Targeting of C-Terminal Binding Protein (CtBP) by ARF Results in p53-Independent Apoptosis

Abstract

ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.

A short acidic motif in ARF guards against mitochondrial dysfunction and melanoma susceptibility

“…As cellular senescence and aging are associated with deterioration of mitochondrial function [28][29][30] , we asked if mitochondrial dysfunction could provide the signal that triggered translocation of ARF to mitochondria during replicative senescence. Consistent with this idea, pre-senescent and senescent cells contained dysfunctional mitochondria according to several criteria, including reduced signals from MitoTracker Red and DiOC 6 (fluorescent dyes that accumulate in mitochondria with an intact membrane potential (DC m )), reduced ATP production, activation of AMP-activated protein kinase (AMPK; a sensor of bioenergetic stress), lower mitochondrial DNA (mtDNA) copy number and a tendency towards lower levels of the mitochondrial protein cyclophilin D (Fig. 2a,b and Supplementary Figs 5 and 6).…”

“…The spectrum of cancers developing in mice lacking Arf, Trp53 and Mdm2 is wider than in mice lacking Trp53 and Mdm2 (ref. 5), and reconstitution of ARF can arrest proliferation of Trp53-null cells 6,7 . Numerous reports based on cell culture experiments have implicated ARF in the regulation of diverse cellular processes, including ribosomal biosynthesis, the DNA damage response pathway, cell cycle arrest, senescence, apoptosis and autophagy [8][9][10][11][12] , suggesting that the tumour-suppressive functions of ARF may be both cell type and context dependent.…”

CtBPs promote mitotic fidelity through their activities in the cell nucleus

“…CtBPs have a well-described pro-survival role; ctbp1 À/À / ctbp2 À/À fibroblasts are hypersensitive to stress-induced apoptosis (Grooteclaes et al, 2003), and small interfering RNA (siRNA) to CtBP causes p53-independent apoptosis in cancer-derived cell lines Paliwal et al, 2006;Bergman et al, 2009). CtBPs can promote cell survival through multiple mechanisms; including the suppression of anoikis (Grooteclaes et al, 2003), repression of specific pro-apoptotic genes (Kovi et al, 2010) and, as we have demonstrated, maintenance of mitotic fidelity (Bergman et al, 2009).…”

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

“…siRNAs were used at very a low concentration (5 nM) to avoid unwanted off-target effects. To confirm the specificity of the CIBZ siRNA, we also monitored the levels of Bax and Noxa proteins, which have pro-apoptotic activities and are reported to be targets of apoptosis involving CtBP (17,19). No effects on the expression of these proteins were observed when CIBZ was knocked down (data not shown), further suggesting that the reduction of the CIBZ level was an antisense-specific effect.…”