SPOP mediates apoptosis and protects against necroptosis by regulating ubiquitination of RIPK1 and RIPK3
Yuzhong Ye, Changjie Yue, Zaosong Zheng, Hailong Ruan, Yuanpeng Zhang, Qi Miao, Xiaoping Zhang, Wen Xiao, Lei Liu
Yuzhong Ye, Changjie Yue, Zaosong Zheng, Hailong Ruan, Yuanpeng Zhang, Qi Miao, Xiaoping Zhang, Wen Xiao, Lei Liu
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Research Article Cell biology Oncology

SPOP mediates apoptosis and protects against necroptosis by regulating ubiquitination of RIPK1 and RIPK3

Abstract

Apoptosis and necroptosis are 2 distinct destinies of cells stimulated with TNF-α; however, it remains unclear how apoptosis and necroptosis are differentially regulated. This study validates the key regulatory role of speckle-type POZ protein (SPOP) in balancing apoptosis and necroptosis. SPOP promotes the polyubiquitination and degradation of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), thereby inhibiting necrosome formation and decreasing cellular sensitivity to necroptosis. Conversely, SPOP interacted with RIPK1 independently of its E3 ubiquitin ligase activity, protecting it from ubiquitination and degradation, thereby enhancing RIPK1 expression and cellular sensitivity to apoptosis. Inhibiting RIPK1 kinase activity with 7-Cl-O-Nec-1 impeded both SPOP-mediated apoptosis and SPOP deficiency–mediated necroptosis. Besides, inhibition or loss of RIPK3 rescued SPOP deficiency–mediated necroptosis. Pancancer analyses indicated that the SPOP/RIPK1/RIPK3 axis is dysfunctional in a variety of tumors. In 3 representative tumor types with high expression of SPOP and RIPK1, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, and breast invasive carcinoma, this regulatory mechanism remains applicable. Based on these findings, a combination therapy using the second mitochondria-derived activator of caspases (Smac) mimetic SM164 and sunitinib was developed, demonstrating a more pronounced efficacy than sunitinib monotherapy, and this sensitizing effect was dependent on the expression level of RIPK1. These results suggest that the combination of Smac mimetics with tyrosine kinase inhibitors holds potential clinical value for tumors with dysregulated SPOP/RIPK1/RIPK3 signaling.

Authors

Yuzhong Ye, Changjie Yue, Zaosong Zheng, Hailong Ruan, Yuanpeng Zhang, Qi Miao, Xiaoping Zhang, Wen Xiao, Lei Liu

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Figure 6

RIPK1 expression is associated with the sensitivity to sunitinib and SM164 cotreatment in tumor.

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RIPK1 expression is associated with the sensitivity to sunitinib and SM1...
(A) After knockdown of RIPK1 in 786-O, HepG2, and MDA-MB-231 cells, the protein levels of apoptotic markers mediated by TNF-α+SM164 (TS) stimulation were detected. (B) WT and RIPK3-overexpressing 786-O, HepG2, and MDA-MB-231 cells were treated with DMSO or TSZ for 6 hours; subsequently WCL was harvested for IB analysis. (C) Cell viability assay to assess the sensitivity of 786-O, HepG2, and MDA-MB-231 cells to SM164 treatment. (D) Cell viability assay for the effect of RIPK1 knockdown on the sensitivity of 786-O, HepG2, and MDA-MB-231 cells to sunitinib treatment. (E) Cell viability assay to assess the effect of combined use of SM164 on sunitinib sensitivity in 786-O, HepG2, and MDA-MB-231 cells. (F) Cell viability assay for the effect of RIPK1 knockdown on the sensitivity of 786-O, HepG2, and MDA-MB-231 cells to sunitinib alone, and sunitinib combined with SM164 treatment. (G) WT and RIPK1-knockdown 786-O, HepG2, and MDA-MB-231 cells were treated with DMSO, sunitinib alone, SM164 alone, or sunitinib combined with SM164 for 24 hours; subsequently WCL was harvested for IB analysis. Cell Counting Kit-8 (CCK-8) experiments were performed in triplicate and repeated 3 times independently. Data are presented as mean ± SD. The significance of differences was revealed based on paired Student’s t test.