Fallopian Tube Secretory Epithelial Cells Research Articles

Abstract A69: The PAX8 cistrome in benign and malignant Mullerian epithelia.

Abstract Background: PAX8 is a highly evolutionarily conserved transcription factor with essential roles in both Mullerian development as well as high grade serous ovarian cancer. While PAX8 has emerged as a common clinical tool to identify Mullerian primary tumors, the transcriptional network directed by PAX8 in Mullerian epithelia is unknown. In this study, we sought to understand how PAX8 interacts with chromatin in the context of both benign fallopian tube secretory epithelial cells as well as in high grade serous carcinomas. Methods: This study examined PAX8 using six cell lines: three immortalized fallopian tube secretory cell lines (FT33TAg, FT194, FT246) generated by our laboratory and three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) with high genomic fidelity to human tumors according to The Cancer Genome Atlas. First, we used chromatin immunoprecipitation combined with DNA sequencing (ChIP-Seq) to map PAX8 binding sites along the genome. Overlapping peaks among the cell lines were aligned to define a minimum consensus binding motif for PAX8. Unsupervised hierarchical clustering then segregated the peaks into groups. The nearest gene within 50000 base pairs to each peak was identified. Next, we used a series of RNAi experiments to knockdown PAX8 in all six cell lines using three independent siRNA sequences plus a pool and examined the effect on the transcriptome in each cell line using RNA-seq. Finally, we used pathway analysis software and gene ontology tools to map the signaling networks directed by PAX8. Results: Among the six cell lines, there were 11,361 unique PAX8 peaks by ChIP-seq. Approximately half of all PAX8 binding sites were located in intergenic sequences and about a 40% were in intronic sequences. The consensus DNA binding motif was almost identical between benign and malignant cell lines and was consistent with a predicted PAX homology motif. Unsupervised clustering identified 3 sets of peaks: 1) a highly conserved set of peaks among all six cell lines comprised of 558 peaks; 2) a common set of peaks common to benign cell lines but absent in cancer cell lines comprised of 3013 peaks; and 3) a very heterogeneous pattern of DNA binding unique to each cancer cell line ranging from few PAX8 peaks (JHOS4) to more than 3000 PAX8 peaks (both KURAMOCHI and OVSAHO). By gene ontology analysis, the common set of peaks for all six cell lines clustered around apoptosis and programmed cell death. Using the combination of RNAi with RNA-seq, few genes (ranging from 1 – 32 genes) were affected by loss of PAX8 in the fallopian tube cell lines or JHOS4, while more than 200 genes were altered in KURAMOCHI and OVSAHO. While in KURAMOCHI these genes were more likely to involve metabolic processes, in OVSAHO these genes were more likely to involve cell cycle regulation. Conclusions: PAX8 expression is shared among benign fallopian tube secretory cells and high grade serous carcinoma, but PAX8 function appears to be context dependent. While there is a shared set of PAX8 binding sites in benign and malignant Mullerian epithelia, PAX8 appears to be essential for the expression of very few targets in benign cells compared to cancer. In addition, PAX8 appears to have greater access to chromatin in the setting of cancer as evidenced by an increase in the number of PAX8 binding sites, while at the same time losing affinity for a set of binding sites seen only in benign fallopian tube cells. Together, these data suggest a dynamic reorganization of chromatin in the process of malignant transformation. Citation Format: Kevin M. Elias, Megan Emori, Myles Brown, Ronny Drapkin. The PAX8 cistrome in benign and malignant Mullerian epithelia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A69.

Abstract A13: Transferrin facilitates the formation of DNA-double strand breaks via transferrin receptor 1 in fallopian tube epithelial cells.

Abstract Objectives: High-grade serous ovarian cancer (HGSOC) is now believed to arise from fallopian tube epithelium (FTE), and p53 signatures are indicated to be the early oncogenic change in HGSOC. Although accumulation of DNA-double strand breaks (DNA-DSBs) are frequently observed in p53 signatures, the mechanism of DNA-DSBs formation in FTE has not been revealed yet. Hydroxyl radicals, which strongly induce DNA-DSBs, are the most active reactive oxygen species (ROS) in a living organism, and hydroxyl radicals are produced in a Fenton reaction catalyzed by free iron ions. Focusing on transferrin, which is a transporter of iron ion and exists abundantly in the follicular fluid or retrograde menstrual blood, we inspected the role of transferrin and transferrin receptor family in DNA-DSBs formation at FTE. Materials and methods: The expressions of transferrin receptor 1(TfR1) and 2 (TfR2) in human FTE were assessed by immunohistochemistry. Immortalized fallopian tube secretory epithelial cells (kindly provided by Dr. Ronny Drapkin, Dana-Farber Cancer institute) and A2780 ovarian cancer cells were cultured with holo-transferrin or vehicle, and the extent of DNA-DSBs was compared. γH2AX was adopted as a marker of DNA-DSBs. ROS were also measured to verify whether transferrin promotes a Fenton reaction. The involvement of TfR1 and TfR2 were assessed with siRNA knockdown strategy. Further, the ex vivo study were performed using murine fallopian tubes. Results: In immunohistochemistry, both TfR1 and TfR2 were ubiquitously positive in human FTE. Transferrin administration significantly increased the γH2AX expression in these cells and led ROS formation. In addition, transferrin treatment also amplified hydrogen peroxide-inducing γH2AX expression. TfR1 knockdown cancelled the uptake of transferrin, subsequent γH2AX expression and ROS formation but TfR2 knockdown didn't. Also we confirmed that transferrin treatment facilitated γH2AX formation in murine FTE ex vivo. Conclusion: We identified transferrin-TfR1 axis facilitates DNA-DSBs by promoting a Fenton reaction. It is possible that FTE exposed to the extracellular transferrin highly concentrated in the follicular fluid or retrograde menstrual blood is deeply involved with the carcinogenesis of HGSOC. Citation Format: Shogo Shigeta, Masafumi Toyoshima, Kazuyuki Kitatani, Masumi Ishibashi, Toshinori Usui, Nobuo Yaegashi. Transferrin facilitates the formation of DNA-double strand breaks via transferrin receptor 1 in fallopian tube epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A13.

Overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating chemokine expression.

We previously found that the scaffold adapter GRB2-associated binding protein 2 (GAB2) is amplified and overexpressed in a subset of primary high-grade serous ovarian cancers and cell lines. Ovarian cancer cells overexpressing GAB2 are dependent on GAB2 for activation of the phosphatidylinositol 3-kinase (PI3K) pathway and are sensitive to PI3K inhibition. In this study, we show an important role of GAB2 overexpression in promoting tumor angiogenesis by upregulating expression of multiple chemokines. Specifically, we found that suppression of GAB2 by inducible small hairpin RNA in ovarian cancer cells inhibited tumor cell proliferation, angiogenesis and peritoneal tumor growth in immunodeficient mice. Overexpression of GAB2 upregulated the secretion of several chemokines from ovarian cancer cells, including CXCL1, CXCL2 and CXCL8. The secreted chemokines not only signal through endothelial CXCR2 receptor in a paracrine manner to promote endothelial tube formation, but also act as autocrine growth factors for GAB2-induced transformation of fallopian tube secretory epithelial cells and clonogenic growth of ovarian cancer cells overexpressing GAB2. Pharmacological inhibition of inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ), but not PI3K, mechanistic target of rapamycin (mTOR) or mitogen-activated protein kinase (MEK), could effectively suppress GAB2-induced chemokine expression. Inhibition of IKKβ augmented the efficacy of PI3K/mTOR inhibition in suppressing clonogenic growth of ovarian cancer cells with GAB2 overexpression. Taken together, these findings suggest that overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating expression of CXCL1, CXCL2 and CXCL8 that is IKKβ-dependent. Co-targeting IKKβ and PI3K pathways downstream of GAB2 might be a promising therapeutic strategy for ovarian cancer that overexpresses GAB2.

Transferrin facilitates the formation of DNA double-strand breaks via transferrin receptor 1: the possible involvement of transferrin in carcinogenesis of high-grade serous ovarian cancer.

Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Role of miR-182 in response to oxidative stress in the cell fate of human fallopian tube epithelial cells.

High grade serous ovarian carcinoma (HGSC) is a DNA instable tumor and its precursor is commonly found originating from the fimbriated end of the fallopian tube secretory epithelial (FTSE) cells. The local stresses via ovulation and related inflammation are risks for HGSC. In this study, we examined the cellular and molecular responses of FTSE cells to stress. We found that excess intracellular reactive oxygen species (ROS) in normal FTSE cells upregulated a subset of microRNA expression (defined as ROSmiRs). Most ROSmiRs' expression and function were influenced and regulated by p53, and together they drove the cells into stress-induced premature senescence (SIPS). However, ROS-induced miR-182 is regulated by β-catenin, not by p53. In normal FTSE cells, miR-182 overexpression triggers cellular senescence by p53-mediated upregulation of p21. Conversely, in cells with p53 mutations, miR-182 overexpression no longer enhances p21 but functions as an "Onco-miR". p53 dysfunction is a prerequisite for miR-182-mediated tumorigenesis. In addition, we found that human follicular fluid could significantly induce intracellular ROS in normal FTSE cells. These findings suggest that ROS and p53 mutations may trigger a series of events, beginning with overexpressing miR-182 by ROS and β-catenin, impairing the DNA damage response, promoting DNA instability, bypassing senescence and eventually leading to DNA instable tumors in FTSE cells.

YAP induces high-grade serous carcinoma in fallopian tube secretory epithelial cells.

Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from Fallopian tube secretory epithelial cells (FTSECs). However, the molecular mechanisms underlying the initiation and progression of HGSC derived from FTSECs remains unclear. In the present study, we found that the Hippo/YAP signaling pathway plays a critical role in the initiation and progression of Fallopian tube and ovarian HGSC. Importantly, YAP was overexpressed in inflammatory and cancerous Fallopian tube tissues. Further, overexpression of wild-type YAP, or constitutively active YAP in immortalized FTSECs, induced cell proliferation, migration, colony formation, and tumorigenesis. Moreover, the Hippo/YAP and the fibroblast growth factor (FGF) signaling pathways formed an autocrine/paracrine positive feedback loop to drive the progression of the FTSECs-derived HGSC. Evidence in this study strongly suggests that combined therapy with inhibitors of YAP (such as verteporfin) and FGFRs (such as BGJ398) can provide a novel therapeutic strategy to treat Fallopian tube and ovarian HGSC.

Abstract POSTER-TECH-1106: Rational design of molecularly targeted therapy for GAB2-driven ovarian cancers

Abstract Studies of cancer genomes indicate that high-grade serous ovarian cancers, the most aggressive subtype accounting for over 70% of all ovarian cancer deaths, are characterized by widespread genomic regions of copy number alterations. Our previous study that systematically assessed 455 amplified genes using open reading frame-based expression approaches for their abilities to transform immortalized human cells has identified the signaling adaptor GAB2 (GRB2-associated binding protein 2) as a potent tumor-promoting gene (*Dunn,*Cheung et al, PNAS 2014). GAB2 gene is located at the chromosome 11q14.1 region that is amplified in approximately 38% of primary ovarian tumors and the amplification event has been associated with poor prognosis in patients. This study investigates the signaling mechanisms by which GAB2 amplification promotes ovarian tumor growth. This will provide us insights into the design of a rational therapeutic strategy. Experimental procedures: We established immortalized human fallopian tube secretory epithelial (FTSE) cells that stably overexpressed GAB2 or a control vector. Gene expression profiling and cytokine arrays were applied to identify signaling pathways altered by GAB2 overexpression. The biological roles of these signaling molecules were assessed in human ovarian cancer cell lines harboring GAB2 amplification/overexpression using a combination of cell-based and in vivo approaches. Summary of data: Increased TNFa signaling was observed in GAB2-overexpressing FTSE cells compared to cells expressing a control vector. This resulted in IKKb-dependent induction of multiple cytokines/chemokines, including IL8, CXCL1 and CXCL2 and others. Suppression of IL8 or CXCL1/2 individually significantly inhibited anchorage-independent growth of GAB2-overexpressing FTSE cells. Suppression of GAB2 in ovarian cancer cells also led to downregulation of IL8, CXCL1 and CXCL2 mRNAs and significantly impaired ovarian tumor growth. We are currently testing multiple IKKb small molecule inhibitors for the ability to suppress IL8 and CXCL1/2 expression. We also plan to evaluate their efficacies alone or in combination with chemotherapy in inhibiting tumor angiogenesis and tumor growth in GAB2-amplified tumors. Conclusions: Overexpression of GAB2 promotes IKKb-dependent induction of multiple cytokines that may enhance tumor angiogenesis and growth of ovarian cancers. We are currently evaluating several IKKb small molecule inhibitors as potential agents to increase the therapeutic efficacy of conventional chemotherapy for ovarian cancers. Funding support: Pilot Study Award, Marsha Rivkin Center for Ovarian Cancer Research (H.W.C.), The V Foundation for Cancer Research (H.W.C.), Liz Tiberis Early Career Award, Ovarian Cancer Research Fund (H.W.C.), American Cancer Society Institutional Citation Format: Hiu Wing Cheung, Chris Duckworth. Rational design of molecularly targeted therapy for GAB2-driven ovarian cancers [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1106.

Abstract 1991: FOXM1 expression in epithelial ovarian cancer coincides with gene amplification and functional loss of p53 and Rb and drives G2-M progression and target gene expression

Abstract Introduction: According to The Cancer Genome Atlas (TCGA), the FOXM1 transcription factor network is activated in 84% of high-grade serous ovarian cancers (HGS-OC), a common and lethal subtype of epithelial ovarian cancer (EOC). FOXM1 plays a key role in cell cycle progression and genomic stability, and is overexpressed in various malignancies. The goal of this study was to understand the genetic determinants of FOXM1 expression in EOC, and to define its functional role in relevant cell models. Methods: We analyzed FOXM1 gene amplification data in HGS-OC and other cancers using cBioPortal. We determined FOXM1 expression and its relationship to clinico-pathology in a large set of primary EOC tissues. We used primary and immortalized murine (MOSE) and human ovarian surface epithelial (hOSE) cells to determine the contribution of p53, Rb, and E2F1 to FOXM1 expression. We used FOXM1 knockdown and overexpression approaches to dissect its functional contribution to cell cycle progression and target gene activation in OSE and EOC cells. We used human EOC cell lines to assess genetic mechanisms driving the expression of the three known FOXM1 splice variants. Results: The FOXM1 locus was amplified in ∼12% of HGS-OC, greater than any other tumor type examined and suggestive of selective pressure in HGS-OC. FOXM1 mRNA was frequently upregulated in EOC, in association with advanced stage and grade. In MOSE cells, combined knockout of Rb1 and Trp53 synergistically induced Foxm1. In agreement, hOSE cells immortalized with SV40 T antigen (IOSE-SV) showed dramatically higher FOXM1 expression as compared to hOSE immortalized with hTERT. Knockdown experiments revealed that FOXM1 induction in IOSE-SV is partially dependent on E2F1. FOXM1 knockdown in IOSE-SV blocked G2-M transition and downregulated known targets of FOXM1, including CCNB1 and PLK1. Conversely, FOXM1 overexpression in primary hOSE upregulated FOXM1 target genes. FOXM1 expression in EOC cell lines was heterogeneous and the highest expression was observed in cell lines with FOXM1 gene amplification and TP53 and RB1 mutations. The level of expression of FOXM1 splice variants in EOC cell lines was consistently FOXM1c > FOXM1b > FOXM1a, indicating that transcriptionally active isoforms show higher expression. Conclusions: Our data reveal that genetic alterations in TP53 and RB1, along with FOXM1 gene amplification, are critical determinants of FOXM1 expression in EOC. FOXM1 functionally contributes to cell cycle progression in EOC models, likely via the transactivation of gene targets driving G2-M transition. Current studies are dissecting the role of post-translational modifications in FOXM1 activation in EOC, and extending work into relevant human fallopian tube secretory epithelial cell systems. Finally, our findings support the development of FOXM1-targeted therapies for EOC. Citation Format: Carter J. Barger, Wa Zhang, Kunle Odunsi, Adam R. Karpf. FOXM1 expression in epithelial ovarian cancer coincides with gene amplification and functional loss of p53 and Rb and drives G2-M progression and target gene expression. [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 1991. doi:10.1158/1538-7445.AM2015-1991

Spontaneous Transformation of Murine Oviductal Epithelial Cells: A Model System to Investigate the Onset of Fallopian-Derived Tumors.

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOELOW) was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH). The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperitoneal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers, such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOEHIGH and MOELOW were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53, and FOXM1 with reduced expression of p21. MOEHIGH were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.

PAX8 expression in ovarian surface epithelial cells.

High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells, a considerable body of evidence suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in fallopian tube secretory epithelial cells, but there are conflicting reports about PAX8 expression in OSECs. The purposes of this study were to comprehensively characterize PAX8 expression in a large series of OSECs and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 messenger RNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44% to 71% of OSECs. Calretinin and E-cadherin were frequently coexpressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P = .028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P = .003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs, and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs, PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs.

Cyclin E1 Deregulation Occurs Early in Secretory Cell Transformation to Promote Formation of Fallopian Tube–Derived High-Grade Serous Ovarian Cancers

The fallopian tube is now generally considered the dominant site of origin for high-grade serous ovarian carcinoma. However, the molecular pathogenesis of fallopian tube-derived serous carcinomas is poorly understood and there are few experimental studies examining the transformation of human fallopian tube cells. Prompted by recent genomic analyses that identified cyclin E1 (CCNE1) gene amplification as a candidate oncogenic driver in high-grade serous ovarian carcinoma, we evaluated the functional role of cyclin E1 in serous carcinogenesis. Cyclin E1 was expressed in early- and late-stage human tumor samples. In primary human fallopian tube secretory epithelial cells, cyclin E1 expression imparted malignant characteristics to untransformed cells if p53 was compromised, promoting an accumulation of DNA damage and altered transcription of DNA damage response genes related to DNA replication stress. Together, our findings corroborate the hypothesis that cyclin E1 dysregulation acts to drive malignant transformation in fallopian tube secretory cells that are the site of origin of high-grade serous ovarian carcinomas.

Mouse Models Identify a Potential Ovarian Cancer Cell of Origin

Abstract High-grade serous ovarian carcinoma can originate from fallopian tube secretory epithelial cells.

A better mimic

Mice in which p53, PTEN and BRCA1 (or BRCA2) are inactivated specifically in fallopian tube secretory epithelial cells develop tumours that are highly similar to human high-grade serous ovarian carcinomas.

Abstract PR02: Cyclin E1 deregulation occurs early during fallopian tube tumorigenesis and promotes secretory cell transformation

Abstract The fallopian tube has been implicated as the dominant site of origin for high-grade serous ovarian carcinoma. However, the molecular pathogenesis of fallopian tube-derived serous cancer is poorly understood and there are few experimental studies examining the transformation of human fallopian tube cells. Recent genomic analyses have identified CCNE1 (Cyclin E1) gene amplification as a potential oncogenic driver of high-grade serous ovarian carcinoma development. Here, we examine the oncogenic role of Cyclin E1 in serous carcinogenesis, first by characterizing its expression in both late-stage and early-stage human tumor samples, and secondly, by generating an experimental model of Cyclin E1-mediated transformation using primary human fallopian tube secretory epithelial cells. Using fluorescent in situ hybridization and immunohistochemical analyses, we show that Cyclin E1 is strongly expressed in a subset of late-stage high-grade serous carcinomas harboring CCNE1 amplification and that aberrant Cyclin E1 expression can occur very early during serous tumorigenesis, evident in non-invasive tubal carcinoma and putative precursor lesions. To examine the effects of constitutive Cyclin E1 expression in untransformed cells, we immortalized human fallopian tube secretory epithelial cells with TERT (telomerase reverse transcriptase) and mutant TP53R175H (representing early genetic alterations in serous tumorigenesis), followed by overexpression of CCNE1 or a vector control. Using a series of in vitro assays, we show that inappropriate Cyclin E1 expression imparts malignant characteristics to immortal fallopian tube secretory epithelial cells, including accelerated proliferation, loss of contact inhibition, and clonal growth ability. In addition, we use a combination of DNA damage assays and PCR array profiling to demonstrate that Cyclin E1 induces DNA double-strand and single-strand breaks, accompanied by up-regulation of DNA damage response genes involved in managing DNA replication stress. In sum, our results suggest that constitutive Cyclin E1 expression drives over-proliferation of untransformed but p53-compromised fallopian tube secretory epithelial cells, accompanied by an accumulation of DNA damage and altered expression of DNA damage response genes. Our data supports the hypothesis that Cyclin E1 promotes fallopian tube transformation by inducing replication stress-associated DNA damage, thus leading to chromosomal instability, and that CCNE1 amplification is a major driver of Cyclin E1 overexpression in high-grade serous carcinoma. This abstract is also presented as Poster A1. Citation Format: Alison M. Karst, Paul M. Jones, Natalie Vena, Azra H. Ligon, Joyce F. Liu, Michelle S. Hirsch, Dariush Etemadmoghadam, David D. Bowtell, Ronny Drapkin. Cyclin E1 deregulation occurs early during fallopian tube tumorigenesis and promotes secretory cell transformation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR02.

FOXO3a loss is a frequent early event in high-grade pelvic serous carcinogenesis

Serous ovarian carcinoma is the most lethal gynecological malignancy in Western countries. The molecular events that underlie the development of the disease have been elusive for many years. The recent identification of the fallopian tube secretory epithelial cells (FTSECs) as the cell-of-origin for most cases of this disease has led to studies aimed at elucidating new candidate therapeutic pathways through profiling of normal FTSECs and serous carcinomas. Here, we describe the results of transcriptional profiles that identify the loss of the tumor suppressive transcription factor FOXO3a in a vast majority of high grade serous ovarian carcinomas (HGSOCs). We show that FOXO3a loss is a hallmark of the earliest stages of serous carcinogenesis and occurs both at the DNA, RNA and protein levels. We describe several mechanisms responsible for FOXO3a inactivity, including chromosomal deletion (chromosome 6q21), upregulation of miRNA-182 and destabilization by activated PI3K and MEK. The identification of pathways involved in the pathogenesis of ovarian cancer can advance the management of this disease from being dependant on surgery and cytotoxic chemotherapy alone to the era of targeted therapy. Our data strongly suggest FOXO3a as a possible target for clinical intervention.

In vitro three-dimensional modeling of fallopian tube secretory epithelial cells

BackgroundFallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane.ResultsWe have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples.ConclusionsThis 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.

Primary culture and immortalization of human fallopian tube secretory epithelial cells

Primary human fallopian tube secretory epithelial cell (FTSEC) cultures are useful for studying normal fallopian tube epithelial biology, as well as for developing models of fallopian tube disease, such as cancer. Because of the limited ability of primary human FTSECs to proliferate in vitro, it is necessary to immortalize them in order to establish a cell line that is suitable for long-term culture and large-scale in vitro experimentation. This protocol describes the isolation of FTSECs from human fallopian tube tissue, conditions for primary FTSEC culture and techniques for establishing immortal FTSEC lines. The entire process, from primary cell isolation to establishment of an immortal cell line, may take up to 2 months. Once established, immortal FTSECs can typically be maintained for at least 30 passages.

<em>Ex Vivo</em> Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

<em>Ex Vivo</em> Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Modeling high-grade serous ovarian carcinogenesis from the fallopian tube

High-grade serous ovarian carcinoma (HGSOC) is a lethal disease for which improved screening and treatment strategies are urgently needed. Progress in these areas is impeded by our poor understanding of HGSOC pathogenesis. Most ovarian cancer research is based on the hypothesis that HGSOC arises from ovarian surface epithelial cells. However, recent studies suggest that >50% of high-grade serous carcinomas involving the ovary likely arise from fallopian tube epithelium. Therefore, limiting HGSOC research to modeling based on ovarian surface epithelium alone is inadequate. To address the need for a fallopian tube-based model of HGSOC, we have developed a system for studying human fallopian tube secretory epithelial cell (FTSEC) transformation. Our model is based on (i) immortalization of FTSECs isolated from primary samples of normal, nondiseased human fallopian tubes, (ii) transformation of FTSECs with defined genetic elements, and (iii) xenograft-based tumorigenic assays. We use our model to show that FTSECs immortalized with human telomerase reverse transcriptase (hTERT) plus SV40 large T and small T antigens are transformed by either oncogenic Ras (H-Ras(V12)) or c-Myc expression, leading to increased proliferation, clonogenicity, and anchorage-independent growth. Additionally, we demonstrate that FTSECs remain susceptible to c-Myc-mediated transformation in the absence of viral oncoproteins, by replacing SV40 large T and small T antigens with sh-p53, mutant CDK4 (CDK4(R24C)), and sh-PP2A-B56γ. Importantly, all transformed FTSECs gave rise to high-grade Müllerian carcinomas that were grossly, histologically, immunophenotypically, and genomically similar to human HGSOC. With this model, we will now be able to assess the transformative effects of specific genetic alterations on FTSECs in order to characterize their respective roles in HGSOC development.