The Cellular Apoptosis Susceptibility Protein (CAS) Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis and Cell Proliferation

Prashant Monian,Xuejun Jiang

doi:10.1074/jbc.m115.685008

Abstract

A signature event during the cell intrinsic apoptotic pathway is mitochondrial outer membrane permeabilization, leading to formation of the apoptosome, a caspase activation complex. The cellular apoptosis susceptibility protein (CAS) can facilitate apoptosome assembly by stimulating nucleotide exchange on Apaf-1 following binding of cytochrome c. We report here that CAS expression itself is up-regulated during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and knockdown of CAS renders cells resistant to TRAIL. We find that TRAIL induces up-regulation of CAS in a posttranscriptional, caspase-8-dependent manner through degradation of cIAP1, an E3 ligase that targets CAS for ubiquitin-dependent proteasomal degradation. We identified a novel signaling pathway whereby caspase-8 engages a feedforward cascade that leads to CAS up-regulation and amplifies the apoptotic signal. Furthermore, in silico analysis revealed that expression of CAS is up-regulated at both the mRNA and DNA levels in human breast tumors, consistent with its role in promoting cell proliferation. Overexpression of various oncogenes led to CAS up-regulation in non-transformed cells. Intriguingly, oncogene-induced CAS up-regulation also resulted in greater susceptibility to TRAIL-induced cell death, consistent with its proapoptotic function. These findings suggest that CAS plays contrasting roles in proliferation and apoptosis and that overexpression of CAS in tumors could serve as a potential biomarker to guide therapeutic choices.

Highlights

Binds to adaptor protein Apaf-1 and triggers assembly of the apoptosome, a heptameric caspase activation complex [2, 3]
Overall cell viability following tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-treatment was greater in cellular apoptosis susceptibility protein (CAS)-siRNA transfected cells (Fig. 1, c and d). These results clearly indicate that CAS facilitates TRAIL-induced cell death
Because CAS clearly plays a functional role in TRAIL-induced cell death, we sought to explore the mechanism of TRAIL-induced CAS up-regulation

Summary

Experimental Procedures

Cell Culture—MCF-10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, EGF (20 ng/ml), hydrocortisone (0.5 ␮g/ml), cholera toxin (100 ng/ml), insulin (10 ␮g/ml), and penicillin-streptomycin. 293T and HT-29 cells were cultured in DMEM high-glucose supplemented with 10% FBS, L-glutamine (2 mM), and penicillin-streptomycin. Lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4 °C and incubated with HA-agarose beads In Vivo Ubiquitination Assays—For assays in MCF10A cells, cells stably expressing GFP-His-CAS were lysed in 100 ␮l of radio-immunoprecipitation assay buffer supplemented with 25 ␮M MG132, 10 mM N-ethylmaleimide (Sigma), and protease inhibitors. In Vitro Ubiquitination Assay—His-tagged cIAP1 proteins were cloned into the pET28A vector, expressed in BL21-DE3 cells (Novagen), and purified from bacterial lysates using NiNTA-agarose beads. Following a final wash in IP buffer, bound proteins were eluted by boiling in 1ϫ SDS sample buffer and subjected to immunoblotting with the indicated antibodies.

Results

Discussion

Proliferation Prashant Monian and Xuejun Jiang