The glycosyltransferase ST6Gal-I, which adds α2-6–linked sialic acids to substrate glycoproteins, has been implicated in carcinogenesis; however, the nature of its pathogenic role remains poorly understood. Here we show that ST6Gal-I is upregulated in ovarian and pancreatic carcinomas, enriched in metastatic tumors, and associated with reduced patient survival. Notably, ST6Gal-I upregulation in cancer cells conferred hallmark cancer stem-like cell (CSC) characteristics. Modulating ST6Gal-I expression in pancreatic and ovarian cancer cells directly altered CSC spheroid growth, and clonal variants with high ST6Gal-I activity preferentially survived in CSC culture. Primary ovarian cancer cells from patient ascites or solid tumors sorted for α2-6 sialylation grew as spheroids, while cells lacking α2-6 sialylation remained as single cells and lost viability. ST6Gal-I also promoted resistance to gemcitabine and enabled the formation of stably resistant colonies. Gemcitabine treatment of patient-derived xenograft tumors enriched for ST6Gal-I–expressing cells relative to pair-matched untreated tumors. ST6Gal-I also augmented tumor-initiating potential. In limiting dilution assays, subcutaneous tumor formation was inhibited by ST6Gal-I knockdown, whereas in a chemically induced tumor initiation model, mice with conditional ST6Gal-I overexpression exhibited enhanced tumorigenesis. Finally, we found that ST6Gal-I induced expression of the key tumor-promoting transcription factors, Sox9 and Slug. Collectively, this work highlighted a previously unrecognized role for a specific glycosyltransferase in driving a CSC state. Cancer Res; 76(13); 3978–88. ©2016 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. Gemcitabine, as a single agent or in combination therapy, remains the frontline chemotherapy despite its limited efficacy due to de novo or acquired chemoresistance. There is an acute need to decipher mechanisms underlying chemoresistance and identify new targets to improve patient outcomes. Here we report a novel role for the ST6Gal-I sialyltransferase in gemcitabine resistance. Utilizing MiaPaCa-2 and BxPC-3 PDAC cells, we find that knockdown (KD) of ST6Gal-I expression, as well as removal of surface α2-6 sialic acids by neuraminidase, enhances gemcitabine-mediated cell death assessed via colonogenic assays and cleaved caspase 3 expression. Additionally, KD of ST6Gal-I potentiates gemcitabine-induced DNA damage, as measured by comet assays and γH2AX foci. ST6Gal-I KD also alters mRNA expression of key gemcitabine metabolic genes, RRM1, RRM2, hENT1 and DCK, leading to an increased Gemcitabine Sensitivity Ratio (GSR), an indicator of gemcitabine toxicity. Gemcitabine resistant MiaPaCa-2 cells display higher ST6Gal-I levels than treatment-naïve cells, along with reduced GSR, suggesting that chronic chemotherapy selects for clonal variants with more abundant ST6Gal-I. Finally, we examined Suit2 PDAC cells and Suit2 derivatives with enhanced metastatic potential. Intriguingly, three metastatic and chemoresistant subclones, S2-CP9, S2-LM7AA and S2-013, exhibit upregulated ST6Gal-I relative to parental Suit2 cells. ST6Gal-I KD in S2-013 cells increases gemcitabine-mediated DNA damage, indicating that suppressing ST6Gal-I activity sensitizes inherently resistant cells to gemcitabine. Together, these findings place ST6Gal-I as a critical player in imparting gemcitabine resistance and as a potential target to restore PDAC chemoresponse.

One of the least-investigated areas of brain pathology research is glycosylation, which is a critical regulator of cell surface protein structure and function. β-Galactoside α2,6-sialyltransferase ( ST6GAL1 ) is the primary enzyme that α2,6 sialylates N-glycosylated proteins destined for the plasma membrane or secretion, thereby modulating cell signaling and behavior. We demonstrate a potentially novel, protumorigenic role for α2,6 sialylation and ST6GAL1 in the deadly brain tumor glioblastoma (GBM). GBM cells with high α2,6 sialylation exhibited increased in vitro growth and self-renewal capacity and decreased mouse survival when orthotopically injected. α2,6 Sialylation was regulated by ST6GAL1 in GBM, and ST6GAL1 was elevated in brain tumor-initiating cells (BTICs). Knockdown of ST6GAL1 in BTICs decreased in vitro growth, self-renewal capacity, and tumorigenic potential. ST6GAL1 regulates levels of the known BTIC regulators PDGF Receptor β ( PDGFRB ), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin, which were confirmed to bind to a lectin-recognizing α2,6 sialic acid. Loss of ST6GAL1 was confirmed to decrease PDGFRB α2,6 sialylation, total protein levels, and the induction of phosphorylation by PDGF-BB. Thus, ST6GAL1 -mediated α2,6 sialylation of a select subset of cell surface receptors, including PDGFRB , increases GBM growth.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 β-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRAS ^G12D had greatly accelerated PDAC progression compared with mice expressing KRAS ^G12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1’s role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. Inhibitors targeting the programmed cell death 1 (PD-1) immune checkpoint have improved MCC patient outcomes by boosting antitumor T cell immunity. Here, we identify PD-1 as a growth-promoting receptor intrinsic to MCC cells. In human MCC lines and clinical tumors, RT-PCR–based sequencing, immunoblotting, flow cytometry, and immunofluorescence analyses demonstrated PD-1 gene and protein expression by MCC cells. MCC–PD-1 ligation enhanced, and its inhibition or silencing suppressed, in vitro proliferation and in vivo tumor xenograft growth. Consistently, MCC–PD-1 binding to PD-L1 or PD-L2 induced, while antibody-mediated PD-1 blockade inhibited, protumorigenic mTOR signaling, mitochondrial (mt) respiration, and ROS generation. Last, pharmacologic inhibition of mTOR or mtROS reversed MCC–PD-1:PD-L1–dependent proliferation and synergized with PD-1 checkpoint blockade in suppressing tumorigenesis. Our results identify an MCC–PD-1–mTOR–mtROS axis as a tumor growth–accelerating mechanism, the blockade of which might contribute to clinical response in patients with MCC.