Normal Human Ovaries Research Articles

FRI394 Anti-inflammatory Protein Neuregulin-1 (NRG1) And ErbB3 Receptor In Human Antral Follicle And Endometrium

Abstract Disclosure: I. Chowdhury: None. S. Banerjee: None. A. Rodriguez: None. Neuregulin 1 (NRG1) is a member of the EGF-like factor family and has recognized pro-survival and anti-inflammatory roles in neurodevelopmental and cardiac function, neurological and psychiatric disorders, cancer, and development. However, the physiological role of NRG1 in the human ovary and uterus has not been well documented. Therefore, we assessed the expression pattern of NRG1 and its receptor ErbB3 in normal human ovary and uterus tissue samples by immunohistochemistry (IHC). ErbB3 is one of the bona fide receptors for NRG1. NRG1 and ErbB3 were colocalized with steroidogenic acute regulatory protein (StAR) in normal human ovarian tissue samples, whereas NRG1 and ErBB3 were colocalized with CD105 (Endoglin) and epithelial cadherin (E-cadherin) in human uterine tissue samples. Our results showed strong immunoreactive signals of NRG1, ErbB3, and StAR in antral follicles, whereas weak or lack of immunoreactive signals in preantral follicles. The strong immunoreactive signals of NRG1 and ErbB3 were observed in granulosa cells and theca interstitial cells of antral follicles. In the uterus, a differential pattern of NRG1, ErbB3, CD105, and E-cadherin expressions were noticed. Interestingly, strong immunoreactive signals of NRG1, ErbB3, CD105, and E-cadherin in the luminal epithelium, and superficial and deep glands of endometrium were observed, whereas weak immunostaining in myometrium was noticed. These findings suggest that NRG1 and ErbB3 are important intracellular autocrine-paracrine anti-inflammatory factors that may govern antral follicular development and uterine implantation. Nothing to Disclose: SB; AR; IC. Sources of Research Support: This study was supported in part by National Institutes of Health Grants 1SC1 GM130544, G12-RR03034, and P50-HD28934. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR18386 from NIH/NCRR. Presentation: Friday, June 16, 2023

Differential epithelial and stromal LGR5 expression in ovarian carcinogenesis

Lgr5 has been identified as a marker of the stem/progenitor cells in the murine ovary and oviduct by lineage tracing. However, little is known regarding LGR5 expression or its functional significance in human ovary tissues. Here, using RNA in situ hybridization and/or immunohistochemistry, we thoroughly investigated LGR5 expression in normal human ovaries, fallopian tubes and various ovarian tumors. We discovered that LGR5 expression is negligible in the human ovary surface epithelium, whereas ovarian stromal cells normally express low levels of LGR5. Remarkably, fallopian tube epithelium, inclusion cysts and serous cystadenomas with a Müllerian phenotype expressed high levels of LGR5, and LGR5 expression was restricted to PAX8+/FOXJ1− secretory cells of the tubal epithelium. Strong stromal LGR5 expression without epithelial LGR5 expression was consistently observed in the path from serous cystadenoma to serous borderline tumor to low grade serous carcinoma (LGSC). Unlike LGSC, high grade serous carcinoma (HGSC), clear cell carcinoma, endometrioid carcinomas displayed various epithelial-stromal LGR5 expression. Notably, high levels of LGR5 expression were observed in serous tubal intraepithelial carcinoma, which slightly declined in invasive HGSC. LGR5 expression was significantly associated with improved progression-free survival in HGSC patients. Moreover, in vitro assays demonstrated that LGR5 expression suppressed tumor proliferation and migratory capabilities. Taken together, these findings indicate a tumor-suppressive role for LGR5 in the progression of HGSC.

Impact of Environmentally Relevant Concentrations of Bisphenol A (BPA) on the Gene Expression Profile in an In Vitro Model of the Normal Human Ovary.

Endocrine-disrupting chemicals (EDCs), including the xenoestrogen Bisphenol A (BPA), can interfere with hormonal signalling. Despite increasing reports of adverse health effects associated with exposure to EDCs, there are limited data on the effect of BPA in normal human ovaries. In this paper, we present a detailed analysis of the transcriptomic landscape in normal Human Epithelial Ovarian Cells (HOSEpiC) treated with BPA (10 and 100 nM). Gene expression profiles were determined using high-throughput RNA sequencing, followed by functional analyses using bioinformatics tools. In total, 272 and 454 differentially expressed genes (DEGs) were identified in 10 and 100 nM BPA-treated HOSEpiCs, respectively, compared to untreated controls. Biological pathways included mRNA surveillance pathways, oocyte meiosis, cellular senescence, and transcriptional misregulation in cancer. BPA exposure has a considerable impact on 10 genes: ANAPC2, AURKA, CDK1, CCNA2, CCNB1, PLK1, BUB1, KIF22, PDE3B, and CCNB3, which are also associated with progesterone-mediated oocyte maturation pathways. Future studies should further explore the effects of BPA and its metabolites in the ovaries in health and disease, making use of validated in vitro and in vivo models to generate data that will address existing knowledge gaps in basic biology, hazard characterisation, and risk assessment associated with the use of xenoestrogens such as BPA.

Mechanomimetic 3D Scaffolds as a Humanized In Vitro Model for Ovarian Cancer.

The mechanical homeostasis of tissues can be altered in response to trauma or disease, such as cancer, resulting in altered mechanotransduction pathways that have been shown to impact tumor development, progression, and the efficacy of therapeutic approaches. Specifically, ovarian cancer progression is parallel to an increase in tissue stiffness and fibrosis. With in vivo models proving difficult to study, tying tissue mechanics to altered cellular and molecular properties necessitate advanced, tunable, in vitro 3D models able to mimic normal and tumor mechanic features. First, we characterized normal human ovary and high-grade serous (HGSC) ovarian cancer tissue stiffness to precisely mimic their mechanical features on collagen I-based sponge scaffolds, soft (NS) and stiff (MS), respectively. We utilized three ovarian cancer cell lines (OVCAR-3, Caov-3, and SKOV3) to evaluate changes in viability, morphology, proliferation, and sensitivity to doxorubicin and liposomal doxorubicin treatment in response to a mechanically different microenvironment. High substrate stiffness promoted the proliferation of Caov-3 and SKOV3 cells without changing their morphology, and upregulated mechanosensors YAP/TAZ only in SKOV3 cells. After 7 days in culture, both OVCAR3 and SKOV3 decreased the MS scaffold storage modulus (stiffness), suggesting a link between cell proliferation and the softening of the matrix. Finally, high matrix stiffness resulted in higher OVCAR-3 and SKOV3 cell cytotoxicity in response to doxorubicin. This study demonstrates the promise of biomimetic porous scaffolds for effective inclusion of mechanical parameters in 3D cancer modeling. Furthermore, this work establishes the use of porous scaffolds for studying ovarian cancer cells response to mechanical changes in the microenvironment and as a meaningful platform from which to investigate chemoresistance and drug response.

Female reproductive tract has low concentration of SARS-CoV2 receptors.

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.

Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA

BackgroundVery few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133).MethodsWe overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation.ResultsmiPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass.ConclusionmiPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries.

Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. RNA was isolated from 5 normal human ovaries (52-79years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75years of age), and 4 PCOS ovaries (18-35years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

Metformin Abrogates Age-Associated Ovarian Fibrosis.

The ovarian cancer risk factors of age and ovulation are curious because ovarian cancer incidence increases in postmenopausal women, long after ovulations have ceased. To determine how age and ovulation underlie ovarian cancer risk, we assessed the effects of these risk factors on the ovarian microenvironment. Aged C57/lcrfa mice (0-33 months old) were generated to assess the aged ovarian microenvironment. To expand our findings into human aging, we assembled a cohort of normal human ovaries (n = 18, 21-71 years old). To validate our findings, an independent cohort of normal human ovaries was assembled (n = 9, 41-82 years old). We first validated the presence of age-associated murine ovarian fibrosis. Using interdisciplinary methodologies, we provide novel evidence that ovarian fibrosis also develops in human postmenopausal ovaries across two independent cohorts (n = 27). Fibrotic ovaries have an increased CD206+:CD68+ cell ratio, CD8+ T-cell infiltration, and profibrotic DPP4+αSMA+ fibroblasts. Metformin use was associated with attenuated CD8+ T-cell infiltration and reduced CD206+:CD68+ cell ratio. These data support a novel hypothesis that unifies the primary nonhereditary ovarian cancer risk factors through the development of ovarian fibrosis and the formation of a premetastatic niche, and suggests a potential use for metformin in ovarian cancer prophylaxis.See related commentary by Madariaga et al., p. 523.

Abstract 4296: Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells

Abstract Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4296.

Polyinosinic-polycytidylic acid induces innate immune responses via Toll-like receptor 3 in human ovarian granulosa cells.

The ovary can be infected by a variety of viruses, which may come from the female reproductive tract or the peritoneum. The innate immune responses to viral infection in the human ovary are poorly understood. The present study demonstrated that human ovarian granulosa cells had innate immune activity in response to viral RNA challenge through Toll-like receptor 3 (TLR3) activation. TLR3 was constitutively expressed in the human ovary and predominantly located in granulosa cells of developmental follicles at all stages. Polyinosinic-polycytidylic acid [poly (I:C)], a synthetic viral double-stranded RNA analog, induced innate immune responses in human ovarian granulosa cells and affected endocrine function. Poly (I:C) significantly upregulated proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β and type I interferon (IFN-α/β), and the innate immune responses were significantly reduced by blocking TLR3 signaling. Furthermore, poly (I:C) induced antiviral genes expression, including 2'-5'-oligoadenylate synthetase, Mx GTPase 1, IFN-stimulating gene 15 and double-stranded RNA-activated protein kinase R. In contrast, the expression of P450 aromatase and inhibin was dramatically inhibited by poly (I:C). Both silencing of TLR3 and neutralizing TNF-α reversed the inhibitory effect of poly (I:C) on P450 aromatase and inhibin expression. Our study demonstrates that granulosa cells play a potential role in innate immune protection against viral infection in the normal human ovary, and the innate immune response perturbs cell endocrine function.

Poly (I:C) and LPS induce distinct immune responses by ovarian stromal fibroblasts

Despite its anatomical location, the ovary is a site of pathogen exposure in the human female reproductive tract (FRT). However, the role of ovarian stromal fibroblasts in immune protection is unclear. We generated a population of ovarian stromal fibroblasts derived from normal human ovaries that expressed the pattern recognition receptors TLR3, TLR4, RIG-I, & MDA5. Poly (I:C) and LPS, respective mimics of viral and bacterial infections, selectively upregulated antiviral gene expression and secretion of chemokines and antimicrobials. Poly (I:C) exclusively stimulated the expression of interferon (IFN) β, IFNλ1, and the IFN-stimulated gene OAS2. Poly (I:C) also significantly increased secretion of elafin, CCL20, and RANTES, but had no effect on SDF-1α. In contrast, LPS had no effect on IFN or ISG expression but significantly increased secretion of RANTES and SDF-1α. Secretions from poly (I:C)-treated fibroblasts had both greater anti-HIV activity and induced higher levels of CD4 + T cell chemotaxis than those from LPS-treated cells. Our studies demonstrate a potential key role for ovarian fibroblasts in innate immune protection against incoming pathogens in the normal ovary.

Abstract AP17: OVARIAN TUMOR STIMULATION CONVERTS NORMAL OVARY AND OMENTAL MESENCHYMAL STEM CELLS INTO TUMOR–PROMOTING CARCINOMA–ASSOCIATED MESENCHYMAL STEM CELLS

Abstract BACKGROUND: Ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs) are multipotent cells which influence the creation of the ovarian tumor microenvironment. CA-MSCs strongly promote ovarian cancer growth, cancer stem cell-like cells and chemotherapy resistance. CA-MSCs lack tumor associated somatic mutations, however compared to normal MSCs, CA-MSCs have a unique expression profile which mediates their pro-tumorigenic properties. The origin of CA-MSCs is unknown. We hypothesized that CA-MSCs are derived from local tissue MSCs reprogrammed by the ovarian tumor to become pro-tumorigenic CA-MSCs. METHODS: We define a “CA-MSC-like expression profile” of 6 genes significantly and reproducibly differentially expressed between normal human MSCs (from bone marrow (BM), omentum (OM) or ovary (OV)) and patient-derived CA-MSCs. Three normal MSC populations were grown with human ovarian cancer cells and analyzed to determine if tumor stimulation can induce a CA-MSC-like expression profile. Tumor stimulated MSCs were functionally analyzed to determine if acquisition of a CA-MSC-like expression profile correlates with development of pro-tumorigenic functions. Methylation array analysis of normal MSCs versus CA-MSCs was performed to explore the role of epigenetic regulation in the formation a CA-MSC. RESULTS: Tumor stimulation of normal OV and OM MSCs but not BM MSCs yield expression changes which approximate the CA-MSC-like expression profile. In vivo tumor stimulation of normal OM MSCs most effectively induces a CA-MSC-like expression profile. The development of a CA-MSC-like expression profile correlates with gain of pro-tumorigenic functions including induction of cancer cell chemotherapy resistance, enrichment of the ovarian cancer stem cell-like pool and enhancement of tumor growth. In contrast, tumor stimulated BM MSCs--which do not develop a CA-MSC-like expression profile--fail to gain pro-tumorigenic functions. Further, methylation array analysis demonstrates significant global differences in methylation patterns of CA-MSCs versus normal MSCs, indicating that epigenetic alterations may drive the development of the CA-MSC phenotype. CONCLUSIONS: Collectively we demonstrate that normal OV and OM MSCs are capable of becoming CA-MSCs through in vivo tumor stimulation with development of a distinct CA-MSC-like expression profile with corresponding pro-tumorigenic functions. These findings highlight the critical influence of the tumor on the normal host environment, manipulating normal cells to become cancer-supporting cells. The differential capacity of OV and OM MSCs but not BM MSCs to become CA-MSCs also highlights the heterogeneity of MSCs and may factor into the intraperitoneal tropism of ovarian cancer spread. Ultimately, understanding how tumor stimulation drives the development of CA-MSCs may identify powerful targets to disrupt the formation and function of the ovarian tumor microenvironment. Citation Format: Coffman, LG and Buckanovich, RJ. OVARIAN TUMOR STIMULATION CONVERTS NORMAL OVARY AND OMENTAL MESENCHYMAL STEM CELLS INTO TUMOR–PROMOTING CARCINOMA–ASSOCIATED MESENCHYMAL STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP17.

CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions

Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 105-fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.

PO-13 - Production of a functional thrombopoietin by a human ovarian cancer cell line

Hemostatic abnormalities are frequently noticed in patients with malignant diseases. These complications include platelets disorders. The role of platelets in cancer extends beyond thrombocytosis and thrombosis, and also platelets promote cancer growth and metastatic dissemination. In the physiology, platelet production is regulated by thrombopoietin, which is mainly secreted by the liver. We, previously, reported that thrombopoietin could be secreted by the ovarian adenocarcinoma cell line, OVCAR-3. Our main purpose is to analyze the gene expression of thrombopoietin in ovarian cancer cells and to assess its functionality. The thrombopoietin gene expression in ascitic cells from patients with ovarian carcinomatosis, as well as, in three cancer cell lines, including OVCAR-3 cells, performed using reverse transcription PCR, real-time PCR and gene sequencing, Normal human ovary and liver tissues are used as controls. The functionality of thrombopoietin on the basis of the viability of a thrombopoietin-dependent cell line (Ba/F3) using a co-culture method. Similarly to liver and ovary tissues, all cancer cells lines express the three TPO-1 (full length TPO), TPO-2 (12bp deletion) and TPO-3 (116pb deletion) variants. By flow cytometry, we show that thrombopoietin production by OVCAR-3 could be increased when cells are stimulated by activated protein C. Lastly, Our results confirm that activated protein C may act, in a paracrine fashion, to boost thrombopoietin production. We report, for the first time, that thrombopoietin secreted by ovarian cancer cells is functional. Hence, thrombopoietin produced by tumor cells may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

50. Efficacy of an Improved Cancer Stroma-Targeted Oncolytic Adenovirus on Human Derived Gynecologic Tumor Samples

Gynecologic cancers originated in the female reproductive organs, including the cervix, ovaries, uterus, fallopian tubes, vagina and vulva. In 2013, it was estimated that 91,730 women would be diagnosed with a gynecologic cancer. Cervical cancer accounted for approximately 13%, ovarian cancer 24% and uterine cancer 54% of cases of cancer (American Cancer Society, Inc). We have recently developed a stroma-targeted CRAd also responsive to the tumor microenvironment, AdF512v4, that contain a chimeric promoter that combines a 0.5 Kb of the SPARC promoter, a Hypoxia-Response Element (HRE) and a NFkB-response element (NFkB). The chimeric promoter drives the expression of delta-RB E1A and the CRAd was pseudotyped with a chimeric fiber 5/3. We also constructed the non-replicative version of AdF512v4 (AdF512v4-luc). We have previously demonstrated the activity of the new promoter in ovarian cancer cell lines. First, we assessed the promoter activity following transduction of cervix cancer cell lines (HeLa, CaSki and SiHa) with the non-replicative adenovirus. We observed 10-35 fold induction of luciferase activity under hypoxic condition (0.1% O2) or/and TNF treatment (5 ng/ml). Next, we measured the efficacy of the CRAd in the 3 cervical cancer cell lines under normal oxygen levels (21% O2), hypoxic condition or TNF treatment. By using the MTS assay we observed that the larger lytic capacity was obtained in the presence of TNF treatment. In order to establish the potential clinical utility of AdF512v4 we assayed its replication capacity in a most rigorous preclinical setting by using slices obtained from fresh tissue explants of human tumors. AdF512v4 replicated in three out of six human cervix cancers, one tumor from uterus and three of four human ovarian carcinomas with no lytic effect on the paired normal tissues. It is of note that Ad-wt 5/3 could replicate in both malignant and normal ovarian samples. In conclusion, AdF512v4 demonstrated a strong killing effect on cervix and ovarian cancer cells in vitro, as well as a replication capacity in fresh human ovary, uterus and cervix cancer explants. AdF512v4 appears safe since no replication was observed in normal human ovary raising its potential use in the clinics.aaa

Frequent and increased expression of human METCAM/MUC18 in cancer tissues and metastatic lesions is associated with the clinical progression of human ovarian carcinoma

ObjectivesHuman METCAM/MUC18 (huMETCAM/MUC18), a cell adhesion molecule, plays an important role in the progression of several epithelial cancers; however, its role in the progression of epithelial ovarian cancers is unknown. To initiate the study we determined expression of this protein in normal and cancerous ovarian tissues, cystadenomas, metastatic lesions, and ovarian cancer cell lines. Materials and methodsImmunoblotting and immunohistochemical (IHC) methods were used to determine huMETCAM/MUC18 expression in lysates of frozen and formalin-fixed, paraffin-embedded tissue sections of normal human ovaries, and ovarian (benign) cystadenomas, carcinomas and metastatic lesions. We also determined expression levels of several downstream effectors of METCAM/MUC18 in these tissues. ResultsHuMETCAM/MUC18 levels in ovarian carcinomas and metastatic lesions were significantly higher than in normal tissues and cystadenomas. IHC results showed that expression of huMETCAM/MUC18 in normal tissues and cystadenomas was mostly absent from epithelial cells, but in carcinomas and metastatic lesions it was localized to epithelial cells. In higher pathological grades of ovarian cancer and metastatic lesions, the percentage of cells stained in IHC was increased. Thirty percent of normal tissues weakly expressed the huMETCAM/MUC18 antigen, but 70% of cancer tissues and 100% of metastatic lesions expressed the antigen. Expression levels of several downstream effectors of huMETCAM/MUC18, Bcl2, PCNA and VEGF, were elevated in cancerous tissues, however, not that of Bax. The phospho-AKT/AKT ratio was elevated in metastatic lesions. ConclusionUpexpression of huMETCAM/MUC18 may be a marker for the malignant potential of ovarian carcinomas. Progression of ovarian cancer may involve increased signaling in anti-apoptosis, proliferation, survival/proliferation pathway, and angiogenesis.

Abstract 1534: Identification of LMX1B as a novel oncogene in human ovarian cancer

Abstract Ovarian cancers are thought to result from the accumulation of multiple genetic aberrations that transform ovarian and/or fallopian tube surface epithelial cells, allowing for their abnormal growth, proliferation and metastasis. We carried out genome-wide copy-number analysis using array comparative genomic hybridization on a panel of mouse ovarian cancer (OVCA) cell lines previously established in our laboratory. We identified a recurrent focal amplification on mouse chromosomal region 2qB, which contains the LIM homeodomain containing transcription factor 1B (Lmx1b) gene. LMX1B is not expressed in normal human ovary, but is expressed in many human OVCA cell lines and primary tumors. High expression of LMX1B correlates with poor outcome. To clarify the role of LMX1B in ovarian carcinogenesis, we transduced LMX1B into a panel of mouse and human OVCA cell lines and demonstrated that LMX1B strongly promotes migration of cancer cells in culture and accelerates xenograft growth in nude mice. Conversely, knockdown of LMX1B in a human cell line with endogenous high expression of LMX1B inhibits cell migration in vitro and tumor growth in vivo. Microarray analysis of cells overexpressing LMX1B identified NF-κB pathway as a potential mediator of tumor progression and subsequent treatment of NF-κB inhibitor decreased the migratory capacity of these cells. Thus, our data demonstrate that LMX1B functions as an oncogene in OVCA pathogenesis. Citation Format: Gayatry Mohapatra, Lei He, Lankai Guo, Vinod Vathipadiekal, Petra Sergent, Whitfield Growdon, Bo Rueda, David Engler, Sandra Orsulic, Michael Birrer. Identification of LMX1B as a novel oncogene in human ovarian cancer. [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 1534. doi:10.1158/1538-7445.AM2014-1534

YAP regulates cell proliferation, migration, and steroidogenesis in adult granulosa cell tumors.

The Hippo signaling pathway has been implicated as a conserved regulator of organ size in both Drosophila and mammals. Yes-associated protein (YAP), the central component of the Hippo signaling cascade, functions as an oncogene in several malignancies. Ovarian granulosa cell tumors (GCT) are characterized by enlargement of the ovary, excess production of estrogen, a high frequency of recurrence, and the potential for malignancy and metastasis. Whether the Hippo pathway plays a role in the pathogenesis of GCT is unknown. This study was conducted to examine the expression of YAP in human adult GCTs and to determine the role of YAP in the proliferation and steroidogenesis of GCT cells. Compared with age-matched normal human ovaries, GCT tissues exhibited higher levels of YAP expression. YAP protein was predominantly expressed in the nucleus of tumor cells, whereas the non-tumor ovarian stromal cells expressed very low levels of YAP. YAP was also expressed in cultured primary human granulosa cells and in KGN and COV434 GCT cell lines. siRNA-mediated knockdown of YAP in KGN cells resulted in a significant reduction in cell proliferation (P<0.001). Conversely, overexpression of wild type YAP or a constitutively active YAP (YAP1) mutant resulted in a significant increase in KGN cell proliferation and migration. Moreover, YAP knockdown reduced FSH-induced aromatase (CYP19A1) protein expression and estrogen production in KGN cells. These results demonstrate that YAP plays an important role in the regulation of GCT cell proliferation, migration, and steroidogenesis. Targeting the Hippo/YAP pathway may provide a novel therapeutic approach for GCT.

Expression of SDF-1 and CXCR4 transcript variants and CXCR7 in epithelial ovarian cancer.

Chemokine stromal cell-derived factor-1 (SDF-1) and its receptors, CXCR4 and CXCR7, have been implicated in epithelial ovarian cancer progression and metastasis. However, limited data are available on the expression levels of SDF-1 and CXCR4 variants and CXCR7 in human epithelial ovarian cancer. The present study aimed to characterize the expression pattern and levels of SDF-1, CXCR4 and CXCR7 in normal human ovaries and epithelial ovarian cancer. The expression of SDF-1 and CXCR4 transcript variants and CXCR7 was determined by quantitative polymerase chain reaction (qPCR). Plasma SDF-1α levels were determined by commercially available EIA kits and cancer antigen 125 (CA 125) levels were quantified by automated microparticle enzyme immunosorbent assay. High expression levels of SDF-1 transcript variant 1 were identified in ovarian cancer and control ovaries. By contrast, in both groups the expression levels of SDF-1 transcript variants 3 and 4 were extremely low. Furthermore, SDF-1 variant 1 levels were notably higher in epithelial ovarian cancer than in control ovaries, while data for the remaining transcripts were similar in both groups. CXCR4 transcript variant 2 and CXCR7 expression levels in normal and neoplastic ovaries were similar. In both groups, CXCR4 transcript variant 2 was not detected. Plasma SDF-1α levels were notably higher in females with epithelial ovarian cancer than in the control ovaries. Elevated levels of blood SDF-1α were found prior to surgery, 6 days after surgery and following completion of the first chemotherapy course. These increases were independent of the type of epithelial ovarian cancer. Our results suggest that the expression of SDF-1 and the genes controlling alternative splicing are elevated in epithelial ovarian cancer, leading to an increased formation of SDF-1 variant 1. Elevated plasma SDF-1α levels in epithelial ovarian cancer patients are not associated with the presence of tumors and/or metastases, however reflect a general response to the disease.

FOXL2, GATA4, and SMAD3 Co-Operatively Modulate Gene Expression, Cell Viability and Apoptosis in Ovarian Granulosa Cell Tumor Cells

Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.