Loss of RhoB Expression in Human Lung Cancer Progression Julien Mazieres, 1 Teresita Antonia, 2 Ghislaine Daste, 3 Carlos Muro-Cacho, 2 Delphine Berchery, 1 Vanessa Tillement, 1 Anne Pradines, 1 Said Sebti, 2 and Gilles Favre 1 1 Institut National de la Sante ´ et de la Recherche Me ´dicale U563, Department of Therapeutic Innovation and Molecular Oncology. Claudius Regaud Institute, Toulouse, France; 2 Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Departments of Interdisciplinary Oncology and Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida; and 3 G. Daste, Department of Pathology, Purpan Hospital, Toulouse, France ABSTRACT Purpose: RhoB is a low molecular weight GTPase be- longing to the Ras protein superfamily. Whereas most Rho proteins have been shown to have a positive role in prolif- eration and malignant transformation, the specific role of RhoB appears more divergent. We reported previously that RhoB inhibits cell proliferation in various human cancer cells. Here, we studied the specific role played by RhoB in human lung cancer. Experimental Design: We analyzed the expression of RhoB protein by immunostaining in human lung tissues ranging from normal to invasive carcinoma from different histological types in two large independent studies of, re- spectively, 94 and 45 samples. We then studied the cellular effect of RhoB overexpression in a model of lung cancer (A549, adenocarcinoma) and tumorigenicity in nude mice. Results: We showed in both studies that RhoB protein was expressed in normal lung and decreased dramatically through lung cancer progression (P < 0.01). Interestingly, RhoB expression was lost in 96% of invasive tumors and reduced by 86% in poorly differentiated tumors compared with the nonneoplastic epithelium. Moreover, the loss of expression of RhoB correlated significantly with tumor stage and proliferative index, whereas no correlation was found between RhoB and p53 or Bcl-2 expression. We then showed that ectopic expression of RhoB in lung cancer cell line A549 suppressed cell proliferation, anchorage-independent growth, and xenograft tumor growth in nude mice. Conclusions: RhoB loss of expression occurs very fre- quently in lung carcinogenesis, reinforcing its putative tu- mor suppressive activity, and raising the value of its poten- tial use in cancer therapy. INTRODUCTION Lung cancer is the leading cause of cancer-related deaths in the world (1). Despite advances in surgery, chemotherapy, and radiation therapy, survival rates have changed little in the last decade, and long-term survival remains dramatically poor. It has been established that lung cancer arises as a consequence of the accumulation of multiple somatic genetic changes involving critical genes of which the protein products control cell motility, proliferation, differentiation, and apoptosis (2). Identification and characterization of these genetic changes that drive lung cancer development and progression is of high interest for helping clinicians in early diagnosis and for developing novel targeted therapy (3). Ras proteins regulate important cellular functions ranging from cell differentiation to cell proliferation. They function as molecular switches, cycling between the inactive GDP- and the active GTP-bound states (4). The oncogene ras plays a pivotal role in malignant transformation. Ras is found mutated to a GTPase-deficient form that lead to constitutive activation of signaling pathways and uncontrolled proliferation in 50% of lung adenocarcinoma (5). Closely related family members of Ras, such as Ras-homologous (Rho) small guanosine triphos- phatases (GTPases) are also involved in the regulation of a variety of cellular processes such as organization of the actin cytoskeleton (6), genotoxic stress-induced signaling (7), and malignant transformation (8). Recent studies additionally con- firmed the role of Rho proteins in cancer by showing their involvement in cell transformation, survival, invasion, metasta- sis, and angiogenesis (9 –11). Moreover, analyses in human tumors demonstrate overexpression of several Rho proteins in breast cancers (12), of RhoA in testicular germ cell tumors (13), and of RhoC in pancreatic adenocarcinoma (14), in melanoma (15), and in breast cancer (16). Whereas most Rho proteins have been shown to have a positive role in proliferation and malignant transformation, the specific role of RhoB in these processes appears more divergent. Among the Rho family members, RhoB displays some particu- lar characteristics such as its localization to early endosomes (17), its role in intracellular transport of cell-surface receptors (18), and its rapid up-regulation by growth factors and genotoxic stress (19) that suggest that RhoB might have a special function in transformed cells. Recent studies indicated that RhoB might exert a tumor-suppressive role in growth control and transfor- mation. We showed that ectopic expression of RhoB in human tumor cells leads to an inhibition of tumor growth in nude mice (20). Furthermore, inactivation of RhoB in knock-out mice Received 9/3/03; revised 12/5/03; accepted 1/2/04. Grant support: French “Ministe `re de l’Enseignement Supe ´rieur et de la Recherche,” grants from the “Groupe de Recherche de l’Institut Clau- dius Regaud,” the “Ligue Nationale de Lutte contre le Cancer” Equipe Labe ´lise ´e La Ligue, and the United States National Cancer Institute Frant (NCDDG CA67771). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Gilles Favre, Institut National de la Sante ´ et de la Recherche Me ´dicale U563, Department of Therapeutic Innovation and Molecular Oncology, Claudius Regaud Institute, 20-24, rue du pont Saint-Pierre, 31052 Toulouse, France. Phone: 33-5-61-42-42-23; Fax: 33-5-61-42-46-31; E-mail: favre@icr.fnclcc.fr. 2742 Vol. 10, 2742–2750, April 15, 2004 Clinical Cancer Research Research. on April 19, 2017. © 2004 American Association for Cancer clincancerres.aacrjournals.org Downloaded from