Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis

Abstract

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'. Cell Death and Differentiation (2012) 19, 1317-1327 doi:10.1038/cdd.2012 published online 10 February 2012 FLIP is an anti-apoptotic protein that blocks the activation of apoptosis mediated by death receptors, such as Fas, TRAIL receptor 1 (TRAIL-R1/DR4) and TRAIL-R2 (DR5).1 By binding to the adaptor protein FADD, FLIP inhibits apoptosis by blocking the processing and activation of procaspase 8 (FLICE) by death receptor complexes termed DISCs (death-inducing signalling complexes). 2 We previously reported that FLIP inhibits apoptosis induced by chemotherapeutic agents 3 and that high FLIP expression is an independent adverse prognostic biomarker in colorectal cancer (CRC). 4 These and other studies have indicated that inhibition of FLIP constitutes a promising therapeutic strategy for the treatment of CRC. Ku70 and its binding partner Ku80 are critical components of the non-homologous end joining (NHEJ) DNA repair machinery.5 Ku70 is regulated by acetylation, which is mediated by the histone acetyl transferases (HATs); CREBbinding protein (CBP) and PCAF, and its acetylation can be enhanced by treating cells with histone deacetylase (HDAC) inhibitors.6 Ku70 acetylation disrupts its DNA-binding activity and sensitises cells to DNA-damaging agents. 7 In addition, cytoplasmic Ku70 binds to and regulates the pro-apoptotic Bcl-2 family member, Bax.6 Ku70 simultaneously inhibits Bax degradation via the ubiquitin proteasome system (UPS) and prevents its translocation to the mitochondria. 8 Moreover, it has been reported that Ku70 may have intrinsic deubiquitinating (DUB) activity.8 The Ku70-Bax complex is disrupted by Ku70 acetylation, which promotes Bax translocation to mitochondria and apoptosis induction. Herein, we report a novel interaction between FLIP and Ku70 that regulates FLIP stability. This interaction is acetylation-dependant and is disrupted by HDAC inhibitors with activity against HDAC6. Disruption of the Ku70-FLIP interaction subsequently leads to FLIP degradation by the UPS and induction of c...

FLIP: A Targetable Mediator of Resistance to Radiation in Non–Small Cell Lung Cancer

“…In this study, we demonstrate that HDAC inhibitors enhance the in vitro and in vivo efficacy of IR in NSCLC models. Our group previously demonstrated that HDAC inhibitors downregulate FLIP in colorectal cancer models at the post-translational level by disrupting its interaction with Ku70, resulting in FLIP ubiquitination and degradation via the ubiquitinproteasome system (17). We also previously demonstrated that HDAC inhibitors effectively suppress FLIP expression at the transcriptional and post-transcriptional levels in H460 and A549 NSCLC cells (11).…”

“…Scrambed control (SC) siRNA, FLIP, caspase-8 and caspase-9 siRNAs were obtained from Dharmacon (Chicago, IL) and transfected as previously described (14,17).…”

“…Cells were harvested and lysed in RIPA buffer and Western blotting was carried out as previously described (17). Antibodies used: FLIP (NF6) and caspase-8 (12F5) (both Alexis Biochemicals, San Diego, CA), Caspase 9 (Cell Signaling Technology, Beverly, MA, USA PARP (eBioscience, San Diego, CA), β-actin (Sigma-Aldrich, UK).…”

“…Cell death, cell cycle distribution and cell surface death receptor expression were determined as previously described (14,17).…”

Molecular Mechanisms of Apoptosis and Roles in Cancer Development and Treatment

“…Kerr et al reported the Ku70 /FLIP interaction affects FLIP protein stability by preventing its polyubiquitination. In addition, they showed that SAHA disrupts the FLIP/ Ku70 complex via increasing the acetylation of Ku70, which consequently triggers FLIP polyubiquitination and degradation by the proteasome (Kerr et al, 2012). C-FLIP has been demonstrated to be down-regulated by several compounds such as actinomycin D (Hernandez et al, 2001), cycloheximide (Kaminskyy et al, 2013), and anisomycin (Mawji et al, 2007a); the first is an RNA synthesis inhibitor, and the second and third are protein synthesis inhibitors.…”