ZNF488 Enhances the Invasion and Tumorigenesis in Nasopharyngeal Carcinoma Via the Wnt Signaling Pathway Involving Epithelial Mesenchymal Transition

Abstract

Purpose: The purpose of this study was to investigate the function of Zinc finger protein 488 (ZNF488) in nasopharyngeal carcinoma (NPC).

Materials and methods: The endogenous expression of ZNF488 in NPC tissues, normal nasopharyngeal epithelium tissues and NPC cell lines were detected by quantitative reverse transcription polymerase chain reaction. ZNF488 over-expressing and knock-down NPC cell line models were established through retroviral vector pMSCV mediated over-expression and small interfering RNA (siRNA) mediated knock-down. The invasion and migration capacities were evaluated by wound healing and transwell invasion assays in ZNF488 over-expressing and control cell lines. Soft-agar colony formation and a xenograft experiment were performed to study tumorigenic ability in vitro and in vivo. Immunofluorescence and western blotting analysis were used to examine protein changes followed by ZNF488 over-expression. Microarray analysis was performed to explore gene expression profilings, while luciferase reporter assay to evaluate the transcriptive activity of Tcf/Lef.

Results: ZNF488 was over-expressed in NPC tissues compared with normal tissues, especially higher in 5-8F and S18, which are well-established high metastatic NPC clones. Functional studies indicate that over-expression of ZNF488 provokes invasion, whereas knock-down of ZNF488 alleviates invasive capability. Moreover, over-expression of ZNF488 promotes NPC tumor growth both in vitro and in vivo. Our data further show that over-expression of ZNF488 induces epithelial mesenchymal transition (EMT) by activating the WNT/β-catenin signaling pathway.

Conclusion: Our data strongly suggest that ZNF488 acts as an oncogene, promoting invasion and tumorigenesis by activating the Wnt/β-catenin pathway to induce EMT in NPC.

Keywords: Carcinogenesis; Epithelial-mesenchymal transition; Invasion; Nasopharyngeal carcinoma; Wnt signaling pathway; ZNF488.

Fig. 1.

Zinc finger protein 488 (ZNF488) is highly expressed in nasopharyngeal carcinoma (NPC). (A) Expression levels of ZNF488 mRNA in NPC tissues and normal nasopharyngeal epithelial tissues. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. (B, C) Expression levels of ZNF488 mRNA (B) or protein (C) in a series of NPC cell lines and immortalized or normal nasopharyngeal epithelial cells. Data are presented as the mean±standard deviation, and p-values were calculated with Student’s t test.

Fig. 2.

Over-expression of Zinc finger protein 488 (ZNF488) promotes invasion and migration in nasopharyngeal carcinoma cells. (A) Expression levels of ZNF488 protein after stably transfected with control or ZNF488-expressing plasmids were detected by western blotting analysis. (B) Effect on migration by wound healing assays. (C) Effect on invasion by transwell assays. (D) The knock-down efficiency of endogenous ZNF488 expression on protein levels were determined by western blotting analysis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (E) 5-8F and S18 cells were transfected with two individual ZNF488 siRNAs or a scrambled control followed by transwell invasion assays. Data are presented as the mean±standard deviation, and chance was ruled out with Student’s t test.

Fig. 3.

Zinc finger protein 488 (ZNF488) induces cancer stem cell-like self-renewal properties to enhance tumorigenesis. (A) Expression of stem cell markers, ABCG2, NANOG, and SOX2 by reverse transcription and quantitative polymerase chain reaction analysis. (B) ZNF488–over-expressing and control cell lines were subjected to anchorage-independent colony formation in soft agar (×20). (C) Representative picture of xenograft tumors in nude mice inoculated with CNE1 stably expressing ZNF488 or vector control. (D) Growth curves of tumor volumes in nude mice. (E) Statistical analysis of tumor weights. Data are presented as the mean±standard deviation, and p-values were calculated with Student’s t test. *p < 0.05, **p < 0.01.

Fig. 4.

Zinc finger protein 488 (ZNF488) activates the WNT/β-catenin pathway to induce the epithelial mesenchymal transition (EMT) transcription factors Tcf/Lef which is capable of triggering EMT. (A) The protein level of E-cadherin, α-catenin, vimentin, and N-cadherin were detected by western blotting analysis. (B) Immunofluorescence staining to dectect the changes of E-cadherin, ZO-1, vimentin, and N-cadherin. (C) Luciferase reporter assays using TopFlash/FopFlash reporter plasmids to monitor the activity of Tcf/Lef transcription factor. (D) Western blotting analysis to detect the expression of β-catenin, phospho-GSK3β (Ser9), GSK3β, Slug, and Snail. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Data are presented as the mean±standard deviation, and p-values were calculated with Student’s t test.