The Effects And Mechanisms Of Glycosyltransferases GALNT6 And FUT4 Promoting Bad Prognosis In Colon Cancer,Breast Cancer And Lung Adenocarcinoma

Objective:Glycosylation is the most common post-translational modification process for proteins.Abnormal glycosylation plays an important role in the development and progression of human malignant tumors.Glycosylation is defined as the enzymatic process that produces glycosidic linkages of saccharides to other saccharides,proteins or lipids.In general,glycosylation is classified as N-glycosylation or O-glycosylation depending on whether a glycosidic bond is formed initially between the polysaccharide and the protein at the nitro group or the oxy group.According to the different groups of terminal modifications,glycosylation modifications are divided into:sialylation,O-glycan truncation,mannosylation and fucosylation.Mucin-type O-glycosylation is the most common and important type of protein O-glycosylation.The initial step of Mucin-type O-glycosylation is catalyzed by the polypeptide N-acetylgalactosaminyltransferases(GALNTs,GalNAc-Ts),and GalNAc is transfered to the Ser/Thr residues of glycoproteins.Mucin-type O-glycosylation occurs in the Golgi complex,which is regulated by the expression and distribution of GALNT members.Studies have confirmed that abnormal Mucin-type O-glycosylation plays a key regulatory role in cell proliferation,apoptosis,differentiation,adhesion,migration,invasion,angiogenesis and invasion of the immune system,and even affects a variety of clinical recurrence and prognosis.As recent studies reported,GALNTs have been shown to be abnormally expressed in cancers and are significantly associated with poor prognosis.Irregular GALNTs expression may play the same or diametrically opposite role in different malignancies.High expression of GALNT6 was positively correlated with TNM stage of lung adenocarcinoma,and could be applied as an independent prognostic factor for predicting poor OS.However,in pancreatic cancer,high expression of GALNT6 is an independent prognostic factor indicating better prognosis.The expression of all GALNTs in a variety of malignancies was analyzed in ONCOMINE,an online tumor gene expression database.GALNT6 was significantly overexpressed in colon cancer samples,and its increased expression and its consistency in each data set were the most significant of all GALNTs in all malignancies.However,the association of GALNT6 in colorectal cancer with prognosis and its effect on malignant biological behavior has not been reported.Tumor stem cells(CSCs)are present in hematological malignancies and solid malignancies and are subgroups of tumor cells with self-renewal and differentiation potential.These small groups of cells endow cancer the ability of drug resistance and metastasis.The key to preventing recurrence and metastasis is to determine the molecular targets which regulate the maintenance of CSCs and their self-renewal.Aldehyde dehydrogenase(ALDHs)are highly expressed in a variety of solid malignancies and hematological malignancies in stem cells,and are universal stem cell markers.It has been reported that GALNTs-mediated mucin-type O-glycosylation regulates the self-renewal capacity of bladder cancer,breast cancer,and hepatocellular carcinoma stem cells by promoting or inhibiting the activation of key factors in stem cell access,suggesting that GALNTs are associated with the stemness of CSCs.The relationship between GALNT6 or other GALNTs members and colon CSCs,as well as the relationship between CSCs markers such as CD44 and ALDH,has not been reported.GALNTs affect its activation of downstream signaling pathways by altering the level of protein glycosylation.Analysis of the SWISS-PROT database predicts that more than 50%of the protein in all proteins is glycosylated.However,only a small amount of GALNTs substrate proteins were validated in affecting cancer malignant phenotypes.It was reported in different types of cancers that differences in GALNTs expression have a protein expression profile.GALNTs expression differences have protein expression profiles.It has been reported that GALNT6 contributed to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1.Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells.The same group reported that FN was O-glycosylated and thereby stabilized by GALNT6.Knockdown of fibronectin abrogated the disruptive proliferation caused by introduction of GALNT6 into epithelial cells.However,it is unhnown whether GALNT6 plays the role in the similar way in triple-negative breast cancer cells in which MUC1 and FN expression are not dominant,and whether there are other one or more substrate proteins involved in this process.Aberrant glycosylation plays a significant role in tumor progression.Fucosylation is one of the most important type of glycosylation.Alterations in the fucosylation of cancer cells closely correlate with proliferation,apoptosis,migration,invasion,angiogenesis and drug resistance in various cancers including LUAD.FUT4 is a key enzyme for synthesizing tumor associated carbohydrate antigen including Lewis A,B,X,and Y,which catalyzes the transfer of L-fucose from GDP-fucose to the substrates.FUT4 is highly expressed in several types of cancers,including leukemia,gastric and breast cancers,and is positively correlated with tumor progression.Few evidence of the association between FUT4 and cancer prognosis has been reported.Early study reported that increased gene expression of FUT4 and/or FUT7 predicted shorter survival in lung cancer.However,both staging system and treatment strategies have undergone dramatical changes in the past two decades.Therefore,it is necessary to reevaluate the role of FUT4 in the prognosis of LUAD.Based on the expression of GALNT6 in a variety of malignancies in online tumor gene expression profile database ONCOMINE,there are an absolute number of studies showing increased expression of GALNT6 in colon and breast cancer.In the first and the second part of this study,we investigated the expression of GALNT6 and its prognostic value in colon and breast cancer,respectively.GALNT6 plays a critical role in the malignant phenotype of colon cancer cell lines RKO and HCT-116,and breast cancer cell lines MDA-MB-231,MCF-7 and MDA-MB-468.Based on the online database we estimated the important signaling pathways of GALNT6 in colon cancer.We investigated the mechanism of GALNT6 to breast cancer malignant phenotype through mucin-type O-glycosylation of new substrates.The third part of this study evaluated the prognostic role of FUT4 in lung adenocarcinoma by online data,and used bioinformatics methods to speculate on the important signaling pathways of FUT4 in regulating lung adenocarcinoma.At the same time,pathological specimens were used to validate the mechanism of FUT4 in lung adenocarcinoma.Methods:1.mRNA expression levels of GALNT6 and FUT4 in colon cancer and lung adenocarcinoma and corresponding normal tissue were evaluated by online dataset ONCOMINE and TCGA,respectively.2.Online data set SurvExpress study was used for meta-analysis,evaluating the relationship between GALNT6 expression and the OS or DFS of colon cancer or breast cancer.TCGA and Kaplan-Meier Plotter were used for predicting and external validating the relationship between FUT4 expression and the OS of lung adenocarcinoma.3.Based on lung adenocarcinoma cohort from the First Hospital of China Medical University,the expression and prognosis of FUT4 in lung adenocarcinoma were verified by immunohistochemistry.4.Correlation between FUT4and clinical pathology parameters was analysed.5.Uni-and multi-variant analysis of FUT4 and clinicopathological parameters on the prognosis of lung adenocarcinoma were performed,and a nomogram and calibration curves were constructed.6.Gene set enrichent analysis(GSEA)was performed based on TCGA dataset of colon cancer and lung adenocarcinoma to find the KEGG pathway associated with differential expression of GALNT6 and FUT4,respectively.7.Correlation between FUT4 and immunosuppressive molecules were analysed based on TCGA lung adenocarcinoma cohort.8.Based on the lung adenocarcinoma cohort of the First Hospital of China Medical University,verify the correlation between FUT4 and immunosuppressive molecule PD-1 was verified by immunohistochemistry.9.siRNA targeting GALNT6 and GALNT6 overexpression plasmids which were stably transfected into colon cancer and breast cancer cell lines.10.GALNT6 knockdown and overexpression lentivirus which were stably infected into colon cancer and breast cancer cell lines.11.Cell proliferation was measured using MTT assay.12.Cell proliferation ability was observed with Colony-Formation Assay.13.Cell migration and invasion ability was detected by Transwell Assay.14.Transient transfection was performed using Lipofectamine 2000reagent.15.The expression of CD44~+ALDH~+was detected by flow cytometry.16.Relative levels of ALDH1A1,ALDH1B1,ALDH2,ALDH5A1,ADLH6A1 and ALDH7A1 mRNA were measured by Real-time PCR.17.Western blot was used to detect the expression of GALNT6,AKT,p-AKT,E-cadherin,Vimentin,ZEB1,slug andβ-actin.18.The level of substrate glycosylation was detected by VVA immunoprecipitation.19.Statistical analysis.All values are expressed as means±SD.The differences of the results between two groups were evaluated by Student’s t-test.P<0.05 was considered to be statistically significant.Results:1.Results from online datasets ONCOMINE suggested that a large number of studies have shown that mRNA levels of GALNT6 in colon cancer tissue were significantly higher than normal tissue.2.Colon studies from online data set SurvExpress was used for meta-analysis.The results showed that GALNT6 overexpression was positively correlated with both OS and DFS in colon cancer.3.MTT and colony formation results suggest that silencing GALNT6 can inhibit colon cancer cell proliferation,while overexpression of GALNT6 can promote colon cancer cell proliferation.4.Transwell migration assays were performed.Results of migration assays revealed that GALNT6 knockdown inhibited migration compared with control colon and cells,and the migration of GALNT6-overexpressing colon cancer cells was significantly increased.5.The gene set enrichment analysis(GSEA)was performed using TCGA colon cancer data set to clarify the molecular mechanism of GALNT6 high expression leading to poor prognosis of colon cancer.GSEA results showed that GALNT6overexpression was positively associated with KEGG_valine_leucine_isoleucine degradationpathway,KEGG_propanoatemetabolicpathway,KEGG_butanoate_metabolism pathway and KEGG_O_glycan_pathway.6.GSEA results suggested that overexpression of GALNT6 promotes the malignant phenotype of colon cancer cells by increasing CSC markers expression,including ALDH1B1,ALDH2,ALDH5A1,ALDH6A1,ALDH7A1.Real-time PCR assay was used to detect the mRNA level of ALDHs in the colon cancer cells before and after overexpression of GALNT6.ALDHs were all significantly upregulated,consistent with the results of GSEA.The percentage of CD44~+/ALDH1A1~+stem cells in HCT-116 cells before and after overexpression of GALNT6 was significantly up-regulated detected by flow cytometry.The above studies have shown that GALNT6 has the ability to promote colon cancer stem cell ability.7.A large number of studies from online datasets ONCOMINE have shown that mRNA levels of GALNT6 in breast cancer tissue were significantly higher than normal tissue.8.Breast cancer studies from online data set SurvExpress was used for meta-analysis.The results showed that GALNT6 overexpression was positively positively correlated with OS in breast cancer.9.MTT and colony formation assays were performed and results of both assays revealed that GALNT6 kncokdown inhibited proliferation compared with control breast cancer cells.These results indicated that GALNT6 promoted the proliferation of breast cancer cells.10.Transwell assays were performed.Results of migration and invasion assays revealed that GALNT6 knockdown inhibited migration and invasion compared with control breast cancer cells.These results indicated that GALNT6 promoted metastasis of breast cancer cells.11.Transwell migration assays were performed.GALNT6 knockdown of overexpressing cells inhibited the supernatant promoting migration of GALNT6 low-expressing cells compared with control supernatant.The results revealed that the supernatant of GALNT6 overexpressing breast cancer cells promoted migration of GALNT6 low-expressing cells.12.We use Western blot to detect the expression levels of EMT-related protein and transcription factor.Compared with control group,GALNT6 knockdown significantly downregulated the mesenchymal marker Vimentin,and upregulated the epithelial marker E-cadherin,and downregulated the transcription factor ZEB1 and Slug.These results indicated that GALNT6 induced breast cancer cells EMT and enhanced migration ability.13.We use Western blot to detect the activation of signal pathways.The phosphorylation of PI3K/AKT were significantly decreased in GALNT6 knockdown breast cancer cell lines which compared with control cells.This result indicated that GALNT6 promoted the proliferation and metastasis of breast cancer cells via upregulating PI3K/AKT.14.Silencing GALNT6 resulted in a significant down-regulation of breast cancer Mucin-type O-glycosylation.15.Results of qualitative glycoproteomics on secreted protein of MDA-MB-231 cells indicated that GALNT6 may promote malignant phenotype of breast cancer by promoting Mucin-type O-glycosylation of AHSG,Hornerin,α2Μand CBFA2T2.16.The lung adenocarcinoma cohort from TCGA showed that the mRNA level of FUT4in lung adenocarcinoma tissues was significantly higher than that in normal tissues.17.The lung adenocarcinoma cohort from TCGA showed that the high expression of FUT4was significantly positively correlated with the poor clinical outcome with lung adenocarcinoma,and was externally validated in another online dataset Kaplan-Meier Plotter.18.The immunohistochemical results of lung adenocarcinoma of cohort from the First Hospital of China Medical University suggests that the expression of FUT4 in lung adenocarcinoma is significantly positively correlated with the poor OS of lung adenocarcinoma.19.The correlation analysis between FUT4 and clinicopathological parameters showed that FUT4 was significantly positively correlated with pN stage and pTNM stage in lung adenocarcinoma.20.Results of uni-and multi-variant analysis of FUT4 and clinicopathological parameters for the prognosis of lung adenocarcinoma suggest that FUT4 is consistent with pT stage and pTNM staging,and is an independent prognostic factor for lung adenocarcinoma.FUT4 can effectively predict the prognosis of lung adenocarcinoma.21.The GSEA results based on TCGA data indicated that FUT4was significantly correlated with ERBB pathway and multiple immune pathways.22.TCGA data showed that FUT4 was significantly positively correlated with proliferation marker Ki67,immunosuppressive molecule PD-1,and MDSC and Treg markers.23.Immunohistochemical results verificated FUT4 and PD-1 showed a significant positive correlation.Conclusion:1.GALNT6 is highly expressed in colorectal cancer tissues,FUT4 is highly expressed in lung adenocarcinoma,and their high expression are positively correlated with corresponding cancers.2.FUT4 is an independent factor for predicting poor prognosis in patients with lung adenocarcinoma.3.GALNT6 promotes colon cancer and breast cancer cell proliferation,migration and invasion.4.GALNT6 expression is positively correlated with propanoate metabolism,methylbutyrate metabolism,branched-chain amino acid degradation and polysaccharide synthesis pathway.5.GALNT6 increased the expression of ALDHs in colon cancer cells,and increase the proportion of ALDH~+CD44~+colon CSCs.6.GALNT6 expression was positively correlated with mucin type O-glycosylation in breast cancer cells.7.GALNT6 can promote EMT of breast cancer cells and activate PI3K/AKT signaling pathway downstream of MDA-MB-231 breast cancer cells.8.FUT4 is involved in PD-1-related immunosuppression and leads to worse survival in patients with lung adenocarcinoma.