Human Ovarian Surface Epithelium Research Articles

Abstract A054: Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC)

Abstract Low-grade chronic inflammation and persistent oxidative stress are hallmarks of malignant transformation. High-grade serous ovarian carcinoma (HGSOC) is a fatal malignancy of women, and the median age of its incidence is 60-65 years. Our earlier studies showed enhanced expression of interleukin 16 (IL-16, an inflammatory cytokine), its receptor CD9, and glucose-regulated protein (GRP78, a marker of cellular stress and inhibitor of DNA damage repair mechanism) in postmenopausal ovaries and patients with HGSOC as compared with premenopausal subjects. It is possible that these age-associated increases in IL-16, CD9, and GRP78 expression may contribute to HGSOC-related epigenetic modifications of gH2A histone family member X (gH2AX, a marker for double-stranded DNA breaks), and histone deacetylase 1 (HDAC1), involved in DNA repair. The goal of this study was to examine if age-associated such changes lead to HGSOC-associated malignant transformation and if so, to determine mechanisms involved in such transformation. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies, and immunoassays based on the review of their hematoxylin and eosin staining and final pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old), and ovarian high-grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without recombinant human IL-16 (rhIL-16) and recombinant human GRP78 (rhGRP78), and small interfering RNA-GRP78 (siGRP78) for 24 hours or 48 hours. Nuclear fractionations from treated- or untreated HOSE cells or OVCAR3 (HGSOC cell line) cells were extracted. Results: Compared to premenopausal women, a subset of postmenopausal women exhibited significantly elevated expression (P<0.01) of inflammatory markers IL-16, and its receptor CD9, as well as a marker of oxidative stress (GRP78). Additionally, markers of epigenetic changes, including gH2AX and HDAC1, presented similar patterns of increase in their expression. These observations were also consistent in HGSOC patients. Enhanced expression of GRP78, gH2AX, HDAC1, and CD9 in rhIL-16 treated HOSE cells like OVCAR3 cells and their reduction in siGRP78 treated HOSE cells suggests that age-associated increase in IL-16 in postmenopausal women may be a risk factor for malignant transformation and GRP78 may be a mechanism for IL-16-induced HGSOC development. Thus, IL-16 and GRP78 represent novel targets that may be used to develop novel targeted therapies to prevent HGSOC. Expression of gH2AX and HDAC1 increased in rhGRP78-treated HOSE cells. Conclusion: The results of this study suggest that age-associated increases in IL-16 (a marker of chronic inflammation) and GRP78 (a marker of oxidative stress) may contribute to the predisposition for HGSOC-associated malignant transformation. IL-16 and GRP78 offer potential targets for the prevention of HGSOC in women. Support: The Portes Foundation Citation Format: Jessica Ramirez, Animesh Barua. Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A054.

IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

Abstract A015: Age-associated molecular changes may predispose the ovary to malignant transformation leading to ovarian cancer (OVCA)

Abstract Background: Ovarian cancer (OVCA) is a fatal malignancy of women which in most cases is a disease of postmenopausal women. Thus, age-associated changes in women may predispose them to malignancy. Persistent high levels of follicle stimulating hormone (FSH) is a feature of aging and we have shown that aging was associated with increased expression of inflammatory cytokine interleukin 16 (IL-16) and glucose regulatory protein (GRP78), a marker of cellular stress. It is possible that age-associated increased expression of IL-16 and GRP78 might be involved in production of mutagenic 8-OXO-2dG or its cognate enzyme OGG1 and malondialdehyde (MDA) as well as markers of DNA repair mechanisms. The goal of this study was to determine the mechanism of age-associated malignant transformation in the ovary. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies and immunoassays based on the review of their hematoxylin and eosin staining and final Pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old) and ovarian high grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without FSH for 24 hours, and their cytoplasmic and nuclear fractionations together with that of OVCAR3 (HGSC cell line) cells were extracted. Expression of markers of inflammation (IL-16), oxidative stress (SOD2, GRP78), DNA adducts (8-OXO-2dG/OGG1, MDA) and epigenetics (HDAC1, gH2AX) and were examined. Significant differences in expression of different markers among different age groups and ovarian HGSC, and treated or control OVCAR3 cells were determined by ANOVA and t-tests. Results: Compared with premenopausal women, expression of markers of inflammation and oxidative stress including IL-16, GRP78, SOD2, 8-OXO-2dG/OGG1, MDA as well as markers of epigenetic changes including HDAC1 and gH2AX was significantly higher (<0.0001) in a subset of postmenopausal women. Similar patterns of expression were also observed in patients with ovarian HGSC. Nuclear expression of mutagenic DNA adducts (OGG1, MDA) and inhibitor of DNA damage repair mechanism (GRP78) enhanced during aging. Thus, age-associated inflammation and oxidative stress are predispositions to malignant transformation. This assumption is based on the observation from the treatment of HOSE cells with FSH which showed increased expression of the above markers as also observed in OVCAR3 cells. Conclusion: Expression of IL-16, GRP78, SOD2, 8-OXO-2dG/OGG1, MDA and HDAC1 increased in ovary during aging. These results suggest that aging is associated with persistent inflammation and oxidative stress in ovarian tissues, which predisposes these tissues to malignant transformation leading to OVCA development. Support: NIH/NCI: CA210370 Citation Format: Jessica Ramirez, Elizabeth Paris, Sanjib Basu, Animesh Barua. Age-associated molecular changes may predispose the ovary to malignant transformation leading to ovarian cancer (OVCA) [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A015.

Microtentacle Formation in Ovarian Carcinoma.

Simple SummaryOvarian cancer commonly spreads throughout the peritoneal cavity by exfoliation of malignant cells into ascites. Chemoresistance remains an important therapeutic obstacle. Microtentacles (McTNs) are microtubule-based protrusions that may influence the metastatic potential and chemoresistance profile of free-floating cells. In this study, we define the various microtentacle morphologies that can be observed in detached ovarian cancer cells, and their clustering behavior in relationship to histology, α-tubulin post-translational modifications, β-tubulin isotype, modulators of cortical stiffness, and sensitivity to clinically relevant microtubule-targeting agents. McTNs represent a new therapeutic target for this disease, and an understanding of their biology could have implications for the refinement of therapies, including intraperitoneal drug delivery. Background: The development of chemoresistance to paclitaxel and carboplatin represents a major therapeutic challenge in ovarian cancer, a disease frequently characterized by malignant ascites and extrapelvic metastasis. Microtentacles (McTNs) are tubulin-based projections observed in detached breast cancer cells. In this study, we investigated whether ovarian cancers exhibit McTNs and characterized McTN biology. Methods: We used an established lipid-tethering mechanism to suspend and image individual cancer cells. We queried a panel of immortalized serous (OSC) and clear cell (OCCC) cell lines as well as freshly procured ascites and human ovarian surface epithelium (HOSE). We assessed by Western blot β-tubulin isotype, α-tubulin post-translational modifications and actin regulatory proteins in attached/detached states. We studied clustering in suspended conditions. Effects of treatment with microtubule depolymerizing and stabilizing drugs were described. Results: Among cell lines, up to 30% of cells expressed McTNs. Four McTN morphologies (absent, symmetric-short, symmetric-long, tufted) were observed in immortalized cultures as well as ascites. McTN number/length varied with histology according to metastatic potential. Most OCCC overexpressed class III ß-tubulin. OCCC/OSC cell lines exhibited a trend towards more microtubule-stabilizing post-translational modifications of α-tubulin relative to HOSE. Microtubule depolymerizing drugs decreased the number/length of McTNs, confirming that McTNs are composed of tubulin. Cells that failed to form McTNs demonstrated differential expression of α-tubulin- and actin-regulating proteins relative to cells that form McTNs. Cluster formation is more susceptible to microtubule targeting agents in cells that form McTNs, suggesting a role for McTNs in aggregation. Conclusions: McTNs likely participate in key aspects of ovarian cancer metastasis. McTNs represent a new therapeutic target for this disease that could refine therapies, including intraperitoneal drug delivery.

Clerodane Diterpenoids from an Edible Plant Justicia insularis: Discovery, Cytotoxicity, and Apoptosis Induction in Human Ovarian Cancer Cells

Objectives: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. Methods: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/β)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. Conclusions: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.

Metformin Reduces NGF-Induced Tumour Promoter Effects in Epithelial Ovarian Cancer Cells.

Epithelial ovarian cancer (EOC) is a lethal gynaecological neoplasm characterized by rapid growth and angiogenesis. Nerve growth factor (NGF) and its high affinity receptor tropomyosin receptor kinase A (TRKA) contribute to EOC progression by increasing the expression of c-MYC, survivin and vascular endothelial growth factor (VEGF) along with a decrease in microRNAs (miR) 23b and 145. We previously reported that metformin prevents NGF-induced proliferation and angiogenic potential of EOC cells. In this study, we sought to obtain a better understanding of the mechanism(s) by which metformin blocks these NGF-induced effects in EOC cells. Human ovarian surface epithelial (HOSE) and EOC (A2780/SKOV3) cells were stimulated with NGF and/or metformin to assess the expression of c-MYC, β-catenin, survivin and VEGF and the abundance of the tumor suppressor miRs 23b and 145. Metformin decreased the NGF-induced transcriptional activity of MYC and β-catenin/T-cell factor/lymphoid enhancer-binding factor (TCF-Lef), as well as the expression of c-MYC, survivin and VEGF in EOC cells, while it increased miR-23b and miR-145 levels. The preliminary analysis of ovarian biopsies from women users or non-users of metformin was consistent with these in vitro results. Our observations shed light on the mechanisms by which metformin may suppress tumour growth in EOC and suggest that metformin should be considered as a possible complementary therapy in EOC treatment.

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

Abstract 3571: Oxidative stress and the ovarian surface epithelium: TGF-β1 stimulation of IOSE cell lines reveals novel upregulation of NADPH oxidase 4, a membrane bound hydrogen peroxide producing enzyme

Abstract Normal ovarian surface epithelium (OSE) has been widely studied as the origin of ovarian cancer. Repetitive ovulation has been implicated in cancer initiation based on damage and inflammation incurred to the OSE upon continuous cycles of tear and repair. Transforming growth factor-beta (TGF-β) plays a role in this normal ovulatory cycle, having been shown to inhibit human OSE proliferation and induce apoptosis, which may prevent the over-proliferation of cells. NADPH oxidase 4 (NOX4), a membrane-associated hydrogen peroxide-producing enzyme, has previously been shown to be sensitive to TGF-β1, with increased expression stimulated in smooth muscle cells and fibroblasts. Here we describe for the first time TGF-β1 induced upregulation of NOX4 in immortalized OSE (IOSE) cells (IOSE121, IOSE144, IOSE364, IOSE386 and IOSE397). This time-dependent increased expression (&amp;gt;5 fold at 2 ng/ml TGF-β1, 24 h) was simulated by direct overexpression of NOX4 in IOSE397 cells and has demonstrated that increased NOX4, in the absence of further growth factor related effects, affords induction of p18 and p21. In addition, using immunohistochemical evaluation of human ovarian cancers in tissue microarrays (n=17 patients), we observed significantly increased expression of NOX4, suggesting that NOX4 upregulation may contribute to the malignant transformation of ovarian cells. Citation Format: Jennifer L. Meitzler, Becky A. Diebold, Smitha Antony, Yongzhong Wu, Agnes Juhasz, Iris Dahan, Guojian Jiang, Jiamo Lu, Stephen Wang, Mariam M. Konaté, Krishnendu Roy, James H. Doroshow. Oxidative stress and the ovarian surface epithelium: TGF-β1 stimulation of IOSE cell lines reveals novel upregulation of NADPH oxidase 4, a membrane bound hydrogen peroxide producing enzyme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3571.

Transcription-Related Dynamics from Immune Disability into Endogenous Innovation.

So far, thymus involution in adults is believed to be irreversible, and endogenous innovation for thymus‐related immunodeficiency remains to be an intractable puzzle. With the expectation of addressing this dilemma, human ovarian surface epithelium (OSE) has been reengineered as epithelial‐mesenchymal transition (EMT)‐tridimensional‐spheroid biologics (ETSB) using a dynamic EMT‐3D‐floating system along with 160 Gy X‐ray‐amelioration, which inoculates subcutaneously into aging rhesus and athymic Balb/cnu/nu mice. Herein, it is bioinformatically validated that ETSB can reset Clock/Arntl‐Per3/Tim molecule rhythm dynamics to re‐prime thymus residual (parathyroid or fatty‐like invalid vesicles yet no thymic architecture) to evolutionary transcription with overall cortex‐medulla endogenized by TECs undergoing MET/EMT reversion. Rhythm dynamics immediately resettles the bHLH‐LTβR‐NFκB‐RelA/B loop as a cascade to provoke the core immune microenvironment for multifunctional innovation of dynamic TCR orchestration, with harmonious naïve T‐subsets and TRECs renewals (P < 0.005). Subsequently, peripheral biological burden and tumor metastasis dynamics are addressed by innovative TCR‐defense/attack dynamics quickly (P < 0.005 vs Control), yet without autoimmune indication to hosts. Moreover, a functional blockade of core‐rhythm dynamics deeply impedes the endogenous innovation of invalid thymus residual. Thus this study may help pioneer a prospective strategy to innovate panoramic central‐peripheral immune microenvironments and defense dynamics for immune‐deficient/aging victims.

COX2 is induced in the ovarian epithelium during ovulatory wound repair and promotes cell survival†.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation, the wound is repaired, however, this process is poorly understood. In epithelial tissues, wound repair is mediated by an epithelial-to-mesenchymal transition (EMT). Transforming Growth Factor Beta-1 (TGFβ1) is a cytokine commonly known to induce an EMT and is present throughout the ovarian microenvironment. We, therefore, hypothesized that TGFβ1 induces an EMT in OSE cells and activates signaling pathways important for wound repair. Treating primary cultures of mouse OSE cells with TGFβ1 induced an EMT mediated by TGFβRI signaling. The transcription factor Snail was the only EMT-associated transcription factor increased by TGFβ1 and, when overexpressed, was shown to increase OSE cell migration. A polymerase chain reaction array of TGFβ signaling targets determined Cyclooxygenase-2 (Cox2) to be most highly induced by TGFβ1. Constitutive Cox2 expression modestly increased migration and robustly enhanced cell survival, under stress conditions similar to those observed during wound repair. The increase in Snail and Cox2 expression with TGFβ1 was reproduced in human OSE cultures, suggesting these responses are conserved between mouse and human. Finally, the induction of Cox2 expression in OSE cells during ovulatory wound repair was shown in vivo, suggesting TGFβ1 increases Cox2 to promote wound repair by enhancing cell survival. These data support that TGFβ1 promotes ovulatory wound repair by induction of an EMT and activation of a COX2-mediated pro-survival pathway. Understanding ovulatory wound repair may give insight into why ovulation is the primary non-hereditary risk factor for ovarian cancer.

Abstract 1774: Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC)

Abstract Resistance to platinum-based chemotherapy is a major problem in the clinical management of recurrent HGSOC. While the use of poly ADP-ribose polymerase inhibitors (PARPi) have made an impact in BRCA1/2 mutated and homologous recombinant-deficient HGSOC, resistance and limited response-rates to PARPi persist. Alisertib (AL) an inhibitor of AAK can inhibit entry and progression of cells through mitosis as well as the interaction with N-myc and its degradation (Front. Oncol. doi:10.3389/fonc.2015.00189). In models of HGSOC from patients with recurrent platinum-resistant disease, cisplatin (CP) treatment does not lead to cell cycle traverse perturbations and significant accumulation of cells in G2-M. Since a defect in the G2-M DNA damage checkpoint is a potential mechanism of resistance to cisplatin, we sought to test the hypothesis that inhibition of AAK that is involved in centrosome maturation and entry into mitosis would enhance the anti-tumor activity of cisplatin in platinum-resistant HGSOC. To test this hypothesis, we treated HGSOC cell lines from patients with recurrent platinum-refractory disease with increasing doses of CP (1- 2.5 μM), of AL (0.05 μM) or the combination and evaluated effects on cell proliferation, apoptosis and generation of reactive oxygen species (ROS). The sequential treatment of four different HGSOC cell lines with CP for 3 hours followed by AL for 48 hours and recovery in drug-free medium for 96-120 hours led to a synergistic increase in apoptosis (p&amp;lt;0.01) and a synergistic decrease in cell survival (p&amp;lt;0.001) compared to treatment with either drug alone. The enhanced apoptosis and decreased cell survival with (CP→AL) treatment was accompanied by a significant increase (p&amp;lt;0.05) in ROS. In contrast, sequential treatment with AL followed by CP (AL→CP) led to an antagonistic response. The synergistic effect of (CP→AL) was accompanied by a modest increase in mRNA and protein levels of AAK. However, protein levels of N-myc protein, which interacts with AAK and is degraded, were not altered. Interestingly, (CP→AL) treatment did not elicit the synergistic cytotoxic response or increased ROS in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. In platinum-refractory HGSOC cells that are wild-type for BRCA1/2 and resistant to olaparib, treatment with the combination of 0.01 μM AL and 1 μM olaparib for 48 hours resulted in an additive cytotoxic response. Based on the mutation landscape of the HGSOC cell panel evaluated, the synergistic cytotoxicity with the CP→AL treatment was independent of BRCA1/2 or TP53 mutation status. In summary, CP resistance in HGSOC due to a defective G2-M DNA damage response, can be potentially circumvented by sequential treatment with CP followed by AL. Citation Format: Ram N. Ganapathi, Eric J. Norris, Ashley P. Sutker, Hala Soliman, Mahrukh K. Ganapathi. Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC) [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 1774.

Overexpression and cytoplasmic localization of caspase-6 is associated with lamin A degradation in set of ovarian cancers

BackgroundIn most women with ovarian cancer, the diagnosis occurs after dissemination of tumor cells beyond ovaries. Several molecular perturbations occur ahead of tumor initiation including loss of lamin A/C. Our hypothesis was that the loss of nuclear structural proteins A type lamins (lamin A/C) transcribed from LMNA gene and substrate for active caspase-6 maybe one of the molecular perturbations. Our objective is to investigate the association between the loss of lamin A/C and the overexpression of caspase-6 in ovarian cancer cells.MethodWestern blotting and immunofluorescence were used to analyze the expression of lamin A/C and active caspase-6 in normal human ovarian surface epithelial (HOSE) cells, immortalized human ovarian surface epithelial cells and a set of seven ovarian cancer cell lines (including OVCAR3, OVCAR5, and A2780). The activity of caspase-6 was measured by densitometry, fluorescence and flow cytometry. Immunohistochemistry was used to evaluate the expression of caspase-6 in set of ovarian cancer tissues previously reported to have lost lamin A/C.ResultsThe results showed that HOSE cells expressed lamin A/C and no or low level of active caspase-6 while cancer cells highly expressed caspase-6 and no or low level of lamin A/C. The inhibition of caspase-6 activity in OVCAR3 cells increased lamin A but has no effect on lamin C; active caspase-6 was localized in the cytoplasm associated with the loss of lamin A.ConclusionOverexpression and cytoplasmic localization of caspase-6 in ovarian cancer cells may be involved in lamin A degradation and deficiency observed in some ovarian cancer cells.

Abstract A22: Functional analysis of PGE2 pathway members EP4 and MRP4 in ovarian cancer

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. Most cases of ovarian cancer present in late stages, leading to recurrent disease that is incurable and often fatal due to innate or acquired chemoresistance; therefore, new therapies are desperately needed. Cyclo-oygenases, COX-1 and COX-2, are enzymes that catalyze the production of the prostaglandin E2 (PGE2), a lipid mediator that is functionally linked to progression of many cancers including breast and ovarian cancer. PGE2 is exported from the cell via multidrug resistance-associated protein 4 (MRP4) where it acts in a paracrine and autocrine manner by activating a family of four G-protein coupled receptors (EP1-4) that are linked to different intracellular signaling pathways. EP2 and EP4 can activate PKA/cAMP, PI3K, and ERK pathways. COX-1 and COX-2 have been shown to be overexpressed in primary ovarian cancer as well as in many ovarian cancer cell lines. We hypothesized that the EP4 receptor is overexpressed in ovarian cancer; that binding of its cognate ligand, PGE2, will drive ovarian cancer progression; and that inhibition of the EP4-mediated signaling will lead to inhibition of ovarian cancer growth and metastasis. In order to test this hypothesis, we analyzed the expression of the EP4 receptor in a human ovarian cancer tissue microarray (TMA) as well as human ovarian cancer cell lines. Immunohistochemical analysis of EP4 on the TMA composed of varying histologies, including serous, endometrioid, and clear cell, as well as normal ovarian tissue revealed that EP4 was expressed in 38.7% of ovarian cancer patients, whereas EP4 was not expressed in the 10 normal ovarian tissue samples. Additionally, in comparison to immortalized human ovarian surface epithelial (HOSE) cells, EP4 is overexpressed in many of the cell lines analyzed, including OVCAR-3, COAV-3, SKOV3, and Kuramochi cells. Blockade of the EP4 receptor via antagonist or siRNA results in a decrease in proliferation, migration, and invasion in ovarian cancer cell lines. In addition to overexpression of EP4, MRP4 was also found to be overexpressed in ovarian cancer cell lines when compared to HOSE cells. Given the role of MRP4 in exporting PGE2 and affecting drug resistance/ sensitivity in breast cancer cell lines, we hypothesize that MRP4 will similarly play a role in modulating the amount of PGE2 in the microenvironment in ovarian cancer, thereby affecting signaling via of EP4 receptor. Functional testing of dual EP4/ MRP4 inhibition may provide much-needed new therapies for the treatment of ovarian cancer. Citation Format: Mc Millan Ching, Cong Fan, Dana Roque, Gautam Rao, Paul Staats, Amy Fulton, Jocelyn Reader. Functional analysis of PGE2 pathway members EP4 and MRP4 in 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 A22.

Abstract 1490: Molecular function of the read-through transcript PRR5-ARHGAP8

Abstract Background: Ovarian carcinoma is one of the most fatal malignancies in females. From the analysis of RNA-seq we have previously conducted to study the transcriptome of ovarian carcinoma upon knockdown of transcription factor hepatic nuclear factor 1β (HNF-1β), PRR5-ARHGAP8 was identified as the only read-through transcript not targeted for the nonsense-mediated decay pathway. Read-through transcription refers to the splicing of the intergenic region from two adjacent genes resulting in a chimeric transcript comprising of one or more exons from both genes. Although several of such transcripts are found in the transcriptome, their significance remains an enigma. Our aim was to ascertain the differential expression of the read-through transcript PRR5-ARHGAP8 in ovarian cancers and to determine its molecular functions. Materials &amp; Methods: The deregulation of PRR5-ARHGAP8 was validated by RT-qPCR in several ovarian carcinoma cell lines. The expression of this fusion transcript and the individual genes were studied in immortalised human ovarian surface epithelial (HOSE) cell lines, ovarian carcinoma cell lines, and matched tumour and non-tumour pairs from frozen patient samples. Overexpression of this transcript was achieved by transient transfection in HEK293T cells to study its effect on proliferation using MTT assays. RNA and protein lysates were also obtained at several time points to evaluate the pathways affected by RT-qPCR and western blot, respectively. Results: There was an approximate 3- to 9-fold upregulation of PRR5-ARHGAP8 transcript with a decrease in the expression of PRR5 and ARHGAP8 transcripts in OVMANA, OVISE and KK ovarian carcinoma cell lines upon the knockdown of HNF-1β. Moreover, higher transcript levels of PRR5, ARHGAP8 and PRR5-ARHGAP8 were observed in ovarian carcinoma cell lines compared to HOSE cell lines and in ovarian cancer samples compared to the non-tumour counterparts. Overexpression of PRR5-ARHGAP8 in HEK293T cells resulted in the decrease of phosphorylated Akt protein expression and a reduction of cell viability. RT-qPCR analysis showed a decrease in E-cadherin and an increase in vimentin expression upon PRR5-ARHGAP8 overexpression. Discussion: The present data suggests PRR5-ARHGAP8 is highly abundant in ovarian carcinomas and may mediate its effects on proliferation via the MAPK pathway and on migration by regulating the epithelial to mesenchymal transition. Overall, this study provides novel insights into the biological relevance of read-through transcription in carcinogenesis. Citation Format: Jasmeen K. Sethi, Oscar G. Wong, Esther S. Wong, Annie N. Cheung. Molecular function of the read-through transcript PRR5-ARHGAP8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1490.

Metformin prevents nerve growth factor-dependent proliferative and proangiogenic effects in epithelial ovarian cancer cells and endothelial cells.

Background:Epithelial ovarian cancer (EOC) is characterized by exacerbated angiogenesis regulated by proangiogenic and growth factors. Nerve growth factor (NGF) is overexpressed in EOC where it promotes proliferation as well as survival and is considered a proangiogenic factor. Metformin, a drug commonly used in the treatment of diabetes, is attributed to antineoplastic effects, but the underlying mechanisms remain unknown. Given that current therapies yield modest results in EOC patients, the aim of this study was to determine the effects of metformin on NGF-enhanced proliferation of EOC cells and the angiogenic potential of endothelial cells.Methods:A2780 (EOC), HOSE (human ovarian surface epithelial) and EA.hy926 (endothelial) cells were treated with NGF and metformin. Cell viability, cell proliferation and cell cycle were evaluated in all three cell lines, and the angiogenic potential in endothelial EA.hy926 cells.Results:NGF enhanced cell proliferation in A2780, HOSE and EA.hy926 cells (p < 0.05), while metformin treatment decreased cell proliferation in A2780 and EA.hy926 cells (p < 0.05). Moreover, the NGF-enhanced angiogenic score in EA.hy926 cells was prevented by metformin (p < 0.05).Conclusions:Given that NGF plays a significant role in EOC progression, our current findings suggest that metformin holds considerable promise as an adjuvant treatment in ovarian cancer.

17\u03b2-Estradiol sensitizes ovarian surface epithelium to transformation by suppressing Disabled-2 expression

Estrogen replacement therapy increases the risk of human ovarian cancer and exogenous estradiol accelerates the onset of ovarian cancer in mouse models. This study uses primary cultures of mouse ovarian surface epithelium (OSE) to demonstrate that one possible mechanism by which estrogen accelerates the initiation of ovarian cancer is by up-regulation of microRNA-378 via the ESR1 pathway to result in the down-regulation of a tumour suppressor called Disabled-2 (Dab2). Estrogen suppression of Dab2 was reproducible in vivo and across many cell types including mouse oviductal epithelium and primary cultures of human ovarian cancer cells. Suppression of Dab2 resulted in increased proliferation, loss of contact inhibition, morphological dysplasia, and resistance to oncogene-induced senescence – all factors that can sensitize OSE to transformation. Given that DAB2 is highly expressed in healthy human OSE and is absent in the majority of ovarian tumours, this study has taken the first steps to provide a mechanistic explanation for how estrogen therapy may play a role in the initiation of ovarian cancer.

Abstract 3308: Mitophagy induction and interference with cancer-specific apoptosis in SHetA2-treated cells

Abstract Background: SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) compound that exhibited in-vivo cancer therapeutic and chemoprevention activity, lack of mutagenicity or teratogenicity, and a pharmacologic profile suitable for an oral drug. First-in-human clinical trials of SHetA2 for ovarian cancer prevention and cervical cancer treatment are in development. Autophagy is induced in cancer cells by a wide variety of therapeutic drugs and commonly interferes with the drugs efficacies. Because SHetA2 binds to mortalin and induces mitochondrial damage and apoptosis in cancer, but not in healthy cells, we hypothesized that this drug induces mitochondria-selective autophagy (mitophagy) in cancer, but not healthy cells; and that the mitophagy interferes with SHetA2-induction of apoptosis. Methods: Cultures of human ovarian and cervical cancer cell lines and human ovarian surface epithelium (HOSE) were treated with various concentrations and combinations of SHetA2, 3-methyladenine (3-MA), Beclin-1 siRNA, and caspase inhibitors. Autophagy was evaluated by electron microscopy (EM) and fluorescent imaging of LC3B processing in cells and by Western blot (WB) analysis of p62, Beclin-1 and LC3B processing. Mitophagy was measured by EM of cells and WB analysis of BNIP3 total levels and co-immunoprecipitation with LC3B in protein extracts. Apoptosis was measured by Annexin-V and propidium iodide staining of cells and by WB analysis of caspase 9 and 3 and PARP-1 cleavage. Result: Induction of autophagy by SHetA2 treatment was demonstrated by the development of double-membraned autophagosomes and accumulation of the processed LC3B II in cancer, but not in HOSE cells. Mitophagy was demonstrated by the presence of mitochondria inside the autophagosomes and by reduction of total BNIP3 in association with increased BNIP3/LC3B complexes in SHetA2 treated cells. Autophagic flux was demonstrated by gradual reduction of p62. Conversion of autophagy to apoptosis was associated with reappearance of basal p62 levels and cleavage of Beclin-1, PARP-1, and caspases 9 and 3 about 24 hours after initiation of treatment. Interference of the autophagy with apoptosis was demonstrated by increased levels of apoptosis in cells co-treated with SHetA2 and autophagy inhibitors (3-MA or Beclin-1 siRNA) along with reduced apoptosis in cells co-treated with SHetA2 and an autophagy inducer (rapamycin). This was observed in cell lines harboring mutant or no p53, but not in an ovarian cancer cell line with wild type p53. Conclusion: SHetA2-induction of autophagy may be initiated by mitochondrial damage and reduces, but does prevent, SHetA2-mediated apoptosis in ovarian and cervical cancer cell lines, but not in a cell line with wild type p53 or in HOSE cells. Combination of SHetA2 with an autophagy inhibitor may improve the therapeutic efficacy in clinical trials of patients harboring mutant or loss of p53 protein. Funded by: RO1CA196200 and RO1CA200126 Citation Format: Vishal Chandra, Andrew Long, Chioniso Patience Masamha, Doris Mangiaracina Benbrook. Mitophagy induction and interference with cancer-specific apoptosis in SHetA2-treated cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3308. doi:10.1158/1538-7445.AM2017-3308

Abstract 4525: Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance

Abstract Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor that when induced regulates the expression of many genes involved in cytoprotective stimuli, which attenuates apoptosis and improves survival.. Increased expression of HIF-1α gene (HIF1A) has been found in several carcinomas, including ovarian cancer. Ovarian cancers are generally refractory to platinum-based chemotherapy. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells after chemotherapy are poorly defined. Genomic instabilities, such as copy number variation(CNV), may play an important role in chemoresistance and have been implicated in many complex diseases, like cancer.. We analyzed CNV data that is publically available through the Cancer Genome Atlas and others. Of particular interest was the transcription factor HIF1A which plays an integral role in oxidative stress response such as those induced by chemotherapy reagents. The present study provides evidence for the rare escape of tumor cells from drug-induced cell death by entering a non-cycling senescent state. We report the adaptive response of human ovarian surface epithelium cells to CoCl2, a chemical hypoxia-mimicking agent resulting in a senescent-like state of chemoresistant cells. The effect of the treatment was evaluated on CNV of HIF-1α gene expression, cell proliferation, survival, and tumor invasiveness. We show here that CNV duplication events of HIF1α results in an oxidative stress response in cells leading to chemoresistance through the induction of cellular senescence. Understanding the molecular events associated with chemoresistance will ultimately lead to better patient treatment and outcomes. Note: This abstract was not presented at the meeting. Citation Format: Noelle L. Cutter, Tyler Walther, Kimberly H. Doyle, Ryan Frank. Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4525. doi:10.1158/1538-7445.AM2017-4525

Effect of estradiol on the expression of angiogenic factors in epithelial ovarian cancer.

Ovarian cancer presents a high angiogenesis (formation of new blood vessels) regulated by pro-angiogenic factors, mainly vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). An association between endogenous levels of estrogen and increased risk of developing ovarian cancer has been reported. Estrogen action is mediated by the binding to its specific receptors (ERα and ERβ), altered ERα/ERβ ratio may constitute a marker of ovarian carcinogenesis progression. To determine the effect of estradiol through ERα on the expression of NGF and VEGF in epithelial ovarian cancer (EOC). Levels of phosphorylated estrogen receptor alpha (pERα) were evaluated in well, moderate and poorly differentiated EOC samples (EOC-I, EOC-II, EOC-III). Additionally, ovarian cancer explants were stimulated with NGF (0, 10 and 100 ng/ml) and ERα, ERβ and pERα levels were detected. Finally, human ovarian surface epithelial (HOSE) and epithelial ovarian cancer (A2780) cell lines were stimulated with estradiol, where NGF and VEGF protein levels were evaluated. In tissues, ERs were detected being pERα levels significantly increased in EOC-III samples compared with EOC-I (p<0.05). Additionally, ovarian explants treated with NGF increased pERα levels meanwhile total ERα and ERβ levels did not change. Cell lines stimulated with estradiol revealed an increase of NGF and VEGF protein levels (p<0.05). Estradiol has a positive effect on pro-angiogenic factors such as NGF and VEGF expression in EOC, probably through the activation of ERα; generating a positive loop induced by NGF increasing pERα levels in epithelial ovarian cells.

Role of CYP1A1 in the biological activity of methylated resveratrol analogue, 3,4,5,4′-tetramethoxystilbene (DMU-212) in ovarian cancer A-2780 and non-cancerous HOSE cells

The role of CYP1A1 and CYP1B1 enzymes in the biotransformation and biological activity of the methylated resveratrol analogue, 3,4,5,4′-tetramethoxystilbene (DMU-212) is still elusive. Our recently published data have shown that one of the metabolites of DMU-212, 3′-hydroxy-3,4,5,4′-tetramethoxystilbene (DMU-214) exerts more potent cytotoxic effects in A-2780 ovarian cancer cell line, as compared to the parent compound. Hence, this study aims to elucidate whether the biological activity of DMU-212 is related to its biotransformation to DMU-214. Furthermore, we aimed to assess which enzymes of CYP1 family are involved in the biotransformation of DMU-212. The human ovarian cancer cell lines A-2780, A-2780CYP1A1(−) and non-cancerous human ovarian surface epithelial (HOSE) cells were employed in the present study.In contrary to other authors’ suggestions we have found that CYP1A1 is the major enzyme of CYP1 family involved in the metabolic activation of DMU-212. Since the distinctly weaker anti-proliferative effects of DMU-212 against HOSE and A-2780CYP1A1(−) cells have been associated with the lack of the expression of CYP1A1, we suggest that the biological activity of the parent compound may be related to its metabolic activation to DMU-214 and the level of this enzyme.