<p>Supplementary Table 1. Primers and siRNAs used. Supplementary Table 2. Antibodies used. Supplementary Figure S1. Effect of different proteasome inhibitors on AIRAP expression in M10 melanoma cells. Supplementary Figure S2. Intracellular localization of AIRAP in bortezomib-treated M10 melanoma cells. Supplementary Figure S3. Specificity of AIRAP silencing in human melanoma cells. Supplementary Figure S4. AIRAP-knockdown enhances the anticancer activity of bortezomib long-term treatment in melanoma cells. Supplementary Figure S5. AIRAP-silencing promotes PARP-cleavage and reduces colony-forming ability in different melanoma cell lines. Supplementary Figure S6. AIRAP-silencing inhibits colony-forming ability in A375 melanoma cells. Supplementary Figure S7. AIRAP-knockdown inhibits SK-MEL-28 melanoma spheroid growth.</p>
ARTICLE ABSTRACT
The zinc-finger AN1-type domain-2a gene, also known as AIRAP (arsenite-inducible RNA-associated protein), was initially described as an arsenite-inducible gene in Caenorhabditis elegans and mammalian cells. Differently from the AIRAP worm homologue, aip-1, a gene known to play an important role in preserving animal lifespan and buffering arsenic-induced proteotoxicity, mammals have a second, constitutively expressed, AIRAP-like gene (AIRAPL), recently implicated in myeloid transformation. We have identified human AIRAP as a canonical heat-shock gene, whose expression, differently from AIRAPL, is strictly dependent on the proteotoxic-stress regulator heat-shock factor 1 (HSF1). AIRAP function is still not well defined and there is no information on AIRAP in cancer. Herein we show that bortezomib and next-generation proteasome inhibitors ixazomib and carfilzomib markedly induce AIRAP expression in human melanoma at concentrations comparable to plasma-levels in treated patients. AIRAP-downregulation leads to bortezomib sensitization, whereas AIRAP-overexpression protects melanoma cells from the drug, identifying AIRAP as a novel HSF1-regulated marker of chemotherapy resistance. More importantly, this study unexpectedly revealed that, also in the absence of drugs, AIRAP-silencing hinders melanoma clonogenic potential and spheroid growth, promoting caspase activation and apoptotic cell death, an effect independent of AIRAPL and linked to downregulation of the antiapoptotic protein cIAP2. Interestingly, AIRAP was found to interact with cIAP2, regulating its stability in melanoma. Taken together, the results identify AIRAP as a novel HSF1-dependent regulator of prosurvival networks in melanoma cells, opening new therapeutic perspectives in chemoresistant melanoma treatment.
The findings identify ZFAND2A/AIRAP as a novel stress-regulated survival factor implicated in the stabilization of the antiapoptotic protein cIAP2 and as a new potential therapeutic target in melanoma.
