Murine Ovarian Surface Epithelium Research Articles

Abstract 928: USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy and one of the most common causes of cancer mortality among women worldwide. Ubiquitin-Specific Peptidase 13 (USP13) gene copy is strongly amplified in human epithelial ovarian cancer, and high USP13 expression is correlated with poor survival outcomes, yet its pathological contribution to ovarian tumorigenesis remains unknown. Here, we crossed a conditional Usp13 overexpressing knock-in mouse with a conditional knockout of Trp53 and Pten mouse, and generated a novel ovarian cancer genetically engineered mouse model (GEMM), which closely recapitulates the genetic changes driving ovarian cancer in humans. Overexpression of USP13 with deletion of Trp53 and Pten in murine ovarian surface epithelium accelerated ovarian tumorigenesis and led to decreased survival in mice. Notably, USP13 greatly enhanced peritoneal metastasis of ovarian tumors with frequent development of hemorrhagic ascites. The primary and metastatic tumors exhibited morphology and clinical behavior similar to human high-grade serous ovarian cancer. Co-inhibition of USP13 and AKT significantly decreased the viability of the primary murine ovarian cancer cells isolated from the GEMM. USP13 also increased the tumorigenic and metastatic abilities of primary murine ovarian cancer cells in a syngeneic mouse study. These findings suggest a critical role of USP13 in ovarian cancer development and reveal USP13 as a potential therapeutic target for ovarian cancer. Citation Format: Juntae Kwon, Hyungja Choi, Anna D. Ware, Bernadette Cecilia Morillo, Haiyang Wang, Cecil Han. USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 928.

USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model

Epithelial ovarian cancer is the most lethal gynecologic malignancy and one of the most common causes of cancer mortality among women worldwide. Ubiquitin-Specific Peptidase 13 (USP13) gene copy is strongly amplified in human epithelial ovarian cancer, and high USP13 expression is correlated with poor survival outcomes. Yet, its pathological contribution to ovarian tumorigenesis remains unknown. We crossed a conditional Usp13 overexpressing knock-in mouse with a conditional knockout of Trp53 and Pten mouse and generated a novel ovarian cancer genetically engineered mouse model (GEMM), which closely recapitulates the genetic changes driving ovarian cancer in humans. Overexpression of USP13 with deletion of Trp53 and Pten in murine ovarian surface epithelium accelerated ovarian tumorigenesis and led to decreased survival in mice. Notably, USP13 greatly enhanced peritoneal metastasis of ovarian tumors with frequent development of hemorrhagic ascites. The primary and metastatic tumors exhibited morphology and clinical behavior similar to human high-grade serous ovarian cancer. Co-inhibition of USP13 and AKT significantly decreased the viability of the primary murine ovarian cancer cells isolated from the GEMM. USP13 also increased the tumorigenic and metastatic abilities of primary murine ovarian cancer cells in a syngeneic mouse study. These findings suggest a critical role of USP13 in ovarian cancer development and reveal USP13 as a potential therapeutic target for ovarian cancer.

Abstract A26: PTEN and colonization of the ovary in metastasis of fallopian tube-derived ovarian cancer

Abstract It is now clear that high-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube epithelium (FTE), making the role of the ovary in these tumors unclear. Therefore, the objectives of this research were to 1) determine if colonization of the ovary was an important step in the spread of FTE-derived tumors to the peritoneum and 2) explore the role of ovarian rupture, independent of hormonal changes, in tumor cell colonization of the ovary. First, 50,000 murine oviductal epithelial (MOE) cells stably expressing PTENshRNA+KRASG12V were allografted into the left ovarian bursa (IB) or peritoneal space (PS). IB-grafted mice developed tumors, with all mice requiring sacrifice within 90 days. Tumors were detected throughout the peritoneum and in the contralateral ovary. In contrast, PS-grafted mice failed to develop tumors for the duration of the experiment (150 days). To determine if rupture of the ovary was important in colonization of the ovary, an ex vivo attachment assay was developed in which ovaries was left intact or cut with a scalpel to rupture the ovarian surface (ovulation mimetic) without associated hormonal changes. Ovulation mimetic ovaries had significantly more MOE GFP and OVCAR8 RFP cells attached, particularly in the wounded areas. To confirm that increased attachment was due to exposure of the ovarian stroma, MOE GFP cells were plated on monolayers of murine ovarian surface epithelium (MOSE), monolayers of murine ovarian stroma (MOST), or three-dimensional (3D) type I collagen gels (the primary extracellular matrix protein in the ovarian stroma). MOE GFP cells attached to MOST more than MOSE and to 3D collagen than MOST, supporting the contention that FTE cells adhere to the extracellular matrix (ECM), such as collagen, exposed during ovulation. We next sought to test the ability of cells to survive in an environment rich in 3D collagen. 3D collagen reduced the viability of normal epithelial cells (MOSE, MOE, and IOSE80) but had no effect on HGSOC cells (OVCAR3, OVCAR4, OVCAR5, OVAR8, and OVSAHO), indicating that transformation of FTE is important for survival in the collagen-rich ovarian stroma. RNA-seq showed that 3D collagen decreases genes associated with focal adhesion, regulation of actin cytoskeleton, and ECM-receptor interaction, supporting the idea that normal epithelial cells respond poorly to a microenvironment rich in 3D collagen. In contrast, genes associated with p53 signaling and suppression of MAPK/AKT were increased when cells were grown on 3D collagen. However, plating cells on 3D collagen did not lead to stabilization of p53 and expression of mutant p53 had no effect on viability of MOE cells on 3D collagen. KRASG12V tended to increase viability, but only PTENshRNA completely rescued viability on 3D collagen as compared to 2D. PTENshRNA had no effect on adhesion to 3D collagen, but it did increase invasion through collagen. Cells expressing PTENshRNA showed increased attachment to 3D ovaries that were wounded to mimic ovulation; however, the AKT inhibitor MK2206 did not reduce this adhesion. Interestingly, after 7 days of culture on 3D collagen, MOE PTENshRNA cells formed spheroids while controls did not, leading to the hypothesis that spheroids may mediate metastasis from the fallopian tube to the ovary. In support of this contention, spheroids were observed in the fallopian tube of PAX8+/cre PTENflox/flox mice, which develop fallopian tube-derived ovarian cancer. Furthermore, OVCAR8 RFP spheroids preferentially attached to wounded sites mimicking ovulation relative to intact ovaries. In conclusion, FTE cells attached to exposed ECM of the ovary and colonization of the ovary enhances further spread of FTE-derived tumors beyond the reproductive tract. Inhibition of ovarian colonization may be a new approach to inhibit peritoneal spread of cancer in women at high risk of developing HGSOC. Citation Format: Matthew Dean, Vivian Jin, Angela Russo, Joanna E. Burdette. PTEN and colonization of the ovary in metastasis of fallopian tube-derived ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A26.

Impact of oviductal versus ovarian epithelial cell of origin on ovarian endometrioid carcinoma phenotype in the mouse.

Endometrioid carcinoma (EC) is a relatively indolent ovarian carcinoma subtype that is nonetheless deadly if detected late. Existing genetically engineered mouse models (GEMMs) of the disease, based on transformation of the ovarian surface epithelium (OSE), take advantage of known ovarian EC driver gene lesions, but do not fully recapitulate the disease features seen in patients. An EC model in which the Apc and Pten tumour suppressor genes are conditionally deleted in murine OSE yields tumours that are biologically more aggressive and significantly less differentiated than human ECs. Importantly, OSE is not currently thought to be the tissue of origin of most ovarian cancers, including ECs, suggesting that tumour initiation in Müllerian epithelium may produce tumours that more closely resemble their human tumour counterparts. We have developed Ovgp1-iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)-regulated Cre recombinase in oviductal epithelium - the murine equivalent of human Fallopian tube epithelium. Ovgp1-iCreERT2 ;Apcfl/fl ;Ptenfl/fl mice treated with TAM or injected with adenovirus expressing Cre into the ovarian bursa uniformly develop oviductal or ovarian ECs, respectively. On the basis of their morphology and global gene expression profiles, the oviduct-derived tumours more closely resemble human ovarian ECs than do OSE-derived tumours. Furthermore, mice with oviductal tumours survive much longer than their counterparts with ovarian tumours. The slow progression and late metastasis of oviductal tumours resembles the relatively indolent behaviour characteristic of so-called Type I ovarian carcinomas in humans, for which EC is a prototype. Our studies demonstrate the utility of Ovgp1-iCreERT2 mice for manipulating genes of interest specifically in the oviductal epithelium, and establish that the cell of origin is an important consideration in mouse ovarian cancer GEMMs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Divergent Roles of PAX2 in the Etiology and Progression of Ovarian Cancer.

PAX2 is an essential transcription factor for development. Aberrant PAX2 expression in adult tissues is associated with carcinogenesis and experimental evidence shows that PAX2 generally exhibits oncogenic properties. Although PAX2 is not expressed in normal ovaries, it is highly expressed in low malignant potential and low-grade epithelial ovarian tumors, suggesting that PAX2 induction in ovarian surface epithelium (OSE) may contribute to transformation. Herein, we provide evidence that expression of PAX2 in normal murine OSE cells (mOSE) enhances their proliferation and survival and, with loss of p53, induces tumorigenicity. PAX2 expression in murine ovarian cancer cells enhanced or inhibited tumorigenicity, depending on the model system. In RM cells (mOSE transformed by K-RAS and c-MYC), PAX2 expression inhibited p53 and induced pERK1/2 and COX2, resulting in enhanced angiogenesis and decreased apoptosis of tumors arising from these cells. However, in a murine model of high-grade serous ovarian cancer (STOSE), PAX2 expression improved animal survival by reducing proliferation and metastasis, which correlated with increased Htra1 and decreased COX2. Thus, PAX2 may not be a classical oncogene or tumor suppressor but instead can act in either role by differential regulation of COX2 and/or HTRA1.

Abstract 226: Parallel microfiltration (PMF): A novel method to screen cell mechanotype

Abstract The mechanical phenotype of cells (mechanotype) is emerging as a valuable label-free biomarker. Cells are visco-elastic materials whose mechanotype is altered in diseases from malaria to cancer. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and in response to chemotherapy treatment. Malignant cells across different types of cancers are consistently softer than benign cells, exhibiting a Young's modulus that is ∼60-70% lower both in vitro and in situ. Cell mechanotype also grades metastatic potential: highly invasive human ovarian carcinoma cells are five-fold more deformable (softer) than less invasive cells. Further, chemotherapeutic drugs can alter mechanotype: human leukemia cells exhibit a nearly two orders of magnitude increase in cell elastic modulus after being treated with dexamethasone and daunorubicin. These published studies suggest the potential of cell mechanotype as a biomarker in cancer prognosis and for identifying effective drug treatments. However, in order to exploit cell mechanical properties for fundamental research and clinical applications a simple and scalable method to measure cell mechanotype is required. Here we describe a parallel microfiltration (PMF) assay, which enables the mechanotype of multiple samples to be simultaneously measured by driving cell suspensions through porous membranes using uniform air pressure. The relative deformability of a cell sample is quantified by the fraction of sample retained above the porous membrane. Based on experimental results and theoretical considerations, we have developed a model that provides a physical explanation of PMF and allows us to relate our experimental data to cell deformability. We employed PMF to study changes in mechanotype in ovarian carcinoma cells. We show that epithelial-to-mesenchymal transition (EMT), a process associated with cancer progression and drug resistance, yields softer cells: OVCA433 cells engineered to overexpress key EMT inducers (Snail, Slug, or Zeb) had much lower retention rates than their epithelial precursors. Similarly, cisplatin-resistant ovarian cancer cell lines (OVCAR5-cisR, SKOV3-cisR) exhibiting features of EMT, such as loss of E-cadherin expression and induction of vimentin, were more deformable than cisplatin-sensitive controls. We also compared normal p53-/- murine ovarian surface epithelial (MOSE) cells with HRasV12-transformed MOSE cells, which lose E-cadherin. PMF confirmed dramatically lower retention rates for the transformed cells relative to normal precursors. We conclude that EMT results in increased cell deformability and that PMF is a robust tool to identify changes in deformability. In summary, we have developed a novel method to assess cell mechanotype that is compatible with high-throughput screening approaches and can complement and enhance existing technology for evaluating cancer cell phenotype and response to drugs. Citation Format: Wolf-Ruprecht Wiedemeyer, Dongping Qi, Navjot Kaur Gill, Chintda Santiskulvong, Oliver Dorigo, JianYu Rao, Barbie Taylor-Harding, Amy C. Rowat. Parallel microfiltration (PMF): A novel method to screen cell mechanotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 226. doi:10.1158/1538-7445.AM2015-226

Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression.

The FOXM1 transcription factor network is frequently activated in high-grade serous ovarian cancer (HGSOC), the most common and lethal subtype of epithelial ovarian cancer (EOC). We used primary human EOC tissues, HGSOC cell lines, mouse and human ovarian surface epithelial (OSE) cells, and a murine transgenic ovarian cancer model to investigate genetic determinants of FOXM1 overexpression in EOC, and to begin to define its functional contribution to disease pathology. The Cancer Genome Atlas (TCGA) data indicated that the FOXM1 locus is amplified in ~12% of HGSOC, greater than any other tumor type examined, and that FOXM1 amplification correlates with increased expression and poor survival. In an independent set of primary EOC tissues, FOXM1 expression correlated with advanced stage and grade. Of the three known FOXM1 isoforms, FOXM1c showed highest expression in EOC. In murine OSE cells, combined knockout of Rb1 and Trp53 synergistically induced FOXM1. Consistently, human OSE cells immortalized with SV40 Large T antigen (IOSE-SV) had significantly higher FOXM1 expression than OSE immortalized with hTERT (IOSE-T). FOXM1 was overexpressed in murine ovarian tumors driven by combined Rb1/Trp53 disruption. FOXM1 induction in IOSE-SV cells was partially dependent on E2F1, and FOXM1 expression correlated with E2F1 expression in human EOC tissues. Finally, FOXM1 functionally contributed to cell cycle progression and relevant target gene expression in human OSE and HGSOC cell models. In summary, gene amplification, p53 and Rb disruption, and E2F1 activation drive FOXM1 expression in EOC, and FOXM1 promotes cell cycle progression in EOC cell models.

Mutant p53 expression in fallopian tube epithelium drives cell migration.

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

Abstract 678: The Ets factor ETV5 regulates E2F target genes and mediates drug resistance

Abstract E2F transcription factors are downstream mediators of several oncogenic drivers, including CDK/cyclin complexes, MYC, and mitogen-activated kinase (MAPK) signaling. E2F transcribe genes involved in G1-S phase transition and couple cell cycle progression with DNA repair. Prominent E2F target genes include CDK1 (CDC2), cyclin E1 (CCNE1) and BRCA1. Cyclin E1 is part of a positive feedback loop: its transcription is induced by activator E2F. Cyclin E1 protein then binds to CDK2, and the complex inactivates the RB protein, a potent inhibitor of activator E2F. The CCNE1 gene is frequently amplified in high-grade serous ovarian cancer (HGSOC, 20%) and other cancers. In the TCGA dataset on HGSOC, E2F target genes, including BRCA1, are co-expressed with CCNE1, and CCNE1-amplified HGSOC overexpress BRCA1. Moreover, a recent study suggests that CCNE1-amplified HGSOC are critically dependent on BRCA1. Loss of BRCA1 function results in synthetic lethality specifically in this subset. This finding provides an explanation for the mutually exclusive relationship between CCNE1 amplification and BRCA1 mutation. Based on the functional link between cyclin E1 and BRCA1, we devised a targeting strategy for cyclin E1-dependent ovarian cancers using pharmacological CDK2 inhibitors. Mutations in BRCA1 and BRCA2 sensitize cancer cells to platinum-based chemotherapy. We hypothesized that CDK inhibitors (CDKi) would induce loss of BRCA function and chemosensitize cyclin E1-dependent HGSOC. Indeed, we found that the CDK1/2 inhibitor, Dinaciclib, sensitized cyclin E1-transformed p53-null murine ovarian surface epithelial (MOSE) cells to cisplatin, whereas no sensitization was observed in HRASV12-driven MOSE cells. Similarly, progression of human CCNE1-amplified OVCAR3 xenografts was blocked by combined treatment with Dinaciclib and cisplatin but not by either single agent. Mechanistically, Dinaciclib treatment resulted in transcriptional downregulation of both BRCA1 and cyclin E1. Comparison of CDKi-sensitive and CDKi-resistant ovarian cancer cell lines revealed that the Ets family transcription factor, ETV5, was highly expressed in resistant lines. ETV5 is a downstream mediator of MAPK signaling and known oncogene in prostate cancer. Using inducible shRNA, we show that genetic depletion of ETV5 in RAS-driven ovarian cancer cells reduces tumorigenicity, accompanied by transcriptional downregulation of cyclin E1 and BRCA1. Thus, ETV5 regulates a subset of E2F target genes. These findings suggest that Ets family transcription factors can compensate for loss of E2F function induced by targeted agents, such as CDKi. Functional compensation on the level of receptor tyrosine kinases has been described as a mechanism of drug resistance. Our study demonstrates similar compensatory mechanisms among transcription factors that mediate oncogenic signaling. Citation Format: Barbie Taylor-Harding, Hasmik Agadjanian, Paul-Joseph Aspuria, Dong-Joo Cheon, Takako Mizuno, Danielle Greenberg, Jenieke R. Allen, Sandra Orsulic, Christine Walsh, Beth Y. Karlan, W. Ruprecht Wiedemeyer. The Ets factor ETV5 regulates E2F target genes and mediates drug resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 678. doi:10.1158/1538-7445.AM2014-678

Biomechanical profile of cancer stem-like/tumor-initiating cells derived from a progressive ovarian cancer model

We herein report, for the first time, the mechanical properties of ovarian cancer stem-like/tumor-initiating cells (CSC/TICs). The represented model is a spontaneously transformed murine ovarian surface epithelial (MOSE) cell line that mimics the progression of ovarian cancer from early/non-tumorigenic to late/highly aggressive cancer stages. Elastic modulus measurements via atomic force microscopy (AFM) illustrate that the enriched CSC/TICs population (0.32±0.12kPa) are 46%, 61%, and 72% softer (P<0.0001) than their aggressive late-stage, intermediate, and non-malignant early-stage cancer cells, respectively. Exposure to sphingosine, an anti-cancer agent, induced an increase in the elastic moduli of CSC/TICs by more than 46% (0.47±0.14kPa, P<0.0001). Altogether, our data demonstrate that the elastic modulus profile of CSC/TICs is unique and responsive to anti-cancer treatment strategies that impact the cytoskeleton architecture of cells. These findings increase the chance for obtaining distinctive cell biomechanical profiles with the intent of providing a means for effective cancer detection and treatment control. From the Clinical EditorThis novel study utilized atomic force microscopy to demonstrate that the elastic modulus profile of cancer stem cell-like tumor initiating cells is unique and responsive to anti-cancer treatment strategies that impact the cytoskeleton of these cells. These findings pave the way to the development of unique means for effective cancer detection and treatment control.

PTEN loss and HOXA10 expression are associated with ovarian endometrioid adenocarcinoma differentiation and progression

Epithelial ovarian cancer is a heterogeneous disease that is subdivided into five major histotypes but the mechanisms driving their differentiation are not clear. Mutations in adenomatous polyposis coli (APC) and β-catenin are commonly observed in the human ovarian endometrioid adenocarcinoma (OEA) patients. However, the mechanisms subsequent to APC deletion in ovarian tumorigenesis have not been well characterized. We have conditionally deleted APC in the murine ovarian surface epithelium (OSE) and showed that its loss leads to development of epithelial inclusion cysts. High-grade OEAs with tightly packed villoglandular histology were observed in older APC-deleted mice. Phosphatase and tensin homolog (PTEN) expression was elevated in the early lesions but lost after progression to the more advanced tumors. Knockdown of APC or expression of a gain-of-function β-catenin similarly induced human OSE cells to develop tumors with endometrioid histology in xenografts. Expression of HOXA10 was induced in both the advanced APC-deleted murine tumors and in the tumor xenografts of human OSE cells with knocked-down APC. These results show that reduced APC activity is sufficient to induce formation of epithelial inclusion cysts and support OEA development and suggest that induced HOXA10 expression and loss of PTEN are key mechanisms driving endometrioid histotype differentiation and progression.

Abstract 1496: Changes in white adipose tissue progenitor populations with ovarian cancer

Abstract Ovarian cancer is the fourth leading cause of cancer death in women and has the highest mortality rate of all gynecological malignancies. Among the greatest risk factors are age, reproductive history, hormone therapy, and more recently suggested, obesity. Currently, the mechanism for how obesity contributes to ovarian cancer is unknown, but there is potential for adipose tissue-associated cells to be key players in this pathogenesis. The stromal vascular fraction (SVF) of white adipose tissue is a rich source of various stem and progenitor cell populations, which can be recruited by cancer cells to enhance cancer progression and metastatic potential. Studies have shown that there is an increase in progenitor populations within the adipose tissue with obesity and age, two risk factors for ovarian cancer. While it has been shown that SVF cells can contribute to cancer, it has not been determined what impact cancer progression has on adipose tissue. It is plausible that ovarian cancer has systemic effects, which may influence cell populations within adipose tissue to promote metastases. Our lab has developed a spontaneously transformed murine ovarian surface epithelial (MOSE) cell model that becomes increasingly more aggressive with repeated passaging. Late passage cells rapidly form tumors when injected into mice, while earlier passage cells do not. Using this model, we investigated the effects of ovarian cancer on systemic changes in progenitor populations. Firefly luciferase-expressing MOSE-L TICv cells, a highly aggressive, metastatic tumor-initiating cell variant were injected into the peritoneal cavity of C57BL/6 mice and stem/progenitor populations and various immune cell populations within the perigonadal white adipose tissue, blood, ascites, and omentum were immunophenotyped by flow cytometry. Changes in gene expression profiles were also analyzed using quantitative real-time PCR. Interestingly, peritoneal dissemination changed the profile and composition of progenitor populations in the various tissues, suggesting that ovarian cancer does have a more widespread impact, affecting the blood and perigonadal adipose tissue. Additionally, co-culture studies demonstrate that MOSE cells and SVF associated cells positively interact enhancing tumor spheroid growth and provide survival signals. This suggests a synergistic relationship between the MOSE and SVF cells. These discoveries may provide a mechanistic link for the detrimental impact of obesity on ovarian cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1496. doi:1538-7445.AM2012-1496

Abstract 401: Early immune modulatory events during ovarian cancer outgrowth in the omental fat band

Abstract Ovarian cancer is the fourth most deadly cancer in women. Typically originating in the layer of epithelial cells surrounding the ovary, cancer cells exfoliate, disseminating throughout the peritoneal cavity. The omental fat band (OFB) has been described as a secondary lymphoid organ, composed of heavily vascularized fatty tissue interspersed with immune cell aggregates. The OFB is also the site of preferential tumor cell adhesion and invasion within the peritoneal cavity, and is removed at the time of surgical debulking in ovarian cancer patients as a preventative measure. Animal studies have shown that ovarian cancer cells home to the omentum as early as 20 minutes post-injection, however the early signaling events that contribute to immune evasion and neoplastic outgrowth have not been fully elucidated. Our lab has developed a spontaneously transformed murine ovarian surface epithelial (MOSE) cell model that, following long-term passaging mimics the progressive stages of ovarian cancer from non-tumorigenic MOSE-E (early) cells to MOSE-L (late) cells which readily disseminate and establish tumors throughout the peritoneum after injection. We have developed two cell variants of the MOSE-L cell line, which differ in their in vivo aggressiveness. The heterogeneous EGFP-expressing MOSE-L IVP2 cell line results in a slowly developing disease state with administration of 1 x 106 cells. In contrast, the MOSE-L firefly luciferase-expressing (FFL) TICv is a highly aggressive tumor-initiating cell variant that results in rapidly disseminating disease with as few as 100 implantable cells. Here we have utilized these cell lines to evaluate early and late changes within the OFB immune microenvironment in order to shed light on both beneficial anti-tumorigenic immune signals, as opposed to signals involved in immune evasion, tumor cell seeding/outgrowth and establishment of aggressive disease states. Specifically, MOSE-E, EGFP∼MOSE-L IVP2 and MOSE-L FFL TICv cells were injected intraperitoneally (IP) into C57BL/6 mice and at 24 h or 4 wks post-injection subsets of mice were euthanized and blood, peritoneal washings, OFB, spleens, perigonadal fat depots were collected for further analysis. Here we describe changes to the OFB and peritoneal serous exudate as a function of tumor cell dissemination using flow cytometry for immunophenotyping and quantitative real-time PCR for gene expression profiling. Preliminary results confirm that ovarian tumor initiating cells preferentially invade the omentum early during cancer metastasis, and are capable of escaping innate immune surveillance mechanisms. In contrast, MOSE-E cells readily home to the OFB early, but are cleared within 7 days. The completion of this study should provide new insights into the initial immunomodulatory events associated with early ovarian cancer seeding and outgrowth within the OFB, a tissue crucial in early ovarian cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 401. doi:1538-7445.AM2012-401

β-catenin/Tcf-signaling appears to establish the murine ovarian surface epithelium (OSE) and remains active in selected postnatal OSE cells

BackgroundWnts are a family of secreted signaling molecules involved in a number of developmental processes including the establishment of cell fate, polarity and proliferation. Recent studies also implicate wnts in epithelial adult stem cell maintenance, renewal and differentiation. Wnts transduce their signal through one of three signaling pathways. The best studied, the wnt/β-catenin pathway, leads to an increase in intracellular β-catenin which acts as a co-transcription factor with members of the Tcf/Lef family. A number of wnts are expressed in the ovary, specifically in the membrana granulosa and ovarian surface epithelium (OSE). We investigated the spatio-temporal pattern of β-catenin/Tcf expression in the OSE using responsive transgenic (TopGal) mice.ResultsThe generated β-galactosidase response (lacZ+) identified the cell population that overlies the medio-lateral surface of the indifferent gonad at embryonic day (E) 11.5. From E12.5 onwards, lacZ expression disappeared in cells covering the testis but remained with ovary development. LacZ+ OSE cells were present throughout embryonic and postnatal ovarian development but demonstrated an age-dependent decrease to a small proportion when animals were weaned and remained at this proportion with aging. Flow cytometric (FACS) and ovarian section analyses showed lacZ+ cells constitute approximately 20% of OSE in postnatal (day 1) mice which fell to 8% in 5 day-old animals while in prepubertal and adult mice this accounted for only 0.2% of OSE. Apoptosis was undetected in OSE of neonates and β-catenin/Tcf-signaling cells were proliferative in neonatal mice indicating that neither cell death nor proliferation failure was responsible for the proportion alteration. It appeared that lacZ+ cells give rise to lacZ- cells and this was confirmed in cell cultures. The DNA-binding dye DyeCycle Violet was used to set up the side population (SP) assay aimed at identifying subpopulations of OSE cells with chemoresistance phenotype associated with ABCG2 transporter activity. FACS analysis revealed lacZ+ cells exhibit cytoprotective mechanisms as indicated by enrichment within the SP.ConclusionsThe study raises the possibility that wnt/β-catenin-signaling cells constitute a progenitor cell population and could underlie the pronounced histopathology observed for human ovarian cancer.

Abstract 799: Obesity-associated modulation of the immune microenvironment of the omental fat band during peritoneal seeding of ovarian cancer

Abstract Ovarian cancer is the leading cause of death by gynecological malignancy in the Western world, the fourth most deadly cancer in women. Typically originating from the layer of epithelial cells surrounding the ovary, cancer cells exfoliate to disseminate throughout the peritoneal cavity. The omental fat band (OFB) has been described as a secondary lymphoid organ composed of heavily vascularized fatty tissue interspersed with immune cell aggregates, or “milky spots”. It is also the site of preferential tumor cell adhesion and invasion within the peritoneal cavity, and is removed at the time of surgical debulking in ovarian cancer patients as a preventative measure. Several studies have suggested that obesity is a risk factor for ovarian cancer, but it is unclear whether the obese state directly influences peritoneal dissemination of ovarian cancer. Here, we set out to elucidate the obesity-related changes in the immune microenvironment of the OFB in the homeostatic state as well as following cancer cell adhesion and outgrowth. Our lab has developed a spontaneously transformed murine ovarian surface epithelial (MOSE) cell model that mimics the progressive stages of ovarian cancer following long-term passaging. Importantly, these cells can be used to study the immune microenvironment of tumor cells in an immunocompetent host. EGFP-expressing MOSE-L (late) cells that had been selected in vivo for an aggressive phenotype were injected i.p. into 6 month-old C57Bl/6 mice and changes to the OFB immune microenvironment were evaluated after 6, 24 hrs and 4 weeks. Furthermore, mice fed a high- or low-fat diet (40 or 5% calories as fat, respectively) were used to determine the effects of diet-induced obesity on cancer cell seeding and outgrowth. Immunofluorescence microscopy, real-time PCR and flow cytometry were used to determine tumor cell attachment and growth and identify changes in OFB immune cell populations. Early seeding (24 hrs post-injection) of EGFP-MOSE-L cells at the OFB was increased in high-fat fed mice. Preliminary data from mRNA expression profiling identified genes in the OFB that were i) obesity-associated, ii) a direct response to tumor cell seeding or iii) were exacerbated in the obese microenvironment following tumor cell seeding. This suggests that obesity-associated signaling events could be polarizing the OFB towards a pro-tumorigenic microenvironment. There were also distinct differences in the immune cell populations within the OFB as function of both obesity and time post-tumor cell seeding. These results indicate that ovarian cancer cells preferentially populate the OFB early during peritoneal dissemination and are capable of escaping immune surveillance mechanisms. This study sheds light on the obesity-induced changes in immunomodulatory events associated with tumor seeding and outgrowth in the peritoneal cavity. Supported by NIH CA118846 to EMS and PCR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2011-799

Abstract 3661: The impact of tumor-infiltrating natural killer cells on ovarian cancer progression

Abstract Cancers adopt diverse strategies to safeguard their survival by evading tumor surveillance. Natural killer (NK) cells and CD8 T-lymphocytes are capable of destroying tumor cells thus playing a critical role in tumor immunity. In previous studies, we showed that NK cell derived TRAIL is essential for the cytotoxic activity of the DR5 agonistic antibody AD5-10, since depletion of NK cells abolished the antitumor activity of AD5-10 in a xenograft ovarian cancer (OC) mouse model. In the present study, we investigated whether NK cells are associated with clinical outcome as well as with the immune infiltrate showing anti-tumor immunity in OC. We analyzed NK cell infiltration in human primary ovarian tumor tissues using immunohistochemical analysis of a representative population of OC patients on a tissue microarray. NK tumor-infiltrating cells were detected within tumor tissues, whereas CD3 tumor-infiltrating T-lymphocytes were almost undetectable. NK cells in addition inversely correlated with FIGO stage (r=−0.51, p&amp;lt;0.001) and tumor grade (r=−0.3, p=0.03). These data indicate that the presence of intratumoral NK cells correlates with improved clinical outcome in OC. When we analyzed the cytotoxic capacity of human NK cells against OC cells in vitro, we found that human NK cells were capable to induce apoptosis in a concentration-and time-dependent manner in several OC cell lines. Interestingly, the addition of the DR5 agonistic antibody AD5-10 sensitized TRAIL resistant OC cell lines to NK cells mediated cytolysis. To study tumor surveillance of OC in vivo, we are currently setting up mouse models. Transformed murine ovarian surface epithelial (MOSE) cell lines derived from C57BL/6 mice have been implanted intraperitoneally into syngenic wild type, RAG2 -/-, RAG2 -/- γ -/-, NK depleted, CD8 depleted C57BL/6 recipient mice. The outcome of these studies will be presented. In summary, our findings point at a critical role for NK cell mediated tumor surveillance for OC development. Importantly, NK cell infiltration correlates with prognosis in human ovarian carcinoma. Thus, our study may open novel therapeutic opportunities by interfering with host immunity in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3661. doi:10.1158/1538-7445.AM2011-3661

Comparison of Expression Profiles in Ovarian Epithelium In Vivo and Ovarian Cancer Identifies Novel Candidate Genes Involved in Disease Pathogenesis

Molecular events leading to epithelial ovarian cancer are poorly understood but ovulatory hormones and a high number of life-time ovulations with concomitant proliferation, apoptosis, and inflammation, increases risk. We identified genes that are regulated during the estrous cycle in murine ovarian surface epithelium and analysed these profiles to identify genes dysregulated in human ovarian cancer, using publically available datasets. We identified 338 genes that are regulated in murine ovarian surface epithelium during the estrous cycle and dysregulated in ovarian cancer. Six of seven candidates selected for immunohistochemical validation were expressed in serous ovarian cancer, inclusion cysts, ovarian surface epithelium and in fallopian tube epithelium. Most were overexpressed in ovarian cancer compared with ovarian surface epithelium and/or inclusion cysts (EpCAM, EZH2, BIRC5) although BIRC5 and EZH2 were expressed as highly in fallopian tube epithelium as in ovarian cancer. We prioritised the 338 genes for those likely to be important for ovarian cancer development by in silico analyses of copy number aberration and mutation using publically available datasets and identified genes with established roles in ovarian cancer as well as novel genes for which we have evidence for involvement in ovarian cancer. Chromosome segregation emerged as an important process in which genes from our list of 338 were over-represented including two (BUB1, NCAPD2) for which there is evidence of amplification and mutation. NUAK2, upregulated in ovarian surface epithelium in proestrus and predicted to have a driver mutation in ovarian cancer, was examined in a larger cohort of serous ovarian cancer where patients with lower NUAK2 expression had shorter overall survival. In conclusion, defining genes that are activated in normal epithelium in the course of ovulation that are also dysregulated in cancer has identified a number of pathways and novel candidate genes that may contribute to the development of ovarian cancer.

Conditional Inactivation of Brca1, p53 and Rb in Mouse Ovaries Results in the Development of Leiomyosarcomas

Epithelial ovarian cancer (EOC) is thought to arise in part from the ovarian surface epithelium (OSE); however, the molecular events underlying this transformation are poorly understood. Germline mutations in the BRCA1 tumor suppressor gene result in a significantly increased risk of developing EOC and a large proportion of sporadic EOCs display some sort of BRCA1 dysfunction. To generate a model in which Brca1-mediated transformation can be studied, we previously inactivated Brca1 alone in murine OSE, which resulted in an increased accumulation of premalignant changes, but no tumor formation. In this study, we examined tumor formation in mice with conditionally expressed alleles of Brca1, p53 and Rb, alone or in combination. Intrabursal injection of adenovirus expressing Cre recombinase to inactivate p53 resulted in tumors in 100% of mice. Tumor progression was accelerated in mice with concomitant inactivation of Brca1 and p53, but not Rb and p53. Immunohistologic analyses classified the tumors as leiomyosarcomas that may be arising from the ovarian bursa. Brca1 inactivation in primary cultures of murine OSE cells led to a suppression of proliferation that could be rescued by concomitant inactivation of p53 and/or Rb. Brca1-deficient OSE cells displayed an increased sensitivity to the DNA damaging agent cisplatin, and this effect could be modulated by inactivation of p53 and/or Rb. These results indicate that Brca1 deficiency can accelerate tumor development and alter the sensitivity of OSE cells to chemotherapeutic agents. Intrabursal delivery of adenovirus intended to alter gene expression in the ovarian surface epithelium may, in some strains of mice, result in more rapid transformation of adjacent cells, resulting in leiomyosarcomas.

Mouse Model of Human Ovarian Endometrioid Adenocarcinoma Based on Somatic Defects in the Wnt/β-Catenin and PI3K/Pten Signaling Pathways

One histologic subtype of ovarian carcinoma, ovarian endometrioid adenocarcinoma (OEA), frequently harbors mutations that constitutively activate Wnt/beta-catenin-dependent signaling. We now show that defects in the PI3K/Pten and Wnt/beta-catenin signaling pathways often occur together in a subset of human OEAs, suggesting their cooperation during OEA pathogenesis. Deregulation of these two pathways in the murine ovarian surface epithelium by conditional inactivation of the Pten and Apc tumor suppressor genes results in the formation of adenocarcinomas morphologically similar to human OEAs with 100% penetrance, short latency, and rapid progression to metastatic disease in upwards of 75% of mice. The biological behavior and gene expression patterns of the murine cancers resemble those of human OEAs with defects in the Wnt/beta-catenin and PI3K/Pten pathways.

Conditional inactivation of Brca1 in the mouse ovarian surface epithelium results in an increase in preneoplastic changes

Epithelial ovarian cancer (EOC) is thought to arise from the ovarian surface epithelium (OSE); however, the molecular events underlying this transformation are poorly understood. Germline mutations in the BRCA1 tumor suppressor gene result in a significantly increased risk of developing EOC and a large proportion of sporadic EOCs display some sort of BRCA1 dysfunction. Using mice with conditional expression of Brca1, we inactivated Brca1 in the murine OSE and demonstrate that this inactivation results in the development of preneoplastic changes, such as hyperplasia, epithelial invaginations, and inclusion cysts, which arise earlier and are more numerous than in control ovaries. These changes resemble the premalignant lesions that have been reported in human prophylactic oophorectomy specimens from women with BRCA1 germline mutation. We also report that inactivation of Brca1 in primary cultures of murine OSE cells leads to a suppression of proliferation due to increased apoptosis that can be rescued by concomitant inactivation of p53. These observations, along with our finding that these cells display an increased sensitivity to the DNA-damaging agent cisplatin, indicate that loss of function of Brca1 in OSE cells impacts both cellular growth control and DNA-damage repair which results in altered cell behavior manifested as morphological changes in vivo that arise earlier and are more numerous than what can be attributed to ageing.