The Tumor-Associated Glycosyltransferase ST6Gal-I Regulates Stem Cell Transcription Factors and Confers a Cancer Stem Cell Phenotype

Abstract

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.

Fig. 1. ST6Gal-I is upregulated in ovarian and pancreatic carcinoma and correlates with patient survival

(A–C) ST6Gal-I expression by IHC in: (A) normal ovarian epithelium and stroma; (B) ovarian tumor with ST6Gal-I-positive tumor cells (arrow), adjacent to ST6Gal-I-negative, normal-appearing epithelium (arrowhead); and (C) normal fallopian tube epithelium. Scale bars=50µm. ST6Gal-I expression correlated with (D) progression free and (E) overall survival. (F–H) IHC for ST6Gal-I in: (F) normal pancreas with acinar (star) and ductal (arrow) cells, and pancreatic islets (arrowhead); (G) pancreatic adenocarcinoma; (H) pancreatic PDX tumor. Scale bars=50µm.

Fig. 2. ST6Gal-I is enriched in metastatic tumors

(A–B) Immunoblot and densitometry (normalized to β-actin) for ST6Gal-I in primary ovarian tumors. (C–D) Immunoblot and densitometry for unmatched metastatic tumors. (E–F) IHC images of a primary and metastatic tumor stained for ST6Gal-I. Scale bars=50µm. (G) Percentage of ST6Gal-I-positive cells within primary and metastatic tumors. Values = means and S.E.; *denotes p<0.05, Student’s t-test. (H) Tumors grouped into low, middle, or high percentage of ST6Gal-I-positive cells.

Fig. 3. ST6Gal-I promotes survival of CSC spheroids

(A–B) Viability of MiaPaCa2 (A) and BxPC3 (B) empty vector (EV) and ST6Gal-I knockdown (ST6-KD) cells grown in CSC culture for two weeks. (C) Viability of SKOV3 EV, ST6-KD or ST6Gal-I overexpressing (ST6-OE) cells in CSC culture. Values = means and S.E. for 3 independent experiments, p<0.05; Student’s t test. (D) Immunoblot of SKOV3 cells cultured in CSC vs standard 10% FBS-containing media for 1 week. (E–F) ST6Gal-I immunoblotting and densitometry for SKOV3 (E) or MiaPaCa2 (F) cells cultured in CSC media for 1, 5, or 14 days. (G) ST6Gal-I expression (arrowhead) in ovarian ascites tumorspheroid. (H) SNA high (SNA-H) and SNA low (SNA-L) ascites cells grown in CSC culture. (I) Spheroids per field. (J) Ovarian PDX tumor SNA-H and SNA-L grown in CSC culture. (K) Viability of OV4 EV or ST6-OE cells incubated in cell-free ascites. Values = means and S.E., p<0.05; Student’s t-test. Scale bars=50µm.

Fig. 4. ST6Gal-I confers gemcitabine resistance

(A) MiaPaCa2 EV and ST6-KD cells were immunoblotted for cleaved caspase-3 after a 24-hr treatment with 100nM Gem. (B) CellTiter-Glo assays were conducted to assess the viability of MiaPaCa2 EV and ST6-KD cells treated with Gem for 3 or 5 days. * = p<0.05 by student’s t-test. (C) Viability of CSC spheroids treated with 100 nM Gem for 5 days. (D) Adherent MiaPaCa2 and BxPC3 cells exposed to 100 nM Gem for 10 days. Insets show live (green) and dead (red) cells. (E) ST6Gal-I expression in pair-matched, pancreatic PDX tumors from saline- or Gem-treated mice. Scale bars = 500µm (left panel, each pair) and 50 µm (right panel, each pair).

Fig. 5. ST6Gal-I contributes to tumor initiation

(A–C) Tumor incidence and growth for mice injected with 10⁶ (A), 10⁵ (B), or 10⁴ (C) cells. *p<0.05. (D–E) ST6Gal-I expression in: (D) mPanINs from Kras^G12D mice; (E) normal murine pancreas. Scale bars=50µm. (F) ST6Gal-I expression in intestines of Rosa-ST6Gal-I⁺/Cre⁻ mice (wildtype) or ST6Gal-I⁺/Cre⁺ transgenic mice (ST6Gal-I knock-in). (G) Representative colon tissues following AOM-DSS treatment. (H) Percent area of tumor tissue per colon in wildtype (WT) or ST6Gal-I knock-in (ST6Gal-I) mice. (I) Tumor number per colon. Values = means and S.E.; p<0.05, Student’s t-test.

Fig. 6. ST6Gal-I regulates Sox9 and Slug expression

(A–D) Sox9 expression is repressed by ST6Gal-I knockdown. (E) Sox9 is induced by forced ST6Gal-I expression. (F) Suit2 cancer cells transduced with increasing amounts of ST6Gal-I lentivirus exhibit a dose-dependent Sox9 increase. (G) Sox9 is induced in HEK293 transduced with ST6Gal-I-expressing adenovirus. (H) Slug expression is induced in OV4 ST6-OE cells, but (I) decreased in SKOV3 ST6-KD cells.