and subcloned into pcDNA3-Myc vector. To establis h stable transfectants, plasmids were cotransfected with pBabe-puro or pEF6-Myc-His (Invitrogen) empty vector and selection was performed with puromycin (puro) or blasticidin S HCl (Bsd). The p53R175H lysine mutants were prepared by site directed mu tagenesis (Stratagene) using pcDNA3-p53 as the template, cloned into the retroviral pKI ve ctor between the BglII/ XhoI restriction sites and transfected into 293 Ampho cells to produce recombinant retrovirus es. The lentiviral pLK0.1 empty, scrambled, Ku70 sh RNA (TRCN0000039611) and p53 shRNA (TRCN0000010814) constructs were purchased from Sigma. The shRNA lentivirus was
136 produced in 293FT cells using the ViraPo wer kit from Invitrogen as per the manufactures recommendations. Reagents LAQ824 and SAHA were kindly provided by Novartis Pharmaceuticals Inc. (East Hanover, NJ) and Merck & Co., Inc. (Flemington, NJ), respectively. Anti-Ku70 monoclonal and polyclonal antibodies and anti-Myc polyclonal antibodies were purchased from Santa Cruz Biotechnology. Anti-p53 (CM1) polyclonal and monoclonal (DO-1) antibodies, anti-Bax polyclonal and monoclonal antibodies, anti-actin, tubulin and Myc monoclonal antibodies were descri bed previously (Yamaguchi et al., 2004; Yamaguchi and Wang, 2006). Anti-Myc or Flag agarose beads were purchased from Sigma. Anti-histone H3 and anti-acetylated p53 (K382) antib odies were purchased from Cell Signaling. Anti-HSP60 and anti-Lamin B antibodies were purchased from BD Biosciences and Calbiochem, respectively. Tandem Affinity Purification of p53QS Binding Protein The p53QS cDNA was subcloned into the Ec oRI and XhoI sites of pcDNA3-TAP vector (Wang et al., 2004). The N-terminal TAP-tagged p53QS was stably expressed in p53-/HCT116 cells and treated with 200 nM L AQ824 for 20 hours. The cells were lysed in TAP lysis buffer (0.5% Triton X-100, 0.5 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 nM LAQ824) and sonicated. After centrifugation, the cell lysate was subjected to purification/ mass spectrometry analysis as described previously (Wang et al., 2004).
137 GST-Ku70/Bax/p53 In Vitro Binding Assay GST or GST-Ku70 conjugated Glutathione Sepharose 4B were resuspended in 1% Chaps buffer and 100 l were aliquoted into separate tubes. Approximately 0.5 g Bax was added with or without 200 ng purified non-acetylated or acetylated p53 and brought up to a total volume of 300 l with 1% Chaps buffer, then incubated with rocking at 4 C for 2 hours. Beads were spun down at 1,000g for 1 minute, supernatant was removed and beads were washed 3X with 1 mL ice-cold 1% Chaps buffer. Laemmli buffer was then added to the beads, boiled for 3 minutes, vortexed, and then run on an SDS-PAGE. Immunoprecipitation Immunoprecipitation of active Bax by anti-Bax 6A7 antibody and coimmunoprecipitation of Ku70 and Bax were carrie d out as described previously (Li et al., 2007; Yamaguchi et al., 2004). For co-immunopr ecipitation of Ku70 and p53, cells were treated or untreated with HDACi for the i ndicated times and lysed in 1% Chaps lysis buffer. After sonication and centrifugation, the lysates were immunoprecipitated with Myc monoclonal, Ku70 monoclonal or p53 (CM1) polyclonal antibodies followed by immunoblot analysis with the indicated anti bodies. For Figure 80, 500 g proteins were incubated overnight with 4 l of anti-Bax se rum, 1 g of Myc (Sigma) or 1 g of Ku70 (Santa Cruz) polyclonal antibodies. Samples we re further incubated with 15 l of Protein A agarose at 4C for 2 hours, washed three times and analyzed by immunoblot.
138 Subcellular Fractionation To isolate the mitochondria-enriched hea vy membrane (HM) fraction, cells were homogenized in isotonic mitochondrial buffer (210 mM sucrose, 70 mM mannitol, 10 mM Hepes pH 7.4, 1 mM EDTA) contai ning protease inhib itor cocktail and centrifuged at 1,000 g for 10 min to discard nuclei and unbroken cells. The resulting supernatant was centrifuged at 10,000 g for 15 min to pellet the heavy membrane fraction and the supernatant wa s centrifuged further at 100,000 g for 30 min to obtain cytosolic fraction, which was transferred to new tubes and the NaCl and CHAPS concentrations were adjusted to 150 mM a nd 1%, respectively. To isolate the nuclear fraction, cells were resuspended in 5 vol ume of Buffer A (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA, 0.5 mM DTT, and prot ease inhibitor cocktail). After incubation on ice for 15 min, Triton X-100 was added to a final concentration of 0.2%. The lysates were then vortexed for 5 sec followed by centrifugation at 10,000 x g for 10min. The crude nuclear pellets were washed with Buffer A twice, resuspended in Buffer B (10 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM ED TA, 0.5 mM DTT, 0.5% Triton X-100, and protease inhibitor cocktail) and sonicated. The sonicated lysate s were then centrifuged at 15,000 x g for 10 min and the supernatant was used as nuclear fraction. For Ku70/Bax/p53 co-immunoprecipitation experiments, the nuclear a nd heavy membrane fractions were lysed in 1% Chaps and sonicated. Caspase Assay, ROS Measurement and Annexin V staining Caspase-3 activity was measured using the fluorometric caspase-3 activity assay kit according to the manufacturer's instructions (Sigma). ROS level was determined as
139 described previously (Chandel et al., 2001). Briefly, cells treated or untreated with HDACi for 12 to 16 hours were incu bated in PBS containing 10 M dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes) for 10 min at room temperature and lysed in ROS lysis buffer. H2DCFDA oxidation into DCF was measured using a spectrofluorometer (excitation 485 nm, emission 535 nm). Data are normalized by protein concentration and expressed as rela tive change from untr eated cells. For flow cytometry measurement of ROS, cells were treated or untreated with 200nM of LAQ for 14 hours then trypsinized, washed with PBS once and incubated in PBS containing 10 M H2DCFDA for 20 minutes at room temperatur e. The cells were analyzed by flow cytometry and the results were analyzed by BD FACSDiva software (BD Bioscience). The results were shown as a change of m ean area of fluorescence signals (measured by the software). Annexin V-FITC/PI or Anne xin V-APC/7AAD staini ng was completed as per the manufacturers recommendations (BD Biosciences) and analyzed by BD FACSCalibur and FlowJo software. Luciferase Assays Cells were seeded on 24-well plates a nd grown to 70% confluency. Cells were then transfected with 100 ng of reporter c onstructs and 10 ng internal control renilla luciferase internal control plasmid pR L-SV40 using Lipofectamine2000 transfection protocol. 24 hours after transfect ion cells were treated with 5 M SAHA or 200 nM LAQ for an additional 12 hours. Cells were then ly sed and subjected to Dual-Luciferase assay as per the manufacturers r ecommendations (Promega).
140 Protein Purification Human Ku70 cDNA was subcloned into pGEX-4T-1 vector using BamHI and XhoI restriction enzyme cloning sites. pGEX-4T-1-Ku70 or empty vector was transformed into the BL21 strain of E. coli and grown overnight at 37 C in LB. One mL of this turbid culture was used to inoculat e 200 mL of LB which was then grown to an OD600 between 0.6-0.8. At this time, IPTG was a dded to a final concentration of 0.5 mM and then incubated for 6 hours at room temperature with shaking. E. coli was isolated by centrifugation at 25,000g for 10 minutes and resuspended in 2 mL PBS supplemented with protease inhibitors (Sigma). The resulting E. coli suspension was sonicated, and Triton X-100 was added to a final concentra tion of 1% and incuba ted for 30 minutes at room temperature with rocking. Cell debris was removed by centrifugation at 20,000g for 20 minutes at 4 C. The supernatant was collected and incubated with Glutathione Sepharose 4B (Amersham) for 30 minutes at room temperature with rocking. After wash 3X with 1 mL PBS and 1X with 1 mL 1% Chaps lysis buffer, the GST protein was eluted with 10 mM of reduced glutathione in 50 mM Tris-HCl (pH=8.0) and dialyzed against PBS (pH=7.4). Recombinant full-length Bax protein wa s purified using the IMPACT system (New England Biolabs) as previously de scribed (Suzuki et al., 2000). The pYTB1-Bax construct, which expresses a Bax and intein tag fusion protein, was transformed into E. coli BL21. Recombinant proteins were isol ated by chitin affinity chromatography according to the manufacturers protocol. Th e Bax protein was cleaved off from the intein tag by dithio threitol and dialyzed in 10 mM HEPES (pH=7.4), 100 mM NaCl, 0.2 mM EDTA.
141 Purification of non-acetylated and acetylated p53 proteins was performed as previously described (Piluso et al., 2005). Briefly, Sf21 armyworm cells were co-infected with HA-p53 and either empty or p300-6XHis baculoviruses. Forty-eight hours after infection, nuclear lysates were prepare d, and total HA-p53 was purified with 12CA5 monoclonal antibody conjugated to protein A-Sepharose (Pierc e) and washed three times in ten volumes of buffer D (20mM Hepe s, pH 7.9, 20% glycerol, and 0.2mM EDTA) containing 0.5 M KCl. A final fourth wash used buffer D containing 0.1 M KCl. HA-p53 was then eluted with synthetic 12CA5 peptide. HA-p53 isolated from HA-p53/p3006XHis co-infected lysate was subsequently depleted of non-acetylated p53 using Pab421protein A beads that specifically recognizes non-acetylated p53. In Vitro Disruption of Endogenous Bax/Ku70 Complex Bax was immunoprecipitated from HCT116 p53-/cells using polyclonal Bax antibody immobilized on Protein A agarose be ads. Immune complexes were washed 3X with 1mL ice-cold 1% Chaps lysis buffer and then resuspended in 20 l 1% Chaps lysis buffer. Approximately 200 ng purified non-acety lated or acetylated p53 was then added and incubated with shaking at room temperature for 10 minutes. The immune complexes were then spun down and the supernatant was collected to detect unbound proteins. The beads were again washed 3X with 1 mL ice-cold 1% Chaps lysis buffer and then resuspended in Laemmli buffer. The supernat ant and immune complexes were analyzed for released and bound proteins, respectively.
142 Immunostaining Cells were cultured in chamber slides and treated with SAHA for 18 hours. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 15 min, and incubated with 3% BSA for 1 h. The cells were then incubated with the primar y antibodies, anti-Myc monoclonal antibody (Sigma) plus anti-Ku70 polyclonal antibody (Santa Cruz), or anti-HSP60 monoclonal antibody (BD Bioscience) plus anti -Bax polyclonal antibody (Cell signaling), overnight at 4 C, washed, and further incubate d with anti-mouse IgG-FITC and antirabbit IgG-Texas Red secondary antibodies. Nu clei were stained with mount medium containing DAPI. Fluorescence images were captured with a fluorescence microscope.
143 Chapter Five: Scientific Significance & Future Directions Interestingly, even with the vast amount s of data accumulated on the topic of Bax activation in the re gulation of apoptosis, there persists a relative uncertainty as to the exact molecular mechanisms that govern the process. One of the most prominent questions is how can BH3-only molecules such as Bim directly promote Bax activation if the molecular interaction betw een the proteins is undetectable in most systems? This could be due to a transient hit-and-run in teraction where no stable complex is created. This may also be the byproduct of the assa y system employed. However, other factors such as membrane lipid composition, structural forces and membrane curvature may also play a role. The one thing that is clear is that Bax is an exquisitely re gulated protein. Cells seem to exist on the edge of apoptos is induction with an tiapoptotic molecules preventing the aberrant activation of Bax. Wh en an apoptotic insu lt does occur and the insult is strong enough, Bax can become activat ed by a number of different proteins and mechanisms to initiate cell death. Although most researchers have been hoping to discover an all-encompassing pathway of apoptos is to exploit, it has become clear that apoptosis depends on many differe nt factors such as cell type apoptotic stimulus and the microenvironment. Research into the multiple modes of Bax regulation will therefore likely be paired with individualized treatment s in the future based on known pathways of Bax activation, the compliment of Bax regulator y elements expressed in the target cell population, and the mechanism of action of the drug.
144 Src Regulation of Anoikis Contextual clues from the extracellular matr ix provide survival signals to adherent cells, and once the cell becomes dissociated from these signals it dies by anoikis. Oncogenic signaling can lead to the inhibi tion of anoikis, a ne cessary regulatory mechanism to overcome in order for cancer s of attachment dependent origin to metastasize. Anoikis inhibition can promote cell survival after detachment from their normal ECM, while detached in the circ ulatory and lymphatic system and after attachment to the incorrect ECM. Indeed, the research pr esented above illustrates the robust ability of Src oncogenic kinase to prevent anoikis through several independent mechanisms that ultimately converg e on regulation of Bax activation. Src activation of classic Akt and Erk1/2 survival signaling pathways regulate the expression profiles of Bcl-2 family proteins Mcl-1 and Bim. Speci fically, we have found that forced detachment of non-transformed NIH3 T3 cells results in a dramatic decrease in Mcl-1 and increase in Bim expression. Src transformation represses these two events which promotes cell survival during detachment. Src-Akt sign aling results in suppression of BimEL transcription, likely through phos phorylation of Foxo3a which retains this positive regulator of Bim transcription in the cytoplasm. Furthermore, Src-Erk1/2 signaling can also suppress Bim expression through phosphorylation which promotes an increase in its proteasomal degradation. Inte restingly, our research has identified Mcl-1 as a critical regulator of anoi kis. This protein is maintain ed at high expression levels by cellular attachment and by Src-Akt signaling which functionally suppresses the kinase activity of GSK-3 which in turn prevents Mcl-1 phosphorylation at serine 159 and its targeting to the proteasome. Oncogene i nduced elongation of the half-life of Mcl-1
145 potently prevents anoikis. These findings are further confirmed in that cells are not dramatically sensitized to a noikis by combined inhibition of Bcl-XL and Bcl-2 through ABT-737 treatment, but repression of Mcl-1 expression alone potently enhances the anoikis response. Also, knockdown of Mcl-1 ca n dramatically reduce the propensity of cancer cells to metastasize in vivo As a novel compound designed to potent iate apoptotic responses ABT-737 has illustrated the need to target Mcl-1, if pres ent at appreciable levels, in order to be effective. It is therefore nece ssary to continue the search fo r novel inhibitors of Mcl-1 to provide enhanced treatment strategies that can target individual or groups of Bcl-2 family proteins. One can envision the use of Mcl-1 sma ll molecule inhibitors in the treatment of cancers that could prevent metastatic progres sion. Unfortunately, inhibition of Mcl-1 expression alone is unable to promote a noikis which also requires the increased expression of proapoptotic Bim to activate Ba x. Development of Bax activating factors is also needed for therapeutic potentiation of apoptosis. Thes e may include factors that promote the expression of BH 3-only proteins, which coul d possibly be accomplished through inhibition of Foxo3a cytosolic re tention. Also, small molecule compounds capable of displacing helix 9 and/or promoting Bax confor mational activation could also prove beneficial. However, such compounds w ould likely require tumor specific targeting to avoid toxicity to normal cells. Another option, assuming the p53 pathway is intact, is to combine traditional chemotherapeutic agents that function through DNA damage and those that inhibit Bcl-2, Bc l-XL or Mcl-1. This would provide both the induction of proapoptotic molecules as well as the inhi bition of their repression and may prove effective in some cancers. Ultimately, promo tion of Bax activity specifically in tumor
146 cells remains highly promising but it is evid ent that enhanced therapeutic options still need to be developed. Furthermore, our studies have also disc overed that Src can prevent anoikis in a manner independent of Akt and Erk1/2 regula tion of Mcl-1 and Bim expression. Our lab has previously discovered a novel Bax interact ing factor, Bif-1, that has the potential to enhance Bax activation in response to ce llular stresses. However, the molecular mechanisms regulating the interaction between Bax and Bif-1 are not clearly defined. We have discovered that Bif-1 is a direct phosphorylation target of Src kinase. Interestingly, the phosphorylation of Bif-1 mediated by Src was found to inhibit its interaction with Bax during cellular detachment and this was a ssociated with lower anoikis potential. This finding illuminates another mechanism by wh ich Src can prevent anoikis through its direct kinase activity. Phosphorylation of Bif-1 by Sr c at tyrosine 80 occurs in the internal amphipathic helix domain which is essential for bi nding and tubulating membranes. Since phosphorylation imparts a negative charge to this region it may affect the helix organization and/or function of this critical domain. This may provide clues as to the causation of Bif-1 association with Bax. It should be determined what effects phosphorylation of Bif-1 by Src has on its me mbrane binding and tubulation potential and then determine if these properties influence the association between Bif-1 and Bax. It is possible that the association between these tw o proteins relies on the ability of Bif-1 to modify membrane structure whic h could in turn lead to Bax activation. If it is found that Bif-1 regulation of membrane dynamics is required for Bax binding and activation, the functional impacts should be delineated to determine at what step Bax activation is
147 affected. This could be found to be im portant for Bax conformational alteration, recruitment to the mitochondrial membrane or the insertional step. Furthermore, as Bif-1 is involved in autophagy the functional impact s on this pathway should also be evaluated. Bif-1 may be required for autophagosome maturation by regulation of membrane dynamics, a process that may potentially be impa cted through Src, or Src family kinase, mediated phosphorylation at tyrosine 80. Src kinase inhibitors are therefore prom ising therapeutic agents against certain cancers that rely on this signaling for metast atic progression. Inhibi tion of Src not only prevents survival signaling through downstream mediators such as Akt and Erk1/2 that regulate Mcl-1 and Bim, assuming other muta tions that independently regulate these pathways are not activated, but also prev ent the inhibition of other anti-anoikis mechanisms such as phosphorylated Bif-1. Elucid ation of other direct targets of Src that can prevent anoikis n eed to be determined to fully understand its role in anoikis prevention. Furthermore, it should be noted that many cancers have multiple oncogenic alterations that may bypass or enhance Src si gnaling. Src expression systems are useful at delineating the pathways that are controlled, but it should be recognized that independent activation of other signaling pathways in cancer may prevent significant therapeutic responses in a clinical setting. Ku70 and p53 Interaction Interestingly, the expression of transa ctivation deficient p53 can enhance HDACiinduced apoptosis. Whereas activatio n of p53 and its stabilization through hyperacetylation by HDACi treatment can c onceivably impact transcription of
148 proapoptotic genes, the discovery of transa ctivation incompetent p53 mediated apoptosis in response to HDACi is somewhat less intuit ive. Exploring the regul ation of this event we have discovered the novel interaction be tween p53 and Ku70. This interaction results in the release of Bax from its inhibitory complex with Ku70 upon acetylation of p53 at three critical C-terminus ly sine residues. Indeed, knockdow n of Ku70 in p53-null but not p53R175H H1299 cells robustly enhanced apoptosis, which indicates that Bax retention by Ku70 inhibits HDACi-induced apoptosis in the absence of acetylated p53. Although acetylation of Ku70 has been shown to be important for the release of Bax and the induction of HDACi-induced apoptosis, it remain s to be determined what role acetylation of Ku70 plays in p53-mediated disruption of the Ku70/Bax complex. Increasing evidence implicates the Ku70/ Bax complex as a critical cellular determinant of apoptosis that can be disrupt ed by HDACi treatment as well as proteins such as NBS1 and p18-cyclin E fragment cau sed by genotoxic stress (Iijima et al., 2008; Mazumder et al., 2007; Subramanian et al., 2007 ). Therefore, it is likely that many proteins that bind Ku70 as well as direct Ku70 modification can disrupt Bax and promote apoptosis. However, our findings are important to the understanding of the role p53 plays in apoptosis independent of its transcriptional activity. It remains to be determined if other cellular stresses that promote p53 acet ylation such as DNA damage can also promote the dissociation of Bax from Ku70 to enhance the apoptotic response. This newly identified apoptotic function of acet ylated p53 may work in concert or independently of its previously described ro le at the mitochondria in the inhibition of Bcl-XL and Bcl-2 to activate Bax. Cell lines that exhibit higher levels of Ba x in the nucleus may be more susceptible
149 to acetylated p53 disruption from the Ku70 complex and may have a larger role in the initiation of apoptosis. A lower prevalence of Bax in the nuclear compartment may exhibit delayed apoptosis due to the time lapse required for the translocation of acetylated p53 to the cytoplasm. Also, since p53 was previously shown to activate Bax directly (Chipuk et al., 2004), the prevalence of nuclear Bax could result in its activation and recruitment to the mitochondria independently of its dissoci ation with Ku70 as described here. Indeed, p53 association with nuclear Ba x has already been found to correlate with cisplatin induced apoptosis in cells expre ssing wild type p53 (Raffo et al., 2000). These results should also be confirmed by additional experiments including those that use mutant p53 expressing cell lines. It also remains to be determined whether nuclear Bax specifically associates with Ku70 in the nucleus. Immunodepletion of Ku70 could be performed to determine the amount of Bax associated with Ku70 in the nucleus. Furthermore, Bax localization should be assessed in Ku70 knockdown and knock-out cell lines because Ku70 may be required for nuclear Bax. Another issue is the role of Ku80 in this complex as it is commonly associated with Ku70 in the nuclear compartment. It s hould be determined what effects nuclear Ku70/Ku80 complex formation has on the inhibition of nuclear Bax activation in both acetylated p53-dependent and independent models. Reciprocally, does nuclear Bax modify the function of Ku70 in NHEJ or V(D)J recombination? Bax bound to Ku70 could act as a direct surveillance mechanism of DNA damage and re pair processes. If genotoxic stresses are found to activate Bax in the nucleus then nuclear Bax could theoretically act more efficiently in the apopt otic cascade as signals would not have to amplify through transcription and translation relaying signals from the nucleus to the
150 cytoplasm to activate Bax. Furthermore, Ku-mediated NHEJ DNA repair has been found to be inhibited by Bcl-2 (Wang et al., 2008). It is possible that Bax somehow plays a role in this process, and release of Bax from K u70 might block the inhib itory effect of Bcl-2 on Ku thereby allowing Ku to act in DNA repa ir. Similarly, the binding potential between Ku70, Bax, and Bcl-2 should be evaluated to determine the functional effects of their presence and/or absence in Ku70-me diated NHEJ and V(D)J recombination. There also remains the issue of how this novel interaction between p53 and Ku70 impacts their respective functions. There is the possibility that binding to Ku70 can change p53 DNA binding affinity, transactivatio n or transrepression capabilities or other protein-protein interactions. It is tempting to speculate that Ku70 lo calization to sites of DNA damage may recruit p53 to signaling comp lexes that enhance its activation. This interaction may tether the inde pendent functions of these two proteins as a mechanism of DNA integrity surveillance. Further research is required to answer these and many other questions that arise out of the association between th ese two important proteins. HDAC Inhibitor Regulation of Apoptosis Histone acetyltransferases and hi stone deacetylases regulate cellular transcriptional machinery by controlling the leve ls of acetylation of the core histones as well as non-histone proteins including transcri ption factors such as p53 (Glozak et al., 2005; Minucci and Pelicci, 2006). HDAC i nhibitors (HDACi) SAHA and LAQ824 are potent inhibitors of class I and II HDACs and are currently under development as chemotherapeutic agents. The FDA has even approved SAHA for the treatment of cutaneous T cell lymphoma, a form of non-H odgkins lymphoma (Mar tinez-Iglesias et
151 al., 2008). HDAC inhibitors are considered to be promising chemotherapeutic agents due to their ability to induce diffe rentiation, cell cycle arrest a nd apoptosis in a variety of cancer cells through altered e xpression of various cell cycl e and apoptosis proteins (Bhalla, 2005; Minucci and Pe licci, 2006). HDACi mediated hyperacetylation of core histones causes transcriptional act ivation of genes which aid in their proapoptotic and cell cycle arrest capabilities (Johnstone, 2002). Reactive oxygen species (ROS) generation also contributes to the apoptotic response in curred by HDACi. The genome wide effect of HDACi and their varied non-hist one targets has made the eluc idation of their mechanism of action challenging. As our understanding increases of how th is class of anti-neoplastic drugs works so does their potential fo r beneficial use in the clinic. Our experiments have helped shed lig ht onto a novel mechanism of HDACiinduced apoptosis that relies on acetylated p53-dependent disruption of Bax/Ku70 complexes. We have also found that this effect of p53 is independent of its transcriptional activity. This has potential therapeutic implications for the treatment of neoplastic malignancies using HDAC inhibitors as a plat form. Mutations of p53 are one of the most common occurrences found in cancer and cause these cancers to be refractory to traditional chemotherapeutic agents that i nduce genotoxic stress. It could therefore be envisioned that HDAC inhibitors might be used in personalized therapies to enhance their effectiveness. Analysis of a patients p53 st atus in cancer could identify those patients that would respond favorably to HDACi ther apy based on expression of p53, which will likely be independent of its mutational stat us. However, those patients that lack p53 expression would be theorized to respond much less favorably to this treatment. To confirm that this hypothesis is correct, additional analysis of p53 expression and
152 mutational status in patient samples should be correlated to clini cal responses to HDACi treatments. Similarly, in vivo experiments using p53-/and mutant p53 knock-in mice could also be used to compare cancer progre ssion and survival rate s to determine if p53 status imparts a survival advantage after challenge with HDACi treatment. Positive results in these experiments would then be justification for moving experiments into the clinic to determine if this personalized car e can increase the efficacy of HDAC inhibitors.
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About the Author Nicholas Taylor Woods attended the Univ ersity of Nebraska at Lincoln from 2000-2004 where he earned his Bachelor of Scie nce degree in Biochemistry. During this time he gained valuable laboratory experience in the labs of Dr. Donald Lee, Dr. Han Asard, and Dr. Mohammad Koohmaraie resear ching plant genetics, redox biochemistry, and microbiology, respectively. After graduati on, he joined the Cancer Biology Ph.D. program at the H. Lee Moffitt Cancer Center and Research Institute. There he joined the lab of Dr. Hong-Gang Wang and completed hi s dissertation work in the field of apoptosis. The focus of his res earch projects was the regula tion of Bax activation in the initiation of apoptosis.