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Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization.

ABSTRACT: Although mutations in the gene encoding the RNA splicing factor SF3B1 are frequent in multiple cancers, their functional effects and therapeutic dependencies are poorly understood. Here, we characterize 98 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations, identifying hundreds of cryptic 3' splice sites common and specific to different cancer types. Regulatory network analysis revealed that the most common SF3B1 mutation activates MYC via effects conserved across human and mouse cells. SF3B1 mutations promote decay of transcripts encoding the protein phosphatase 2A (PP2A) subunit PPP2R5A, increasing MYC S62 and BCL2 S70 phosphorylation which, in turn, promotes MYC protein stability and impair apoptosis, respectively. Genetic PPP2R5A restoration or pharmacologic PP2A activation impaired SF3B1-mutant tumorigenesis, elucidating a therapeutic approach to aberrant splicing by mutant SF3B1. SIGNIFICANCE: Here, we identify that mutations in SF3B1, the most commonly mutated splicing factor gene across cancers, alter splicing of a specific subunit of the PP2A serine/threonine phosphatase complex to confer post-translational MYC and BCL2 activation, which is therapeutically intervenable using an FDA-approved drug.See related commentary by O'Connor and Narla, p. 765.This article is highlighted in the In This Issue feature, p. 747.

SUBMITTER: Liu Z

PROVIDER: S-EPMC8188517 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Although endometrial cancer is the most common cancer of the female reproductive tract, we have little understanding of what controls endometrial cancer beyond the transcriptional effects of steroid hormones such as estrogen. As a result, we have limited therapeutic options for the ~62,000 women diagnosed with endometrial cancer each year in the United States. Here, in an attempt to identify new prognostic and therapeutic targets, we focused on a new area for this cancer-alternative mRNA splicing-and investigated whether splicing factor, SF3B1, plays an important role in endometrial cancer pathogenesis. Using a tissue microarray, we found that human endometrial tumors expressed more SF3B1 protein than non-cancerous tissues. Furthermore, SF3B1 knockdown reduced in vitro proliferation, migration, and invasion of the endometrial cancer cell lines Ishikawa and AN3CA. Similarly, the SF3B1 inhibitor, Pladienolide-B (PLAD-B), reduced the Ishikawa and AN3CA cell proliferation and invasion in vitro. Moreover, PLAD-B reduced tumor growth in an orthotopic endometrial cancer mouse model. Using RNA-Seq approach, we identified ~2000 differentially expressed genes (DEGs) with SF3B1 knockdown in endometrial cancer cells. Additionally, alternative splicing (AS) events analysis revealed that SF3B1 depletion led to alteration in multiple categories of AS events including alternative exon skipping (ES), transcript start site usage (TSS), and transcript termination site (TTS) usage. Subsequently, bioinformatics analysis showed KSR2 as a potential candidate for SF3B1-mediated functions in endometrial cancer. Specifically, loss of SF3B1 led to decrease in KSR2 expression, owing to reduced maturation of KSR2 pre-mRNA to a mature RNA. Importantly, we found rescuing the KSR2 expression with SF3B1 knockdown partially restored the cell growth of endometrial cancer cells. Taken together, our data suggest that SF3B1 plays a crucial oncogenic role in the tumorigenesis of endometrial cancer and hence may support the development of SF3B1 inhibitors to treat this disease.

Project description:BackgroundCircular RNAs play an important role in tumor genesis and progression, but they have not been sufficiently studied in patients with nasopharyngeal carcinoma (NPC).MethodsThe circular RNA, circCAMSAP1, was screened in NPC cells by RNA sequencing analysis. The expression of circCAMSAP1 in NPC tissues was examined by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization. Wound-healing, transwell, MTT and flow cytometry assays, and nude mouse tumor models were used to explore the effect of circCAMSAP1 on proliferation and metastasis of NPC in vitro or in vivo. The downstream proteins regulated by circCAMSAP1 were screened using mass spectrometry. The interaction between circCAMSAP1 and the SERPINH1 mRNA was identified using the circular RNA immunoprecipitation method and the luciferase reporter assay. The interaction between SERPINH1 and transcription factor c-Myc was verified through Co-immunoprecipitation (Co-IP) and immunofluorescence. The effect of c-Myc on the generation of circCAMSAP1 was examined through RT-qPCR and chromatin immunoprecipitation. Finally, the splicing factors that promote the production of circCAMSAP1 were explored by RT-qPCR and RNA immunoprecipitation (RIP).ResultsWe found that circCAMSAP1 was highly expressed in NPC tissues and promoted NPC proliferation and metastasis. Additionally, circCAMSAP1 promoted SERPINH1 expression through improved SERPINH1 mRNA stability by binding to the 3'-untranslated region (3'UTR) of SERPINH1. Highly expressed SERPINH1 reduced the ubiquitination-degradation rate of c-Myc, causing increased tumorigenesis. Meanwhile, c-Myc, cooperating with splicing factor 10 (SRSF10), could also promote CAMSAP1 pre-mRNA transcription and back-splicing, forming a positive feedback of circCAMSAP1 production, resulting in the proliferation and metastasis of NPC.ConclusionsOur findings revealed that circCAMSAP1 promotes NPC proliferation and metastasis by binding to the 3'UTR of SERPINH1, suggesting that the positive feedback of circCAMSAP1-SERPINH1-c-Myc may serve as a prognostic biomarker or therapeutic target in patients with NPC.

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Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization.

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