Study On The Role Of Polypeptide N- Acetylgalactosaminyltransferase 4 In Human Breast Carcinomas:Potential Implication In Tumor-associated Antigens Synthesis

Protein glycosylation is a major posttranslational modification which plays key roles in differentiation, development and many biological functions. It generally can be divided into major subtypes:N-glycosylation, O-glycosylation and glycosaminoglycans. Among these subtypes, the less studied and most complex is the O-GalNAc or mucin-type glycosylation, regulated by about 20 isoenzymes playing simultaneous and overlapping functions. Alterations in the glycosylation process have been linked to carcinogenesis because inducing impairment in protein regulation or even changing cells signaling network hence modification in cells behavior and phenotypes. For this reason nowadays, many studies are focus in cancer related glycans.Purpose studyThe purpose of this study is to investigate the roles of Polypeptide N-acetylgalactosaminyltransferase 4 or GalNAc-T4 in the regulation of breast cancer, through its potential implication in the O-GalNAc biosynthetic pathway.Experimental procedureThis study was divided in three main parts:Part 1:Clinical analysis of the role of GALNT4 in breast tumors.In this section we use human tissue microarrays which contain 192 human breast tissues with different grades and also normal and cancer adjacent tissues. GALNT4 belongs to a family of N-acetylgalactosaminyltransferases, which catalyze the transfer of GalNAc to Serine or Threonine residues in the initial step of mucin-type O-linked protein glycosylation. This step forms the Tn antigen which can be further elongated by other glycosyltransferases. The Tn antigen was reported to be over expressed in many tumor cells and this resulted of an inhibition of its possible elongation. This second and crucial step is controlled by a chaperone COSMC (C1galT1Cl) which activates the T synthase (C1galT1). For this reason we use three cores of the same arrays and each of them was stained using GALNT4, vicia villosa lectin (WL) which detects the Tn antigen, and COSMC. Slides were then analyzed in order to observe the different expressions.Part 2:Role of GaINAc-T4 in the regulations of Tumor-Associated AntigensWhen our first part was describing the clinic-pathological role of GALNT4, this part came to confirm our hypothesis by the use of different type of breast cancer cell lines to first screen the expression of GALNT4 and after check its biological function through different assays. Its endogenous expression was also depleted in order to understand its potential implication in the O-glycosylation pathway, through regulation of glycosyltransferases activation.Part 3:Role of GALNT4 in regulation of estrogen receptor ERa transcription networkBy screening the expression of GALNT4 in different breast cancer cells we found a possible correlation between its expression and the estrogen status of the cells. We knockdown GALNT4 expression by siRNA approach and then check the expression of the estrogen receptor and its potential cofactor and targets in order to understand the mechanism by which GALNT4 might act in this process.ResultsThe analysis of the tissues staining depicted a high correlation in the tumor grades and the Tn antigen expression. Surprisingly GalNAc-T4 and COSMC showed a relative similar pattern of staining increasing in the early carcinoma but did not show any significant strong staining in the high grades. This raises the hypothesis of another mechanism responsible of the expression of the Tn antigen rather than decrease or mutation in COSMC expression, but also emphasis the possible role of GALNT4 inhibiting directly the T synthase, hence even if the expression of COSMC increase, it will be unable to form the corel structure. This could explain the increase of the Tn antigen in the high grades of breast carcinoma.In coterminous to our first part, the second part of this study reveals that GALNT4 negatively correlates with ClgalT1 and COSMC and rescuing its expression resulted in COSMC decreased expression. And this can be the reason of the attenuation in many biological activities of the cells (decrease in proliferation and G0-G1 cell cycle arrest). We also found an increase in the T antigen expression, in the GALNT4 knockdown cells, which decrease upon GALNT4 rescued. At the same time it promotes the expression of ST6GalNAc-I hence a possible increase in STn antigen expression.In the last part we found that knockdown of GALNT4 expression in human breast cancer cells attenuated the protein expression of Estrogen Receptor ERα, FOXA1 and Cyclin D1. Further, our immunoprecipitation assays depicted the possibility of FOXA1 to undergo O-GalNAc modifications with a decrease of GalNAc residues in the GALNT4 knockdown cells, and also impairment in the FOXAl-ERa association. Rescuing GALNT4 expression could restore the interaction as well as the glycosylation of FOXA1.ConclusionIn summary, this study highlights the role of GALNT4 in the O-GalNAc glycosylation pathway through regulation of some key glycosyltransferases activities, and this might explain the synthesis of tumor-associated antigens (Tn, STn antigens). By exploring its potential role in the estrogen pathway, we found that GalNAc-T4 putatively modulates the estrogen regulatory network through FOXA1 glycosylation in human breast cancer cells.