Deciphering The Role And Molecular Mechanism Of DIRAS2 And SETD2 In Renal Cell Carcinoma

Background: Renal cell carcinoma is one of the most common and lethal human urological malignancies in the world.Major subtypes are clear cell renal cell carcinoma(cc RCC,75%),papillary renal cell carcinoma(p RCC,16%)and chromophobe renal cell carcinoma(ch RCC,7%).Renal cell carcinoma is a polygene-related tumor which is difficult to be diagnosed at early stage.Unfortunately,the molecular biological mechanism of its pathogenesis and development has not been clarified so far.Studies have shown that RCC is not only related to genetic factors,but also related to epigenetic regulation.Alterations in histone methylation are reported in several types of cancers and have also been examined in RCC.The GTP-binding Ras-like protein 2(DIRAS2)is one of GTPases forming a distinct subgroup of the Ras family,and is upregulated in RCC.As an epigenetic factor,SETD2 has been increasingly identified as a common mutation across cancer types,including RCC.However,the specific mechanism of DIRAS2 and SETD2 in RCC remains largely undefined.Objective: Our project mainly divided into two parts to explore the molecular biological mechanism of DIRAS2 and SETD2 genes in the formation and development of renal cell carcinoma.It would help us understand the development and progression of renal cell carcinoma and provide a promising strategy to treatment and early diagnosis.Methods: To determine the role of DIRAS2 in RCC,we first analyzed the expression level of DIRAS2 using tumor databases and biopsy specimens.Then,cell experiments were performed to explore the tumor-promoting function of DIRAS2 in vivo and in vitro.Finally,molecular experiments were conducted to explore the molecular mechanism of DIRAS2 in RCC.To explore a possible role of SETD2 in kidney development and tumorigenesis,we first generated Setd2-floxed mice and deleted the SETD2 in tubular epithelial cells.The immunohistochemical staining and western blotting were performed to analyze the mouse phenotype.Then,RNA-seq and Ch IPseq analysis were utilized to explore the changes of relevant signaling pathways and gene expression caused by the deletion of SETD2.Finally,rescue experiments were performed to detemin the molecular mechanism of SETD2 deficiency in the occurrence of RCC.Results: Part 1: We performed searches and meta-analysis on microarray data sets from the Oncomine(www.oncomine.com)and TCGA(www.cancer.gov)databases for possible targets whose expressions are altered significantly in renal cell carcinoma.Interestingly,DIRAS2 was one of the top hits in our searches.We reported in this part that DIRAS2 is upregulated in clear cell renal cell carcinoma with mutated VHL gene and exerts an activating effect on the MAPK pathway,thereby promoting renal cell carcinoma cells proliferation,migration,invasion and clone formation in the absence of VHL gene.Meanwhile,DIRAS2 activity is regulated by VHL,which can promote the ubiquitination and degradation of DIRAS2 protein.Part 2: We established a mouse model of polycystic kidney disease driven by c-MYC and a mouse model of clear cell renal cell carcinoma driven by c-MYC and deficiency of SETD2 to determine the importance of SETD2 during the transition from polycystic kidney disease to renal cell carcinoma.We reported a new tumor suppressing mechanism of SETD2 in inhibiting β-catenin activity on transcriptional and posttranscriptional levels by competing with the β-catenin for binding at promoters of a subset of β-catenin-regulated genes and maintaining transcription levels of the members of “β-catenin destruction complex” through H3K36me3 modification.Thus,SETD2 deficiency leads to up-regulated Wnt/β-catenin signaling and enhanced epithelia–mesenchymal transition,which accelerates the transition from polycystic kidney disease to renal cell carcinoma.Conclusion: Our findings for the first time revealed previously unrecognized the role of DIRAS2 in promoting renal cell carcinoma formation and the role of SETD2 in multi-levelly regulating β-catenin activity during the transition from polycystic kidney disease to renal cell carcinoma.In addition,an autochthonous cancer models of cc RCC that recapitulate its cardinal molecular and cellular features during the transition from PKD to RCC was successfully established.