We report that loss of IMP-1 inhibits expression of let-7 miRNA oncogenic protein targets, Cdc34 and K-Ras, and the let-7 repressor Lin-28B, and concomitantly suppresses colon cancer cell proliferation, anchorage-independent growth, and triggers caspase-mediated cell death in both monolayer and 3D culture. By gene profiling analysis and PCR analysis we have discovered a novel pro-apoptotic target, CYFIP2, which is upregulated by IMP-1 knock-down. Importantly, we have identified a new function of IMP-1 to bind to K-Ras mRNA and regulate cell survival and K-Ras expression via inhibition of CYFIP2. We further show that a gain of IMP-1 expression occurs in human colon cancer cells and correlates with K-Ras levels in patient colon tumors.
To date, little is known about the molecular mechanisms that are involved in the complex regulation of IMP-1 in mammalian cells (26
). Constitutive activation of β-catenin/Tcf has been shown to upregulate IMP-1 levels in colon cancers and is necessary for the stabilization and induction of c-Myc
). In addition, a recent study demonstrated that overexpression of the anti-tumorigenic protein 15S
-Lox-2) suppresses human prostate carcinoma cell growth and proliferation by downregulation of IMP-1 (39
). Our novel finding that loss of c-Myc reduces IMP-1 expression and re-expression of IMP-1 increases c-Myc levels suggests a new potential feedback mechanism whereby c-Myc and IMP-1 may reciprocally modulate expression of each other in colon cancer cells. Oncogenic c-Myc induction has been shown to repress members of the let-7 family in human cancer cells and IMP-1 is a direct let-7 target (3
). Consistent with this, inhibition of IMP-1 by targeted knock-down of c-Myc resulted in an increase in mature let-7 miRNA expression, suggesting that c-Myc regulation of IMP-1 is mediated in part by let-7 in colon cancer cells. Furthermore, loss of IMP-1 inhibited expression of let-7 oncogenic targets, Cdc34, K-Ras, and Lin-28B, but did not alter the endogenous levels of c-Myc, pri- or mature let-7 miRNAs (data not shown), suggesting that IMP-1 is regulated by additional genes involved in promoting the cancer cell phenotype.
We showed that loss of IMP-1 suppressed colon cancer cell proliferation and anchorage-independent growth, and increased trypan blue uptake, caspase and lamin-mediated cell apoptosis. By gene profiling microarray analysis, we also identified a new IMP-1 target, CYFIP2
, a direct target of the tumor suppressor p53 that is sufficient for caspase activation and colon cancer apoptosis (Supplementary Table I
mutations are relatively frequent and appear in colonic adenomatous polyps and colon cancers of mice and humans (40
). Importantly, we observed that knock-down of IMP-1 or it's upstream regulator, β-catenin/Tcf, decreased levels of K-Ras protein levels in colon cancer cells. Conversely, IMP-1 expression rescues inhibition of K-Ras by β-catenin siRNA. In addition, we found that IMP-1 binds directly to the coding region and 3′UTR of K-Ras
mRNA. Furthermore, we also showed that CYFIP2 knock-down reverts Caspase-3 and Parp-mediated apoptosis, and inhibition of endogenous K-Ras expression due to loss of IMP-1. These findings indicate that K-Ras may be a novel downstream target of IMP-1 signaling and is regulated via direct interaction with K-Ras
mRNA and the suppression of CYFIP2. Interestingly, IMP-1 was upregulated in patient colon tumors and correlated with K-Ras expression, suggesting that gain of IMP-1 maybe a novel mechanism to increase endogenous K-Ras levels.
Overexpression of IMP-1 has been reported in a variety of cancers, suggesting an important role of IMP-1 in cancer development (41
). Constitutively active β-catenin/Tcf transcription factor upregulates IMP-1 in colon cancer cells, and elevated levels of IMP-1 correlate with β-catenin/Tcf signaling in primary colorectal tumors (24
). We found that nuclear β-catenin levels were significantly increased in colon cancers compared to normal colonic mucosa, however, there was no correlation between IMP-1 and β-catenin expression in tumors, suggesting that IMP-1 dysregulation may occur independently of β-catenin in vivo
In summary, we report that IMP-1 binds to K-Ras
mRNA and plays an important role in regulating K-Ras expression potentially via repression of the pro-apoptotic protein, CYFIP2, downstream of β-catenin/Tcf, and promotes colon cancer cell proliferation, anchorage-independent growth, and survival (Supplementary Fig.S2
). Furthermore, we show that human colon tumors express high levels of IMP-1 relative to normal colonic epithelium and present new evidence that IMP-1 positively correlates with K-Ras in colon cancers. Based on our novel findings, we propose that IMP-1 maybe a candidate for targeted therapeutic intervention in human cancers with dysregulation of K-Ras expression and signaling.