Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MARK/ERK signaling pathway

Abstract

Ovarian cancer is one of the most common cancers with a high mortality rate in women. The major reason is a lack of reliable biomarkers that caused most ovarian cancer patients diagnosed at late-stage, which is strongly accompanied with a high rate of metastasis, recurrence, and chemoresistance. Tumor metastasis is the most difficult obstacles in the clinical management of this disease. Different from other solid tumors, peritoneal metastasis is the common route of metastasis in advanced ovarian cancer. Ovarian cancer patients with such route of metastasis have a poor prognosis. Emerging evidence has suggested that epigenetic alteration rather than somatic mutation is the dominant driver of cancer development and progression. Hence, a deeper knowledge of such epigenetic mechanisms underlying the progression of ovarian cancer will assist in exploring better cancer therapeutic regimes to cure this cancer. Our laboratory has previously reported that human growth factor receptor-bound protein 7 (GRB7), a multidomain adapter protein involved in receptor tyrosine kinase signaling such as MAPK pathway and regulates diverse biological processes, is aberrantly upregulated and correlated with advanced-stage and high-grade ovarian cancer. Of note, a discrepancy between mRNA and protein levels of GRB7 is found in some ovarian cancer cells, suggesting there is an abnormal post-transcriptional regulation for GRB7 in ovarian cancer cells. In this study, both in vitro and in vivo tumorigenic assays were used to determine that GRB7 enhances tumor formation and metastatic dissemination. Its upregulation is correlated with poor survival of human ovarian cancer, emphasizing its clinical relevance. On the other hand, microRNAs (miRNAs) have been characterized as emerging post-transcriptional regulators and are involved wildly in the regulation of gene expression. By in silico and experimental approaches, miR-193a-3p and miR-193b-3p were shown to directly targeting 3’ UTR of GRB7, exhibited distinct expression patterns in ovarian cancer cells. Indeed, only miR-193a-3p shows an inversed correlation with GRB7 in ovarian cancer cells, as well as in an ovarian cancer tissue array (OVC1-021, Pantomics). Further studies identified that miR-193a-3p modulates not only GRB7 but also other key components (ERBB4, SOS2, and KRAS) of MAPK signaling pathway. On the other hand, miR-193a-3p exhibited a significant stepwise reduction from low to high grade ovarian cancer, and its downregulation could be restored by a DNA methyltransferase inhibitor (5-Aza-dc) in ovarian cancer cells. Methylation-specific PCR (MS-PCR) and bisulfite pyrosequencing revealed that miR-193a-3p was gradually hypermethylated during the progression of ovarian cancer. Enforced expression or doxycycline-induction of miR-193a-3p could inhibit cell proliferation, cell migration, and cell invasion. Consistently, the conditional induction of miR-193a-3p significantly inhibit MAPK signaling and abrogate the tumorigenic properties like metastatic colonization in mouse models. Taken together, this study suggests that epigenetic silencing of miR-193a-3p activates GRB7 and MAPK/ERK signaling pathway during the development and progression of ovarian cancer. This study also proposes a paradigm confirming that epigenetic alterations play dominantly in promoting tumor progression of advanced ovarian cancer.