Loss of RhoB Expression Promotes Migration and Invasion of Human Bronchial Cells Via Activation of AKT1 Emilie Bousquet, 1,2,3 Julien Mazie `res, 1,2,3,4 Maud Privat, 1,2,3 Virginie Rizzati, 1,2,3 Anne Casanova, 1,2,3 Adeline Ledoux, 1,2,3 Eliane Mery, 3 Bettina Couderc, 2,3 Gilles Favre, 1,2,3 and Anne Pradines 1,2,3 1 InstitutNational dela Sante et dela Recherche MedicaleU563-De´partement Innovation The´rapeutiqueet Oncologie Mole´culaire; 2 Universite´ de Toulouse,UPS, Centrede Physiopathologie Toulouse Purpan; 3 Institut Claudius Regaud,De´partement de Biologie; 4 CHU Toulouse, Service de Pneumologie, Ho ˆpital Larrey, Toulouse, France Abstract Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Previously, we reported an inverse correlation between RhoB expression and the progression of the lung cancer, occurring between preinvasive and invasive tumors. Herein, we mim- icked the loss of RhoB observed throughout lung oncogenesis with RNA interference in nontumoral bronchial cell lines and analyzed the consequences on both cell transformation and invasion. Down-regulation of RhoB did not modify the cell growth properties but did promote migration and invasive- ness. Furthermore, RhoB depletion was accompanied by modifications of actin and cell adhesion. The specific activation of the Akt1 isoform and Rac1 was found to be criticalforthisRhoB-mediatedregulationofmigration.Lastly, we showed that RhoB down-regulation consecutive to K- RasV12 cell transformation is critical for cell motility but not for cell proliferation. We propose that RhoB loss during lung cancer progression relates to the acquisition of invasiveness mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT and Rac1 pathways rather than to tumor initiation. [Cancer Res 2009;69(15):6092–9] Introduction Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Despite optimal therapy, disease progression is inevitable in the majority of patients diagnosed at an advanced stage. This highlights the need for a better understanding of the molecular mechanisms involved in lung cancer progression. The Rho family of small GTPases have been widely implicated in cell transformation, survival, invasion, migration, metastasis, and angiogenesis(1).Analysesinhumantumorshaveshownchangesin Rho expression, such as the up-regulation of RhoA in testicular germ cell tumors (2) and RhoC in melanoma (3). RhoB, in contrast with its close relatives RhoA and RhoC, has a negative effect on oncogenesis, thereby likely acting as a tumor suppressor (4). We (5–7) and others (8, 9) have previously shown that ectopic expression of RhoB suppresses cell tumorogenesis. Knockout of the rhoB gene in mice increased the frequency of chemically induced tumors (10). Moreover, we reported in two independent immunohistochemical studies that RhoB protein expression decreased dramatically through lung cancer progression (5). Loss of RhoB expression has also been reported in head and neck carcinomas (11), glioblastomas (12), numerous lung cancer cell lines (13), and lung tumor tissues (14). The purpose of this work was to decipher the precise role played by RhoB in the acquisition of the tumor phenotype in lung cancer. Our previous results suggest that RhoB might be involved in the processes of proliferation and transformation (5). On the other hand, we reported that the loss of RhoB expression occurred between preinvasive and invasive stages of the tumor (5), suggesting that RhoB might be more involved in the regulation of lung cancer invasiveness than in the processes of proliferation and transformation. To specifically address this hypothesis, instead of analyzing the effect of forced overexpression of RhoB, as performed until now, we chose to examine the physiopathologic situation. With RNA interference, we mimicked the loss of RhoB expression observed in tumoraltissuesinbothnontumoralbronchialcelllines(BEAS-2Band HBE-135) and K-RasV12–transformed BEAS-2B cells and analyzed the consequences on cell transformation, migration, and invasion. Materials and Methods Cell culture and transfection. Human bronchial epithelial cell lines BEAS-2B (ATCC CRL-9609) and HBE-135 cells (ATCC CRL-2741) were grown in DMEM supplemented with 10% FCS or in keratinocyte serum-free medium (Invitrogen) supplemented with epidermal growth factor, bovine pituitary extract, insulin, and hydrocortisone, respectively, at 37jC in a humidified incubator with 5% CO 2 . BEAS-2B cells transformed by K-RasV12 were obtained by transfection with pZip-KRasV12 plasmid encoding the constitutively activated mutant of K-Ras (a kind gift of Channing Der, North Carolina) using the Jet PEI method, as indicated by the supplier (PolyPlus Transfection), followed by selection with 1 mg/mL G418 (Invitrogen). Transient transfection of small interfering RNA (siRNA; Eurogentec) was performed using Oligofectamine (Invitrogen) following the manufacturer’s instructions. The sequences used were two siRNAs against the 3¶ untranslated region of RhoB siB1 (5¶GGCAUUCUCUAAAGCUAUG3¶) and siB2 (5¶GCUAAGAUGGUGUUAUUUA3¶) at 20 nmol/L, siAkt1-1 (5¶GAGCGG- GAGGAGUGGACA3¶), siAkt1-2 (5¶CCAAGGAGAUCAUGCAGC3¶), siAkt2-1 (5¶GGGCUAAAGUGACCAUGA3¶), siAkt2-2 (5¶CCACAAGCGUGGUG- AAUA3¶), siAkt3-1 (5¶GCAAAAUGCCAGUUAAUG3 ¶), and siAkt3-2 (5¶AGAGAAGGCAAGUGGAAA3¶) at 5 nmol/L (all AKT isoform sequences from ref. 15), siRac1 (5¶CACCACUGUCCCAACACUC3¶) at 10 nmol/L, and siNeg (5¶UGGUCACAAAGUCGCAUGA3¶, aleatory sequence from Eurogentec) at a concentration depending on the experiment. Adenoviral constructs and transduction protocol. Replication- defective (DE1, E3) adenoviral (Ad) vectors expressing RhoB under the transcriptional control of the cytomegalovirus promoter were constructed with the AdEasy System (MP Biomedical), as described previously (16). For rescueexperiments,140 Â 10 3 cellswereplatedona35-mmdish24hbefore Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Anne Pradines, Institut Claudius Regaud, 20-24 rue du Pont Saint Pierre, 31052 Toulouse Cedex, France. Phone: 33-5-61-42-42-26; Fax: 33-5-61-42- 46-31; E-mail: pradines.anne@claudiusregaud.fr. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-4147 Cancer Res 2009; 69: (15). August 1, 2009 6092 www.aacrjournals.org Cell, Tumor, and Stem Cell Biology Downloaded from http://aacrjournals.org/cancerres/article-pdf/69/15/6092/2866915/6092.pdf by guest on 21 June 2022