Decreased expression of CD63 tetraspanin protein predicts elevated malignant potential in human esophageal cancer

Abstract

The tetraspanin CD63 has been described to have critical roles in multiple biological processes, including tumorigenesis and metastasis in several types of cancer. However, its role in esophageal carcinoma (EC) has not been reported. In the current study, immunohistochemistry was used to investigate CD63 expression in 106 esophageal cancer samples, 49 adjacent esophagus tissues and 17 normal esophagus mucosa tissues. The results revealed that the overexpression of CD63 was observed in esophageal cancer samples and negatively correlated with tumor stage and lymph node metastasis. To further evaluate the role of CD63 in esophageal carcinoma, the invasiveness of EC cells was analyzed using matrigel invasion assays and wound healing assays in vitro. Furthermore, it was found that CD63 knockdown increased the invasiveness of TE-1 cells through the upregulation of matrix metalloproteinase (MMP) expression via promoting epithelial-mesenchymal transition. The current data therefore suggested that low levels of CD63 expression may be involved in the tumor progression of esophageal carcinoma.

Keywords: epithelial-mesenchymal transition; esophageal carcinoma; invasiveness; lymph nodes metastasis; tetraspanin CD63; tumor stage.

Figure 1.

(A) Representative IHC analysis of CD63 expression in human esophageal cancer tissues, adjacent esophagus tissues and 17 normal esophagus mucosa tissues. (B) Bar graph representing the range of CD63 IHC staining score in esophageal cancer tissues (n=106), matched adjacent esophagus tissues (n=49) and normal esophagus mucosa tissues (n=17), **P<0.01. IHC, immunohistochemistry.

Figure 2.

Bar graph representing the range of CD63 IHC staining scores in earlier stage (I and II) and advanced stage (III and IV) esophageal carcinoma tissues. **P<0.01. IHC, immunohistochemistry.

Figure 3.

Bar graph representing the range of CD63 IHC staining score in EC tissues with or without metastatic lymph nodes. **P<0.01. IHC, immunohistochemistry.

Figure 4.

Effect of CD63 on esophageal cancer cell invasiveness. (A) Western blot analysis confirmed a marked reduction of CD63 protein following CD63 small interfering RNA transfection in esophageal cancer cells. (B and C) In the Matrigel invasion assay, following treatment for 48 h, the number of CD63 knockdown TE-1 cells that migrated to the bottom surface of the membrane was greater compared with TE-1 cells transfected with empty plasmid and the control TE-1 cells. (D and E) Wound healing was observed 48 h after treatment, and the open wound area of CD63 knockdown TE-1 cells was larger than the TE-1 cells transfected with empty plasmid and the control TE-1 cells. **P<0.01. NC, negative control; siCD63, small interfering RNA targeting CD63.

Figure 5.

Western blot analysis of the expression of E-cadherin, vimentin, MMP-2 and MMP-9 following treatment with small interfering RNA targeting CD63. The epithelial marker E-Cadherin was downregulated and the mesenchymal maker vimentin was upregulated by CD63 knockdown. MMP-2 and MMP-9 expression levels were increased by CD63 knockdown. MMP, matrix metalloproteinase; NC, negative control; siCD63, small interfering RNA targeting CD63.