Four specimens (the AsPC-1 cell line, and three xenografts) had genomic profiles that were particularly informative in delimiting the amplicon boundaries within 7q21-q22 (Fig. 1A). The smallest common region of gain spanned just 325 Kb within cytoband 7q22.1, and contained just two RefSeq [14] genes, SMURF1 (SMAD specific E3 ubiquitin protein ligase) and KPNA7 (karyopherin alpha 7). SMURF1 is a known inhibitor of TGFb signaling (by promoting degradation of its receptor TGFbRI, and signaling mediator SMAD4 [15,16]), a pathway frequently disrupted in pancreatic cancer. Given an obvious connection to pancreatic carcinogenesis, we therefore focused subsequent efforts on SMURF1. Consistent with an oncogenic role, SMURF1 transcript (measured by microarray) was significantly elevated in cell lines/ xenografts with 7q22.1 gain/amplification (as were several co- amplified neighboring genes) (Fig. 1B). To evaluate SMURF1 amplification in primary pancreatic tumors, we also carried out FISH on a tissue microarray containing 105 pancreatic cancer cases. Four of 95 (4.2%) evaluable cases exhibited SMURF1 amplification (locus/centromere ratio .2.5) (Fig. 1C), comparable to our CGH findings for early-passage xenografts. We were unable to identify a suitable antibody and staining conditions to evaluate SMURF1 expression by immunohistochemistry. SMURF1 amplification promotes cell invasion and anchorage-independent growth To evaluate possible oncogenic functions of SMURF1, we first used RNAi to knockdown SMURF1 expression in the relevant context of gene amplification, using AsPC-1 cells. Transfection of four different small interfering RNAs (siRNAs), or a pool of the four together, each led to reduced SMURF1 protein levels (by Western blot), compared to a non-targeting siRNA pool (Fig. 2A). Knockdown of SMURF1 did not alter cell proliferation, measured by WST-1 assay (Fig. 2B,C), and by BrdU incorporation (Fig. 2D), but led to significantly decreased cell invasion through Matrigel, measured by Boyden chamber assay (Fig. 2E). Decreased invasion was seen with each of the four siRNAs targeting distinct SMURF1 sequences, while the growth rate of the cells remained unchanged within the same time period (Fig. 2C), strongly supporting the specific role of SMURF1 in the invasiveness phenotype. Given its known, antagonistic function in the TGFb pathway, we also sought to evaluate the effect of SMURF1 knockdown on TGFb signaling, using a TGFb responsive transcriptional reporter (p3TP-Lux) [17]. Knockdown of SMURF1 did not enhance TGFb pathway-mediated transcription in AsPC-1 cells (Fig. 2F). Of note, however, AsPC-1 cells (like most pancreatic cancers) harbor a mutated SMAD4, here SMAD4 (R100T) [18], charac- terized to be inactivating [19,20]. Therefore, AsPC-1 cells are likely incapable of a TGFb pathway transcriptional response. More generally, these findings suggest that the main effect(s) of SMURF1 amplification/overexpression are likely mediated through pathways distinct from TGFb signaling. To evaluate longer-term phenotypes, we also stably transfected a short hairpin RNA (shRNA) targeting SMURF1. Stable knockdown of SMURF1 in AsPC-1 cells, confirmed by Western blot (Fig. 3A), significantly reduced anchorage independent growth (soft agar colonies), compared to a non-targeting shRNA control (Fig. 3B). We also sought to evaluate the effect of siRNA knockdown in other pancreatic cancer cell lines. We chose two cell lines, BxPC-3 cells which (like AsPC-1 cells and most pancreatic tumors) have mutated SMAD4 (here by homozygous deletion) [21], and Hs700T cells which are wildtype for SMAD4 and have an intact TGFb growth-inhibitory pathway (Fig. S1). Notably, neither of these lines SMURF1 Amplification in Pancreatic Cancer harbors focal amplification of SMURF1 (AsPC-1 cells are the only established line with focal amplification), nor elevated SMURF1 protein levels (Fig. 4A). Knockdown of SMURF1 (validated by Western blot; Fig. 4B) led to modestly reduced cell proliferation in BxPC-3 cells (Fig. 4C), and more so in TGFb-growth inhibitory pathway-intact Hs700T cells (Fig. 4D). SMURF1 knockdown also resulted in reduced cell invasion in BxPC-3 cells (Fig. 4E), though not significantly so. However, given that SMURF1 is neither focally amplified nor overexpressed in these lines, a simple explanation for the discordant phenotypes (compared to AsPC-1) is that SMURF1 may not function as an oncogenic driver in these cell contexts. Finally, in complementary, overexpression studies, we trans- fected SMURF1 cDNA (expressed from a CMV promoter) into NIH-3T3 mouse fibroblasts. Overexpression of SMURF1, con- firmed by Western blot (Fig. 5A), led to a significant loss of contact inhibition (i.e. increased foci), compared to a vector control (Fig. 5B). Notably, transfection of a catalytically-inactive mutant of SMURF1 (C699A) [22] did not reduce contact inhibition (Fig. 4B), indicating that this oncogenic activity is dependent on the E3 ubiquitin protein ligase activity of SMURF1. Discussion Here, we set out to pinpoint and discover the oncogene driving 7q21-q22 amplification in pancreatic cancer. High-resolution genomic profiling of pancreatic cancer cell lines and early-passage xenografts defined a 325 Kb minimal amplicon spanning SMURF1. Transcript levels of SMURF1 were elevated in specimens with gain/amplification, and by FISH we confirmed SMURF1 amplification in primary pancreatic cancers. Using complementary approaches of knockdown (in focally-amplified AsPC-1 cells) and overexpression (in NIH-3T3 cells), we determined that SMURF1 amplification/overexpression does not alter cell proliferation, but promotes cell invasion, anchorage- independent growth, and loss of contact inhibition, of which at least the latter is dependent on its catalytic activity. SMURF1 was initially an intriguing oncogene candidate because of its known connection to TGFb signaling. The TGFb pathway, at least early in tumor development, is growth sup- pressive [23]. Normally, TGFb binds to its receptors (TGFbRI, TGFbRII), leading to the phosphorylation of signal transducers SMAD2/SMAD3, which then shuttle to the nucleus and in complex with SMAD4 mediate transcription. Key transcriptional responses include induction of CDKN2B (p15Ink4b) and CDKN1A (p21Cip1), and repression of MYC, together leading to G1 cell- cycle arrest. The TGFb growth suppressive pathway is commonly disrupted in pancreatic cancer, most often through mutation/ deletion of SMAD4, but also through inactivation/loss of TGFbRI and TGFbRII [4]. SMURF1 is a HECT-domain E3 ubiquitin ligase (E3 ubiquitin ligases carry out the third and substrate-specific step in protein ubiquitination). SMURF1 promotes the nuclear export of TGFb pathway inhibitor SMAD7 (increasing its availability), and the destruction of TGFbRI and SMAD4 (through ubiquitination- mediated degradation) [15,16]. All these activities should serve to antagonize TGFb signaling, and together provide a strong rationale for SMURF1 amplification/overexpression in pancreatic cancer. It was notable then, that SMURF1 knockdown in AsPC-1 cells did not enhance TGFb-pathway transcription (though perhaps not surprising, given the inactivating mutation of SMAD4). Therefore, the oncogenic activities of SMURF1 must act at least in part independently of its functions in TGFb signaling (at least at the pathway level of SMAD4). To this end, SMURF1 has also PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23924