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This file contains: Figure S1: Apoptosis determined by annexin V-FITC/7-AAD. Figure S2: NO prevents TNFa-induced classical NF-kB signaling pathway. Figure S3: Detection of S-nitrosylation of cIAP1 with the biotin-switch assay. Figure S4: Example of MALDI-TOF-MS spectra of S-nitrosylated cIAP1. Figure S5: SW480 stable cell line expressing 6myc-cIAP1-C571/574H. Figure S6: Characterization of apoptosis induced by GTN/TNFa. Figure S7: Functional analysis of the ubiquitin E3 ligase activity of cIAP1 mutants. Figure S8: NO prevents TNFa-mediated complex I to promote complex II. Table S1: TNFa concentration in media from human macrophages treated with 5-Fluorouracil/oxaliplatin. Supplementary Materials and Methods.

TNFα is a prominent proinflammatory cytokine and a critical mediator for the development of many types of cancer such as breast, colon, prostate, cervical, skin, liver, and chronic lymphocytic leukemia. Binding of TNFα to TNFR1 can lead to divergent signaling pathways promoting predominantly NF-κB activation but also cell death. We report here that the nitric oxide (NO) donor glyceryl trinitrate (GTN) converts TNFα, generated from immune cells or cancer cells stimulated by chemotherapy, into a prodeath mediator in colon and mammary cancer cells. GTN-mediated S-nitrosylation of cIAP1 on cysteines 571 and 574 inhibited its E3 ubiquitin ligase activity, which in turn reduced Lys63-linked ubiquitination of RIP1 and initiated assembly of a death complex. These findings provide insights into how NO can harness advantageous aspects of inflammation in cancer and provide new therapeutic strategies.Significance: Combination of an NO donor with chemotherapeutic drug–induced TNFα represents a potentially valuable anticancer strategy. Cancer Res; 78(8); 1948–57. ©2018 AACR.