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 TGFβ signaling. The TGFβ pathway, at least early in tumor development, is growth suppressive 
. Normally, TGFβ binds to its receptors (TGFβRI, TGFβRII), 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 TGFβ growth suppressive pathway is commonly disrupted in pancreatic cancer, most often through mutation/deletion of SMAD4
, but also through inactivation/loss of TGFβRI
and TGFβRII 
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 TGFβ pathway inhibitor SMAD7 (increasing its availability), and the destruction of TGFβRI and SMAD4 (through ubiquitination-mediated degradation) 
. All these activities should serve to antagonize TGFβ 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 TGFβ-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 TGFβ signaling (at least at the pathway level of SMAD4). To this end, SMURF1 has also been shown to dissolve tight junctions (by degradation of RhoA) during epithelial-mesenchymal transition 
, and focal adhesions (by degradation of talin heads) to potentiate cell migration 
. 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 characterized the 7q21-q22 amplicon in pancreatic cancer. Suzuki et al. 
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
, 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 
. Suzuki et al.
went on to show that SMURF1 overexpression in two pancreatic cancer cell lines enhanced colony growth on tissue culture plastic. Nonetheless, 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. 
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
, 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
, 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. 
). 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 
. Indeed, several small molecule inhibitors (including against MDM2, a regulator of TP53) are presently being evaluated 
. Our findings identify SMURF1 as a possible new target for molecularly-directed therapy against the devastating disease of pancreatic cancer.