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Background: E2F5 is a transcription factor that is overexpressed in the early stages of ovarian cancer and has been suggested as a potential biomarker for early detection. In this study, we aimed t...

High-grade epithelial ovarian cancer is a fatal disease in women frequently associated with drug resistance and poor outcomes. We previously demonstrated that a marine-derived compound MalforminA1 (MA1) was cytotoxic for the breast cancer cell line MCF-7. In this study, we aimed to examine the effect of MA1 on human ovarian cancer cells. The potential cytotoxicity of MA1was tested on cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) ovarian cancer cell lines using AlamarBlue assay, Hoechst dye, flow cytometry, Western blot, and RT-qPCR. MA1 had higher cytotoxic activity on A2780S (IC50 = 0.23 µM) and A2780CP (IC50 = 0.34 µM) cell lines when compared to cisplatin (IC50 = 31.4 µM and 76.9 µM, respectively). Flow cytometry analysis confirmed the cytotoxic effect of MA1. The synergistic effect of the two drugs was obvious, since only 13% of A2780S and 7% of A2780CP cells remained alive after 24 h of treatment with both MA1 and cisplatin. Moreover, we examined the expression of bcl2, p53, caspase3/9 genes at RNA and protein levels using RT-qPCR and Western blot, respectively, to figure out the cell death mechanism induced by MA1. A significant down-regulation in bcl2 and p53 genes was observed in treated cells compared to non-treated cells (p &lt, 0.05), suggesting that MA1 may not follow the canonical pathway to induce apoptosis in ovarian cancer cell lines. MalforminA1 showed promising anticancer activity by inducing cytotoxicity in cisplatin-sensitive and cisplatin-resistant cancer cell lines. Interestingly, a synergistic effect was observed when MA1 was combined with cisplatin, leading to it overcoming its resistance to cisplatin.

Objectives: New compounds are needed to overcome the resistance to commonly used cytotoxic chemotherapy for epithelial ovarian cancer. Marine sponges are a rich source of diverse chemical compounds and hymenialdisine has been found to have antiproliferative effects. This study aimed to investigate the cytotoxic effect of hymenialdisine in cisplatin-sensitive and cisplatin resistant ovarian cancer cell lines. Methods: This study took place at Sultan Qaboos University, Muscat, Oman between August and November, 2019. The anti-cancer effects of hymenialdisine or cisplatin were assessed using treating cells with different concentrations of hymenialdisine and cisplatin. Cell viability was determined using the AlamarBlue® Assay. Results: The half-maximal inhibitory concentration (IC50) of cisplatin was estimated at 31.4 μM for A2780S and 76.9 μM for A2780CP, whereas the IC50 of hymenialdisine was evaluated at 146.8 μM for A2780S cells. Despite the higher concentrations of hymenialdisine (up to 300 μM), IC50 could not be determined for the A2780CP cell line. Conclusion: When compared to cisplatin, hymenialdisine was less toxic against both A2780S and A2780CP ovarian cancer cell lines. Keywords: Gynecologic Neoplasm; Ovarian Cancer; Cisplatin; Hymenialdisine; Oman.

Background The adhesion molecule, FAT4, has a tumor suppressor function with a critical role in the epithelial-to-mesenchymal-transition (EMT) and anti-malignant growth in several cancers. No study has investigated yet its role in epithelial ovarian cancer (EOC) progression. In the present study, we examined the role of FAT4 in proliferation and metastasis, and its mechanisms of interaction in these processes. Methods We have performed cell viability, colony formation, and invasion assays in ovarian cancer cells treated with siRNA to knockdown FAT4 gene expression. The regulatory effects of FAT4 on proteins involved in apoptotic, Wnt, Hippo, and retinoblastoma signaling pathways were evaluated by Western blotting following FAT4 repression. Also, 426 ovarian tumor samples and 88 non-tumor samples from the Gene Expression Profiling Interactive Analysis (GEPIA) database were analyzed for the expression of FAT4. Pearson’s correlation was performed to determine the correlation between FAT4 and the E2F5, cyclin D1, cdk4, and caspase 9 expressions. Results Lower expression of FAT4 was observed in ovarian cancer cell lines and human samples as compared to non-malignant tissues. This down-regulation seems to enhance cell viability, invasion, and colony formation. Silencing FAT4 resulted in the upregulation of E2F5, vimentin, YAP, β-catenin, cyclin D1, cdk4, and Bcl2, and in the downregulation of GSK-3-β, and caspase 9 when compared to control. Furthermore, regulatory effects of FAT4 on the EMT and aggressive phenotype seem to occur through Hippo, Wnt, and cell cycle pathways. Conclusion FAT4 downregulation promotes increased growth and invasion through the activation of Hippo and Wnt-β-catenin pathways.

Breast and ovarian cancer are heterogeneous diseases. While breast cancer accounts for 25% of cancers worldwide, ovarian cancer accounts for 3.5% of all cancers and it is considered to be the most lethal type of cancer among women. In Oman, breast cancer accounts for 25% and ovarian cancer for 4.5% of all cancer cases. Various risk factors, including variable biological and clinical traits, are involved in the onset of breast and ovarian cancer. Although highly developed diagnostic and therapeutic methods have paved the way for better management, targeted therapy against specific biomarkers has not yet shown any significant improvement, particularly in triple-negative breast cancer and epithelial ovarian cancer, which are associated with high mortality rates. Thus, elucidating the mechanisms underlying the pathology of these diseases is expected to improve their prevention, prognosis and management. The aim of the present study was to provide a comprehensive review and updated information on genomics and proteomics alterations associated with cancer pathogenesis, as reported by several research groups worldwide. Furthermore, molecular research in our laboratory, aimed at identifying new pathways involved in the pathogenesis of breast and ovarian cancer using microarray and chromatin immunoprecipitation (ChIP), is discussed. Relevant candidate genes were found to be either up- or downregulated in a cohort of breast cancer cases. Similarly, ChIP analysis revealed that relevant candidate genes were regulated by the E2F5 transcription factor in ovarian cancer tissue. An ongoing study aims to validate these genes with a putative role as biological markers that may contribute to the development of targeted therapies for breast and ovarian cancer.

Introduction/Background FBXW7, a tumor suppressor gene plays a critical role in cell cycle progression under the regulation of the transcription factor E2F5. While FBXW7 is silenced in several cancers; its role in ovarian cancer pathogenesis is still unclear. This study explored FBXW7 promotor methylation status in ovarian cancer and its functional significance. Methodology FBXW7 expression at mRNA and protein levels were assessed in OVSAHO and MCAS cells using RT-qPCR and western blotting respectively. Expression of FBXW7 was high in MCAS and low in OVSAHO cells. FBXW7 gene methylation status was analyzed in cell lines (MCAS & OVSAHO) as well as in 23 EOC patients (5 normal, 8 benign, 4 borderline, 5 high grade tumors) by both methylation-specific PCR and MS-HRM analysis. Results In addition to OVSAHO cells, more than 60% of patients analyzed showed methylation. To evaluate its functional importance, FBXW7 expression was restored in OVSAHO cells by treatment with the demethylation agent, 5’-aza-2’deoxycitidine. Western blotting in FBXW7-restored OVSAHO cells and FBXW7-positive cell line (MCAS) showed an upregulation in caspases-2, 3, 7, BAX, P53, BAD, Cyclin D1, pNFkB and pGSK3-beta, whereas Bcl-2, Bcl-x, pBad, STAT3, GSK3-beta and NFkB were downregulated; indicating cells to undergo apoptosis via STAT3 pathway. Conclusion This study described an alternative mechanism for FBXW7 inactivation, namely promoter specific methylation in ovarian cancer and identified FBXW7 as a potential target for guiding the development of therapeutic strategies against ovarian cancer. Disclosure Nothing to disclose.

Introduction/Background Epithelial ovarian cancer (EOC) accounts for about 70% of all types of ovarian cancer.It is a heterogeneous disease and has variable prognosis. In a previous study, chromatin immunoprecipitation (ChIP) was used to study expression of E2F5, a transcription factor, and revealed that FAT4, is regulated by E2F5. FAT4, a tumor suppressor gene, is an upstream regulator of the Hippo pathway and its expression in EOC remains unclear. Objective We aimed to investigate the involvement of FAT4 gene in EOC by quantifying its expression in MCAS, OVSAHO, A2780cp and A2780s ovarian cancer cell lines and compare to HOSE, a normal ovarian cell line. Methodology We evaluated the differential expression of FAT4 and E2F5 in five ovarian cancer cell lines using qRT-PCR and western blotting. We knocked down FAT4 and E2F5 in MCAS and OVSAHO using siRNA and monitored it at the mRNA and protein levels. The effect on viability and invasion following knockdown were assessed by alamar blue and invasion assay. The mechanism linking FAT4 and E2F5 was evaluated by assessing proteins involved in Hippo, Retinoblastoma and apoptotic pathways. Results MCAS and OVSAHO showed higher FAT4 expression compared to A2780s and A2780cp. FAT4 and E2F5 were knocked down significantly using siRNA in MCAS (FAT4: p Conclusion Down regulation of FAT4 in ovarian cancer cell lines promotes growth and invasion via the modulation of apoptosis, Rb and Hippo pathways. Disclosure Nothing to disclose.

Introduction/Background Ovarian cancer is one of the fatal cancers in women, often associated with drug resistance. Therefore, finding effective drugs, including naturally derived compounds is urgently needed. We have previously examined the effect of gallic acid (GA) on breast cancer cell line MCF-7 and found a significant anti-cancer activity. We aimed to test the anti-cancer potential of GA monohydrate and six different derivatives of GA on cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) human epithelial ovarian cancer cell lines and on HOSE6-3 normal epithelial ovarian cell line. The effect of cisplatin and GA in combination was also examined. Methodology Cells were exposed to different concentration of GA (0–100 µg/ml), and cisplatin (0–24 µg/ml) and cell viability was tested via AlamarBlue and CCK08 assays. Apoptosis was determined by Hoechst stain. Results GA monohydrate decreased cell viability in a dose- and time-dependent manner in all cell lines. GA monohydrate was demonstrated to have the highest cytotoxicity to both A2780S and A2780CP cell lines compared to all six derivatives. The highest cytotoxicity of GA was observed in A2780S (IC-50=19.4 µg/ml), followed by A2780CP cells (IC-50=35.6 µg/ml), whereas the least cytotoxicity was observed in HOSE6-3 (IC-59=49.3 µg/ml). Similarly, cisplatin displayed a higher cytotoxicity on A2780S (IC-50=9.4 µg/ml) than on A2780CP (IC-50=23.1 µg/ml). However, cisplatin was more toxic to HOSE6-3 than ovarian cancer cells (IC-50=8.8 µg/ml). There was no significant difference in cytotoxicity between cisplatin and GA either combined or used separately. Apoptosis was estimated to be 49% and 47% in A2780S and A2780CP cell lines respectively at a GA concentration of 25 µg/ml. Conclusion GA monohydrate exhibited significant cytotoxicity in ovarian cancer cell lines independent of cisplatin, suggesting a cytotoxic effect of GA on ovarian cancer. The growth inhibitory effect of GA, especially on resistant ovarian cancer cells, merits further investigation. Disclosure We are greatly thankful for His Majesty Sultan Qaboos Trust Fund Grant (SR/MED/MEDE/17/01). Also, we would like to thank Ms Buthaina Al Dhahli for her endless support and help.

Introduction/Background Epithelial Ovarian Cancer (EOC) is the most lethal disease among gynecologic cancers. It is usually diagnosed at late stages with a poor outcome due to its asymptomatic nature. In Oman, the incidence rate for 2013 was estimated at 2.9 per 100,000. There is an urgent need to identify biomarkers for an early detection of EOC. we used chromatin immune precipitation (ChIP) to identify downstream genes regulated by the E2F5 transcription factor (TF) in ovarian cancer cell lines (MCAS and OVSAHO). Results revealed that SOCS-2 was regulated by the TF E2F5. SOCS (1–8) family have a role in JAK/STAT transcription pathway. Any disruption in this pathway may lead to many diseases such as cancers. Some of these SOCS genes found to act as biomarker for some cancers. SOCS2 was recently shown to act as an early biomarker for prostate and colorectal cancer. The aim of this study is to explore the role of SOCS2 gene in EOC by monitoring the expression of the gene in stages variety of EOC tissues and cell lines. Methodology We examined the expression of SOCS2 in tissues and cell lines using the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to monitor the differential expression of SOCS2 in cancerous and normal tissues. Results Significant down-regulation of SOCS2 was found in borderline tissues but it was not significant in other cancer stages such as benign and malignant tissues. Although all examined cell lines (MCAS, OVSAHO and 2008OV) showed a significant downregulation of the gene. SOCS2 methylation was investigated using MSP technique. It was revealed that SOCS2 is methylated in OVSAHO cell line but not in MCAS cells while cancerous tissues showed partially methylation. Conclusion The down regulation of SOCS2 gene in EOC is not caused by methylation; other mechanisms might be involved. Disclosure Nothing to disclose.

Introduction/Background High grade epithelial ovarian cancer is often associated with drug resistance and poor outcomes. Previously, we demonstrated significant cytotoxic efficacy of a marine compound MalforminA1 against MCF-7 breast cancer cell line. Methodology We tested the anti-cancer potential of MalforminA1on cisplatin-sensitive (A2780s) and cisplatin-resistant (A2780cp) ovarian cancer cell lines using AlamarBlue assay, flow cytometry, western blot and RT-qPCR. Results MalforminA1 had significantly higher cytotoxic activity when compared to cisplatin in both A2780s and A2780cp cell lines (IC500.23µMand 0.34µM compared to 31.4 µM and 76.9 µM p Conclusion MalforminA1 showed promising anti-cancer activity by introducing apoptosis, and therefore warrants further investigation. Disclosure We are greatly thankful for the support through His Majesty Trust Fund Grant (SR/MED/MEDE/17/01).

Objectives The SOCS3 gene is a key regulator for JAK/STAT pathway, responsible for inflammation and proliferation response, was found to be regulated by E2F5 and its down regulation seems to have a role in the pathogenesis of different tumors including gastric and pancreatic cancers. Our aim to investigate the involvement of SOCS3 gene in EOC by monitoring its expression in ovarian cancer cell lines and EOC tissue samples, then to compare results to normal ovarian tissue samples. We also examine methylation status of both, ovarian cell lines and ovarian tissue specimen. Methods Real time qPCR was used to assess gene expression using Taq Man gene expression assay. Five cell lines (MCAS, OVSAHO, OV 2008, A2780s and A2780cp) and 19 tissue samples with different histopathology types (6 normal, 4 benign, 5 borderlines and 4 high grade serous) were examined. Methylation status analysis was performed by methylation specific PCR Results OVSAHO, OV2008 and A2780s cell lines showed down regulation of SOCS3 expression when compared to normal. Benign, borderline and high grades samples, displayed also significant down regulation of SOCS3 expression. Analysis of methylation pattern showed no hyper-methylation in both cell lines and tissues. Conclusions Down regulation of SOCS3 gene expression was detected in ovarian cancer cell lines and EOC tissue samples, suggesting a putative role of SOCS3 in the pathogenesis of EOC. Other epigenetics mechanisms such as micro-RNAs are involved in the regulation of SOCS3 expression in addition to hyper-methylation and therefore, further study is needed to uncover these mechanisms.

Epithelial ovarian cancer (EOC) is a deadly q disease and the related statistics are alarming with 15000 lives claimed in USA each year. EOC is a subtle disease since the majority of patients at presentation are diagnosed with a higher stage disease characterized by an aggressive potential. Early diagnosis seems to be of importance since complete cure can reach up to 90% if the disease is diagnosed at an early stage. Therefore, biological markers to detect patients at an early stage of the disease progression are urgently needed. In this study, the focus was centered on the transcription factor E2F5 used as biomarker and highly expressed at early stages of EOC pathogenesis. Therefore, we hypothesize that downstream genes regulated by this transcription factor might be also involved in the early events leading to ovarian cancer and can therefore potentially serve as useful biomarkers. Chromatin Immuno-Precipitation (ChIP) was performed using two ovarian cancer cell lines SK-OV-3 and OVCAR-3 and an antibody against E2F5 transcription factor. The enriched chromatin was cloned and sequenced before using available databases for BLAST and identification of downstream regulated genes. A short list of relevant genes, all involved in cancer, was obtained and analysis for their putative role in early detection of EOC is ongoing. The selected genes will be also validated using frozen human ovarian tissues available in our institution. ChIP is a robust technology to identify genes regulated by E2F5 with a putative potential to play a crucial role in the early steps of ovarian cancer pathogenesis. Citation Format: Yahya Tamimi, Ikram Burney, Moza Al-Kalbani, Ritu Lakhtari, Roseline Godbout, Mansour Al-Moundhri. Identification of putative genes involved in early steps of epithelial ovarian cancer pathogenesis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1130.