ppGalNAc-T4-catalyzed O-Glycosylation of TGF-β type Ⅱ receptor regulates breast cancer cells metastasis potential

Abstract

GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex posttranslational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of-function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial–mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-β) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knockout decreased O-GalNAc modification of TGF-β type Ⅰ and Ⅱ receptor (TβR Ⅰ and Ⅱ) and led to the elevation of TGF-β receptor dimerization and activity. Importantly, a peptide from TβR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TβR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31 A mutation enhanced TGF-β signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-β receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment. GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex posttranslational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of-function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial–mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-β) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knockout decreased O-GalNAc modification of TGF-β type Ⅰ and Ⅱ receptor (TβR Ⅰ and Ⅱ) and led to the elevation of TGF-β receptor dimerization and activity. Importantly, a peptide from TβR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TβR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31 A mutation enhanced TGF-β signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-β receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment. Breast cancer, one of the most commonly diagnosed malignancies, accounts for 24.2% of female cancers and is the leading cause of cancer-related death among women worldwide (1Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J. Clin. 2018; 68: 394-424Crossref PubMed Scopus (51165) Google Scholar). Despite advances in early diagnosis and treatment, a high rate of metastasis remains the underlying cause of death in the majority of patients (2Denny L. de Sanjose S. Mutebi M. Anderson B.O. Kim J. Jeronimo J. Herrero R. Yeates K. Ginsburg O. Sankaranarayanan R. Interventions to close the divide for women with breast and cervical cancer between low-income and middle-income countries and high-income countries.Lancet. 2017; 389: 861-870Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Therefore, elucidation of the underlying molecular mechanisms of breast cancer metastasis and development of efficient prognostic and therapeutic biomarkers are urgently needed. The epithelial-to-mesenchymal transition (EMT) process is necessary for tumor progression toward metastatic disease, during which cells lose polarity and cell–cell junctions and gain mesenchymal properties. EMT promotes cancer cell motility and dissemination (3Brabletz T. EMT and MET in metastasis: where are the cancer stem cells?.Cancer cell. 2012; 22: 699-701Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 4Yang J. Weinberg R.A. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis.Dev. Cell. 2008; 14: 818-829Abstract Full Text Full Text PDF PubMed Scopus (2450) Google Scholar). The transforming growth factor-β (TGF-β) signaling pathway evidently increases tumor malignancy in advanced cancer by inducing EMT in response to TGF-β (5Pickup M. Novitskiy S. Moses H.L. The roles of TGFbeta in the tumour microenvironment.Nat. Rev. Cancer. 2013; 13: 788-799Crossref PubMed Scopus (660) Google Scholar). Signal transduction occurs upon ligand binding with heteromeric complexes of TGF-β type Ⅰ and R Ⅱ receptor (TβR Ⅰ and TβR Ⅱ) to induce Smad2/3 phosphorylation, which activates Smad4 accumulation in the nucleus to regulate downstream transcription factors (6Ikushima H. Miyazono K. TGFbeta signalling: a complex web in cancer progression.Nat. Rev. Cancer. 2010; 10: 415-424Crossref PubMed Scopus (918) Google Scholar). These targeted genes, especially SNAI1 and TCF8, induce EMT as E-cadherin repressors (7Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7581) Google Scholar). Recently, evidence has that aberrant glycosylation is involved in cancer EMT and metastasis L. and the of glycosylation in cancer cells epithelial–mesenchymal PubMed Scopus Google Scholar, L. K. K. of glycosylation in cancer Cancer. 2017; PubMed Scopus Google the roles of these modifications as a of posttranslational is a process of of to that is by the of and glycosylation catalyzed by is a of and roles in cancer and metastasis M. L. cancer metastasis and 2010; PubMed Scopus Google Scholar, I. L. glycosylation promotes PubMed Scopus Google Scholar). GalNAc-type is as one of the most abundant and modifications the majority of and and is involved in in cancers Huang J. the pathway cancer cells to and PubMed Scopus Google Scholar, R. M. The polypeptide promotes the migration of cancer by 2013; PubMed Scopus Google Scholar, T. S. K. of 13: PubMed Scopus Google Scholar, T. K. T. K. T. K. T. roles of glycosylation by polypeptide in 2010; PubMed Scopus Google Scholar). O-GalNAcylation is by a of to polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), and substrate H. L.A. of a of the polypeptide 2012; 22: PubMed Scopus Google Scholar). O-GalNAcylation and ppGalNAc-Ts potential associated with and tumor and at high in most and metastatic are involved in invasion J. H. of GalNAc-type in the promotes cancer cell S. A. 2013; PubMed Scopus Google Scholar, and PubMed Scopus Google Scholar). in glycosylation in L. F. A. S. K. aberrant promotes in Cancer. 14: PubMed Scopus Google and of was to be associated with in S. J. of in and aberrant J. Cancer. PubMed Scopus Google Scholar). of and invasion and the of cancer cells as a tumor J. L. H. T. H. J. F. H. A. suppresses progression of J. Cancer 2018; Google Scholar). expression is associated with and a higher tumor in and the malignant of H. R. S. J. M. the malignant of by the PubMed Scopus Google Scholar). cell the potential by the of which be a therapeutic Huang Huang migration and invasion of cell by glycosylation and PubMed Scopus Google Scholar). to be to EMT and as involved in and EMT H. H. and expression of a novel Full Text Full Text PDF PubMed Scopus Google Scholar, H. M.A. S. H. and of a close of for Full Text Full Text PDF PubMed Scopus Google Scholar, L. K. H. K. of in transition S. A. PubMed Scopus Google Scholar, S. A. for the of in the of by PubMed Scopus Google and was to be early of EMT J. J. Epithelial-mesenchymal transition in a and in Cancer. PubMed Scopus Google Scholar). a role in the invasion and migration of breast cancer cells by the activity of and the expression of EMT T. A. J. L. tumor invasion and migration in breast cancer cell PubMed Scopus Google Scholar). and from the which high to and roles in signaling in J. M. and of N-acetylgalactosaminyltransferases with roles in PubMed Scopus Google Scholar). Recently, that ppGalNAc-T4 in L. J. promotes cell and of cancer of J. 2018; 10: Google Scholar, cancer cell via of 2017; PubMed Scopus Google Scholar, J. S. L. F. A. J. M. The polypeptide expression in cancer and invasion and J. 2018; PubMed Scopus Google Scholar). ppGalNAc-T4 was and and inducing of cells in vitro H. L. H. J. J. of in 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). is evidence that ppGalNAc-T4 naked peptide from J. L.A. the functions of polypeptide by substrate Full Text Full Text PDF PubMed Scopus Google that to H. J. J. M.A. of a that in of the Full Text Full Text PDF PubMed Scopus Google where glycosylation has catalyzed by and J. of a polypeptide that Full Text Full Text PDF PubMed Scopus Google Scholar, H. M.A. J. J. H. The domain of Full Text Full Text PDF PubMed Scopus Google Scholar). However, the exact molecular and potential of ppGalNAc-T4 in breast the role of ppGalNAc-T4 in breast cancer was investigated. Loss-of-function studies that ppGalNAc-T4 the migration and invasion of breast cancer cells by Importantly, for the that ppGalNAc-T4 TGF-β signaling by O-GalNAcylation of TGF-β type Ⅱ receptor at and attenuated dimerization of TGF-β in the of TGF-β signaling and EMT in breast cancer cells. Together, these results a novel mechanism of ppGalNAc-T4 breast cancer cell migration and Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment. the substrate the and mechanism of ppGalNAc-T4 in cancer progression be from the the expression of ppGalNAc-T4, the expression tumor and was was one of which expression in breast to molecular breast cancer be A and growth factor and to a of breast cancer, a of and PubMed Scopus Google Scholar). A and a a rate with high metastasis and L. of breast cancer are at and with receptor J. Cancer. 2018; PubMed Scopus Google Scholar). ppGalNAc-T4 is involved in the of breast cancer and to of expression a web for cancer and expression and 2017; PubMed Scopus Google Scholar, A. J. to the of breast cancer of Cancer 2010; PubMed Scopus Google Scholar). The results that in was in breast with that in the breast expression of was found between A and the expression between breast tumor and A high of was with a high expression the of in breast cancer progression Further, the expression of ppGalNAc-T4 in breast cancer cells and and cells and and the cell lines ppGalNAc-T4 expression higher in than in The and expression of ppGalNAc-T4 in breast cancer cell lines with the of These a between ppGalNAc-T4 and metastasis potential in breast cancer cells. breast tumor metastasis is a in and are breast cells with high and and the role of ppGalNAc-T4 in breast cancer, ppGalNAc-T4 in cells and ppGalNAc-T4 in cells by cells ppGalNAc-T4 and a mesenchymal ppGalNAc-T4 knockout These cell that ppGalNAc-T4 in ppGalNAc-T4 the and E-cadherin and the mesenchymal at the and and which are transcription and ppGalNAc-T4 and E-cadherin TCF8, and SNAI1 and The expression of and E-cadherin was by These factors the expression in cells and which a role of ppGalNAc-T4 between the transition of and mesenchymal the of ppGalNAc-T4 in cell metastasis and cell motility was and cell junctions ppGalNAc-T4 was The results in cells ppGalNAc-T4 was a of the EMT process The of the TGF-β to 2013; PubMed Scopus Google Scholar, M. A. of EMT by in 2012; PubMed Scopus Google TGF-β signaling in the nucleus to a of transcription factors M. the mechanisms and of EMT during cancer 2012; PubMed Scopus Google among which and SNAI1 by ppGalNAc-T4 in the the role of ppGalNAc-T4 in cell metastasis and cells with TGF-β by of the EMT process and TGF-β signaling activity. with the of and and as as the accumulation of Smad4 decreased ppGalNAc-T4 was in cells. the and and accumulation of Smad4 ppGalNAc-T4 was in cells the EMT process was impaired by of ppGalNAc-T4, and these by knockout of ppGalNAc-T4 Together, these that ppGalNAc-T4 is for EMT in breast cancer cells. the of ppGalNAc-T4 in TGF-β signaling ppGalNAc-T4 with TGF-β type Ⅰ and R Ⅱ receptors (TβR Ⅰ and TβR Ⅱ) was The is for and Ⅰ R Ⅱ in cells by to the Ⅰ and Ⅱ was found to to Ⅰ and Ⅱ the cell with and with as as ppGalNAc-T4 Ⅰ and Ⅱ to with These results that ppGalNAc-T4 with TβR Ⅰ and Ⅱ in which the for the O-GalNAcylation of TGF-β TGF-β transduction occurs via the between TGF-β and the heteromeric complex of TβR Ⅰ and TβR glycosylation has to regulate the binding activity of TGF-β with receptors J. Kim TGF-β is by glycosylation of the type TGF-β J. 2012; PubMed Scopus Google Scholar, R. growth factor-β receptors and and 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). ppGalNAc-T4 signaling, these TGF-β receptors are by ppGalNAc-T4-catalyzed The is for which results in the and of GalNAc-type knockout T. K. S. of the O-GalNAc J. 2013; PubMed Scopus Google and cell lines to and the O-GalNAc substrate the O-GalNAcylation of TβR Ⅰ and R Ⅱ, to in a The by TβR Ⅰ R Ⅱ corresponding to TβR Ⅰ and R Ⅱ in that TβR Ⅰ and R Ⅱ and cells with TGF-β receptors and and O-GalNAc modification TβR Ⅰ and R Ⅱ was ppGalNAc-T4 knockout in the cell the of ppGalNAc-T4 in TβR Ⅰ and R Ⅱ a was to the TβR Ⅰ and R Ⅱ found that O-GalNAcylation of TβR Ⅰ and R Ⅱ enhanced the dimerization between ppGalNAc-T4 TβR Ⅰ and R Ⅱ the O-GalNAcylation and the between in cell TβR Ⅰ and R Ⅱ to be by ppGalNAc-T4 These that TGF-β receptors are by O-GalNAcylation in a and the TGF-β receptor complex is by that are as and J. Rev. PubMed Scopus Google Scholar, and PubMed Scopus Google Scholar, of the PubMed Scopus Google Scholar). that TβR Ⅰ and TβR Ⅱ proven to be by ppGalNAc-T4, extracellular of TGF-β receptors for TβR Ⅰ and for TβR Ⅱ) to ppGalNAc-T4 O-GalNAcylation of the extracellular The extracellular of TGF-β receptors in and cells. with and revealed that of TβR Ⅰ and R Ⅱ by ppGalNAc-T4 knockout The in cells results the of ppGalNAc-T4 in the extracellular domain the mechanism of ppGalNAc-T4-catalyzed O-GalNAcylation TGF-β in vitro O-GalNAc the peptide by in the O-GalNAc and the results from a most of the TβR Ⅰ and R Ⅱ extracellular O-GalNAcylation for the modification These as of O-GalNAcylation S. L. K. Huang H. J. of the O-GalNAcylation catalyzed by in PubMed Scopus Google Scholar). was proven that the glycosylation of by ppGalNAc-T4 glycosylation by ppGalNAc-Ts with substrate which is in and naked as O-GalNAc was as a and and the and ppGalNAc-T4 from the by with affinity in a naked Ⅱ peptide as the and with advanced at and with the that the peptide from extracellular domain of TβR Ⅱ be by ppGalNAc-T4. The of the that ppGalNAc-T4 activity with these in and as the O-GalNAcylation the TβR Ⅱ that the glycosylation with one at the glycosylation with at with these in the These that a naked peptide from the TGF-β type Ⅱ receptor is a in vitro substrate of ppGalNAc-T4 and that potential glycosylation and in the TβR Ⅱ of and ppGalNAc-T4 activity with naked TβR Ⅱ peptide was and by to the and the results that the and of higher than of ppGalNAc-T4 for the Ⅱ These that ppGalNAc-T4 a higher affinity toward the Ⅱ peptide than ppGalNAc-T2. ppGalNAc-T4 may a role in TβR Ⅱ in cells. the of TβR Ⅱ O-GalNAcylation a TβR Ⅱ peptide in which to of the are in S31 S31 and to the O-GalNAcylation the in vitro was in S31 A and A that one be at in and in in the a of one in that ppGalNAc-T4 catalyzed the of from to was in and that was the in of and potential glycosylation of TβR Ⅱ, site TβR Ⅱ A and for and and O-GalNAcylation the S31 A a decreased O-GalNAcylation with the was modification between the A and the These results that Ser31, was the O-GalNAcylation site for TβR Ⅱ in breast cancer cells the of the O-GalNAcylation site of TβR Ⅱ in TGF-β signaling in breast cancer cells. the results in that the S31 A the between TβR Ⅰ and TβR Ⅱ, role for O-GalNAcylation in TGF-β and cells TβR Ⅱ was to TβR Ⅱ which high and TβR Ⅱ was to be and A in TβR Ⅱ expression. TGF-β treatment, the of and in the S31 A and in the and A that TGF-β signaling was the O-GalNAcylation of TβR Ⅱ at cells TGF-β signaling the role of ppGalNAc-T4 in the process. O-GalNAcylation the of the TβR Ⅰ and TβR Ⅱ molecular and upon TβR Ⅰ and TβR Ⅱ and the is in The of between the TβR Ⅰ of of TβR Ⅰ and and TβR Ⅱ of complex was as the to the which revealed the between the and the The at of TβR Ⅱ the and corresponding to between TβR Ⅰ and at of TβR Ⅱ to that of TβR Ⅱ and The the between the and a The between the receptor complexes decreased the at of TβR Ⅱ O-GalNAcylation of the TGF-β type Ⅱ receptor at results in of the TβR Ⅰ and TβR Ⅱ complex and TGF-β signaling in breast cancer cells. a role of O-GalNAc glycosylation in breast cancer cell metastasis that O-GalNAc glycosylation of TGF-β type Ⅰ and R Ⅱ receptors is ppGalNAc-T4. of ppGalNAc-T4 in decreased O-GalNAcylation of TβR Ⅰ and R Ⅱ, which dimerization and the TGF-β signaling pathway These the of the toward mesenchymal with migration and invasion of breast cancer cells. identified a naked peptide from the extracellular domain of TβR Ⅱ that be by ppGalNAc-T4 in was as the O-GalNAcylation site via in vitro and in cell Together, a novel mechanism of ppGalNAc-T4-catalyzed TGF-β receptor that suppresses breast cell migration and invasion via the EMT process. O-GalNAc is associated with cancer and and has a of T. The and PubMed Scopus Google Scholar). expression of ppGalNAc-Ts in and and cancer ppGalNAc-T4 expression was with tumor progression J. S. L. F. A. J. M. The polypeptide expression in cancer and invasion and J. 2018; PubMed Scopus Google Scholar). the of ppGalNAc-T4 expression the malignant of L. H. J. J. in patients with cell cell PubMed Scopus Google Scholar). a expression of ppGalNAc-T4 was in and breast tumor and high expression of ppGalNAc-T4 was associated with breast cell ppGalNAc-T4 expression a necessary to in the ppGalNAc-T4 may as a cancer by substrate as TβR Ⅱ, in O-GalNAcylation the and of substrate to cancer cell the mechanisms of breast cancer the of ppGalNAc-T4 and revealed the role of ppGalNAc-T4 in tumor to has that the glycosylation and regulate the activity of H. L. H. J. J. of in 2017; Full Text Full Text PDF PubMed Scopus Google has that naked peptide is a substrate for ppGalNAc-T4 J. L.A. the functions of polypeptide by substrate Full Text Full Text PDF PubMed Scopus Google has that roles in glycosylation of and H. J. J. M.A. of a that in of the Full Text Full Text PDF PubMed Scopus Google Scholar). These results that ppGalNAc-T4 is one of the which to by the strategy was to the of ppGalNAc-T4. strategy a cell in which the the molecular for is for strategy which be and by and the be by to TGF-β receptors be and by of that at a of TGF-β receptors in cells are that TGF-β receptors and is to the expression of ppGalNAc-Ts and which involved in TGF-β receptors which is in the with in was to be with ppGalNAc-T4 knockout the of these cells for the results of in and cell that ppGalNAc-T4 the O-GalNAc modification of TβR Ⅰ and R TβR Ⅰ and R Ⅱ are that as receptors of TGF-β signaling, which is and in advanced cancers J. TGFbeta in Rev. 2012; 13: PubMed Scopus Google Scholar). was that TβR Ⅱ has glycosylation at and which to the cell by impaired signaling pathway J. Kim TGF-β is by glycosylation of the type TGF-β J. 2012; PubMed Scopus Google Scholar). cells EMT, TβR Ⅱ TβR Ⅰ to Smad2/3 and transcription that ppGalNAc-T4 TβR Ⅰ and R Ⅱ O-GalNAc which and suppresses downstream the that TβR Ⅰ and R Ⅱ are by ppGalNAc-T4 and TGF-β signaling activity. of ppGalNAc-T4 in and Smad4 accumulation in breast cancer cells Correspondingly, ppGalNAc-T4 negatively with with and EMT, which in enhanced and was in breast cancer cells with ppGalNAc-T4 cell of the O-GalNAcylation site of TβR Ⅱ O-GalNAc which TGF-β signaling activity and cell metastasis potential in breast identified a role for ppGalNAc-T4 in breast cancer cell metastasis potential by O-GalNAc TβR Ⅰ and R However, the mechanism of O-GalNAcylation TGF-β receptors remains to be Here, potential the modification may in accumulation in the which ligand O-GalNAcylation the of TβR Ⅰ and R Ⅱ the cell which the and between binding ppGalNAc-T4 TβR Ⅰ and R Ⅱ molecular and The at of TβR Ⅱ the and the between TβR Ⅰ and the at of TβR Ⅱ than of the of TβR Ⅱ the between the receptor complexes decreased the at of TβR Ⅱ may be one of the mechanisms by which ppGalNAc-T4 EMT in breast cancer cells. ppGalNAc-Ts a of O-GalNAc the in substrate and expression in H. functions of the polypeptide 2012; PubMed Scopus Google and expression of of the polypeptide from Full Text Full Text PDF PubMed Scopus Google Scholar). ppGalNAc-T4 is in that is the identified that is in vitro to in the of which and H. J. J. M.A. of a that in of the Full Text Full Text PDF PubMed Scopus Google Scholar). has that ppGalNAc-T4 activity toward catalyzed by H. M.A. J. J. H. The domain of Full Text Full Text PDF PubMed Scopus Google Scholar). naked as a the activity is J. L.A. the functions of polypeptide by substrate Full Text Full Text PDF PubMed Scopus Google Scholar). the peptide from TβR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4, which is ppGalNAc-T4 substrate has that ppGalNAc-T4 to in the are in a between the of TβR Ⅱ and the The for may be that the mechanism of for naked peptide be and is between Further, peptide of ppGalNAc-Ts are and be to the of ppGalNAc-Ts A of ppGalNAc-Ts substrate is for substrate of the ppGalNAc-T4-catalyzed as a the of the ppGalNAc-T4-catalyzed and in the as of the that ppGalNAc-T4 the peptide as ppGalNAc-T2. potential of as results a substrate peptide for the ppGalNAc-T4 in vitro for and activity with the TβR ppGalNAc-T4 a higher affinity than in properties. These that ppGalNAc-T4 is for TβR Ⅱ than in cells. the and expression of in cancer of the ppGalNAc-Ts as ppGalNAc-T4, be and a role in the O-GalNAc modification of TβR Ⅱ in and cells. the O-GalNAcylation of TβR Ⅱ, and site by and O-GalNAc modification at was proven by and glycosylation at was by the the with advanced be a glycosylation a of a The results with the peptide in in the cell in cells the O-GalNAcylation of the S31 A was and the S31 A the A the downstream TGF-β The results of the in with of the cell and was identified as site of TβR Ⅱ by ppGalNAc-T4. The in cells the O-GalNAcylation of TβR Ⅰ by ppGalNAc-T4 and in vitro of glycosylation in the TβR Ⅰ extracellular domain that ppGalNAc-T4 activity that ppGalNAc-T4-catalyzed of TβR Ⅰ may glycosylation by may naked TβR Ⅰ are catalyzed by ppGalNAc-T4 in are to the O-GalNAcylation in TβR identified a posttranslational mechanism of EMT O-GalNAc glycosylation of TβR Ⅰ and R Importantly, O-GalNAc at in TβR Ⅱ is a in signaling and a naked peptide from TβR Ⅱ is to be a substrate of ppGalNAc-T4. the of and in tumor Together, novel the role of O-GalNAc glycosylation in cancer cell metastasis is a to expression The Cancer and The is patients with breast cancer and the the expression of in of breast cancer and the via and breast cancer cells and cells lines from of the of and from cells in with cells in with was in with and and in with was in with and was with cells in at in induce EMT, was with to cells. A was and a and extracellular domain of and A and was and of for is which has proven to TβR Ⅱ expression. cells the and as H. J. S. J. by increases metastatic potential of cell via 2018; PubMed Scopus Google Scholar). The and Ⅰ from TβR Ⅱ, from from cells with and The cell with and by by with the targeted was with at for The H. J. H. Huang M. during 2017; PubMed Scopus Google and the was with by with and The by and with cell was and was to the The was by for genes, with as The of the upstream and downstream are in to and the in expression was the in for at and with in for and with for at with ppGalNAc-T4, in at with and with to targeted for at with and with for at cells with cells The are and the of by cells. cells by for and a cell was by in Further, the cell was and the the site was by the and The was and and by at the are and are and The cell of to the migration and invasion of cells. 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