ST6Gal-I sialyltransferase promotes chemoresistance in pancreatic ductal adenocarcinoma by abrogating gemcitabine-mediated DNA damage

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.

Keywords: H2A histone family; chemoresistance; drug metabolism; gemcitabine; glycosylation; member X (H2AFX); pancreatic cancer; pancreatic ductal adenocarcinoma (PDAC); β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1).

Figure 1.

Cells with ST6Gal-I knockdown exhibit reduced survival upon exposure to gemcitabine. A and B, ST6Gal-I KD in MiaPaCa-2 (A) and BxPC-3 (B) cells as depicted by immunoblotting. EV is the control. MiaPaCa-2 (C) and BxPC-3 (D) cells have reduced ST6Gal-I activity as indicated by decreased surface α2–6 sialylation. α2–6 sialylation was measured by staining cells with the SNA lectin followed by flow cytometry. E and F, representative images of MiaPaCa-2 and BxPC-3 cells showing that gemcitabine (Gem)-treated KD cells have reduced colony-forming potential compared with gemcitabine-treated EV cells. G and H, MiaPaCa-2 and BxPC-3 KD cells display a significantly reduced survival fraction post-gemcitabine treatment compared with the control EV populations. Graphs depict results from three independent experiments with each independent experiment performed in triplicate. Error bars represent S.E. * denotes p < 0.05 as measured by Student's t test.

Figure 2.

Removal of surface sialic acids reverses the protective effect of ST6Gal-I on gemcitabine-induced cell death. A, mean fluorescence intensity (MFI) values for cells stained with either SNA or MAA lectin. Lectin staining was conducted on UT cells or cells incubated with A. ureafaciens neuraminidase (neura) for 30 min. B, EV and KD cells were treated with or without gemcitabine (Gem) for 24 h and monitored for cleavage (activation) of caspase 3. C, cells were pretreated with neuraminidase for 30 min and then incubated with gemcitabine for 24 h. Immunoblots of cleaved caspase 3 (cl. casp 3) show that neuraminidase treatment sensitized EV, but not KD, cells to gemcitabine. D, cells were incubated with anti-TNFR1 function–blocking antibody and gemcitabine for 24 h. The TNFR1-blocking antibody potentiated gemcitabine-induced cell death and eliminated differences in EV and KD apoptotic responses. inhib, inhibitor.

Figure 3.

ST6Gal-I knockdown increases the GSR. MiaPaCa-2 KD (A) and BxPC-3 KD (B) cells treated with gemcitabine (Gem) have a significantly increased GSR compared with gemcitabine-treated EV cells. Graphs depict results from three independent experiments with each experiment performed in triplicate. Error bars represent S.E. * denotes p < 0.05 as measured by Student's t test.

Figure 4.

Gemcitabine-treated ST6Gal-I KD cells have increased DNA damage as measured by comet assay. Representative images depict increased tail lengths in MiaPaCa-2 KD (A) and BxPC-3 KD (B) cells. MiaPaCa-2 KD (C) and BxPC-3 KD (D) cells treated with gemcitabine (Gem) had a significantly increased tail moment compared with gemcitabine-treated EV cells. Graphs depict results from three independent experiments. Error bars represent S.E. * denotes p < 0.05 as evaluated by Student's t test.

Figure 5.

Greater DNA damage in KD versus EV cells as indicated by γH2AX foci. Cells were stained with anti-γH2AX antibody, and the number of foci per cell was counted. Cells were grouped into categories of low DNA damage (<5 foci), moderate DNA damage (5–15 foci), or severe DNA damage (>15 foci). A, representative images of the three categories. Images (B) and quantification (C) of γH2AX foci in MiaPaCa-2 cells are shown. Increased DNA damage in gemcitabine (Gem)-treated KD versus EV cells is demonstrated by a significantly increased number of cells with severe damage and a decreased number of cells with low damage. Images (D) and quantification (E) of γH2AX foci in BxPC-3 show greater DNA damage in gemcitabine-treated KD versus EV cells. Graphs depict results from three independent experiments. Error bars represent S.E. * denotes p < 0.05 as measured by Student's t test.

Figure 6.

ST6Gal-I levels are elevated in cells with stable gemcitabine resistance. MiaPaCa-2 cells were exposed to long-term gemcitabine (Gem) treatment to obtain stably resistant lines. Two independent lines were developed by growing cells in either 50 or 100 nm gemcitabine. A, endogenous ST6Gal-I expression is enriched in a dose-dependent manner in stably resistant lines as compared with the parental (Par), treatment-naïve population. B, 100 nm gemcitabine-resistant cells have reduced GSR relative to parental MiaPaCa-2 cells. C, treatment with 100 nm gemcitabine for 24–48 h does not induce up-regulation of ST6Gal-I. The graph depicts results from three independent experiments with each experiment performed in triplicate. Error bars represent S.E. * denotes p < 0.05 as evaluated by Student's t test.

Figure 7.

A metastatic, chemoresistant Suit-2 subclone with up-regulated ST6Gal-I can be sensitized to gemcitabine by ST6Gal-I knockdown. A, Suit-2 cells with no detectable ST6Gal-I compared with three Suit2-derived metastatic subclones, S2-CP9, S2-LM7AA, and S2-013. All of the metastatic derivatives have increased ST6Gal-I expression as measured by immunoblotting. B, ST6Gal-I was knocked down in the S2-013 line, and reduced expression was verified by immunoblotting. C, reduction in SNA staining in KD cells, indicative of reduced surface α2–6 sialylation. D, S2-013 KD cells have increased gemcitabine (Gem)-induced cell death relative to EV cells as indicated by increased expression of cleaved caspase 3. S2-013 KD cells have decreased clonogenic potential (E) and significantly reduced survival fraction (F). The graph depicts results from three independent experiments with each experiment performed in triplicate. Error bars represent S.E. * denotes p < 0.05 as measured by Student's t test.

Figure 8.

S2-013 KD cells exhibit enhanced gemcitabine-induced DNA damage. A, representative images of gemcitabine (Gem)-treated S2-013 cells evaluated by comet assay. B, gemcitabine-treated S2-013 KD cells depict significantly increased tail moment compared with gemcitabine-treated EV cells. C, representative images of S2-013 cells stained for γH2AX foci. D, quantification of γH2AX foci in Suit2-013 EV and KD cells treated with or without gemcitabine. Graphs depict results from three independent experiments. Error bars represent S.E. * denotes p < 0.05 as measured by Student's t test.