Abstract
Ras proteins (HRas, KRas and NRas) are common oncogenes that require membrane association for activation. Previous approaches to block/inhibit Ras membrane association were unsuccessful for cancer treatment in human clinical studies. In the present study we utilized a new approach to decrease Ras membrane association in hepatocellular carcinoma (HCC) cell lines via inhibition of an enzyme aminoacylase 3 (AA3; EC 3.5.1.114). AA3 expression was significantly elevated in the livers of HCC patients and HCC cell lines. Treatment of HepG2 cells with AA3 inhibitors, and HepG2 and HuH7 with AA3 siRNA significantly decreased Ras membrane association and was toxic to these HCC cell lines. AA3 inhibitors also increased the levels of N-acetylfarnesylcysteine (NAFC) and N-acetylgeranylgeranylcysteine (NAGGC) in HepG2 and Huh7 cell lines. We hypothesized that AA3 deacetylates NAFC and NAGGC, and generated farnesylcysteine (FC) and geranylgeranylcysteine (GGC) that are used in HCC cells for the regeneration of farnesylpyrophosphate and geranylgeranylpyrophosphate providing the prenyl (farnesyl or geranylgeranyl) group for Ras prenylation required for Ras membrane association. This was confirmed experimentally where purified human AA3 was capable of efficiently deacetylating NAFC and NAGGC. Our findings suggest that AA3 inhibition may be an effective approach in the therapy of HCC and that elevated AA3 expression in HCC is potentially an important diagnostic marker.