Extracellular galectin-3 facilitates colon cancer cell migration and is related to the epidermal growth factor receptor

Abstract

We previously found that galectin-3 enhanced DLD-1 cell migration through the K-Ras-Raf-Erk1/2 pathway, but the effect of extracellular galectin-3 on cancer cell migration and its interaction with the epidermal growth factor receptor (EGFR) remained unknown. We aimed to determine the effect of extracellular galectin-3 on colon cancer cell migration and its correlation with the EGFR expression. Western blotting was performed to analyze galectin-3 secretion, shRNA was used to stably knock down galectin-3 expression and a migration assay was performed to evaluate colon cancer cell migration. Tissues from eighty patients with four different stages of colon cancer were obtained and compared to normal colon tissue. The galectin-3 knockdown colon cancer cells exhibited decreased migration, which was restored by recombinant galectin-3. An EGFR blocking antibody decreased colon cancer cell migration. The addition of recombinant galectin-3 increased phosphorylated EGFR expression within minutes and enhanced the internalization of the EGFR from the cell membrane to the cytoplasm, particularly upon EGF stimulation. Extracellular galectin-3 increased colon cancer cell migration, which correlated with the EGFR. Targeting galectin-3 may have a synergistic effect on EGFR-targeted therapy.

Keywords: Colorectal carcinoma; EGFR; cell migration; galectin-3.

Figure 1

Extracellular galectin-3 correlated with the migration of different colon cancer cell lines and facilitated colon cancer cell migration. A. Caco2 cells secreted more galectin-3 than DLD-1 cells according to the western blot analysis; B. Caco2 cells migrated faster (as detected by performing a wound healing assay) than the DLD-1 cells. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” which denote a P value of < 0.05. C. Recombinant galectin-3 dose-dependently enhanced DLD-1 cell migration. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” which denote a P value of < 0.05. Bars represent the mean ± SD of three independent experiments.

Figure 2

Lactose and galectin-3 antibody inhibit DLD-1 colon cancer cell migration. (A) Migration rate was dose-dependently inhibited by lactose and anti-galectin-3 Ab (B2C10) (B). Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” and “**” which denote a P value of < 0.05 and < 0.01, respectively.

Figure 3

Extracellular recombinant galectin-3 rescues galectin-3 knockdown DLD-1 cell migration. (A) shRNA knockdown of galectin-3 shown by WB and ICC. (B) Stable knockdown of galectin-3 decreased the lamellipodia formation, migration rate (C) and tumor growth by iRFP (D). Recombinant galectin-3 restored the galectin-3 knockdown-induced decrease in lamellipodia formation (B) and cell migration (E). Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” which denote a P value of < 0.05.

Figure 4

Inhibition of extracellular galectin-3 decreased colon cancer cell migration. Modified citrus pectin dose-dependently blocked galectin-3-induced DLD-1 cell migration. Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” and “**” which denote a P value of < 0.05 and < 0.01, respectively.

Figure 5

Galectin-3 interacts with EGFR via carbohydrate recognition and effects migration. A. Interaction between EGFR and galectin-3 was shown by IP, followed by IB. B. According to the in situ PLA, the addition of galectin-3 induced fluorescent resonance between EGFR and galectin-3, which was blocked by lactose but not the EGFR inhibitor. C. Addition of recombinant galectin-3 increased EGFR phosphorylation within 10 minutes. D. EGFR inhibitor significantly decreased DLD-1 cell migration. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” which denote a P value of < 0.05.

Figure 6

Recombinant-galectin-3 enhanced EGFR internalization. A. Recombinant galectin-3 promoted EGFR internalization from the membrane to the cytoplasm as shown by WB of the membrane and cytoplasm EGFR expression. B. Addition of galectin-3 to galectin-3 knockdown colorectal cancer cells decreased the membrane EGFR by flow cytometry. Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed using the Student’s t-test and the statistical significance is indicated by “*” which denote a P value of < 0.05. N.S.: not significant.

Figure 7

Correlation between higher Gal-3 expression and augmented EGFR and CEA expression in advanced stages of colon cancer. Advanced stages of colon cancer had higher Gal-3, EFGR, and CEA expression than earlier stage cancer or normal colonic tissue. White bar represents 100 μm. Statistical analysis was performed using one-way analysis of variance and subsequent Bonferroni post-hoc tests, the statistical significance is indicated by “*” and “**” which denote a P value of < 0.05 and < 0.01, respectively.