Cancer metastasis is a multi-step process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases 2 (TIMP2) in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly downregulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.1%; 23/56) in human HCCs as compared to the corresponding non-tumorous livers and was significantly associated with direct liver invasion into the adjacent liver parenchyma and poorer survival outcomes of the HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced the cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Furthermore, using orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line MHCC-97L not only significantly reduced the incidence of tumor microsatellite formation and venous invasion in the primary hepatic tumor xenografts, but also pulmonary metastasis, suggesting that both extrahepatic and intrahepatic metastasis in HCC was suppressed by TIMP2 expression. Mechanistically, TIMP2 suppression in a hypoxic environment was induced through a regulatory feedback circuit consisting of HIF-1á, miR-210 and HIF-3á. Taken altogether, our findings established that TIMP2 was frequently downregulated in human HCCs and its downregulation was associated with aggressive behavior and poorer patients’ outcome. Its suppression was under the regulation of a novel feedback circuit consisting of HIF-1á/ miR-210/ HIF-3á. Overall, our study has provided solid in vitro and in vivo evidence that TIMP2 is an important regulator of ECM degradation and HCC metastasis. These findings demonstrated that perturbation of the dynamic HIF-1a signaling circuit by miR-210 inhibitor abolished TIMP2 downregulation and suppressed HCC cell invasion. They also support the notion that targeting against HIF-1á signaling is a promising direction to tackle the hypoxic responses in HCC elicited by transarterial chemo-embolization (TACE) treatment to HCC patients.

Citation Format: Alan KL Kai, Lo Kong Chan, Regina CL Lo, Joyce Lee, Carmen CL Wong, Jack CM Wong, Irene OL Ng. Downregulation of TIMP2 via HIF-1á/miR-210/HIF-3á regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 689.