XIAP promotes melanoma growth by inducing tumour neutrophil infiltration.

Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer.

The X-linked inhibitor of apoptosis protein (XIAP) is involved in melanoma invasion by regulating cell migration and survival.

BACKGROUND: The X-linked inhibitor of apoptosis (XIAP) is a potent cellular inhibitor of apoptosis, based on its unique capability to bind and to inhibit caspases. However, XIAP is also involved in a number of additional cellular activities independent of its caspase inhibitory function. The aim of this study was to investigate whether modulation of XIAP expression affects apoptosis-independent functions of XIAP in melanoma cells, restores their sensitivity to apoptosis and/or affects their invasive and metastatic capacities. METHODS: XIAP protein levels were analyzed by immunohistochemical staining of human tissues and by Western blotting of melanoma cell lysates. The effects of pharmacological inhibition or of XIAP down-regulation were investigated using ex-vivo and transwell invasion assays. The biological effects of XIAP down-regulation on melanoma cells were analyzed in vitro using BrdU/PI, nucleosome quantification, adhesion and migration assays. In addition, new XIAP binding partners were identified by co-immunoprecipitation followed by mass spectrometry. RESULTS: Here we found that the expression of XIAP is increased in metastatic melanomas and in invasive melanoma-derived cell lines. We also found that the bivalent IAP antagonist birinapant significantly reduced the invasive capability of melanoma cells. This reduction could be reproduced by downregulating XIAP in melanoma cells. Furthermore, we found that the migration of melanoma cells and the formation of focal adhesions at cellular borders on fibronectin-coated surfaces were significantly reduced upon XIAP knockdown. This reduction may depend on an altered vimentin-XIAP association, since we identified vimentin as a new binding partner of XIAP. As a corollary of these molecular alterations, we found that XIAP down-regulation in melanoma cells led to a significant decrease in invasion of dermal skin equivalents. CONCLUSION: From our data we conclude that XIAP acts as a multifunctional pro-metastatic protein in skin melanomas and, as a consequence, that XIAP may serve as a therapeutic target for these melanomas.

IAP antagonization promotes inflammatory destruction of vascular endothelium.

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Ubiquitin C-terminal hydrolase-L1 potentiates cancer chemosensitivity by stabilizing NOXA.

The BH3-only protein NOXA represents one of the critical mediators of DNA-damage-induced cell death. In particular, its involvement in cellular responses to cancer chemotherapy is increasingly evident. Here, we identify a strategy of cancer cells to escape genotoxic chemotherapy by increasing proteasomal degradation of NOXA. We show that the deubiquitylating enzyme UCH-L1 is a key regulator of NOXA turnover, which protects NOXA from proteasomal degradation by removing Lys(48)-linked polyubiquitin chains. In the majority of tumors from patients with melanoma or colorectal cancer suffering from high rates of chemoresistance, NOXA fails to accumulate because UCH-L1 expression is epigenetically silenced. Whereas UCH-L1/NOXA-positive tumor samples exhibit increased sensitivity to genotoxic chemotherapy, downregulation of UCH-L1 or inhibition of its deubiquitylase activity resulted in reduced NOXA stability and resistance to genotoxic chemotherapy in both human and C. elegans cells. Our data identify the UCH-L1/NOXA interaction as a therapeutic target for overcoming cancer chemoresistance.