SPOP Functions As A Dual Modulator Of Apoptosis And Necroptosis By Regulating RIPK1/RIPK3 And Its Application In Metastatic Renal Cancer

In China,the last decade has witnessed the mortality and incidence of renal cell carcinoma(RCC)progressively growing,posing a severe threat to the well-being of the population.RCC rarely presents with obvious clinical symptoms in its early stages,and about a quarter of patients are confirmed as metaststic RCC at the first pathologic diagnosis.The prognosis of metaststic RCC is very poor,and targeted therapy,represented by sunitinib,is currently recommended as the first-line treatment for metaststic RCC.However,the efficacy of targeted therapy depends on the development of drug resistance,which is an important challenge in clinical practice.The resistance to targeted drugs mainly involves resistance of regulated cell death.Apoptosis and necroptosis are two distinct destinies of cell death stimulated with TNFα;however,it remains unclear how apoptosis and necroptosis are differentially regulated.The research of the regulatory mechanism of apoptosis and necroptosis and the application of related drugs are of great value in discovering clinical therapeutic targets.Speckle-type POZ protein(SPOP)plays a critical role in various tumors,but its role in cell death stimulated by TNFαhas not been reported.Based on the regulatory mechanism of apoptosis and necroptosis,this study found and proved the role of SPOP,and explored the anti-tumor effect of SM164 in RCC.Objective:To investigate the effect of SPOP on apoptosis and necroptosis induced by TNF-αand the mechanism of interaction in between.What’s more,to search for the representative cancer type with significant differential expression of SPOP and its regulatory genes,and further confirm the above mechanism in this cancer type.The effects of the apoptosis inducer SM164 on the antitumor effects of sunitinib were subsequently investigated in vivo and in vitro in response to this mechanism.Methods:In the first part,SPOP or receptor-interacting seronine protein kinase 3(RIPK3)was knocked out in mouse embryonic fibroblasts.Different combinations of inducers,corresponding inhibitors or knockout of RIPK3 were then used to block the apoptotic or necroptotoc pathway.Various inducers and/or inhibition methods were employed to examine alterations in cell viability,apoptotic and necroptotic markers,and functional complexes.CCK-8 and Western blotting(WB)were utilized in the detection process.Furthermore,cycloheximide,plasmid transfection and immunoprecipitation were applied to investigate the regulatory mechanism of SPOP on RIPK1 and RIPK3.The investigation also delved into the domain responsible for the binding of RIPK3 to SPOP,along with the underlying ubiquitination mechanism.In the second part,to identify the representative tumor,the gene expression data of SPOP/RIPK1/RIPK3 were extracted from the pan-cancer data set,and then employed in an analysis of the expression discrepancies across various clinical and pathological stages,as well as between tumor and normal tissues.To confirm the expression level and interaction of SPOP/RIPK1/RIPK3 in representative tumor cell lines and tissues,RT-q PCR and western blotting were employed.The influencing factors of overall survival were analyzed with tumor prognostic data.Finally,in the third part,a RCC cell line stably low-expressing RIPK1 was established,and CCK-8 and WB were employed to evaluate the impact of RIPK1 and SM164 on the sensitivity of RCC cells to sunitinib.Subsequently,tumor models were created under the skin of nude mice,with the aim of examining the potential anti-tumor impact of sunitinib and SM164.Immunohistochemistry was utilized to detect alterations in apoptosis markers,while HE staining was performed to identify any potential toxicity of the indicated drugs on the heart,liver,and kidney.Results:The deletion of SPOP inhibits apoptosis and mediates necroptosis,which are mediated by RIPK1 and RIPK3.SPOP mediates the formation of complex IIb by promoting the ubiquitination of RIPK3 and inhibiting the ubiquitination of RIPK1.RIPK3is most likely bound to the MATH domain of SPOP and ubiquitinated by the E3 ubiquitin ligase.Pan-cancer analysis revealed the expression profile of SPOP/RIPK1/RIPK3 in 34types of tumors,and renal clear cell carcinoma(KIRC)was screened as a representative tumor with high expression of SPOP and RIPK1.Also,the interaction of SPOP with RIPK1/RIPK3 is still working in RCC,and m RNA levels of SPOP can serve as an independent prognostic marker for KIRC.SM164 increased the sensitivity of RCC cell lines and corresponding subcutaneous tumors to sunitinib in a RIPK1-dependent manner and enhanced the anti-tumor effect of sunitinib by inducing apoptosis in tumor cells.No obvious drug toxicity was observed in heart,liver and kidney by HE staining.Conclusions:SPOP binds to RIPK1 and RIPK3 to inhibit the ubiquitination of RIPK1 and promote the ubiquitination of RIPK3,thus promoting the apoptotic pathway and inhibiting the necroptotic pathway.Renal clear cell carcinoma(KIRC)can be regarded as the representative tumor with high expression of SPOP and RIPK1,where the mechanism of SPOP regulating RIPK1 and RIPK3 still exists.The m RNA level of SPOP may be an independent prognostic marker for KIRC.SM164 could promote anti-tumor effects of sunitinib in vivo and in vitro in a RIPK1-dependent manner.