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
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.
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