Effects of ADAM10 upregulation on progression, migration, and prognosis of nasopharyngeal carcinoma

Abstract

A disintegrin and metalloprotease 10 (ADAM10) is a typical member of the ADAMs family, which has been reported to be upregulated in various types of cancers and contribute to cancer progression and metastasis. However, little is known about the role of ADAM10 in nasopharyngeal carcinoma (NPC). The purpose of this study is to explore ADAM10 expression status and its biological functions in NPC. We first examined the expression of ADAM10 in NPC tissues and cell lines by immunohistochemistry, Western blotting, PCR, and immunofluorescence analysis. We observed that ADAM10 was significantly elevated in NPC and its expression level was correlated with T classification (P = 0.044), distant metastasis (P = 0.016), TNM clinical stage (P = 0.013), and proliferation marker Ki-67 expression (P = 0.001). Patients with NPC with high expression of ADAM10 had shorter overall survival rates. In addition, knockdown of ADAM10 by RNAi was found to inhibit the CNE-2 cell proliferation and migration. Our findings hinted that overexpression of ADAM10 promotes the progression and migration of NPC, which makes it a potential therapeutic target for the treatment of tumor metastases in NPC.

Keywords: ADAM10; migration; nasopharyngeal carcinoma; prognosis; progression.

Figure 1

Expression of types of a disintegrin and metalloprotease (ADAM) in nasopharyngeal carcinoma (NPC) tissues and cell lines. (a–d) mRNA levels of ADAM9, ADAM10, ADAM12, and ADAM17 in 20 paired NPC tissues by quantitative PCR. (e–h) mRNA levels of ADAM9, ADAM10, ADAM12, and ADAM17 in NPC cell lines by quantitative PCR. CNE‐1, CNE‐2, 5‐8F, and 6‐10B are human NPC cell lines; NP‐69 is an immortalized normal nasopharyngeal epithelial cell line. The same experiment was repeated at least three times.*P < 0.05.

Figure 2

Expression of a disintegrin and metalloprotease 10 (ADAM10) in nasopharyngeal carcinoma (NPC) tissues. (a) Protein levels of ADAM10 in 8 of 20 paired NPC (T) and non‐cancerous nasopharyngeal tissues (N) by Western blotting. (b) Quantitative results of Western blot analysis. (c) Immunohistochemistry analysis of ADAM10 and Ki‐67 expression in NPC tissues (original magnification, ×400). β‐actin was used as a loading control. Scale bar = 50 μm. The same experiment was repeated at least three times.

Figure 3

Relationship between Ki‐67 proliferation index and a disintegrin and metalloprotease 10 (ADAM10) expression in nasopharyngeal carcinoma. Scatterplot of Ki‐67 versus ADAM10 with regression line showed a correlation using the Spearman's correlation coefficient (P < 0.01).

Figure 4

Kaplan–Meier survival curves of patients with nasopharyngeal carcinoma based on a disintegrin and metalloprotease 10 (ADAM10) expression status. Patients in the high expression group had significantly poorer prognosis than those in the low expression group (P < 0.05, log–rank test).

Figure 5

Expression of a disintegrin and metalloprotease 10 (ADAM10) in nasopharyngeal carcinoma cell lines. (a–c) Western blot and immunofluorescence analysis of ADAM10 expression in CNE‐1, CNE‐2, 5‐8F, 6‐10B, and NP‐69 nasopharyngeal carcinoma cell lines. NP69 is an immortalized normal nasopharyngeal epithelial cell line. The same experiment was repeated at least three times. β‐actin was used as control. *P < 0.05. Scale bar = 50 μm.

Figure 6

Expression of a disintegrin and metalloprotease 10 (ADAM10) and proliferating cell nuclear antigen (PCNA) in proliferating nasopharyngeal carcinoma CNE‐2 cells. (a) Flow cytometry quantitation of cell cycle progress in CNE‐2. Cells synchronized at G₀/G₁ progressed into the cell cycle when serum was added for the indicated times. (b) CNE‐2 cells were serum‐starved for 72 h. Following release into serum, cell lysates were prepared and the expressions of ADAM10 and PCNA were analyzed by Western blot. (c) Quantitative results of Western blot analysis. β‐actin was used as a loading control. Mean ± SEM of three independent experiments. *^#P < 0.05 vs S72 h.

Figure 7

Knockdown of a disintegrin and metalloprotease 10 (ADAM10) reduced CNE‐2 nasopharyngeal carcinoma cell proliferation. (a–c) Interference efficiency was detected by Western blot analysis and PCR. (d) Cell proliferation was measured by CCK‐8 assay. (e) Cell cycle analysis by flow cytometry in CNE‐2 cells with ADAM10 downregulation. Data are mean ± SEM. The same experiment was repeated at least three times. *^P < 0.05 vs control.

Figure 8

Knockdown of a disintegrin and metalloprotease 10 (ADAM10) decreased the migration of CNE‐2 nasopharyngeal carcinoma cells. (a) CNE‐2 cells transfected with ADAM10 siRNAs showed a slower wound closure rate than cells transfected with non‐specific siRNA or control. A representative image from three independent experiments is shown. (b) Wound‐healing assays showed the relative migration distance of cells. (c,d) Knockdown of ADAM10 inhibited cell migration by Transwell assays. (d) Number of cells that invaded through the membrane was counted in 10 fields under ×20 objective lens. Bars, SD. (n = 3; P < 0.05). (e) Western blot analysis of E‐cadherin, N‐cadherin, and vimentin in CNE‐2 cells transfected with ADAM10 siRNAs and non‐specific siRNA. (f) Western blot analysis of E‐cadherin, N‐cadherin, and vimentin in NP69 cells transfected with GV362–ADAM10 and mock GV362 vector. The same experiment was repeated at least three times. *P < 0.05.