High SRC-1 and Twist1 expression predicts poor prognosis and promotes migration and invasion by inducing epithelial-mesenchymal transition in human nasopharyngeal carcinoma

Abstract

Steroid receptor coactivator 1 (Src-1) and Twist1 are aberrantly upregulated in a variety of tumors and play an important role in tumor progression. However, the exact role of Src-1 and Twist1 in nasopharyngeal carcinoma (NPC) is uncertain. In this study, we investigated the possible prognostic value and biological effect of Src-1 and Twist1 in NPC. Src-1 and Twist1 expression was detected in a cohort of NPC patients (n = 134) by qRT-PCR. Kaplan-Meier survival analysis was used comparing overall survival (OS) and progression-free survival (PFS). Multivariate analysis was performed using the Cox proportional hazard regression model. Biologic effect of Src-1 and Twist1 in NPC cell lines was evaluated by western blot, colony formation assay, soft agar assay, scratch wound healing assay, transwell invasion assay and tumor xenografts growth. We have found that Src-1 and Twist1 were aberrantly upregulated in human NPC tissues, and associated with advanced tumor stage, distant metastasis and unfavorable prognosis. Knockdown of Src-1 or Twist1 in human NPC cell line CNE-1 suppressed colony formation, anchorage-independent growth, cell migration, invasion and tumor xenografts growth, while enforced expression of Src-1 or Twist1 in human NPC cell line HNE-2 promotes anchorage-independent growth, cell migration and invasion. In addition, Src-1 and Twist1 could suppress E-cadherin expression and increase Vimentin expression, thus suggested that Src-1 and Twist1 enhanced the malignant behaviors of NPC cells via inducing epithelial-mesenchymal transition (EMT). Our data indicated that Src-1 and Twist1 could be possible prognostic biomarkers and potential therapy targets for patients with NPC.

Fig 1. Src-1 and Twist1 is aberrantly up-regulated in nasopharyngeal carcinoma tissues.

(A) the mRNA expression of Src-1 and Twist1 in tumor samples and adjacent normal tissues of the 134 NPC patients was evaluated by qRT-PCR, then relative mRNA expression Log2 (T/N) was calculated. (B) Src-1 or Twist1 expression in tumor samples (T) and paired normal tissues (N) of selected NPC patients was evaluated by western blot. (C, D) Representative images of IHC staining and statistical results for low and high expression of Src-1 or Twist1 in tumor tissues of NPC patients. Scale bars = 50μm. (E) Protein expression of Src-1 and Twist-1 in a panel of nasopharyngeal carcinoma cell lines.

Fig 2. Kaplan-Meier survival analysis of NPC patients according to Src-1 and Twist1 expression.

Overall survival according to Src-1 expression (A), Twist1 expression (B) and both Src-1/Twist1 expression (C). Progression-free survival according to Src-1 expression (D), Twist1 expression (E) and both Src-1/Twist1 expression (F).

Fig 3. Knockdown of Src-1 or Twist1 expression in CNE-1 upregulates E-cadherin expression and downregulates Vimentin expression.

(A, B) 2.5×10⁵ CNE-1 cells were seeded in 6-well-plates and transduced with sh-Src-1, sh-Twist1 or a negative control (sh-NC) for 4 days, then cell lysates were analyzed by western blot. (C, D) quantification of western blot bands in A and B. Protein expression was normalized to GAPDH. Lysates of parental CNE-1 cells were used as positive control. ***P<0.05 comparing with sh-NC group.

Fig 4. Knockdown of Src-1 or Twist1 expression in CNE-1 suppressed colony formation, anchorage-independent growth, cell migration and invasion.

(A) 500 CNE-1 cells transduced with sh-Src-1, sh-Twist1 or sh-NC control were seeded in 6-well-plates, allowed grow for 2 weeks and stained with crystal violet. (B) Colony numbers from A. (C) 5000 CNE-1 cells transduced with sh-Src-1, sh-Twist1 or sh-NC control were seeded in soft agar and cultured for 3 weeks, then stained with 1 mg/ml MTT. (D) colony numbers from C. (E) 1×10⁵ CNE-1 cells transduced with sh-Src-1, sh-Twist1 or sh-NC control were used for transwell cell invasion assay, then stained with Giemsa dye and imaged after incubating for 24hr. (F) percentage of invasion cells from E comparing with sh-NC. (G) CNE-1 cells transduced with sh-Src-1, sh-Twist1 or sh-NC control were used for scratch wound healing assay, photos of the plates were took at 0h and 48h. Scale bars = 50um.(H) Relative cell migration from G analyzed by Digimizer software system. (I) cell proliferation of sh-Src-1, sh-Twist1 or sh-NC transduced CNE-1 cells was evaluated by cell viability assay. ***P<0.05 comparing with sh-NC group.

Fig 5. Knockdown of Src-1 or Twist1 expression suppressed tumor xenografts growth in CNE-1.

2×10⁶ CNE-1 cells expressing either sh-Src-1, sh-Twist1 or sh-NC cotrol were subcutaneously injected in the armpits of nude mice (n = 5). Tumor size were measured every two days (A), and four weeks after implantation, tumor xenografts were dissected out (B) and weighed (C). ***P<0.05 comparing with sh-NC group.

Fig 6. Enforced Src-1 or Twist1 expression in HNE-2 promotes anchorage-independent growth, migration, invasion and lung metastasis.

(A) 2.5×10⁵ HNE-2 cells were seeded in 6-well-plates and transduced with Src-1 or Twist1 expression lentivirus for 4 days, then cell lysates were analyzed by western blot. Cells transduced with empty vector (EV) were used as control. (B) Quantification of western blot bands in A. Protein expression was normalized to GAPDH. (C) 5000 HNE-2 cells transduced with Src-1, Twist1 or EV control were seeded in soft agar and cultured for 3 weeks, than stained with 1 mg/ml MTT. (D) colony numbers from C. (E) 1×10⁵ CNE-1 cells transduced with Src-1, Twist1 or EV control were used for transwell cell invasion assay, then stained with Giemsa dye and imaged after incubating for 24hr. (F) percentage of invasion cells from E comparing with EV control. (G) HNE-2 cells transduced with Src-1, Twist1 or EV control were used for scratch wound healing assay, photos of the plates were took at 0h and 48h. Scale bars = 50um (H) Relative cell migration from G analyzed by Digimizer software system. (I-J) 3×10⁶ HNE-2 cells transduced with Src-1, Twist1 or EV control were injected into the tail vein of nude mice. Represent images of tumors metastasized to lung were shown (I). Number of tumors in lung was calculated (J). ***P<0.05 comparing with EV group.