ABSTRACT: Development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. We have previously demonstrated that constitutively photomorphogenic 1 (COP1), which regulates p53 activity by ubiquitination, is frequently overexpressed in human HCC. Here we examined whether molecular targeting of COP1 by small interfering (si) RNA can affect the course of HCC progression. COP1-1 was selected as the most effective target siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Interestingly, the growth inhibition occurred both in HCC cells that retain wild-type p53 or express mutant p53 (Y220C or R249S). Next we have determined to investigate the molecular mechanisms underlying the phenotypic changes. Given the recent findings that COP1 functions as a negative regulator of p53, we addressed whether the phenotypic changes caused by COP1 silencing were due to alterations in p53 and/or p21 status. Indeed, in COP1-depleted HepG2 cells expressing wild type p53, induction of apoptosis was associated with restoration of p53 function as judged by a marked increase in the levels of p53 and its target p21, suggesting that cell death was p53-dependent. However, the COP1 silencing in Huh7 cells, which carry Y220C mutation, caused a strong induction of apoptosis without changing p53 levels. To further address this issue, we next looked for the global transcriptional changes underlying the antitumor effects of COP1 silencing in HCC cells [with different p53 status]. For this purpose, Huh7 and HepG2 were treated with NCsiRNA and COP1-1siRNA for 48 hours and subjected to illumina microarray analysis. We found that the number of differentially expressed genes which displayed more than a 2-fold change (P < 0.01 by Bootstrap t-test) was 462 (167 up- and 295 down-regulated genes) and 522 (179 up- and 343 down-regulated genes) in COP1siRNA-treated Huh7 and HepG2 cells, respectively. As expected, the expression levels of RFWD2 (COP1) mRNA were significantly reduced in both treated HCC cell lines. Consistent with phenotypic changes, COP1-depleted Huh7 cells also displayed changes in p53-associated group of genes functionally involved in regulation of apoptosis, growth and differentiation including CASP6, GLIPR1, FHL2, GADD45A, HMOX1 BCL6, FOXO3, GDF15, and SCNN1A genes. Finally, we generated the common COP1 knockdown gene signature which included 78 genes. The Ingenuity Pathway Analysis analysis revealed that the 5 top putative networks with high score (>19) were strongly associated with NF-κB, HNF4α, TNF, and p53 pathways, suggesting that a common subset of molecular alterations in the diverse oncogenic pathways may cooperatively result in growth inhibition of HCC cells with wild type and mutant p53. Analysis of COP1 knockdown gene expression signatures by microarray revealed that the anti-proliferative effect was driven by a common subset of molecular alterations including p53-associated functional network. Systemic delivery of a modified COP1siRNA by stable-nucleic-acid-lipid-particles (SNALP) significantly suppressed neoplastic growth in liver, without unwanted immune response in an orthotopic xenograft model. These findings provide the first proof of principle that COP1 is a promising target for systemic therapy of HCC.
