birinapant and Melanoma

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.. 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.. 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.. 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.

Topics: Apoptosis; Caspases; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Dipeptides; Humans; Indoles; Inhibitor of Apoptosis Proteins; Melanoma; Neoplasm Invasiveness; Protein Binding; Vimentin; X-Linked Inhibitor of Apoptosis Protein

Antagonists of inhibitors of apoptosis proteins (IAPs), alone or in combination with genotoxic therapeutics, have been shown to efficiently induce cell death in various solid tumors. The IAP antagonist birinapant is currently being tested in phase II clinical trials. We herein aimed to investigate the antitumor efficacy of dacarbazine in vitro, both as a single agent and in combination with birinapant, in melanoma cell lines. Covering clinically relevant drug concentration ranges, we conducted a total of 5,400 measurements in a panel of 12 human melanoma cell lines representing different stages of disease progression. Surprisingly, functionally relevant synergies or response potentiation in combination treatments was not observed, and only one cell line modestly responded to birinapant single treatment (approximately 16% cell death). Although we did not study the underlying resistance mechanisms or more complex in vivo scenarios in which dacarbazine/birinapant response synergies may still possibly manifest, our findings are nevertheless noteworthy because IAP antagonists were demonstrated to strongly enhance responses to DNA-damaging agents in cell lines of other cancer types under comparable experimental conditions in vitro.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Death; Cell Line, Tumor; Dacarbazine; Dipeptides; Humans; Indoles; Melanoma; Mutation; Skin Neoplasms

Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas.. Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models.. When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line.. Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis Regulatory Proteins; Baculoviral IAP Repeat-Containing 3 Protein; Cell Line, Tumor; Cell Proliferation; Dipeptides; Disease Models, Animal; Humans; Indoles; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Melanoma; Mice; Mitochondrial Proteins; Molecular Mimicry; Spheroids, Cellular; Tumor Burden; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays