XIAP as a Target of New Small Organic Natural Molecules Inducing Human Cancer Cell Death.
ABSTRACT: X-linked inhibitor of apoptosis protein (XIAP) is an emerging crucial therapeutic target in cancer. We report on the discovery and characterisation of small organic molecules from Piper genus plants exhibiting XIAP antagonism, namely erioquinol, a quinol substituted in the 4-position with an alkenyl group and the alkenylphenols eriopodols A-C. Another isolated compound was originally identified as gibbilimbol B. Erioquinol was the most potent inhibitor of human cancer cell viability when compared with gibbilimbol B and eriopodol A was listed as intermediate. Gibbilimbol B and eriopodol A induced apoptosis through mitochondrial permeabilisation and caspase activation while erioquinol acted on cell fate via caspase-independent/non-apoptotic mechanisms, likely involving mitochondrial dysfunctions and aberrant generation of reactive oxygen species. In silico modelling and molecular approaches suggested that all molecules inhibit XIAP by binding to XIAP-baculoviral IAP repeat domain. This demonstrates a novel aspect of XIAP as a key determinant of tumour control, at the molecular crossroad of caspase-dependent/independent cell death pathway and indicates molecular aspects to develop tumour-effective XIAP antagonists.
Project description:Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.
Project description:The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation.
Project description:X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases 3, 7 and 9, and mitochondrial Smac (second mitochondria-derived activator of caspase) release during apoptosis inhibits the activity of XIAP. In this study we show that cytosolic XIAP also feeds back to mitochondria to impair Smac release. We constructed a fluorescent XIAP-fusion protein by labelling NH(2)- and COOH-termini with Cerulean fluorescent protein (C-XIAP-C). Immunoprecipitation confirmed that C-XIAP-C retained the ability to interact with Smac and impaired extrinsically and intrinsically activated apoptosis in response to tumour necrosis factor-related apoptosis-inducing ligand/cycloheximide and staurosporine. In C-XIAP-C-expressing cells, cytochrome c release from mitochondria proceeded normally, whereas Smac release was significantly prolonged and incomplete. In addition, physiological expression of native XIAP prolonged or limited Smac release in HCT-116 colon cancer cells and primary mouse cortical neurons. The Smac-binding capacity of XIAP, but not caspase inhibition, was central for mitochondrial Smac retention, as evidenced in experiments using XIAP mutants that cannot bind to Smac or effector caspases. Similarly, the release of a Smac mutant that cannot bind to XIAP was not impaired by C-XIAP-C expression. Full Smac release could however be provoked by rapid cytosolic C-XIAP-C depletion upon digitonin-induced plasma membrane permeabilization. Our findings suggest that although mitochondria may already contain pores sufficient for cytochrome c release, elevated amounts of XIAP can selectively impair and limit the release of Smac.
Project description:X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.
Project description:Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-? (TNF?) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNF? and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is "druggable". A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.
Project description:X-linked inhibitor of apoptosis (XIAP) is an inhibitor of apoptotic cell death that protects cells by caspase-dependent and independent mechanisms. In a screen for molecules that participate with XIAP in regulating cellular activities, we identified apoptosis-inducing factor (AIF) as an XIAP binding protein. Baculoviral IAP repeat 2 of XIAP is sufficient for the XIAP/AIF interaction, which is disrupted by Smac/DIABLO. In healthy cells, mature human AIF lacks only the first 54 amino acids, differing significantly from the apoptotic form, which lacks the first 102 amino-terminal residues. Fluorescence complementation and immunoprecipitation experiments revealed that XIAP interacts with both AIF forms. AIF was found to be a target of XIAP-mediated ubiquitination under both normal and apoptotic conditions, and an E3 ubiquitin ligase-deficient XIAP variant displayed a more robust interaction with AIF. Expression of either XIAP or AIF attenuated both basal and antimycin A-stimulated levels of reactive oxygen species (ROS), and when XIAP and AIF were expressed in combination, a cumulative decrease in ROS was observed. These results identify AIF as a new XIAP binding partner and indicate a role for XIAP in regulating cellular ROS.
Project description:X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of cell death that functions by suppressing caspases 3, 7, and 9. Here we describe the establishment of Jurkat-derived cell lines stably overexpressing either full-length XIAP or a truncation mutant of XIAP that can only inhibit caspase 9. Characterization of these cell lines revealed that following CD95 activation full-length XIAP supported both short- and long-term survival as well as proliferative capacity, in contrast to the truncation mutant but similar to Bcl-x(L). Full-length XIAP was also able to inhibit CD95-mediated caspase 3 processing and activation, the mitochondrial release of cytochrome c and Smac/DIABLO, and the loss of mitochondrial membrane potential, whereas the XIAP truncation mutant failed to prevent any of these cell death events. Finally, suppression of XIAP levels by RNA interference sensitized Bcl-x(L)-overexpressing cells to death receptor-induced apoptosis. These data demonstrate for the first time that full-length XIAP inhibits caspase activation required for mitochondrial amplification of death receptor signals and that, by acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation.
Project description:<h4>Background</h4>In various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated.<h4>Methods</h4>To better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression.<h4>Results</h4>Our data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits.<h4>Conclusion</h4>Our results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.
Project description:X-linked inhibitor of apoptosis (XIAP) is the most potent endogenous caspase inhibitor preventing cell death via caspase-9, -7 and -3 (initiator and executioner caspase pathways). Using short hairpin RNA (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones have been developed. XIAP mRNA levels were established by RT-PCR, the four X (XIAP knockdown) clonal cell lines show 82-93% reduction in XIAP mRNA when compared to the four L (luciferase control) cell lines. Immunoblot analysis showed a 67-89% reduction in XIAP protein in X cell lines compared to L. RNA was analysed by microarray and XIAP knockdown was confirmed in 7 probe sets, there was no significant compensation of other IAP family members. XIAP knockdown induced a 2-fold increase in the basal level of apoptosis without modification of caspase 3/7 activity. Finally, XIAP knockdown sensitises cells to radiotherapy by 20%, to recombinant TRAIL by a 3-fold factor, and to paclitaxel and docetaxel by >2 fold factor. Future work should focus on targeted agents such as rhTRAIL in combination with strategies to down regulate XIAP. XIAP antisense is now in clinical development in oncology. Overall design: Human HCT116 colon cancer cell lines were transfected with either XIAP (X) or luciferase (L) shRNA. The expression profiles of both were compared at early (p4) and late (p8) passage.
Project description:Dysregulation of apoptosis plays an important role in tumour progression and resistance to chemotherapy. The X-linked inhibitor of apoptosis (XIAP) is considered to be the most potent caspase inhibitor of all known inhibitor of apoptosis-family members. Only recently, an antagonist of XIAP has been identified, termed Smac/DIABLO. To explore the relevance of antiapoptotic XIAP and proapoptotic Smac/DIABLO for tumour progression in renal cell carcinomas (RCCs), we analysed XIAP and Smac/DIABLO mRNA and protein expression in the primary tumour tissue from 66 RCCs of all major histological types by quantitative real-time PCR, Western blot and ELISA. X-linked inhibitor of apoptosis and Smac/DIABLO mRNA expression was found in all RCCs. Importantly, the relative XIAP mRNA expression levels significantly increased from early (pT1) to advanced (pT3) tumour stages (P=0.0002) and also with tumour dedifferentiation (P=0.04). Western blot analysis confirmed the tumour stage-dependent increase of XIAP expression on the protein level. In contrast, mRNA and protein expression levels of Smac/DIABLO did not significantly change between early and advanced tumour stages or between low and high tumour grades. Consequently, the mRNA expression ratio between antiapoptotic XIAP and proapoptotic Smac/DIABLO markedly increased during progression from early (pT1) to advanced (pT3) tumour stages. Moreover, RCCs confined within the organ capsule (pT1 and pT2) exhibited a significantly lower XIAP to Smac/DIABLO expression ratio when compared with RCCs infiltrating beyond the kidney (pT3; P=0.01). Thus, our investigation demonstrates that the delicate balance between XIAP and Smac/DIABLO expression is gradually disturbed during progression of RCCs, resulting in a relative increase of antiapoptotic XIAP over proapoptotic Smac/DIABLO, thereby probably contributing to the marked apoptosis resistance of RCC.