The Role And Its Underlying Mechanism Governed By SALL4 In The Malignant Phenotypes Of Clear Cell Renal Cell Carcinoma

【Background】Renal cell carcinoma,stemming from the renal tubular epithelium,is one of the top ten leading malignancies.As a most frequent subtype of renal cell carcinoma,clear cell renal cell carcinoma(cc RCC)comprises approximately 70% of kidney cancers.Mutation of the von Hippel-Lindau(VHL)tumor suppressor is observed in roughly 80% of cc RCC tumors and identified as one of the genetic determinants driving cc RCC initiation and progression.Development of metastatic spread and radiochemoresistance contributes to a poor prognosis,as evidenced by a dismal 8-12% five-year overall survival of metastatic cc RCC patients.It is universally acknowledged that cc RCC is a highly vascularized malignancy and therapies targeting angiogenesis are initially efficacious in tumor regression.Unfortunately,it is inevitable that acquisition of drug resistance occurs within a year and renders this treatment invalid in most patients.Deepening the understanding of the molecular dependencies and vulnerabilities of cc RCC will contribute to developing new therapeutic strategies for patients who fail to respond to conventional treatment.SALL4(spalt-like protein 4),a transcription factor containing zinc-finger domains,plays a critical role in regulating pluripotency of embryonic stem cells(ESCs)and sustaining ESCs self-renewal.SALL4 is indispensable for embryogenesis,but rarely expressed in most adult tissues.Nevertheless,aberrant expression of SALL4 is observed in numerous malignancies.The re-expressed SALL4 is shown to be responsible for tumor growth,metastasis and treatment resistance through activating multiple signaling pathways.However,it is not known whether SALL4 is involved in the pathogenesis of cc RCC.Here,we investigate the relationship between SALL4 and cc RCC tumorigenesis.【Objectives】To study the expression level and biological function of SALL4 in cc RCC and explore the molecular mechanism by which SALL4 regulates cancer progression,aiming at offering potential diagnostic markers and therapeutic targets for cc RCC.【Methods】Analyses of publicly available datasets and clinical specimen were performed to evaluate the expression level of SALL4 in cc RCC and determine the correlation between SALL4 and clinical pathological parameters,such as histopathological grade and AJCC stage.The correlation between SALL4 expression and cc RCC patients’ overall survival was determined by Kaplan-Meier survival analysis.Univariate and multivariate Cox regression analyses were performed to determine the prognostic value of SALL4 for cc RCC patients.Lentiviral transduction was conducted to establish cell lines with stable SALL4 downregulation.The cells were then subjected to colony formation assay,CCK-8assay,SA-β-gal staining,flow cytometry analysis and in vivo tumorigenicity assay to determine the influence of SALL4 on cc RCC cell colony formation ability,proliferation,cell senescence,cell cycle distribution and tumorigenic potential.The effect of SALL4 on migration and invasion of cc RCC cells was analyzed by wound healing assay and Transwell assay.Pearson correlation analysis was performed to determine the relationship between SALL4 expression and individual gene transcription.Integrative database analysis was conducted to explore the association of SALL4 with multifarious molecular signatures and the involvement of SALL4 in biological interaction network in cc RCC.A series of in vitro experiments were performed in HUVEC cells with stable SALL4 downregulation to determine the effect of SALL4 on endothelial cell function.Endothelial cells treated with tumor-derived conditioned medium were subjected to a series of in vitro experiments to evaluate the effect of SALL4 in cc RCC on tumor angiogenesis.Western blotting,immunofluorescence staining and public database analysis were performed to investigate the effect of SALL4 on Akt/GSK-3β/VEGFA axis,the correlation of VHL mutation with SALL4 expression and the association of SALL4 DNA methylation with its expression level.【Results】Compared with normal kidney tissues,SALL4 expression was increased in cc RCC.SALL4 expression was positively correlated with T stage,histopathological grade and AJCC stage.Kaplan-Meier survival analysis showed that patients harboring higher SALL4 level exhibited poorer overall survival.Univariate and multivariate Cox regression analysis indicated that SALL4 could be an independent prognostic factor of overall survival for cc RCC patients.Results from in vitro and in vivo experiments demonstrated that SALL4 promoted cc RCC cell proliferation,colony formation ability,cell cycle progression,migration,invasion and tumorigenicity and inhibited cell senescence.Further investigation revealed a widespread association of SALL4 with individual gene transcription and the involvement of SALL4 in biological processes such as endothelial development and vasculogenesis.On a genome-wide scale,SALL4 m RNA level was significantly associated with multiple molecular signatures,including gene expression,protein level(RPPA),micro RNA expression,DNA methylation,somatic copy number,and somatic mutation.SALL4 was able to promote endothelial cell proliferation,migration and tube formation.In the context of cc RCC,SALL4 promoted tumor vascularization by recruiting endothelial cells.In addition,we found that SALL4 could exert its tumor-promoting effect via the activation of Akt/GSK-3β/VEGFA axis.The VHL mutation was closely related to the expression of SALL4.Compared with cc RCC harboring wild-type VHL,the level of SALL4 in VHL-mutant cc RCC tissues was significantly increased.Nevertheless,no significant difference of SALL4 expression was observed among cc RCC patients harboring different VHL mutation types.Further analysis of the association between VHL point mutations and SALL4 expression showed that the level of SALL4 in cc RCC tissues harboring VHL＿p.L89 H point mutation was significantly increased.DNA methylation analysis indicated that compared with normal kidney tissues,the DNA methylation level of SALL4 was significantly decreased and negatively correlated with SALL4 expression in cc RCC tissues.【Conclusions】Upregulated SALL4 is closely related to cancer progression and poor prognosis in cc RCC.SALL4 can promote the proliferation,metastasis and tumor vascularization of cc RCC through activating Akt/GSK-3β/VEGFA signaling.VHL mutation and DNA hypomethylation may be involved in the upregulation of SALL4 in cc RCC.