Pea3 expression promotes the invasive and metastatic potential of colorectal carcinoma

AIM: To investigate the function of Pea3 in colorectal carcinoma (CRC) invasion and metastatic potential.

AIM:To investigate the function of Pea3 in colorectal carcinoma(CRC)invasion and metastatic potential.METHODS:The expression of Pea3 during clinical progression of human CRC was investigated using Oncomine Research Edition.To assay Pea3 expression in established CRC cell lines,we performed western blotting of cell lysates.We employed sh RNA-mediated knockdown of Pea3 in HCT116(HCT)and LS174T CRC cells which was confirmed by real-time quantitative PCR(q PCR)and western blotting.Transwell invasion assays,MTS proliferation assays,anoikis assays,and fluorometric matrix metalloprotease(MMP)assays wereperformed to determine the effects of Pea3 knockdown on invasion,proliferation,anoikis and MMP activity in CRC cells in vitro.Alterations in epithelial-mesenchymal transition(EMT)and matrix metalloprotease(MMP)m RNA levels were determined by q PCR.CRC cells were injected into the flanks of nude mice to generate xenografts and tumor growth monitored with serial calliper measurements.To assay metastatic potential,CRC cells were injected into the spleen of nude mice,and histological analysis performed on the livers 21 d later.RESULTS:We demonstrated that reduction of Pea3expression in CRC cells significantly impaired their invasive capacity(HCT.sh Pea3,0.28±0.04 fold,P<0.01;LS.sh Pea3,0.15±0.04 fold;SW.sh Pea3,0.23±0.03,P<0.01),reduced anoikis resistance(HCT.sh Pea3 75.4%±1.9%viable cells vs HCT.sh Ctrl 88.6%±0.6%viable cells,P<0.01;LS.sh Pea3 71.7%±0.5%viable cells vs LS.Ctrl 89.6%±0.3%viable cells,P<0.005,but had no effect on proliferation(HCT.sh Ctrl AUC 5098±123 vs HCT.sh Pea3 5689±151,P<0.05;LS.sh Ctrl AUC 5600±324.1 vs LS.sh Pea3 6423±400,P<0.05).In vivo,HCT.sh Pea3 and HCT.sh Ctrl tumour xenografts grew at a similar rate(HCT.sh Pea3 2.64±0.82 fold vs HCT.sh Ctrl 2.88±0.80 fold,P>0.05).In keeping with a pro-metastatic function for Pea3 in CRC,several EMT markers and MMPs were downregulated in sh Pea3-expressing cells,suggesting that Pea3 may exert its effects through these processes.A reduction in overall MMP activity was observed in HCT.sh Pea3 cells compared to their control counterparts(HCT.sh Pea30.61±0.04 fold,P<0.005).This translated in vivo to the complete absence of metastases in the livers of mice that were grafted with CRC cells lacking Pea3.Conversely,CRC cells expressing Pea3 formed liver metastases in all mice.CONCLUSION:Our study implicates Pea3 as a mediator of metastases,and provides a biological rationale for the adverse prognosis associated with elevated Pea3 expression in human CRC.