Abstract No. 555 uPAR and MMP-9 expression is coupled to HIF-1alpha and invasiveness of hepatocellular carcinoma and CRLM tumors

Abstract

Changes in the microenvironment coupled with extracellular matrix remodeling is essential for solid tumors to survive and metastasize. Unraveling the causal mechanisms allows for identification of novel targets and determination of efficacy of targeted treatments. The upregulation of uPAR and MMP-9 are known to be associated with tumor invasiveness, angiogenesis and tumor growth. The purpose of this study is to evaluate whether uPAR and MMP-9 expression is coupled to HIF-1α and whether suppression decreases the aggressiveness of HCC and CRLM tumors. Hepatocellular carcinoma (MCA-RH777), colorectal liver metastasis (CC-531) and immortalized liver (Clone 9) cell lines, grown in DMEM medium at 37°C in a humidified atmosphere with 5% CO2 in normoxic and < 1% O2 hypoxic conditions, were assessed for the expression of HIF-1α, uPAR and MMP-9 by immunocytochemistry. Cells cultured in matrigel-coated transwell were assessed for invasiveness under normoxic and hypoxic conditions. Cells were treated with a HIF-1α inhibitor (R59949) and doxorubicin, and matrigel invasion assays were performed in vitro. Further, tumor sections explanted from rabbit and rat models of HCC and CRLM were stained for the expression of uPAR, MMP-9 and HIF-1α, and compared to normal liver sections after treatment with doxorubicin, R59949 or in combination. HCC and CRLM cells presented striking increases in HIF-1α, uPAR and MMP-9 expression under hypoxic conditions. Percentage of hypoxia induced invasive cells were higher among CRLM cells compared to HCC cells at 12h in Matrigel invasion assays. R59949 significantly reduced the invasion of both CRLM and HCC cells under hypoxic conditions. Immunohistochemistry revealed that rat and rabbit HCC tumors and rat CRLM tumors have abundant levels of HIF-1α, uPAR and MMP-9 compared to the respective normal liver sections. Increase of CRLM and HCC cell invasion under hypoxic conditions is coupled with uPAR and MMP-9 expression.