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Novel Roles for 185delAG M utant BRCA1 in O varian Cancer P athology by Rebecca J. Linger A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology and Cell Biology College of Medicine University of South Florida Major Professor: Patricia A. Kruk, Ph.D. Santo V. Nicosia, M.D. Jin Q. Cheng, M.D., Ph.D. David E. Birk, Ph.D. Mumtaz Rojiani, Ph.D. Date of Approval: November 10, 2010 Keywords: BRAT, Matrix metalloprotein ase 1, ovarian surface epithelium, breast cancer, mutation Copyright 2010, Rebecca J. Linger
Dedication I dedicate this dissertation to my wonderful husband, who has been a bottomless well of patience, strength, encouragement, and love throughou t its completion. I love you. I would also like to thank my parents, who have supported my every dream since they first taught me how to have them, and have believed in me every step of the way. I would like to thank Dr. Kruk, my mentor, for accepting me i nto her lab and giving me the opportunity to continue my education at USF. Thank you for teaching me to be a better scientist and writer than I ever could have been without you. Lastly, I would like to thank my lab mates, Nicole, Christina, and Kamisha, fo r keeping me going and keeping me laughing even on the worst days. I could not have survived this journey without you.
Acknowledgements I would like to express my thanks to my committee members for guidance in my project and for sharing their wealth of scientific knowledge. I would also like to thank Dr. Michael Barber and Dr. Eric Bennett for welcoming me into the program, as well as the rest of the Medical Sciences program and Pathology and Cell Biology department al staff who have provided support for me during my time at the University of South Florida.
i Table of Contents List of Tables ................................ ................................ ................................ ..................... iv List of Figures ................................ ................................ ................................ ...................... v List of Abbreviations ................................ ................................ ................................ ....... vii Abstract ................................ ................................ ................................ .............................. xi Chapter 1: Background ................................ ................................ ................................ ........ 1 Ovarian Cancer ................................ ................................ ................................ ........ 1 Clinical c haracteristics of o varia n c ancer ................................ .................... 1 Origins of o varian c ancer ................................ ................................ ............. 2 Models of o varian c ancer ................................ ................................ ............. 6 Risk Factors for o varian c ancer d evelopment ................................ .............. 7 BRCA1 ................................ ................................ ................................ ..................... 7 Introduction ................................ ................................ ................................ .. 7 The BRCA1 g ene p roduct ................................ ................................ ............ 8 Types of BRCA1 m utations ................................ ................................ ......... 9 Loss of f unction m utations ................................ ................................ ......... 1 1 Gain of f unction m utat ions ................................ ................................ ........ 1 4 The r ole of g ain of f unction m utations for d evelopment, c el l ular p roliferation, c hemosensitivity, a poptosis, and g ene r egulation .......... 16 Clinical i mpact of g ain of f unction m utations ................................ ........... 20 MMP1 ................................ ................................ ................................ .................... 2 2 Introduction ................................ ................................ ................................ 2 2 Structure and f unction ................................ ................................ ................ 23 Regulat ion ................................ ................................ ................................ .. 26 MMPs and c ancer ................................ ................................ ...................... 28 Rationale ................................ ................................ ................................ ................ 3 0 Central Hypothesis ................................ ................................ ................................ 3 3 Specific Aims ................................ ................................ ................................ ........ 3 3 References ................................ ................................ ................................ .............. 3 4 Chapter 2: The 185delAG BRCA1 Mutation Enhances MMP1 Expression in Human Ova rian Surface Epithelial Cells ................................ ................................ ..... 4 7 Introduction ................................ ................................ ................................ ............ 4 7 Meth ods ................................ ................................ ................................ .................. 49 Cell c ulture and t ransfection ................................ ................................ ...... 49
ii Microarray ................................ ................................ ................................ .. 5 0 Western b lot ................................ ................................ ............................... 5 1 RT PCR ................................ ................................ ................................ ...... 5 2 Enzy me linked i mmunosorbant a ssay ................................ ....................... 5 3 Luciferase a ssay ................................ ................................ ......................... 5 4 Statistics ................................ ................................ ................................ ..... 5 4 Results ................................ ................................ ................................ .................... 5 4 BRAT alters expression of genes involved i n multiple cellular processes ................................ ................................ .............................. 5 4 BRAT enhances MMP1 gene expr ession in HOSE 118 cells ................... 5 5 BRAT increases expression and secretion of pro MMP1 by HOSE 118 cells ................................ ................................ ............................... 5 7 MMP1 and maspin ar e independent targets of BRAT ............................... 57 BRAT mediated MMP1 modulation is c Jun dependent ........................... 6 1 AP 1 sites in the MMP1 promoter mediate enhanced MMP 1 expression in BRAT cells ................................ ................................ .... 6 5 Increased pro MMP is detectable in BRAT mutation carrier deriv ed cellular conditioned media ................................ ...................... 7 0 Discussion ................................ ................................ ................................ .............. 7 0 References ................................ ................................ ................................ .............. 81 Chapter 3: Impact of BRAT on A poptosis, G ene R egulation, and M igration of H uman B reast C ancer C ells ................................ ................................ ......................... 8 7 Introduction ................................ ................................ ................................ ............ 8 7 Methods ................................ ................................ ................................ .................. 9 0 Cell c ulture and t ransfection ................................ ................................ ...... 9 0 Cell Viability Assay 92 Cell v iability a ssay ................................ ................................ ..................... 91 Western b lot ................................ ................................ ............................... 9 1 RT PCR ................................ ................................ ................................ ...... 9 1 Scrape a ssay ................................ ................................ ............................... 9 2 Statistics ................................ ................................ ................................ ..... 9 3 Results ................................ ................................ ................................ .................... 9 3 Endogenous BRCA1 and exogenous BRAT expression levels in normal breast epith elial and breast cancer cells ................................ ... 9 3 BRAT does not significantly impact proliferation or chemosensitivity of normal breast or breast cancer cells ..................... 9 4 BRAT does not significantly impact maspin expression in normal breast epithelial or breast cance r cells ................................ ............... 10 1 BRAT does not significantly alter MMP1 expression levels in normal breast epithelial or human breast cancer cells ....................... 10 2 BRAT does not significantly impact migr ation of breast ca ncer cells ................................ ................................ ................................ .... 10 5 Discussion ................................ ................................ ................................ ............ 10 5 References ................................ ................................ ................................ ............ 11 3 Chapter 4: Conclusions ................................ ................................ ................................ .... 118
iii Origins of BRC A1 A ssociated O varian C ancer ................................ .................. 118 Future S tudies ................................ ................................ ................................ ...... 1 19 Significance ................................ ................................ ................................ .......... 12 4 References ................................ ................................ ................................ ............ 12 7 About the Author ................................ ................................ ................................ ... End Page
iv List of Tables Table 1.1. Studies supporting loss or gain of function mutation as mechanisms of enhanced BC and OC risk. ................................ ................................ ............... 1 2 Table 2.1. S election of genes determined to be different ially regulated in BRAT cells ................................ ................................ ................................ .................. 5 6
v List of Figures Figure 1.1. BRCA1 mutations and their cellular and physiologic impact. ........................ 10 Figure 1.2. MMP1 domain structure and potential substrates of importance in OC ........ 24 Figure 1.3. Maspin in part mediates the enhanced apoptotic response of BRAT cells to STS treatment. ................................ ................................ .................... 3 2 Figure 2.1. MMP1 mRNA is increased i n BRAT expressing HOSE cells. ...................... 5 8 Figure 2.2. BRAT increases cellular pro MMP1 and tota l secreted MMP1 in BRAT cells ................................ ................................ ................................ ..... 59 Figure 2.3. MMP1 and maspin are parallel targets of BRAT. ................................ ........... 6 2 Figure 2.4. BRAT mediated MMP1 m odulation is c Jun dependent. ............................... 6 3 Figure 2.5. AP 1 sites in the MMP1 promoter mediate enhanced M MP1 expression in BRAT cells. ................................ ................................ .............. 6 6 Figure 2.6. Increased pro MMP1 is detectable in BRAT mutation carrier derive d cellular conditioned media. ................................ ................................ ............ 7 1 Figure 2.7. ETs 1 protein leve ls are elevated in BRAT cells ................................ ............ 7 4 Figure 2.8. Constitutively active Akt reverses BRAT mediated MMP1 up regulation. ................................ ................................ ................................ ....... 7 6 Figure 3.1. Wild t ype BRCA1 levels in normal human breast epithe lial and breast cancer cells. ................................ ................................ ................................ .... 9 5 Figure 3.2. Transfection efficiency of normal breast epithelial and breast cancer cell lines. ................................ ................................ ................................ ......... 9 6 Figure 3.3. BRAT is efficiently expressed in SKBr3 cell s ................................ ................ 9 7 Figure 3.4. BRAT does not significantly impact growth or chemosensi tivity of breast cancer cells ................................ ................................ ........................... 99
vi Figure 3.5. BRAT does not significantly impact maspin expre s sion in breast cancer cells. ................................ ................................ ................................ .. 10 3 Figure 3.6. MMP1 expression is not altered by BRAT in normal breast epithe lia l or breast cancer cells. ................................ ................................ ................... 10 6 Figure 3.7. Migration of SKBr3 cells is not si gnificantly impacted by BRAT ............... 10 7 Figure 4.1. Pro ditioned media of BRAT cells ............ 12 1
vii List of Abbreviations (aa) Amin o acids AKT1/PKB Protein kinase B AP1 Activator protein 1 APC Adenomatous polyposis coli APMA 4 Aminophenylmercuric acetate BARD1 BRCA1 associated RING domain 1 BCL 2 B cell lymphoma 2 BCL 2A1 BCL 2 related protein A1 BRAT BR CA1 185delAG A mino T erminal truncated protein BRCA1 Breast and Ovarian Cancer Susceptibility Gene 1 BRCT BRCA1 C terminal CA 125 Cancer antigen 125 cDNA Complementary DNA cIAP1 Cellular inhibitor of apoptosis 1 CREB cAMP response element binding Ct Threshold c ycle ECL Enhanced chemiluminescence substrate ECM Extracellular matrix EGF Epidermal growth factor
viii EGFR Epidermal growth factor receptor ELISA Enzyme linked immunosorbant assay EMMPRIN Extracellular Matrix Metalloprotease Inducer ERK Extracell ular signal regulated kinase ETS 1 v ets erythroblastosis virus E26 oncogene homolog 1 FBS Fetal bovine serum FOL1 Folate receptor alpha GFP Green fluorescent protein h Hours Her2 Human epidermal growth factor receptor 2 HGF Hepatocyte grow th factor HOSE Human ovarian surface epithelium HRP Horseradish peroxidase HUVEC Human umbilical vein endothelial cell IGF 1 Insulin like growth factor 1 IGF 1R Insulin like growth factor 1 receptor IL 1 Interleukin 1 JNK/SAPK c Jun N terminal kinase/stress activated protein kinase kDA Kilodalton kRAS Kirsten rat sarcoma viral oncogene homolog LATS1 Large tumor suppressor 1 LOH Loss of heterozygosity LPA Lysophosphatidic acid
ix MAPK Mitogen activated protein kinase M CSF Macrophage c olony stimulating factor MMP1 Matrix metalloprotease 1 mRNA messenger RNA MSH2 Human mutS homolog 2 NES Nuclear export signal NFKB2 Nuclear factor kappa B 2 NLS Nuclear localization signal OC Ovarian cancer OVX1 OC cell line antigen P21 C yclin dependent kinase inhibitor protein 21 P300 E1A binding protein 300 P53 Tumor protein 53 PAR 1 Protease activated receptor 1 PBS Phosphate buffered saline PDGF Platelet derived growth factor PEA3 Polyoma enhancer activator protein 3 PI3K Phosphatidylinositol 3 kinase PTEN Phosphatase and tensin homolog PVDF Polyvinylidene fluoride RQ Relative mean mRNA expression SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis siRNA Small interfering RNA
x SNP Single nucle otide polymorphism STS Staurosporine TIMP Tissue inhibitor of metalloprotease TPD52 Tumor protein D52 uPA Urokinase plasminogen activator uPAR Urokinase plasminogen activator receptor Wt Wild type XIAP X linked i nhibitor of apoptosis protein
xi Abstract Familial history is the strongest risk factor for developing ovarian cancer (OC), and a significant contributor to breast cancer risk. Most hereditary breast cancers and OCs are associated with mutation of the tu mor suppressor Breast and Ovarian Cancer Susceptibility Gene 1 (BRCA1). Studying risk associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Recent studies have shown novel BRCA 1 mutant functions that may contribute to breast and OC initiation and progression independent of the loss of wtBRCA1. Previously, we have found that normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT ( BR CA1 185delAG A mino T erminal truncated protein), exhibit enhanced chemosensitivity and up regulation of the OC associated serpin, maspin. In the current study, I identify an additional target of the BRAT mutation, matrix metalloprotease 1 (MMP1), a key player in invasio n and metastasis. BRAT expressing HOSE cells exhibit increased MMP1 messenger RNA (mRNA) by real time PCR and protein by Western blotting Pro MMP1 levels are also higher in conditioned media of BRAT expressing cells and HOSE cell lines derived from BRAT m utation carriers. c Jun is critical for BRAT mediated MMP1 up regulation, as siRNA kno ckdown diminishes MMP1 levels. Luciferase reporter constructs reveal that a ctivator Protein 1 (AP1) sites throughout the distal end of the promoter contribute to BRAT med iated MMP1
xii expression, and basal activity is mediated in part by an AP1 site at ( 72). Reporters containing a single nucleotide polymorphism (SNP) associated with OC risk and progression yield increased activity that is further enhanced in BRAT cells. Inte restingly, BRAT mediated changes in chemosensitivity and gene regulation are not recapitulated in a normal breast epithelial or breast cancer cell model. This suggests tissue specific mutant BRCA1 functions may contribute to breast and ovarian tissue speci ficity of BRCA1 mutation associated cancer risk and also to differential breast and ovarian cancer risk and penetrance associated with specific mutations. Early molecular and cellular changes such as MMP1 up regulation in the ovarian surface epithelium of BRCA1 mutation carriers may promote OC initiation and progression and represent a step forward on the continuum of cellular malignancy. Further investigation is warranted, as elucidating these early changes will aid in identification of potential screening and treatment strategies.
1 Chapter 1: Background Ovarian Cancer Clinical characteristics of o varian c ancer. According to the American Cancer but ranks 5th in cancer related deaths  The dea dliness of this disease can be attributed to multiple challenges, including lack of a conclusive screening method, the fact that early stages of OC are virtually asymptomatic, and lack of a clear molecular profile. These challenges have limited experimenta l and clinical research to little progress in increasing the survival rate for this deadly disease in the last 7 decades  Because early stage OC is difficult to detect, much investigation has been done to find early OC markers for use in screening. Some potential markers under investigation include osteopontin, macrophage colony stimulating factor (M CSF), OVX1 (OC cell line antigen), lysophosphatidic acid (LPA)  B cell lymphoma 2 ( BCL 2)  angiostatin  and the current gold standard, cancer antigen 125 (CA 251)  Analyzing the proteome signatures of OC patient serum has also been suggested as a screening tool, as well as a way to improve assignment of prognosis and treatment strategy  Other screening methods include pelvic exam and trans vaginal ultrasound  though so far, screening of this type does not significantly decrease mortality 
2 The clinic al behavior of OC presents a formidable challenge to improving long term survival. Metastasis plays a major role in this difficulty. The majority of OC diagnoses are made in Stage 3 and 4, when the primary tumor has metastasized and patient survival falls below 30%  In contrast, stage 1 OCs, which are confined to the ovary, have a survival rate approaching 95%  Typically, late stage patients present with symptoms of abdominal pain and distention because of accumulating fluid and extensive tumor growth within the peritoneum. Cancer encompasses the ovari es, and shed tumor cells are found in the intraperitoneal fluid (ascites). These cells disseminate easily throughout the peritoneal cavity, and have frequently formed metastases in the omentum and peritoneum, as well as through the peritoneum and into the stroma of other organs  First line treatment involves debulking surgery and chemotherapy. Sixty to eighty percent of patients benefit significantly from systemic or intraperitoneal treatment with a platinum based drug, specifically carboplatin, which is often combined wi th a taxane  Ultimately, the majority of patients eventually develop resistance and experience disease recurrence within 18 months  Patients who progress further and those who do not respond to first line platinum based therapy with a remission greater than 6 months may be treated with alternative drugs, such as Gemcitabine, Bevacizumab, or Tamoxifen  though r esponses are uniformly poor. Despite these treatment efforts, second line therapy is generally accepted as a palliative strategy. Origins of ovarian c ancer. To better detect and treat OC, much investigation has sought the origins of OC. Ninety percent of OCs arise from the surface epithelium of the ovary  while the remainder are categorized as sex cord stromal tumors, germ cell tumors, or indeterminat e tumors  Hereafter, OC will refer to epithelial ovarian cancer.
3 HOSE is a non stratified squamous or cuboidal epithelium that exhibits apical microvilli, cilia, simple desmosomes, and incomplete tight junctions and expresses keratins, integrins, and N cadherin  HOSE shares a common developmental progenitor with the mesothelial layer lining the peritoneal c avity. Both types of cells arise from the mesodermally derived celomic epithelium, which lines the embryonic celomic cavity. Specifically, the celomic epithelium covering the paired gonadal ridges proliferates on the surface to form the HOSE. Some cells al so migrate into the gonadal ridges through the stroma and differentiate to become granulosa cells  The Mullerian ducts, which later develop into the oviducts, uterus, cervix, and upper vagina, also arise from the celomic epithelium  The embryologic origin of HOSE is extremely important to understanding O C. In contrast to most cancer types, in which malignant cells become less differentiated as the tumor progresses, malignant HOSE cells acquire a more mature, more differentiated phenotype than normal HOSE, and resemble specific types of Mullerian epithelia Serous tumors are the most common type of epithelial OC and are comprised of cystic, solid, and papillary regions  The epithelium resembles that of the Fallopian tube, as cells are columnar and ciliated, and form papillae  Mucinous tumors are characterize d by stratified cervical or intestinal like epithelial cells that express mucin and form cysts and glands  E ndometrioid tumors are solid tumors with fluid filled cysts, and the cells resemble endometrial epithelium  Clear cell OCs exhibit tubular, solid, papillary or mixed growth, and are comprised of cells full of clear cytoplasm and peg shaped cells with apical nuclei 
4 The mesodermal origin of the HOSE is also important for understanding OC because it imparts upon HOSE cells a unique bi potential epithelial mesenchymal hybrid phenotype, which may contribute to the ability of HOSE cells to develop a malignant phenotype. For example, HOSE cells co express the mesenchymal marker vimentin with epithelial keratins  On some substrates in culture, HOSE cells become contractile  fibroblast like, invasive, down regulate expression of some integrins, and secrete proteases and basal lamina and stromal components such as laminin, and collagen I, III, and IV  The potential of HOSE cells to exhibit mesenchymal characteristics such as these are likely important to their normal physiologic function of restoring the normal HOSE and extracellular matrix (ECM) structure after ovulatory rupture. This abili ty to adapt to and modify the ECM could impart an advantage to HOSE cells in invasion and metastases. Though it is clear that the embryologic development of HOSE is important for OC initiation and progression, the early stages of OC are still largely uncle ar. Unlike some cancer types, such as colon cancer, a clear stage by stage progression from normal tissue to pre cancerous neoplasia to malignant disease has not been defined. One theory is that OCs arise from the metaplastic epithelium of surface invagina tions and inclusion cysts, which are epithelium lined cysts within the ovarian stroma that have lost contact with the surface of the ovary. These regions increase in frequency with age. Much of the evidence supporting the theory that inclusion cysts and in vaginations are the origin of OC comes from prophylactically removed normal ovaries from patients with a known OC associated BRCA1 mutation or a strong family history of OC. In some studies, this tissue exhibits more invaginations in the ovary surface, dys plasia, hyperplasia, and/ or surface
5 papillae compared to normal ovaries [16 18] though contrary findings have been reported [19, 20] Additionally, occlusion cysts and cyto logic changes are more common in the unaffected ovary of OC patients, and occlusion cysts frequently express markers associated with malignant OC, such as CA 125, E cadherin, and tumor protein 53 (p53) [11, 21 23] An alternative hypothesis is that serous OCs actually originate from the Fallopian tube epithelium (Reviewed in  ). The histologic similarity of tubal intraepithelial carcinomas to serous OC and frequency of ext ensive intraperitoneal tumor growth at diagnosis makes identification of primary tubal intraepithelial carcinoma difficult. Nonetheless, this hypothesis merits consideration, as several studies have reported genetic changes common to serous OC and putative tubal intraepithelial carcinoma in normal fallopian epithelium and benign regions of hyperplasia  For example, in prophylactically removed fallopian tubes of BRCA1 mutation carriers, regions of p53 overexpress ion frequently coincide with and have been found adjacent to tubal intraepithelial carcinoma, suggesting that these regions may represent a pre malignant precursor  Further, in at least one study, primary fallo pian tube carcinomas have been found more frequently in BRCA1 mutation carriers  Further study will prove valuable in determining the contribution of tubal intraepithelium to serous OC in these patients. Finall y, in contrast to the inclusion cyst and Fallopian tube hypotheses, other studies suggest low grade OCs, especially mucinous and endometrial, evolve from benign/ borderline neoplasia (Reviewed in  ).
6 Models of ovarian c ancer. Models of OC have been difficult to develop. Most animals do not develop spontaneous OC, with the exception of some strains of mice, rats, and hens. In these animals, the disease manifests with a low frequency and highly variable phenotype  Other mouse models require manipulation, such as suppression of the large tumor suppressor 1 (Lats1) gene  Normal HOSE can be immortalized with Large T antigen, and transformed with the human papilloma virus E6/E7 genes or the addition of E cadherin to the Large T Antigen  however, characteristics of OC cell models vary with the oncogene used to transform the cells  Trans formed cells can then be injected subcutaneously or under the murine bursal membrane of the ovary to create animal models. These models have weaknesses, however, as humans do not have a bursal membrane, and as with most xenograft models, the host animal mu st be immunocompromised  Resulting tumors in animal models frequently do not reflect the histologic subtype of the original tumor, resulting instead in undifferentiated tumors  ). In contrast, intraperitoneal injecti on of cell lines derived from late stage human OC provides an excellent model for understanding OC invasion and metastasis  as OC metastases are most commonly found within the peritoneal cavity and represent the biggest barrier to impr oving OC survival. Another major challenge to creating animal and cell models of OC is the lack of a clear molecular profile. Mutations, amplifications, and deletions have been found in multiple pathways, though each alteration is found in only a fractio n of OC cases. Increased phosphatidylinositol 3 kinase (PI3K), Akt1, Akt2, estrogen receptor, and insulin like growth factor 1(IGF 1)/IGF 1 Receptor (IGF 1R) activity have been
7 demonstrated in OC, as well as the loss of the tumor suppressor phosphatase and tensin homolog (PTEN) (Reviewed in  ). Kirsten rat sarcoma viral oncogene homolog (kRas) is overexpressed in 30% of OCs, human epidermal growth factor receptor 2 (Her2) in 34%, and a subset of OCs exhibit p53 mu tation or overexpression  OC cells often exhibit acquired telomerase activity  Despite these findings, however, no apparent universal culprit in OC init iation or progression has been elucidated. R isk factors for ovarian cancer d evelopment. An effective strategy to increase the proportion of OC cases diagnosed earlier, and therefore better OC survival, is to analyze risk factors for OC development. Environ mental factors such as use of talc on the genitals, cigarette smoking, radiation exposure, the use of certain medications, and diet have been associated with an increased OC risk  Physiologic factors may also pr omote OC, for example obesity or history of another gynecologic disorder such as pelvic inflammatory disease, polycystic ovarian syndrome, or endometriosis  Further, hormonal factors such as reproductive history and contraceptive use impact OC risk significantly. Early menarche and late menopause increase risk, while parity and breast feeding, as well as oral contraceptive use decrease OC risk  The importance of hormon e replacement therapy to OC risk is debated [33, 34] The most informative risk factor, however, is current or past OC diagnosis of a first degree relative. BRCA1 Introduction. Family history is the strongest risk factor for development of OC and a major risk factor for development of breast cancer  Understanding how risk associated mutations contribute to cancer initiation and progression will provide insight
8 into molecular mechanisms and aid in better risk assessment, prophylaxis, and treatment for carriers. The majority of hereditary OCs a nd a significant proportion of hereditary breast cancers are associated with mutation of the BRCA1 gene [35, 36] The BRCA1 g ene p roduct. The 220 kDa, 1863 aa predominantly nuclear BRCA1 protein, which shuttles bet ween the nuclear and cytoplasmic compartments, has multiple functions in the cell [37, 38] BRCA1 plays an important role in the DNA damage response, as evidenced by the fact that BRCA1 null mice die early in embryonic development and exhibit c hromosomal aberrations which are exacerbated by a p53 mutation  (Reviewed in    are cyclic, and BRCA1 plays a role in the cell cycle as well, by regulating key cell cycle controllers, including cyclin dependent kinase inhibitor (p21), and by physically interacting with cell cycle regulators (Reviewed in  ). BRCA1 can also recruit chromatin modifying proteins, such as histone acetyltransferases and histone deacetylases and directly interact with other transcription factors to alter their function (Reviewed in  ). For example, BRCA1 binds and modulates phosphorylation of p53 to enhance its transactivation function  [45 ] Lastly, BRCA1 is capable of ubiquitin ligase activity when heterodimerized with BRCA1 associated RING domain 1 (BARD1)  The loss of these cellular functions of BRCA1 may contribute to cancer by promoting genomic instability and accumulation of cancer causing mutations  a process further accelerated by p53 mutation, a common characteristic of BRCA1 mutant OCs  BRCA1 mutation carriers have a 30% risk of developing OC during their lifetime  and a 50 80% risk of developing breast cancer before the age of 70 
9 Types of BRCA1 mutations. All types of mutations have been reported in the 80 kb BRCA1 gene, including frameshift, nonsense, missense, in frame insertions and deletions, splice altering mutations, mutations in the untranslated regions, as well as silent mutations. The majority of risk associated mutations ar e frameshift or nonsense mutations that result in a premature stop codon and truncated protein product (NIH Breast Cancer Information Core). Risk associated truncation mutations are found throughout the entire BRCA1 coding sequence (Figure 1. 1 ) and result in mutant proteins that vary in length and structural impairment. For example, the nonsense mutation Y1853X, which lacks the last 11 amino acids, is only missing a small portion of the second (BRCA1 C terminal) BRCT repeat, while the 39 amino acid 185delA G mutant A smaller percentage of risk associated BRCA1 mutations are point mutations classified as missense mutations. Like truncation mutations, missense mutations occur throughout the entire BRCA1 coding se quence (Figure 1.1.)  though it is difficult to determine the clinical importance of these mutations because of their rarity and because  The functional significance of the BRCA1 RING and BRCT domains as well as the substantial conservation of their sequences fuel speculation that man y missense mutations in these areas are likely to be linked to cancer predisposition. Nonetheless, several missense mutations have already been linked to breast and/or OC predisposition including C61G, M1775K, and P1749R. BRCA1 is thought to act as a class cellular functions is thought to occur through bi allelic inactivation. Carriers of mutations
10 Figure 1.1. BRCA1 mutations and their cellular and physiologic impact. A. Domain structure of BRCA1 protein and location of risk associated mutations. NES (Nuclear export signal), NLS (Nuclear localization signal). B. BRCA1 mutations categorized by cellular processes in which each has been found to lack function or exhibit function different from wt. Nomenclature used for each mutation was that used in the original research article, or a structural description, if designation was not descriptive of mutation or mutant structure. (Linger and Kruk, 2010)
11 have one germline hit (the inherited mutated copy of BRCA1) and, in the tumor, a second somatic hit usually through loss of heterozygosity (LOH)  The observed phenotype of enhanced breast and OC risk is generally thought to result from loss of some or all wild type (wt) functions of the BRCA1 gene product. However, countless studies have revealed the complexities of signaling molecule and transcription factor interactions, as well as cellular adaptations in response to the unique selective pressures of tumor initiation and progression. Therefore, it is important to investigat e all possible molecular mechanisms by which a mutation may contribute to the disease phenotype. Mutant proteins may antagonize wt proteins in a dominant negative manner resulting in loss of remaining wt function [51 ] or they may engage in unique molecular interactions and manifest novel functions independent of the loss of wt protein function  Likewise, BRCA1 mutations may contribute to cancer risk though loss of wt BRC A1 function or through gain of function associated with mutant BRCA1 proteins. Loss of function mutations. As mentioned previously, several lines of evidence suggest loss of wt BRCA1 function as a common mechanism for enhanced breast and OC risk (Table 1.1.). Similar to BRCA1 knockout mice and cell lines, elevated levels of aneuploidy and LOH indicative of an impaired DNA damage response have been noted in breast cancer tissue from mutation carriers compared to control breast cancers, as well as in the h uman BRCA1 truncated breast cancer cell line, HCC1937 (Reviewed in  ). In structural protein studies, Tischkowitz et al. suggested that structural alterations in the BRCT phosphopeptide binding pocket caused by the BRCA1 M1775K missense mutation contributed to enhanced breast and OC risk through diminished transactivation
12 Table 1.1. Studies supporting loss or gain of function mutation as mechanisms of enhanced BC and OC risk. Mutation Result of Mutation In vitro In vivo Model system Endpoint Summary Reference Loss of Function Various X NA # genetic changes Mutant BCs more chromosomal gain/ los s events vs control BCs Tirkkonen et al., 1997 P1749R C64G T826K M1775R Missense P>R Missense C>G Missense T>K Missense M>R X Breast cancer DNA Damage HCC1937 cells; Mutants did not Scully et al., 1999 5382InsC Truncated: 1828 aa X Breast cancer DNA Damage, chemosensitivity wt BRCA1 rescued hyper recombination, chemosensitivity of MCF7 cells; Mutants did not Cousineau and Belmaaza, 2007 P1749R Q1756InsC Y1853 STOP Missense P>R Truncated: 1828 aa Truncated: 1852 aa X COS, colon cancer Gene regulation wt BRCA1 increased p21 expression in COS, cancer cells; Mutants did not Somasundaram et al., 1997 1835S TOP 340STOP Truncated: 1834 aa Truncated: 339 aa X X Breast cancer Cell growth, tumor growth wt BRCA1inhibited growth, tumor growth in nude mice; Mutants did not Holt et al., 1996 Gain of Function 5677InsA Truncated: 1852 aa X P rostate cancer Proliferation Mutant inhibited proliferation more efficiently than wt BRCA1 Fan et al., 1998 N terminal 602 amino acids Synthetic mutant: 602 aa X X Mouse ovarian epithelium Proliferation, chemosensitivity, tumorigenesis Mutant BRCA1 e nhanced proliferation, chemosensitivity, tumorigenesis; wt BRCA1 suppressed Sylvain et al., 2002 5677InsA N terminal 302 aa N terminal 771 aa Truncated: 1852 aa Synthetic mutant: 302 aa Synthetic mutant: 771 aa X Prostate cancer Proliferation, che mo sensitivity 5677InsA and wt BRCA1impaired proliferation, enhanced chemosensitivity; Synthetic truncations decreased sensitivity Fan et al., 2001 185delAG Truncated: 39 aa X Ovarian epithelium Apoptosis 185delAG decreased cIAP1, XIAP, P Akt, and e nhanced cleaved caspase 3, apoptosis after drug treatment 5382InsC 5677InsA Truncated: 1828 aa Truncated: 1852 aa X Breast, Ovarian Cancer Apoptosis Coexpression of mutants with wt BRCA1 a poptosis Thangaraju et al., 2000 5083del19 Truncated: 1669 aa X X HeLa Gene regulation Mutant increased periostin mRNA, protein, and mutation carrier serum, BC tissue Quaresima et al., 2008 ( L inger and Kruk, 2010)
13 and binding to other DNA damage response proteins  Likewise, Williams et al. found that decrea sed stability of BRCA1 missense and truncation mutants resulting from aberrant protein folding contributed to loss of BRCA1 function and enhanced cancer risk  Expression of mutant BRCA1 constructs in the absenc e of wt BRCA1 frequently HCC1937 breast cancer cell line, which lacks wt BRCA1 and carries two 5382InsC BRCA1 alleles that code for a frameshift and premature stop signal at codon 1829, and transfection of several BRCA1 mutants into these cells failed to alter radiation sensitivity  In agreement, ad dition of wt BRCA1 expression into breast cancer cell lines that exhibit low wt BRCA1 expression due to the presence of a single wt BRCA1 allele inhibited growth. However, expression of the risk associated truncation mutants 1835STOP and 340STOP as w ell as the syntheti 1092, failed to alter cell growth, tumor formation and tumor progression in nude mice  Lastly, introduction of wt BRCA1 into HCC1937 breast cancer cells and IGROV 1 OC cells inhibited tumor initiation and growth, while a synthetic BRCA1 mutant lacking the last 542 amino acids did not  Interestingly, Cousineau and Belmaaza hypothesize that reduced gene dosage of wt BRCA1 in mutation carriers is so lely responsible for altered DNA damage repair, subsequent mutation accumulation, and increased cancer risk. Using MCF7 breast cancer cells that harbor a single copy of wt Cousi neau and Belmaaza showed that transfection of MCF7 cells with wt BRCA1
14 diminished hyper recombination and chemosensitivity while addition of the 5382InsC BRCA1 mutation affected neither endpoint  These st udies further support a role for loss of wt BRCA1 function as a contributing factor to enhanced breast and OC risk. It is important to note that many of the aforementioned studies attempted to delineate BRCA1 mutant function in model systems lacking normal levels of wt BRCA1, which makes it difficult to discriminate between the contribution of BRCA1 mutants and loss of wt BRCA1 for disease risk. However, several studies utilizing a wt BRCA1 background clearly support the loss of BRCA1 wt function for cancer risk. For example, though overexpression of wt BRCA1 in several wt BRCA1cancer cell lines and COS cells up regulated p21 expression, several synthetic deletion and truncation mutants and risk associated BRCA1 mutants, including P1749R, Q1756InsC (aka 5382 InsC), and Y1853STOP (aka 5677InsA), a frameshift mutation resulting in a premature stop codon that lacks the last 11 amino acids  failed to alter p21 expression  Gain of function mutations. While mutations resulting in a premature stop codon are typically susceptible to nonsense mediated messenger RNA (mRNA) decay, mounting evidence suggests mutant mRNA and proteins are not uniformly degraded. Perrin Vidoz et al. found that several BRCA1 mutations were unaffected by mRNA decay, including 185delAG and 5382InsC  two of the most common risk associated BRCA1 mutations  Truncation mutant mRNAs may avoid decay by translation reinitiation at a methionine codon downstream of the premature stop codon  and consequently, may contribute aberrant gene pr oducts coding for truncation proteins exhibiting varying degrees of protein stability that may impart novel cellular functions  It is important to consider that detection of some mutant BRCA1 proteins in clinical
15 samples has proven unsuccessful due to technical challenges such as cro ss reactivity of antibodies with wt BRCA1, however, validation studies of mutant proteins in tissue samples are ongoing and will provide a framework within which to view experimental studies of mutant function. BRCA1 mutant proteins may participate in nove l protein protein interactions as a result of aberrant cellular localization. Rodriguez et al. found that exogenous missense and truncation mutants lacking a small portion of the BRCA1 C terminus, including 5382InsC, exhibited aberrant cytoplasmic localiza tion in breast cancer cells, while larger truncations resulted in enhanced nuclear localization of mutants  Aberrant localization may result from m utation or loss of the nuclear localization or export signals, impaired recognition of these signals as a result of improper protein folding, or altered interaction with binding partners that impact BRCA1 localization, such as BARD1  Mutant BRCA1 proteins may convey unique phenotypes by inhibiting normal function of wt BRCA1 in a dominant negative manner by binding BRCA1 and inhibiting its interacti on with other proteins, or by sequestering BRCA1 binding partners. Likewise, mutant proteins may also convey unique functions by interacting with novel proteins and/or regulating alternative genes. Indeed, a significant proportion of BRCA1 associated breas t cancer tissue samples  as well as primary cells from mutation carrier derived OC cell xenograft tumors  exhibit loss of the wt BRCA1 allele concomitant with increased mutant allele copy number. Conseq uently, mutant BRCA1 proteins have been shown to impact a range of cellular functions including development, proliferation, chemosensitivity, apoptosis, and gene regulation (Table 1 .1. ).
16 Role of g ain of f unction mutations for development, cellular prolife ration, chemosensitivity, apoptosis and gene r egulation. Essentially all BRCA1 knockouts are embryonic lethal in mice (Reviewed in  ), however, mice homozygous for a specific synthetic mutation truncating the BRCA1 protein by half are viable, though highly suscept ible to multiple tumor types, including lymphomas, sarcomas, and carcinomas/ adenocarcinomas of the colon, endometrium, lung, liver, and mammary gland  Interestingly, introduction of a synthetic BRCA1 truncation mutant encoding the first 300 BRCA1 amino acids inhibits mammary gland differentiation and structural formation during murine development, despite the presence of wt BRCA1  Likewise, when injected into the cleared murine mammary fat pad, pr imary human breast epithelial cells W1777Stop, (which mimics the human 1835STOP mutation), undergo limited differentiation and branching and develop extensive hyperplasia  The 5677InsA insertion mutation, resulting in a frameshift and premature stop signal at codon 1853, inhibits proliferation of DU145 human prostate cancer cells expressing a low level of wt BRCA1 more efficiently than exogenous wt B RCA1  while a synthetic N terminal mutant was found to inhibit physical interaction of wt BRCA1 and cyclin D1  In contrast, an exogenous C terminal fragment of BRCA 1 can enhance normal breast epithelial cell growth, possibly by acting in a dominant  Similarly, while overexpression of wt BRCA1 in the ID8 mouse ovari an epithelial cell line diminished proliferation, chemosensitivity, and tumorigenicity of intraperitoneally injected cells, expression of a synthetic truncation mutant encoding the first 602 amino
17 acids of BRCA1 yielded enhanced proliferation and chemosens itivity. Further, when injected intraperitoneally, cells expressing the mutant were significantly more tumorigenic  It should be noted, however, that BRCA1 mutants have also been shown to exhibit some residual wt growth function as a result of remaining intact exhibited a failed G2 M checkpoint  while breast cancer cells expressing only the 53 82InsC mutant maintained an intact G2 M checkpoint  In DU145 prostate cancer cells expressing low levels of wt BRCA1, Fan et al. reported that overexpression of wt BRCA1 or 5677InsA increased topoisomerase inhibit or cytotoxicity, which could be reversed by transfection of synthetic mutants Eco RI (amino acids (aa) 1 302) and Kpn I ( aa 1 771), yielding chemoresistant cells  Likewise, in the HCC1937 breast cancer cell mo del system lacking endogenous wt BRCA1, addition of exogenous wt BRCA1 enhanced chemoresistance, which was reversed by co transfection of Eco RI and Kpn I  This suggests that mutants can, at least in part, over turn wt BRCA1 function, thereby supporting a role for gain of function BRCA1 mutations. The 185delAG (BRAT) mutation, which imparts upon carriers a 66% lifetime risk of developing OC  arises from the deletion o f two nucleotides (AG) in the second exon of the BRCA1 gene. This deletion results in a reading frame shift that produces a premature stop signal at codon 39 and a truncated protein product. Using SV 40 transfected HOSE cells from women with the BRAT mutat ion, we found that mutant cells exhibited enhanced apoptosis and caspase 3 activation in response to staurosporine  possibly related to diminished levels of phospho Akt/protein kin ase B, X linked
18 inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 (c IAP1)  To rule out the possible contribution of wt BRCA1 haploinsufficiency to altered apoptosis in 185del AG cells, BRAT was expressed in wt BRCA1 HOSE cells. In agreement with our earlier studies, BRAT enhanced caspase3 mediated apoptosis and diminished levels of phospho Akt, cellular inhibitor of apoptosis 1 (cIAP1), and x linked inhibitor of apoptosis 1 (XI AP)  In more recent studies, we found that BRAT upregulated expression of maspin  a tumor suppres sor important in apoptosis, invasion, and metastasis that is uniquely overexpressed in several tumor types, including OC  Maspin expression has been correlated with cisplatin sensitivity in OC cell lines and lo nger progression free and overall survival times in OC patients  and may be involved in BRAT mediated enhanced chemosensitivity  Lastly, Thangaraju et al. found that co expression of 5382InsC and 5677InsA with wt BRCA1 inhibited the wt  Several studies support a role for BRCA1 mutants in gene regulation. For example, wt BRCA1 and 5677InsA inhibited exogenous estrogen receptor alpha transactivation, but co transfection of Bam HI, Kpn I, and Eco RI reversed this phenomenon  1863), which encodes a protein less than a third the length of wt, inhibited wt BRCA1 mediated activation of a p53 reporter  Likewise, using the mouse mammary gland specific expressi on of wt BRCA1, a risk associated mutation that truncates the protein at amino acid 340, or a BRCA1 splice variant that omits the N terminal 72 amino acids, Hoshino et al. showed that the splice variant mediated hyperproliferation and enhanced lobule forma tion in the mammary gland. In addition, tumorigenesis and death were accelerated in mice
19 expressing the splice variant  In separate studies, Quaresima and colleagues performed microarray analysis on HeLa cells stably expressing vector, wt BRCA1, or the founder mutation 5083del19, which encodes a BRCA1 protein missing the last 193 amino acids, and, consequently both BRCT domains, and found differential regulation of multiple genes, including up regulation of peri ostin. Further, periostin levels were also increased in serum and breast cancer tissue from a small number of patients carrying this mutation  In other studies, expression of a synthetic truncation mutant maint aining the first third of the BRCA1 protein enhanced p53 expression in 1D8 mouse epithelial OC cells and down regulated constituents of the c Jun N terminal kinase/stress activated protein kinase (JNK/SAPK) and mitogen activated protein kinase/ extracellul ar signal regulated kinase (MAPK/ERK) pathways  Finally, the missense mutation Ser1841Asn, which is associated with enhanced breast cancer risk, up regulates tumor protein D52 (TPD52) and the folate receptor a lpha (FOL1) in HeLa cells  This regulation is clinically relevant since expression of these genes correlates with tumor progression in breast   and OCs   Taken together, these studies support a gain of function role for some mutations. The presence or absence of a mutant function as well as its impact on the cell is likely very specific to each mutation and factors impacting mutant function, including mutant protein size, loss/ maintenance of various domains, or structural changes resulting in novel domains. These studies must also be viewed in a cautionary manner. Gain and loss of function experiments provide valuable insight into the mechanism of BRCA1 mutant functions, however, until the presence of stable mutant proteins is validated clinically, it
20 is necessary to remain mindful of the limitations as well as the promise of these types of experimental studies. Clinical impact of gain of function m utations. Studies investigating the effect of BRCA1 m utant proteins in the context of wt BRCA1 are clinically important. They represent the genotypic and phenotypic state of disease free mutation carriers before loss of both wt BRCA1 alleles. Novel functions mediated by mutant proteins have been shown in var ious model systems to significantly impact proliferation and apoptosis, and therefore, have the potential to influence cancer initiation, progression, and ultimately prognosis for patients carrying mutations. While some mutants may retain specific wt BRCA1 functions, others may enhance the risk of cancer development by antagonizing is warranted, as a better understanding of the function of specific mutations could greatly improve risk assessment and prognostic value for mutation carriers. A better understanding of BRCA1 mutant functions may also help identify novel drug targets for treatment and prophylaxis of mutation carriers. Novel interacting proteins and signaling path ways as well as downstream target genes may reveal as yet unidentified players in BRCA1 mutation associated breast and OC. Data from our lab suggests that genes important for cancer initiation and progression, such as maspin, are differentially regulated i n normal human ovarian epithelial cells expressing the BRAT mutation  Further, compared to sporadic breast cancer tissue, BRCA1 mutation associa ted breast cancer samples reveal more chromosomal aberrations in specific regions, potentially containing additional tumor suppressors important in BRCA1 dependent tumor initiation and progression  An understan ding of specific interacting
21 proteins, signaling pathways, and target genes involved in the mechanism of enhanced breast and OC risk conveyed by each mutation provides the opportunity for mutation specific personalized therapy for mutation carriers. Simila r mutations may also share common functions and respond to similar therapeutic strategies. Further, targeting functions of BRCA1 mutants that likely contribute to pre malignancy, cancer initiation, and early stages of tumor growth holds great promise for e ffective prophylactic measures that are less invasive than oophorectomy and mastectomy. It is interesting to speculate that cells heterogeneous for risk associated mutations, though non tumorigenic in their current state, may represent an initial step tow ard cellular transformation, though additional changes may be necessary for these cells to become malignant. Likewise, early changes that may promote malignant transformation, including enhanced telomeric instability, have been observed in cell lines gener ated from normal ovarian surface epithelial cells of women with a strong family history of OC  (Reviewed in  ). As mentioned previously, several studies have found more frequent occurrence of deep invaginations in t he ovary surface, dysplasia, hyperplasia, and/ or surface papillae in high risk prophylactically removed ovaries versus normal ovaries    su carriers. The possibility of independent mutant BRCA1 functions does not exclude the contribution of other oncogenes, tumor suppressors, or invasion/ metastasis promoting proteins. Conv ersely, these early changes are likely to facilitate further cellular changes that manifest in the aggressive phenotype seen clinically in hereditary breast and OC. Though the specificity of BRCA1 mutations for increasing the risk of breast and OC is well established, few studies have investigated differences in risk and etiology
22 between BRCA1 mutation associated breast and OC. This question warrants investigation, as the lifetime risk for development of breast cancer is higher than that for OC   Further, families with predisposition to both breast and OC exhibit variation in the ratio of breast to OC occurrence  and this ratio is dependent in part on the location of the mutation within the BRCA1 gene (Reviewed in  ). Disparate risk levels of breast and OC suggest mutant proteins may mediate different functions in different tissues, which in turn may exhibit tissue specific degrees of importance for the specific functions lost or gained as a result of each mutation. In conclusion, it is clear from a wide range of model systems and endpoints that BRCA1 mutations are capable o f significant physiologic impacts. Further, molecular and phenotypic changes are evident in mutation carriers. These changes may result from loss of wt BRCA1 function, gain of function mutations, or both. Consequently, further experimental and clinical stu dies of mutant BRCA1 proteins are warranted, and will provide a better understanding of mutation associated breast and OC and improve the strength of prognosis and efficacy of prophylaxis and treatment for mutation carriers. MMP1 Introduction. The aggress ive clinical course of OC illustrates the importance of invasion and metastasis in OC progression. One of the normal physiologic processes that cancer cells frequently exploit to attain these capabilities is cleavage of extracellular matrix (ECM) component s and other substrates by matrixmetalloproteases (MMPS). This adaptation that can ultimately promote tumor growth, invasion, and metastasis through
23 multiple mechanisms (Reviewed in  Preliminary microarray data from our lab suggest that the gene encod ing MMP1 may be differentially regulated in HOSE cells expressing BRAT. Structure and f unction. MMPs are a family of proteases that utilize Zinc at their active site to cleave a wide variety of substrates. Specifically, MMP1 cleaves fibrillar collagens, bu t has also been shown in vitro to cleave other substrates, including ECM constituents, pro growth factors, growth factor receptors, and cell cell adhesion mediators  The N terminal 18 30 amino acids of MMPs comprise a signal peptide (Figure 1.2.), which directs translation of the protein to the endop lasmic reticulum, and is cleaved off before secretion  (amino acid 73 of MMP1), that occupies the active site zinc with a su lfhydryl group, thereby rendering the enzyme inactive  The catalytic domain is comprised of a five stranded beta sheet and two alpha helices  160 170 amino acids, and contains the sequence HELGHXXGXXH  The three histidines within this sequence bind the catalytic zinc molecule and are crucial for enzymatic function  MMPs bind one structural zinc and a calcium as well. The hinge, or linker, domain follows. This region is variable in lengt h and sequence in MMPs and is thought to be involved in disrupting the triple helix structure of collagen I to increase accessibility of the cleavage site. Lastly, the structure of the C terminal domain varies, and contains a transmembrane spanning region in some MMPs. MMP1 is classified structurally by its C terminus as a simple hemopexin domain family member. This domain, comprised of four repeat regions that form a four bladed beta propeller  is similar in s equence to vitronectin, and may mediate
24 Figure 1.2. MMP1 domain structure and potential substrates of importance in OC.
25 binding to ECM components, and/or help lock collagen into the active site for more efficient cleavage  MMPs are synthesized and secreted as zymogens. In the inactive form, the cysteine residue within the pro domain inhibits ac tivation by binding the catalytic zinc molecule, preventing its interaction with a water molecule that is crucial for catalysis  Pro MMPs can be activated by mercurial compounds (such as 4 aminophenylmercury acetate (APMA)), other thiol reactive reagents, reactive ox ygen species, heat treatment, and other compounds by occupation of the cysteine and disruption of the cysteine zinc interaction. Biologically, MMPs are activated by other MMPs and proteases, such as trypsin, through cleavage and partial removal of the pro domain, thereby freeing the active site zinc. MMP1 subsequently autocleaves to remove an additional portion of the pro domain, however, this 42 kilodalton (kDa) MMP1 is only about 20% active. MMP1 is fully activated by cleavage at residue 80 into a 41 kDa product. This may be achieved by MMP2, 3, 7, 10, or 11. In the absence of one of these proteases, MMP1 may further autocleave at residue 81 or 82, resulting in a 30 40% active enzyme  MMPs cleave a variety of physiologically important substrates, but do not cleave a t a strict consensus sequence. In fact, there are few specific characteristics shared by substrates. MMP1 is one of a small group of proteases capable of cleaving native, or fibrillar, collagens, which are the most abundant collagens in interstitial connec tive tissue  The ovarian stroma is composed mostly of collagens I and III and fibronectin, while fibulin 1, perlecan and other heparin sulfate proteoglycans (syndecans and glypicans), lecticans, decorin, and hyaluronan chains are also present (Reviewed in
26  ). At the surface of the ovary, beneath the HOSE lies the basal lamina, which is comprised primarily of networks of collagen type IV and laminin, as well as nidogen/ entactin and perlecan [10 4] A dense network of collagen fibrils called the tunica albuginea underlies the basal lamina. MMP1 substrates with potential importance in OC pathology are shown in Figure 1.2. As frequent metastasis is a highly important feature of OC, and the omentum is the most common site of OC metastases  the occurrence of MMP1 substrates in the omental ECM and stroma are also listed. Though these substrates have been identified in vitro, their importance in vivo is sti ll being investigated. Regulation. MMPs are regulated by proteolytic activation, as mentioned previously, as well as through pericellular localization. MMPs can be regulated by localized availability of activators, such as the membrane associated urokinase plasminogen activator (uPA), while other MMPs are membrane bound. MMP family members are also regulated by expression. Transcriptional regulation of MMP 1 occurs in part through activator protein 1 (AP1)  and v ets erythroblastosis virus E26 oncogene homolog 1 (Ets)  sites in the promoter. The most proximal AP1 site is located at ( 73) and is necessary for basal transcription, though other AP1 and Ets sites upstream also contribute to basal transcription. IL 1 and TNFalpha mediated up regulation occurs through c Jun and an AP1 site as well, though there may also be promoter regions through which IL 1 negatively impacts expression. Expression of MMP1 is also modulated by other cytokines and growth factors. For example, platelet derived growth factor (PDGF) and EFG up regulate MMP1, while  Epidermal growth factor (EGF) an
27 MMP1 mRNA stability [109, 110] These impacts are cell type specific, however, and factors may exert alternative context dependent effects. MMPs are also up regulated by Extracellular Matrix Metalloprotease Inducer, or EMMPRIN. EMMPRIN a member of the immunoglobulin superfamily, is heavily glycosylated and localizes to the cell surface. Staining for EMMPRIN is frequently observed at the leading invasive edge of tumors  EMMPRIN w as originally isolated from the membranes of cancer cells, however, it is secreted by and affects both cancer and normal stromal cells. Transfection into breast cancer cells increases tumorigenicity in nude mice and expression of MMP2 and 9 in tumors  Expression is elevated in malignant ovarian tumors compared to normal tissue  and correlates with poor survival in serous OC [ 114] The mechanism by which EMMPRIN induces MMP expression has not been completely elucidated, though clustering of EMMPRIN with itself and caveolin 1 has been implicated, as well as signaling through p38 MAPK and JNK   MMPs are regulated extracellularly by the binding of several protein types. The cules that bind MMPs in a 1:1 ratio and displace the water molecule usually associated with the catalytic zinc  Specifically, MMP1 is inhibited by all four TIMP proteins (1 4)  The importance of TIMPs in some types of cancer has been illustrated. Melanoma cells overexpressing T IMP3 exhibit decreased invasive ability and undergo apoptosis  TIMP secretion from stromal cells also influences MMP activi ty greatly, as brain metastases of fibrosarcoma cells were significantly diminished in mice with brain specific overexpression of TIMP1  MMPs are also inhibited by the large serum
28 MMP  and the complex is subsequently endocytosed  MMPs and c ancer. MMPs are involved in normal physiologic pr ocesses, including wound healing, mammary gland and uterine involution, and cervical dilation  In the ovary, MMPs are up regulated in the pre ovulatory follicle  and are secreted by ovarian surface epithelial cells from lysosomes  Both sources of proteases may aid in release of the oocyte during ovulation. Conversely, MMPs have been implicated in cancer. Mutations and gene amplifications are not generally reported, but MMP expression increases i n multiple cancer types, including ovarian  Microarray data suggest MMP12 may be a useful serum marker for breast cancer  while MMPs 2 and 19 may be useful biomarkers for OC  Specifically, MMP1 expression has been associated with poor prognosis in breast, colorectal, and esophageal cancer [124 126] MMPs are crucial for invasion of tumor cells into and through the bas ement membrane, thereby promoting metastasis  Cleavage of ECM constituents may reveal binding sites in the ECM that modulate migration and adhesion. MMPs also cleave and release pro growth factors that may promote tumor cell growth (Reviewed in  ). Kenny et al. found that adhesion of SkOV3 OC cells to mouse peritoneum and a 3 D peritoneal model decreased after MMP2 inhibition or knock down, and that MMP2 expression was enhanced in adherent cells  MMP family mem bers also participate in multiple novel mechanisms of cancer progression, including angiogenesis, apoptosis, and the immune response to cancer
29 (Reviewed in  ). The importance of MMPs in angiogenesis is especially relevant to OC. ECM remodeling allows m igration of endothelial cells through the ECM. MMPs also cleave and release pro and anti angiogenic factors from the ECM (Reviewed in  ). Interestingly, recent studies have also demonstrated the ability of aggressive OC cells to form vascular like st ructures in 3D culture, and integrate into the tumor MMP1 have been detected within the vascular structures  and inhibiting MMPs diminishes the formation of vascular structures by the OC cells  In addition to interactions of tumor cells with the ECM, stromal tumor cell interactions are known to be important in multiple facets of tumor progression, including paracrine growth signaling and angiogenesis. For example, normal sheep ovarian stromal cells were found to inhibit the growth of OC cell lines in vitro an d in vivo  Conversely, tumors formed from subcutaneously injected OC cells exhibited regions of host stromal tissue integrated into the tumor that could promote further tumor growth  Mouse ovarian stromal cells stimulated anchorage independent growth of HOSE cells  Co culture also enhanced estrogen stimulated growth of rabbit OSE cells  It is possible that TIMPS or MMP regulators may be secreted by ovarian stromal cells in vivo. This phenomenon has been examined in detail in a melanoma cell model that highly expresses MMP1. Melanoma cells exhibited invasion through a type I collagen matrix only when co cultured with fibroblasts or treated with fibroblast conditioned media. MMP3 and an unidentified serine protease secreted by the fibroblasts cleaved and activated MMP 1 s ecreted by the melanoma cells. The activated MMP1 subsequently facilitated invasion 
30 The specific pathological role for matrix metalloprotease 1 in OC has not yet been made clear. Knockout mice of several other MMPs have been studied, and reveal a general theme of decreased tumorigenesis and angiogenesis  Unfortunately, however, there is not a sufficiently similar homologue to MMP1 in the mouse genome, so an MMP1 knockout mouse has not yet been created. Howeve r, using intraperitoneal injection of human OC cells into mice, Agarwal et al. implicate MMP1 in the activation of protease activated receptor 1 (PAR 1) and reveal the importance of this pathway to ascites formation, metastasis, and angiogenesis  In a 1998 study utilizing a leukocyte gene library, a single nucleotide nucleotide was found to increase the efficacy of an Ets (erythroblast osis virus E26 oncogene homolog) transcription factor binding site and increase MMP1 expression  A later study found that the percentage of OC patients with the SNP was disproportionately high compared to norm al women, and that tumors with the 2G allele expressed greater than seven times the MMP1 expression levels of tumors without a 2G allele, suggesting that MMP1 is indeed important in OC pathology  Further, presence of the 2G poly morphism was associated with decreased disease free and overall survival in OC patients  Rationale We have previously found that BRAT plays an important role in the enhanced apoptotic response of immortalized HOSE cells to drug treatment. Immortalized HOSE cell lines derived from BRAT mutation carriers exhibit enhanced caspa se 3 activation and apoptosis as a result of diminished levels of apoptotic inhibitors XIAP and cIAP1 and
31 reduced Akt activation [78, 79] Enhanced chemosensitivity is mediated by the BRAT mutation, independently of the loss of wt BRCA1, as transfection of BRAT into wt HOSE and OC cells recapitulates the effects on XIAP, caspase 3, and apoptosis [79, 80] .These findings are in agreement with studies illustrating better initial chemotherapeutic response in BRCA1 mutation carrier OC patients compared to control patients [138, 139] More recently, we have identified the tumor suppressor maspin as a downstream target of BRAT. Maspin mRNA and protein levels are increased in wt 118 HOSE cells transiently and stably expressing BRAT. Maspin up regulation is transcriptional, and occurs in part through c Jun, as maspin promoter activity is significantly decreased by c Jun knockdown as well as trunca tion of the maspin promoter and elimination of several transcription factor binding sites, including an AP1 site  c Jun is critical for enhanced BR AT cell chemosensitivity as well. c Jun knockdown decreases caspase 3 cleavage  Preliminary data also suggest maspin plays a role in BRAT cell chem osensitivity, as maspin knockdown results in diminished caspase 3 cleavage as well (Figure 1.3.,  ). These findings are clinically relevant, as cytop lasmic maspin correlates with cisplatin sensitivity in OC cell lines, and treatment response and overall survival in OC patients  It is clear from these studies that the BRAT mutation mediates unique molecular and cellular changes in HOSE and OC cells independent of the loss of wt BRCA1 function, and that these changes have great potential for physiologic impact in carriers of the BRAT mutation. Consequently, it is worthwhile to pursue identification of addition al BRAT targets and cellular processes important in OC initiation and progression. The
32 Figure 1.3. Maspin, in part, mediates the enhanced apoptotic response of BRAT cells to sta urosporine (STS) treatment. Stable BRAt cells were transfected with Si Con or Si Maspin, and 48 hours later, were treated with 1 M STS. Lysates were collected six hours after treatment. Samples were analyzed for cleaved caspase 3, pro caspase 3, maspin, and actin protein levels via
33 185delAG mutation is associated with increased risk of breast cancer risk as well as OC, however mutation associated breast and OC occur with different penetrance in fam ilies and individuals. Therefore, it is also important to determine whether the cellular functions of BRAT are tissue specific and could contribute to epidemiologic and etiologic differences between 185delAG associated breast and OC. Central Hypothesis I hypothesize that expression of the 185delAG BRCA1 mutant protein product, BRAT, alters the regulation and/or activity of several potentially important players in BRAT associated OC pathology, including matrix metalloprotease 1. I hypothesize that BRAT mediated MMP1 up regulation involves Akt and the transcription factor c Jun, and occurs in part t hrough specific AP1 sites in the MMP1 promoter I also hypothesize that BRAT mediates differential effects on apoptosis, gene regulation, and migration in normal HOSE and ovarian cancer cells compared to normal breast epithelial and breast cancer cells. B RAT mediated molecular and cellular changes in ovarian surface epithelial cells may serve, then, as an intermediate state in the transformation of these cells from normal to malignant. Specific Aims To address these hypotheses, I propose execution of three specific aims. 1. Confirm MMP1 as a downstream target of BRAT in immortalized human ovarian surface epithelial cells. 2. Determine the signaling pathways and transcription factors involved in BRAT mediated MMP1 up regulation. 3. Demonstrate the specific ity of BRAT function in ovarian compared to breast migration in normal human breast epithelial cells and breast cancer cells.
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47 Chapter 2: The 185delAG BRCA1 M utation E nhances MMP1 E xpression in H uman O varian S urface E pithelial C ells Introduction Ovarian cancer is the 9th m ost common cancer in women, but ranks 5th in cancer related deaths  The deadliness of this disease can be attributed in part to the fact that early stages of OC are not well understood and are virtually asymptomatic. The majority of OC diagnoses are made in Stage 3 and 4, whe n the primary tumor has metastasized and patient survival falls below 30%  In stark contrast, stage 1 OCs, which are confined to the ovary, have a survival rate approaching 95%  As evidenced by this, improving OC survival will require a better understanding of early stages of the disease, as wel l as the mechanisms by which OCs become invasive and metastatic. Studies investigating hereditary OC risk and early stage disease have determined that the majority of hereditary OCs are associated with mutation of the BRCA1 gene  Carriers of a BRCA1 mutation have a 30% 40% risk of developing OC during their lifetime [3, 4] The gene product of the BRCA1 gene is the multifunctional tumor suppressor BRCA1, which is involv ed in cell cycle, DNA damage response, chromatin remodeling  and ubiquitin ligase activity  Founder mutations of BRCA1 occur at a high frequency in genetically isolated p opulations, such as the Ashkenazi Jewish population. The 185delAG founder mutation, BRAT, is the deletion of two nucleotides
48 (AG) in the second exon of the BRCA1 gene. This deletion results in a reading frame shift and likely results in translation of a pr emature stop signal at codon 39 and a truncated protein product. Though the BRAT mutation is detected in clinical samples and solidly correlates with OC risk, cross reactivity of antibodies with wt BRCA1 has made detection of the truncated protein in clini cal samples unsuccessful. However, recent studies suggest that mutant BRCA1 proteins may contribute to cancer by unique gain of function activities, independent of the loss of normal BRCA1 function, by influencing proliferation, apoptosis, and gene regulat ion (Reviewed in  ). For example, we have previously found that expression of BRAT in normal HOSE cells increases expression of the tumor suppressor maspin, which is uniquely up regulated in OC [8, 9] Preliminary microarray data reveal an additional downstream target of BRAT, the matrix metalloprotease 1 gene. MMP1 is a member of the matrix metalloprotease family of enzymes, which utilize Zinc at the active site to cleave a broad spectrum of substrates, including ECM constituents, pro grow th factors, growth factor receptors, and cell cell adhesion mediators  MMPs are secreted as zymogens and activated upon cleavage and removal of their auto inhibitory pro domain by other MMPs or serine proteases  Specifically, MMP1, also known as interstitial collagenase I, is capable of cleavi ng several substrates of potential importance for OC, including collagens I and III. In vitro, MMP1 also cl eaves fibronectin, and laminin, which are present in the ovarian and omental extracellular matrix, as well as pro MMP2 and 9 and pro [12 14] In the normal ovary, MMPs are up regulated in the pre ovulatory foll icle  and may be secreted by HOSE cells from lysosomes  to aid in release of the oocyte during ovulation. MMP mutations and gene amplifications are not generally reported, but
49 MMP expression is increased in multiple cancer types, including ovarian  For example, Kenny et al. demonstrate that adhesion of SkOV3 OC cells to mouse peritoneum decreases after MMP2 inhibition or knock down, and that MMP2 expression is enhanced in adherent cells  Agarwal et al. implicate MMP1 in the a ctivation of protease activated receptor 1 (PAR 1) and demonstrate the importance of this pathway for OC ascites formation, metastasis, and angiogenesis  In this chapter, I demonstrate that the BRAT mutation in creases MMP1 expression and pro MMP1 secretion by HOSE cells, and that this up regulation occurs through c Jun and specific AP 1 sites in the MMP1 promoter. Further, I demonstrate that immortalized HOSE derived from BRAT mutation carriers also express sign ificantly elevated levels of pro MMP1. In accordance with these data, I hypothesize that enhanced MMP1 expression and secretion by the OSE of patients carrying the BRAT mutation may represent an initial step toward cellular malignancy and ultimately mutati on associated OC initiation and progression. Methods Cell culture and t ransfection. The SV 40 Large T Ag transfected HOSE cells: wt BRCA1 confirmed: (HOSE 118  ), negative family history of OC: HOSE 121, IMCC5, a nd 185delAG confirmed: (3261 77a, 3261 77b  1816 686a, and 1816 686b  ) were cultured in Medium 199/ MCDB 105 (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS) and gentamicin. Multiple stable BRAT clones wer e generated by transfection of 2 3 million HOSE 118 cells with 2 2.5ug of PCDNA3.1 or Flag BRAT  Stable lines were maintained in 1 mg/ml G418 selection media and confirmed to express BRAT by RT PCR  All cells were incubated at 37C with 5% CO 2 Two and
50 a half million cells were transiently transfected as previously described  using Program X 005, Kit V, and the Nucleofecto r device (Amaxa/ Lonza, Walkersville, MD) with 1.5 2.5 ug of plasmid (BRIT, Flag BRAT  pGL4.74 Renilla Luciferase/TK (Promega, Madison, WI), human MMP1 luciferase reporters (1G and 2G wt (full length), 1G and 2 G ( 3292), 1G and 2G ( 2942), ( 1546), and ( 517)   or green fluorescent protein (GFP). The control plasmid BRIT was generated by inserting a frameshift mutation to abrogate the BRAT sequence from amino acids 22 38 in the BRAT plasmid, and has been previously characterized as an appropriate control  The full length MMP1 promoter consists of the 4372 base pairs directly upstream of MMP1 gene start codon, and extends to +63. The reporter plasmids in the truncation series are truncated from t 1607) of the full length promoter in which the AP1 site at the indicated location has been mutated and rendered non functional. For kn ockdown studies, cells were co transfected with 2ug Smartpool small interfering RNA (siRNA) targeting c Jun (Si c Jun) or Smartpool non targeting control siRNA (Si Con) from Dharmacon (Chicago, IL). Microarray. HOSE 118 cells were serum starved for 24 hou rs (h), and transfected with 3ug of indicated transfectant (PCDNA3.1 or flag BRAT) using Lipofectamine reagent (Life Technologies, Inc., Grand Island, NY)  Twenty four hours after transfection, cells were colle cted in Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA was purified using the RNeasy cleanup procedure (Qiagen Inc., Valencia, CA). The quality of total RNA was assessed by agarose gel electrophoresis and A260/A280 ratio or by analysis on the Agilen t 2100 Bioanalyzer.
51 Five micrograms of total RNA from each sample was processed for microarray analysis. The poly(A) RNA was specifically converted to complementary DNA (cDNA) and then amplified and labeled with biotin following the procedure initially de scribed by Van Gelder et al.  Hybridization with the biotin labeled RNA, staining, and scanning of the chips followed the prescribed procedure outlined in the Affymetrix technical manual and has been previously described  Probe arrays: The oligonucleotide probe arrays were the Affymetrix U133A human arra ys. These arrays consist of 22,215 probe sets, which target known and suspected genes as well as a number of suspected splice variants. Data Analysis: Scanned output files were visually inspected for hybridization artifacts and then analyzed using Affymetr ix Microarray 5.0 software. Signal intensity was scaled to a trimmed mean intensity of 500 prior to output. For direct comparison analysis the MAS 5.0 software was used to identify differentially s ided signed rank test was used to assess the behavior of all oligonucleotide probes in each probeset  Probe sets that yielded a change p value less than 0.005 were identified as changed (increased or decreased ). The analyses of replicate experimental pairs were intersected to produce the final list. Using the MAS 5.0 calculated signal value the list was further reduced by removing probesets with low expression value in all samples and where the average change i n signal value between the control condition and the experimental condition was less than 1.4 fold. Western blot. Cells were washed in phosphate buffered saline (PBS), trypsinized, pelleted, and washed 1 2 times in cold PBS. Cells were lysed for 30 minutes on ice in modified CHAPS buffer, and lysate was centrifuged at 115,000 xg, at 4 degrees C, for 1h.
52 polyacrylamide gel electrophoresis (SDS PAGE). Proteins were transferred to Polyvin ylidene fluoride (PVDF) membranes, dried, and blocked in 5% milk in Tween 20 Tris buffered Saline. Blots were incubated in their respective antibodies overnight, followed by incubation with a horseradish peroxidase (HRP) conjugated secondary (Fisher, Pitts burgh, PA), and developed via enhanced chemiluminescence substrate (ECL) (Pierce/ Fisher, Pittsburgh, PA). Antibodies: c Jun (1:1000) Cat. #9165, Phospho c Jun (1:500) Cat. #9261, Cell Signaling Technology (Beverly, MA), Actin clone AC 40 (1:10,000) Cat. # 4700 Sigma (St. Louis, MO), MMP1 (2ug/mL) Cat. #MAB901 R&D Systems, Inc. (Minneapolis, MN), Ets 1 (1:500) Cat. #sc 56674 Santa Cruz (Santa Cruz, CA). RT PCR. RNA samples were isolated using TRIzol reagent from Invitrogen (Carlsbad, CA) per manufacturers pr otocol and DNAse treated. For semi quantitative PCR, 1 microgram total RNA, oligo(dT), and reverse transcriptase were used to generate single strand cDNA as previously described  The cDNA samples were amplifi ed using the Applied Biosystems GeneAmp RNA PCR Core Kit (Foster City, CA). Primers used were: Flag BRAT sense (CGATGACAAAATGGATTTATCTGC), Flag BRAT antisense (GAGACAGGTTCCTTCATCAACTCC), MMP1 sense (GAGCAAACACATCTGAGGTACAGGA), MMP1 antisense (TTGTCCCGATGAT CTCCCCTGACA)  actin (98 base pairs) sense (GGGAATTCAAAACTGGAACGGTGAAGG), and actin (98 base pairs) antisense (GGAAGCTTATCAAAGTCCTCGGCCACA). The amplified products were separated
53 by electrophoresis on a 10% poly acrylamide gel, stained with SYBR Green (Lonza, Rockland, ME), and photographed with the Kodak EDS 120 Digital Analysis System. The net intensity of each band was normalized to the respective endogenous control band. For real time PCR, 100 ng total RNA was reverse transcribed to generate single strand cDNA as previously described  The cDNA samples were amplified using Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems Step One Plus instrume nt. RQ (relative mean mRNA expression level) is calculated by the Step One software version 2.0. Using standard curves constructed for target and endogenous control genes, an arbitrary quantitative gene expression value is determined from the threshold cyc le (Ct) for each gene for each sample. Target gene values are normalized to control gene values, and fold difference is determined by dividing by the designated reference/ calibrator sample. Enzyme linked immunosorbant a ssay. For conditioned media analysi s, media containing 0.1% FBS was added to cells 24 h after transfection/ plating. Twenty four hours later, cells were counted and media was collected and centrifuged to remove debris, aliquoted, and stored at ( 80)C. To assess MMP1 activity, MMP1Emzyme Li nked Immunosorbant Assay (ELISA) Enzyme Activity Assay (R&D Systems, Inc, Minneapolis, MN) was performed on un concentrated conditioned media samples in detectable by a ddition of APMA MMP activating agent to all sample wells and standards. Fluorescence was read on a Fluorostar Galaxy plate reader. Resultant values were derived from a standard curve and expressed as the mean MMP1 concentration of triplicate samples +/ st andard error. When cell viability varied significantly, MMP1 concentration
54 was normalized to average cell number at time of conditioned media collection. MMP2 and 3 ELISAs (R&D Systems, Inc, Minneapolis, MN) were performed in triplicate on un concentrated Luciferase assay. Stable 118 PCDNA3.1 or BRAT cells were transfected with 0.15ug Renilla Luciferase reporter and 1.5ug MMP1 luciferase reporter. Twenty four hours later, cells were collected in Promega Pass ive Lysis Buffer and subjected to 2 freeze thaw cycles. Lysates were centrifuged at 10,600 xg for 1 minute at 4 degrees C, and supernatant was collected. Luciferase activity was assessed using a manual luminometer and the Promega Dual Luciferase Assay Syst em according to manufacturer protocol. For knockdown reporter studies, siRNA was co transfected and cells were collected 48 hours after transfection. Statistics. For real time PCR, error bars illustrate RQmin and RQmax, which are calculated as: RQave div confidence interval, for 5 degrees freedom) and RQave times (standard deviation^ This range represents the confidence interval at the 95% confidence level. For ELISA replicates. For ELISA comparison of MMP1 in conditioned media in cell lines, data did not follow a normal dis tribution; therefore, a nonparametric Wilcoxon test was performed. Results BRAT alters expression of genes involved in multiple cellular processes. Several studies supporting the importance of independent BRCA1 mutant functions
55 report modulation of gene e xpression by mutants. The periostin gene was up regulated in HeLa cells stably expressing the risk associated BRCA1 mutation 5083del19, which lacks the C terminal 193 amino acids, but not wt BRCA1, while increased periostin levels were also found in breast cancer tissue and serum of patients carrying the mutation  Similarly, mRNA and protein of D52 (TD52) and the folate receptor alpha (FOL1) were elevated in Hela cells expressing the Ser1841Asn breast cancer ris k associated BRCA1 mutation  and a synthetic BRCA1 truncation mutant comprising the N terminal third of the protein up regulated p53 gene expression and down regulated constituents of the JNK/SAPK and MAPK/ERK pathways in mouse epithelial OC cells  Preliminary microarray analysis of HOSE 118 cells (wt BRCA1), transiently transfected with the BRAT mutation revealed numerous differentially regulated genes (Table 2.1.), including elevated expression of the gene encoding matrix metalloprotease 1. BRAT enhances MMP1 gene expression in HOSE 118 cells. MMP family members are important for invasion and metastasis and are overexpressed in OC  In n PAR 1 activation in OC  a polymorphism in the MMP1 promoter that enhances expression  was found more frequently in OC patients compared to normal women. Further, tumors with the allele expressed seven times more MMP1 than tumors without the allele  Therefore, I decided to further explore MMP1 as a downstream target of BRAT in HOSE cells. To confirm that BRAT enhances MMP1expression transcriptionally, I performed semi quantitative RT PCR to measure the MMP1 mRNA level in HOSE 118 cells stably expressing BRAT or the control vector PCDNA3.1. While vector expressing cells exhibited undetectab le levels of MMP1 by this method, MMP1 mRNA levels were
56 Table 2.1. Selection of genes determined to be differentially regulated in BRAT cells. Gene Change PCDNA3.1 vs BRAT Average fold change IL1 alpha I 4.61 BCL2A1 I 4.55 IL1 be ta I 3.84 NFKB2 I 3.60 MMP1 I 2.70 IL6 I 2.50 IL11 I 2.06 Collagen I alpha 1 D 1.84 Collagen III alpha 1 D 2.65
57 significantly enhanced in both BRAT clones 6 and 7 (Figure 2.1. A). MMP1 mRNA levels were also evaluated by real time RT PCR, and BRAT cells exhibited MMP1 mRNA levels 16 18 fold higher than PCDNA3.1 cells (Figure 2.1. B). This data supports the hypothesis that BRAT enhances transcription of the MMP1 gene in HOSE 118 cells. BRAT increases expression and secretion of pro MMP1 by HOSE 118 cells. To evaluate the impact of BRAT expression on intracellular protein levels of MMP 1, Western blotting was performed on lysates of stable HOSE 118 BRAT and PCDNA3.1 cells. As expected, active (cleaved) MMP 1 was not detectable in lysates, however levels of pro MMP1 were 5 21 fold higher in BRAT clones 6 and 7 compa red to PCDNA3.1 cells (Figure 2.2. A.). To determine whether BRAT expression also results in increased pro MMP1 protein secretion, the MMP1 Enzyme Activity Assay was utilized to measure enzyme activity in conditioned media of cultured cells. Addition of a chemical MMP activating reagent, 4 Aminophenylmercuric acetate (APMA), allows measurement of total MMP1 (pro and active). Endogenously activated MMP1 was not detectable in un concentrated conditioned media of HOSE 118 cells, however, total MMP1 levels were 6 8 fold higher in conditioned media of BRAT clones 6 and 7 compared to PCDNA3.1 cells (Figure 2.2. B). BRAT mediated up regulation was specific to M MP1, as MMP2 and 3 were undetectable in conditioned media of transiently transfected BRIT or BRAT cells (Figure 2.2. C). These data indicate that BRAT specifically enhances expression and secretion of pro MMP1 by HOSE 118 cells. MMP1 and maspin are indepe ndent targets of BRAT. As mentioned previously, maspin was determined to be up regulated in BRAT expressing HOSE cells  Maspin is a member of the serine protease inhibitor (serpin) family that was
58 Figure 2.1. MMP1 mRNA is increased in BRAT expressing HOSE cells. Cells stably expressing BRA T or the PCDNA vector were plated at equal densities and collected. RNA was isolated, DNAse treated, and reverse transcribed. A. Semi quantitative PCR was performed for MMP1 and actin. PCR products were electrophoresed on a 10% acrylimide gel, stained with SYBR green, and imaged. B. Real time PCR was performed in triplicate for MMP1 and actin using SYBR green detection. RQ (relative mean mRNA expression level) was calculated by the Step One software version 2.0. Using standard curves constructed for target and endogenous control genes, an arbitrary quantitative gene expression value is determined from the Ct for each gene for each sample. Target gene values are normalized to control gene values, and fold difference is determined by dividing by the designate d reference/ calibrator sample.
60 Figure 2.2. BRAT increases cellular pro MMP1 and total secreted MMP1 in BRAT cells. A. Cells stably expressing PCDNA or BRAT were plated at equal densities, collected, and lysed. Protein lysate was separated using SD S PAGE and Western blotting was performed using the indicated antibodies. Relative band intensity was determined by dividing the pro MMP1 band intensity by the actin band intensity for each lane. B. Stable cells were plated at equal densities, allowed to a ttach, and then serum starved in media containing 0.1% fetal bovine serum for 24 hours. Conditioned media was collected and debris and dead cells were removed by centrifugation. Total MMP1 levels in un concentrated conditioned media were determined in trip licate by an MMP1 ELISA activity assay using the addition of the MMP activating reagent APMA. Graph indicates averages +/ standard error. C. Cells were transiently transfected with indicated transfectant and conditioned media was collected as described. T otal MMP levels in un concentrated conditioned media were determined in triplicate by ELISA kits. Graphs illustrate averages +/ standard error, except positive control human dermal fibroblast and human umbilical vein endothelial cell (HUVEC) conditioned m edia.
61 identified by its diminished expression in breast tumor samples compared to normal breast tissue  Maspin is non inhibitory due to a shortened and non conserved reactive site loop, the serpin domain responsible for protease inh ibition  Maspin expre ssion is low or absent in normal HOSE [9, 33] but in OC samples correlates with high tumor grade and shorter overall survival  Exogenous maspin expression inhibits migra tion, angiogenesis, invasion, and metastasis in multiple cancer models in vitro and in vivo, including breast, prostate, and OC cells [9, 34 36] Because maspin has been identified as a target of BRAT and maspin mod ulates migration, invasion, and metastasis, I decided to test whether maspin impacts MMP1 expression in BRAT cells. MMP1 mRNA levels were not significantly impacted by maspin knockdown (Figure 2.3), suggesting maspin and MMP1 are parallel targets of BRAT. BRAT mediated MMP1 modulation is c Jun dependent. MMPs are regulated extracellularly by pericellular localization  proteolytic activation, and binding of tissue inhibitors of metalloproteases (TIMPS)  as well as in tracellularly by transcription. To begin to elucidate the mechanism of MMP1 gene up regulation in BRAT cells, I chose to evaluate the importance of c Jun, as this transcription factor was previously implicated in BRAT mediated maspin up regulation in HOSE 118 cells  and because multiple AP1 sites in the MMP1 promoter are important in gene regulation (Reviewed in [38 40] ). HOSE 118 cells were transiently co transfected with BRIT or BRAT and Si Con or Si c Jun. BRIT/BRAT expression was confirmed by semi quantitative PCR (Figure 2.4. A). Greater than 95% knockdown of c Jun was confirmed by Western Blot (Inset in F igure 2.4. B). c Jun knockdown diminished MMP1 mRNA levels by 60% in BRIT cells, and by 80% in BRAT cells (Figure 2.4. B). Further, c Jun
62 Figure 2.3. MMP1 and maspin are parallel targets of BRAT. HOSE 118 cells stably expressing PCDNA or BRAT were transiently transfected with siRNA targeting maspin. Cells were collected after 48 hours, and RNA was isolated, DNAse treated, revers e transcribed, and real time PCR was performed in triplicate for MMP1 and actin using SYBR green detection. Semi quanitative PCR was performed for maspin and actin, and PCR products were run on a 10% acrylimide gel, stained with SYBR green, and imaged.
64 Figure 2.4. BRAT mediated MMP1 modulation is c Jun dependent. A. 118 HOSE cells were transien tly transfected with indicated transfectant and cells were collected 48 hours after transfection. RNA was isolated, DNAse treated, reverse transcribed, and semi quantitative PCR was performed for BRIT/BRAT and actin. PCR products were electrophoresed on a 10% acrylimide gel, stained with SYBR green, and imaged. B. Protein lysates were collected in parallel for knock down analysis by Western blot. Real time PCR was performed in triplicate for MMP1 and actin using SYBR green detection. C. Conditioned media wa s collected in parallel. MMP1 ELISA activity assay was performed in triplicate as described, however, because cell viability differed significantly, cell counts were performed upon conditioned media collection, and total MMP1 levels were normalized to cell number. Graph illustrates averages +/ standard error.
65 knockdown also reduced total MMP1 level in conditioned media by 69% in PCDNA cells, and 81% in BRAT cells (Figure 2.4. C). Interestingly, c Jun protein levels were elevated in BRAT cells (1.4 fold) (Figure 2.4. B), suggesting that BRAT mediated MMP1 up regulation may occur at least, in part, through increased c Jun levels. AP 1 sites in the MMP1 promoter mediate enhanced MMP1 expression in BRAT cel ls. Specific AP1 sites in the MMP1 promoter have been shown to impact MMP1 gene expression. The AP1 site at ( 72) is important for basal as well as induced MMP1 expression [41, 42] In ad dition, an AP1 site at ( 1602) is important for regulation, and this region is of critical importance in OC. A single nucleotide polymorphism in the was found to increase the efficacy of an Ets transcription factor binding site adjacent to the AP1 site at ( 1602), and increase MMP1 expression in normal human fibroblasts and melanoma cells  Interestingly, compared to the 1G allele, the 2G allele was present in a higher proportion of OC patients compared to normal controls  To determine whether BRAT mediates 1G/2G allele specific regulation of the MMP1 promoter, a set of MMP1 luciferase reporter constructs was obtained (Figure 2.5. A). Each construct contains the full length MMP1 promoter with eithe r the 1G or 2G allele at ( 1607) in control of the firefly luciferase gene. Activity of the 1G wt reporter was 1.7 fold higher in stable BRAT cells compared to stable PDCNA cells, while activity of the 2G wt reporter was greater than 2 fold higher in BRAT cells (Figure 2.5. B). Further, the 2G wt reporter construct mediated significantly more activity than the 1G promoter in both PCDNA (3.4 fold) and BRAT cells (4.4 fold). The importance of the ( 72) and ( 1602) AP1 sites was also determined
68 Figure 2.5. AP 1 sites in the MMP1 promoter mediate enhanced MMP1 expression in BRAT cells. A. Human MMP1 promoter luciferase reporter constructs. B. Cells stably expressing PCDNA or BRAT were transiently transfected with the indicated MMP1 reporter construct an d a Renilla constitutive luciferase reporter plasmid for normalization. Lysates were collected, subjected to two freeze thaw cycles, and assayed in triplicate on a manual normal ized to Renilla luciferase activity for each triplicate, averaged, and results are expressed +/ standard error. C. Stable cells were also co transfected with non targeting control siRNA or siRNA targeting c Jun, collected, and assayed similarly. Protein l ysates were collected in parallel for knock down analysis.
69 by utilizing 1 G and 2G full length promoter constructs in which the indicated AP1 site, ( 72) or ( 1602), was mutated to be non functional (Figure 2.5. A). In reporter constructs containing either 1G or 2G version of the SNP, MMP1 promoter activity was diminished signi ficantly by mutation of either AP1 si te, ( 72) or ( 1602) (Figure 2. 5 B). These data suggest that AP1 sites at ( 72) and ( 1602) are critical for BRAT mediated enhancement of MMP1 expression. Further, the presence of an Ets binding site adjacent to the di stal AP1 site further augments c Jun mediated MMP1 transcription. To dissect the contribution of other regions of the promoter to BRAT mediated MMP1 up regulation, a second set of luciferase reporter constructs was utilized. The constructs consist of a ser firefly luciferase gene (Figure 2.5. A). The full length (FL), ( 3292), and ( 2942) constructs have the 2G version of the SNP, while the smaller constructs, ( 1546) and ( 517) lack this region. SiRNA against c Jun was used concomitantly to validate the importance of c Jun for BRAT mediated MMP1 promoter activity mediated by each construct. Activity of the ( 329 2) truncation, in which an OCTA3 site, a PEA 3 site, an AP1 site, and several silencers are lost, was decreased by 24% compared to the full length reporter (Figure 2.5. C). Truncation to ( 2942) results in loss of cAMP response element binding (CREB) and p olyoma enhancer activator protein (PEA3) sites from the promoter. Transfection of ( 2942) yielded 18% lower activity than the full length promoter as well. Notably, c Jun knockdown in BRAT cells reduced full length, ( 3292), and ( 2942) reporter activity 6 1, 62, and 66.5% respectively, confirming the importance of c Jun for BRAT mediated MMP1 expression. Similarly, c Jun knockdown significantly diminished remaining activity of the ( 1546) and ( 517) constructs. These
70 data suggest that proximal portions of t he MMP1 promoter are important for BRAT mediated up regulation, and that AP1 sites throughout the promoter are necessary for MMP1 expression in BRAT cells. Increased pro MMP is detectable in BRAT mutation carrier derived cellular conditioned media. To eva luate the clinical impact of BRAT on MMP1 levels, BRAT mutation carrier derived immortalized HOSE cell lines were utilized. MMP1 levels in conditioned media of immortalized HOSE cell lines from the ovaries of women carrying a BRAT mutation were compared to lines without a family history of OC or with a confirmed wt genotype. Total conditioned media MMP1 levels were significantly higher in BRAT carrier HOSE cell lines (3261 77a, 3261 77b, 1816 686a, and 1816 686b) compared to non carrier HOSE cell lines (121 118, IMCC5) (Figure 2.6.). These data suggest that BRAT mediated MMP1 up regulation is detectable in cells of patients carrying this mutation. Discussion We have previously shown a role for the 185delAG BRCA1 mutation, BRAT, in STS induced apoptosis of normal HOSE a nd OC cells  as well as in the up regulation of the OC associated serpin, maspin, in normal cells  H ere, I identify a novel downstream target of BRAT, MMP1, and show that MMP1 is up regulated transcriptionally through a mechanism involving c Jun. Further, ELISA reveals higher total MMP1 secretion by HOSE cell lines derived from BRAT mutation carriers. I nterestingly, several of the genes differentially regulated in BRAT expressing HOSE cells encode proteins that localize to the extracellular space and are potentially important in OC (collagen I, and collagen III, IL 6, IL 1alpha, IL 1beta, and MMP1).
71 Figure 2.6. Increased pro MMP is detectable in BRAT mutation carrier derived cellular conditioned media. Cells were plated in triplicate at similar densities and conditioned media was collected as de scribed. MMP1 ELISA activity assay was performed in triplicate as described, however, because cell viability differed significantly, cell counts were performed upon conditioned media collection, and total MMP1 levels were normalized to cell number. Graph i llustrates averages +/ standard error.
72 Coll agens I and III are present in the extracellular matrix of the ovary and/or the omentum [12, 13] IL  and up  another potential BRAT target and mitogenic factor for HOSE cel ls  while IL 6 can enhance invasion of OC cell lines  MMP1 cleaves collagens I and III and can also cleave pro laminin, which are present in the ovarian and omental ECM, and pro MMP 2 and 9, which cleave multiple extracellular matrix constituents  Taken together, these targets of BRAT could promote motility, invasion, and metastasis of normal HOSE and potential tumor cells of mutation carriers. c ulation has been well established [41, 42] and in my model system, c Jun is crucial for BRAT mediated MMP1 up regulation. Increased c Jun protein levels in BRAT cells likely contribute to MMP1 up regulation. In agr eement, c Jun mRNA levels increase significantly prior to TNF 1 induced MMP1 up regulation  Future studies may reveal additional c Jun responsive genes differentially regulated by BRAT. I have begun to elucidate specific AP1 sites and other MMP1 promoter elemen ts necessary for BRAT mediated MMP1 up regulation. Truncation of the full length MMP1 promoter to ( 3292) significantly decreases reporter activity in BRAT cells, which indicates the importance of one or more distal promoter elements for activity. Indeed, binding sites for AP1 and PEA3, which has been shown to transactivate the MMP1 promoter  are located here. In some experiments, activity of the ( 2942) construct was marginally higher. Loss of a PEA3 site at this position could make available additional PEA3, which has been shown to synergize with c Ju n  BRAT cell reporter
73 activity of the three lar gest constructs was dramatically decreased by c Jun knockdown. Reduced activity was comparable to that of the full length promoter in PCDNA3.1 cells, confirming the importance of c Jun for BRAT mediated MMP1 expression. The greatest loss in reporter activi ty relative to full length occurs by truncation of the promoter to ( 1546) or ( 517), by which several elements are eliminated, including two AP1 sites. Specific disruption of the ( 1602) AP1 site significantly decreases reporter activity as well. I n agreement with the literature (Reviewed in  ), one or more of the most proximal AP1 sites ( 1062, 891, 562, 436, 181, 72) contribute to basal BRAT mediated MMP1 promoter activ ity, as c Jun knockdown significantly diminishes remaining activity of the ( 1546) and ( 517) constructs. Further, specific disruption of the ( 72) AP1 site also abrogates reporter activity. Taken together, these data reveal the necessity of c Jun in BRAT mediated MMP1 up regulation. The presence of the 2G MMP1 promoter polymorphism increases promoter activity  and is associated with decreased disease free and overall survival in OC patients  As expected, BRAT cells exhibited significantly higher activity than PCDNA3.1 cells when measu ring 1G and 2G versions of the promoter. In agreement with previous findings, the 2G wt reporter construct mediated significantly more activity than the 1G promoter in both PCDNA3.1 and BRAT cells. Interestingly, the enhancement in promoter activity mediat ed by the additional G nucleotide was even more apparent in BRAT cells, and Ets 1 levels were increased in BRAT cells compared to PCDNA cells (Figure 2.7.). Greater MMP1 2G promoter activity in BRAT cells may occur through cooperation of Ets 1 with increas ed c Jun, as Ets 1 and c Jun physically interact and can synergistically transactivate promoter expression  Further, AP1 and
74 Figure 2.7. Ets 1 protein levels are elevated in BRAT cells. Cells stably expressing PCDNA or BRAT were plated at eq ual densities, collected, and lysed. Protein lysate was separated using SDS PAGE and Western blotting was performed using the indicated antibodies. Relative band intensity was determined by dividing the Ets 1 band intensity by the actin band intensity for each lane.
75 Ets 1 sites are found in the Ets 1 promoter  therefore increased Ets 1 levels in BRAT cells may o ccur in part through increased c Jun transactivation. Alternatively, Ets 1 may represent an additional independent signaling pathway alternatively regulated in BRAT cell. It would be interesting to determine whether BRAT mutation associated OCs exhibit a h igh frequency of 2G alleles, as the combination of this allele with elevated c Jun protein could potentially augment OC progression or metastasis by up regulating MMP1. I have determined that maspin and MMP1 are parallel targets of BRAT, as maspin does not significantly impact MMP1 expression in BRAT cells. Interestingly, MMP1 is activated in vitro in a stepwise manner by uPA and MMP3 (stromelysin)  and maspin has been shown to negatively regulate uPA activity by enhancing uPA/uPAR internalization and by inhibiting activation of pro uPA  appear to regulate MMP1 expression, it may be important in regulation of MMP1 activity. Indeed, DU145 prostate cancer c ells stably expressing maspin exhibit decreased collagen cleavage in collagen degradation assays  We have previously found significantly lower levels of phospho Akt concurrent with enhanced apoptosis in BRAT c ells treated with cytotoxic drugs  It would be interesting to determine whether diminished Akt activity is necessary for MMP1 up regulation in BRAT cells, and the signaling pathways involved. Indeed, preliminar y data suggest Akt signaling is involved in BRAT mediated MMP1 up regulation. Expression of a constitutively active Akt construct diminishes MMP1 mRNA expression and total MMP1 in conditioned media of BRAT cells (Figure 2.8.). Interestingly, Akt inhibition has been shown to enhance activation of the MAPK pathway  which in turn
76 Figure 2.8. Constitutively active Akt reverses BRAT mediated MMP1 up regulation. A. 118 HOSE cells were transi ently transfected with indicated transfectant and collected 24 hours later. RNA was isolated, DNAse treated, reverse transcribed, and real time PCR was performed in triplicate for MMP1 and actin using SYBR green detection. Protein lysates were collected in parallel for analysis of CA Akt expression. B. Conditioned media was collected in parallel. MMP1 ELISA activity assay was performed in triplicate as described, however, because cell viability differed significantly, cell counts were performed upon conditi oned media collection, and total MMP1 levels were normalized to cell number. Graph illustrates averages +/ standard error.
77 enhances activity of the transcription factor c Jun. It will be interesting to determine in future studies the mechanism by which Akt mod ulates MMP1 expression. Normal cell lines and prophylactically removed ovaries from BRCA1 mutation carriers provide model systems in which to study early genetic, cellular, and histologic steps in OC initiation and progression. Immunohistochemical analysis revealed intermediate levels of Ki67 and p53 staining for prophylactically removed ovaries, while normal ovaries had the lowest staining and OC specimens exhibited the highest staining  Not all of the prophyl actically removed ovaries were from patients with confirmed BRCA1 mutations, however, and no correlation was found between any of the markers and presence of a BRCA1 mutation  In contrast, Piek et al. found no histologic change s in prophylactically removed ovaries compared to normal ovaries, and no difference in staining of Ki67, p21, p27, p53, Cyclin A, cyclin D1, Her2, ERalpha, and PR. Only Bcl 2 expression was significantly higher than in normal ovaries  Kirkpatrick et al. devised a multiphoton microscopy method to image ovarian surface epithelium and collagen fibrils of the ECM immediately after oophorectomy. Changes in collagen fibril organization similar to cancer specimens wer e detected in high risk ovaries  Similarly, abnormal regions of OSE, such as invaginations, inclusions cysts, papillary and stratified areas, adenomas, and microscopic adenocarcinomas, lacked a detectable basem ent membrane, as defined by collagen IV and laminin staining  These studies suggest that alteration in the ECM integrity may be a characteristic of BRCA1 mutant OC pathology. Early molecular and cytological cha nges have also been seen in cell lines generated from normal OSE cells of women with a strong family history of OC,
78 including irregular cellular morphology and organization in culture, elevated CA 125 and E cadherin, and amplification of various signaling pathways such as hepatocyte growth factor (HGF) and the PI3K pathway (Reviewed in  ). Immortalized HOSE from these patients also exhibited higher telomeric instability when compared to cells from patients withou t a strong family history  Our investigation reveals BRAT mutation associated phenotypic changes, as mutation carrier derived cell lines exhibit elevated MMP1secretion. Cleavage of ECM components, cell cell adhesion molecules, growth factors/ receptors, and other proteases is a crucial step in cancer cell migration, invasion, and metastasis. Consequently, up regulation and enhanced secretion of pro MMP1 from the HOSE of BRAT mutation carriers could prime these cells for transformation and metastasis. Cleavage of pro growth factors and release of ECM bound growth factors potentially promote survival and, therefore, accumulation of cancer promoting mutations. ECM remodeling also alters cell ECM contacts, which influence cell survival and migration as well. Final ly, as mentioned previously, OCs are highly prone to metastasis through cell shedding, and MMPs are critical for OC cell attachment at metastatic sites and establishment of metastases  Though MMP inhibitors hav e been largely ineffective in clinical trials, this may be in part because they are administered after metastases are already established  In contrast, animal models reveal preventative treatment diminishes tumor development  Confirming the imp ortance of MMP1 in a subset of OCs could improve survival by identifying patient populations that will respond best to treatment strategies targeting MMPs.
79 It is important to remember that in vivo, stromal cells, epithelial cells, and endothelial cells al l contribute to the MMPs found in the extracellular environment  This conc Instead, the potential exists for even greater enhancement of motility and invasive capability. MMP1 can be activated by multiple other MMPs that may be present in the ECM from an y cell source. Further, MMP1 activates several other proteases that cleave substrates it cannot. For example, MMP1 aids in activation of MMP2, which cleaves collagen IV, a major constituent of the ovarian basement membrane. Regardless of the mechanism, alt eration of MMP activity likely has far reaching consequences for the local ECM and epithelial stromal interplay in vivo. In these cells, BRAT mediated changes conducive to development of a malignant phenotype may have begun. It is interesting to speculate that BRAT cells, though non tumorigenic in their current state  may represent a step forward on the continuum of cellular malignancy. Loss of DNA damage repair through BRCA1 mutation and LOH as well as gain of function mutant activities such as gene regulation both likely contribute to further accumulation of genetic changes that promote OC progression and are characteristic of late stage BRCA1 mutation associated OC. I have identified another downstream target of the 185delAG BRCA1 mutant, BRAT. MMP1 expression is increased in BRAT cells transcriptionally in a c Jun dependent mechanism, and BRAT cells exhibit increased MMP1 secretion. Mutation associated changes early in OC development could poise cells in norm al tissue of mutation carriers for transformation or acquisition of invasive or metastatic ability.
80 Further exploration of these changes can increase our understanding of early steps of OC development and help identify potential screening and treatment str ategies.
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87 Chapter 3: Impact of BRAT on A poptosis, G ene R egu lation, and M igration in N ormal B reast E pithelial and B reast C ancer C ells Introduction Though BRCA1 mutation carriers have a slightly elevated risk for cervical, uterine, and pancreatic cancer  the most significant cancer risk associated with a BRCA1 mutation is for breast and OC  Though it is not known why the disease manifests preferentially in these tissues, several hypotheses have be en put forth: 1) BRCA1 mutation results in impaired DNA damage repair, accumulation of DNA damage, and apoptosis. Cells in the breast and ovary, however, could have a mechanism by which to delay apoptosis and accumulate mutations, resulting in cell surviva l and tumorigenesis  ; 2) Higher rates of LOH may occur in the breast and ovary  which could result in loss of wt function and emphasize the importance of mutant BRCA1 functions in these tissues; and 3) Increased risk for breast and OC may result in part from the influences of hormone signaling. Estrogen is clear ly important for normal breast development and lactation and also in sporadic breast cancer. Expression of estrogen receptor alpha, which mediates the proliferative effects of estrogen for breast epithelial cells, is over expressed in more than half of spo radic breast cancers  and selective estrogen receptor modulators, such as tamoxifen, are highly effective in tre ating breast cancers that express estrogen receptor alpha. Despite the fact that most BRCA1
88 associated breast tumors are estrogen receptor alpha negative  evidence indicates an interplay between BRCA1 and estrogen signaling. BRCA1 inhibits estrogen receptor dependent tra nsactivation function and down regulates the co activator p300 (E1A binding protein 300) (Reviewed in  ). Consequently, aberrant estrogen signaling in the breast and ovary, either directly or indirectly through paracrine signaling, may contribute to cancer in these tissues by enhancing survival despite impaired DNA damage repair due to BRCA1 mu tation (Reviewed in  ). Very few epidemiologic studies have attempted to determine whether specific BRCA1 mutations confer differential risk for breast or OC. Individuals who carry the same BRCA1 mutation may develop either breast or OC, whi le some individuals develop both. In population studies, families with predisposition to both breast and OC exhibit variation in the ratio of breast to OC occurrence  possibly as a result of the mutation they sh are. These observations suggest specific mutations have tissue specific functions that contribute to risk of cancer in the breast and/or ovary. Neuhausen et al. did not observe a significant difference in proportion of OC and breast cancer incidence mediat ed by specific mutations  In contrast, utilizing a population of 191 Ashkenazi Jewish OC patients, Moslehi and colleagues compared the estimated risk for breast or OC to 75 years of age mediated by three founder mutations. The three mutations conferred a similar risk of breast cancer, however risk of OC was greater in 185delAG mutation carriers compared to 5382InsC and 6174delT carriers (odds ratio 36.6, 20.8, and 14.2 respectively)  Differential risk of OC mediated by specific mutations may result from gain of function activity mediated by BRCA1 mutants in the ovary, while similar functions are not conferred, or have less impact in the breast.
89 Studies estimating risk levels for specific mutations are inherently difficult to interpret, however, because risk estimates are dependent on the study population. For example, risk estimations are higher in studies involving high risk families, and depend on biologic factors su ch as prophylactic oophorectomy or mastectomy and age  Environmental factors such as diet or oral contraceptive use further complicate risk assessmen t. As a result, estimated ranges of risk associated with BRCA1 mutation for ovarian or breast cancer are wide, and statistical differences in risk between specific mutations are not typically reported  Larger studies examining mutations grouped by their location within the BRCA1 gene have attempted to strengthen epidemiologic analysis. Such studies have reported differential ovarian and breast cancer risk based on the location of truncation mutations 4191) contribute to a signif icantly higher proportion of breast to OC incidence [7, 11] Conversely, Thompson et al. identified a region between nucleotides 2401 and 4190 in which mutations contribute to a higher proportion of OC to breast can cer  The location of a truncation mutation within the gene determines the predicted truncated protein size, which potentially influences the function of the mutant within the breast or ovary and, for some mutants, the relative ratio of breast to OC associated with a specific mutation. Indeed, clinical disease characteristics have been shown previously to be portions of the adenomatous polyposis coli (APC) g ene result in a less severe version of familial adenomatous polyposis 
90 I have previously observed that the BRAT mutation confers molecular and cellular changes in HOSE that may promote OC development. In order to understand the unique functions may preferentially promote OC, I examined the cellular and molecular impact of BRAT in normal breast epithelial and breast cancer cells. I hav e investigated several cellular and molecular processes previously found to be influenced by BRAT in normal HOSE or OC cells, including apoptosis and gene regulation, as well as cell migration. Methods Cell culture and t ransfection. MCF7, SkBr3, and MDA M B 231 human breast cancer cells were cultured in Medium 199/ MCDB 105 (Sigma, St. Louis, MO) with 10% fetal bovine serum and gentamicin. MCF10A normal human breast epithelial cells were cultured in DMEM/F12 (Mediatech, Manassas, VA) supplemented with 15mM HEPES, insulin 10ug/mL, EGF 20ng/mL (Sigma, St. Louis, MO), choleratoxin 100ng/mL (Biomol, Plymouth Meeting, PA), hydrocortisone 0.5ug/mL (BD Biosciences, Sparks, MD), L glutamine (MP Biomedicals, Solon, OH), glucose, sodium bicarbonate, 10% fetal bovine s erum and gentamicin. All cells were incubated at 37C with 5% CO 2 Two million cells were transiently transfected as previously described  using Program X 005 (for MCF7 cells), E 09 (for SkBr3 cells), X 013 (for 231 cells), T 024 (for MCF10A cells), Kit V, and the Nucleofector device (Amaxa/ Lonza, Walkersville, MD) with 2.5 3.5 ug of plasmid (GFP, PCDNA3.1 or Flag BRAT  ).
91 Cell viability a ssay. Twenty four hours afte r transfection, cells were trypsinized, counted, and plated at sub confluence in triplicate in 96 well plates with media containing vehicle or 2 10uM cisplatin. After 1 hour, cells were assayed using the CellTiter 96 AQueous One Solution Cell Proliferatio n MTS (Promega, Madison, WI) colorimetric microplate reader (Bio then assayed every 24 hours for 72 hours. Re sults were expressed as the mean absorbance standard error. Western blot. Cells were PBS washed, trypsinized, pelleted, and washed 1 2 times in cold PBS. Cells were lysed for 30 minutes on ice in modified CHAPS buffer, and lysate was centrifuged at 115, 000 separated via 10% SDS PAGE. Proteins were transferred to PVDF membranes, dried at 37 C for 1 hour, and blocked in 5% milk or bovine serum albumin in Tween 20 TBS. Blots were incubated in their respectiv e antibodies overnight, followed by incubation with an HRP conjugated secondary (Fisher Scientific), and developed via ECL (Pierce). Antibodies: BRCA1 (Calbiochem), cleaved caspase 3, caspase 3 (Cell Signaling Technology, Beverly, MA), actin (clone AC 40, Sigma, St. Louis, MO), and maspin (BD Biosciences, San Jose, CA). RT PCR. RNA samples were isolated using TRIzol reagent from Invitrogen quantitaive PCR, one microgram total RNA, oligo( dT), and reverse transcriptase were used to generate single strand cDNA as previously described  The cDNA samples were amplified using the Applied Biosystems GeneAmp RNA PCR Core Kit (Foster City,
92 CA). Primers used were: Flag BRAT sense (CGATGACAAAATGGATTTATCTGC), Flag BRAT antisense (GAGACAGGTTCCTTCATCAACTCC), MMP1 sense (GAGCAAACACATCTGAGGTACAGGA), MMP1 antisense (TTGTCCCGATGATCTCCCCTGACA)  actin (98 base pairs) se nse (GGGAATTCAAAACTGGAACGGTGAAGG), and actin (98 base pairs) antisense (GGAAGCTTATCAAAGTCCTCGGCCACA) Maspin sense (GGAGGCCACGTTCTGTAT) and Maspin antisense (CCTGGCACCTCTATGGA). The amplified products were separated by electrophoresis on a 10% polyacrylamid e gel, stained with SYBR Green (Lonza, Rockland, ME), and photographed with the Kodak EDS 120 Digital Analysis System. The net intensity of each band was normalized to the respective endogenous control band. For real time PCR, one hundred ng total RNA was reverse transcribed to generate single strand cDNA as previously described  The cDNA samples were amplified using Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems Step One Plus instrumen t. RQ (relative mean mRNA expression level) was calculated by the Step One software version 2.0. Using standard curves constructed for target and endogenous control genes, an arbitrary quantitative gene expression value was determined from the Ct for each gene from each sample. Target gene values were normalized to control gene values, and fold difference was determined by dividing by the designated reference/ calibrator sample. Scrape Assay. Following transfection, cells were plated at near confluence in 60millimeter dishes. Sixteen to twenty four hours later, cells were washed one time with PBS, and a scrape was made down the center of the dish using a sterile rubber cell
93 scraper. Cells were washed 2 times with PBS, and imaged immediately and every 24 hou rs for 72 hours using an Olympus 1X71 microscope with D870 camera. Statistics. Samples for MTS assays were run in triplicate, and the data were Results Endogenous BRCA1 and exogenous BRAT expression levels in normal breast epithelial and breast cancer cells. Etiologic studies have revealed a possible role for hormones and their receptors in BRCA1 associated breast cancer. For example, earlier age at first pregnancy, parity, a nd later first menarche decrease breast cancer risk in non mutation carriers, however, studies have shown opposite or absent trends in BRCA1 mutation carriers  In contrast, breast feeding reduces risk and oral contraceptive use increases risk similarly to non mutation carriers  Prophylactic oophorectomy, which eliminates a major source of estrogen production before menopause, significantly reduces breast cancer risk in mutation carriers as well  For this reason, multiple breast cancer cell lines, estrogen receptor positive and negative, were chosen for analysis. Each cell line represents human breast adenocarcinoma cells isolated from pleural effusion fluid, including: MCF7 cells are estrogen receptor posi tive, tumorigenic and metastatic in nude mice [16 18] SkBr3 cells are tumorigenic, estrogen receptor negative, and overexpress Her2   MDA MB 231 cells are tumorigenic and estrogen receptor negative  and used as a model for highly aggressive breast cancer. One normal breast epithelial cell line, MCF10A was also analyzed. MCF10A cells are a spontaneously immortalized sub population of normal,
94 non tumorigenic breast epithelial cells derived from a patient wi th fibrocystic disease  that are frequently utilized for comparison with breast cancer cell lines. The breast cancer cell lines util ized exhibit varying levels of BRCA1 expression. MCF7 cells are reported to have genomic loss of one BRCA1 allele  and to express low levels of BRCA1 mRNA and protein  SkBr3 cells are also re ported to express low basal levels of BRCA1  The studies reporting BRCA1 levels in these cell lines have utilized different methods of detection (i.e. RT PCR for mRNA level, Western blotting for protein level), and different reagents. To determine the relative expression of BRCA1 in the cell lines utilized, lysates were collected from each cell line and Western blotting was performed. SkBr3 cells expressed the highest levels of BRCA1, followed by MCF7 cells, MDA MB 231 cells, and MCF10A cells (Figure 3.1). Optimal transfection conditions for cell number, amount of DNA, and electroporation parameters were elucidated by transfection of GFP and followed by microscopic or flow cytometric analysis of the percentage o f transfected cells (transfection efficiency). Transfection efficiencies were approximately 40 70% (Figure 3.2.). Transfection of BRAT was then performed under the optimized conditions for each cell line and confirmed by semi quantitative PCR. Figure 3.3. illustrates a representative figure confirming the 120 nucleotide BRAT PCR product efficiently expressed in SkBr3 cells from 24 72 hours after transfection. Expression was confirmed in each remaining cel l line (data not shown). BRAT does not significantly impact proliferation or chemosensitivity of normal breast or breast cancer cells. Previous studies from our lab have shown that expression of BRAT in normal HOSE and OC cells increases the sensitivity o f these cells
95 Figure 3.1. Wild type BRCA1 levels in normal human breast epithelial and breast cancer cells. Cells were plated at equal densities, colle cted, and lysed. Protein lysate was separated using SDS PAGE and Western blotting was performed using the indicated antibodies. Relative band intensity was determined by dividing the BRCA1 band intensity by the actin band intensity for each lane.
96 Figure 3.2. Transfection efficiencies of normal breast epithelial and breast cancer cell lines. Indicated cell lines were transfected with GFP using the appropriate Amaxa nucleofector program. After 24 hours, cells were imaged using fluorescent microscopy, and p ercent GFP positive cells was estimated. Representative fluorescence images at 10X magnification for each cell line are pictured with the concomitant phase contrast image. For flow cytometry, cells were collected by centrifugation and resuspended in PBS. L iving cells were gated for GFP positivity.
97 Figure 3.3. BRAT is efficiently expressed in SkBr3 cells. SkBr3 cells were transiently transfected with BRAT plasmid, and cells were collected 24 72 hours after transfection. RNA was isolated, DNAse treated, re verse transcribed, and semi quantitative PCR was performed for BRAT. PCR products were electrophoresed on a 10% acrylimide gel, stained with SYBR green, and imaged.
98 to cytotoxic drug treatment  Therefore, to test whether BRAT impacts chemosensitivity of breast cancer cells and normal breast epithelial cells, the MTS assay was performed on vehicle and cisplatin treated cells. No significant change in cell viability was observed in MCF7 cells treated with vehicle or 10uM cisplatin up to 72 hours after transfection (Figure 3.4. A). Similar resul ts were observed in MDA MB 231 and MCF10A cells (data not shown). SkBr3 cells transfected with BRAT exhibited cell viability similar to control PCDNA transfected cells (Figure 3.4. B). However, cisplatin mediated cytotoxicity was 17% greater in SKBr3 BRAT cells at 24 hours. The decrease in BRAT cell viability was most pronounced at 48 hours (24%), and a moderate decrease was still evident at 72h (15%) (Figure 3.4. B). The enhanced chemosensitivity observed in BRAT HOSE cells was previously found to involve induction of apoptosis through caspase 3 activation [13, 24] Therefore, to further investigate the effect of BRAT on chemosensitivity of SkBr3 cells and determine whether caspase 3 is involved, Western blotting wa s performed to measure the level of cleaved (activated) caspase 3. No significant difference was observed in cleaved caspase 3 levels of BRAT transfected SkBr3 cells compared to control PCDNA SkBr3 cells wh en treated with vehicle or cisplatin (Figure 3.4. C). This data suggests that BRAT does not enhance apoptosis by caspase 3 activation in SkBr3 breast cancer cells. The transfection efficiency of 118 HOSE was previously found to be greater than 97%  Transfection of MCF10A and breast cancer cells was efficient, but not as high.(Figure 3.2.). To rule out the possibility that BRAT failed to modula te chemosensitivity because of lower transfection efficiency and, therefore, copy number in transiently transfected cells, stable cell lines were generated. SkBr3 cells were transfected
100 Figure 3.4. BRAT does not significantly impact grow th or chemosensitivity of breast cancer cells. A. MCF7 or B. SkBr3 cells were transiently transfected with PCDNA or BRAT and plated in 96 well plates. Proliferation was measured via MTS assay at indicated time points on triplicate samples of untreated or 1 0 M cisplatin treated cells. Graphs illustrate average absorption at 450 nm SE. C. SkBr3 cells were transiently transfected with PCDNA or BRAT and plated. Cells were treated with 10 uM cisplatin for the indicated time point, lysed, and protein lysate wa s electrophoresed on a 10% SDS PAGE gel. After transfer to PVDF membrane, blotting was performed with indicated antibodies. Relative band intensity was calculated by dividing cleaved caspase 3 band intensity by actin band intensity for each lane.
101 with PCDNA or BRAT and maintained in G418 selection media. BRAT expres sion was confirmed by semi quantitative PCR to be similar to or higher than that of 118 stable cells (Data not shown). SkBr3 cells stably expressing BRAT exhibited 14% decrease in viability compared to PCDNA3.1 cells after 48 hours of cisplatin treatment, though viabilities were similar to PCDNA cells at other time points (data not shown). This data suggests that the BRAT mutation mediates ovary specific effects on cell growth and chemosensitivity. BRAT does not significantly impact maspin expression in nor mal breast epithelial or breast cancer cells. In addition to its impacts on chemosensitivity in HOSE cells, BRAT was previously found to increase expression of the 42 kDa serine protease inhibitor, maspin. Maspin is unable to undergo the conformational cha nge necessary for normal serpin function  Maspin was identified by its diminished expression in breast tumor samples compared to normal breast tissue  Interestingly, maspin expression is low in normal HOSE, but is uniquely up regul ated in OC and correlates with high tumor grade and shorter overall survival [27, 28] maspin has the potential to impact several imp ortant processes in breast cancer cells. Increased maspin is associated with enhanced drug sensitivity in breast cancer models  Maspin has been shown to increase adhesion and diminish invasion and metastasis [ 30, 31] of breast cancer cells as well. Therefore, I next sought to determine whether BRAT impacts maspin expression in normal human breast epithelial cells and breast cancer cells as it does in HOSE cells.
102 To this end, I first performed Western blotting to determine basal levels of maspin in each cell line. As expected, maspin expression was robust in normal MCF10A cells and undetectable in MCF7, SkBr3, and MDA MB 231 breast cancer cells (Figure 3.5. A). Transfection of BRAT into MCF10A cells did not sign ificantly alter maspin mRNA levels as measured by semi quantitative PCR (Figure 3.5. B). Further, transfection of BRAT into SkBr3 cells did not result in detectable maspin expression (Figure 3.5. C), suggesting that BRAT does not significantly modulate mas pin expression in normal human breast epithelial cells, nor does BRAT activate maspin expression in human breast cancer cells. BRAT does not significantly alter MMP1 expression levels in normal breast epithelial or human breast cancer cells. My data indica te elevation of the gene encoding matrix metalloproteinase 1 (MMP1) in HOSE cells transfected with BRAT (Chapter 2). MMP1 is clearly important for breast cancer progression. MMP1 gene expression is elevated in breast tumor tissue from tumors that metastasi zed to bone compared to tissue from non metastatic breast cancer  Further, inhibition of MMP1 enhances apoptosis and inhibits metastasis of human breast cancer cells from the mammary fad pat of nude mice  Because BRAT regulates MMP1 expression in normal HOSE cells, and because of a possible role for MMP1 in breast cancer progressio n, I evaluated the impact of BRAT on MMP1 expression in normal breast epithelial and human breast cancer cells. MMP1 expression was undetectable in MCF10A, SkBr3, and MCF7 cells by real time PCR, and transfection of BRAT did not result in increased express ion (data not shown). Similarly, MMP1 expression was not detected by semi quantitative PCR in
103 Figure 3.5. BRAT does not significantly impact maspin expressi on in breast cancer cells. A. Cells were plated at equal densities, collected, and lysed. Protein lysate was separated using SDS PAGE and Western blotting was performed for maspin. MCF10A (B.) or SkBr3 (C.) cells were transiently transfected with BRAT pla smid. Cells were collected 24 72 hours after transfection. RNA was isolated, DNAse treated, reverse transcribed,
104 and semi quantitative PCR was performed for maspin and actin. PCR products were electrophoresed on a 10% acrylimide gel, stained with SYBR gree n, and imaged.
105 MCF10A or SkBr3 cells (Figure 3.6.). While a 185 nt PCR product representing MMP1 mRNA was detectable in MDA MB 231 cells, levels were not significantly altered b y expression of BRAT (Figure 3.6.), suggesting MMP1 up regulation is a tissue specific function of the BRAT mutation. BRAT does not significantly impact migration of breast cancer cells. Though BRAT did not appear to significantly impact apoptosis or regulation of targets previously identified in HOSE, it has not yet been determined whether BRAT impacts migration in any cell system. MMP1 is a major contributor to extracellular matrix remodeling, cell migration, invasion, and metastasis, therefore, in addition to MMP1 regulation, I chose to investigate whether BRAT modulates cell migration of breast cancer cells. To this end, a scrape assay was performed. For MDA MB 231 and MCF7 cells, images of scraped edge appeared similar for PCDNA and BRAT transfected cells (data not shown). SkBr3 control PCDNA cells exhibited slightly mo re frequent patches of cells growing within the scraped area compared to BRAT cells (Figure 3.7.), however, changes were minimal. These data suggest BRAT does not significantly alter breast cancer cell migration. Discussion BRCA1 m utation associated breast cancers are significantly more aggressive than their sporadic counterparts. Mutation associated tumors are frequently ductal carcinomas  highly proliferative  of a higher grade [35, 36] exhibit enhanced genomic instability  and result in poorer survival than sporadic breast cancer  Like BRCA1 mutation associated OC, BRCA1 associated breast cancer cells are more sensitive to cisplatin treatment  BRCA1 associated tumors are also more frequently estrogen receptor alpha, progesterone receptor, and Her2 negative  characteristics that
106 Figure 3.6. MMP1 expression is not alter ed by BRAT in normal breast epithelial or breast cancer cells. Indicated cells were transiently transfected with BRAT plasmid. Cells were collected 24 hours after transfection. RNA was isolated, DNAse treated, reverse transcribed, and semi quantitative PCR was performed for MMP1 and actin. PCR products were electrophoresed on a 10% acrylimide gel, stained with SYBR green, and imaged.
107 Figure 3.7. Migration of SkBr3 cells is not significantly impacted by BRAT. SkBr3 cells were plated at near confluence, a llowed to attach overnight, and a single continuous scrape was made down the center of the dish. Detached cells were removed and remaining adherent cells were washed with PBS and imaged at 4X immediately and every 24 hours for 72 hours.
108 are frequently associated with more aggressive disease and preclude treatment with anti est rogens. In contrast to BRCA1 associated OCs, BRCA1 associated breast cancers exhibit a unique gene expression profile, including up regulation of DNA repair genes, such as Rad51 and human mutS homolog 2 (MSH2)  and genes associated with a basal like tumor histology, such as cytokeratin 5 and 17 (Reviewed in  ). Because of the aggressive clinical nature of BRCA1 mutation assoc iated breast cancers, decisions about screening and prophylaxis for mutation carriers are critical. Therefore, it is important to understand clinical characteristics specific to each mutation. For example, classification of mutations as having low penetran ce for breast cancer would afford carriers the choice to avoid radical prophylactic procedures, such as mastectomy. Instead of grouping all BRCA1 mutations, clinical data such as tumor histology, breast/ ovarian specificity, and aggressiveness should be an alyzed for each mutation. Further, the molecular changes that mediate tumor initiation and progression are likely dependent on BRCA1 mutant function, and are, therefore, specific to each mutation. In addition to epidemiologic data for each BRCA1 mutation, study of mutant functions in cell and animal model systems will reveal mechanisms by which each mutation promotes cancer in the breast or ovary and aid in identification of targets for more effective treatment of mutation associated breast and OC. To this end, specific BRCA1 mutants have been shown previously to impact important cellular processes in breast epithelial and breast cancer models through gain of function or dominant negative activities. For example, mouse mammary gland specific expression of w t BRCA1 delays mutagen induced tumors, however, expression of a BRCA1 splice variant lacking the
109 first 72 amino acids accelerates tumorigenesis and death of transgenic mice  A synthetic BRCA1 truncation mutant encoding the first 300 BRCA1 amino acids inhibits mammary gland differentiation and development in wt mice  HCC1937 breast cancer cells are homozygous for the 5382InsC mutation, carry a p53 mutation with concom itant loss of the wt allele, and exhibit deletion of PTEN and other genetic aberrations implicated in breast cancer  When transfected with wt BRCA1, these cells exhibit chemo resistance, however, resistance is reversed upon co transfection of C terminal BRCA1 mutants  Co expression of 5382InsC and 5677InsA with wt BRCA1 in breast cancer cells exerts a dominant negative effect by inhibiting the wt enhance apoptosis  Of the three breast cancer cell lines tested, only the SkBr3 breast cancer cell line exhibited an increase in cisplatin sensitivity. Interestingly, this cell line over expresses Her2, a memb er of the EGF receptor family that has no specific ligand, but preferentially dimerizes with and activates other family members. This dimerization amplifies activation of MAPK and PI3K pathways and promotes survival, proliferation, migration, invasion, met astasis, and angiogenesis  Her 2 is over expressed in 25 30% of all breast and OCs  and correlates with poor progno sis  As mentioned previously, Her2 is frequently absent in BRCA1 mutation associated breast cancers, however, data is unavailable for each mutation. It would be interesting to examine whether Her2 over expression is associated with some BRCA1 mutations and not others in tumor samples as well as whether Her2 promotes or cooperates with mutant BRCA1 functions in mutation associated breast and ovarian mod el systems.
11 0 In contrast, some BRCA1 mutants do not mediate significant activity in breast cancer models independent of the loss of wt BRCA1 function. HCC1937 cells are more sensitive to ionizing radiation compared to breast cancer cells without a known BRC A1 mutation, but restoration of wt BRCA1 partially reverses this sensitivity. Interestingly, expression of other BRCA1 mutants, M1775R, T C64G, and P1749R, does not reverse sensitivity  While wt BRCA1 reduces hyper recombination and enhances homologous DNA repair in MCF7 cells, which have a single wt BRCA1 copy, the 5382InsC mutation does not mediate these functions  A breast epithelial cell line has been established that harbors one wt BRCA1 copy and one copy of the 185delAG mutation  Analysis of this line revealed no detectable difference in growth, anchorage independence, response to ionizing radiation and hydrogen peroxide treatment, or tumorigenicity compared to breast epithelial cells with two wt copies of BRCA1  Though most studies investigate mutant function in only one model system, Holt and colleagues compared the effect of two BRCA1 truncation mutants of 340 and 1835 amino acids on growth of breast and OC cells. In agreement with my findings, cell type specific effects were observed. The mutants did not significantly impact growth of breast cancer cells, however, growth inhibition was observed in three OC cell lines  You et al. found cell type specific BRCA1 mutant functions, as well. While a synthetic BRCA1 mutant lacking the N terminal 302 amino acids enhanced apoptosis and inhibited growth of MCF10A cells, growth of HeLa cells was unaffected  Reproductive organs and breast tissues are sometimes grouped together under a
111 these tissues are overlooked. Tissue s pecific effects in the context of cancer are a concept that has been demonstrated before. For example, tamoxifen is categorized as a selective estrogen receptor modulator because it exerts growth inhibitory effects on breast cancer cells and stimulatory ef fects on the uterine lining [52 ] These disparate impacts are likely related to the complement of transcription factors and co regulators expressed in each tissue type. Likewise, similar differences likely dictate the function of BRCA1 mutant proteins by providing different expression and stoichiometry of interacting proteins as well as redundancy for pathways inhibited by mutant function. Changes in gene regulation are observed in BRCA1 mutation carriers, and likely contribute to tissue specific mutant function as well. Indeed, compar ative genomic hybridization revealed that amplifications or deletions of specific chromosome regions was significantly more frequent in breast tumors from mutation carriers versus control tumors  Conversely, Ja zaeri et al. found similar patterns of gene expression in BRCA1/2 mutant ovarian tumors compared to sporadic tumors  Whether these changes in expression are correlative or occur as a result of BRCA1 mutation, t here is great potential for differential mutant BRCA1 protein function as a result of differential tissue specific gene expression. Lastly, splice variants of the BRCA1 gene have been confirmed  It has been suggested that tissue of wt protein expression  Tissue specific alteration of the balance of mutant and wt protein or repression of the mutant protein in this manner could result in significant impacts on cell physiology. Indeed, BRC a significantly lower proportion of OC compared to breast cancer incidence 
112 However, it has been difficult to determine differences in risk associated with spec ific mutations because of limited sample numbers. It is clear from this study, then, that the BRAT mutation confers tissue specific effects on apoptosis and gene regulation. Tissue specific mutant functions may contribute significantly to differential bre ast and OC risk and penetrance. It remains to be determined whether other BRCA1 mutations confer similar functions and, therefore, similarly impact cancer risk in the breast and ovary. Elucidating the mechanism by which mutations confer enhanced cancer ris k in the breast and ovary will allow for a better understanding of mutation associated cancer etiology and therefore better treatment for mutation carriers. Further, this information will improve prognostic accuracy and allow physicians and patients to mak e better decisions regarding treatment and prophylaxis. We have previously reported BRAT mediated changes in normal HOSE and OC cells that modulate cytotoxicity and regulation of genes potentially important in BRAT mpact on these cellular processes in normal apoptosis and regulation of MMP1 and maspin are specific to the ovary, and the BRAT mutation likely increases risk of cancer in the breast by an alternative and yet undiscovered mechanism.
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118 Chapter 4: Conclusions Origins of BRCA1 associated Ovarian Cancer A better understanding is needed of the mechanism by which normal HOSE of BRCA1 mutation carriers becomes malignant. Models of several cance r types, such as changes that promote tumorigenesis. Because OC is frequently diagnosed at late stage, identification and characterization of a pre malignant state has prove n difficult, however, studies suggest histologic and cytologic changes in non tumor ovarian tissue of patients with a family history of OC or a confirmed BRCA1 mutation. For example, more frequent deep invaginations in the ovary surface, dysplasia, hyperpl asia, and/ or surface papillae have been observed in prophylactically removed ovaries versus normal ovaries [1 3] These abnormal regions are hypothesized to be the origin of epithelial OC. The role of BRCA1 mutatio ns in the development of histologically abnormal regions is not yet understood, though the high frequency of these regions in mutation carriers suggests a role for BRCA1 mutants. Indeed, carcinoma was found to originate in inclusion cysts of several prophy lactically removed ovaries of mutation carriers, and the same p53 mutation was found in tumor tissue and tumor adjacent dysplastic and normal surface epithelium  This evidence supports the hypothesis of morphologically abnormal regions of HOSE as BRCA1 associated OC precursors, and for p53 mutation
119 and BRCA1 LOH as early events in BRA1 mutation associated OC development [4 ] fallopian tube epithelium from BRCA1 mutation carriers, though this phenomenon was detected in a similar proportion of BRCA1 mutation carriers and control patients [5 7] HOSE cells stably expressing BRAT have previously been characterized as non tumorigenic by lack of growth in soft agar and lack of telomerase activity  Though BRAT ce lls do not exhibit malignant characteristics, it is possible that changes conducive to development of a malignant phenotype have begun. I propose that BRAT cells, like the ovarian surface epithelium of BRCA1 mutation carriers, may represent forward movemen t on the continuum of cellular malignancy. Additional changes or mutations may be necessary for cells to become malignant. Indeed, the model I propose does not exclude the contribution of other oncogenes, tumor suppressors, or invasion/ metastasis promotin g proteins. Loss of DNA damage repair through BRCA1 mutation and LOH as well as gain of function mutant activities such as gene regulation both likely contribute to further accumulation of genetic changes that promote OC progression and are characteristic of late stage BRCA1 mutation associated OC. Future Studies The role of BRAT and other BRCA1 mutants in early pre malignant changes conducive to OC development requires further investigation. First, it is crucial to identify other BRCA1 mutants that mediate gain of function or dominant negative activity and the specific pathways and contexts in which they function. Other BRCA1 mutations found to elude nonsense mediated RNA decay, such as 5382insC, 5677insA, 188del11, and Arg1835ter  are promisi ng avenues for further study. Interestingly, the 188del11
120 mutation results in a STOP codon at codon 39 and a predicted protein product of similar size to BRAT, and may, therefore, mediate similar functions and specificity for ovarian cells. The stability o f BRCA1 mutant mRNAs should also be tested in breast and ovarian cell lines, because previous studies were performed in lymphoblastoid cells  Similarly, it is critical to continue seeking BRCA1 mutant proteins in cell lines and clinical speci mens, as some mutant proteins may exhibit instability  As demonstrated by our studies, tissue specificity is an important factor in BRAT function. It is likely that other mutants besides BRAT mediate tissue specific effects. HOSE 118 cells would be an ideal model in which to determine the impact of other BRCA1 mutations that exhibited gain of function activity in other model systems. For example, 5083del19, which increased periostin expression in HeLa cells and breast cancer tissue  may reg ulate other genes in the ovary. Cellular processes previously demonstrated to be altered by BRCA1 mutants provide direction for identifying additional downstream targets. For example, several of the genes found by microarray to be differentially regulated in BRAT expressing HOSE cells encode proteins that localize to the extracellular space and are potentially important in OC motility, invasion, and metastasis (collagen I, and collagen III, IL 6, IL 1alpha, IL 1beta, and MMP1). IL 1 found at higher levels in conditioned media of BRAT cells compared to PCDNA cells (Figure 4.1). As mentioned previously, IL 1 enhances MMP1 mRNA expression and stability  and promotes HOSE cell proliferation indirectly by up regulating the  Further, as demonstrated by our s tudies, BRCA1 mutation carrier derived normal cell lines can reveal mutant functions that promote early
121 Figure 4.1. Pro HOSE 118 cells were transiently transfected with indicated transfectan t. Cells were serum starved in media with 0.1% FBS for 24 hours. Conditioned media was collected, concentrated 47 fold, and run on a 10% SDS PAGE gel. Membrane was blotted with
122 p re malignant changes and transformation (Chapter 2), while expression of BRCA1 mutations in cancer cell models can reveal mutant functions that impact cancer progression through apoptosis  growth, invasion, or metastasis. as up regulation of both maspin and MMP1 involves c importance in enhanced chemosensitivity of BRAT cells, preliminary data also indicate a role for Akt in MMP1 up involvement in BRAT mediated MMP1 up regulation, and determine whether the MAPK signaling cascade is also affected by BRAT. It remains to be determined whether these and ot her as yet undiscovered functions of BRAT will share common mechanisms or signaling pathways. Cell models allow mechanistic studies of BRCA1 mutant function at the molecular level, however, discerning the physiologic impact of BRCA1 mutant functions in vi vo is vital to establishing clinical relevance. Staining of BRAT mutation associated OC tissue and adjacent normal HOSE from patients is currently ongoing to determine whether MMP1 expression is elevated in these tissues compared to tissue from patients wi thout a family history of OC. Though sample size will be limited, this data will confirm the clinical relevance of BRAT mediated MMP up regulation. To execute additional in vivo studies, a transgenic mouse model heterozygous for BRAT or other risk associa ted BRCA1 mutations could be created to confirm the presence of pre malignant changes similar to those found in human mutation carriers. BRCA1 heterozygous knockout mice do not spontaneously develop mammary or ovarian tumors  possibly because the mouse lifespan is not long enough to accumulate the
123 additional genetic changes necessary to achieve tu morigenesis  BRCA1 knockout mouse models also fail to recapitulate BRCA1 mutant protein funct ions that likely contribute to tumorigenesis. In contrast, a transgenic BRCA1 mutant mouse model would address this shortcoming. Indeed, BRCA1 variants besides wt contribute significantly to mammary tumor development, as BRCA1 knockout mice that retain the develop tumors that are morphologically and genotypically distinct from knockout models designed to eliminate the entire BRCA1 gene  BRCA1 mutation associated breast and OCs exhibit frequent chromosomal aberrations and it is clear that accumulation of additional mutations is necessary for tumorigenesis. For example, mouse mammary gland s pecific BRCA1 knockout mice that harbor a p53 mutation develop tumors with shorter latency  It would be informative to investigate latency and tumor characteristics of BRCA1 mutant transgenic mice with concomit ant overexpression of oncogenes previously shown to be important in OC, such as Ras, epidermal growth factor receptor (EGFR), or Her2, or in the context of a p53 mutant background. Transgene expression specific to the reproductive epithelium has been achie ved using the Mullerian inhibitory substance type II receptor  Further, hormonal factors in BRCA1 mutation associated OC development could be addressed by observing the effect of hormone treatment or reproducti on on cancer incidence of heterozygous BRCA1 mutant mice. Recapitulation of breast cancers and OCs similar to those of mutation carriers could reveal targets and pathways of potential importance for treatment of BRCA1 mutation carriers. The utility of a tr ansgenic BRCA1 mutant mouse models has some limitations, however. Specifically for BRAT, interpretation would be complicated by the lack of a
124 mouse homolog to the human MMP1 gene. An animal model with a longer lifespan and greater potential for stochastic mutation accumulation than the mouse would be beneficial as well. The rabbit ovary may be useful for this endeavor, as the normal histology of the rabbit ovarian surface has been described  Further, in contrast to humans, a bursal membrane surrounds the murine ovary, whi ch potentially alters the stromal/tumor microenvironment and acquisition of invasive and metastatic capabilities by tumor cells. In addition to animal models, mining previously collected epidemiologic data could further support a role for BRAT or other BRC A1 mutants in development of OC. For example, if mutation designation were available, other genetic alterations observed in prophylactically removed ovaries or BRCA1 mutation associated OCs could hint at downstream targets of specific BRCA1 mutants or addi tional mutations that promote tumorigenesis in carriers of that mutation. Significance The scope of importance of BRAT and its impacts are broad in the field of OC pathology and treatment. Functions or targets of the BRAT protein may be common to other sim ilar BRCA1 mutants. The similarity of breast cancer to OC ratios for BRCA1 truncation mutants of similar sizes suggests BRCA1 mutants may share common mechanisms of action. If there are common themes, this discovery could advance understanding of and treat ment of many high risk patients. Information gleaned about BRCA1 mutation associated OCs may also be applied to sporadic cancers. Differences between sporadic and BRCA1 mutation associated OC are less well defined than for breast cancer. Unlike BRCA1 mutat ion associated breast
125 cancers, which are frequently more proliferative and higher grade [19 21] reports disagree as to whether stage and grade differ significantly between BRCA1 associated and sporadic OC [22, 23] In contrast to BRCA1 associated breast cancers, BRCA1 OCs lack a unique molecular profile compared to sporadic OCs [24, 25] BRCA1 mutation associated and sporadic OCs may s hare common downstream targets, such as Akt, c Jun, or MMP1. Common signaling mediators shared between BRCA1 mutation associated and sporadic OC increase the utility of drugs targeting these pathways by increasing the population of patients that will likel y respond. Most studies reveal that BRCA1 associated OCs are more frequently categorized as serous carcinomas and rarely as mucinous [23, 26] The histologic subtypes of OC not only have different clinical impacts (ie treatment response and survival rates), but are thought to arise from distinct precursor regions, undergo distinct stepwise histologic changes, and occur though deregulation of distinct molecular pathways (Reviewed in  ). Subty pe specific pre malignant changes may be similar in BRCA1 and sporadic OCs. Indeed, surface invaginations and inclusion cysts, which are hypothesized to be the origin of some types of OC, occur more frequently in BRCA1 mutation carriers, but are present in the ovaries of the general population and increase with age  contributi on to OC and breast cancer risk may have limited application to prognosis nd treatment of the general population. Regardless, increasing knowledge about BRAT will greatly benefit confirmed mutation carriers and patients through better risk assessment, deci sions about prophylaxis and treatment, and treatment efficacy.
126 I have identified MMP1 as a novel target of the BRAT BRCA1 mutation in HOSE. I have determined that BRAT increases MMP1 gene expression and enhances cellular and secreted pro MMP1 in a c Jun d ependent manner involving several AP1 sites and gene regulation are specific for the ovary. Taken together, these early molecular changes could poise cells for transformat ion or acquisition of invasive or metastatic ability. Further exploration of these changes can increase our understanding of early steps of OC development and help identify potential screening and treatment strategies.
127 References 1. S alazar H, Godwin AK, Daly MB, Laub PB, Hogan WM, Rosenblum N, Boente MP, Lynch HT & Hamilton TC (1996) Microscopic benign and invasive malignant neoplasms and a cancer prone phenotype in prophylactic oophorectomies. J Natl Cancer Inst 88 1810 1820. 2. Wer ness BA, Afify AM, Bielat KL, Eltabbakh GH, Piver MS & Paterson JM (1999) Altered surface and cyst epithelium of ovaries removed prophylactically from women with a family history of ovarian cancer. Hum Pathol 30 151 157. 3. Casey MJ, Bewtra C, Hoehne LL, Tatpati AD, Lynch HT & Watson P (2000) Histology of prophylactically removed ovaries from BRCA1 and BRCA2 mutation carriers compared with noncarriers in hereditary breast ovarian cancer syndrome kindreds. Gynecol Oncol 78 278 287, doi: 10.1006/gyno.2000.5 861S0090 8258(00)95861 X [pii]. 4. Pothuri B, Leitao MM, Levine DA, Viale A, Olshen AB, Arroyo C, Bogomolniy F, Olvera N, Lin O, Soslow RA, et al. (2010) Genetic analysis of the early natural history of epithelial ovarian carcinoma. PLoS One 5 e10358, doi : 10.1371/journal.pone.0010358. 5. Shaw PA, Rouzbahman M, Pizer ES, Pintilie M & Begley H (2009) Candidate serous cancer precursors in fallopian tube epithelium of BRCA1/2 mutation carriers. Mod Pathol 22 1133 1138, doi: modpathol200989 [pii]10.1038/modpa thol.2009.89. 6. Lee Y, Miron A, Drapkin R, Nucci MR, Medeiros F, Saleemuddin A, Garber J, Birch C, Mou H, Gordon RW, et al. (2007) A candidate precursor to serous carcinoma that originates in the distal fallopian tube. J Pathol 211 26 35, doi: 10.1002/pa th.2091. 7. Folkins AK, Jarboe EA, Saleemuddin A, Lee Y, Callahan MJ, Drapkin R, Garber JE, Muto MG, Tworoger S & Crum CP (2008) A candidate precursor to pelvic serous cancer (p53 signature) and its prevalence in ovaries and fallopian tubes from women with BRCA mutations. Gynecol Oncol 109 168 173, doi: S0090 8258(08)00007 3 [pii]10.1016/j.ygyno.2008.01.012. 8. O'Donnell JD, Johnson NC, Turbeville TD, Alfonso MY & Kruk PA (2008) BRCA1 185delAG truncation protein, BRAt, amplifies caspase mediated apoptosis in ovarian cells. In Vitro Cell Dev Biol Anim 44 357 367, doi: 10.1007/s11626 008 9122 0. 9. Perrin Vidoz L, Sinilnikova OM, Stoppa Lyonnet D, Lenoir GM & Mazoyer S (2002) The nonsense mediated mRNA decay pathway triggers degradation of most BRCA1 mRNAs b earing premature termination codons. Hum Mol Genet 11 2805 2814. 10. Anczukow O, Ware MD, Buisson M, Zetoune AB, Stoppa Lyonnet D, Sinilnikova OM & Mazoyer S (2008) Does the nonsense mediated mRNA decay
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About the Author Rebecca Linger completed her undergraduate studies at West Virginia Wesleyan College, where she graduated with a B.S. degree in Biology and a minor in Chemistry. During this time, she received a Mary Babb Randolph Cancer Center Undergraduate Research Fellowship at West Virginia University, where she completed a su mmer research project. Rebecca received her M.S. in Molecular Physiology and Biological Physics from the University of Virginia, where she was awarded a Congressionally Directed Medical Research Programs Department of Defense Breast Cancer Research Predoct oral Traineeship. She was also invited to the American Association for Cancer Research Edward A. Smuckler Memorial Pathobiology of Cancer Workshop in Aspen, CO. Rebecca joined the Medical Science Ph.D. Program in the USF College of Medicine in 2007. She pr esented her research at the 2010 American Association for Cancer Research Annual Meeting in Washington, D.C. and at USF Health Research Day in 2009 and 2010, where she was awarded Outstanding Poster Presentation in 2010. Rebecca was active in the Associati on of Medical Science Graduate Students, and served as the Student Leadership Award for ou tstanding leadership and service.
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Novel roles for 185delag mutant brca1 in ovarian cancer pathology
h [electronic resource] /
by Rebecca Linger.
[Tampa, Fla] :
b University of South Florida,
Title from PDF of title page.
Document formatted into pages; contains X pages.
Dissertation (PHD)--University of South Florida, 2010.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
ABSTRACT: Familial history is the strongest risk factor for developing ovarian cancer (OC), and a significant contributor to breast cancer risk. Most hereditary breast cancers and OCs are associated with mutation of the tumor suppressor Breast and Ovarian Cancer Susceptibility Gene 1 (BRCA1). Studying risk-associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Recent studies have shown novel BRCA1 mutant functions that may contribute to breast and OC initiation and progression independent of the loss of wtBRCA1. Previously, we have found that normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT (BRCA1 185delAG Amino Terminal truncated protein), exhibit enhanced chemosensitivity and up-regulation of the OC-associated serpin, maspin. In the current study, I identify an additional target of the BRAT mutation, matrix metalloprotease 1 (MMP1), a key player in invasion and metastasis. BRAT-expressing HOSE cells exhibit increased MMP1 messenger RNA (mRNA) by real time PCR and protein by Western blotting. Pro-MMP1 levels are also higher in conditioned media of BRAT-expressing cells and HOSE cell lines derived from BRAT mutation carriers. c-Jun is critical for BRAT-mediated MMP1 up-regulation, as siRNA knockdown diminishes MMP1 levels. Luciferase reporter constructs reveal that activator Protein 1 (AP1) sites throughout the distal end of the promoter contribute to BRAT-mediated MMP1 expression, and basal activity is mediated in part by an AP1 site at (-72). Reporters containing a single nucleotide polymorphism (SNP) associated with OC risk and progression yield increased activity that is further enhanced in BRAT cells. Interestingly, BRAT-mediated changes in chemosensitivity and gene regulation are not recapitulated in a normal breast epithelial or breast cancer cell model. This suggests tissue-specific mutant BRCA1 functions may contribute to breast and ovarian tissue specificity of BRCA1 mutation-associated cancer risk and also to differential breast and ovarian cancer risk and penetrance associated with specific mutations. Early molecular and cellular changes such as MMP1 up-regulation in the ovarian surface epithelium of BRCA1 mutation carriers may promote OC initiation and progression and represent a step forward on the continuum of cellular malignancy. Further investigation is warranted, as elucidating these early changes will aid in identification of potential screening and treatment strategies.
Advisor: Patricia A. Kruk, Ph.D.
Matrix metalloproteinase 1
Ovarian surface epithelium
x Medical Sciences
t USF Electronic Theses and Dissertations.