www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 28 Research Paper Eukaryotic translation initiation factor 3B accelerates the progression of esophageal squamous cell carcinoma by activating β-catenin signaling pathway Fengkai Xu1,*, Cheng-Zhi Xu2,*, Jie Gu1,*, Xiaoming Liu3, Ronghua Liu3, Enyu Huang3, Yunfeng Yuan1, Guangyin Zhao1, Jiahao Jiang1, Chen Xu4, Yiwei Chu3, Chunlai Lu1, Di Ge1 1 Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, Shanghai, P. R. China 2 Department of Otolaryngology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China 3 Department of Immunology and Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University, Shanghai, P. R. China 4 Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, P. R. China *These authors contributed equally to this work Correspondence to: Di Ge, email: [email protected] Chunlai Lu, email: [email protected] Yiwei Chu, email: [email protected] Keywords: esophageal squamous cell carcinoma, eukaryotic translation initiation factors 3B, β-catenin signaling pathway Received: October 22, 2015 Accepted: April 28, 2016 Published: May 30, 2016 ABSTRACT Introduction: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors. Eukaryotic translation initiation factors 3B (EIF3B) is considered to influence tumor proliferation, invasion, apoptosis and cell cycle, which act together to promote the progression of tumors. However, the role of EIF3B in ESCC is unknown. This study aims to explore the clinical and biological role of EIF3B in ESCC. Results: EIF3B expressions were up-regulated in both ESCC tissues and cell lines. Overexpression of EIF3B was associated with tumor depth, lymph node metastasis and advanced TNM stage. Importantly, patients with high EIF3B expression suffered shorter overall and disease-free survival. Knockdown of EIF3B could inhibit cell proliferation and invasion, promote cell apoptosis, and interfere the cell cycle in vitro. EIF3B-knockdown cells could form smaller subcutaneous tumors in vivo. Finally, we demonstrated EIF3B could activate β-catenin signaling pathway. Methods: Immunohistochemical staining and Western blot were performed to detect the EIF3B expression in ESCC patient tissues and cell lines. The association between EIF3B expression and patients’ prognosis was analyzed by Kaplan-Meier and Cox regression. Then, CCK-8, colony-formation, Transwell and wound-healing assay were performed to compare the bio-functional change after knockdown of EIF3B. Flow cytometry was applied to analyze the change of cell apoptosis and cycle induced by EIF3B knockdown. Tumor xenograft assay was done to verify the in-vitro results. Conclusions: EIF3B might serve as a novel marker for predicting prognosis of ESCC patients and as a potential therapeutic target, individually or together with other subunits of EIF3 complex. www.impactjournals.com/oncotarget 43401 Oncotarget INTRODUCTION of EIF3B in 8 pairs of fresh primary ESCC tumors and corresponding para-cancerous tissues. We observed that Esophageal squamous cell carcinoma (ESCC) is the EIF3B expression in ESCC tissues was significantly one of the most aggressive malignant tumors throughout higher than that in para-cancerous ones (Figure 1B). the world. In China, the incidence of ESCC greatly Moreover, we investigated the EIF3B protein level in one outnumbers that of esophageal adenocarcinoma [1]. In human normal esophageal epithelial cell line (HEEC) and spite of the recent improvements achieved in diagnostic three ESCC cell lines (EC109, TE1 and KYSE510). As and therapeutic methods, no significant improvements shown in Figure 1C, the EIF3B expression in EC109 and have been achieved in the 5-year survival rate due to KYSE510 was higher than that in HEEC (Figure 1C and high rate of early metastasis [2, 3]. Thus, it is quite urgent Supplementary Figure 1). Therefore, we picked up EC109 to discover the mechanism deeply under the initiation and KYSE510 for further bio-functional studies. and progression of ESCC so as to gain new therapeutic To investigate the correlations between EIF3B strategies for ESCC. expression and clinicopathological features, we employed The progression of ESCC could be influenced by statistical analysis and found that high expression of numerous factors, among which protein synthesis plays EIF3B was positively associated with tumor depth, lymph an important role. Meanwhile, the control of translation node metastasis and TNM stage, significantly (P < 0.05) is one of the important regulation points, which is the (Table 1). EIF3B expression was also elevated with the up- early step in the protein synthesis. Eukaryotic translation grade of tumor histological differentiation level, although initiation factors (EIF) are the indispensable components the difference did not reach the criterion of significance for the formation of an initiation complex that participate (Figure 1D), which indicated that high expression of EIF3B in assembling the 80S-mRNA-Met-tRNA complex to was positively correlated with the poor differentiation initiate the protein synthesis [4]. Among the family of tumor. During the follow-up, recurrence or metastasis of EIF, EIF3 is the largest factor and composed of 13 occurred in 99 patients and the metastatic areas included subunits: A-M, contributing to the maintenance of 40S supraclavicular lymph node, mediastinal lymph node, liver, subunits and assembling of 43S pre initiation complex. In lung, skeleton and brain. Besides, 95 patients were died different solid malignant tumors, different EIF3 subunits of ESCC. In univariate analysis, patients with high EIF3B have been reported to show shift expression [5, 6]. expression suffered low DFS and OS compared with the Eukaryotic translation initiation factors 3B (EIF3B), ones with low EIF3B expression (P < 0.05). Graphic serving as a scaffolding subunit, has been verified to pattern of Kaplan-Meier curves suggested that prognosis be overexpressed in colon cancer, bladder and prostate was poor for patients with high EIF3B expression cancer , glioblastoma, etc. and also made difference in the (Figure 1E and 1F). Tumor depth, lymph node metastasis biological characteristics of cancer cells [7–9]. However, and TNM stage were significantly correlated with patients’ no related research has been done in ESCC. DFS and OS (p < 0.01) (Table 2). In multivariate analysis, In this study, we demonstrated that EIF3B “lymph node metastasis” was identified as an independent expression was up-regulated in ESCC cells and negatively factors in patients’ prognosis (Table 3). correlated with the prognosis of patients. Knock-down of EIF3B expression with small interfering RNA (siRNA) EIF3B promotes the cell proliferation and could significantly inhibit the progression of ESCC invasion of ESCC cells both in vitro and in vivo. EIF3B accelerated the progression of ESCC via the activation of β-catenin To explore the importance of EIF3B expression signaling pathway. for the progression of ESCC further, we constructed 3 pairs of siRNA to knock down the EIF3B expression. RESULTS The efficiency of the transfection was high according to the Cy3-modified expression under light microscope and EIF3B is overexpressed in ESCC and negatively fluorescence microscope (Supplementary Figure 2). The correlated with the prognosis effect of the knockdown was validated through Western blot and qRT-PCR analyses. As shown in the Figure 2A In the 154 ESCC tissue samples, we applied the IHC and 2B, the EIF3B-siRNA-3 showed the best effect of to detect the expression of EIF3B and found that EIF3B knockdown and thus picked up for further study. expression was nearly undetectable in normal epithelium We applied CCK-8 and colony-forming assay to of esophagus. However, EIF3B exhibited cytoplasmic study the proliferative change after knockdown of EIF3B staining in ESCC tissues (Figure 1A). According to the and found that, compared with the normal control (NC) evaluation criteria described in the method, the number groups, both two cell lines with knockdown of EIF3B of cases with low (score 0, 1) and high (score 2, 3) showed low proliferative ability, which indicated that expression of EIF3B was 75 and 79, respectively. Next, EIF3B promoted the proliferation of ESCC (Figure 2C we performed Western blot assay to detect the expression and 2D). In addition, in vivo, tumor xenograft assay was www.impactjournals.com/oncotarget 43402 Oncotarget performed to verify that EIF3B made a difference in the in vitro (Figure 2E). Furthermore, we applied IHC assay to proliferation ability. The results showed that NC groups could detect the EIF3B expression in each sample and found that form significantly larger tumors than the EIF3B knockdown EIF3B expressions were higher in NC group than those in groups did, which was consistent to the previous results knockdown group (Figure 2F and Supplementary Table 1). Figure 1: EIF3B expression is overexpressed in ESCC and negatively associated with patents’ prognosis. (A), IHC (× 200 for a–e and × 400 for e–h) analysis of EIF3B expression in ESCC tissues from 154 enrolled patients. Typical images of EIF3B expression level from score 0 to 3 were exhibited. (B and C), Western blot assay was performed to detect the EIF3b expression in ESCC tumors, corresponding para-cancerous tissues and cell lines. β-actin was used as an internal reference. (D), percentage of EIF3B expression level