MACC1 Down-Regulation Inhibits Proliferation and Tumourigenicity of Nasopharyngeal Carcinoma Cells through Akt/β-Catenin Signaling Pathway

et al. (2013) MACC1 Down-Regulation Inhibits Proliferation and Tumourigenicity of Nasopharyngeal Carcinoma Cells through Akt/b-Catenin Signaling Pathway. PLoS ONE 8(4): e60821. doi:10.1371/journal.pone.0060821 MACC1 Down-Regulation Inhibits Proliferation and Tumourigenicity of Nasopharyngeal Carcinoma Cells through Akt/b-Catenin Signaling Pathway Fengjiao Meng 0 Hui Li 0 Huijuan Shi 0 Qingxu Yang 0 Fenfen Zhang 0 Yang Yang 0 Lili Kang 0 Tiantian Zhen 0 Sujuan Dai 0 Yu Dong 0 Anjia Han 0 Salvatore V Pizzo, Duke University Medical Center, United States of America 0 1 Department of Pathology, the First Affiliated Hospital and Zhongshan School of Medicine, Sun Yat-Sen University , Guangzhou , China , 2 Department of Pathology, Huizhou Municipal Central Hospital , Huizhou , China The present study was aimed at investigating the expression of metastasis-associated in colon cancer 1 (MACC1) in nasopharyngeal carcinoma (NPC), its relationship with b-catenin, Met expression and the clinicopathological features of NPC, and its roles in carcinogenesis of NPC. Our results showed that MACC1 expression was higher in NPC cells and tissues than that in normal nasopharyngeal cells and chronic inflammation of the nasopharynx tissues, respectively. MACC1 expression was closely related to the clinical stage (p = 0.005) and the N classification (p,0.05) of NPC. Significant correlations between MACC1 expression and Met expression (p = 0.003), MACC1 expression and b-catenin abnormal expression (p = 0.033) were found in NPC tissues. MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, and colony formation, but induced apoptosis in NPC cells compared with the control group. Furthermore, MACC1 down-regulation inhibited phosphorylated-Akt (Ser473) and b-catenin expression in NPC cells, but phosphorylated-Erk1/2 expression was not altered. Further study showed that phosphotidylinsitol-3-kinase inhibitor downregulated b-catenin and Met expression in NPC cells. There was a significant relationship between MACC1 expression and phosphorylated-Akt expression (p = 0.03), b-catenin abnormal expression and phosphorylated-Akt expression (p = 0.012) in NPC tissue, respectively. In addition, Epstein Barr virus-encoded oncogene latent membrane protein 1 upregulated MACC1 expression in NPC cells. Our results firstly suggest that MACC1 plays an important role in carcinogenesis of NPC through Akt/b-catenin signaling pathway. Targeting MACC1 may be a novel therapeutic strategy for NPC. - Funding: This study was supported by National Natural Science Foundation of China (81272636; 81201582) and Guangdong Province Natural Science Foundation (8151008901000125; S2011010004853). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Nasopharyngeal carcinoma (NPC) is relatively rare in the Western world, but more common in Southern China and Southeast Asia, with an annual incidence rate of about 20 per 100,000 people in endemic areas[1]. The epidemiologic evidence implies that environment risk factors, Epstein- Barr virus (EBV) infection and genetic susceptibility play crucial roles in carcinogenesis of NPC[2]. EBV-encoded oncogene latent membrane protein 1 (LMP1) is an important oncogene, which is involved in the activation of signal transduction pathways, such as the nuclear factor-kB, signal transducer and activator of transcription, and activator protein 1, which mediates various biological functions including proliferation, apoptosis, differentiation, and metastasis[3]. The Wnt/b-catenin signaling pathway is tightly regulated and has important functions in development, tissue homeostasis, and regeneration. Oncogenic activation of the Wnt/b-catenin signaling pathway by mutation in adenomatous polyposis coli or bcatenin, which results in the cytoplasmic and nuclear b-catenin accumulation and in b-catenin/T-cell factor (TCF) 4regulated transcription of TCF target genes such as cyclin D1 and c-Myc, is mandatory for the initial neoplastic transformation of intestinal epithelium[4]. Our previous study has reported that LMP1 increases nuclear b-catenin accumulation and its transcriptional activity in NPC[5]. b-catenin knockdown dramatically inhibited cellular growth, migration and invasion, but induced apoptosis of NPC cells[6]. Metastasis-associated in colon cancer-1(MACC1), a newly identified key regulator of hepatocyte growth factor(HGF)-MET signaling, predicts colon cancer metastasis[7,8]. Recently, MACC1 expression has been found in lung cancer[9,10], hepatocellular carcinoma[11], ovarian carcinoma[12], and gastric carcinoma[13]. Overexpression of MACC1 associates with the progression of these carcinomas and prognosis of the patients with these carcinomas. To our knowledge, there is no report on MACC1 expression and its roles in carcinogenesis of NPC in the literature. Our current paper firstly reported MACC1 expression, its relationship with b-catenin, Met expression, and the clinicopathological features of NPC. Moreover, MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, and colony formation, but induced apoptosis in NPC cells, which might through down-regulating phosphorylated-Akt (p-Akt) and bcatenin expression. Materials and Methods Cell lines and cell culture All NPC cell lines kindly supplied by the Cancer Center of Sun Yat-sen University, China were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 100 units/ml of penicillin, and 0.1 mg/ml of streptomycin (Sigma, St. Louis, MO). CNE1 is an EBV-negative and well differentiated human NPC cell line, CNE2, HNE-1, and SUNE1 are EBV-negative and poorly differentiated human NPC cell line. C666-1 is EBV-positive poorly differentiated human NPC cell line. NP69 is normal nasopharyngeal cell line kindly supplied by the Cancer Center of Sun Yat-sen University, China. Small interfering RNA (siRNA) duplexes were synthesized and purified by Ribobio Inc. (Guangzhou, Guangdong, China). The siRNA sequences for MACC1 used were: sense 59- CAC CAU AGC UUG CAA AGU A dTdT-39, antisense 59UAC UUU GCA AGC UAU GGU G- dTdT-39. The siRNA sequences for Met used were: sense 59 CCA AUG ACC UGC UGA AAU U dTdT 39, antisense 59AAU UU C AGC AGG UCA UUG G- dTdT- 39. Transfection of siRNAs was done using Lipofectamine 2000 reagent (Invitrogen Co., Carlsbad, CA) according to the manufacturers instructions. Patient information and tissue specimens A total of 85 samples of paraffin-embedded, archived NPC tissues and 24 samples of chronic inflammation of the nasopharynx between 1998 to 2003 were collected from Department of Pathology, the first Affiliated Hospital, Sun Yat-sen University, China. For the research purposes of these clinical materials, prior patients consents and approval from the Institution (...truncated)