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. Microarray was performed on human Ref-8v3 microarrays (illumina) as recommended by the manufacture. In detail, total RNAs were isolated 48 h after the transfection of NCsiRNA or COP1-1siRNA to Huh7, HepG2 or Hep3B cells. Biotin-labeled cRNA was linearly amplified according to manufacturerâs specification (AMIL1791; Ambion, Austin, TX). As input, 200 ng total RNA from tumor was used for the in vitro transcription (IVT) reactions which were incubated for 16 h at 37 ºC. The efficiency of this single round amplification was measured by NanoDrop (ND1000, Thermo Scientific). Hybridization, washing, detection (Cy3-streptavidin, Amersham Biosciences, GE Healthcare) and scanning were performed on an Illumina iScan system (Illumina) using reagents and following protocols supplied by the manufacturer. Briefly, the biotinylated cRNA (750 ng/sample) was hybridized on Sentrix whole genome beadchips human Ref-8v3 for 18 h at 58ºC while rocking (5 rpm). The beadchip covers ~ 24,000 RefSeq transcripts. Image analysis and data extraction were performed automatically using Illumina GenomeScan Software. To explore the functional relationships among the genes with altered expression in the HCC cells treated with COP1-1siRNA, a pathway analysis was carried out with the Ingenuity Pathway Analysis tool (Ingenuity Systems). Using the approach, we examined functional associations among genes and generated the gene networks with high significance on the basis that they had more of the interconnected genes present than would be expected by chance. The significance of each network was estimated by scoring system provided by Ingenuity. The scores are determined by the number of differentially expressed genes within each of the networks and the strength of the associations among network members. Once over-represented genes that are functionally relevant in gene networks are identified, we validated their functional association by using the independent pathway analysis tool PathwayStudio (Ariadne Genomics).
