Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

Abstract

Collaborator of ARF (CARF) was cloned as an ARF-interacting protein and shown to regulate the p53–p21WAF1–HDM2 pathway, which is central to tumor suppression via senescence and apoptosis. We had previously reported that CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis, however, the mechanisms governing this phenomenon remained unknown. Thus, we examined various cell death and survival pathways including the mitochondrial stress, ataxia telangiectasia mutated (ATM)–ATR, Ras–MAP kinase and retinoblastoma cascades. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, it protected the cells against genotoxicity; CARF knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γH2AX, leading to induction of mitotic arrest and eventual apoptosis. We also show that the CARF-silencing-induced apoptosis in vitro translates to in vivo. In a human tumor xenograft mouse model, treatment of developing tumors with short hairpin RNA (shRNA) against CARF via an adenovirus carrier induced complete suppression of tumor growth, suggesting that CARF shRNA is a strong candidate for an anticancer reagent. We demonstrate that CARF has a vital role in genome preservation and tumor suppression and CARF siRNA is an effective novel cancer therapeutic agent.