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

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Abstract

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.

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Acknowledgments

We thank Claudia Coerper-Ochsman for excellent technical assistance. This work was founded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (grant MA 1181/7-1 to H.K. and C.M.; and Project number 73111208 - SFB 829 (to P.Z. and C.M.).

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Author notes

  1. Ouissam Ayachi and Meltem Barlin equally contributed

Authors and Affiliations

  1. Department of Dermatology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

    Ouissam Ayachi, Meltem Barlin, Cornelia Mauch & Paola Zigrino

  2. Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), Center for Molecular Medicine Cologne (CMMC), CECAD, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

    Pia Nora Broxtermann & Hamid Kashkar

  3. Department of Dermatology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Kerpener Strasse 62, 50937, Cologne, Germany

    Paola Zigrino

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  1. Ouissam Ayachi
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Supplementary Fig. 1

Targeting XIAP. (A) the real-time PCR analysis of XIAP transcript expression in BLM cells. S26 was used as loading control. (B, C) Western blot analysis of the cell lysates. 50 μg of protein extracted from control and sh-XIAP clones was analyzed for XIAP, c-IAP1, c-IAP 2, survivin and MITF expression. β-actin was used as loading control and Ponceau staining of the membrane shows transfer efficiency. (D) Proteins extracted from control and XIAP down-regulated melanoma cell lines clones were analyzed for expression of MITF as a measure of the differentiation status of the cells. Ponceau staining of the membrane was used as loading control and transfer efficiency (∆, crisp/cas9 knockout clones; si, transient down-regulation of XIAP using siRNA). (E) Cells were serum starved for 24 h, afterward medium ± 100 ng/ml TRAIL was added. After 24 h incubation Annexin-V (AV)-allophycocyanin (APC) and PI, stained cells were analyzed using flow cytometry. Quantification of % number of positive cells is shown in the graphs. (p = *0,01, ***0,0001) (PPTX 819 kb)

Supplementary Fig. 2

Migration of shXIAP- and scr-BLM cells Monolayers formed on uncoated and fibronectin (10 μg/ml) coated plates, after mitotic inactivation, were scratched and migration of cells into the generated open space was monitored over time (A). Alternatively, after mitotic inactivation, cells were seeded sparsely and migration of single cells monitored for 24 h (B). The graphs represent average measurements. * p < 0.05. (PPTX 298 kb)

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Ayachi, O., Barlin, M., Broxtermann, P.N. et al. The X-linked inhibitor of apoptosis protein (XIAP) is involved in melanoma invasion by regulating cell migration and survival. Cell Oncol. 42, 319–329 (2019). https://doi.org/10.1007/s13402-019-00427-1

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