PIM1 Mediates Epithelial-Mesenchymal Transition By Targeting Smads And C-Myc In The Nucleus And Potentiates Clear-Cell Renal-Cell Carcinoma Oncogenesis

Background Emerging evidence has shown that the PIM serine/threonine kinase family,including PIM1,PIM2 and PIM3,is associated with tumour progression towards metastasis.PIM1,an attractive molecular target,has been identified as a potential prognostic biomarker for haematological and epithelial malignancies.However,to date,the potential regulatory roles and molecular mechanisms by which PIM1 affects the development and progression of cancers,including clear-cell renal-cell carcinoma(ccRCC),remain largely unknown.Objective The objective of this study is to investigate the expression profile and role of PIM1 in ccRCC and provide a possible new therapeutic opportunity for ccRCC patients.Methods(1)Immunohistochemical staining was performed on 75 specimens of ccRCC tissue microarray including cancer tissues and surrounding tissues and paraffin-embedded tissues of 56 patients with renal cell carcinoma collected from the hospital using PIM1 monoclonal antibody.Then,the results of staining were analyzed and the differential expression of PIM1 was detected.The localization of PIM1 protein was determined,and the relationship between PIM1 differential expression and survival prognosis was analyzed.Additionally,the expression of PIM1 in ccRCC cell lines was detected by immunoblotting assays and real-time fluorescence quantitative PCR assays.(2)We detected whether the stable downregulation of PIM1 mediates the proliferation,migration,invasion,angiogenesis and epithelial-mesenchymal transition(EMT)of ccRCC cells in vitro.(3)Immunoblotting assays,coimmunoprecipitation assays and small molecule inhibitors were performed to search the potential regulatory roles and molecular mechanisms by which PIM1 affects the development and progression of ccRCC.The relationship between PIM1 and the expression of EMT-inducing transcription factors,including ZEB1,ZEB2,Snail1,Snail2 and Twist proteins,was detected,and the relationship between PIM1 and Smad signaling pathway was determined.Furthermore,the relationship between Smads and c-Myc was analyzed following stable downregulation of PIM1.(4)Subcutaneous tumor formation assay in athymic nude mice was used to detect the proliferation of ccRCC cell line following the stable downregulation of PIM1 in vivo.Results(1)In our study,we present the first evidence that PIM1 is aberrantly overexpressed in human ccRCC tissues and cell lines and positively correlated with progression of human ccRCC.(2)Depletion of PIM1 attenuated ccRCC cell proliferation,colony formation,migration,invasion and angiogenesis,suggesting that PIM1 expression may be a cancer-promoting event in ccRCC.(3)Mechanistically,we observed that PIM1 could interact with Smad2 or Smad3 in the nucleus and subsequently phosphorylate Smad2 and Smad3 to induce the expression of transcription factors,including ZEB1,ZEB2,Snail1,Snail2 and Twist,to promote epithelial-mesenchymal transition(EMT).(4)In addition,PIM1-mediated phosphorylation of c-Myc activates the expression of the above transcription factors to synergistically promote EMT but does not activate Smads.Conclusion(1)Our results demonstrate that aberrant expression of PIM1 contributes to ccRCC development and progression.(2)Moreover,our data reveal a potential molecular mechanism in which PIM1 mediates crosstalk between signalling pathways,including different Smad proteins and c-Myc,which target downstream transcription factors(ZEB1,ZEB2,Snail1,Snail2 and Twist)to trigger EMT.(3)Together,our data suggest that PIM1 may be a potential therapeutic target for ccRCC patients.