Regulatory Role And Mechanism Of CKAP4 Palmitoylation Modification In Colorectal Cancer

Regulatory role and mechanism of CKAP4 palmitoylationmodification in colorectal cancer Background:Colorectal cancer(CRC),as the third most common tumor in the world,seriously threatens people’s life and health.In addition to the traditional surgical treatment in the past,the search for biomarkers of colorectal cancer has become an important direction of research.The development of colorectal cancer is an intricate process,which is influenced by many factors,such as microenvironment,epigenetic disorders,etc.Protein translational modifications(PTMs)in epigenetics has become one of the important mechanisms involved in the development of colorectal cancer.Protein Spalmitoylation is a reversible post-translational modification that binds a 16-carbon palmitoic acid to a cysteine residue via a thioester bond.It is a dynamic,cyclic process that exists in all eukaryotes and plays an important role in regulating protein stability,subcellular localization,membrane transport,and effector protein interactions.The development of many cancers is closely related to the palmitoylation/de-palmitoylation cycle,which relies on the regulation of palmitoylation and de-palmitoylation enzymes.Cytoskeleton-associated protein 4(CKAP4)is a non-glycosylated type II transmembrane protein that is involved in a variety of physiological functions such as cell proliferation,migration,and stabilization of the endoplasmic reticulum,and has recently been found to act as an activating receptor on the cell surface.More and more studies have shown that CKAP4 is associated with multiple cancer types,and it is expected to be a biological marker for tumor diagnosis and molecular targets.Therefore,exploring the expression regulation and functional role of CKAP4 in colorectal cancer may provide new ideas for cancer diagnosis and treatment strategies of CKAP4.Based on the above background,this study found and verified that CKAP4 is modified by S-palmitoylation in colorectal cancer tissues and cells through acyl-biotin exchange(ABE)method and liquid chromatography mass spectrometry.Additionally,the expression level of CKAP4 in colorectal cancer tissues differs from that in normal tissues.Consequently,it is postulated that CKAP4 potentially governs the initiation and progression of colorectal cancer through S-palmitoylation.To elucidate the intricate mechanism underlying this process,a comprehensive investigation has been undertaken.Objective:The S-palmitoylation protein in colorectal cancer was identified by ABE method and liquid chromatography mass spectrometry to clarify the expression and biological role of CKAP4 palmitoylation in colorectal cancer.The aim is to reveal the potential mechanism of CKAP4 palmitoylation in colorectal cancer,and to provide relevant evidence for elucidating the pathogenesis of colorectal cancer.Methods:1.Enrichment and purification of S-palmitoylated protein from human colorectal cancer tissues and cell lines by ABE method and streptavidin magnetic beads.2.The S-palmitoyl protein in colorectal cancer was identified by liquid chromatogram-mass spectrometry.3.The bioinformatics approach was employed to analyze the biological functions and pathways associated with S-palmitoyl protein.4.The protein that underwent S-palmitoylation,as detected through mass spectrometry,was subsequently confirmed using Western blot analysis.5.SW480 and SW620 cell lines with silenced ZDHHC2(si-ZDHHC2),overexpression of CKAP4(OE-CKAP4)and point mutation CKAP4(C100A)were transfected by liposome transfection technique.6.RT-q PCR was used to determine the m RNA expression in the cells.7.The enzymatic activity of PPT1 was assessed in SW480 cells that were subjected to treatment with DC661,using an enzyme-linked immunoassay.8.The immunofluorescence technique was utilized to identify the lysosomal deacidification in SW480 cells subsequent to the administration of DC661.9.Immunofluorescence was employed to ascertain the expression and membrane localization of CKAP4 in cells treated with SW480 using 2-BP and DC661.10.Immunofluorescence was employed to detect the cellular membrane localization of both wild-type CKAP4 and point-mutant CKAP4.11.The levels of related proteins in the cell membrane and cytoplasm were determined using Western blot analysis.12.The identification of proteins that interact with CKAP4 was achieved by employing co-immunoprecipitation(Co-IP)and liquid chromatography-mass spectrometry techniques.13.The establishment of a subcutaneous tumor formation model of colorectal cancer in nude mice was accomplished.14.CCK-8,Ed U,cell clonal formation,and Ki-67 immunohistochemistry were employed to assess the proliferation of colorectal cancer cells and subcutaneous tumors.To evaluate the migration and invasion of colorectal cancer cells and subcutaneous tumors,Transwell and N-cadherin immunohistochemistry techniques were utilized.Additionally,flow cytometry and CCND1 immunohistochemistry were employed to examine the cell cycle and apoptosis of colorectal cancer cells and subcutaneous tumors.15.The protein levels of β-catenin,p-GSK3β and GSK3β were determined by Western blot.Results:1.ABE method and streptavidin magnetic beads can effectively accumulate Spalmitoylated proteins in rectal cancer tissues and cells.2.In the study of colorectal cancer,the application of mass spectrometry successfully detected a total of 21 proteins with potential S-palmitoylation.Subsequent Western blot analysis provided confirmation that CKAP4 indeed exhibited Spalmitoylation.3.The expression of CKAP4 is comparatively diminished in human colorectal cancer as opposed to normal tissue.4.According to mass spectrometry data and palmitoylation site prediction software,the palmitoylation site of CKAP4 was determined to be Cys100,and no palmitoylation occurred in CKAP4 after point mutation was verified by Western blot.5.DC661 has demonstrated significant efficacy in inhibiting the enzymatic activity of PPT1,thus establishing its potential as a viable inhibitor of PPT1.6.The palmitoacyltransferase inhibitor 2-BP can effectively inhibit the palmitoylation of CKAP4,while the total protein level of CKAP4 is slightly decreased.7.Depalmitoylation enzyme inhibitor DC661 can increase the levels of CKAP4 palmitoylation protein and total protein after treatment of colorectal cancer cell lines.8.CKAP4 membrane localization function was inhibited after 2-BP treatment and transfection of point mutant CKAP4 plasmid.The localization function of CKAP4 membrane was increased after DC661 treatment.9.Co-IP,liquid chromatography-mass spectrometry and immunofluorescence experiments proved that CKAP4 and ZDHHC2 had interaction.10.The outcomes of the Ed U and clonal formation experiments provided evidence that the upregulation of CKAP4 exerted a suppressive impact on the proliferation of colorectal cancer cells subsequent to treatment with DC661.Furthermore,the results obtained from the cell scratch and Transwell experiments revealed that the overexpression of CKAP4 subsequent to DC661 treatment hindered the migratory and invasive abilities of colorectal cancer cells.Furthermore,flow cytometry analysis revealed that CKAP4 overexpression following DC661 treatment had an impact on the apoptosis and cell cycle progression of colorectal cancer cells.11.The subcutaneous tumor formation model in nude mice provided evidence that the expression of CKAP4 was elevated following administration of DC661,resulting in a significant suppression of colorectal tumor growth.12.In comparison to the control group,the expression levels of GSK3β in the overexpression(OE-CKAP4)and DC661 treated(DC661)groups exhibited no significant alterations.Conversely,the expression levels of β-catenin and p-GSK3βdecreased,suggesting that both the OE-CKAP4 and the treatment with DC661 yielded comparable effects.Therefore,it can be deduced that both interventions have the capacity to hinder the Wnt/β-catenin signaling pathway.13.In comparison to the control group,the expression of GSK3β in the CKAP4 overexpression group(OE-CKAP4)and CKAP4 point mutation group(C100A)exhibited no significant changes.However,the expression levels of β-catenin and pGSK3β exhibited a notable decrease in the OE-CKAP4 group.Conversely,the expression levels of β-catenin,GSK3β,and p-GSK3β in the C100 A group did not display significant alterations.It is suggested that palmitoylation regulates CKAP4 protein,which in turn affects the activation of Wnt/β-catenin signaling pathway.Conclusions:1.CKAP4 is inhibited in colorectal cancer;2.The stability of CKAP4 protein can be regulated by ZDHHC2-mediated palmitoylation modification.The application of depalmitoylation enzyme inhibitors(DC661)increased the level of CKAP4 protein,and then affected the biological behaviors of colorectal cancer cells such as proliferation,migration,invasion,cell cycle and apoptosis.3.The palmitoylation of CKAP4 regulates its membrane localization function and influences colorectal tumor growth through the Wnt/β-catenin signaling pathway.