Search

Inhibitor of apoptosis (IAP) proteins play pivotal roles in cellular survival by blocking apoptosis, modulating signal transduction, and affecting cellular proliferation. Through their interactions with inducers and effectors of apoptosis IAP proteins can effectively suppress apoptosis triggered by diverse stimuli including death receptor signaling, irradiation, chemotherapeutic agents, or growth factor withdrawal. Evasion of apoptosis, in part due to the action of IAP proteins, enhances resistance of cancer cells to treatment with chemotherapeutic agents and contributes to tumor progression. Additionally, IAP genes are known to be subject to amplification, mutation, and chromosomal translocation in human malignancies and autoimmune diseases. In this review we will discuss the role of IAP proteins in cancer and the development of antagonists targeting IAP proteins for cancer treatment., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival, which makes them attractive targets for therapeutic intervention in cancers. Herein, we describe the structure-based design of IAP antagonists with high affinities and selectivity (>2000-fold) for c-IAP1 over XIAP and their functional characterization as activators of apoptosis in tumor cells. Although capable of inducing cell death and preventing clonogenic survival, c-IAP-selective antagonists are significantly less potent in promoting apoptosis when compared to pan-selective compounds. However, both pan-IAP- and c-IAP-selective antagonists stimulate c-IAP1 and c-IAP2 degradation and activation of NF-kappaB pathways with comparable potencies. Therefore, although compounds that specifically target c-IAP1 and c-IAP2 are capable of inducing apoptosis, antagonism of the c-IAP proteins and XIAP is required for efficient induction of cancer cell death by IAP antagonists.

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.

Melanoma inhibitor of apoptosis (ML-IAP) is a potent inhibitor of apoptosis, which is highly expressed in melanomas and likely contributes to their resistance to chemotherapeutic treatments. Herein, we show that the lineage survival oncogene microphthalmia-associated transcription factor (MITF) is a critical regulator of ML-IAP transcription in melanoma cells. The ML-IAP promoter contains two MITF consensus sites, and analysis of MITF and ML-IAP mRNA levels revealed a high correlation in melanoma tumor samples and cell lines. In reporter assays, MITF promoted a strong stimulation of transcriptional activity from the ML-IAP promoter, and MITF bound the endogenous ML-IAP promoter in melanoma cells by chromatin immunoprecipitation and electrophoretic mobility shift assay. Strikingly, small interfering RNA (siRNA)-mediated knockdown of MITF in melanoma cells led to a dramatic decrease in ML-IAP mRNA and protein levels, establishing that ML-IAP expression in melanoma cells is MITF dependent. Additionally, cyclic AMP-mediated induction of MITF expression in melanocytes resulted in increased ML-IAP expression, suggesting that melanocytes can express ML-IAP when MITF levels are heightened. Disruption of MITF by siRNA led to a decrease in melanoma cell viability, which could be rescued by ectopic expression of ML-IAP. Collectively, these findings implicate MITF as a major transcriptional regulator of ML-IAP expression in melanomas, and suggest that ML-IAP contributes to the prosurvival activity of MITF in melanoma progression.

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.