HEY Cell Lines Research Articles

Regulation of cancer cell ferroptosis by PTRF/Cavin-1

Ovarian cancer, marked by high rate of recurrence, novel therapeutic strategies are needed to improve patient outcome. One of the potential strategies is inducing ferroptosis in ovarian cancer cells. Ferroptosis is an iron-dependent, lipid peroxidation-driven mode of cell death primarily occurring on the cell membrane. PTRF, an integral component of the caveolae structures located on the cell membrane, is involved in a multitude of physiological processes, including but not limited to, endocytosis, signal transduction, and lipid metabolism. This study elucidates the relationship between PTRF and ferroptosis in ovarian cancer, offering a fresh perspective for the development of new therapeutic strategies. We knocked down PTRF employing siRNA in the ovarian cancer cell lines HEY and SKOV3, following which we stimulated ferroptosis with Erastin (Era). Our research indicates that the lack of PTRF sensitizes cancer cells to ferroptosis, likely by altering membrane stability and tension, thereby affecting signal pathways related to ferroptosis, such as lipid and atherosclerosis, fluid shear stress, and atherosclerosis. Our findings provide new insights for developing new treatments for ovarian cancer.

Abstract 644: PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines

Abstract Ovarian cancer is the most lethal malignant disease of female. Treatment options for ovarian cancer are mostly the use of chemotherapeutics, surgery, or radiotherapy. However, due to resistance to drugs, relapse occurs and thus 5 years survival rate is only 47%. The tumor suppressor protein p53 works as a transcription factor to transcribe several proteins such as p21, p16 and PUMA that lead to halt cell cycle and also induce apoptosis when there is a damage in DNA. Mutation in several regions of p53 (mutp53) is quite common in cancers and can lead to the accumulation of p53 with loss of functions or sometimes gain of oncogenic functions. Thus there is a search for drugs that can degrade the mut53 and can restore the functions of normal p53. In this research, it was observed that the treatment of ovarian cancer cell lines (ES-2, HEY and OVCAR3) with atypical protein kinase C-iota (aPKCɩ) specific inhibitors ICA-1S can cause the degradation of mutp53 (53.6%, N=6 trials). Treatment with ICA-1S also lowered the level of phosphorylated p53 (ser15, 87%, N=3 trials). However, the level of MDM2 protein (murine double minute 2), a known p53 E3 ligase was not significantly (98%, N=2 trials) affected by ICA-1S in ES-2 cell line with mut p53 as well as p21 was also not affected. However level of PUMA was decreased both in ES-2 and HEY cell lines. Immunoprecipitation (IP) with aPKCɩ specific beads showed the aPKCɩ is associated with p53, though reverse IP with p53 beads was not able to show the association. Application of proteasome inhibitor MG-132 at a single dose of 0.5µM could not completely inhibit the degradation of mut53 and WTp53 in ICA-1S treated ES-2 cell line and HEY cell line, respectively, indicating the drug may be affecting the transcription of p53 as well. It is therefore postulated that aPKCɩ can phosphorylate p53, stabilizing it and may be affecting the transcription of p53 as well in those cell lines. Further research (kinase activity assay, immunoprecipitation, immunofluorescence) is going on to reveal the mode of this degradation. Citation Format: Mahfuza Marzan, Nuzhat Nowshin Oishee, Abigail Olatunji, Mildred Acevedo-Duncan. PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 644.

Identification of estrogen response-associated STRA6+ granulosa cells within high-grade serous ovarian carcinoma by single-cell sequencing

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is a pathologic subtype of ovarian cancer (OC) with a more lethal prognosis. Extensive heterogeneity results in HGSOC being more susceptible to treatment resistance and adverse treatment effects. Revealing the heterogeneity involved is crucial. MethodsWe downloaded the single-cell RNA-seq (scRNA) data from GEO database and performed a scRNA analysis for cell landscape of HGSOC by using the Seurat package. The highly expressed genes were uploaded into the DAVID and KEGG database for enrichment analysis, and the AUCell package was used to calculate cancer-associated hallmark score. The SCENIC analysis was used for key regulons, the estrogen response enrichment scores in TCGA-OV RNA-seq dataset were calculated by using the GSVA package. Besides, the expression of STRA6 and IRF1 and the cell invasion and migration in si-STRA6 OC cells were detected by using the quantitative reverse transcription (qRT)-PCR method and Transwell assay respectively. ResultsWe successfully constructed a single-cell atlas of HGSOC and delineated the heterogeneity of epithelial cells therein. There were five epithelial cell subpopulations, GLDC + Epithelial cells, PEG3+ leydig cells, STRA6+ granulosa cells, POLE2+ Epithelial cells, and AURKA + Epithelial cells. STRA6+ granulosa cells have the potential to promote tumor growth as well as the highest estrogen response early activity through the biological pathways analysis of highly expressed genes and estrogen response score of ssGSEA. We found that IRF1 and STRA6 expression was remarkably upregulated in the OC cancer cell line HEY. Silencing of STRA6 markedly decreased the invasion and migration ability of the OC cancer cell line HEY. ConclusionThere is extreme heterogeneity of epithelial cells in HGSOC, and STRA6+ granulosa cells may be able to promote cancer progression. Our findings are benefit to the heterogeneity identification of HGSOC and develop targeted therapy strategy for HGSOC patients.

Chemoprotective effect of baicalin against cyclophosphamide induced ovarian toxicity in mice via inhibition of TGF-β

Ovarian toxicity is most common gynecologically related malignancy, arising for most cases owing to the advanced stage of diagnosis. The aim of the current study was to explore the anticancer potential of baicalin against cyclophosphamide (CP) induced ovarian toxicity in mice and explore the possible mechanism. ovarian cancer cells (Hey, SKOv3ip and HO891PM) were treated with different doses of baicalin and examined via flow cytometry and cell proliferation assay. Subcutaneous administration of CP (200 mg/kg) was used to induce the ovary toxicity and mice were received the oral administration of baicalin. Oxidative, pro-inflammatory, inflammatory, apoptosis parameters, progesterone, estrogen hormones and histopathological were also estimated at end of the study. Baicalin increased the apoptosis and caused the cell cycle arrest at the G2/M stage in ovarian cancer cells. Baicalin significantly (P < 0.001) reduced the level of TGF-β in the HO8910PM, SKOv3ip and Hey cell lines. Baicalin significantly (P < 0.001) increased the body weight and reduced the tumor volume in mice. Baicalin significantly (P < 0.001) increased the level of estrogen and progesterone. Baicalin significantly (P < 0.001) reduced the level of malonaldehyde (MDA) and increased the level of superoxide dismutase (SOD) and catalase (CAT). Baicalin significantly (P < 0.001) decreased the level of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and inflammatory parameter such as nuclear kappa B factor (NF-κB), respectively. Baicalin significantly (P < 0.001) reduced the level of the caspase-3. Baicalin, act as the potential agent against the ovarian toxicity by alteration of TGF-β and inflammatory pathways.

Polystyrene nanoparticle exposure accelerates ovarian cancer development in mice by altering the tumor microenvironment

Microplastics and nanoplastics are ubiquitous pollutants, widely spread in the living and natural environment. Although their potential impact on human health has been investigated, many doubts remain about their effects in carcinogenic processes. We investigated the potential effects and its molecular mechanisms of polystyrene nanoplastics (PS-NPs) on epithelial ovarian cancer (EOC) using the human EOC cell line HEY as an in vitro cell model and mice as a mammalian model. In vivo exposure to PS-NPs (100 nm; 10 mg/L) via drinking water significantly accelerated EOC tumor growth in mice. In in vitro tests the PS-NPs reduced the relative viability of EOC cells in a dose-dependent manner. Histological analysis showed increased mitotic counts in EOC tumor tissues of PS-NP exposed mice. PS-NP exposure significantly affected gene expression and disturbed many metabolic pathways in both cultured EOC cells and EOC tumor tissue in mice. Gene functional and pathway analysis indicated that immune-related responses and the tumor microenvironment pathway were significantly enriched, which may be attributed to disturbed expression of thrombomodulin (THBD) and its regulators. It may be concluded that PS-NP exposure caused a significant acceleration of EOC tumor growth in mice and a dose-dependent decrease in the relative viability of EOC cells by altering the tumor growth microenvironment. This offers new insights into the mechanisms underlying PS-NP effects on EOC.

Overexpression of NPTX2 Promotes Malignant Phenotype of Epithelial Ovarian Carcinoma via IL6-JAK2/STAT3 Signaling Pathway Under Hypoxia.

BackgroundEpithelial ovarian cancer (EOC) is the main subtype of ovarian cancer and shows an aggressive phenotype and poor prognosis. Neuronal pentraxin II (NPTX2) is a member of the neuronal pentraxin family and plays a contradictory role in different tumors. However, there has been no report about the possible role and effect of NPTX2 in EOC.MethodsBioinformatics analysis, qPCR, western blotting and immunohistochemistry were used to detect the expression of NPTX2 in EOC. Lentivirus-based transfection for NPTX2 overexpression or knockdown was performed on the EOC cell lines A2780, HEY, SKOV3 and OVCAR-3. The effect of NPTX2 on the malignant phenotype of EOC was examined through methods of MTS assay, Edu assay, transwell assay, western blotting analysis, qPCR analysis, luciferase reporter assay and xenograft experiment.ResultsEOC tissues showed higher NPTX2 expression than the normal tissues with poor prognosis. NPTX2 overexpression can promote the proliferation, invasion, migration and tumorigenesis of EOC via IL6-JAK2/STAT3 signaling pathway. Moreover, hypoxia-inducible factor-1(HIF-1) can promote the transcription and expression of NPTX2 under the hypoxic environment. NPTX2 knockdown abolished the hypoxia-induced malignant phenotypes in ECO.ConclusionsThe above results suggest that NPTX2 may play a novel role in ovarian cancer’s malignant phenotype and act as a promising treatment target for EOC molecular therapy.

ALPL regulates the aggressive potential of high grade serous ovarian cancer cells via a non-canonical WNT pathway

The ALPL gene is linked to hypophosphatasia, a rare genetic disease. Owing to the inverse relationships between ALPL expression and both the International Federation of Gynecology and Obstetrics (FIGO) stages and histological grades assigned to patients with serous ovarian cancer (SOC), this study was designed to explore the role and possible mechanisms of ALPL in cell motility of high grade SOC (HGSOC). The effects of ALPL overexpression on migration and invasion were detected in HGSOC cell lines SKOV3 and HEY. Gene ontology analysis for differential genes with ALPL overexpression identified several biological processes, including EMT, correlated with cell motility. Genes potentially implicated in EMT and associated with ALPL were screened using The Cancer Genome Atlas (TCGA) database. The WNT receptor Frizzled2 (FZD2) was identified and its role in HGSOC cell motility and survival was investigated. It was found that forced expression of ALPL could inhibit migration, invasion, and EMT in HGSOC cells. It also reduced the expression of FZD2 and its ligand WNT5A, accompanied by suppressed expression of their downstream target phosphorylated-STAT3 (pSTAT3). These effects were initiated via the FZD2 knockdown using siRNA and reversed by recombinant WNT5A protein. The relationship between FZD2 expression and poor HGSOC patient survival was also investigated. This data supports that ALPL might restrict the function of WNT5A-FZD2-STAT3 axis, a non-canonical WNT pathway for promoting EMT progression, which results in attenuated migration and invasion in HGSOC cells and improves survival in patients with SOC.

Adenosine enhances cisplatin sensitivity in human ovarian cancer cells.

Ovarian cancer is the deadliest gynecologic cancer due to lack of early effective diagnosis and development of resistance to platinum-based chemotherapy. Several studies reported that adenosine concentrations are higher in tumor microenvironment than in non-tumor tissue. This finding inspired us to study the role of adenosine in ovarian cancer cells and to investigate if adenosine pathways offer new treatment options urgently needed to prevent or overcome chemoresistance. The ovarian cancer cell lines HEY, A2780, and its cisplatin-resistant subline A2780CisR were used in this study. Expression and functional activity of adenosine receptors were investigated by RT-PCR, Western blotting, and cAMP assay. A1 and A2B adenosine receptors were expressed and functionally active in all three cell lines. Adenosine showed moderate cytotoxicity (MTT-IC50 values were between 700 and 900μM) and induced apoptosis in a concentration-dependent manner by increasing levels of sub-G1 and cleaved PARP. Apoptosis was diminished by QVD-OPh, confirming caspase-dependent induction of apoptosis. Forty-eight hours pre-incubation of adenosine prior to cisplatin significantly enhanced cisplatin-induced cytotoxicity in a synergistic manner and increased apoptosis. SLV320 or PSB603, selective A1 and A2B antagonists, was not able to inhibit adenosine-induced increase in cisplatin cytotoxicity or apoptosis whereas dipyridamole, a nucleoside transporter inhibitor, completely abrogated both effects. Mechanistically, adenosine increased pAMPK and reduced pS6K which was prevented by dipyridamole. In conclusion, application of adenosine prior to cisplatin could be a new therapeutic option to increase the potency of cisplatin in a synergistic manner and thus overcome platinum resistance in ovarian cancer.

Abstract 5137: Tryptophan catabolism in ovarian cancer

Abstract Serous epithelial ovarian cancer metastasizes by direct seeding to form disseminated tumors within the peritoneal cavity and has an extremely poor clinical outcome with a 46.5% five-year survival rate. Resistance to anoikis (detachment-induced cell death) is a critical factor in the progression of this disease. We previously demonstrated that triple-negative breast cancer (TNBC) upregulates tryptophan-2,3-dioxygenase (TDO2), a rate limiting enzyme in tryptophan catabolism, under anchorage-independent conditions or by stimulation with an NFκB stimulating cocktail (IL1-β, TNFα). Indoleamine-pyrrole 2,3-dioxygenase (IDO1) is also a rate-limiting enzyme in the tryptophan catabolism pathway, &amp; both TDO2 and ID01 can be expressed by tumors. Kynurenine, a metabolite of tryptophan catabolism acts in an autocrine fashion through the aryl hydrocarbon receptor to provide anti-apoptotic signals that enhance survival under anchorage independent conditions. Through paracrine action, kynurenine can suppress anti-tumor cytotoxic T-cell function. We hypothesize that tryptophan catabolism promotes ovarian cancer progression through these two mechanisms, primarily through TDO2. We mined the publically available Tothill Ovarian Cancer patient cohort (n=293) for correlations between TDO2 or IDO1 &amp; ovarian cancer outcomes. The anoikis resistant serous ovarian cancer cell lines HEY, OV-1847, OVCA-420 &amp; OVCA-433 were used to test levels of enzymes involved in tryptophan catabolism in vitro at baseline, in attached versus suspended conditions &amp; following NFκB stimulation with IL1-β and TNFα. Three enzymes in the pathway; TDO2, IDO1 &amp; Kynureninase (KYNU) were quantified by qRT-PCR &amp; immunohistochemistry. Using a cytokine/chemokine array and follow-up ELISA, we also analyzed factors secreted by ovarian cancer cells surviving in suspension culture as compared to attached. In the ovarian cancer patient cohort, high TDO2 significantly correlated with higher stage disease (p=0.0033), increased recurrence rates (p&amp;lt;0.0001) &amp; lower survival rates (p=0.0034), whereas IDO1 did not show significant correlations. The expression of tryptophan catabolizing enzymes was significantly increased in the suspended condition when compared to attached &amp; following NFκB stimulation. Ovarian cancer cell lines in suspension have increased levels of IL-1α, G-CSF, MIF and IL-6 on arrays, &amp; IL-6 was confirmed by ELISA. Data mining of patient specimens indicates that TDO2 may be the more relevant enzyme responsible for tryptophan metabolism in ovarian cancer since high TDO2 is correlated with higher clinical stage, disease progression &amp; in recurrent disease, suggesting that TDO2 may facilitate disease progression. Tryptophan catabolizing enzymes are elevated in ovarian cancer cells under anchorage independent condition or inflammatory stimuli. Based on these findings, we believe that clinical trials targeting TDO2 in addition to IDO may be warranted for serous ovarian cancer. Citation Format: Lynelle P. Smith, Lucas M. Harrell, Jessica L. Christenson, Benjamin Bitler, Jill Slansky, Jennifer K. Richer. Tryptophan catabolism in ovarian cancer [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 5137.

Abstract 789: Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells

Abstract Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process. Materials &amp; Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT). Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry. Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers. Funding: FONDECYT 1120292, 3150028 &amp; 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001 Citation Format: Andres Valdivia, Dusan Racordon, Raul Aravena, Gabriel Mingo, Alejandra Sandoval, Maria Loreto Bravo, Mauricio A. Cuello, Sumie Kato, Rafaela Erices, Carolina Ramirez, Pamela Gonzalez, Beatriz Sanchez, Alejandro H. Corvalan, Gareth I. Owen. Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer 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 789. doi:10.1158/1538-7445.AM2017-789

Chikusetsusaponin IVa methyl ester induces G1 cell cycle arrest, triggers apoptosis and inhibits migration and invasion in ovarian cancer cells

BackgroundPanacis Japonici Rhizoma (PJR) is one of the most famous Chinese medical herbs that is known for exhibiting potential anti-cancer effects. PurposeThis study aims to isolate and investigate the anti-cancer potential of saponins from PJR in ovarian cancer cells. MethodsThe compounds were separated by comprehensive chromatographic methods. By comparison of the 1H- and 13C NMR data, as well as the HR-ESI-MS data, with the corresponding references, the structures of compounds were determined. MTT assay was performed to evaluate cell viability, along with flow cytometry for cell cycle analysis. JC-1 staining, Annexin V-PI double staining as well as Hoechst 33; 342 staining were used for detecting cell apoptosis. Western blot analysis was conducted to determine the relative protein level. Transwell assays were performed to investigate the effect of the saponin on cell migration and invasion and zymography experiments were used to detect the enzymatic activities. ResultsEleven saponins were isolated from PJR and their anti-proliferative effects were evaluated in human ovarian cancer cells. Chikusetsusaponin IVa methyl ester (1) exhibited the highest anti-proliferative potential among these isolates with the IC50 values at less than 10 µM in both ovarian cancer A2780 and HEY cell lines. Compound 1 induced G1 cell cycle arrest accompanied with an S phase decrease, and down-regulated the expression of cyclin D1, CDK2, and CDK6. Further study showed that compound 1 effectively decreased the cell mitochondrial membrane potential, increased the annexin V positive cells and nuclear chromatin condensation, as well as enhanced the expression of cleaved PARP, Bax and cleaved-caspase 3 while decreasing that of Bcl-2. Moreover, compound 1 suppressed the migration and invasion of HEY and A2780 cells, down-regulated the expression of Cdc42, Rac, RohA, MMP2 and MMP9, and decreased the enzymatic activities of MMP2 and MMP9. ConclusionThese results provide a comprehensive evaluation of compound 1 as a potential agent for the treatment of ovarian cancer.

Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer.

BackgroundOvarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis.MethodsThe expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice.ResultsWe report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts.ConclusionThe expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients.

A critical role of Oct4A in mediating metastasis and disease-free survival in a mouse model of ovarian cancer.

BackgroundHigh grade epithelial ovarian cancer (EOC) is commonly characterised by widespread peritoneal dissemination and ascites. Metastatic EOC tumour cells can attach directly to neighbouring organs or alternatively, maintain long term tumourigenicity and chemoresistance by forming cellular aggregates (spheroids). Cancer stem-like cells are proposed to facilitate this mechanism. This study aimed to investigate the role of Oct4A, an embryonic stem cell factor and known master regulator of pluripotency in EOC progression, metastasis and chemoresistance.MethodsTo investigate the expression of Oct4A in primary EOC tumours, IHC and qRT-PCR analyses were used. The expression of Oct4A in chemonaive and recurrent EOC patient ascites-derived tumour cells samples was investigated by qRT-PCR. The functional role of Oct4A in EOC was evaluated by generating stable knockdown Oct4A clones in the established EOC cell line HEY using shRNA-mediated silencing technology. Cellular proliferation, spheroid forming ability, migration and chemosensitivty following loss of Oct4A in HEY cells was measured by in vitro functional assays. These observations were further validated in an in vivo mouse model using intraperitoneal (IP) injection of established Oct4A KD clones into Balb/c nu/nu mice.ResultsWe demonstrate that, compared to normal ovaries Oct4A expression significantly increases with tumour dedifferentiation. Oct4A expression was also significantly high in the ascites-derived tumour cells of recurrent EOC patients compared to chemonaive patients. Silencing of Oct4A in HEY cells resulted in decreased cellular proliferation, migration, spheroid formation and increased chemosensitivity to cisplatin in vitro. IP injection of Oct4A knockdown cells in vivo produced significantly reduced tumour burden, tumour size and invasiveness in mice, which overall resulted in significantly increased mouse survival rates compared to mice injected with control cells.ConclusionsThis data highlights a crucial role for Oct4A in the progression and metastasis of EOC. Targeting Oct4A may prove to be an effective strategy in the treatment and management of epithelial ovarian tumours.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0417-y) contains supplementary material, which is available to authorized users.

Abstract 2395: Leptin induces an IL-6 mediated interaction between macrophages and ovarian cancer cells that prompted invasiveness and migration

Abstract Introduction: The current knowledge in ovarian epithelial carcinogenesis grants a major role for chronic inflammation in driving the multiple steps in the natural history of the most deadly gynecological cancer. Different environmental stimuli have been associated with an increased ovarian cancer risk. Growing evidence has become to support the potential role of obesity as one of the conditioning factors. Leptin, an adipokine, controlling metabolism and energy expenditure and normally circulating at higher serum levels among overweight women, has been syndicated as the pro-inflammatory mediator in obesity. So far, few are known about the role of and how leptin modulate cells to cells interactions among the different components of tumor microenvironment. Goal: In the present study we investigated the leptin effect in prompting a pro-inflammatory microenvironment, the communication between macrophages and ovarian cancer cells and the impact of this interaction in cancer cell migration and invasion. Methods: HEY ovarian cancer cell line and the THP-1 monocyte cell line were used. THP-1 cells were differentiated to macrophages by using PMA (Phorbol 12-myristate 13-acetate) 200 nM for 24h. Upon differentiation macrophages were exposed to a time course of leptin (100 ng/ml, 6h to 24h). Upon control or leptin exposure, conditioned medium was collected from macrophages (MCM), and used to measure IL-6 release to the medium and to treat HEY cell line for 24h. Secreted IL-6 was used as inflammation marker and measured by ELISA assay. Protein extracts were obtained from HEY cells upon control and MCM exposure and used to quantify epithelial mesenchymal transition (EMT) marker vimentin by Immunoblotting and Immunocytochemistry. The effects of leptin and MCM in HEY migration and invasion was assessed by Boyden chamber assay without or with matrigel, respectively. Results: Upon leptin treatment both HEY and macrophages significantly increased IL-6 release to the medium. More importantly, incubation of MCM with HEY cells resulted in a synergistic increase of IL-6 release, (from a basal release of 13,4±2 pg/mL to 2,3×104 pg/mL upon MCM exposure). Moreover, HEY cells exposure to MCM resulted in phenotypic changes that were associated to changes in vimentin expression (mainly upon exposure to 6h leptin incubated MCM). Finally, this exposure to leptin-incubated MCM significantly increased migration and invasion of HEY cells. Conclusions: Our findings support a role of leptin in prompting a pro-inflammatory microenvironment that involved an increase in IL-6 release from the macrophages. These changes induced by leptin in macrophages resulted in a cytokine secretion pattern, which consequentially modified cancer cells phenotype favoring migration and invasion and a more aggressive epithelial ovarian cancer behavior. (Funding: FONDECYT 1120292 and 3140335) Citation Format: Sumie Kato, Lorena Abarzua-Catalan, Isidora Solar-Costabal, Karen García, Jorge Branes, Gareth I. Owen, Mauricio A. Cuello. Leptin induces an IL-6 mediated interaction between macrophages and ovarian cancer cells that prompted invasiveness and migration. [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 2395. doi:10.1158/1538-7445.AM2015-2395

Utilizing camelid-derived single domain antibodies to enhance immunotherapy of ovarian cancer

growth, and cancer cells are well known to be “glutamine-addicted.” Glutamine is converted by glutaminase to glutamate, and glutamate is converted by glutamate dehydrogenase (GDH) to α-ketoglutarate, which enters the tricarboxylic acid cycle for the production of adenosine triphosphate (ATP). In addition, glutamine generates the antioxidant glutathione, which removes reactive oxygen species (ROS) and regulates critical cell signaling pathways. Our objective was to explore the effects of glutamine on ovarian cancer (OC) cell growth and metabolism. Methods: We used the OC cell lines HEY, SKOV3, and IGROV1 and assessed dell proliferation by MTT assay. Cell cycle progression and apoptosis were evaluated by Cellometer. Western immunoblotting was performed to evaluate the effects of glutamine on downstream targets of the mTOR and MAPK pathways and proteins related to cellular stress. Glucose uptake, GDH activity, lactate, ATP, and ROS production were all assessed by enzyme-linked immunosorbent assay. The effect of mTOR pathway inhibition on glutaminestimulated cell proliferation was assessed by 1) treatment with rapamycin, an mTOR inhibitor, and 2) knockdown of S6 by siRNA transfection. Results: Glutamine stimulated cell proliferation in a dose-dependent manner in the three OC cell lines. In addition, glutamine increased GDH activity, glucose uptake, and lactate and ATP production and resulted in increased phosphorylation of S6 and p42/44. As expected, glutamine starvation had the opposite effect on the OC cell lines, leading to inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, reduced glucose uptake, and decreased lactate and ATP production. Glutamine depletion also induced ROS production and increased expression of the cellular stress proteins PERK, PARP, Bip, and calnexin. Inhibition of S6 by siRNA or rapamycin resulted in inhibition of glutamine-induced cell proliferation as well as a decrease in GDH activity. Conclusions: Glutamine potently increased cell proliferation, glucose uptake, and ATP production whereas glutamine depletion had the opposite effects on OC cells. Glutamine-induced cell proliferation appeared dependent on the mTOR pathway. These studies suggest that targeting glutamine may be a promising therapeutic strategy for ovarian cancer.

Abstract 3762: Downregulation of TRPC3 enhances sensitivity to cisplatin and inhibits motility in epithelial ovarian cancer

Abstract Epithelial ovarian cancer (EOC) remains the most lethel gynecological malignancy. In the absence of an effective screening strategy, two thirds of patients are diagnosed in advanced clinical stage. Despite chemotherapy with cisplatin and paclitxel, primary or acquired chemoresistance faciltates survival of cancer cells, resulting in disease recurrence and death. Our previous studies indicate that ovarian cancer patients whose tumors overexpress transient receptor potential channel C3 (TRPC3) protein tend to relapse early and have shorter overall survival. In vitro studies indicated that follicle-stimulating hormone (FSH) induced resistance to cisplatin-induced apoptosis could be partially reversed by blocking expression of TRPC3 in EOC cell lines. To enhance sensitivity to platinum compounds, we have investigated the relationship between resistance to cisplatin and TRPC3. We have now found that expression of TRPC3 in recurrent cancer following chemotherapy is higher than in specimens from primary cytoreductive surgery. The expression of TRPC3 in the cisplatin-resistant EOC line A2780cp is higher than in the sensitive parental cell line A2780. An in vitro study showed that transient treatment of the EOC cell line HEY with cisplatin upregulated TRPC3 mRNA and membrane localized protein. Induced cisplatin-resistant cell line HEYcp augmented the expression of TRPC3 and enhanced invasive capacity in cell migration assays. Knockdown of TRPC3 expression in EOC cell lines with specific siRNA or miRNA decreased cell migratory ability and enhanced cisplatin-induced apoptosis when compared to EOC cells treated with scrambled control RNA. Our data indicate that TRPC3 may play an important role in cisplatin-resistance of EOC and could serve as a therapeutic target to resensitize cancer cells to cisplatin in culture and in xenografts. Citation Format: Xiang Tao, Hongyan Jin, Zhenbo Zhang, Robert C. Bast, Youji Feng, Yinhua Yu. Downregulation of TRPC3 enhances sensitivity to cisplatin and inhibits motility in epithelial 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 3762. doi:10.1158/1538-7445.AM2014-3762

Abstract 4427: Multiple cation-selective transporters contribute to the anti-proliferative effects of metformin in ovarian cancer cell lines.

Abstract PURPOSE: Recent studies show that the anti-diabetic drug metformin exhibits anticancer effects against a broad spectrum of cancers including ovarian cancer. Because metformin is highly hydrophilic with a net positive charge at all physiologic pH values, it requires cation-selective transporters to enter into cells and interact with its intracellular target 5’ adenosine monophosphate-activated protein kinase. The expression pattern and level of these transporters in cancer cells would determine the intracellular concentrations and subsequently the anti-proliferative activity of metformin. This study aims to investigate metformin transporter expression and the impact of these transporters on metformin uptake and anti-proliferative activity in human ovarian cancer cell lines that are routinely used to assess potential anti-cancer agents against this cancer type. METHODS: Total RNA from human ovarian cancer cell lines (IG, SKOV3 and HEY) was subjected to real-time PCR to determine expression levels of the common cation transporters organic cation transporter (OCT) 1-3, plasma membrane monoamine transporter (PMAT), multidrug and toxin extrusion transporters (MATE) 1 and 2. Uptake of [14C]metformin (50μM) in the presence or absence of the cation-selective transporter inhibitors famotidine and 1-methyl-4-phenylpyridinium (MPP+) was assessed to determine the contributions of transporters to metformin uptake in these cell lines. RESULTS: OCT3 mRNA was highly expressed in IG cells, with low levels of OCT2, PMAT and MATE1. Metformin uptake into IG cells was significantly (P&amp;lt;0.05) inhibited by 5mM MPP+ (which inhibits all cation-selective transporters at this concentration), and by 200 μM famotidine (which is a more selective inhibitor of OCT3 than OCT2 at this concentration). In SKOV3 cells where OCT2 was the predominant transporter with low levels of OCT3, PMAT and MATE1, metformin uptake was significantly (P&amp;lt;0.05) inhibited by 5 mM MPP+ and 200 μM famotidine. Cation-selective transporter expression was negligible in the HEY cell line in which metformin uptake was lower than uptake in IG and SKOV3 cells, and was not inhibited by either of the two inhibitors. CONCLUSIONS: The ovarian cancer cell lines used in this study are utilized as models to test anticancer agents against ovarian cancer. In order to use these cell lines to investigate the anti-proliferative activity of metformin so as to predict its anticancer effects in ovarian cancer, the expression of metformin transporters and metformin uptake in relation to the expression of these transporters needs to be elucidated. Studies on determining the IC50 values of metformin in ovarian cancer cell lines, as well as cation-selective transporter expression studies in human ovarian tumor tissue samples are currently in progress. Citation Format: Ruth S. Everett, Yunhui Zhang, Innocent M. Ononiwu, Victoria L. Bae-Jump, Dhiren R. Thakker. Multiple cation-selective transporters contribute to the anti-proliferative effects of metformin in ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4427. doi:10.1158/1538-7445.AM2013-4427

MiR-200c and HuR in ovarian cancer

BackgroundMicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.MethodsExpression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3′UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.ResultsIn a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3′UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3′UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.ConclusionThis study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.

Activation of the Canonical WNT Signaling Pathway Promotes Ovarian Surface Epithelial Proliferation Without Inducing β‐Catenin/Tcf‐Mediated Reporter Expression

In response to activation of the canonical WNT signaling pathway, β-catenin cooperates with Lef/Tcf (lymphoid enhancer factor/T-cell factor) transcription factors to drive expression of Wnt target genes. The canonical WNT signaling pathway is involved in development, wound repair, and tumorigenesis. Studies examining the involvement of the canonical WNT signaling pathway in the development of ovarian surface epithelium (OSE) and ovarian carcinogenesis, however, have recently begun to emerge. In this study, we investigated the modulation of β-catenin and β-catenin/Tcf-signaling activity within the OSE using responsive transgenic mice and examined the response of primary OSE cells and ovarian cancer cell lines to activation of the canonical WNT signaling pathway. β-catenin was localized to the lateral membrane of the ovarian epithelium. Stimulation of primary OSE cells in vitro with LiCl or Wnt3a led to GSK-3β inhibition and stabilization of β-catenin but failed to induce β-catenin/Tcf-mediated lacZ expression. Furthermore, E-cadherin expression was downregulated and the proliferative potency of OSE cells increased. Of four ovarian cancers cell lines screened, only the HEY cell line demonstrated induction of luciferase reporter upon canonical WNT stimulation. These observations suggest that in ovarian adenocarcinoma, dysregulated WNT signaling may not always be indicative of β-catenin/Tcf-mediated transcriptional activity.

Abstract A6: Comparison of a hypoxia gene signature in matched ovarian primary tumors and peritoneal metastasis

Abstract Ovarian cancer is characterized by genomic and microenvironmental heterogeneity, both of which contribute to tumor progression, metastasis, and drug resistance. Hypoxia, or low oxygen tension, is one microenvironmental factor that influences genomic instability and promotes angiogenesis and metastasis. We investigated the gene expression levels of hypoxia-regulated genes in matched ovarian serious carcinoma primary and peritoneal metastasis from five patients. The hypoxia gene set used was based on a published metagene hypoxia signature established using breast and head and neck cancer samples by Buffa et al, 2010. To ensure that the genes selected were also hypoxia-regulated in ovarian tumor cells, we grew the Hey and Ovcar-3 ovarian cancer cell lines under normoxia, 1.0% and 0.2% oxygen for 4, 16, and 48 hours. RNA was extracted and cDNA prepared for quantitative real-time polymerase chain reaction (qRT-PCR). Each sample was analyzed in triplicate and normalized for loading using ribosomal protein, large, PO (RPLP0). Relative gene expression was calculated by comparing hypoxia treated samples to the normoxic control grown for the same length of time. The Hey cells were also grown in an intraperitoneal xenograft model and tumor tissue harvested following 22 days of growth. Primary tumor gene expression was normalized to the Hey cell line grown in normoxia. The metastatic tissue was also compared relative to each matched primary sample. From an initial list of 16 genes, we identified 9 genes that demonstrated robust increases in gene expression across treatment conditions in vitro in both OVCAR 3 and Hey cells: vascular endothelial growth factor (VEGF-A), solute carrier family 2 (SLC2A1; Glut-1), phosphoglycerate mutase 1 (PGAM1),lactate dehydrogenase A (LDHA), prolyl 4-hydroxylase, alpha polypeptide (P4HA1), adrenomedullin (ADM), v-yc myelocytomatosis viral related oncogene ( NDRG1; N-myc), aldolase A (ALDOA), carbonic anhydrase 9 (CA9). Xenograft tissue showed high overexpression of all 9 of these hypoxia regulated genes when normalized to the Hey normoxia cell line control grown in vitro, with CA9 levels being maximally elevated. On average, primary tumors showed highest expression levels of VEGF and CA9 when normalized to the in vitro Hey cell normoxia control. When the primary tumor samples were compared to matched metastatic tissue there was a strong correlation between expression patterns of VEGF-A and SLC2A1. Two of five matched samples showed over 3 fold elevation in VEGF and SL2A1 in the metastatic tissue relative to primary tumor. Our data suggests that there may be significant heterogeneity between the primary and metastatic tumors which could lead to sampling bias when investigating hypoxia regulated genes or gene products such as VEGF-A and SL2A1 as prognostic or therapeutic biomarkers. Citation Format: Amanda F. Baker, Scott Malm, Cindy Laughren, Setsuko K. Comparison of a hypoxia gene signature in matched ovarian primary tumors and peritoneal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A6.