Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma Hong Lok Lung 1,2 , Arthur Kwok Leung Cheung 1,2 , Yue Cheng 2,3 , Fung Mei Kwong 1,2 , Paulisally Hau Yi Lo 1,2 , Evan Wai Lok Law 1,2 , Daniel Chua 1 , Eugene R. Zabarovsky 4 , Nancy Wang 5 , Sai Wah Tsao 6 , Eric J. Stanbridge 7 and Maria Li Lung 1,2 1 Centre for Cancer Research and Department of Clinical Oncology, University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China 2 Department of Biology and Center for Cancer Research, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (SAR), People’s Republic of China 3 Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 4 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden 5 Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 6 Department of Anatomy, University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China 7 Department of Microbiology and Molecular Genetics, University of California, Irvine, CA THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G 0 /G 1 phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC. THY1 (CD90) is a cell surface glycoprotein of 25–37 kDa localized on the outer leaflet of cell membranes, enriched in lipid raft microdomains. Human THY1 maps to chromosome 11q22.3. It is expressed on various cell types, including human fibroblasts, neurons, blood stem cells and endothelial cells as well as murine T cells. 1 THY1 is involved in T cell activation and the other nonimmunologic functions including inhibition of neurite outgrowth, apoptotic signaling, leukocyte and melanoma cell adhesion and migration and fibroblast proliferation and migration. 2,3 Taken together, these func- tions suggest that THY1 is an important mediator of cell–cell and cell–matrix interactions. In T cells, it is reported to func- tion in cell adhesion with the bone marrow stroma. 1 Surface expression of THY1 promotes focal adhesion in fibroblasts. 2 In cancers, THY1 was initially found to be differentially expressed in tumorigenic and nontumorigenic hybrid clones derived from the transfer of chromosome 11 into the human ovarian cancer cell line SKOV-3. 4 The later functional studies also suggest that THY1 is associated with tumor suppression in human ovarian cancer. 5 A recent study shows that loss of THY1 expression is correlated with poor survival rate in neu- roblastoma patients. 6 THY1 is also involved in the adhesion of melanoma cells to the human dermal microvascular endothe- lial cells. 7 THY1 is a cell surface marker, which has been used to identify local and circulating liver cancer stem cells. 8,9 Nasopharyngeal carcinoma (NPC) is a malignancy that is common in Southern China among the ethnic Chinese. Epstein-Barr virus, genetic predisposition, dietary and envi- ronmental factors are all believed to be involved in NPC Key words: THY1, nasopharyngeal carcinoma, tumor suppressor gene, antiinvasive, tetracycline-regulated gene expression Grant sponsor: Research Grants Council of the Hong Kong Special Administrative Region, People’s Republic of China; Grant number: CA03/04.SC01; Grant sponsors: the Swedish Cancer Society, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Swedish Institute, the Royal Swedish Academy of Sciences, INTAS, Karolinska Institute DOI: 10.1002/ijc.25047 History: Received 8 Jul 2009; Accepted 29 Oct 2009; Online 17 Nov 2009 Correspondence to: Maria Li Lung, Department of Clinical Oncology, University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China, Tel: 852-28199783; Fax: þ852-28195872, E-mail: mlilung@hku.hk Cancer Cell Biology Int. J. Cancer: 127, 304–312 (2010) V C 2009 UICC International Journal of Cancer IJC