Can-13-0036 1..11

Dysregulation in patterns of alternative RNA splicing in cancer cells is emerging as a significant factor in cancer pathophysiology. In this study, we investigated the little known alternative splice isoform survivin-3B (S-3B) that is overexpressed in a tumor-specific manner. Ectopic overexpression of S-3B drove tumorigenesis by facilitating immune escape in a manner associated with resistance to immune cell toxicity. This resistance was mediated by interaction of S-3Bwith procaspase-8, inhibiting death-inducing signaling complex formation in response to Fas/ Fas ligand interaction. We found that S-3B overexpression also mediated resistance to cancer chemotherapy, in this case through interactions with procaspase-6. S-3B binding to procaspase-6 inhibited its activation despite mitochondrial depolarization and caspase-3 activation. When combined with chemotherapy, S-3B targeting in vivo elicited a nearly eradication of tumors. Mechanistic investigations identified a previously unrecognized 7-amino acid region as responsible for the procancerous properties of survivin proteins. Taken together, our results defined S-3B as an important functional actor in tumor formation and treatment resistance. Cancer Res; 73(17); 1–11. 2013 AACR. Introduction Alternative splicing is an important mechanism for the generation of the variety of proteins indispensable for cell functions. In normal cells, it is finely regulated to orchestrate cellular processes. During cancer development, myriad defects occur throughout cell transformation. Among these defects, alternative splicing misregulation seems to be an important factor. Misregulation of alternative splicing can be observed in cancer cells in the absence of genomic mutations of the genes concerned (1). In cancer cells, aberrant alternative splicing can give rise to variants that are specifically expressed in cancer tissues and completely absent from normal ones (2). Among alternative spliced genes, birc5, or survivin, is important as its expression is highly deregulated in cancer (3). First described in 1997 (4), survivin undergoes alternative splicing that can induce the expression of five different transcripts with different functions: survivin, survivin-DEx3, survivin-2B (5), survivin-3B (S-3B; ref. 6), and survivin-2a (7). Although survivin was first described as a tumor-specific gene, over the years it has come to light that it is also expressed in normal cells where its expression is necessary for cell-cycle progression (8–10). In the present work, we highlighted that S-3B was a cancerspecific isoform. In addition, overexpression of this variant induced tumorigenesis through the inhibition of tumor-directed immune-cell cytotoxicity by inhibiting death-inducing signaling complex (DISC) formation. Moreover, the expression of S-3B gave a strong advantage to cancer cells exposed to cancer treatment by blocking cell death through the inhibition of procaspase-6 activation. In vivo, the targeting of S-3B, in association with chemotherapy, greatly improved tumor response to treatment. Finally, we identified a C-terminal 7amino acid domain in S-3B as a new protein domain responsible for protein functions. Materials and Methods Cell lines All of the cell lines used (A549, BT474, BT474c, BT483, Calu-3, CCRF-CEM, HBL100, HCC1419, HCC1569, HCC1954, HCC2218, HCT116, HT29, Hs578T, M113, M125, M44, M6, MCF-7, MDA-MB-231, MDA-MB-468, MelC, NCI-H1703, NCIH1781, NCI-H1975, NCI-H322, NCI-H460, NCI-H520, siHa, SKBr-3, SKOV3, SNB19, SW1116, SW48, SW620, T47D, U373, U87-MG, UACC-812, and Widr) were purchased from American Type Culture Collection (ATCC) or Oncodesign. They were routinely grown in accordance with ATCC-recommended culture conditions. Sensitive and resistant cell lines were purchased from Oncodesign. Authors' Affiliations: Departments of Tumor Biology and Pathology, Unit of Molecular Biology, Radiotherapy, and Tumor Biology and Pathology, Unit of Pathology, Preclinical Imaging Platform, Centre Georges-François Leclerc; Institut National de la Sant e et de la Recherche Medicale (INSERM) U866; and Universit e de Bourgogne UFR Sciences et Techniques ICMUB UMR CNRS 6302, Dijon, France. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Lizard-Nacol and R. Boidot contributed equally to this work Corresponding Author: Romain Boidot, Unit of Molecular Biology, Centre Georges François Leclerc, 1, rue du Professeur Marion, 21079 Dijon, France. Phone: 33-3-80-73-75-00, ext. 3185; Fax: 33-3-80-73-77-82; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-0036 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 10, 2017. © 2013 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst July 15, 2013; DOI: 10.1158/0008-5472.CAN-13-0036