SMURF1 Amplification in Pancreatic Cancer Figure 2. SMURF1 knockdown in amplified AsPC-1 cells reduces invasion but not growth. (A) Four different siRNAs targeting SMURF1, and a pool of all four, lead to reduced SMURF1 levels (by Western blot) compared to a non-targeting control siRNA pool). Residual SMURF1 levels, here normalized to GAPDH, are indicated. (B) SMURF1 knockdown (using siRNA pool) does not reduce cell proliferation/viability, measured by WST1 assay, and done in triplicate (mean +/2 1SD shown). (C) SMURF1 knockdown using four different siRNAs does not significantly alter cell proliferation/ viability, measured three days post transfection. Done in triplicate (mean +/2 1SD shown). (D) SMURF1 knockdown does not reduce cell-cycle progression (S-phase), measured by BrdU incorporation (1.5 hr and 4 hr pulse labeling), done in triplicate (mean +/2 1 SD shown). (E) SMURF1 knockdown (using siRNA pool and individual siRNAs) inhibits cell invasion through Matrigel. Boyden chamber assay done in triplicate and harvested three days post transfection (mean +/2 1SD shown); *, P,0.05 (Student’s t-test). (F) SMURF1 knockdown does not enhance TGFb pathway-mediated transcription. AsPC-1 cells were co-transfected with siRNAs and p3TP-Lux reporter, done in triplicate, and firefly/Renilla luciferase ratios shown (mean +/2 1SD shown). Panc1 cells (with wildtype SMAD4) +/2 TGFb serve as a positive control. doi:10.1371/journal.pone.0023924.g002 been shown to dissolve tight junctions (by degradation of RhoA) during epithelial-mesenchymal transition [24], and focal adhesions (by degradation of talin heads) to potentiate cell migration [25]. Additional studies should clarify the key SMURF1 substrates linked to invasiveness and anchorage-independent growth in pancreatic cancers with 7q22 amplification. During the progress of this work, two other studies character- ized the 7q21-q22 amplicon in pancreatic cancer. Suzuki et al. [26] by genomic profiling of cell lines identified the amplicon in AsPC- 1 cells, with the amplicon peak spanning 11 genes. Further efforts focused on two genes, TRRAP and SMURF1, with significantly elevated expression when amplified. However, in contrast to our study, they reported that knockdown of SMURF1 inhibited AsPC- 1 cell proliferation. Notably, though, they evaluated only one siRNA. Given our results that four independent siRNAs knocked down SMURF1 levels comparably and decreased invasion without affecting cell proliferation, we suggest that their finding might reflect a non-specific or specific off-target RNAi effect. Indeed, growth inhibition is a common non-specific effect, triggered by a type I interferon response to siRNA [27]. Suzuki et al. went on to PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23924