Figure 3. Knockdown of amplified SMURF1 in AsPC-1 cells reduces anchorage-independent growth. (A) A stably transduced shRNA targeting SMURF1 leads to reduced SMURF1 levels (by Western blot) compared to a non-targeting control shRNA. Residual SMURF1 levels, here normalized to GAPDH, are indicated. (B) Stable SMURF1 knockdown reduces anchorage-independent growth (i.e. soft agar colonies). Assay done in triplicate (mean +/2 1SD shown); *, P,0.05 (Student’s t-test). doi:10.1371/journal.pone.0023924.g003 show that SMURF1 overexpression in two pancreatic cancer cell lines enhanced colony growth on tissue culture plastic. Nonethe- less, our findings based on knockdown in the physiologically- relevant context of focal SMURF1 amplification suggest that the main oncogenic function of SMURF1 relates to promoting cell invasiveness rather than proliferation. In another recent study, Laurila et al. [28] by FISH analysis of cell lines delimited the 7q21-q22 amplicon to 0.77 Mb spanning 10 genes (including SMURF1), but focused their efforts on ARPC1A and ARPC1B, subunits of the Arp2/3 complex functioning in actin polymerization. Using RNAi, they found that knockdown of either reduced cell motility, and knockdown of ARPC1A also reduced cell invasion. Though our minimal amplicon excluded ARPC1A and SMURF1 Amplification in Pancreatic Cancer ARPC1B, it is nonetheless possible that their amplification contributes to motility/invasion in tumors where they are amplified. It is not uncommon to find multiple driver oncogenes within tumor amplicons (e.g. ref. [29]). Indeed, our own studies do not resolve whether KPNA7, within our 325 Kb minimal amplicon, might also have an oncogenic role (along with SMURF1). To summarize, by genomic profiling and functional analysis we identified SMURF1 as an amplified oncogene driving cell invasiveness in pancreatic cancer. Perhaps of most significance, as an enzyme SMURF1 represents a tractable drug target. Other E3 ubiquitin ligases have been linked to cancer, and because of their substrate specificity E3 ubiquitin ligases are thought to be attractive targets for therapy [30]. Indeed, several small molecule inhibitors (including against MDM2, a regulator of TP53) are presently being evaluated [31]. Our findings identify SMURF1 as a possible new target for molecularly-directed therapy against the devastating disease of pancreatic cancer. Materials and Methods Specimens Pancreatic cancer cell lines, described previously [12], and NIH-3T3 cells were obtained from the American Type Culture Collection (Manassas, VA). Pancreatic cancer xenografts, which effectively enrich the tumor epithelial fraction for DNA analysis, were generated as described [32] at the Johns Hopkins Hospital, with approval from the Institutional Review Board (IRB) (protocol ID 05-04-14-02) and Animal Care and Use Committee (protocol ID MO05M466). Briefly, a 1 mm3 piece of the primary tumor was soaked in Matrigel (Collaborative Biomedical Research), then implanted subcutaneously in a nu/nu mouse. Engrafted tumors were harvested when they reached 1–2 cm in diameter, and tumor cell enrichment confirmed by H&E-stained frozen section. DNA and RNA were isolated using the Qiagen (Valencia, CA) AllPrep kit. Eleven of the 48 xenografts were previously profiled by lower- resolution CGH on cDNA arrays [6]. Array CGH CGH was done using Agilent (Santa Clara, CA) catalogue 244K CGH arrays. DNAs were labeled as described [33], then hybridized (vs. a pool of eight sex-matched normal leukocyte DNAs) following the manufacturer’s instructions. Arrays were scanned using an Agilent G2505B scanner, and data extracted and normalized using Agilent Feature Extraction software (version 9.1) with default settings. Copy number alterations were called using cghFLasso [34], and low-level gains and higher-level ampli- fications defined by cghFLasso tumor/normal ratios .1.3 and .3.0, respectively. CGH data detailed herein are available at GEO (GSE19852); a complete description of the dataset is in preparation. Expression profiling Expression profiling was done using Agilent catalogue 44K Whole Human Genome arrays. RNAs were labeled using the Quick Amp Labeling kit (Agilent), then hybridized (vs. a universal RNA reference pool of 11 cancer cell lines [35]) following the manufacturer’s instructions. After scanning and data extraction (as above), expression data were normalized (mean-centered) by array and by gene, and mean-centered log2 ratios reported. FISH A tissue microarray containing 105 pancreatic ductal adeno- carcinoma cases (archived at Stanford University, and used with PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23924