α2,3-sialyltransferase type I regulates migration and peritoneal dissemination of ovarian cancer cells

Abstract

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecologic cancers due to advanced stage presentation, peritoneal dissemination, and refractory ascites at diagnosis. We investigated the role of α2,3-sialyltransferase type I (ST3GalI) by analyzing human ovarian cancer datasets and human EOC tissue arrays. We found that high expression of ST3GalI was associated with advanced stage EOC. Transwell migration and cell invasion assays showed that high ST3GalI expression enhanced migration of EOC cells. We also observed that there was a linear relation between ST3GalI expression and epidermal growth factor receptor (EGFR) signaling in EOC patients, and that high ST3GalI expression blocked the effect of EGFR inhibitors. Co-Immunoprecipitation experiments demonstrated that ST3GalI and EGFR were present in the same protein complex. Inhibition of ST3GalI using a competitive inhibitor, Soyasaponin I (SsaI), inhibited tumor cell migration and dissemination in the in vivo mouse model with transplanted MOSEC cells. Further, SsaI synergistically enhanced the anti-tumor effects of EGFR inhibitor on EOC cells. Our study demonstrates that ST3GalI regulates ovarian cancer cell migration and peritoneal dissemination via EGFR signaling. This suggests α2,3-linked sialylation inhibitors in combination with EGFR inhibitors could be effective agents for the treatment of EOC.

Keywords: 3-sialyltransferases type I; epidermal growth factor receptor; epithelial ovarian cancer; soyasaponin I; α2.

Figure 1. ST3GalI is a prognostic factor for tumor migration and peritoneal dissemination of human ovarian cancer

(A) Using Oncomine TCGA ovarian cancer genomics (562 ovarian carcinoma samples analyzed on an Affymetrix Human Genome U133 array; 12,624 measured genes), we compared different ST mRNAs, including α2,3-, α2,6-, and α2,8-linked ST, with survival time using a tercile approach. Patients with an upper one-third mRNA expression were defined as the high subgroup, while others with lower two-thirds mRNA expression were defined as the low subgroup. (B-C) IHC analysis of ST3GalI was performed on commercial human ovarian cancer tissue array samples (Super Bio Chips, CJ2, Korea). The intensity scores were as follows: 0, no staining; 1, weak; 2, moderate; 3, strong. Low ST3GalI included weak, moderate or no staining; high ST3GalI was defined as strong staining. Scale bars representing 20μm were added from an image taken at identical magnification and resolution. The percentage was determined in the early stage (FIGO stage I &II) or late stage (FIGO stage III&IV) disease groups. The Fisher's exact test was used to statistically analyze the percentage for the early and late stages. Kaplan-Meier survival curves were used to analyze OS in low- and high-ST3GalIgroups. (D-E) Transwell migration and matrigel invasion of ES2 human ovarian cancer cells with either ST3GalI knocked-down or over-expressed was assayed. Total numbers of cells in 7 random fields were counted. Data shown are the mean ± SD of 3 separate experiments (*: p< 0.05, **: p<0.01.). Data on the Y-axis represented relative value compared to control. Immunoblots were quantified using Image J software.

Figure 2. ST3GalI interacts with EGFR signaling pathway in ovarian cancer

(A) The correlation of 20 STs and significant cell receptors in the ES2ovarian cancer cell line were analyzed by the L1000 mRNA microarray. Data shown are mean ± SD of separate repeat experiments. (B) The analysis of mRNA expression of ST3GalI and EGFR from Oncomine TCGA (n=562), Bittner (n=241), and Lu (n=50) ovarian cancer genomics is shown. The expression of EGFR was compared between low and high ST3GalI groups using a tercile approach. (C) The mRNA expression of α2,3-sialyltransferases and TKI drug efficiency (Actarea) are shown from the CCLE ovarian cancer dataset. (D-E) Western blot analysis of EGFR and phospho-EGFR in ST3GalI knocked-down or overexpressing cells compared to controls is shown. GAPDH was used as control (same GAPDH as in Figure 1D-1E). (F) The co-immunoprecipitation assay coupled with immunoblotting analysis to evaluate the protein-protein interaction of ST3GalI and EGFR is shown. (G) Immunoblotting of ST3Ga1I immunoprecipitated with anti-EGFR antibodies in SC and ST3GalI knock-down of ES2 cell line are shown. (H) Time course RNA and protein analysis showing EGFR expression during ST3GalI knock-down.

Figure 3. α2,3-sialylation inhibitor SsaI suppresses ovarian cancer tumor migration and peritoneal dissemination

(A) Quantification of ST3GalI in ovarian cancer cells (MOSEC, ES2, and OVCAR3) treated with 100μM SsaI or DMSO control for 72h by western blot; GAPDH was used as internal control. (B) The Transwell migration (upper panel) and invasion assay (low panel) on ovarian cancer cells that were stained with Toluidine blue and Crystal violet solution, respectively. Data shown are the mean ± SD of 3 separate experiments. (C) MOSEC cells (2×10⁶) were injected into the peritoneal cavity of 8weekold female C57BL/6 mice (n=10). ALZET Micro-Osmotic Pumps were filled with either 100μM SsaI or DMSO as control was implanted subcutaneously. The mice were sacrificed after 4 weeks and the amount of ascites was measured. The body weights of mice were recorded after injecting MOSEC cells and implantation of pumps implantation. Data shown are the mean ± SD of 5 mice. Peritoneal seeding and carcinomatosis were analyzed in the SsaI and DMSO control groups. (*: p< 0.05, **: p<0.01, ***: p< 0.001.)

Figure 4. α2,3-sialylation inhibitor SsaI affects EGFR signaling and synergizes with TKI

(A) Quantification of EGFR and phospho-EGFR in ES2 cells treated with 100μM SSaI or DMSO control for 72h; GAPDH was used as control (same GAPDH as in Figure 3). (B) Intraperitoneal tumors from B6 mice treated with either SsaI or DMSO control were stained by the Duolink in situ IHC staining kit to analyze for ST3GalI and EGFR. (C) ES2 cells were treated with 100μM SsaI and 5μm AG1478 (EGFR inhibitor) were subjected to Transwell matrigel invasion assay. Total numbers of cells were counted in 7 to 10 random fields. Data shown are the mean ± SD of 3 separate experiments. (D) Synergy between ST3GalI and EGFR were determined by treating ES2 cells with either SsaI orAG1478 (EGFR inhibitor) or both for 48h followed by determination of their effects on cell proliferation rate. Data shown are the mean ± SD of 3 independent experiments. (E) A proposed mechanism of the interaction between ST3GalI and the EGFR signaling pathway.