Abstract
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 found 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 clonogenic assays and cleaved caspase 3 expression. Additionally, KD of ST6Gal-I potentiates gemcitabine-induced DNA damage as measured by comet assays and quantification of 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, an indicator of gemcitabine toxicity. Gemcitabine-resistant MiaPaCa-2 cells display higher ST6Gal-I levels than treatment-naïve cells along with a reduced gemcitabine sensitivity ratio, 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 up-regulated 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.
Highlights
Reduced ST6Gal-I activity in KD cells was confirmed by flow cytometric analyses of cells stained with SNA (Fig. 1, C and D), a lectin that binds to surface ␣2– 6 sialic acids
To evaluate the role of ST6Gal-I in chemoresponse, MiaPaCa-2 and BxPC-3 empty lentiviral vector (EV) and KD cells were exposed to gemcitabine for 72 h, and cells were plated for clonogenic assays
Replicate wells were seeded with untreated (UT) cells to control for differences in seeding density and to enable calculation of the survival fraction
Summary
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States with a dismal 5-year survival rate of 8% [1, 2]. Given that inherent or acquired resistance to gemcitabine is one of the chief contributors to PDAC lethality, an understanding of the molecular mechanisms regulating tumor cell response to this drug is of paramount importance. PDAC resistance to gemcitabine can occur through multiple mechanisms including alterations in gemcitabine metabolism or activation of DNA repair pathways such as the DNA damage response (DDR). ST6Gal-I protects against gemcitabine-induced DNA damage mechanism involving altered glycosylation of surface proteins. Knockdown of ST6Gal-I expression in MiaPaCa-2, BxPC-3, and S2-013 PDAC cell lines potentiates DNA damage by gemcitabine as measured by comet assays and quantification of ␥H2AX. Removal of surface ␣2– 6 sialic acids by neuraminidase treatment mimics the effect of ST6Gal-I knockdown by sensitizing cells to drug cytotoxicity, supporting a role for ST6Gal-I–mediated sialylation in gemcitabine resistance. These studies implicate ST6Gal-I as a potential therapeutic target to combat gemcitabine resistance in PDAC
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