Snail promotes metastasis of nasopharyngeal carcinoma partly by down-regulating TEL2

Abstract

Background: Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). We previously reported that TEL2, a negative regulator of SERPINE1, could inhibit NPC metastasis to lymph nodes.

Method: A series of in vivo and in vitro assays were performed to elucidate the regulation between Snail and TEL2. TEL2 expression was analyzed in three representative NPC cell lines expressing low levels of Snail (S26, 6-10B, HK1) and two cell lines expressing high levels of Snail (S18, 5-8F). Luciferase and chromatin immunoprecipitation assays were used to analyze the interaction between Snail and TEL2. The roles of the Snail/TEL2 pathway in cell migration and invasion of NPC cells were examined using transwell assays. Metastasis to the lungs was examined using nude mouse receiving NPC cells injection through the tail vein.

Results: Ectopic Snail expression down-regulated TEL2 at the mRNA and protein levels, whereas knockdown of Snail using short hairpin RNA up-regulated TEL2. Luciferase and chromatin immunoprecipitation assays indicated that Snail binds directly to the TEL2 promoter. Ectopic Snail expression enhanced migration and invasion of NPC cells, and such effects were mitigated by TEL2 overexpression. TEL2 overexpression also attenuated hypoxia-induced cell migration and invasion, and increased the number of metastatic pulmonary nodules. Snail overexpression reduced the number of metastatic pulmonary nodules.

Conclusions: TEL2 is a novel target of Snail and suppresses Snail-induced migration, invasion and metastasis in NPC.

Keywords: Metastasis; Nasopharyngeal carcinoma; Snail; TEL2.

Fig. 1

Snail down-regulates TEL2 at the mRNA and protein levels in NPC cells. a Levels of Snail mRNA in the indicated tissues. P, NPC primary tissues (n = 10); L, metastatic tumor tissues in lymph nodes (n = 4). Bars indicate SD. *P < 0.05. b, c Cells were stably transfected with empty vector or Snail-encoding plasmid, and levels of TEL2 and E-cadherin mRNA were measured using qRT-PCR. Data are mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01. d Cells were stably transfected with empty vector or Snail-encoding plasmid, and levels of TEL2 and E-cadherin protein were analyzed by Western blot. e, f In cells stably transfected with plasmids expressing anti-Snail shRNA or scrambled control shRNA (#1, #2), levels of TEL2 and E-cadherin mRNA were analyzed using qRT-PCR. Data are mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01. Knockdown of Snail at the protein level was confirmed in S18 and 5-8F cells by Western blot

Fig. 2

Snail binds directly to the TEL2 promoter in NPC cells. a Schematic illustration of the wild-type TEL2 promoter and its mutants in luciferase reporter assays. b, c S26 cells stably transfected with empty vector or Snail-encoding plasmid were transfected with a luciferase reporter plasmid in which luciferase expression was driven by the wild-type or mutant TEL2 promoter. Luciferase activity was measured as described in Methods. Data are mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01. d Schematic illustration of the location of primers for ChIP analysis. e–g Cells were analyzed in ChIP assays using anti-Snail antibody as described in “Methods”

Fig. 3

Snail is involved in hypoxia-induced TEL2 down-regulation in NPC. a Migration and invasion activity was measured after 24 h in transwell assays under normoxia (Nor, 21% O₂) or hypoxia (Hyp, 1% O₂). Data are mean ± SEM. The number of cells passing through the membrane in each transwell was analyzed in triplicate and repeated three times with similar results. **P < 0.01. b Representative image of the assay in a. Red scale bar, 100 μm. c Levels of Snail, HIF1α and TEL2 proteins were analyzed by Western blot in S26 cells exposed for 24 h to normoxia (21% O₂) or hypoxia (1% O₂). d Levels of Snail, E-cadherin and TEL2 mRNA were measured by qRT-PCR in S26 cells treated as in (c). e Level of Snail protein in S26 cells was analyzed by Western blot after Snail knockdown. Tubulin was used as a loading control. f Levels of TEL2 and HIF1α proteins were measured in the indicated cells after treatment as in c. Tubulin was used as a loading control

Fig. 4

The Snail/TEL2/SERPINE1 axis functions in NPC cells. a Schematic illustration for generating a fusion protein of Snail and TEL2 via a T2A linker. b Levels of Snail, TEL2 and Snail-TEL2 proteins were determined by Western blot in the indicated cells. Tubulin was used as a loading control. c Levels of SERPINE1 mRNA were measured by qRT-PCR and levels of SERPINE1 protein by Western blot. GAPDH served as a control. Data are mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001. d Levels of E-cadherin mRNA were measured by qRT-PCR and levels of E-cadherin protein by Western blot. GAPDH served as a control. Data are mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001. e In NPC tissues, a significant negative correlation was observed between Snail and TEL2 expression. f In NPS tissues, a significant positive correlation was observed between Snail and SERPINE1 expression

Fig. 5

Snail promotes NPC cell migration and invasion mostly by down-regulating TEL2. a–f Migration and invasion activity of indicated cells were measured at 24 h in transwell assays. The number of cells passing through the membrane in each well was analyzed in triplicate and repeated three times with similar results. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. b, d, f Representative images of transwell assays. Red scale bar, 100 μm. e, f The migration and invasion abilities of S26 cells with NC+scr, NC+shSnail or siTEL2+shSnail. The number of cells passing through the membrane in each well was analyzed in triplicate and repeated three times with similar results. Data are mean ± SEM. *P < 0.05, ** P < 0.01. f Representative image of the transwell assays described in e. Red scale bar, 100 μm. g, h TEL2 overexpression attenuated hypoxia-induced migration and invasion in S26 cells. h Representative image of the transwell assays described in g. Red scale bar, 100 μm

Fig. 6

Snail promotes NPC metastasis to lung mostly by down-regulating TEL2 in a nude mouse model. a–c Stably transfected cells were injected into the lateral tail vein of nude mice. a Quantitation of the number of metastases. Data are mean ± SEM (n = 6 per group). b Macroscopic appearance of metastatic lung tumors. c Tumor cross sections with hemotoxylin-eosin staining. Scale bars in (c): 500 μm (left), 50 μm (right). d Proposed model for the regulation and function of TEL2 in NPC metastasis. Snail down-regulates TEL2 during exposure to normoxia or hypoxia, up-regulating SERPINE1, which promotes NPC metastasis