The Effects Of PEAK1-PPP1R12B Signaling Axis On The Growth And Metastasis Of Colorectal Cancer

BACKGROUND: Colorectal cancer(CRC)is one of the most prevalent carcinomas worldwide,and its morbidity and mortality are in the forefront.The malignant growth and metastasis of CRC is a comprehensive biological progression involving multi-steps,multi-stages,and multi-factors interaction.CRC progression and metastasis is influenced by a series of well-characterized processes,such as anti-apoptosis,proliferation,and invasion of tumor cells,epithelial-mesenchymal transition(EMT)and angiogenesis.Protein tyrosine kinase(PTK)and its mediated signaling pathways play an important role among these progresses.PEAK1(Pseudopodium-enriched atypical kinase 1),also known as Sgk269(Sugen kinase 269),is a novel atypical protein kinase,which mainly regulates p130cas/Crk/Rac1 and RAS/Raf/ERK signaling.Recent studies have emerged supporting a functional role for PEAK1 in the growth and distant metastasis of several human malignancies.However,its exact molecular mechanism and potential clinical application value in CRC progression have not been completely explored.OBJECTIVE: The aim of this study is to detect the expression level of PEAK1 mRNA and protein in clinical CRC samples,and the relationship between PEAK1 expression and clinical pathological parameters of CRC patients was discussed.Then,to further detect the expression of PEAK1 in human CRC cell lines,and select the appropriate cell line to construct stable PEAK1-overexpression/knockout cell lines via using lentivirus vectors.Next,to confirm the roles of PEAK1 in CRC cell proliferation,migration,invasion and EMT in vitro.Subsequently,in vivo mouse tumor bearing and metastasis models to further explore the role of PEAK1 in the growth,angiogenesis,and distant metastasis of CRC.Finally,the key molecular signals and potential target genes regulated by PEAK1 in CRC growth and transfer are revealed,and the correlation between the key sites of PEAK1 protein and these molecules is also analyzed.METHODS:1.Analysis of the correlation between PEAK1 expression level and clinical pathological parameters in human CRC tissue27 pairs of frozen CRC tissues and 124 formalin-fixed paraffin-embedded tissues(including 84 tumor and 40 normal tissues)were obtained from the Department of Gastroenterology,the Affiliated Zhongda Hospital of Southeast University,and the Department of Gastrointestinal Surgery,the Affiliated Hospital of Zunyi Medical University.q RT-PCR and Western blot were used to detect the expression of PEAK1 mRNA and protein in paired frozen tissue,respectively.The expression of PEAK1 protein was detected in formalin-fixed paraffin-embedded sections by using immunohistochemistry(IHC),and analyzed the relationship between PEAK1 expression and clinical pathological parameters.2.Building stable PEAK1 overexpression and knockout CRC cell linesThe expression of PEAK mRNA and protein in multiple human CRC cell lines was detected by q RT-PCR and Western blot analyses,and then selected the appropriate cell strains for post-experiment.The stable PEAK1-overexpression LoVo cell line(named LoVo-PEAK1),HCT116 cell line(named HCT116-PEAK1),and their corresponding control cell strains(named LoVo-Ctrl,HCT116-Ctrl,respectively)were built through using Lentiviral vectors bearing PEAK1 gene or empty lenti-vectors.The expression of PEAK1 mRNA and protein was confirmed by q RT-PCR and Western blot analyses,respectively.The stable PEAK1-knockout SW480 cell line(named SW480-KO),HT29 cell line(named HT29-KO),and their corresponding control cell strains(named SW480-Ctrl,HT29-Ctrl,respectively)was constructed by CRISPR-Cas9 system for PEAK1 or negative vector,and q RT-PCR and Western blot analyses were used to detect the expression of PEAK1.3.Detection of the effects of PEAK1 on CRC cell proliferation,migration and EMT processesThe effects of PEAK1 on the proliferation and clonogenic abilities of CRC cells in each groups were detected by CCK-8 and colony formation analyses,respectively.Scratch and transwell experiments were used to verify the effects of PEAK1 on cell migration and invasion.Western blot and cell immunofluorescent staining were used to detect EMT-related indicators(such as epithelial markers E-cadherin and β-catenin,mesenchymal markers N-cadherin and Vimentin)in each groups regulated by PEAK1 expression.4.Analysis of the effects of PEAK1 on CRC growth,angiogenesis and distant metastasisThe effects of PEAK1 on the growth,tumorigenesis and vascular formation of CRC cells were verified through injecting stable PEAK1-overexpression CRC cells into the flanks of nude mice.Stable PEAK1-overexpression CRC cells were intravenously injected into nude mice via the tail vein to construct metastasis animal models,and then detected the roles of PEAK1 in the cell invasive,metastasis and metastatic nodules.5.To find the key signaling pathways and potential target genes involved in the regulation of PEAK1 on CRC growth and metastasisBulk RAN-seq analysis was used to explore the target genes that PEAK1 may regulate,then detected their correlation with endogenous expression in CRC cell lines and clinical specimens.KEGG pathway analysis preliminary indicated key molecular signaling pathways that PEAK1 affected CRC growth and metastasis,and western blot further confirmed these results.The technique of point mutation was used to analyze whether the key sites on the PEAK1 protein were involved in the regulation of these signaling pathways and target genes.6.To explore whether the target gene can reshape PEAK1 induced biology function and signaling transduction of CRC cellIn stable PEAK1-overexpression or knockout CRC cell lines,to explore whether downregulation or upregulation of the target gene could rescue PEAK1-mediated signaling pathway and CRC cell biology function.Finally,small molecule antagonists were used to inhibit the transduction of key signaling pathways,and to further verify whether it could reverse abnormal PEAK1 expression induced the growth and metastasis of CRC cells.RESULTS:1.In 27 pairs of paired frozen CRC tissues,the expression level of PEAK1 mRNA and protein was significantly reduced in cancer tissues.IHC staining further showed that PEAK1 protein in tumor tissues was markedly impaired as compared to expression in normal colorectal tissues,and its expression was obviously associated with tumor size,differentiation status,lymph node metastasis,distant metastasis and clinical stage(P< 0.05).2.Highly metastatic cell lines,such as HCT116 and LoVo expressed low level of PEAK1 mRNA and protein,while primary cancer cell lines SW480 and HT29 expressed high level of PEAK1 mRNA and protein.Subsequently,stable PEAK1-overexpression LoVo and HCT116 cell lines,and stable PEAK1-knockout SW480 and HT29 cell lines were successfully built.3.Cell proliferation,clonogenic,migration and invasion abilities were significantly decreased following ectopic upregulation of PEAK1 in LoVo and HCT116 cells,whereas PEAK1 knockout obviously increased cell proliferation,colony formation,migration and invasion in SW480 and HT29 cells,as compared to those in corresponding control cells.Further studies had shown that PEAK1 upregulation in LoVo and HCT116 cells resulted in low expression of mesenchymal markers,such as N-cadherin and Vimentin,and high expression of epithelial markers,such as E-cadherin and β-catenin,whereas PEAK1 knockout in SW480 and HT29 cells led to the opposite effects.4.PEAK1 overexpression resulted in markedly reduced tumor volumes,growth speed,and exhibited decreased cell proliferation activity and angiogenesis,as compared to those in the control group.Experimental metastasis animal models showed that PEAK1 overexpression induced a significant reduction in liver metastases rate and metastatic nodules.5.The sequencing results showed that 27 differentially expressed genes might be the regulated targets for PEAK1 gene.Further studies showed that the endogenous expression of PPP1R12 B was positively correlated with PEAK1 expression,and PEAK1 could significantly upregulate PPP1R12 B level.The PEAK1-PPP1R12 B axis inhibited the malignant progression of CRC by negatively regulating the Grb2/PI3K/Akt pathway.PEAK1 protein affected CRC growth,metastasis,and the transduction of Grb2/PI3K/Akt signaling partially through Y1188,rather than P1153 site.6.PPP1R12 B knockdown significantly decreased the suppressive effects of PEAK1 on cell proliferation,migration,invasion,and Grb2/PI3K/Akt signaling in stable PEAK1-overexpression LoVo and HCT116 cell lines.However,exogenous PPP1R12 B expression in PEAK1-knockout SW480 and HT29 cells resulted in a marked reduction in PEAK1-induced cell proliferation,migration,and invasion activities,and Grb2/PI3K/Akt signaling pathway.In stable PEAK1-knockout SW480 and HT29 cell lines,the small molecular inhibitors of PI3K/Akt signaling pathway significantly reduced PEAK1 knockout-induced cell proliferation,migration,and invasion activities,as compared to that in the control groups.CONCLUSIONS:1.The expression level of PEAK1 mRNA and protein was downregulated in CRC tissues,and its expression was obviously associated with the growth,metastasis,and clinical stage of CRC.2.PEAK1 inhibited the proliferation,invasion,and metastasis activities of CRC cells in vitro and in vivo.3.PEAK1 and PPP1R12 B formed a positive feedback axis,and negatively regulated the Grb2/PI3K/Akt signaling pathway,which then induced a decreased in CRC growth and metastasis.4.PEAK1 protein regulated the biological function of CRC cells and Grb2/PI3K/Akt signaling pathway partly through regulation of Y1188 site.5.PPP1R12 B expression recapitulated the negative regulatory role of PEAK1 on CRC cell proliferation,migration,invasion,and the transduction of Grb2/PI3K/Akt signaling.