Truncated O-glycan-dependent mechanisms inducing metastatic dissemination in pancreatic cancer

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Pancreatic cancer (PC) is the fourth leading cause of cancer death and often goes undiagnosed until it has already advanced and metastasized. Aberrant changes in O-glycans, such as increased expression of truncated carbohydrate antigens (Tn, sialylated Tn/STn), are commonly observed in PC. However, the mechanistic involvement of these truncated O-glycan structures in PC progression and metastasis is under-explored. Hence, our study is focused on investigating the mechanistic role of truncated O-glycans during early metastatic dissemination in PC. The O-glycosyltransferase Core 1 β1,3-Galactosyltransferase (C1GALT1) catalyzes the second step of mucin-type O-glycan biosynthesis by adding galactose to the first sugar N-acetylgalactosamine (Tn) that forms the Core 1 carbohydrate structure. Such structures are usually elongated to mature O-glycans found on normal tissue, but their extension may be truncated at the Tn-glycan stage during cancer due to inactive C1GALT1 activity. Our preliminary data demonstrated the loss of O-glycosyltransferase activity, C1GALT1, in a subset of (poorly differentiated) human PC tissue. Further, CRISPR/Cas9-based C1GALT1 knockout (KO) in PC cells resulted in aberrant O-glycosylation (increased Tn and STn glycans). Along with glycan alterations presented upon oncogenic glycoproteins (mucin glycoproteins and cancer stem cell markers), our studies also indicate increased tumorigenicity and metastasis of C1GALT1 KO PC cells. We have also observed O-glycan truncation present on CD44, a cancer stem cell marker, in C1GALT1 models. Interestingly, knockout of C1galt1 along with KrasG12D and Trp53R172H/+ mutations in mouse models resulted in early-onset (in 3 weeks) and early distant metastasis (in 10 weeks) of PC. Based on these observations, our major goal is to investigate the mechanistic role of truncated O-glycans in PC progression and metastasis. Based on these observations, we hypothesize that "Truncated O-glycans on cancer-associated glycoproteins (mucins and stemness markers) induce the early onset of progression and metastatic dissemination in pancreatic cancer." To test this hypothesis, the following aims are proposed. The first aim will investigate the functional impact of C1GALT1 expression and aberrant glycosylation profile on cancer-associated glycoproteins in pancreatic cancer. The second aim will elucidate how truncated O-glycans (such as Tn and STn) on membrane-bound mucins and stemness markers facilitate pancreatic cancer metastasis. The third aim will determine, in vivo, the impact of truncated O-glycans in the early onset of pancreatic cancer metastasis using C1galt1 knockout KC and KPC mice. The proposed studies will establish the association of aberrant expression of truncated Tn and STn glycans with differential membrane- bound mucin function during PC progression and metastasis. This study will significantly contribute to our knowledge of mucin glycobiology in cancer. Altogether, this proposed study will also pave the way for developing novel therapeutics for modulating membrane-bound mucin function in PC.