1 FAM198B is Associated with Prolonged Survival and Inhibits Metastasis in Lung 2 Adenocarcinoma via Blockage of ERK-mediated MMP-1 Expression 3 4 Chia-Ying Hsu1,2, Gee-Chen Chang3,4, Yi-Ju Chen5, Yi-Chiung Hsu6,7, Yi-Jing Hsiao1,2, 5 Kang-Yi Su1,2,8, Hsuan-Yu Chen2,7,9, Chien-Yu Lin2,7, Jin-Shing Chen10, Yu-Ju Chen5, 6 Qi-Sheng Hong1,2, Wen-Hui Ku11, Chih-Ying Wu12, Bing-Ching Ho1,2, Ching-Cheng 7 Chiang1, Pan-Chyr Yang2,13,14, Sung-Liang Yu1,2,8,15,16,17 8 9 1Department of Clinical and Laboratory Sciences and Medical Biotechnology, 10 National Taiwan University College of Medicine, Taipei, Taiwan, 2Center of Genomic 11 Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, 3Faculty 12 of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, 13 4Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans 14 General Hospital, Taichung, Taiwan, 5Institute of Chemistry, Academia Sinica, Taipei, 15 Taiwan, 6Department of Biomedical Sciences and Engineering, National Central 16 University, Taoyuan, Taiwan, 7Institute of Statistical Science, Academia Sinica, Taipei, 17 Taiwan, 8Department of Laboratory Medicine, National Taiwan University Hospital, 18 Taipei, Taiwan, 9Graduate Institute of Medicine, Kaohsiung Medical University, 19 Kaohsiung, Taiwan, 10Division of Thoracic Surgery and Department of Surgery, 20 National Taiwan University Hospital and National Taiwan University College of 21 Medicine, Taipei, Taiwan, 11Taipei Institute of Pathology, Taipei, Taiwan, 22 12Department of Pathology & Laboratory Medicine, Taichung Veterans General 23 Hospital, Taichung, Taiwan, 13Department of Internal Medicine, National Taiwan 24 University Hospital, Taipei, Taiwan, 14Institute of Biomedical Sciences, Academia 25 Sinica, Taipei, Taiwan, 15Department of Pathology and Graduate Institute of 26 Pathology, National Taiwan University College of Medicine, Taipei, Taiwan, 27 16Graduate Institute of Clinical Medicine, National Taiwan University College of 28 Medicine, Taipei, Taiwan, 17Institute of Medical Device and Imaging, College of 29 Medicine, National Taiwan University, Taipei, Taiwan 30 31 Running title: FAM198B is a novel tumor suppressor in lung cancer 32 33 Keywords: FAM198B, tumor suppressor, cell invasion, N-glycosylation, prognosis 34 35 Financial support 36 This study was supported by grants from the Ministry of Science and Technology, 37 Taiwan, (NSC 98-2314-B-002-120-MY3, NSC 102-2911-I-002-303, MOST 38 103-2911-I-002-303, MOST 104-2911-I-002-302, 104R8400). Mathematics in 1 1 Biology Group of the Institute of Statistical Science Academia Sinica and Taiwan 2 Biosignature Project of Lung Cancer supported data analysis work. 3 4 Corresponding author 5 Sung-Liang Yu, Department of Clinical Laboratory Sciences and Medical 6 Biotechnology, National Taiwan University College of Medicine; No. 1 Chang-Te 7 Street, Taipei 10048, Taiwan. 8 Telephone: +886-2-2312-3456 ext. 88697 9 Fax: +886-2-2395-8341 10 E-mail: [email protected] 11 12 Conflicts of interest 13 The authors declare no potential conflicts of interest. 14 15 TRANSLATIONAL RELEVANCE 16 Lung adenocarcinoma is the most common malignant tumor worldwide, and the 17 outcome of patients is still unsatisfactory with low survival rates. To reduce cancer 18 mortality caused by recurrence and metastasis, an in-depth understanding of the 19 mechanisms involved in cancer progression is urgently needed. Herein, we 20 demonstrate that FAM198B is a novel tumor suppressor that inhibits cancer 21 metastasis via attenuating pERK/MMP-1 signaling axis, and high FAM198B 22 expression is positively associated with overall survival in lung adenocarcinoma 23 patients in public database and in a 95-Taiwanese cohort. Interestingly, we find that 24 FAM198B is an N-glycoprotein, and the glycosylation can increase the protein 25 stability of FAM198B and is necessary for the metastasis-suppression activity. 26 Collectively, FAM198B represents a novel prognostic marker for predicting survival 27 of lung adenocarcinoma. 28 29 30 31 32 33 34 35 36 37 38 2 1 ABSTRACT 2 Purpose: The comprehensive understanding of mechanisms involved in the tumor 3 metastasis is urgently needed for discovering novel metastasis-related genes for 4 developing effective diagnoses and treatments for lung cancer. 5 Experimental design: FAM198B was identified from an isogenic lung cancer 6 metastasis cell model by microarray analysis. To investigate the clinical relevance of 7 FAM198B, the FAMB198B expression of 95 Taiwan lung adenocarcinoma patients 8 was analyzed by quantitative real-time PCR and correlated to patients’ survivals. The 9 impact of FAM198B on cell invasion, metastasis and tumor growth was examined by 10 in vitro cellular assays and in vivo mouse models. Additionally, the 11 N-glycosylation-defective FAM198B mutants generated by site-directed mutagenesis 12 was used to study protein stability and subcellular localization of FAM198B. Finally, 13 the microarray and pathway analysis were used to elucidate the underlying 14 mechanisms of FAM198B-mediated tumor suppression. 15 Results: We found that the high expression of FAM198B was associated with 16 favorable survival in Taiwan lung adenocarcinoma patients and in a lung cancer 17 public database. Enforced expression of FAM198B inhibited cell invasion, migration, 18 mobility, proliferation and anchorage-independent growth and FAM198B silencing 19 exhibited opposite activities in vitro. FAM198B also attenuated tumor growth and 20 metastasis in vivo. We further identified MMP-1 as a critical downstream target of 21 FAM198B. The FAM198B-mediated MMP-1 downregulation was via inhibition of 22 the phosphorylation of extracellular signal-regulated kinase (ERK). Interestingly 23 Deglycosylation nearly eliminated the metastasis suppression activity of FAM198B 24 due to a decrease of protein stability. 25 Conclusions: Our results implicate FAM198B as a potential tumor suppressor and to 26 be a prognostic marker in lung adenocarcinoma. 27 28 INTRODUCTION 29 Lung cancer is the leading cause of cancer-related deaths worldwide and lung 30 adenocarcinoma is the predominant histological subtype of lung cancer in woman, 31 never-smokers, and younger adults (1-3). Cancer development is a multi-phase 32 process resulting from genomic instability, transcriptional alterations, cancer stemness, 33 abnormal metabolic pathways, epigenetic alteration, tumor-promoting inflammation 34 and evasion of the immune system, which drive the progression of cancer (4-8). 35 Proteolytic degradation of the extracellular matrix (ECM) and the basement 36 membranes surrounding the primary tumor by the matrix metalloproteinase (MMPs) 37 is a critical step for tumor angiogenesis, invasion and metastasis (9). The MMPs have 38 served as potential prognostic markers and therapeutic targets in cancer (10). 3 1 Previously, we established an isogenic lung cancer metastasis cell model and 2 discovered several metastasis-related gene (11-18). However, the comprehensive 3 understanding of mechanisms involved in the tumor metastasis remains to further 4 explore. Thus, the discovery and understanding of novel metastasis-related genes are 5 crucial for developing more effective diagnoses and treatments for lung cancer. 6 The human family with sequence similarity 198, member B (FAM198B) is a 7 novel gene with unknown function and predicted to be a membrane-bound 8 glycoprotein localized on Golgi apparatus (19, 20). FAM198B might play a role in the 9 regulation of mouse and Xenopus embryonic development and is a downstream target 10 of the FGF receptor signaling pathway (21, 22). Nearly one-half of all known 11 eukaryotic proteins are N-glycosylated, which is a ubiquitous posttranslational 12 modification (23) and the alteration of glycosylation has been reported to be 13 associated with tumor proliferation, invasion, metastasis, angiogenesis, receptor 14 activation and intracellular or cell-matrix interactions (24-26). 15 In this study, we identified a novel potential tumor suppressor, FAM198B, from 16 an isogenic lung cancer metastasis cell model by expression microarrays. The clinical 17 relevance of FAM198B was analyzed in both public databases and Taiwanese lung 18 adenocarcinoma patients. The tumor suppression activity of FAM198B was 19 characterized by in vitro and in vivo metastasis and tumorigenesis assays. Microarray 20 and pathway analyses were used to investigate the molecular signaling of FAM198B 21 and the effect of glycosylation on FAM198B stability was also investigated. 22 23 MATERIALS AND METHODS 24 Human lung tumor specimens and RNA extraction 25 A total of 95 frozen tissues were collected from lung adenocarcinoma who underwent 26 complete surgical resection at the Taichung Veterans General Hospital (Taichung, 27 Taiwan) between May, 2000 and June, 2009. After surgical resection, half tumor 28 specimen was immediately frozen within 30 min and half was formalin-fixed, 29 paraffin-embedded (FFPE). The average tumor content and necrosis were 53.09 ± 30 21.10 and 10.35 ± 13.19, respectively (mean ± SD, n=92 for tumor content, n=90 for 31 necrosis). Total RNA was extracted from the frozen tumor tissue specimens by using 32 the TRIzol Plus RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA). The 33 qRT-PCR of TATA-box binding protein (TBP) was used to assess the quality of RNAs. 34 Ct 40 was used as the cutoff value to define undetectable or detectable and specimens 35 with Ct less than 40 were enrolled in this study (27-29). This study was