1A). These data suggest that the translation of SIRT2 is subjected to differential regulation, and SIRT2 MAPK Inhibitor Library screening is expressed at a higher level in HCC cells and tissue. Different from SIRT1, which is localized exclusively in the nucleus in HCC cells,23 both SIRT2 isoforms were predominantly localized in the cytoplasm in HCC cell lines (Fig. 1B), suggesting that SIRT1 and SIRT2 may elicit distinctive functions in HCC. To gain a better understanding of the role of SIRT2 in HCC, we tried to determine the expression level of SIRT2 using clinical specimens

of HCC. We evaluated a panel of commercially available SIRT2 Abs for immunohistochemistry, but the lack of specificity of these Abs precluded the analysis of a SIRT2 expression pattern using HCC tissue microarrays. Alternatively, we determined protein level and significance of SIRT2 expression in 45 pairs of primary HCC and adjacent nontumoral liver. Clinicopathological parameters of these patients are summarized in Table 1. Western blotting analysis

revealed that SIRT2 expression can be detected in the majority of nontumoral liver specimens, but a significant portion MK 2206 of patients showed elevated SIRT2 level in tumor tissues (23 of 45 cases) (Fig. 1C). Moreover, the average level of SIRT2 was found to be significantly higher (P < 0.001) in the tumor group (median, 1.68; quartiles, 1.05-2.24), relative to the nontumoral liver group (median, 1.00; quartiles, 0.66-1.68) (Fig. 1D). However, analysis of SIRT2 messenger RNA (mRNA) levels from these patients by real-time qPCR reveals that average SIRT2 mRNA levels in tumor and nontumoral liver did not differ significantly (data not shown), suggesting that SIRT2 expression in HCC is regulated by transcription-independent mechanisms. Correlative analysis of SIRT2 protein levels with clinicopathologic features suggested significant association between increased SIRT2 expression and the histologic presence of microscopic vascular invasion (P = 0.001) and more-advanced tumor stages (P = 0.004) (Table 1). Up-regulation of SIRT2 in HCC was also found to predict shorter overall survival (P = 0.0499) of patients (Fig. 1E). An earlier

study suggested that there was a role for SIRT2 in the motility of mouse embryonic fibroblasts.18 The GPX6 association between SIRT2 expression and microscopic vascular invasion in HCC also suggested a role of SIRT2 in the cell motility of HCC cells. Therefore, we carried out lentivirus-mediated shRNA knockdown to elucidate the cellular functions of SIRT2. Two independent shRNAs (shSIRT2-1 and shSIRT2-2) showed efficient SIRT2 knockdown in p53 wild-type (WT) (SK-Hep-1 and HepG2) and mutated (PLC5 and Huh-7) HCC cells, compared with scrambled shRNA (shCont)-transduced cells (Fig. 2A). Down-regulation of SIRT2 inhibited the growth of the above HCC cells over a course of 6 days, and this was independent of their p53 status (Fig. 2B and Supporting Fig. 1).