Serous Ovarian Carcinoma Cell Line Research Articles

Structural Elucidation and Prognostic Relevance of 297-11A-Sulfated Glycans in Ovarian Carcinoma

Ovarian carcinoma is usually diagnosed at an advanced stage with peritoneal dissemination and/or lymph node metastasis, and the prognosis for such advanced carcinoma is very poor. Therefore, new biomarkers to predict patient prognosis are needed. Miyamoto et al. previously showed that keratan sulfate (KS) detected by the 5D4 monoclonal antibody was expressed in ovarian carcinoma. However, the detailed structure of such KS was not determined, and the biological significance of this finding remained to be clarified. We previously generated the 297-11A monoclonal antibody, which recognizes galactose (Gal)-6-O-sulfated N-acetyllactosamine (LacNAc) located at the nonreducing terminus. Because the 297-11A epitope overlaps with that of 5D4, here we chose to use the 297-11A antibody as a tool to analyze KS and related structures. We conducted immunohistochemical analysis of 98 ovarian carcinoma cases with 297-11A antibody combined with a series of glycosidases and performed mass spectrometry analysis of the human serous ovarian carcinoma cell line OVCAR-3 to deduce the glycan structure of 297-11A-sulfated glycans. We also performed western blot analysis to assess a potential association of 297-11A-sulfated glycans with the mucin core protein mucin 16 (MUC16; also known as cancer antigen 125 (CA125)). Finally, we examined the relationship between 297-11A expression and patient prognosis. Consequently, 297-11A-sulfated glycans were primarily expressed in serous and endometrioid carcinomas and poorly expressed in mucinous and clear cell carcinomas. We reveal that structurally, 297-11A-sulfated glycans expressed in ovarian carcinoma are O-glycans carrying partially sialylated, Gal-6-O-sulfated LacNAc and that these glycans are likely displayed on MUC16 mucin core proteins. Of clinical importance is that expression of 297-11A-sulfated glycans correlated with shorter progression-free survival in patients. Thus, 297-11A-sulfated glycans may serve as a predictor of ovarian carcinoma recurrence.

Abstract 1117: Investigating PD-1/PD-L1 checkpoint inhibition as a treatment for platinum-resistant ovarian cancer

Abstract Ovarian cancer is the deadliest cancer of the female reproductive system, with the majority of patients experiencing relapse on first-line platinum-based chemotherapies. Identifying potential targets for additional second-line therapies is critical for treating platinum-resistant ovarian cancer. To that end, the evolution of platinum resistance in a cell-based model of ovarian cancer was monitored via characterization of proteomic changes in cancer-derived extracellular vesicles (EVs) after progressive rounds of cisplatin treatment. The content of cancer-derived EVs was analyzed via reverse phase protein array, mass spectrometry, and Western blotting to identify pathways that could be targeted by second-line therapies. Several therapeutically relevant markers were upregulated, including phospho-EGFR (Y1068), multidrug transporter ABCE1, and PD-L1. Monoclonal antibody-based therapies (mAbs) targeting PD-1/PD-L1 have been deployed successfully as second-line treatments for a variety of cancers, but their success has yet to be translated to ovarian cancer. Current mAb-based immunotherapies suffer from several pitfalls including poor tissue penetration, a long-standing issue in ovarian cancer treatment. To offer an alternative to mAb-based immunotherapies, we developed a small interfering peptide consisting of a portion of the PD-L1 interface sequence. This lead peptide underwent several rounds of in silico optimization via molecular dynamics and modeling simulations to identify modifications that could improve affinity for PD-1 or stability in serum. When efficacy was assessed in vitro via Amplified Luminescent Proximity Homogeneous Assay screening, the modifications to the lead peptide were not shown to improve efficacy. However, serum stability studies indicated the modifications dramatically improved peptide stability, with detectable levels still quantifiable via mass spectrometry after 48 hours. The stabilized peptide, titled MN1.4, was chosen for further efficacy testing in cell-based models. A cisplatin-treated daughter line from the OVCAR8 high grade serous ovarian carcinoma cell line was created to model the effects of clinical platinum-based chemotherapy treatments on the most common ovarian cancer subtype. OVCAR8 cells were co-cultured in the presence of stimulated Jurkat T cells to measure markers of T cell activation, including IL-2, via ELISA after treatment with MN1.4 in the presence or absence of EVs derived from cisplatin-treated cells. This model allows for examination of the potential sensitization effects of first-line cisplatin treatment on PD-1/PD-L1 immunotherapy, and how cancer-derived EVs from cisplatin-treated cells can abrogate this effect. We conclude that exosomal PD-L1 may impair responses to PD-1/PD-L1 immunotherapy and should be considered when assessing the viability of this treatment option for ovarian cancer patients. Citation Format: Rachel Carter, Marissa Howard, James Erickson, Paul Russo, Alessandra Luchini, Amanda Haymond, Fatah Kashanchi, Mikell Paige, Lance Liotta. Investigating PD-1/PD-L1 checkpoint inhibition as a treatment for platinum-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1117.

Promising Therapeutic Impact of a Selective Estrogen Receptor Downregulator, Fulvestrant, as Demonstrated In Vitro upon Low-Grade Serous Ovarian Carcinoma Cell Lines.

Few studies have reported hormonal agent use in the treatment of low-grade serous ovarian carcinomas (LGSOCs), which are chemoresistant. Considering the need for novel effective therapies, we investigated the hormone receptor expression and hormonal inhibition efficacy in LGSOCs. Using immunohistochemistry, we assessed the estrogen receptor (ER) expression status in 33 cases of histologically confirmed serous ovarian tumors, including 10, 11, and 12 cases of LGSOCs, serous borderline tumors (SBTs), and serous cystadenomas (SCAs), respectively. The genetic background reported in our previous study was used in the current study. MPSC1 cells, which were established from LGSOCs, were used in cell proliferation assays. We observed a higher ER expression in LGSOCs and SBTs than in SCAs (70%, 81%, and 50%, respectively). Thus, LGSOCs and SBTs exhibit higher ER expression than SCAs. Moreover, the PIK3CA mutation positively correlated with ER expression in LGSOCs (p = 0.0113). MPSC1 cells showed low ER expression on Western blotting. MPSC1 cell proliferation was significantly inhibited by fulvestrant (a selective ER downregulator). The activation of ER and PI3K/AKT signaling pathways may play an important role in LGSOC carcinogenesis. ER downregulation with fulvestrant or combination therapy with PI3K inhibitors is a possible novel treatment for patients with LGSOCs.

Differential and divergent activity of insulin‑like growth factor binding protein 6 in platinum‑sensitive versus platinum‑resistant high‑grade serous ovarian carcinoma cell lines

Insulin-like growth factor binding protein 6 (IGFBP6) is a secreted protein with a controversial role in human malignancies, being downregulated in most types of human cancer, but upregulated in selected tumors. Ovarian cancer (OC) is a human malignancy characterized by IGFBP6 downregulation; however, the significance of its low expression during ovarian carcinogenesis is still poorly understood. In the present study, IGFBP6 expression and activation of its associated signaling pathway were evaluated in two matched OC cell lines derived from a high-grade serous OC before and after platinum resistance (PEA1 and PEA2 cells, respectively). A whole genome gene expression analysis was comparatively performed in both cell lines upon IGFBP6 stimulation using Illumina technology. IGFBP6 gene expression data from human OC cases were obtained from public datasets. Gene expression data from public datasets confirmed the downregulation of IGFBP6 in primary and metastatic OC tissues compared with in normal ovarian tissues. The comparative analysis of platinum-sensitive (PEA1) and platinum-resistant (PEA2) cell lines showed quantitative and qualitative differences in the activation of IGFBP6 signaling. Notably, IGFBP6 enhanced ERK1/2 phosphorylation only in PEA1 cells, and induced more evident and significant gene expression reprogramming in PEA1 cells compared with in PEA2 cells. Furthermore, the analysis of selected genes modulated by IGFBP6 (i.e., FOS, JUN, TNF, IL6, IL8 and EGR1) exhibited an inverse regulation in PEA1 versus PEA2 cells. In addition, selected hallmarks (TNFA_signaling_via_NFKB, TGF_beta_signaling, P53_pathway) and IL-6 signaling were positively regulated in PEA1 cells, whereas they were inhibited in PEA2 cells in response to IGFBP6. These data suggested that dysregulation of IGFBP6 signaling may serve a role in the progression of OC, and is likely associated with the development of platinum resistance.

The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17.

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.

Antitumoral Effect of Plocabulin in High Grade Serous Ovarian Carcinoma Cell Line Models

Ovarian cancer (OC) is a life-threatening tumor and the deadliest among gynecological cancers in developed countries. First line treatment with a carboplatin/paclitaxel regime is initially effective in the majority of patients, but most advanced OC will recur and develop drug resistance. Therefore, the identification of alternative therapies is needed. In this study, we employed a panel of high-grade serous ovarian cancer (HGSOC) cell lines, in monolayer and three-dimensional cell cultures. We evaluated the effects of a novel tubulin-binding agent, plocabulin, on proliferation, cell cycle, migration and invasion. We have also tested combinations of plocabulin with several drugs currently used in OC in clinical practice. Our results show a potent antitumor activity of plocabulin, inhibiting proliferation, disrupting microtubule network, and decreasing their migration and invasion capabilities. We did not observe any synergistic combination of plocabulin with cisplatin, doxorubicin, gemcitabine or trabectedin. In conclusion, plocabulin has a potent antitumoral effect in HGSOC cell lines that warrants further clinical investigation.

Computational-based identification and analysis of globally expressed differential genes in high-grade serous ovarian carcinoma cell lines

Ovarian Cancer (OVCA) is the most occurring gynecological cancer worldwide, often diagnosed at a later stage and ultimate results in a high death rate. To overcome this serious health concern, it is important to understand the molecular mechanisms and equally significant to identify the putative biomarkers as well as the therapeutic drug targets for the early diagnosis and treatment of OVCA. In doing so, a strategy is designed to study the most frequently diagnosed cases of OVCA called as High-Grade Serous Ovarian Carcinoma (HGSOC) cell lines with the combination of computational biology, biostatistics and cancer informatics approaches. This study is directed to investigate the global gene expression profiling, and to perform the analyses of identified global Differently Expressed Genes (DEGs) of OVCA. The microarray dataset (GSE71524) is comprised of tumor and cell line samples of OVCA and it was used for the identification of DEGs in the current study. The STRING database was used to construct Protein-Protein Interaction (PPI) network of DEGs, and hub genes were identified by the CytoHubba. In addition, a functional enrichment analysis of up- and down-regulated DEGs was performed by a bioinformatics database called as DAVID. The microRNAs (miRNAs) and transcription factors (TFs) analyses were conducted with the aid of biological tools, MAGIA and GenCOdis3, respectively. As a result, the genes comprised of CSF1R, TYROBP, PLEK, FGR, ACLY, ACACA, LAPTM5, C1 or f162, IL10RA and CD163 were identified as hub genes. Additionally, miRNA analysis resulted in finding an association of zinc finger protein with OVCA comes out after implementing different algorithms. On the other hand, in the TFs analysis resulted in various DEGs that were enriched by NFAT, NF1 and GABP TFs. In this study, it was observed that ACACA, ACLY and CSF1R DEGs showed significant occurrence in different steps, and therefore, these genes were studied, precisely. Nevertheless, the results may help to discover the potential biomarkers with deep understanding of molecular mechanisms. However, further validation is required to explain the OVCA pathogenesis.

Three-Dimensional Cell Culture Based on Magnetic Fields to Assemble Low-Grade Ovarian Carcinoma Cell Aggregates Containing Lymphocytes.

There is a limited number of established ovarian cancer cell lines matching the low-grade serous histotype available for research purposes. Three-dimensional (3D) culture systems provide in vitro models with better tissue-like characteristics than two-dimensional (2D) systems. The goal in the study was to characterize the growth of a given low-grade serous ovarian carcinoma cell line in a 3D culture system conducted in a magnetic field. Moreover, the culture system was evaluated in respect to the assembly of malignant cell aggregates containing lymphocytes. CAISMOV24 cell line alone or mixed with human peripheral blood mononuclear cells (PBMC) were cultured using a commercially available 3D culture system designed for 24 well plates. Resulting cell aggregates revealed the intrinsic capacity of CAISMOV24 cells to assemble structures morphologically defined as papillary, and reflected molecular characteristics usually found in ovarian carcinomas. The contents of lymphocytes into co-cultured cell aggregates were significantly higher (p < 0.05) when NanoShuttle-conjugated PBMC were employed compared with non-conjugated PBMC. Moreover, lymphocyte subsets NK, T-CD4, T-CD8 and T-regulatory were successfully retrieved from co-cultured cell aggregates at 72h. Thus, the culture system allowed CAISMOV24 cell line to develop papillary-like cell aggregates containing lymphocytes.

Involvement of epigenetic modification of TERT promoter in response to all-trans retinoic acid in ovarian cancer cell lines

BackgroundAll-trans retinoic acid (ATRA) is currently being used to treat hematological malignancies, given the ability to inhibit cell proliferation. This effect seems to be related to epigenetic changes of the TERT (Telomerase Reverse Transcriptase) promoter. When hypomethylated, ATRA-inducible TERT repressors can bind the promoter, repressing transcription of TERT, the rate-limiting component of telomerase. Ovarian carcinomas are heterogeneous tumors characterized by several aberrantly methylated genes among which is TERT. We recently found a hypomethylation of TERT promoter in about one third of serous carcinoma, the most lethal histotype. Our aim was to investigate the potential role of ATRA as an anticancer drug in a sub-group of ovarian carcinoma where the TERT promoter was hypomethylated.MethodsThe potential antiproliferative and cytotoxic effect of ATRA was investigated in seven serous ovarian carcinoma and one teratocarcinoma cell lines and the results were compared to the methylation status of their TERT promoter.ResultsThe serous ovarian carcinoma cell line OVCAR3, harboring a hypomethylated TERT promoter, was the best and fastest responder. PA1 and SKOV3, two cell lines with an intermediate methylated promoter, revealed a weaker and delayed response. On the contrary, the other 5 cell lines with a highly methylated promoter did not respond to ATRA, indicative of ATRA-resistant cells.ConclusionsOur results demonstrate an inverse correlation between the methylation level of TERT promoter and ATRA efficacy in ovarian carcinoma cell lines. Although these results are preliminary, ATRA treatment could become a new powerful, personalized therapy in serous ovarian carcinoma patients, but only in those with tumors harboring a hypomethylated TERT promoter.

Abstract 5876: Exploratory testing of PM060184 compound in high-grade serous ovarian carcinoma cell lines

Abstract Purpose of the Study: Marine sponges have developed mechanisms to protect themselves from a hostile marine microenvironment. One of these mechanisms is the use of biologically active metabolites, explored nowadays for their anticancer properties. PM060184 is one of these compounds, isolated from the Madagascan sponge Lithoplocamia lithistoides. This polyketide is currently under evaluation on clinical trials, and its antitumor activity was previously reported in the preclinical setting in a panel of cell lines and subcutaneous tumor xenografts of different origin. The purpose of the study is to explore the effect of PM060184 in a panel of ovarian high-grade serous (HGS) carcinoma cell lines with different sensitivity to cisplatin and paclitaxel. Experimental Procedures: Cell growth inhibition was analyzed by exposure of increasing concentrations of PM060184 in 96 multiwell plates for 72h, by subsequent confluence evaluation and sulphorhodamine (SRB) staining. Cell cycle experiments were done by propidium iodide (PI) staining after treatment of cell lines at their IC50 value for 72h. Three independent experiments with 6 replicates per condition were performed for both approaches. Celigo Image Cytometer platform was employed for phase contrast and viable cells discrimination with a triple staining (Hoechst, PI and calcein AM), as well as for cell cycle analyses. Results: We have focused on the effect of this drug on a panel of HGS ovarian carcinoma cell lines, previously characterized by their response to cisplatin and paclitaxel. We have observed antiproliferative activity in a concentration-dependent manner, with IC50 values at subnanomolar concentrations in all the cell lines tested. This effect was observed in platinum-sensitive and -resistant cell lines and all of them were also more sensitive to PM060184 than paclitaxel, another tubulin-binding agent usually used for the treatment of ovarian cancer. We have evaluated cell cycle and apoptosis at the IC50 values for each cell line. We have seen that this agent disrupts the cell cycle at different phases, as DNA synthesis and mitosis, depending on the cell line. Additionally, an increase in apoptotic cell death is detected as a sub G1 peak by flow cytometry in most of the cell lines tested. Conclusions: PM060184 is a new tubulin-binding agent with a potent antitumor activity in a panel of HGS ovarian cancer cell lines, with different ranges of sensitivity to cisplatin and paclitaxel. Additionally, this effect is more potent than that exerted by other tubulin-binding compounds, such as paclitaxel. However, the underlying mechanism of PM060184 action on ovarian HGS cell lines will need to be further elucidated. Citation Format: Victoria Heredia-Soto, Andrés Redondo, Alejandro Gallego, María Miguel-Martín, Roberto Crespo, Alicia Hernández, David Hardisson, Jaime Feliu, Carlos Galmarini, Marta Mendiola. Exploratory testing of PM060184 compound in high-grade serous ovarian carcinoma cell lines [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 5876.

CAISMOV24, a new human low-grade serous ovarian carcinoma cell line

BackgroundThe spontaneous immortalization of primary malignant cells is frequently assigned to their genetic instability during in vitro culturing. In this study, the new epithelial ovarian cancer cell line CAISMOV24 was described and compared with its original low-grade serous ovarian carcinoma.MethodsThe in vitro culture was established with cells isolated from ascites of a 60-year-old female patient with recurrent ovarian cancer. The CAISMOV24 line was assessed for cell growth, production of soluble biomarkers, expression of surface molecules and screened for typical mutations found in serous ovarian carcinoma. Additionally, comparative genomic hybridization was employed to compare genomic alterations between the CAISMOV24 cell line and its primary malignant cells.ResultsCAISMOV24 has been in continuous culture for more than 30 months and more than 100 in vitro passages. The cell surface molecules EpCAM, PVR and CD73 are overexpressed on CAISMOV24 cells compared to the primary malignant cells. CAISMOV24 continues to produce CA125 and HE4 in vitro. Although the cell line had developed alongside the accumulation of genomic alterations (28 CNV in primary cells and 37 CNV in CAISMOV24), most of them were related to CNVs already present in primary malignant cells. CAISMOV24 cell line harbored KRAS mutation with wild type TP53, therefore it is characterized as low-grade serous carcinoma.ConclusionOur results corroborate with the idea that genomic alterations, depicted by CNVs, can be used for subtyping epithelial ovarian carcinomas. Additionally, CAISMOV24 cell line was characterized as a low-grade serous ovarian carcinoma, which still resembles its primary malignant cells.

Abstract 4409: Cholesterol pathway determines ovarian cancer drug resistance through transcription factor SREBP2

Abstract Ovarian cancer is the most common cause of gynecological cancer death in women in United States. Up to 70% of all ovarian cancer cases are high-grade serous carcinomas with 5-year survival rates less than 30%. Recent studies have suggested the importance of the cholesterol pathway in multiple cancers including gynecological malignancies. Here we demonstrate that genetic or pharmacological disturbance of cholesterol pathway in high-grade serous ovarian carcinoma cell lines results in significant changes in survival rate after drug treatment, and in protein and RNA expression patterns. SREBP2 is a transcription factor encoded by SREBF2 gene that regulates expression of sterol-regulated genes and thus maintains cholesterol homeostasis. To assess the role of SREBP2 in ovarian cancer we have created a stable OVCAR8 cell line with SREBF2 disrupted using CRISPR technology. This SREBF2-KD line has reduced SREBF2 mRNA and protein expression level that indicates an effective gene knockdown. Activation of SREBF2 is dependent on the cholesterol status of the cell. We observed that expression of the SREBP2 precursor form is significantly reduced in SREBF2-KD line when cells are maintained under low serum conditions. Mutant cells treated with paclitaxel in low, but not high serum or in presence of statin, revealed significantly lower cell viability. SREBF2-KD cells do not survive long-term, high concentrations of paclitaxel, nor do they grow back as efficiently as control cells. RNA expression and proteomic analysis revealed the critical regulators mediating SREBP2 activity in stressed cells. Together, these observations suggest that the cholesterol pathway is critical for ovarian cancer cells not only to resist stresses, such as chemotherapy, but also to return to a high proliferation state upon recurrence. Further studies will provide important targets for developing new drugs for treatment of ovarian cancer. Citation Format: Galina Karashchuk, Nataliya Karashchuk, Signe Caksa, Tyler S. Smith, Alexander S. Brodsky. Cholesterol pathway determines ovarian cancer drug resistance through transcription factor SREBP2 [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 4409. doi:10.1158/1538-7445.AM2017-4409

Establishment and characterization of a platinum- and paclitaxel-resistant high grade serous ovarian carcinoma cell line.

High grade serous ovarian cancer (HGSOC) patients have a high recurrence rate after surgery and adjuvant chemotherapy due to inherent or acquired drug resistance. Cell lines derived from HGSOC tumors that are resistant to chemotherapeutic agents represent useful pre-clinical models for drug discovery. Here, we describe establishment of a human ovarian carcinoma cell line, which we term WHIRC01, from a patient-derived mouse xenograft established from a chemorefractory HGSOC patient who did not respond to carboplatin and paclitaxel therapy. This newly derived cell line is platinum- and paclitaxel-resistant with cisplatin, carboplatin, and paclitaxel half-maximal lethal doses of 15, 130, and 20µM, respectively. Molecular characterization of this cell line was performed using targeted DNA exome sequencing, transcriptomics (RNA-seq), and mass spectrometry-based proteomic analyses. Results from exomic sequencing revealed mutations in TP53 consistent with HGSOC. Transcriptomic and proteomic analyses of WHIRC01 showed high level of alpha-enolase and vimentin, which are associated with cell migration and epithelial-mesenchymal transition. WHIRC01 represents a chemorefractory human HGSOC cell line model with a comprehensive molecular profile to aid future investigations of drug resistance mechanisms and screening of chemotherapeutic agents.

Abstract IA02: Functional role of endogenous retroviruses in cancer immunity

Abstract Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections of germ cells, which integrated into the genome of mammals and are inherited in a Mendelian manner. ERVs belong to the class of retro-elements possessing three main viral genes: group-specific antigen (gag), polymerase (pol) and envelope (env) flanked by two long terminal repeats (LTR). More than 8% of the human genome is considered of retroviral origin. Most ERV genes are protein non-functional due to recombination, mutations and deletions; however some ERV gag, pol and env genes produce functional proteins. Although largely gene defective, the LTRs of ERVs retained their regulatory activities resulting in high transcriptional levels; and in addition represent most of the ~10,000 annotated long non-coding (lnc) RNAs. In somatic cells ERVs are generally silent; however we and others demonstrated ERVs become activated during tumorigenesis. Recently we showed that induction of immune responses in human cancer cells by DNA methyltransferase inhibitors (DNMTi), like 5-azacytidine (Aza) and 2'-deoxy-5-azacytidine (Dac) is primarily due to the activation of (hyper)-methylated ERVs (Chiappinelli et al. 2015, Cell 162: 974-86). Furthermore, the unique genomic architecture of ERVs with 5 and 3 LTRs induced sense and antisense RNAs leading to formation of cytosolic dsRNA, which activated the cellular viral RNA sensing and defense pathway (like TLR3 and MAVS) triggering the type I interferon (IFN) response. This kind of response following ERV activation is called viral mimicry or endogenous viremia. Using ovarian carcinoma (OVCA) cell lines as a model, we showed that Aza significantly induced multiple ERV families and IFN stimulated genes (ISG). The induction was due to the formation of dsRNA, which was abolished with RNAse III (hydrolyzing dsRNA) and TLR3 or MAVS specific inhibition. ISGs were also directly induced by transfected ERV genes. Subsequently, a cohort of primary human serous OVCA tissues was analyzed and a significant positive correlation between ERV and ISG expression could be demonstrated. We and others previously showed that treatment with the DNMTi Aza positively contributed to immune checkpoint blockade therapy in a mouse model and in human cancers. Like exogenous retroviruses, which upregulate immune checkpoint genes (ICG), we also evaluated for a possible connection between ERV induction and ICG expression in primary tumors. Presently, we demonstrate that human serous OVCA tissues express significantly higher ICGs than control ovarian tissues, with VTCN-1 (B7-H4) as the highest expressed ICG. Using serous OVCA cell lines transfected with different ERV genes we could show an early induction of ICGs, primarily LGALS9 and PD-L1. Further analysis of tumor infiltrating immune cells using the same serous OVCA cohort and multiplex immunohistochemistry (IHC), we detect in some tumors high levels of tumor-associated CD79a, CD3+/CD8- and CD3+/CD8+ cells. Moreover, we find a direct link between ISG, ICG and infiltrating immune cells in OVCA. Analyzing other human tumors, e.g. brain tumors, like glioblastoma multiforme (GBM) and giant cell GBM (gcGBM) also demonstrate a significant correlation between high ERV induction and ISG as well as ICG expression. In contrast to serous OVCA, PD1 and VTCN-1 were the highest expressed ICG in GBM and gcGBM, which could also be shown with ERV transfected GBM cell lines. Using IHC gcGBM shows a high level of immune cell infiltration, which associated with giant cells and ICG expression, like PD-L1. Interestingly, the highest ERV expression was found in gcGBM, which correlated with endomitosis and cell fusion leading to giant nuclei and multinucleated cells as also shown with a stable ERV transfected GBM cell line. In summary, DNMTi mediated loss of methylation induced ERVs forming dsRNA in tumor cells and stimulated the innate immune system via ISGs. ERV RNA per se induced ISGs, but also ICGs similar to exogenous retroviruses in order to escape the immune system. Different upregulated ERVs, ISGs and ICGs were found in different cancers, like serous OVCA, GBM and gcGBM indicating diverse tumor and tumor microenvironment signatures. Thus ERVs have an ambivalent role in tumorigenesis, where tumor microenvironmental factors tip the balance being detrimental by generating immune evasion or beneficial for cancer therapy by stimulating the innate immune system. Citation Format: Reiner Strick. Functional role of endogenous retroviruses in cancer immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA02.

Abstract 305: EphB4 and EphrinB2 regulates ovarian cancer progression

Abstract EphB4 belongs to the Eph Receptor Tyrosine Kinase family. EphB4 regulates angiogenesis, neuron migration and breast morphogenesis. Overexpression of EphB4 has been observed in numerous cancer types, including breast, ovarian and prostate cancers. How EphB4 regulates cancer progression is still largely unknown. EphrinB2 is the unique ligand for EphB4. EphrinB2 triggers phosphorylation, activation and down-regulation of EphB4. We analyzed various ovarian cancer cell lines and patient derived xenograft samples by in vitro and in vivo assays including siRNA knockdown and gene expression profiling. A large portion of ovarian cancer models express both EphB4 and ephrinB2. SiRNA targeting of EphB4 induced growth inhibition and induction of apoptosis in several cell lines. Serous ovarian carcinoma cell line, Ovcar-3 which expresses both EphB4 and ephrinB2 was most sensitive to siRNA targeting. Non ephrinB2 expressing cell lines were less sensitive suggesting that sensitivity to EphB4 siRNA targeting is not only dependent on the levels of EphB4 but also the status of ephrinB2. Gene expression profiling studies showed that EphB4 is implicated in signaling transduction, cell attachment and growth. Currently, we are examining how ephrinB2 expression modulates EphB4 signaling and the down-stream effectors of EphB4 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2011-305

Interleukin-10 in serous ovarian carcinoma cell lines.

Interleukin-10, one of the most potent antiinflammatory cytokines, is expressed in ovarian carcinomas in vivo. In contrast to the high levels of IL-10 in ascites and tumour tissue, the expression of this cytokine appears to be a rare event in ovarian carcinoma cell lines in vitro. Virtually nothing is known about the regulation of IL-10 expression in ovarian carcinoma cell lines. We investigated the expression of IL-10 in four cell lines originally derived from ovarian serous adenocarcinoma: OVCAR-3, SKOV-3, CAOV-3 and OAW-42. IL-10- specific mRNA was detected in OVCAR-3 and only this cell line produced IL-10 constitutively under serum-free conditions as well as in serum-containing medium. Our studies on the regulation of IL-10 secretion in OVCAR-3 revealed that (1) proinflammatory stimuli IL-1beta and TNF-alpha, but not LPS, enhance IL-10 secretion, (2) IL-6 has no influence on the release of IL-10, (3) prostaglandin E2 influences neither the spontaneous nor the TNF-alpha- or IL-1beta-stimulated IL-10 production and (4) interferon-gamma inhibits IL-10 secretion. We conclude that only a minority of serous ovarian carcinoma cells maintain the ability to produce IL-10 in vitro. Our data on the regulation of IL-10 production in OVCAR-3 indicate that ovarian carcinoma cells share some, but not all, of the regulatory features typical for the monocytic IL-10 secretion.