Controllo genetico dei regolatori del ciclo cellulare attraverso il complesso SKP2/CKS1 ubiquitina ligasi nel cancro epatico dei roditori e sua associazione con la prognosi dell'epatocarcinoma umano

Abstract

Previous work showed a genetic control of cell cycle during hepatocarcinogenesis. Evidences indicate a posttranscriptional regulation of oncosuppressors by the ubiquitin ligase SKP1/CUL1/F-box protein (SCF) complex. Here we evaluated the role of SCFSKP2 ubiquitin ligase on cell cycle negative regulators in determining the predisposition to HCC, in F344 and BN rats genetically susceptible/resistant to HCC respectively, and in 2 hHCC subtypes. Rats were treated accordingly the “resistant hepatocytes” protocol, we used human control livers, hHCCs with better (hHCCB) and with poor (hHCCP) prognosis. SKP2 function was studied in hHCC cell lines by silencing or inducing SKP2. SKP2 and cell cycle regulators were determined by IP, WB and qPCR in rats and humans. We showed p21WAF1, p27KIP1, p57KIP2, p130 and RassF1A mRNA increases in F344 fast growing lesions, lower/no increase in BN slowly growing lesions. Opposite results occurred at protein levels. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rats. The results of hHCCP are comparable to F344 lesions and those of hHCCB to BN lesions. Proliferation indexes of 60 hHCCs were inversely correlated only with protein levels of cell cycle negative regulators in hHCC, indicating a posttranslational modification. Our results indicate that hHCCP exhibit active ubiquitination of cell cycle negative regulators, suggesting that CKS1-SKP2 ligase might be a promising diagnostic marker