BMP2 promotes proliferation and invasion of nasopharyngeal carcinoma cells via mTORC1 pathway

Abstract

Bone morphogenetic protein-2 (BMP2) is a secreted protein that highly expressed in a variety of cancers and contributes to cell proliferation, migration, invasiveness, mobility, metastasis and EMT. However, its clinical significance and biological function in nasopharyngeal carcinoma (NPC) remain unknown up to now. Up-regulation of BMP2 was first observed in NPC cell lines by a genome-wide transcriptome analysis in our previous study. In this study, BMP2 mRNA was detected by qRT-PCR and data showed that it was upregulated in NPC compared with non-cancerous nasopharynx samples. Immunohistochemistry (IHC) analysis in NPC specimens revealed that high BMP2 expression was significantly associated with clinical stage, distant metastasis and shorter survival of NPC patients. Moreover, overexpression of BMP2 in NPC cells promoted cell proliferation, migration, invasiveness and epithelial-mesenchymal transition (EMT). Mechanistically, BMP2 overexpression increase phosphorylated protein level of mTOR, S6K and 4EBP1. Correspondingly, mTORC1 inhibitor rapamycin blocked the effect of BMP2 on NPC cell proliferation and invasion. In conclusion, our results suggest that BMP2 overexpression in NPC enhances proliferation, invasion and EMT of tumor cells through the mTORC1 signaling pathway.

Keywords: BMP2; mTORC1; metastasis; nasopharyngeal carcinoma.

Figure 1. The expression of BMP2 is up-regulated in NPC and correlates with poor prognosis

(A) qRT-PCR analysis showed that the expression of BMP2 mRNA was significantly higher in NPC tumor tissues (n=22) than non-cancerous nasopharyngitis tissues (n=14) (** p<0.01, independent Student's t-test). (B) The expression level of BMP2 in NPC cell lines (HONE1, SUNE1, CNE2, C666-1, S18, S26, 6-10B and 5-8F) and the normal epithelial NP69 was analyzed by Western blot. NP69 showed a relative low level of endogenous BMP2 protein expression and the protein expression level of BMP2 was obviously higher in S-18 and 5-8F cells with high tumorigenic and metastatic potential compared with their paired subclones S-26 and 6-10B cells with low tumorigenic and metastatic potential. (C) BMP2 immunostaining of representative images of NPC patients with different IHC scores. (D, E, F). Patients with high expression of BMP2 had shorter overall survival (OS) (D), progression-free survival (PFS) (E), and distant-metastasis-free survival (DMFS) (F) than those with low levels of BMP2.

Figure 2. BMP2 enhances NPC cells growth

(A) The over-expression efficiency of BMP2 in S18 and 6-10B cells were confirmed by Western blot, respectively. And CCK-8 assays showed that BMP2 could significantly promote proliferation of S18-BMP2 and 6-10-BMP2 cells compared with the controls. (B) BMP2 knockdown in 5-8F cell via three specific siRNA was confirmed by Western blot and CCK-8 assays showed that BMP2 knockdown reduced the proliferation of 5-8F cell. (C) In colony formation assays, the number of colonies was significantly higher in the S18-BMP2 and 6-10B-BMP2 cells than the controls.

Figure 3. BMP2 promotes NPC cell migration, invasion and epithelial-mesenchymal transition

(A) Wound-healing assays demonstrated that BMP2 upregulated cells had higher migration rate than control cells. (B) Matrigel invasion chambers were used to evaluate the ability of NPC cell invasion. Upregulation of BMP2 significantly increased the number of invaded cell in S18 and 6-10B compared with the controls. (C) silencing expression of BMP2 in 5-8F much reduced the invaded cell numbers. (D) Western blotting revealed that overexpression of BMP2 in S18 and 6-10B cells decreased the expression of epithelial makers (E-cadherin) and increased the expression of mesenchymal markers (N-cadherin, Vimentin and β-catenin) in comparison with the controls. (E) Western blotting showed that knockdown of BMP2 increased the expression of E-cadherin and decreased the expression of N-cadherin, Vimentin and β-catenin.

Figure 4. BMP2 activates mTOR signal pathway in NPC cells

(A) Western blotting analysis revealed that in S18-BMP2 and 6-10B-BMP2 cells, the expression of phosphorylated (active) mTOR, P70S6K and 4EBP1 (p-mTOR, p-p70S6k and p-4EBP1) were upregulated compared to those control cells. (B) The expression of p-mTOR, p-p70S6k and p-4EBP1 were downregulated in 5-8F knockdown BMP2 cell. (C) Proliferation abilities of S-18 and 6-10B cells expressing BMP2 or the empty vector was evaluated by CCK8 assay after pretreatment with or without 20 nM of rapamycin. (D) Invasive abilities of S-18 and 6-10B cells expressing BMP2 or the empty vector was evaluated by transwell assay after pretreatment with or without 20 nM of rapamycin.