DOI: 10.25442/hku.16536177.v1
title: Supporting data for Epigenetic silencing of miR-33b promotes peritoneal metastases via targeting TAK1/FASN/CPT1A/NF-kB signaling

description: High-grade serous ovarian carcinoma (HGSOC) is the most common malignant cancer with the highest mortality rate of ovarian cancer. Due to the lack of early-stage clinical symptoms, most HGSOC patients are diagnosed at an advanced stage with peritoneal metastases, which is significantly correlated with a poor prognosis. The omental tumor microenvironment is a complex milieu driving epigenetic alterations, which trigger ovarian cancer development and metastatic progression. However, molecular mechanisms underlying the metastatic ovarian cancer cells affected by epigenetic alterations within the stressful peritoneal-associated tumor microenvironment remain largely unclear. Stressful factors in the tumor microenvironment, such as hypoxia and metabolic starvation, can induce DNA hypermethylation in tumor cells and contribute to epigenetic silencing of tumor suppressor miRNAs. In this study, miR-33b is frequently downregulated in ovarian cancer cells derived from metastatic omental tumor tissues, while its downregulation could be counteracted by the treatment of 5-Aza-dc (DNA methylating inhibitor). In addition, methylation-specific PCR (MS-PCR) and pyrosequencing assay confirmed the promoter hypermethylation of miR-33b during tumor progression and metastasis of ovarian cancer. Here, we identified that miR-33b is frequently silenced by promoter hypermethylation in HGSOC cells derived from metastatic omental tumor tissues. Our laboratory has previously established an omental conditioned medium (OCM) to imitate the omental tumor microenvironment and found that ovarian cancer cells showed increased cell proliferation, migration, and invasion when co-cultured in OCM. On the other hand, OCM-treated ovarian cancer cells displayed a significant increase in de novo lipogenesis and fatty acid oxidation. In this study, it was shown that restoration of miR-33b mitigates the OCM-promoted oncogenic properties in ovarian cancer cells. Moreover, miR-33b overexpression inhibited OCM-stimulated lipid metabolic activities. These results implied that miR-33b exerts tumor suppressor functions by repressing lipid metabolism and, as a consequence, inhibits oncogenic properties and peritoneal metastases of ovarian cancer cells found in the omentum. Mechanistically, miR-33b directly targets 3’ UTR of transforming growth factor beta-activated kinase 1 (TAK1), which is highly associated with tumor progression and metastasis of ovarian cancer. Indeed, TAK1 shows an inversed correlation with miR-33b in the ovarian cancer tissue array. Notably, miR-33b acts as a tumor suppressor by inhibiting OCM-activated TAK1/NF-κB signaling and regulates OCM-initiated lipid reprogramming by abolishing the expression of fatty acid synthase (FASN) and carnitine palmitoyltransferase 1A (CPT1A) in ovarian cancer cells when cultured in OCM. Besides, depletion of TAK1 prevents OCM-facilitated lipid biosynthesis and fatty acid degradation by repressing the activities of FASN and CPT1A in OCM-cultured ovarian cancer cells. The treatment of FASN or CPT1A inhibitor reduces the OCM-mediated NF-κB signaling activities. These results indicate that miR-33b directly targets TAK1 and results in an inhibition of FASN and CPT1A expression, thereby suppressing phosphorylation of NF-κb signaling and impairing OCM-induced oncogenic behaviors of ovarian cancer cells. In conclusion, this study suggests that the lipid-rich tumor microenvironment is associated with epigenetic silencing of miR-33b in ovarian cancer. miR-33b exerts tumor suppressor functions to prevent ovarian cancer peritoneal dissemination and omental metastasis, at least in part, by targeting the TAK1/FASN/CPT1A/NF-κB signaling axis. <br>