The RING domain in the anti-apoptotic protein XIAP stabilizes c-Myc protein and preserves anchorage-independent growth of bladder cancer cells

Abstract

Edited by Eric R. Fearon X-linkedinhibitorofapoptosisprotein(XIAP)suppressesapoptosis and plays key roles in the development, growth, migration, and invasion of cancer cells. Therefore, XIAP has recently attracted much attention as a potential antineoplastic therapeutic target, requiring elucidation of the molecular mechanisms underlying its biological activities. Here, using shRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, anchorage-independent growth assay, and invasive assay, we found that XIAP's RING domain, but not its BIR domain, is crucial for XIAP-mediated up-regulation of c-Myc protein expression in human bladder cancer (BC) cells. Mechanistically, we observed that the RING domain stabilizes c-Myc by inhibiting its phosphorylation at Thr-58 and that this inhibition is due to activated ERK1/2-mediated phosphorylation of glycogen synthase kinase-3␤ (GSK-3␤) at Ser-9. Functional studies further revealed that c-Myc protein promotes anchorage-independent growth and invasion stimulated by the XIAP RING domain in human BC cells. Collectively, the findings in our study uncover that the RING domain of XIAP supports c-Myc protein stability, providing insight into the molecular mechanism and role of c-Myc overexpression in cancer progression. Our observations support the notion of targeting XIAP's RING domain and c-Myc in cancer therapy. . 4 The abbreviations used are: BC, bladder cancer; XIAP, X-linked inhibitor of apoptosis protein; IAP, inhibitor of apoptosis; CHX, cycloheximide; GSK, glycogen synthase kinase; BIR, baculoviral IAP repeat; MEF, mouse embryo fibroblast; DN, dominant negative; FBS, fetal bovine serum.Figure 4. XIAP RING domain activated ERK1/2 and in turn mediated the phosphorylation of GSK-3␤ at Ser-9. A, the cell extracts from the indicated transfectants were subjected to Western blotting to detect the expression levels of the indicated proteins. GAPDH was used as the protein-loading control. B and C, cell extracts from WT versus ⌬RING MEFs (B) and ⌬RING versus ⌬RING/GFP-RING MEFs (C) were subjected to Western blotting to detect the expression levels of the indicated proteins. GAPDH was used as the protein-loading control. D-G, DN-ERK2 was overexpressed in shXIAP78/⌬BIR cells, and the stable transfectants were then subjected to Western blotting for determination of the expression of the indicated proteins. GAPDH was used as the protein-loading control. H, lysates from WT and ⌬XIAP HCT116 cells were co-immunoprecipitated with anti-XIAP antibody, and immunoprecipitates were then subjected to Western blotting for determination of c-Myc protein. *, significant difference as compared with control transfectant (p Ͻ 0.05). #, significant difference as compared with vector transfectant (p Ͻ 0.05). Error bars, S.D. XIAP RING domain stabilization of c-Myc J. Biol. Chem. (2019) 294(15) 5935-5944 5939 by guest on July 10, 2020 http://www.jbc.org/ Downloaded from Figure 5. XIAP RING domain was required for preserving the anchorage-independent growth and invasion abilities of BC cells. A-D, ...

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