SIK1 Supresses Colorectal Cancer Metastasis And Chemoresistance Via The TGF-β Signaling Pathway

Objective:Metastatic colorectal cancer(CRC)is the cancer with the highest mortality rate worldwide.Among patients with an initial CRC diagnosis,about 15%had liver metastases and 14%had metastases at various sites.Tumor metastasis,the spread of cancer cells from the primary site to distant organs,is a complex multistep process that is influenced by a variety of factors,including Tumor cell properties:The ability of cancer cells to invade surrounding tissues,enter blood vessels or lymphatic vessels,and survive in a new environment is a key factor in tumor metastasis.Tumor microenvironment:The tumor microenvironment can provide physical and metabolic support for tumor cells,facilitating their dissemination to distant organs.Angiogenesis:The formation of new blood vessels in the tumor microenvironment can provide a way for cells to enter the bloodstream and reach distant sites.Inflammation:Chronic inflammation in the tumor microenvironment can promote the development of a supportive environment for tumor cell survival and spread.Host immunity:The host immune system can act to promote or inhibit the spread of tumor cells.Genetic and epigenetic changes:Tumor cells can undergo genetic and epigenetic changes that increase the degree of malignancy and promote the development of a more aggressive phenotype.Surgery,chemotherapy,and radiation therapy can control a variety of localized tumors,but their overall utility in limiting the ability of tumors to metastasize has been dismal.Several factors allow a tumor to acquire drug resistance,including genetic mutations,and resistance mechanisms that actively pump out the drug or modify the drug target so that the drug cannot get in.Tumor heterogeneity:Tumors are composed of heterogeneous cell populations with varying degrees of drug sensitivity,which can lead to the selection of resistant cells.Tumor microenvironment:The tumor microenvironment includes the surrounding stroma and blood vessels,which can provide physical and metabolic support for tumor cells,making tumor cells more resistant to treatment.Inflammation:Chronic inflammation in the tumor microenvironment can promote the development of drug-resistant cells.Epigenetic changes:Changes that regulate gene expression through mechanisms such as DNA methylation,histone modifications,and non-coding RNAs can lead to drug resistance.Tumor resistance is a complex multifactorial process that may involve a combination of these and other mechanisms.In conclusion,the important factors leading to tumor recurrence are distant metastasis and chemotherapy resistance.Therefore,it is urgent to find prognostic markers and therapeutic targets for metastatic and drug-resistant tumors.SIK1 is a serine/threonine kinase protein.It is involved in cell cycle regulation,gluconeogenesis,lipid regulation,muscle growth,differentiation,etc.SIK1 has been reported to be associated with the suppression of malignancy in a variety of tumors,among which SIK1 has been comprehensively reported.SIK1 is involved in the regulation of tumor suppressor effect in lung cancer.SIK1 knockdown can increase the metastatic ability of CRC cells by increasing EMT,suggesting that SIK1 may be a potential tumor suppressor gene.Transforming growth factor-β(TGF-β)induces multiple pleiotropic pathways that are regulated by the cellular environment and integrated with diverse signaling pathways.In tumors,the pleiotropic response of TGF-β leads to a variety of genetic responses,ranging from the inhibition of apoptosis in early tumors to the proliferation,invasion,angiogenesis,and carcinogenesis in late tumors.These responses are closely related to the regulation of EMT,which can promote the transformation of tumor cells into cancer stem cells(CSC)and acquire multi-drug resistance characteristics.The main research direction of SIK1 regulating TGF-β signaling pathway is further study in some non-tumor diseases,but the cancer research is insufficient,and even the specific mechanism has not been determined,which still needs to be further studied.Therefore,the present study aimed to test the hypothesis that SIK1 exerts anti-tumor effects by inhibiting the activation of the TGF-β signaling pathway and subsequently inhibiting EMT and oxaliplatin resistance,thus providing different therapeutic targets for CRC.Methods:1.Use the TCGA database and the bioinformatics analysis website GEPIA to analyze the expression and methylation status of SIK1 in colorectal cancer.2、Conduct initial SIK1 expression detection in CRC cell lines and normal immortalized colon cell line FHC,and select cell lines for subsequent experiments.3.Construct SIK1 knockdown cell lines in HCT116 with high SIK1 expression,and use Transwell,scratch assay,CCK8,and clonogenic assay to investigate the、phenotypic changes in colon cancer cells caused by SIK1 knockdown,and further evaluate the effects of SIK1 on cell proliferation and migration.Additionally,stable overexpression of SIK1 in SW480 and RKO cell lines with low SIK1 expression will be established and subjected to the same experiments to complement the knockdown results.4、Use bioinformatics analysis results to select the TGF-β signaling pathway for Western blot analysis of pathway activation and evaluation of EMT markers in the knockdown and overexpression groups.In addition,the knockdown group will be tested for oxaliplatin(OXA)IC50 to assess its effect on drug resistance.5、Use the TGF-β signaling pathway inhibitor LY2157299 to perform pathway recovery experiments,and use Western blot to evaluate the recovery of TGF-βpathway markers and downstream EMT markers.6.Use the TGF-β signaling pathway inhibitor LY2157299 to perform functional recovery experiments,and use Transwell,scratch assay,and IC50 to test the functions of HCT116 knockdown cells.7、Further study the binding of SIK1 to the key molecule SMAD7 in the TGFβ pathway using co-IP and Western blot experiments.8、Construct subcutaneous tumors in vivo and perform OXA administration to study the drug resistance of tumors.Results:1、The expression of SIK1 is down-regulated in colorectal cancer cells,which may be due to abnormal methylation of DNA promoter,and its expression is generally low in CRC cell lines.2、SIK1 can inhibit the metastatic ability of CRC cells.Overexpression of SIK1 in Transwell and wound healing assays showed an inhibitory effect on the migration of CRC tumor cell lines.3、Downregulation of SIK1 can promote the migration and oxaliplatin resistance of CRC cell line HCT116,and the promotion of the migration function of CRC tumor cell line was expressed in the SIK1 interference cell line HCT116 experimental group.Meanwhile,the interference of SIK1 could promote the drug resistance of CRC cell line HCT116.5、SIK1 may regulate the phenotypic changes of CRC cells through TGF-β signaling pathway.KEGG pathway analysis of GSEA software in TCGA database data showed that SIK1 was highly enriched in TGF-β signaling pathway in CRC.Overexpression of SIK1 significantly inhibited the activation of TGF-β pathway,while knockdown of SIK1 promoted the activation of TGF-β pathway.6、SIK1 can regulate downstream target genes of TGF-β pathway and EMT.Western blot results showed that the phosphorylation of SMAD2,a key molecule in the TGFβ pathway,was significantly inhibited in cell lines overexpressing SIK1,while the downstream target gene Fibronectin(Fibronectin,a key molecule in the Tgf-β pathway)was significantly inhibited.FN)and PAI-1 were inhibited,and EMT was also inhibited.7、SIK1 regulates TGF-β signaling pathway by binding to SMAD7.In the COIP experiment,we observed that SIK1 interacts with SMAD7 and participates in the regulation of TGF-β signaling pathway.8、The regulation of SIK1 on CRC HCT116 was restored by LY2157299 pathway,which was reversed by a TGF-β receptor inhibitor(LY2157299,Galunisertib(Gal)).9.Downregulation of SIK1 can reverse chemoresistance in vivo.HCT116 cell line with stable expression of SIK1 combined with OXA chemotherapy had stronger proliferation ability and higher expression of ki67 than shNC+OXA chemotherapy group.Conclusion:the down-regulation of SIK1 in colorectal cancer(CRC)and its effect on the metastasis and oxaliplatin resistance of CRC cells.The authors found that the expression of SIK1 was significantly down-regulated in CRC tissues and cell lines,and that up-regulation of SIK1 inhibited the migration and wound-healing ability of RKO and SW480 cell lines,while knockdown of SIK1 promoted the migration and oxaliplatin resistance ability of HCT116 cells.The authors also investigated the underlying mechanism and found that SIK1 may regulate the phenotypic changes of CRC cells through the TGF-β signaling pathway and downstream target genes of EMT.This research provides evidence for SIK1 as a potential therapeutic target for CRC.