Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/β-catenin signaling

He et al. BMC Cancer Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/-catenin signaling Weiling He Shanyang He Zuo Wang 0 Hongwei Shen Wenfeng Fang Yang Zhang Wei Qian Millicent Lin Jinglun Yuan Jinyang Wang Wenhua Huang Liantang Wang 0 Zunfu Ke 0 0 Department of Pathology, the First Affiliated Hospital, Sun Yat-Sen University , 58 Zhongshan Road II, Guangzhou, Guangdong 510080 , Peoples' Republic of China Background: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors. Methods: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry. Results: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/-catenin signaling cascade, including GSK-3 and CKI. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients. Conclusions: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC. AEG-1; Epithelial-mesenchymal transition; Non-small cell lung cancer; Wnt; -catenin - Background Lung cancer is the most common malignant tumor in the world, and the leading cause of cancer-related death in human beings [1]. Despite the achievements made in diagnosis and treatment in the recent years, the prognosis of lung cancer patients is still poor and their overall 5-year survival rate is 15% [2]. Although the clinical stage at diagnosis is the key prognostic determinant for lung cancer survival [3], considerable variability in reoccurrence and survival is commonly observed in patients with a similar stage. Thus, the initial diagnosis is extremely important because it could reduce the mortality rate for lung cancer patients [4]. The progress of cancer metastasis depends on the unique mechanisms of cancer cells evading from the primary tissue and spreading into surrounding tissues. Molecular reprogramming, as a part of the epithelialmesenchymal transition (EMT), is considered to be a crucial step in the metastasis process of most carcinomas [5]. During metastatic progression, EMT drives primary epitheliallike tumour cells to acquire invasive potential, such as increased motility and mesenchymal characteristics, triggering dissemination from the tumor and infiltration into the tumor vessel. Then, the EMT-driven cells circulating in the blood flow redifferentiate into primary status via MET during colonization and growth at distant metastatic sites [6,7]. Because of EMTs role in the metastatic process, controlling EMT progress and progression in tumors is now thought to be a promising strategy to inhibit metastasis and to prolong cancer patients survival. Astrocyte-elevated gene-1 (AEG-1), also known as LYRIC (lysine-rich CEACAM1) or metadherin, is originally induced in primary human fetal astrocytes [8]. Recently, numerous reports demonstrated that AEG-1 might play a pivotal role in the pathogenesis, progression, invasion, metastasis and overall patient survival in diverse human cancers [9-12]. This evidence indicates that the upregulation of AEG-1 contributes to malignant progression [13]. Furthermore, AEG-1 overexpression can facilitate migration and invasion of human glioma cells [14], as well as activate Wnt/-catenin signaling via ERK42/44 activation [11]. Although AEG-1 is an oncogene that has been implicated in pathways critical to lung cancer carcinogenesis [15], AEG-1 was also found to control the expression of E-cadherin and Vimentin [16]. The above findings suggest that AEG-1 may mediate the metastasis of lung carcinoma through the regulation of EMT. In this study, we concentrated on elucidating the role of AEG-1 in EMT of NSCLC. We demonstrated that upregulation of AEG-1 was significantly associated with lymph node metastasis and EMT status of NSCLC. We further investigated that AEG-1 could activate Wnt/-catenin signaling by inducing GSK-3 (glycogensynthasekinase 3) phosphorylation via CKI (casein kinase I), consequently enhancing EMT status. Methods Cell culture and tissue specimen selection Lung cancer cell lines, including NCI-H226, NCI-H460, L-78, A549 and Slu-01, were maintained in Dulbeccos modified Eagles medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). AEG-1 overexpression plasmid pcDNA3.1-AEG-1, -catenin overexpression plasmid pcDNA3.1--catenin, AEG-1 siRNA and CKI siRNA (RiboBio, China) were transiently transfected using Lipofectamine 2000 (Invitrogen, USA). A total of 210 cases from 2000 to 2005 coded as lung cancer were collected consecutively from the pathology archives of the Affiliated First Hospital, Sun Yat-sen University. The medical ethics committee of Sun Yat-sen University approved the present retrieval of cancer specimens and the connection with clinical data from our institute. Migration assay Invasive ability was measured by using 24-well BioCoat cell culture inserts (Costar, New York, NY, USA) with an 8-m-porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix (Cultrex, MD, USA). At the end of the assay, cells that did not migrate or invade through the pores were removed with a cotton swab. The invasion ability was determined by counting the cells that migrated to the lower side of the filter. Western blot and immunofluorescence Western blot was carried out as described earlier [17]. Blotted membranes were incubated with the antibodies for AEG-1(Invitrogen, USA), Twist 1, E-cadherin, Vimennt, -catenin, p-GSK-3(Ser-9), GSK-3, CKI and GAPDH (Abcam, Cambridge, UK) in 5% milk/TBST (tris-buffered saline Tween-20). For immunofluorescence microscopy, cells grown on chamber slides were probed with AEG-1 E-cadherin, Vimennt and -catenin. The fluorescein isothiocyanate (...truncated)