SMAD6 Contributes to Patient Survival in Non–Small Cell Lung Cancer and Its Knockdown Reestablishes TGF-B Homeostasis in Lung Cancer Cells Hyo-Sung Jeon, 1 Tatiana Dracheva, 1 Sei-Hoon Yang, 1 Daoud Meerzaman, 2 Junya Fukuoka, 1 Abbas Shakoori, 1 Konstantin Shilo, 3 William D. Travis, 4 and Jin Jen 1 1 Laboratory of Human Carcinogenesis and 2 Laboratory of Population Genetics, Center for Cancer Research, National Cancer Institute, Bethesda,Maryland; 3 DepartmentofPulmonaryandMediastinalPathology,ArmedForcesInstituteofPathology,Washington,Districtof Columbia; and 4 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York Abstract The malignant transformation in several types of cancer, including lung cancer, results in a loss of growth inhibition by transforming growth factor-B (TGF-B). Here, we show that SMAD6 expression is associated with a reduced survival in lung cancer patients. Short hairpin RNA (shRNA)–mediated knockdown of SMAD6 in lung cancer cell lines resulted in reduced cell viability and increased apoptosis as well as inhibition of cell cycle progression. However, these results were not seen in Beas2B, a normal bronchial epithelial cell line. To better understand the mechanism underlying the association of SMAD6 with poor patient survival, we used a lentivirus construct carrying shRNA for SMAD6 to knock down expression of the targeted gene. Through gene expression analysis, we observed that knockdown of SMAD6 led to the activation of TGF-B signaling through up-regulation of plasminogen activator inhibitor-1 and phosphorylation of SMAD2/3. Furthermore, SMAD6 knockdown activated the c-Jun NH 2 -terminal kinase pathway and reduced phosphory- lation of Rb-1, resulting in increased G 0 -G 1 cell arrest and apoptosis in the lung cancer cell line H1299. These results jointly suggest that SMAD6 plays a critical role in supporting lung cancer cell growth and survival. Targeted inactivation of SMAD6 may provide a novel therapeutic strategy for lung cancers expressing this gene. [Cancer Res 2008;68(23):9686–92] Introduction Transforminggrowthfactor-h (TGF-h)belongstoasuperfamily of structurally related polypeptides that are involved in various biological processes, including cell growth, differentiation, angio- genesis, apoptosis, and extracellular matrix remodeling (1). Alter- ationsinTGF-h signalingarelinkedtoavarietyofhumandiseases, including cancer, inflammation, and tissue fibrosis (2, 3). The disruptionofTGF-h signalingoccursinseveralhumancancersand the pathway generally possesses a tumor suppressor function (4). However,ascarcinogenesisproceeds,tumorcellsacquireresistance toTGF-h–inducedgrowtharrest. TGF-h anditssuperfamilymember,bonemorphogenesisprotein (BMP), activate their respective intracellular signaling cascades by binding to the type II receptor followed by the recruitment of the type I receptor. The activated type I receptor phosphorylates the receptorSMADs(R-SMAD),suchasSMAD2andSMAD3,whichthen form a heteromeric complex with the Co-SMAD, SMAD4. The R-SMAD/SMAD4 complex translocates into the nucleus, where it regulates the transcription of target genes (1, 5, 6). Among the TGF-h/BMPtargetgenesaretwoinhibitorySMADproteins,SMAD6 and SMAD7. SMAD6 is generally thought to mediate BMP signals, whereas SMAD7 mediates TGF-h signaling. Both proteins regulate the TGF-h signaling pathway through a negative feedback mechanism (7–9). Recently, SMAD6 and SMAD7 have been shown to play a role in tumorigenesis. SMAD7 overexpression causes malignant conversion in a multistage cancer model (10) and enhanced tumorigenicity in pancreatic cancer (11). Otherwise, stableoverexpressionofSMAD7inhumanmelanomacellsimpairs bone metastasis by blocking the TGF-h signal pathway (12). Similarly,adenoviraldeliveryofSMAD7toJygMC(A)breastcancer cellssignificantlyimpairstheircapacitytometastasizetolungand liver, possibly by altering their adhesive and migratory properties; however,overexpressionof SMAD6 hadnoeffectonmetastasis(13). TheexpressionofSMAD6andSMAD7wasinverselycorrelatedwith thedepthofinvasionintheearlystagesofcarcinogenesis,butthere wasasignificantcorrelationbetweentheexpressionofSMAD6and SMAD7topoorsurvivalesophagealsquamouscellcarcinoma(14). In this study, we observed that SMAD6 expression was associated with poor survival in non–small cell lung cancer (NSCLC)patients.KnockdownofSMAD6restoredTGF-h signaling pathwaybyincreasingSMAD2/3phosphorylationandplasminogen activatorinhibitor-1(PAI-1)activationinlungcancercelllinesbut not minimally transformed normal bronchial epithelial cells, Beas2B. We propose that SMAD6 contributes to lung cancer progression by limiting TGF-h signaling-mediated growth inhibi- tion and that SMAD6 down-regulation restores the TGF-h sensitivity, which led to reduced viability, proliferation, and increased apoptosis in lung cancer. Materials and Methods Cell lines and culture. All lung cancer cell lines and normal bronchial epithelialcellline,Beas2B,wereobtaineddirectlyfromtheAmericanType Culture Collection. All lung cancer cell lines were cultured in RPMI 1640 supplementedwith10%fetalbovineserum(FBS;LifeTechnologies).Beas2B was cultured in BEGM and growth supplements (Cambrex Bio Sciences, Inc.) in a humidified atmosphere with 5% CO 2 . Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for J. Fukuoka: Laboratory of Pathology, Toyama University Hospital, 2630 Sugitani, Toyama 930-0194, Japan. This work is dedicated to the memory of Dr. Anita Roberts. Requests for reprints: Jin Jen, Department of Pulmonary and Critical Care Medicine, Mayo Clinic, Stabile 13-74, 200 First Street SW, Rochester, MN 55905. Phone: 507-284-0526; E-mail: Jen.Jin@mayo.edu. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-1083 Cancer Res 2008; 68: (23). December 1, 2008 9686 www.aacrjournals.org Research Article Research. on June 19, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from