Abstract 4983: GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer

Abstract

Abstract Purpose: MUC4 is one of the membrane-bound mucins that is expressed de novo during pancreatic cancer (PC) and contributes to pathogenesis. Further, MUC4 is aberrantly glycosylated in PC and some of its tumor-promoting effects are, in part, mediated by interactions facilitated by the glycan epitopes, which are generally masked under normal conditions. Mucins predominantly undergo O-glycosylation that is initiated by a family of enzymes known as GALNTs (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase), catalyzing the transfer of first sugar residue (GalNAc) to ser/thr residues in mucin tandem repeat domains. Not much is known about the specific glycosyltransferases (GTs) involved in MUC4 glycosylation. Understanding the specific enzymes regulating differential glycosylation of MUC4 in cancer may be important for developing therapeutic strategies for modulating MUC4 function in PC. Thus, the goal of this study was to identify specific GALNT(s) catalyzing MUC4 glycosylation and investigate its role in differential MUC4 glycosylation in PC. Materials & Methods: PCR array analysis was performed in a panel of MUC4 expressing and non-expressing PC cell lines, normal pancreatic cancer cells and MUC4 expressing normal colon cells to identify differentially expressed GTs. To determine the altered glycosylation of MUC4 in normal versus cancerous conditions, Co-immunoprecipitation (Co-IP) studies were conducted using several carbohydrate-specific antibodies. Stable knockdown of GALNT3 was performed in PC and normal colon cells to understand its involvement in differential MUC4 glycosylation. To determine the functional implications of GALNT3 mediated aberrant glycosylation, various in vitro assays such as colony formation and migration were carried out. Results: PCR array and immunoblot analysis demonstrated GALNT3 as one of the GTs upregulated in MUC4 expressing cells as compared to MUC4 non-expressing PC cells. Interestingly, Co-IP analysis revealed differential expression of carbohydrates epitopes such as STn, SLex, SLea on MUC4 in PC and normal colon cells. GALNT3 knockdown in cancer cells resulted in decreased expression of Tn but simultaneous increase in STn epitope. Interestingly, we observed significant increase in colony formation and migration in GALNT3 knockdown cells. This was associated with increase in proliferation and EMT markers in GALNT3 knockdown PC cells such as N-cadherin, vimentin and phosphorylated ErbBs. Conclusion: Overall, our results suggested a correlation of MUC4 and GALNT3 expression, and knockdown of GALNT3 resulted in altered MUC4 glycosylation rather than complete abrogation, suggesting a possible compensatory increase in other GALNTs and other GTs. Further investigations along these lines will provide insight into specific GTs regulating MUC4 glycosylation and glycan epitope-mediated MUC4 functions in cancer. Citation Format: Seema Chugh, Vinayaga Srinivasan Gnanapragassam, Maneesh Jain, Moorthy Ponnusamy, Surinder Kumar Batra. GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2015-4983