PAK4 Kinase-Mediated LASP1 Phosphorylation Promotes Esophageal Squamous Cell Carcinoma Metastasis

ObjectiveAccording to the 2019 China Cancer Registration Annual report,esophageal cancer ranks sixth in the incidence of malignant tumors in the country,while in the mortality rate of malignant tumors,esophageal cancer ranks fourth.In China,there are obvious regional differences in the incidence of esophageal cancer.Henan Province is one of the areas with high incidence of esophageal cancer in China.At the same time,the main pathological type of esophageal cancer in China is esophageal squamous cell carcinoma(ESCC).The etiology of ESCC is complex,and the specific pathogenesis is unclear.The average 5-year survival rate of ESCC patients is only 15%-20%.Local invasion is the most common mode of metastasis of esophageal squamous cell carcinoma,accompanied by lymph node metastasis.Numbers of patients with esophageal cancer have already exhibit metastasis at the time of diagnosis.The complications from tumor metastasis are the main cause of cancer-associated death.At the same time,there is a lack of specific markers for early diagnosis and treatment of ESCC.p21-activated kinase 4(PAK4),a member of the p21-activated kinase(PAK)family of serine/threonine kinases,was initially identified as an effector of Cdc42 regulating cytoskeleton reorganization.It has been found that PAK4 is involved in a variety of cellular processes including cytoskeleton reorganization,cell migration,cell apoptosis and cell proliferation.PAK4 is the most closely related to human tumors among other PAKs.Studies have shown that PAK4 is highly expressed in liver cancer,breast cancer,prostate cancer and so on.In the previous study,we found that PAK4 is highly expressed in ESCC tissues and cell lines compared with normal esophageal tissue and esophageal epithelial cell line.we also found that PAK4 can promote cell proliferation,migration and invasion of ESCC in vitro and in vivo.Moreover,pull down assay showed that LASP1 may be a potential target of PAK4.LIM and SH3 proteinl(LASP1)was initially identified from a cDNA library of the metastatic lymph nodes of human breast cancer,originally named MLN50.In recent years,growing evidence has shown that LASP1 is involved in a variety of cellular signal transduction and transcriptional regulation and various biological processes.Studies have found that LASP1 is highly expressed in a variety of cancers,promoting cancer metastasis,and its expression level is negatively correlated with the survival rate of patients.According to the previous results,we found that PAK4 can bind and co-localize with LASP1 in vitro and in cells via immunoprecipitation assay and confocal microscopy analysis.However,the relationship between LASP1 and PAK4 and the detail mechanism need to be further identified.In this study,first,rescue experiment was used to determine whether LASP1 is the downstream of PAK4.Then,we used kinase assay in vitro to determine whether PAK4 could phosphorylate LASP1,and the phosphorylation site was determined by mutagenesis of the potential phosphorylation site.The role of p-LASP1(S198)on migration and invasion in ESCC in vitro and in vivo was investigated by overexpression of mutant LASP1 in ESCC cell lines,and p-LASP1(S198)in EMT pathway was also observed.Finally,we observed the effect of p-LASP1(S198)on Snail transcription and protein stability in ESCC cells overexpressing mutant LASP1 to further explore the mechanism of PAK4 promoting the development of EMT pathway by phosphorylating LASP1 in ESCC,which provides theoretical and experimental basis for PAK4 and p-LASP1(S198)to become new targets for clinical diagnosis and treatment of esophageal squamous cell carcinoma.Methods1.Determine whether LASP1 is a downstream target molecule of PAK4.(1)Lentivirus infection was used to knock down the expression level of LASP1 in esophageal squamous cell carcinoma cell line KYSE150 which had been overexpressed with PAK4.The effect of rescue experiment on EGFR and EMT signal pathways was detected by western blotting to determine whether LASP1 is the downstream target molecule of PAK4.(2)Wound healing assay and Transwell invasion assay were used to further determine whether LASP1 is the downstream of PAK4.2.Explore whether PAK4 can phosphorylate LASP1,and determine the phosphorylation site.(1)LASP1 protein was purified and western blotting was used to detect the p-ser and p-thr after in vitro kinase assay of PAK4 and LASP1.(2)The serine at the potential phosphorylation sites was mutated to alanine by DNA site-directed mutagenesis,and the mutant LASP1 protein was purified.(3)In vitro kinase assay was performed using PAK4 and wt-LASP1 or mut-LASP1 to confirm the phosphorylation sites are right or not.3.Observe the effect of p-LASP1(S198)on invasion and metastasis of ESCC,and explore the mechanism of p-LASP1(S198)promoting the progression of ESCC.(1)Subclone pET-28a-mut-LASP1 to pcDNA3.1-mut-LASP1.(2)The cDNA of mut-LASP1 was transfected into KYSE150,KYSE30 and KYSE510 esophageal squamous cancer cells.(3)Wound healing assay was used to investigate the changes of the effect of p-LASP1(S198)on migration ability of ESCC cell lines.(4)Transwell migration assay and Transwell invasion assay was used to check the effect of p-LASP1(S198)on migration and invasion ability of ESCC cell lines.(5)Metastasis assay in vivo was used to verify the metastatic roles for p-LASP1(S198)in ESCC.(6)Western blotting was used to detect the effect of p-LASP1(S198)on EMT signal pathways.(7)RT-qPCR was used to observe the effect of p-LASP1(S198)on the mRNA level of Snail.(8)CHX was used to investigate the effect of p-LASP1(S198)on the protein stability of Snail.Results1.LASP1 is the downstream of PAK4 via the rescue experiment.2.In vitro kinase assay showed that PAK4 can phosphorylate LASP1 at serine 198.3.p-LASP1(S198)can increase migration and invasion of esophageal squamous cancer cells.4.p-LASP1(S198)can promote the migration and invasion of esophageal squamous carcinoma cells through the regulation of EMT signaling pathway.5.p-LASP1(S198)can increase the expression level of Snail by stabilizing Snail.Conclusions1.PAK4 phosphorylates LASP1 at serine 198.2.p-LASP1(S198)can increase the stability of Snail and promote the invasion and metastasis of esophageal squamous cell carcinoma through EMT.