ORIGINAL ARTICLE Transforming growth factor-b decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells S Ehata 1,2 , E Johansson 1 , R Katayama 2 , S Koike 2 , A Watanabe 3 , Y Hoshino 1 , Y Katsuno 1 , A Komuro 1 , D Koinuma 1 , MR Kano 1 , M Yashiro 4 , K Hirakawa 4 , H Aburatani 3 , N Fujita 2 and K Miyazono 1 1 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan; 2 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research (JFCR), 3-10-6 Ariake, Koto-ku, Tokyo, Japan; 3 Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, Japan and 4 Department of Surgical Oncology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahi-machi, Abeno-ku, Osaka, Japan Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characteri- zed the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-b (TGF-b) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-b repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-b, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-b negatively contributes to maintain the CICs within the cancer. Oncogene (2011) 30, 1693–1705; doi:10.1038/onc.2010.546; published online 6 December 2010 Keywords: ABCG2; cancer-initiating cell; diffuse type gastric carcinoma; SP cell; TGF-b Introduction Gastric cancer remains a major public health issue as the fourth most common cancer and the second leading cause of cancer death worldwide (Hohenberger and Gretschel, 2003; Crew and Neugut, 2006). Of the two histological subtypes of gastric cancers, the incidence of proximal, diffuse-type gastric adenocarcinoma has been increasing, particularly in Western countries. Diffuse- type gastric carcinoma is an aggressive type of gastric cancer with poor prognosis, affecting relatively young individuals. The majority of diffuse-type gastric carci- nomas are in advanced stages, for which gastrectomy and chemotherapy are of limited efficacy. Thus, under- standing of the mechanism underlying progression of diffuse-type gastric carcinoma is essential for manage- ment of this tumor. The concept of ‘cancer stem cells’ or ‘cancer-initiating cells (CICs)’ is an attractive explanation for the functional heterogeneity that is commonly observed in many types of tumors. Tumors possess a hierarchical organization of cells, among which a subpopulation of stem-like cells is responsible for sustaining tumor growth (Stingl and Caldas, 2007; Visvader and Linde- man, 2008). As these cells are likely to share many of the properties of normal stem cells, which have a long lifespan, self-renewal ability, resistance to drugs, active DNA-repair activity and resistance to apoptosis, the presence of CICs is thought to be important for progression of tumors and resistance to conventional therapies (Dean et al., 2005). Recently, CICs have been identified in a range of hematopoietic malignancies and solid tumors (Bonnet and Dick, 1997; Lobo et al., 2007). In the maintenance of CICs, the interaction of CICs and ‘niche’ has critical roles, and factors secreted from the tumor microenvironment appear to regulate the main- tenance of CICs in certain niches (Iwasaki and Suda, 2009). However, the factors responsible for regulation of the CIC pool have not been fully investigated. Transforming growth factor-b (TGF-b) is the proto- typic member of a family of secreted proteins that includes three isoforms of TGF-b (TGF-b1, TGF-b2 and TGF-b3), activins and bone morphogenetic proteins (BMPs). TGF-b mediates biological activities in cells through binding to heteromeric complexes of receptors on the cell surface that are composed of TGF-b type I and type II (TbRII) receptors. TGF-b type I receptor activated by TbRII phosphorylates Smad2 and Smad3, which interact with Smad4 and translocate to the Received 10 August 2010; revised 15 October 2010; accepted 20 October 2010; published online 6 December 2010 Correspondence: Professor K Miyazono, Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: miyazono@m.u-tokyo.ac.jp Oncogene (2011) 30, 1693–1705 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc