SPOP regulates prostate epithelial cell proliferation and promotes ubiquitination and turnover of c-MYC oncoprotein

Abstract

The E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) is frequently dysregulated in prostate adenocarcinoma (PC), via either somatic mutations or mRNA downregulation, suggesting an important tumor suppressor function. To examine its physiologic role in the prostate epithelium in vivo, we generated mice with prostate-specific biallelic ablation of Spop. These mice exhibited increased prostate mass, prostate epithelial cell proliferation, and expression of c-MYC protein compared to littermate controls, and eventually developed prostatic intraepithelial neoplasia (PIN). We found that SPOPWT can physically interact with c-MYC protein and, upon exogenous expression in vitro, can promote c-MYC ubiquitination and degradation. This effect was attenuated in PC cells by introducing PC-associated SPOP mutants or upon knockdown of SPOP via short-hairpin-RNA, suggesting that SPOP inactivation directly increases c-MYC protein levels. Gene set enrichment analysis revealed enrichment of Myc-induced genes in transcriptomic signatures associated with SPOPMT. Likewise, we observed strong inverse correlation between c-MYC activity and SPOP mRNA levels in two independent PC patient cohorts. The core SPOPMT;MYCHigh transcriptomic response, defined by the overlap between the SPOPMT and c-MYC transcriptomic programs, was also associated with inferior clinical outcome in human PCs. Finally, the organoid-forming capacity of Spop-null murine prostate cells was more sensitive to c-MYC inhibition than that of Spop-WT cells, suggesting that c-MYC upregulation functionally contributes to the proliferative phenotype of Spop knock-out prostates. Taken together, our data highlight SPOP as an important regulator of luminal epithelial cell proliferation and c-MYC expression in prostate physiology, identify c-MYC as a novel bona fide SPOP substrate, and help explain the frequent inactivation of SPOP in human PC. We propose SPOPMT–induced stabilization of c-MYC protein as a novel mechanism that can increase total c-MYC levels in PC cells, in addition to amplification of c-MYC locus.

Ubiquitin ligases in oncogenic transformation and cancer therapy

“…Turnover of MYC is regulated by multiple cancer-associated E3s and E3 complexes in a tissue-specific manner, including SCF–FBXW7 (REFS 134–136), CRL3–potassium channel tetramerization domain-containing protein 2 (KCTD2) 137 , HUWE1 (REFS 138–140), CRL3–SPOP 141 and SCF–SKP2 (REF. 142).…”

“…Finally, MYC has been identified as a CRL3–SPOP substrate in prostate epithelial cells 141 . SPOP mutations found in prostate and endometrial cancers are thought to disrupt substrate binding, suggesting that SPOP plays a tumour-suppressor role in these tissues 12,141,149 .…”

“…SPOP mutations found in prostate and endometrial cancers are thought to disrupt substrate binding, suggesting that SPOP plays a tumour-suppressor role in these tissues 12,141,149 . Accordingly, prostate-specific biallelic ablation of Spop in mice results in MYC accumulation and promotes the development of prostatic intraepithelial neoplasia 141 .…”

“…SPOP mutations found in prostate and endometrial cancers are thought to disrupt substrate binding, suggesting that SPOP plays a tumour-suppressor role in these tissues 12,141,149 . Accordingly, prostate-specific biallelic ablation of Spop in mice results in MYC accumulation and promotes the development of prostatic intraepithelial neoplasia 141 . CRL3–SPOP has also been implicated in the regulation of steroid receptor co-activator protein 3 (SRC3; also known as NCOA3), androgen receptor (AR) and ETS-related gene (ERG), key oncogenic signalling proteins in prostate cancer 12,150,151 .…”

SPOP and FOXA1 mutations are associated with PSA recurrence in ERG wt tumors, and SPOP downregulation with ERG‐rearranged prostate cancer

“…Previous studies have shown that SPOP mutations and ERG rearrangements are mutually exclusive in prostate cancer, suggesting that both alterations may represent alternative mechanisms resulting in the same oncogenic phenotype in neoplastic prostate cells . SPOP targets ERG, AR, and c‐myc among others, for ubiquitin‐proteasome system degradation and mutations are associated with an increase in the levels of its substrate protein . Different data based on clonality analysis and in the presence of SPOP mutations in high‐grade prostate PIN suggest that SPOP mutations could be also an early and recurrent event in prostate tumorigenesis.…”