Rahul Bhagat

Microsatellite instability, promoter methylation and protein expression of the DNA mismatch repair genes in epithelial ovarian cancer.

The role of defective mismatch repair (MMR) system in ovarian carcinoma is not well defined. The purpose of the study was to determine the relationship between microsatellite instability (MSI), promoter methylation and protein expression of MMR genes in epithelial ovarian carcinoma (EOC). MSI and promoter methylation of MLH1, MSH2 and PMS2 genes were studied using PCR methods in the study cohort. A small subset of samples was used to analyze the protein expression by immunohistochemistry (IHC). MSI was observed in >60% of tumor samples and 47% of normal ovaries. MLH1 was methylated in 37.5% and 64.3% EOCs and LMP tumors. The loss of immunoexpression of MMR genes was not seen in ovarian tumors. There was no correlation between MSI, promoter methylation and protein expression of the MMR genes suggesting that each may function independently. MSI is a common event in ovarian carcinoma and may increase the clinical awareness of the subset of tumors.

Investigating impact of Vascular Endothelial Growth Factor Polymorphisms in Epithelial Ovarian Cancers: A Study in the Indian Population

Epithelial ovarian cancer is one of the increasingly incident malignancies that is notorious because of its evasiveness for early diagnosis and high mortality rates. Epithelial ovarian cancers are highly dependent on pathologic vasculature and Vascular Endothelial Growth Factor is known to be one of the most efficient angiogenic factors. Polymorphisms of the VEGF gene, in this study, were assessed for association with the malignancy and other clinico-pathological factors. 300 case samples and 320 age and mensus status matched controls were inculcated into the study. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were the six single nucleotide polymorphisms that were scrutinized. Genotyping was carried out by polymerase chain reaction and restriction fragment length polymorphism. rs 3025039 showed immense promise as a marker for disease aggression and recurrence and a factor for poor prognosis. rs699947 showed least association with the disease and clinico-pathologic factors studied. rs833061, rs 1570360 showed significant association with some clinico-pathological factors such as bilateral affliction of ovaries and post operative CA-125 levels. rs2010963 associated with presence of ascites in higher volumes. The SNPs under consideration showed no formidable linkage in our study samples. A haplotype analysis (excluding rs699947 and rs1413711) revealed 5 frontrunners being present in >85% of the population with TGGC and CGCC associating significantly as protective and risk factors respectively. These haplotypes showed a dose dependent additive effect of their seeming functionality. This study is unique and a first of its kind carried out in the Indian population of South-east Asia.

GSTP1 expression and promoter methylation in epithelial ovarian carcinoma

Context: GSTP1 is a subgroup of glutathione-S-transferase family, which provides cellular protection against free radical and carcinogenic compounds due to its detoxifying function. Altered GSTP1 activity due to down regulation of enzyme activity and DNA methylation has been reported in many tumors, although data for ovarian cancer are few. In this study, we aimed at determining the expression of GSTP1 in relation to the methylation of the GSTP1 promoter in epithelial ovarian cancer (EOC). Materials and Methods: GSTP1 mRNA expression and GSTP1 enzyme concentration were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay, respectively, in 88 EOCs, 14 low malignant potential (LMP) tumors, and 20 benign tumors. The promoter methylation of GSTP1 gene was evaluated by methylation-specific PCR. Results: Reduced GSTP1 mRNA expression was observed in 49% EOCs, 21.4% LMP, and 45% benign tumors. Significantly lower levels of plasma GSTP1 were observed in all tumor samples compared to normal. GSTP1 promoter methylation was detected in 10 (11.4%) EOCs and 1 (7.3%) LMP tumors. No methylation was observed in benign tumors and normal ovaries. Conclusions: Our results show that there is a significant down regulation of GSTP1 expression while hypermethylation of the GSTP1 gene promoter is not very frequent in EOC. Further studies are needed to study underlying mechanisms leading to decreased expression.

Follicle stimulating hormone is an autocrine regulator of the ovarian cancer metastatic niche through Notch signaling

The association between upregulated Notch and FSH signaling and ovarian cancer is well documented. However, their signaling functions have been predominantly probed independently, and in the context of primary tumor tissues. The aim of this study was to investigate the interactive effects of FSH and Notch signaling on the formation and maintenance of disseminated metastatic ovarian cancer cells. We found that FSH upregulated Notch signaling in the ovarian cancer cells by increasing the expression of Notch receptors and ligands. The proliferation rate of the ovarian cancer cells was also increased in the presence of FSH. Further, high levels of FSH were detected in the ascites of patients with serous ovarian adenocarcinoma. The spheroids from the ascites of the patients, as well as, the spheroids from the ovarian cancer cell lines, expressed FSHβ subunit mRNAs and secreted the hormone into the medium. In contrast, primary ovarian tumor tissues and cell line monolayers expressed very low levels of FSHβ. Ovarian cancer cells cultured as spheroids exhibited higher expression of the FSH receptor (FSHR) and Notch downstream genes than counterpart monolayers. The Single chain Fragment variables (ScFvs) specific for the Negatively Regulatory Region (NRR) of Notch, and antibodies against the extracellular domain of FSHR, in combination, significantly inhibited spheroid formation and cell proliferation in vitro. In conclusion, this study demonstrates the autocrine regulation of ovarian cancer spheroidogenesis through FSH-driven Notch signaling. Further, the Notch and FSHR together are the potential immunotherapeutic targets for the treatment of the ovarian cancer metastasis.

Aberrant promoter methylation and gene expression of H‑cadherin gene is associated with tumor progression and recurrence in epithelial ovarian carcinoma

Background: Loss of expression of cadherins by promoter hypermethylation has been described in many epithelial cancers, and it may play a role in tumor cell invasion and metastasis. Previously, we reported that E-cadherin gene is frequently methylated in epithelial ovarian cancer. Aim: The aim of this study was to compare the promoter hypermethylation of H-cadherin gene in ovarian epithelial neoplasms to better understand the role of epigenetic silencing in carcinogenesis. Materials and Methods: We examined the promoter methylation of the H-cadherin gene in 134 epithelial ovarian carcinomas (EOC), 23 low malignant potential (LMP) tumors, 26 benign cystadenomas and 15 normal ovarian tissues. Methylation was investigated by methylation specific polymerase chain reaction (MSP) and the results confirmed by bisulfite DNA sequencing. Relative gene expression of H-cadherin was done using quantitative reverse transcriptase PCR on 51 EOC cases, 9 LMP tumors, 7 benign cystadenomas with 5 normal ovarian tissues. Results: Aberrant methylation of H-cadherin was present in 20 of 134 (15%) carcinoma cases, 2 of 23 (09%) LMP tumors and 1 of 26 (4%) benign cystadenomas. No methylation was observed in any of the normal ovarian tissues. The mRNA expression level of H-cadherin was significantly down-regulated in EOC and LMP tumors than the corresponding normal tissues, whereas the expression level was normal in benign cystadenomas. A significant correlation of H-cadherin promoter methylation was observed with reduced gene expression in EOC. The prevalence of H-cadherin methylation was associated significantly with stage, histopathological grade, and menopausal status of the patient. H-cadherin methylation also had significant association with recurrence and differentiation of tumor. Conclusion: Our findings suggest an association between H-cadherin methylation, tumor progression and recurrence in EOC.