Evaluation of the prognostic value and functional roles of CD44v6 in gastric cancer

Abstract

Purpose: Tumor stem cell surface marker CD44v6, a member of the CD44 protein family, is causally involved in the metastasis of cancer. Little is known about the functions of CD44v6 in gastric cancer. The aim of this study was to evaluate the prognostic value of CD44v6 and investigate its functional roles.

Methods: The expression of CD44v6 in 208 primary gastric adenocarcinoma patient samples was examined using immunohistochemistry and its correlation with clinicopathological parameters, and 5-year patient survival was assessed. Two pairs of MGC-803 stable cells with either CD44v6 overexpression or knockdown were created. The effect of CD44v6 on cell proliferation, colony formation, migration and apoptosis was investigated using these two pairs of cells.

Results: Overexpression of CD44v6 was observed in all cancer cell lines. The 5-year survival rate of patients with positive CD44v6 expression is significantly worse compared to those with negative expression (38.8 vs. 73.6 %). CD44v6 and TNM stage are two independent prognostic factors of primary gastric adenocarcinoma. The risk factors for the positive CD44v6 expression are location of tumor, depth of invasion, lymph node metastasis, Lauren classification and TNM stage. In MGC-803 cells, CD44 stimulated proliferation and colony formation, antagonized oxaliplatin-induced apoptosis, but did not affect migration.

Conclusion: CD44v6 is an important prognosis marker in gastric cancer. Tissue specificity may affect the functions of CD44v6, and further work is needed to elucidate its regulation.

Fig. 1

Evaluation of CD44v6 expression in gastric cancer cell lines and patient samples. a CD44v6 protein expression in GES-1, MGC-803, BGC-823, SGC-7901 and MKN-45 cells was detected by Western blot. b Positive CD44v6 protein expression is associated with gastric cancer patient survival

Fig. 2

Investigation of the oncogenic functions of CD44v6 overexpression. a Cell lysates were extracted from MGC-803 cell lines stably expressing GFP or GFP-CD44v6, and the expression of GFP-tagged proteins and GAPDH was detected by Western blot. b Proliferation of MGC-803 cell lines stably expressing GFP or GFP-CD44v6 over 5 days was examined using MTT assay. The readings at day 0 were used as 1 for fold change calculation. c Colony-forming ability of MGC-803 cell lines stably expressing GFP or GFP-CD44v6 was investigated by colony formation assay. The upper panel was the representative result images of the relative cell line. The lower panel was the quantitative comparison. The MGC-803 cell lines stably expressing GFP were used as 1 for fold change calculation. d MGC-803 cell lines stably expressing GFP or GFP-CD44v6 were subject to scratch wound-healing assays. The wound status was recorded at 0-, 12- and 24-h post-scratch, and the representative images were presented. e MGC-803 cell lines stably expressing GFP or GFP-CD44v6 were exposed to oxaliplatin at indicated concentrations, and the cell lysates were collected 24-h post-treatment. The expression of cleaved-PARP (c-PARP) and GAPDH was detected by Western blot. The experiments in (b–e) have been repeated three times

Fig. 3

Investigation of the effects of CD44v6 knockdown. a Cell lysates were extracted from MGC-803 cell lines stably expressing scramble RNA or CD44v6-shRNA, and the expression of CD44v6 and GAPDH was detected by Western blot. b Proliferation of MGC-803 cell lines stably expressing scramble RNA or CD44v6-shRNA over 5 days was examined using MTT assay. The readings at day 0 were used as 1 for fold change calculation. c Colony-forming ability of MGC-803 cell lines stably expressing scramble RNA or CD44v6-shRNA was investigated by colony formation assay. The upper panel was the representative result images of the relative cell line. The lower panel was the quantitative comparison. The MGC-803 cell lines stably expressing scramble RNA were used as 1 for fold change calculation. d MGC-803 cell lines stably expressing scramble RNA or CD44v6-shRNA were subject to scratch wound-healing assays. The wound status was recorded at 0-, 12- and 24-h post-scratch, and the representative images were presented. e MGC-803 cell lines stably expressing scramble RNA or CD44v6-shRNA were exposed to oxaliplatin at indicated concentrations, and the cell lysates were collected 24-h post-treatment. The expression of cleaved-PARP (c-PARP) and GAPDH was detected by Western blot. The experiments in (b–e) have been repeated three times