IMP-1 Displays Cross-Talk with K-Ras and Modulates Colon Cancer Cell Survival through the Novel Proapoptotic Protein CYFIP2

Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States. Deregulated expression of the oncogenic transcription factor c-Myc occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumor progression. Importantly, c-Myc expression is elevated in approximately 70% of colon tumors due to defective Wnt signaling (1, 2). Recent evidence indicates that c-Myc induction causes global transcriptional repression of human microRNA (miRNA) genes, including members of the let-7 family that are downregulated in human cancer cells and tumors (3, 4). The let-7 family of miRNAs act as tumor suppressors and inhibit the translational expression of oncogenic mRNAs including K-Ras, c-Myc, Hmga2, and Cdc34, and suppress cancer cell growth, proliferation, and tumor formation in vivo (5–12). K-Ras is frequently mutated in human tumors and plays key roles in regulating diverse cellular pathways important for cell growth, differentiation, and survival (13). Indeed, 40% to 50% of human colon cancers harbor activating mutations in the K-Ras proto-oncogene and is associated with progression from an adenoma to adenocarcinoma. Thus, the K-Ras signaling pathway represents an attractive target for cancer therapy (14–18).

The human c-Myc mRNA coding region determinant-binding protein, also known as insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1), is expressed during early embryonic mammalian development and functions in translational stability by binding and shielding several mRNAs that play critical roles in cell growth and proliferation from proteolytic degradation including c-Myc (19–24). Consistent with its oncofetal function, loss of IMP-1 in mice causes perinatal lethality, dwarfism, and impaired intestinal morphogenesis (25). In striking contrast to normal adult tissues, IMP-1 reexpression has been reported in breast, ovarian, and colorectal tumors (26). Furthermore, IMP-1 is a positive predictor of poor clinical outcome in colon cancer patients (27). Recent work has revealed that the β-catenin/Tcf complex upregulates IMP-1 mRNA and protein expression, necessary for the stabilization and induction of c-Myc and β-TrCP1 mRNAs in CRCs, and maybe involved in the suppression of apoptosis (24, 28). Moreover, increased IMP-1 levels positively correlate with activation of β-catenin/Tcf signaling in primary colorectal tumors (24). Importantly, IMP-1 is a direct let-7 target and promotes cell cycle progression, growth, and migration (29). These studies suggest that IMP-1 plays a role in regulating human cancer progression.

Herein, we report a molecular mechanism by which c-Myc positively modulates IMP-1 expression in colon cancers, in part by negative regulation of let-7 miRNAs. We also show that loss of IMP-1 downmodulates K-Ras expression downstream of β-catenin, and concomitantly inhibits colon cancer cell proliferation, anchorage-independent growth, and survival in monolayer and organotypic (3D) cell culture. Furthermore, we identify a novel proapoptotic gene target, cytoplasmic FMR1-interacting protein 2 (CYFIP2), which is downregulated by IMP-1, and mediates the regulation of cell survival and K-Ras expression in colon cancer cells. In contrast to our knockdown studies, IMP-1 overexpression increases c-Myc and K-Ras expression and colon cancer cell proliferation. For the first time we find that IMP-1 directly interacts with K-Ras mRNA and is highly elevated in colon cancer cells and tumors and positively correlates with K-Ras relative to normal mucosa, thus suggesting a novel interrelationship with K-Ras in vivo.

Caco-2, SW-480, and LIM2405 cells were purchased from the American Type Culture Collection (ATCC) and maintained in accordance with the manufacturer's recommendations. Cells were transfected with human β-catenin siRNA (gift from Dr. Jeffrey Drebin, University of Pennsylvania), SignalSilence c-Myc siRNAs I and II (Cell Signaling Technology), IMP-1 siRNA, CYFIP2 siRNA (Santa Cruz Biotechnology), or scrambled negative control siRNA (Ambion) by using Lipofectamine 2000 reagent (Invitrogen), per manufacturers' instructions. The IMP-1/pMSCV-PIG retroviral vector was purchased from Addgene (plasmid 21659) and used to stably infect LIM2405 cells as described previously (30, 31).

Total RNA was isolated from tissues and cultured cells by using the mirVana miRNA isolation kit (Ambion) and analysis of mRNA expression by reverse transcription PCR (RT-PCR) as described previously (32). Normal human colon was obtained from the Cooperative Human Tissue Network at the Hospital of the University of Pennsylvania [with Institutional Review Board (IRB) approval]. Human primary let-7a3-b intron, CYFIP2, and β-actin PCR products were amplified by the following oligonucleotide primer pairs:

• hlet-7a3-b intron

• 5′-GGGGCCGCCTACACTGAGAAG-3′ (Forward)

• 5′-CTGGGGCACGTGCTGGGAACCT-3′ (Reverse)

• hCYFIP2

• 5′-TGGCGTCATCATTCCGTATCC-3′ (Forward)

• 5′-GTCAGGTCCTCACTCAAGC-3′ (Reverse)

• hβ-actin

• 5′-AGAAATCTGGCACCACACC-3′ (Forward)

• 5′-AGAGGCGTACAGGGATAGCA-3′ (Reverse)

RT-PCR products were resolved by 1% TAE agarose gel electrophoresis.

Quantitative real-time PCR (qRT-PCR) was performed on an Applied Biosystems 7900HT Real-Time PCR System. The reverse transcription was performed by the TaqMan miRNA Transcription kit, followed by quantification of hsa-IMP-1 and mature hsa-let-7a and -7b, using predesigned TaqMan Assays (Applied Biosystems), according to the manufacturer's recommendations. β-Actin or U47 endogenous controls (Applied Biosystems) were used as an internal standard to normalize. PCR reactions were performed in triplicate. Data were analyzed by ABI PRISMs 7000 sequence detection system software (Applied Biosystems).

We purchased the following antibodies: IMP-1 [for immunohistochemistry (IHC)], c-Myc, β-catenin, cleaved Caspase-3 (Asp175) (5A1E) and PARP (Asp214), Lamin A/C (Cell Signaling Technology), β-catenin (for IHC), Cdc34, Cyclin D1 (BD Transduction Laboratories), Ras clone 10 (Upstate), IMP-1, K-Ras (Santa Cruz), Caspase-8 (Enzo Life Sciences), Cyfip2 (Abcam), Lin28B (Abgent), and K-Ras (for IHC; Spring Bioscience).

Immunoblotting was performed as described previously (33). The membranes were stripped by using Blotfresh Western Stripping Reagent (SignaGen) and reprobed for anti–β-actin (Sigma-Aldrich) to confirm equal loading. Relative band intensities were quantified by Adobe Photoshop software, and normalized to the most intense band, for each antibody.

Twelve tissue microarrays (TMA) were constructed with two cores of representative areas of each carcinoma and two cores of normal adjacent mucosa under IRB approval. A uniform cohort of 228 patients (133 male and 95 female) with colon cancer, 88 in stage 2, and 140 in stage 3, which were diagnosed between November 1993 and October 2006. Rectal tumors were excluded from the study. The pathology reports were available in all cases. Hematoxylin and eosin (H&E) slides and paraffin blocks were retrieved from the Surgical Pathology files of the Hospital Clinic, Barcelona. H&E slides from the original surgery and subsequent tumors were examined. All patients were treated with surgery and 5-fluorouracil. Slides were imaged by a Nikon TE2000 Eclipse microscope with a QiCam (Q Imaging) camera and IPLab imaging software (BD Bioscience Bioimaging). H-score is the product of the intensity of the staining (0, 1, 2, or 3) by the percent of stained cells (0 to 100). The distributions of nuclear β-catenin, K-Ras, and IMP-1 intensity scores, percent stained cells, and H-scores were right skewed and did not follow a normal distribution. Thus, Wilcoxon's signed rank tests were used to compare normal versus tumor tissue, paired within each patient. Spearman's correlation coefficients, stratified by tumor type, were used to assess the strength of linear association between nuclear β-catenin, K-Ras, and IMP-1 measures. Spearman's correlation coefficients greater than 0.50 indicate a strong association and those greater than 0.30 indicate a moderate association. All analyses were performed by SAS version 9.2 (SAS Institute).

WST-1 cell proliferation and anchorage-independent growth were performed as described previously (31).

Gene expression profiling and bioinformatics analysis was performed as described previously (33), using total RNA isolated from IMP-1 and scramble control siRNA SW-480 cells.

SW480 cells were transiently transfected with either scramble control or IMP-1 siRNAs, seeded on Type I collagen embedded with human fetal colonic fibroblasts (ATCC), grown in organotypic culture (OTC), harvested, and fixed as previously described (34).

The K-Ras coding cDNA and its 3′UTR subcloned into pGEM-T easy vector were linearized by NcoI and SalI restriction enzymes, respectively. GLI1 expression plasmid and the fragment encompassing nucleotides 2,713 to 3,600 of GLI1 cDNA (35) were used, respectively, as positive and negative controls for the binding assay of K-Ras coding region with IMP-1. The full-length GLI1 subcloned into pOTB7 (ATCC) was linearized immediately 3′ to the target DNA insert by BglII restriction enzyme. Fragments encompassing nucleotides 17 to 597 and 577 to 1,182 of β-TrCP1 cDNA (24) were subcloned into pcDNA3.1, linearized by XbaI restriction enzyme, and used, respectively, as negative and positive controls for the binding assay of the 3′UTR region of K-Ras with IMP-1. In vitro transcription, transfection, whole cell extraction, and UV cross-linking were performed as described previously (35).

We first sought to determine the relative abundance level of endogenous c-Myc protein in 8 human colon cancer cell lines by Western blot analysis and selected Caco-2/15 cells for further c-Myc knockdown and biochemical studies based on its lower abundance of c-Myc relative to the other cell lines surveyed (Fig. 1A). Caco-2/15 cells were transiently transfected with two different c-Myc siRNAs either individually or together. We found that a combination of the two c-Myc siRNAs (1 and 2) more effectively reduced endogenous c-Myc protein expression by 82% compared with transfection with the individual c-Myc siRNA-1 (37%) and -2 (43%), or the scramble control cells (Fig. 1B). Interestingly, we also observed a concomitant decrease in expression of the let-7 miRNA target IMP-1 by 73%, and Cdc34 and total Ras, by 30% and 16%, respectively, compared with control cells (Fig. 1B), suggesting let-7 regulation by c-Myc. To test this, total RNA was isolated from Caco-2/15 cells transfected with either a scramble control or c-Myc siRNAs. By PCR analysis using let-7 intron-specific primers, we detected no change in primary transcript levels of the let-7a3-b bicistronic cluster in c-Myc knockdown cells relative to scramble controls (Fig. 1C). However, qRT-PCR showed an increase in both mature let-7a and let-7b miRNA expression (Fig. 1D), suggesting that c-Myc modulates expression of known let-7 targets, IMP-1, Cdc34, and Ras, in part by negatively regulating endogenous mature let-7 miRNA levels in human colon cancer cells.

Our novel finding that c-Myc siRNA inhibits IMP-1 expression suggests a potential positive feedback mechanism. To explore this possibility further, we first determined the relative mRNA and protein expression level of IMP-1 in several human colon cancer cell lines by qRT-PCR and immunoblot analysis, respectively. We discovered that IMP-1 mRNA was upregulated by 3-fold or more in 8 of 14 human colon cancer cell lines surveyed, relative to normal colonic epithelium (NCE; Fig. 2A). IMP-1 mRNA levels were most notably increased in Caco-2 (511-fold) and SW-480 (38-fold) cells (Fig. 2A). Consistent with this finding, IMP-1 protein was highly expressed in Caco-2, SW-480, and SW-620 cells, and abundant levels of β-catenin, a positive regulator of IMP-1 (Fig. 2B). To examine the biochemical role of IMP-1 in c-Myc signaling, SW480 cells were transiently transfected with control, c-Myc, or IMP-1 siRNAs. By Western blot, we found that loss of IMP-1 decreased endogenous levels of Cdc34 (68%), Lin-28B (53%), a let-7 repressor and mediator of c-Myc–driven cellular proliferation, and K-Ras (60%), relative to scramble controls, but not to the same extent as c-Myc inhibition (36; Fig. 2C). Furthermore, we did not detect any significant changes in c-Myc or mature let-7a or -7b miRNA expression levels (data not shown). To assess for effects of IMP-1 in β-catenin signaling, we transfected SW-480 cells with control scramble, c-Myc, IMP-1, and β-catenin siRNAs. Western blot analysis showed that loss of β-catenin reduced endogenous protein levels of oncogenic c-Myc (89%), IMP-1 (67%), and Cyclin-D1 (98%), relative to scramble control cells (Fig. 2D). Interestingly, K-Ras was also reduced in these cells (90%) to a greater extent than by IMP-1 knockdown (42%), compared with controls (Fig. 2D). Because IMP-1 has been shown to bind and stabilize several mRNAs including oncogenic c-Myc, we hypothesized that it might regulate K-Ras expression by binding to K-Ras mRNA (37). Indeed, protein-RNA binding assays revealed that IMP-1 interacts directly with both the coding region and 3′UTR of K-Ras mRNA, compared with negative controls (Fig. 2E). Taken together, these findings suggests that IMP-1 functions downstream of β-catenin and may modulate K-Ras expression in human colon cancer cells via binding to K-Ras mRNA.

To determine the biological role of IMP-1 in human colon cancer cell lines, we transfected SW-480 cells with scramble control or IMP-1 siRNAs and examined the effects on regulating cell proliferation and anchorage-independent growth. We found that loss of IMP-1 expression reduced colon cancer cellproliferation in WST-1 assays by approximately 2-fold (P = 0.0001) and significantly suppressed both colon cancer colony size formation and number in soft agar assays by 3-fold, compared with scramble controls (P = 0.008; Fig. 3A and B). Intriguingly, transfection with IMP-1 siRNA also increased trypan blue uptake by 2.5-fold (P = 0.013) compared with scramble control cells (Fig. 3C). Loss of IMP-1 also induced cell rounding, membrane blebbing, and floating cells, suggesting the induction of cell death (Fig. 3C). To examine this further, adherent and floating SW-480 cells were collected and harvested 24 and 48 hours posttransfection with scramble or IMP-1 siRNAs. By immunoblot analysis we found that two markers of apoptosis, Caspase-3 and Lamin A/C cleavage products, were both increased in IMP-1 knockdown cells by 2-fold compared with control cells (Fig. 3D). In addition, we also observed a 3.6-fold increase in cleaved Caspase-8 in IMP-1 siRNA-transfected cells but no change in Caspase-9 cleavage (data not shown) relative to scramble controls, suggesting that IMP-1 functions via induction of an extrinsic or mitochondria-mediated cell death pathway (Fig. 3E). Taken together, these data support the notion that IMP-1 is a positive modulator of colon cancer cell proliferation, anchorage-independent growth, and survival.

To identify novel genes that are involved in mediating IMP-1 functions, expression profiling analysis was performed on total RNA isolated from scramble control and IMP-1 siRNA SW-480 cells. Gene expression analysis confirmed that IMP-1 was reduced by approximately 1.6-fold in knockdown cells compared with scramble controls (Supplementary Table S1). We focused on the CYFIP2, which was the most upregulated gene target (2.3-fold; P = 0.00001) by IMP-1 loss compared with control cells (Supplementary Table S1). Importantly, CYFIP2 is a p53-inducible gene that is sufficient for inhibition of colon cancer proliferation, caspase activation, and the induction of apoptosis (38). Consistent with the gene microarray results, PCR analysis confirmed that CYFIP2 mRNA levels were increased by 4.5-fold in cells transfected with IMP-1 siRNA but was reduced or undetectable in scramble controls (Fig. 4A and B). We next asked whether CYFIP2 inhibition by IMP-1 mediates cell survival and K-Ras expression. To test this, SW-480 cells were transiently transected with scramble control, IMP-1, CYFIP2, or a combination of IMP-1 and CYFIP2 siRNAs. By Western blot, we found that cotransfection of IMP-1 and CYFIP2 siRNAs reversed Caspase-3 and PARP cleavage and rescued K-Ras inhibition by IMP-1 loss, similar to levels observed in control cells (Fig. 4C). On the basis of these findings, we propose that IMP-1 promotes colon cancer cell survival and regulates K-Ras expression, in part by suppressing CYFIP2.

As a complementary approach to our knockdown studies and to further investigate the role of IMP-1, we chose to modify LIM2405 cells, which express low endogenous IMP-1 mRNA and protein levels (Fig. 2A and B), with a retrovirus expressing IMP-1 or the vector alone as a control. We found that IMP-1 upregulated endogenous levels of c-Myc and K-Ras by 3.8-fold and 2.6-fold, respectively, relative to vector controls in LIM2405 cells (Fig. 5A). IMP-1 also increased LIM2405 cell proliferation in WST-1 assays by 1.5-fold (P = 0.00125; Fig. 5B and C). We next wanted to examine whether IMP-1 expression can rescue K-Ras inhibition by β-catenin knockdown. To test this, IMP-1 and vector-alone LIM2405 cells were transfected with scramble or β-catenin siRNAs. Western blot analysis showed that K-Ras levels were 1.7-fold more in IMP-1 cells transfected with β-catenin siRNA compared with vector-alone cells (Fig. 5D). These results support the notion that IMP-1 regulates K-Ras expression downstream of β-catenin signaling.

We next examined the effect of IMP-1 on colonic epithelium in OTC. SW480 cells were transiently transfected with IMP-1 or scramble control siRNAs and seeded on Type I collagen matrix embedded with colonic fetal fibroblasts. In contrast to scramble controls, IMP-1 siRNA induced the formation of apoptotic bodies indicative of cells undergoing death (Fig. 6A). Consistent with this notion and our biochemical monolayer culture results, we also detected a 2.4-fold (P = 0.02) increase in cleaved Caspase-3 positive cells relative to scramble control cells by IHC (Fig. 6A and B). Together these findings suggest that IMP-1 plays a key role in modulating cell survival.

On the basis of our biochemical finding that IMP-1 expression is elevated in human colon cancer cell lines, we sought to determine whether there is a correlation between IMP-1, β-catenin, and K-Ras expression levels, in a cohort of 228 paired patient normal and adjacent colon cancer tissue specimens by IHC. In comparison with adjacent normal colonic mucosa, we observed a significant upregulation of nuclear β-catenin, K-Ras, and IMP-1 in paired colon cancers by 9.9-fold, 8.3-fold, and 33.8-fold (P ≤ 0.0001), respectively (Fig. 7A). Importantly, IMP-1 predominantly localized in the cytoplasmic compartment of colon tumors and positively associated with K-Ras intensity (r = 0.35, P ≤ 0.0001) and H-scores (r = 0.32, P ≤ 0.0001) among the tumor tissues (Fig. 7B; Supplementary Fig. S1) compared with adjacent normal colonic mucosa (Fig. 7B). Collectively, our findings suggest that IMP-1 expression is upregulated in human colon cancers and correlates with K-Ras levels.

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 proapoptotic target, CYFIP2, which is upregulated by IMP-1 knockdown. 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 (24, 29). In addition, a recent study showed that overexpression of the antitumorigenic protein 15S-Lipoxygenase (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 reexpression 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, 4, 29). Consistent with this, inhibition of IMP-1 by targeted knockdown 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 S1; ref. 38).

K-Ras mutations are relatively frequent and appear in colonic adenomatous polyps and colon cancers of mice and humans (40). Importantly, we observed that knockdown of IMP-1 or its 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 knockdown 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 (41–44). 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, 28). We found that nuclear β-catenin levels were significantly increased in colon cancers compared with 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 proapoptotic 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 NCE and present new evidence that IMP-1 positively correlates with K-Ras in colon cancers. On the basis of 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.

No potential conflicts of interest were disclosed.

We thank D. Baldwin for the gene expression profiling and J. Tobias for the microarray statistical analysis (Penn Microarray Facility Bioinformatics Group); J. Drebin (Department of Surgery) for the β-catenin siRNA; X. Yang (Department of Cancer Biology) for the Caspase-8 antibody; G.P. Swain, D. Budo, and S. Campbell (Penn Morphology Facility) for IHC; and C. Brensinger for the TMA statistical analysis (Center for Clinical Epidemiology & Biostatistics).

This work was supported in part by NIH DK056645, the National Colon Cancer Research Alliance, the Hansen Foundation (A.K. Rustgi), the American Cancer Society Research Professorship (A.K. Rustgi), and NCI Grant CA121851 (V.S. Spiegelman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.