The Wilms' tumour suppressor gene Wt1 is a key regulator of embryonic development and tissue homeostasis. In humans, mutation in the gene may lead to childhood kidney cancer, severe glomerular kidney diseases, gonadal dysgenesis and, in rare cases, heart diseases. The importance of WT1 in embryonic development is related to its crucial function in the regulation of two cellular plasticity processes: the epithelial to mesenchymal transition (EMT) and the reverse process, the mesenchymal to epithelial transition (MET). WT1 expression persists during the waves of EMT and MET that generate certain mesodermal tissues. In fact, WT1 is a major regulator of both transitions and it is essential for the survival of mesenchyme progenitors. Furthermore, it has been proposed that WT1 is required for the derivation of progenitors from different mesothelia, possibly through an EMT. Progenitors expressing WT1 are believed to differentiate into different cell types, giving rise to coronary vasculature, adipocytes and hepatic stellate cells. In my PhD I aimed to investigate the instructive role of different WT1 isoforms. To address this, the first goal was to generate a single plasmid that would accommodate all necessary components of an inducible system, in order to derive cellular models for the inducible expression of WT1 single isoforms. Second, I aimed to understand the processes that the single variants were sufficient to drive. Therefore, I started with the establishment of two cellular models for the inducible expression of the main four isoforms of WT1 (with or without the exon 5 and/or the KTS, here referred as +/+, +/-, -/+ and -/-). I cloned different plasmids carrying doxycycline inducible WT1 isoforms and derived single stable clones in two epithelial kidney cell lines that do not express WT1: the MDCK and the IMCD3 cells. I then analysed the expression profiles of the clones, using either microarray or RNA sequencing, and performed cellular assays to characterize the cells after WT1 induction. Overall, WT1 induction did not affect cell growth, cell cycle, cell migration or anchorage independent growth, suggesting that the expression of WT1 in these two cell lines does not change their oncogenic potential. The expression analysis of the MDCK cells suggested that the induction of WT1 isoforms activates an inflammatory response, leading to the overexpression of several cytokines. Moreover, the -/+ isoform speciffically caused the upregulation of fibrotic markers and the rearrangement of the actin cytoskeleton. Interestingly, the expression of the mesothelial marker UPK3B increased following the induction of the -/+ isoform. Because the expression of the -/+ variant led to the most signifficant isoform-specific changes in both cell lines, I focused on this isoform for the validation of the transcriptomic data of the IMCD3 cells. I confirmed that the induction of WT1 -/+ in the IMCD3 cells leads to the upregulation of fibrotic markers, increases cell adhesion and activates the AKT and MAPK pathways. Moreover, there was a significant overexpression of different mesothelial markers and, importantly, of key regulators and markers of developmental processes, such as adipogenesis, skeletal and cartilage development, as well as angiogenesis. I then dissected the timing of expression of some specific markers and regulators, analysing the levels of the genes at different time points after WT1 -/+ induction. The preliminary results intimate that WT1 -/+ might induce epithelial cells in the direction of cartilage-skeletal tissue and fat, possibly through a mesothelial intermediate. The data also suggest that the induction of this isoform initiates an EMT, possibly followed by an MET, as the levels of expression of the differentiation markers and regulators increase. To validate the proposed instructive role of WT1, it will be of crucial importance to determine both RNA and protein levels of markers and regulators at even later time points, both in IMCD3 cells and in a model of inducible embryonic stem cells, which is currently under development. In the future, it will be important to address the relevance of these findings in vivo and to dissect the molecular mechanisms.

Prostate cancer presents a significant paradox: it is very common, yet rarely fatal. To wit, the prostate is the most common non-skin tissue for cancer diagnosis in men in the United States. Despite its high incidence, fatal malignancy occurs in only a small fraction of diagnosed men. The fatal cases are characteristically defined by distant spread in the body, also known as metastasis. In order to metastasize a cancer cell must complete several sequential steps. These include degradation of and invasion through the epithelial basement membrane, typically through the loss of static intracellular adhesions with fellow epithelial cells; entrance into the blood stream (intravasation); survival within circulation; exit from the blood stream upon arrival at a new tissue (extravasation); and survival and colonization at the secondary site. At the time of diagnosis, it is not currently possible to accurately predict future metastasis and thereby clinicians cannot delineate those men at high risk for fatal disease from the vast majority of men who are likely to experience an indolent disease course. Consequently, we examined the behavior of cancer cells in several steps of the metastatic cascade. In doing so, we uncovered both molecular and biophysical characteristics of cancer cells that may facilitate successful metastatic dissemination and tumor outgrowth. Epithelial-to-mesenchymal transition (EMT) is physiological process of transdifferentiation that is normally initiated during vertebrate development, but has recently been implicated in tumor development, progression, and metastases. The EMT program results in dramatic changes, including the exchange of epithelial for mesenchymal markers, altered cellular morphology, and gain of motility. EMT-like cellular alterations have been implicated most strongly in the metastasis steps of invasion and survival of cells at primary tumors sites. How EMT-like changes may facilitate survival and growth in the microenvironment of a micrometastatic niche has been less clearly elucidated. Consequently, we evaluated how EMT-like changes may affect the survival and subsequent outgrowth of prostate cancer cell lines following restrictive growth conditions. We observed that EMT-like cells as compared to their more epithelial counterparts displayed enhanced maintenance of their proliferative potential following extended culture in nutrient restriction. This phenotype depended on an EMT-associated increase in autophagy. Notably, the post-stress outgrowth phenotype could be conferred through a paracrine signaling mechanism that may involve autophagy-derived exosome-like extracellular vesicles. These studies demonstrated that EMT-like cells have a resistance to nutrient restriction through enhanced autophagy and may have uncovered a novel autophagy-dependent exosomal secretion pathway. Metastatic efficiency is thought to be strongly limited by the destruction of circulating tumor cells by the hemodynamic shear forces within the vasculature. However, such a persistent belief has little appropriate published experimental evidence. We developed an in vitro assay to expose cells to fluid shear stress (FSS). By monitoring the viability of the cells, we determined that transformed cells had a highly conserved ability to resist even very high FSS. The mechanism depended on the capacity to patch membrane defects, extracellular calcium, and a dynamic cytoskeleton. We also observed a stiffening of cancer cell membranes after exposure to FSS. Taken together, these studies expand the understanding of how cancer cells survive in circulation and indicate that metastatic efficiency is less limited by hemodynamic forces than previously thought. The steps of hematogenous metastasis between intravasation and extravasation necessitate the existence of circulating tumor cells (CTCs). Collection, enumeration, and study of CTCs have the potential to serve as a "liquid biopsy" of the metastatic cascade. In prostate cancer, the enumeration of CTCs by detection of the expression of epithelial markers has displayed limited clinical utility. We hypothesized that the prognostic value of CTC number may be enhanced by detection of cells which have undergone the pro-metastatic EMT-like program. We developed a flow cytometry-based experimental assay for enumeration of CTCs using epithelial (EpCAM) and mesenchymal-like (N-cadherin) surface proteins. We detected from prostatectomy patients before and after surgery events expressing EpCAM, N-cadherin, and both. However, the detection of background events from healthy control subjects was unacceptably high. These studies support the idea of mesenchymal-like tumor cells in circulation, but will require further assay development for reliable conclusions to be drawn. In sum, the work described above has provided descriptive and mechanistic insight to molecular and biophysical properties that may facilitate prostate cancer metastasis. It is our hope that these data will result in the development of relevant preventative, diagnostic, and therapeutic clinical strategies for prostate cancer.

Congenital heart defects (CHDs) occur in 5% of births. While gene mutations have been identified in CHD patients, not much is known about coordinated signaling mechanisms during heart morphogenesis. Endocardial cushions of the atrioventricular canal and outflow tract contribute to the formation of valves and septa in the heart. Epithelial cell to mesenchymal cell transition (EMT) is a key process in cardiac cushions before this tissue undergoes remodeling into valves and septa. Defining complex signaling networks directing cardiac cushion epithelial to mesenchymal transition is essential for understanding the etiology of CHDs. We identified the MAP3Kinases, MEKK3 and MEKK4, as signaling components present during cardiovascular development. MEKK3 is detected in myocardium and endocardium surrounding the cardiac cushions of the atrioventricular canal during heart morphogenesis, while MEKK4 is found in the myocardium, endocardium, and cushion mesenchyme. Functional assays were employed to examine how MEKK3 and MEKK4 kinase activity contributes to endocardial EMT. Addition of dominant negative (dn)-MEKK3 or dn-MEKK4 to endocardial cushion explants, cultures that recapitulate in vivo EMT, causes a significant decrease in mesenchyme formation as compared to controls. Ventricular explant cultures, where the endocardial cells do not normally undergo EMT, provided with constitutively active (ca) MEKK3 activates mesenchyme production. ca-MEKK4 is not sufficient to cause EMT in ventricular endocardium. Furthermore, ca-MEKK3 expression in ventricular explants leads to increased secreted TGFβ2, which mediates mesenchyme formation. Blockade of TGFβ2 in ventricular explant cultures provided with ca-MEKK3 ablates the activation of EMT. In addition to in vitro studies, we show that mice expressing kinase inactive MEKK4 have myxomatous valves characterized by increased proliferation and changes in extracellular matrix molecules such as hyaluronan. We next investigated whether signal transduction is affected in cushions and valves of the MEKK4 kinase inactive mice. Abnormal TGFβ signaling is observed in MEKK4 mutant hearts, which is also seen with Marfan's sydrome. Remarkably, activated MEKK3 is maintained in cardiac cushions of these mice after EMT indicating compensation by MEKK3 for loss of MEKK4 catalytic activity. Our observations define MEKK3 and MEKK4 expression during cardiovascular development and suggest that MEKK3 and MEKK4 have diverse functions during development of heart valves.

Breast cancer is a disease that will be diagnosed in about 1 in 10 women throughout their lifetime. The majority of breast cancers are originated from the epithelial cells of the mammary ducts, and this occurrence can be due to several factors including hereditary and acquired mutation. There are several major breast cancer subtypes, including estrogen receptor-α (ERα)-positive, HER2-enriched and triple-negative (TNBC). Patients diagnosed with ER+ tumors are generally treated with estrogen blockers (e.g., tamoxifen, letrozole and fulvestrant). Patients with HER2+ tumors are commonly administered with drugs that block HER2 signaling (e.g., trastuzumab) or inhibit HER2’s tyrosine kinase activity (e.g., lapatinib). For patients with TNBC, chemotherapies such as taxanes and anthracyclines are standard of care therapies. However, for each breast cancer subtype, a significant number of patients develop resistance to these therapies and eventually die from metastasis, a process which accounts for ~90% of breast cancer mortality. Currently, metastatic breast cancer is incurable, and the short median survival of 3 years for patients with metastatic breast cancer has not significantly changed in over 20 years. Therefore identification of new molecules that are involved in breast cancer metastasis and development of more precisely targeted therapeutic strategies are urgently needed to improve the clinical outcome for this disease. The transforming growth factor pathway beta (TGFβ) pathway has been show to play a key role in metastasis through induction of epithelial-mesenchymal transition (EMT), cell migration and invasion. Over more than a decade, this pathway has been studied across several cancers and it is now better established that it has context-dependent tumor suppressive and oncogenic qualities. In the early stages of breast cancer, TGFβ pathway is a suppressor of benign and early stage tumor growth. However, as disease progresses and corresponding levels of TGFβ ligands become elevated, a “switch” will take place and promote oncogenic phenotypes like EMT and cancer cell stemness which drive metastasis. Long noncoding RNAs (lncRNAs) are an emerging subclass of RNA molecules in cancer biology. LncRNAs are >200nt and can influence target gene expression locally in “cis”, or along a distant chromosome in “trans”, through various mechanisms and interactions with other biological molecules. The contribution of TGFβ-regulated lncRNAs to associated phenotypes like EMT and cancer cell stemness has not been very well studied. The aim of this doctoral dissertation is to address the functional and mechanistic roles of lncRNAs in these processes. Using a well-established TGFβ-induced EMT model (e.g., mouse mammary epithelial cell NMuMG treated with TGFβ, we have identified 3 conserved lncRNAs (lncRNA-HIT, WDFY3-AS2 and TIL) that are significantly upregulated upon TGFβ-induced EMT. They all mediate TGFβ-induced EMT, cell migration and invasion. Overexpression of these lncRNAs is frequently detected during the breast cancer progression and is associated with high grade and late stage of breast cancer as well as metastatic lesion. We have also demonstrated that lncRNA-HIT positively regulates HOXA13 through “cis” mechanism and that WDFY3-AS2 induces WDFY3 and STAT3 expression at mRNA level by direct interaction with hnRNP-R. Interestingly, TIL stimulates C-MYC protein but not mRNA expression by promoting Akt phosphorylation of NF90 leading to its translation from the nucleus to cytosol where NF90 binds to C-MYC mRNA and enhances C-MYC translation. Importantly, we have shown that knockdown of lncRNA-HIT and WDFY3-AS2 significantly reduces breast cancer growth and lung metastasis in orthotopic breast cancer model. These findings indicate that these TGF-induced lncRNAs play critical role in EMT, metastasis, and are relevant in human patient tumors. Therefore, it is important to consider utilizing these molecules for clinical applications like diagnosis, monitoring recurrence, predicting a response to therapy, and even as a direct target for therapeutic intervention.

<p> Downregulation of E-cadherin is a frequent event in epithelial cancers and it correlates with weakened cell-cell adhesion and the induction of an epithelial-to-mesenchymal transition (EMT). It is postulated that E-cadherin downregulation liberates the catenin p120 and allows p120's translocation to the nucleus where it interacts with and functionally regulates the novel BTB/POZ transcription factor, Kaiso. Kaiso mediates transcriptional repression of various tumourigenesis-associated genes via methylated CpG dinucleotides or a sequence-specific Kaiso binding site (KBS). The Kaiso/p120 interaction has been detected in E-cadherin expressing cells of various origins, but is seldom detected in N-cadherin expressing cells or cells that have undergone EMT. We hypothesize that p120 and Kaiso play a role in EMT by modulating the expression of EMT-associated genes. We demonstrated that TGF-β-induced EMT occurs in a dose- and time-dependent manner in NMuMG cells but not in FHL-124 cells. In both cells lines, the Kaiso/p120 interaction occurred irrelevant of EMT induction by TGF-β. In NMuMG cells, the expression of p120 increased with EMT induction, while the expression of Kaiso remained unchanged. Finally, misexpression of Kaiso and p120 in mammary epithelial cells affected TGF-β-mediated EMT induction by delaying the upregulation of the positive mesenchymal markers, N-cadherin and α-SMA.</p> / Thesis / Master of Science (MSc)

Breast cancer is the most common cancer in women and unspayed female dogs. The Epithelial to Mesenchymal Transition (EMT) is a process involved in embryogenesis, carcinogenesis, and metastasis. The Transforming Growth Factor- Beta (TGF-β) pathway and its associated transcription factors are crucial for EMT induction, during which epithelial cells lose their defining characteristics and acquire mesenchymal properties. EMT has been implicated as a driver of metastasis as it allows cells to migrate and invade different organs. Recent evidence indicates that cancer stem cells are required to establish metastatic tumours at distant sites, and that EMT may promote development of cancer cells with stem-cell characteristics, thus, the EMT pathway may be an important molecular determinant of tumour metastasis. The main objective of this project was to characterise TGF-β-induced EMT in breast cancer models. EMT was induced by TGF-β in human, canine and feline breast cancer cell lines, and confirmed by morphological changes and molecular changes at the protein level by Western blot analysis. Changes at the mRNA level were confirmed in human and canine mammary carcinoma cell lines by qRT-PCR; migratory properties were assessed by invasion assays in vitro in feline and canine mammary carcinoma cells. Importantly, we observed that feline and canine mammary carcinoma cells stimulated by TGF-β acquired stem cell characteristics including sphere-forming ability, self-renewal, and resistance to apoptosis, and also enhanced migration potential. Canine cells showed resistance to chemotherapeutic drugs after TGF-β stimulation. These data suggests a link between EMT and cancer stem-cells. Moreover, global changes in microRNA expression were mapped during TGF-β-induced EMT of canine mammary carcinoma cells. This gave significant insight into the regulation of EMT in canine cancer cells and identified several potential targets, which require further investigation. During EMT cells acquire migratory properties and cancer stem-cell characteristics, suggesting that EMT and the stem-cell phenotype are closely related during cell migration and metastasis, therefore making the TGF-β pathway a potential target for the development of novel therapies against cancer and its progression.

acase@tulane.edu / Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance. / 1 / Van Hoang

Epithelial to mesenchymal transition (EMT) is an important process in embryonic development, tissue repair, inflammation, and cancer. During EMT, epithelial cells disassemble cell-cell adhesions, lose apicobasal polarity, and initiate migratory and invasive processes that allow individual cells to colonize distant sites. It is the means by which non-invasive tumors progress into malignant, metastatic carcinomas. In vitro, EMT occurs in two steps. First, cells spread out, increasing in surface area and pushing the colony borders out. Then cells contract, pulling away from neighboring cells and rupturing cell-cell junctions, resulting in individual highly migratory cells. Recent discoveries indicate that calcium signaling is central in EMT. Both previous data with patch clamping and new calcium imaging data show a series of calcium influxes in cells induced to undergo EMT with hepatocyte growth factor (HGF). It has also been shown that blocking calcium signaling prevents EMT from progressing normally. However, it is not known if calcium alone is sufficient to drive EMT behaviors. By experimentally triggering calcium influxes with an optigenetic cation channel, the behaviors that calcium influxes induce can be determined noninvasively, without use of drugs that may have secondary effects. The results of using the optigenetic set up along with live cell imaging are that cells become more motile and disrupt normal epithelial cell-cell adhesions. This behavior is believed to be due to increased cell contractility downstream of calcium signaling, and is dependent on Ca2+/calmodulin-dependent protein kinase II (CaMKII). When cells are pre-treated with CaMKII inhibitor before HGF addition, they undergo the spreading step of EMT without subsequent cellular contraction and rupture of cell-cell junctions. CaMKII is a protein kinase that is activated by binding Ca2+/calmodulin, and is a known downstream component of calcium signaling. CaMKII is known to affect the actin cytoskeleton by both physically bundling actin filaments to increase their rigidity, and through signaling by activation of myosin light chain kinase (MLCK), which has a role in stress fiber formation. Immunofluorescence did not show colocalization of CaMKII with actin, ruling out regulation through actin bundling. However, CaMKII does appear to have a role in stress fiber formation. EMT induced with HGF treatment results in increased numbers of stress fibers as well as trans-cellular actin network formation, both actin structures decorated with non-muscle myosin II (NMII). CaMKII inhibition not only blocks these actin formations, but it also decreases stress fiber levels below basal unstimulated levels in cells that have not been treated with HGF. This suggests that CaMKII has a role in regulating contractility through cellular actin networks, indicating a mechanism for calcium's role in cellular contractility in EMT.

Pulmonary fibrosis is a potentially deadly pathology characterized by excessive deposition of extracellular matrix (ECM), increased tissue stiffness, and loss of tissue structure and function. Recent evidence has suggested epithelial to mesenchymal transition (EMT), the transdifferentiation of an epithelial cell into a mesenchymal fibroblast, is one mechanism that results in the accumulation of myofibroblasts and excessive deposition of ECM. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin mediated interactions with ECM proteins and soluble growth factors including TGFβ. TGFβ, a potent inducer of EMT, can be activated by cell contraction mediated mechanical release of the growth factor from a macromolecular latent complex. Therefore, TGFβ activity and subsequent EMT may be influenced by both the biochemical composition and biophysical state of the surrounding ECM. Based on these knowns it was first investigated how changes in the biochemical composition of the matrix and changes in tissue rigidity together modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin specific interactions with fibronectin (Fn) variants displaying both the RGD and PHSRN binding sites facilitate cell binding through α3β1 and α5β1 integrins, and that these interactions maintain an epithelial phenotype despite engagement of increased tissue rigidities. Conversely, Fn fragments that facilitate cell binding through αv integrins drive TGFβ activation and subsequent EMT even while engaging soft underlying substrates. Adding to the complexity of studying mechanisms that contribute to pulmonary fibrosis, is exposure of the lung to injuries from environmental particulates. Therefore, we investigated how EMT is altered in response to particulate matter (PM). Here we show that PM exposure further drives TGFβ activation, EMT, and increases intracellular levels of reactive oxygen species (ROS). Additionally, cells binding the ECM through α5β1 and α3β1 integrins only partially recover an epithelial phenotype, suggesting ROS may be a secondary driver of TGFβ and EMT. Taken together these results suggest dynamic changes to the ECM microenvironment are major contributors to the control of EMT responses and provide insights into the design of biomaterial-based microenvironments for control of epithelial cell phenotype.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation the wound is repaired, however this process, and the mechanisms to maintain OSE homeostasis after the wound is repaired are poorly understood. We have shown the mouse OSE (mOSE) contains a stem cell population that is expanded by Transforming Growth Factor Beta 1 (TGFB1), a factor present in follicular fluid. These data suggest that components in the follicular fluid such as TGFB1 may promote wound repair and OSE homeostasis through maintenance of the OSE stem cell population. Additionally, TGFB1 may promote wound repair through induction of an epithelial-to-mesenchymal transition (EMT) and activation of pro-survival pathways, as seen in other tissues. To elucidate the mechanism for TGFB1-mediated ovulatory wound repair, mOSE cells were treated with TGFB1, which induced an EMT seen with increased Snai1 expression and cell migration. Snai1 overexpression also increased cell migration and sphere formation (a stem cell characteristic). RNA sequencing results suggest this is at least in part through elevated collagen deposition in SNAI1 overexpressing cells. A TGFB signalling targets array identified Cox2 induction following TGFB1 treatment. Constitutive Cox2 expression did not promote an EMT, but enhanced sphere formation and cell survival. Finally, TGFB1 treatment decreased Brca1 expression, which when deleted from mOSE cells also increased sphere formation. RNA sequencing results suggest that Brca1 deletion promotes stemness through activation of the stem cell genes Ly6a and Lgr5. RNA sequencing was also used to compare mOSE cells cultured as monolayers and as spheroids, with and without TGFB1. These results validate our findings that TGFB1 promotes an EMT partially through Snail induction and the upregulation of Cox2. mOSE cells cultured as spheroids acquire a mesenchymal transcriptional profile that is further enhanced with TGFB1 treatment. These data suggest that TGFB1 may promote ovulatory wound repair and maintain OSE homeostasis through the induction of an EMT, maintenance of the stem cell population and activation of a pro-survival pathway. Interestingly, mOSE spheroids also decrease Brca1 expression and upregulate cancer associated genes such as Pax8 and Greb1. The induction of survival pathways, while simultaneously increasing stemness and repressing Brca1 could render cells more susceptible to transformation. This work provides novel insights as to why ovulation is the primary non-hereditary risk factor for ovarian cancer.