ORIGINAL ARTICLE TGF-b-induced apoptosis of B-cell lymphoma Ramos cells through reduction of MS4A1/CD20 KC Kawabata 1 , S Ehata 1 , A Komuro 1 , K Takeuchi 2 and K Miyazono 1 Transforming growth factor-b (TGF-b) exhibits growth inhibitory effects on various types of tumor cells, including B-cell lymphoma cells. In the present study, the role of TGF-b in the survival of Epstein–Barr virus-negative B-cell lymphoma Ramos cells was investigated. As TGF-b-induced apoptosis of Ramos cells in vitro and in vivo, we attempted to identify novel target gene(s) responsible for their survival. Oligonucleotide microarray analysis and chromatin immunoprecipitation revealed that Smad proteins directly regulated the transcription of membrane-spanning 4-domains, subfamily A, member 1 (MS4A1), also known as CD20, in Ramos cells upon TGF-b stimulation. In addition, immunohistochemical analysis using clinical samples from B-cell lymphoma patients showed an inverse correlation between the expression of MS4A1/CD20 and phosphorylation of Smad3. Although knockdown of MS4A1/CD20 in Ramos cells resulted in an increase of apoptotic cells, Ramos cells stably expressing MS4A1/CD20 were resistant to TGF-b-induced apoptosis. This suggests that MS4A1/CD20 is responsible for TGF-b-induced apoptosis of B-cell lymphoma cells. Moreover, downregulation of MS4A1/CD20 by TGF-b attenuated the effects of the monoclonal anti-MS4A1/CD20 antibody, rituximab, on Ramos cells. Our findings suggest that the sensitivity of B-cell lymphoma cells to rituximab may be affected by TGF-b signaling. Oncogene (2013) 32, 2096–2106; doi:10.1038/onc.2012.219; published online 4 June 2012 Keywords: B-cell lymphoma; apoptosis; TGF-b; MS4A1/CD20; rituximab INTRODUCTION Malignant lymphoma is a group of hematological malignancies, which includes Hodgkin lymphoma and non-Hodgkin lymphoma. Non-Hodgkin lymphoma is generally classified according to origin, that is, B-cell non-Hodgkin lymphoma, which accounts for B80% of all of malignant lymphomas, and T/NK-cell non-Hodgkin lymphoma. 1 Among these subclasses, Burkitt lymphoma, which originates from germinal center B cells, represents highly aggressive non-Hodgkin B-cell lymphoma. Owing to its rapid progression, involvement of the central nervous system, and frequent recurrence after conventional chemotherapy, Burkitt lymphoma is an incurable hematological malignancy. 2 Burkitt lymphoma is divided into the Epstein–Barr virus (EBV)-positive (endemic) subtype and the EBV-negative (sporadic) subtype. 3,4 Most Burkitt lymphoma cells commonly exhibit translocations between the myelocytomatosis (MYC) oncogene and the immunoglobulin heavy (IGH) chain genes. 5,6 Transforming growth factor-b (TGF-b) is a prototypic 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 a wide variety of biological and cellular activities through binding to heteromeric complexes of receptors on the cell surface that are composed of type I and type II receptors. Activin receptor-like kinase (ALK) ALK1 and ALK5 serve as type I receptors for TGF-b, whereas the TGF-b type II receptor (TbRII) acts as the type II receptor for TGF-b. Similarly, ALK1, ALK2, ALK3 and ALK6 function as type I receptors for BMPs, whereas activin type IIA receptor (ActRIIA), ActRIIB and BMP type II receptor serve as type II receptors for BMPs. 7 Upon ligand binding, two type I receptors and two type II receptors form a heteromeric complex, which in turn transduces intracellular signals by phosphorylation of receptor-regulated Smads (R-Smads). Smad2 and Smad3 are TGF-b-specific R-Smads, whereas Smad1, Smad5 and Smad8 are BMP-specific R-Smads. Phosphorylated R-Smads form heteromeric Smad complexes with common-partner Smad, Smad4. Next, the Smad complexes translocate into the nucleus, bind to trans- cription factors, transcriptional co-activators, and/or corepressors and regulate the transcription of target genes. 7–9 In addition to the Smad pathway, non-Smad pathways, including various mitogen-activated protein kinase pathways, are activated by TGF-b. In many epithelial cells, TGF-b inhibits cell cycle progression in the G1 phase through the regulation of p15 INK4B , p21 CIP1/WAF1 and c-Myc. Alterations in TGF-b signaling have been identified in epithelial malignancies. 10,11 TGF-b also has tumor suppressive activity in some types of hematological malignancies. 12 Loss of Smad3 was observed in T-cell acute lymphoblastic leukemia cells. 13 In acute promyelocytic leukemia blasts, a fusion protein, promyelocytic leukemia-retinoic acid receptor a (PML-RARa), ablates the TGF-b signaling pathway. 14 In malignancies in B cells, several reports have stated that TGF-b inhibits the proliferation of tumor cells in vitro. 15,16 However, the role of TGF-b signaling during the development of B-cell malignancy is not fully understood. In the present study, we investigated the role of TGF-b family signaling in the progression of B-cell lymphoma. Human EBV- negative Burkitt lymphoma cells, Ramos, responded to TGF-b and BMP. However, only TGF-b showed suppressive effects on the 1 Department of Molecular Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan and 2 Pathology Project for Molecular Targets, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan. Correspondence: Professor K Miyazono, Department of Molecular Pathology, Graduate School of Medicine, the University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan. E-mail: miyazono@m.u-tokyo.ac.jp Received 9 January 2012; revised 19 April 2012; accepted 1 May 2012; published online 4 June 2012 Oncogene (2013) 32, 2096–2106 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc