LASP2 Suppresses Colorectal Cancer Progression Through JNK/p38 MAPK Pathway Mediated Epithelial-mesenchymal Transition

Background and Objection:Colorectal cancer(CRC)is one of the most common cancers and a crucial cause of cancer-related death worldwide,the morbidity and cancer-related mortality rang the upper third and fourth of all tumors.In recent decades,the incidence of CRC has been fleetly increasing in our country due to the rapid economy development and the so-called western lifestyle.Although the 5-year survival rate of early stage CRC patients can reach 90%through surgical resection,about 40%of CRC patients have been diagnosed with tumor micro metastasis at clinic in China.And tumor metastasis is one of the most important cause of cancer mortality.CRC has seriously affected the life quality and survival time of patients,and also brought heavy financial burdens to the patients and society.Carcinogenesis and metastasis of CRC is a complex process invoving multiple genes in multistep and multistage,including the loss of normal regulation of cell growth in intestinal epithelial cells under multiple tumorigenesis factors,result in disorder of cell cycle,followed by immortalized cell growth,and eventually tumorigenesis.Then the cancer cells escape from the primary tumor site into the surrounding matrix,subsequently into the circulation system and lymphatic system.The cancer cells finally migrate extracascular from the endothelia cell wall for distant invasion as well as angiogenesis,and eventually develop metastases.Although numerous researches have been carried out on the carcinogenesis and metastasis molecular mechanism of colorectal cancer,the milestone has not reached yet.More than one hundred of genes have been reported to be involved in the metastasis of colorectal cancer,accomapnying lots of oncogene activation and anti-oncogene inactivation.Thus,it is urgent to fully understand the molecular mechanisms of CRC occurrence and progression,and to seek effective,sensitive and specific molecular biomarkers of CRC metastesis for early diagnosis,early intervention and outcome improvement,and finally producing clinical values and social benefits.LASP2(LIM and SH3 protein 2)gene is located on human chromosome 10p12.31,containing 39 exons.It can encode nebullete protein and LASP2 is one of the three isoforms,containing 1014 amino acids,and belongs to nebulin protein family together with LASP1(LIM and SH3 protein).They both contain a LIM domain at the amino-terminal and a homology Src region 3(SH3)at the C-terminal of the protein.LASP1 has a significant impact on cell motility as an important cytoskeleton protein,and was demonstrated involving in numerous metastatic cancer such as breast cancer and ovarian cancer.Our previous studies have proved LASP1 was significantly overexpressed on aggressive CRC,and promotes CRC growth aas well as metastasis.LASP2 shares a highly similar structure with LASP1,and is more likely to participate in the cancer progression.However,incipient studies were mainly about the function of LASP2 on cardiac diseases since nebulette was first found overexpressed in myocardium,which was an isoform of the protein encoded by LASP2 gene.Recently,the expression pattern of LASP2 was found to be different from nebulette as another isoform and researches emphasis have switched to its relation with cell adhesion and migration.This study focuses on the expression of LASP2 in CRC and its relationship with clinicpathological characteristics,investigating the role of LASP2 in the occurrence and progression of CRC and the underlying molecular mechanism,The aim of the study is to provide a brand new biomarker for early staging and a potential target for anti-cancer medicine of CRC.MethodsPart 1.The expression of LASP2 in CRC cell lines and tissues1.After extracting mRNA and protein from CRC cell lines,using qPCR and western blot to detect relative LASP2 mRNA and protein expression,respectively.2.Immunohistochemistry(IHC)was used to detect LASP2 expression in 89 paraffin-embedded CRC samples and 72 normal ones.Part 2.The relationship between LASP2 expression and clinicopathological parametersUsing Chi-square test,Kaplan-Meier survival analysis and Cox regression model to analyze the relationship between LASP2 expression and clinicopathological parameters such as cancer staging,metastasis and survival.Part 3.The function of LASP2 in the CRC growth and metastasis1.Consturct LASP2 overexpression vector and transfect to CRC cell lines with low LASP2 experession,using western blot and qPCR to identify the effect.Then evaluate the growth and migration ability through CCK8 and transwell assays.2.Synthesise siRNA to interfere the expression of LASP2 and transfect to LASP2 overexpressed CRC cell lines.Evaluate the impact of LASP2 on CRC cell growth and migration through CCK8 and transwell assays after verify the interference effect.Part 4.Molecular mechanism of LASP2 mediated CRC growth and metastasis1.Western blot was utilized to detect signaling pathway activation and EMT markers after LASP2 interference.2.Specific signaling pathway inhibitors were applied to verify the corresponding signaling pathway was required for LASP2 modulated CRC development.Part 5.The relationship between LASP2 and LASP1 expression in CRC1.Detect LASP1 and LASP2 expression in CRC cell lines and tissues using qPCR.2.Transfect LASP1 overexpression vector and interference siRNA respectively,and then detect LASP1 and LASP2 expression using western blot.3.IHC was used to detect LASP2 epxression in LASP1 overexpressed and depressed CRC samples.Results:Part 1.The expression of LASP2 in CRC cell lines and tissues1.LASP2 expression in CRC cell lines LASP2 expression was low in HT29,RKO and SW480,but high in HCT116,SW620 and SW480/M5.Western blot also revealed similar result,and we chose SW480 as LASP2 low CRC cell line,and HCT 116 as well as SW620 as LASP2 overexpression CRC cell lines for further studies.2.LASP2 expression in CRC tissues IHC results showed that the overexpression rate of LASP2 was 68%(49/72)in normal colorectal tissues,compared with 50.6%(45/89)in colorectal tumors.(p=0.025).3.The relationship between LASP2 expression and clinicopathological parameters LASP2 expression was irrelevant with sex,age,tumor size and T classification(p>0.05),but positive with high tumor differentiation(p=0.023),and negative with N classification(p=0.026)as well as AJCC stage(p=0.015).Although Cox proportional hazards model of multivariate analysis indicated that LASP2 expression level is not an independent prognostic factor for prognosis of CRC patients,univariate analysis revealed it do correlate with the overall survival rate.4.The relationship between LASP2 expression and CRC patient survival Kaplan-Meier survival analysis demonstrated that the CRC patients with high expression level of LASP2 had a better prognosis not only in T3 + T4 stage(log rank=4.384,p=0.036)but also all stages(log rank=5.490,p=0.019).Part 2.The effect of LASP2 expression on CRC biological characteristics.1.The effect of LASP2 transient overexpression transfection on CRC cell proliferation and migration1.1 The verify of LASP2 transient overexpressionAfter tranfecting LASP2 overexpression vector and control vector to SW480,we used western blot to verify that LASP2 was significantly overexpressed in the LASP2 vector group compared with control group(p<0.01)1.2 The impact of LASP2 overexpression on CRC cell proliferation and migrationCCK8 assay showed that the growth of SW480 cells decrased significantly in the LASP2 vector group compared with the control group(p<0.05),indicating LASP2 overexpression can suppress CRC cell growth.Transwell assay showed that the cell number that migrated through transwell chamber was significantly decreased in the LASP2 vector group compared with the control group(p<0.01),indicating that LASP2 overexpression can suppress CRC cell migration.2.The effect of LASP2 transient interference transfecdtion on CRC cell growth and migration2.1 The verify of LASP2 transient silencingWe chose the most effective siRNA to interfere LASP2 expression and transfected it to HCT 116 and SW620.Then we used western blot to detect the expression of LASP2 in the LASP2 silencing group and control group,results showed that LASP2 expression was markedly interfered in the siRNA group compared with control group(p<0.05).2.2 The impact of LASP2 transient silencing on CRC cell proliferation and migrationCCK8 assay showed that the growth of HCT116 and SW620 cells increased significantly in the LASP2 siRNA group compared with the control group(p<0.05),indicating that LASP2 silencing can promote CRC cell growth.Transwell assay showed that the cell number that migrated through transwell chamber was significantly increased in the LASP2 silencing group compared with the control group(p<0.01),indicating that LASP2 silencing can promote CRC cell migration.Part 3.The molecular mechanism of LASP2 regulated CRC cell proliferation and migration1.The impact of LASP2 silencing on JNK/p3 8 MAPK sinaling pathway After transfecting LASP2 siRNA and control to SW620 respectively,western blot was used to detect the expression of MAPK signaling pathway markers.Results showed that phosphorylation of p38,SAPK/JNK,p44/42 were significantly increased in the siRNA group,indicating LASP2 silencing can activate JNK/p38 MAPK signaling pathway.2.The impact of LASP2 silencing on EMT markers Western blot was used to detect the EMT markers expression in the LASP2 silencing group and control group.Results showed that the expression of epithelial markers(ZO0-1,E-cadherin,β-catenin)were markedly decreased and the expression of mesenchymal markers(N-cadherin,vimentin)were significantly increased in the LASP2 silencing group,indicating that LASP2 silencing can induce EMT in CRC cells.3.The reverse effect of signaling pathway inhibitor on LASP2 regulated signaling pathway activation and CRC cell behaviors We added JNK inhibitor and p38 inhibitor to the SW620 cells that have been transfected with LASP2 siRNA,and then detected the expression of p38,JNK,and EMT markers.Results showed that the expression of these molecular markers were markedly recovered compared with the LASP2 siRNA group.And transwell assay showed cells that migrated through the chamber were significantly decreased in the signaling pathway inhibitor group.All these results suggested that JNK/p38 MAPK signaling pathway was required in the LASP2 regulated CRC cell proliferation and migration.Part 4.The relationship of LASP2 and LASP1 expression in CRC1.The relationship of LASP2 and LASP1 expression in CRC cell lines We used qPCR to detect LASP1 and LASP2 mRNA relative expression in CRC cell lines and found that the mRNA expression of LASP1 and LASP2 were negatively correlated in CRC cell lines.Meanwhile,we transfected LASP1 siRNA to SW480 and LASP1 vector to SW620,and then used western blot to detect LASP1 and LASP2 expression.Results indicated that LASP2 protein expression in CRC cell lines were negatively regulated by LASP1.2.The relationship of LASP2 and LASP1 expression in CRC tissues 89 Paraffin-embedded CRC samples were stained with anti-LASP1 or anti-LASP2 antibodies in the same position.IHC results revealed that the expression of LASP1 and LASP2 were negatively correlated in CRC tissues(R=-0.390,P=0.013).ConclusionLASP2 expression is low in CRC cell lines and tissues.It can suppress CRC cell proliferation and migration,and is correlated with tumor differentiation,N classification AJCC stage as well as patient survival,which will add evidences to LASP2 as a novel biomarker in cancer staging and prediction of survival.