SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer Kevin A. Kwei1., A. Hunter Shain1., Ryan Bair1, Kelli Montgomery1, Collins A. Karikari2, Matt van de Rijn1, Manuel Hidalgo3,4, Anirban Maitra2, Murali D. Bashyam5, Jonathan R. Pollack1* 1 Department of Pathology, Stanford University, Stanford, California, United States of America, 2 Department of Pathology, The Johns Hopkins University, Baltimore, Maryland, United States of America, 3 Department of Oncology, The Johns Hopkins University, Baltimore, Maryland, United States of America, 4 Clinical Research Program, Centro Nacional de Investigaciones Oncolo´ gicas, Madrid, Spain, 5 Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, India Abstract Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor b (TGFb) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGFb signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy. Citation: Kwei KA, Shain AH, Bair R, Montgomery K, Karikari CA, et al. (2011) SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer. PLoS ONE 6(8): e23924. doi:10.1371/journal.pone.0023924 Editor: Hana Algu¨ l, Technische Universita¨t M u¨nchen, Germany Received December 14, 2010; Accepted August 1, 2011; Published August 22, 2011 Copyright: 2011 Kwei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the NIH: CA112016 (J.R.P.), CA09151 (K.A.K.), GI Cancer SPORE CA62924 (A.M.); NIH/NCRR CTSA grant UL1 RR025744 to Stanford Spectrum; and the Lustgarten Foundation (J.R.P.). M.D.B. was supported in part by a Biotechnology overseas associateship from the Department of Biotechnology, Ministry of Science and Technology, Government of India. The authors also thank the family of Margaret Lee and the Sol Goldman Pancreatic Cancer Research Center for supporting the xenografting efforts at Johns Hopkins. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pollack1@stanford.edu . These authors contributed equally to this work. Introduction Pancreatic ductal adenocarcinoma (hereafter, pancreatic can- cer) is nearly always fatal, with a five year survival rate less than 5% [1]. It is often disseminated at diagnosis, and can metastasize widely. Early detection can improve survival, but surgical resection is rarely curative [2]. Pancreatic cancer is also largely resistant to conventional chemotherapy. Therefore, new therapies are urgent- ly needed. In particularly, it will be important to discover and validate new targets for molecularly-directed therapy. The molecular genetics of pancreatic cancer are in part known [3,4]. Somatic activating mutations of KRAS (sometimes occurring with gene amplification) are found in .90% of pancreatic cancers. Also common are inactivating mutations/deletions of tumor suppressors CDKN2A (.95% of cancers), TP53 (50–75%), and SMAD4 (also known as DPC4) (55%), an effector of TGFb- mediated growth inhibition. Other gene mutations, each occurring in less than 5% of cancers, impact these and other core cancer signaling pathways [5]. Genomic profiling studies, by array-based comparative genomic hybridization (array CGH), have begun to catalogue DNA amplifications and deletions, pinpointing and revealing novel pancreatic cancer genes (e.g. [6,7]). Among altered loci, we and others previously identified 7q21-q22 as a site recurrently amplified in pancreatic cancer [8–13]. Here, we narrow that locus, and characterize SMURF1 as an oncogene product promoting cell invasion and anchorage-independent growth. Results SMURF1 is focally amplified in pancreatic cancer We had previously identified recurrent amplification at 7q21-q22 in pancreatic cancer cell lines, using CGH on cDNA microarrays [12]. To further delimit the amplicon, and pinpoint the resident oncogene(s), we now carried out additional genomic profiling of a collection of 22 pancreatic cancer cell lines and 58 early-passage pancreatic cancer xenografts, using high-resolution 244K Agilent CGH arrays. The 7q21-q22 locus was focally amplified (tumor/ normal ratios .3-fold) in 1 of 22 (4.5%) cell lines (AsPC-1), and in 1 of 58 (2%) xenografts. Including lower-level gains (ratios .1.3 fold), gain/amplification spanning 7q21-q22 was found in 6 of 22 (27%) cell lines, and in 19 of 58 (33%) xenografts. PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23924