APC mutations cause activation of Wnt/β-catenin signaling, which invariably leads to colorectal cancer. Similarly, overexpressed Dvl proteins are potent activators of β-catenin signaling. Screening a large tissue microarray of different staged colorectal tumors by immunohistochemistry, we found that Dvl2 has a strong tendency to be overexpressed in colorectal adenomas and carcinomas, in parallel to nuclear β-catenin and Axin2 (a universal transcriptional target of Wnt/β-catenin signaling). Furthermore, deletion of Dvl2 reduced the intestinal tumor numbers in a dose-dependent way in the ApcMin model for colorectal cancer. Interestingly, the small intestines of Dvl2 mutants are shortened, reflecting in part a reduction of their crypt diameter and cell size. Consistent with this, mammalian target of rapamycin (mTOR) signaling is highly active in normal intestinal crypts in which Wnt/β-catenin signaling is active, and activated mTOR signaling (as revealed by staining for phosphorylated 4E-BP1) serves as a diagnostic marker of ApcMin mutant adenomas. Inhibition of mTOR signaling in ApcMin mutant mice by RAD001 (everolimus) reduces their intestinal tumor load, similarly to Dvl2 deletion. mTOR signaling is also consistently active in human hyperplastic polyps and has a significant tendency for being active in adenomas and carcinomas. Our results implicate Dvl2 and mTOR in the progression of colorectal neoplasia and highlight their potential as therapeutic targets in colorectal cancer. Cancer Res; 70(16); 6629–38. ©2010 AACR.

Most colorectal cancers are initiated by hyperactivation of the Wnt/β-catenin pathway in the intestinal epithelium, typically by loss-of-function mutations of the APC tumor suppressor (1, 2). APC is a negative regulator of β-catenin; it binds to Axin to promote the phosphorylation of β-catenin by glycogen synthase kinase 3β, thus earmarking it for proteasomal degradation (3). APC truncations such as those typically found in colorectal cancer lack their Axin-binding domain and, therefore, retain only partial function (4); thus, β-catenin accumulates in the cytoplasm and nucleus where it binds to TCF factors to operate a transcriptional switch. Apc mutations in mice also initiate intestinal tumorigenesis (5), and the transcriptional program activated by APC loss resembles that of the normal intestinal crypts, which comprise the intestinal stem cell compartment (2). One of the key APC effector genes in normal crypts and tumorigenesis is c-myc (6).

Loss of APC function mimics β-catenin activation by Wnt signals in normal cells, which critically depends on Dishevelled (Dvl; ref. 7): upon Wnt stimulation, Dvl binds to and recruits Axin to the plasma membrane by virtue of its polymerizing activity (8, 9), thus assembling signalosomes that also contain Frizzled receptor and LRP6 coreceptor and promoting the phosphorylation of the LRP6 cytoplasmic tail (10, 11). The latter acts as a direct inhibitor of glycogen synthase kinase 3β (12, 13), which allows unphosphorylated β-catenin to accumulate and trigger a transcriptional switch, much like APC loss. Notably, if Dvl is expressed at high levels, it potently activates β-catenin, independently of Wnt stimulation; it recruits Axin into cytoplasmic signalosomes (8, 9) and inhibits glycogen synthase kinase 3β through LRP6 phosphorylation (14).

In binding to Axin, Dvl blocks the activity of the Axin-APC complex in downregulating β-catenin; if overexpressed, Dvl could thus synergize with a partially functional Axin-APC complex and further promote Wnt/β-catenin pathway activity. This is the case in Drosophila, in which dishevelled is essential for Wnt pathway activity in hypomorphic APC mutant embryos (15). The same could be true in colorectal cancer cells, which carry hypomorphic APC mutations (4), which delete the Axin-binding domain from APC, and thus allow only indirect association of the two proteins through β-catenin (16); indeed, these cancer cells could be particularly sensitive to Dvl expression levels, and their β-catenin hyperactivation might reflect both their Dvl signaling activity and their APC loss. If so, Dvl and its signaling partners (which include several kinases; ref. 7) could have potential as targets for therapeutic intervention.

To examine the possible role of Dvl in colorectal cancer, we screened a large tissue microarray (TMA) of colorectal tumor samples and found that Dvl2 has a strong tendency to become overexpressed during the tumor progression. Furthermore, we show that lowering the dose of Dvl2 reduces the numbers of intestinal tumors in the ApcMin mouse model, indicating a tumor-promoting role of Dvl2 in the intestine. We also discovered that Dvl2−/− mice have shortened intestines, and we present evidence that this reflects partly fewer intestinal crypts, and partly reduced crypt diameters, suggesting that Dvl2 may promote crypt cell growth. Consistent with this observation, we show that crypts exhibit high levels of phosphorylated 4E-BP1 (p4E-BP1), a key read-out of activated mammalian target of rapamycin (mTOR) signaling that promotes cell growth (17), consistent with earlier results (18). Indeed, we find high p4E-BP1 levels to be a diagnostic marker for nascent polyps and larger intestinal tumors of ApcMin mutant mice, and we confirm that inhibition of this pathway by the rapamycin-like inhibitor RAD001 reduces the tumor numbers in this model (18). Importantly, we find that mTOR signaling is highly active in human hyperplastic polyps, and also within a subset of adenomas and colorectal carcinomas, indicating the therapeutic potential of mTOR inhibitors in colorectal cancer.

Mouse models

Animal care and procedures were done in accordance with the standards set by the United Kingdom Home Office. Dvl2+/− mice (19, 20) were back-crossed into a C57BL/6 background (the genetic background of ApcMin/+; ref. 5) for four successive generations (whereby the final cross was with ApcMin/+), and compound ApcMin/+Dvl2+/+, ApcMin/+Dvl2−/+, and ApcMin/+Dvl2−/− mutants were generated from a back-cross of ApcMin/+ Dvl2−/+ males with Dvl2−/+ females.

Tissue analysis and adenoma scoring

Organ sizes were determined by weighing or, in the case of the small intestine, by measuring the distance between stomach and cecum in intact dissected intestines. Tumors were scored in dissected intestines (including the colon) of 120-day-old mice as described (21). For immunohistochemistry of intestinal preparations, previously described procedures were followed, including fixation by methacarn or formalin (22). Staining was done with α-p4E-BP1 (Cell Signaling Technology) or other antibodies (see below). Crypt-enriched tissue lysates were generated as described (see also Supplementary Information; ref. 23).

Small interfering RNA knockdown, Western blot, and real-time quantitative PCR analysis

Small interfering RNA–mediated depletion of Dvl2, TOPFLASH luciferase reporter assays, Western blot, and real-time quantitative PCR analyses (Supplementary Figs. S1 and S3) were done as described (for details of cell lines used, see Supplementary Information; refs. 8, 24). To detect endogenous Dvl2 (Supplementary Fig. S1C; Fig. 1B), a rabbit antiserum was generated against human Dvl2 (amino acids 78–250), was affinity purified, and was characterized in overexpression and small interfering RNA–mediated depletion experiments (Graeb, data not shown) as described (8, 25).

Figure 1.

Dvl2 and Axin2 are overexpressed in colorectal tumors. A to C, immunohistochemical staining of TMA cores of normal colonic tissues, hyperplastic polyps, adenomatous polyps, and adenocarcinomas, as indicated underneath panels, fixed and stained with antibodies (left); arrowheads, nuclear β-catenin. Scale bars, 100 μm. D, boxplots representing the TMA scoring results; each individual tissue core is represented by a vertical line; boxes straddle the 25th to 75th percentiles (thick horizontal line, 50th percentile); outliers are above or below thin horizontal lines (90th and 10th percentiles, respectively).

Figure 1.

Dvl2 and Axin2 are overexpressed in colorectal tumors. A to C, immunohistochemical staining of TMA cores of normal colonic tissues, hyperplastic polyps, adenomatous polyps, and adenocarcinomas, as indicated underneath panels, fixed and stained with antibodies (left); arrowheads, nuclear β-catenin. Scale bars, 100 μm. D, boxplots representing the TMA scoring results; each individual tissue core is represented by a vertical line; boxes straddle the 25th to 75th percentiles (thick horizontal line, 50th percentile); outliers are above or below thin horizontal lines (90th and 10th percentiles, respectively).

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Colorectal tumor samples

Tissue samples for Western blots (Supplementary Fig. S1C) were collected from patients undergoing elective surgery for colorectal resections in accordance with standard procedures (26); ethical approval for this collection was granted by the United Bristol Hospital Trust Research and Development Ethical Committee (SU/2005/2114 and 05/Q2006/164). For analysis by immunohistochemistry (Supplementary Fig. S2; Figs. 1 and 6), two TMAs were constructed from multiple (24) replicate tissue cores from 64 patients undergoing colectomy resections for colorectal cancer at Addenbrookes Hospital, Cambridge, England4.

4Ibrahim et al., submitted for publication.

Ethical approval was obtained from the Cambridgeshire Local Research Ethics Committee (04/Q0108/125 and 06/Q0108/307). Samples were selected on the basis of availability of paraffin blocks with adequate cellularity. H&E-stained slides of all cases were reviewed, marked, and used to guide the sampling from morphologically representative regions of the tissue blocks. Five-micrometer sections were obtained from paraffin-embedded blocks and were deparaffinized and rehydrated with xylene and alcohol. Antigen retrieval was performed with EDTA buffer (pH 9.0) at 100°C for 20 mintues. The antibodies used were as follows: affinity-purified α-Dvl2 (1:50), α-β-catenin (1:200; BD Transduction Laboratories), α-Axin2 (1:500; Abcam), and α-pS6 (1:100; Cell Signaling Technology). A commercially available α-Dvl2 antiserum was also tested on some samples (1:100; AB5972, Millipore), with similar results as those obtained with our affinity-purified antibody. Antibody detection was done by streptavidin-biotin labeling, and visualization was done with diamino benzidine chromagen (DAKO). All slides were scored by individuals who were blinded to clinical outcome and other experimental data; strength of staining was scored semiquantitatively as negative (0), weakly positive (1), moderately positive (2), or strongly positive (3). β-Catenin staining was scored as percentage of positively labeled nuclei.

Endogenous Dvl2 is expressed at high levels in various colorectal cancer cell lines (Supplementary Fig. S1A). Furthermore, Dvl2 depletion by small interfering RNA reduced the β-catenin–specific transcription (measured by TOPFLASH luciferase reporter assays; ref. 27) by ∼50% (Supplementary Fig. S1B). This suggested that Dvl2 contributes to the β-catenin hyperactivation in colorectal cancer cells and prompted us to examine the Dvl2 expression levels in colorectal tumors. Screening total protein lysates from a small set of human colorectal carcinomas (n = 24) by Western blot analysis, we found that the Dvl2 levels were elevated in approximately one third of the carcinomas compared with their resection margin controls (Supplementary Fig. S1C). We thus proceeded to screen a TMA of 393 tissue cores from 64 patients presenting with colorectal cancer, including subsets of matched normal mucosa, hyperplastic and adenomatous polyps, and staged colorectal carcinomas, by staining them with affinity-purified antibody against Dvl2, and compared this to antibody staining against Axin2, a well-established universal Wnt/β-catenin target gene (28), and β-catenin itself, which accumulates in cell nuclei during the progression of colorectal cancer (29). We found that the number of β-catenin–positive nuclei increased in a stepwise fashion from normal tissue to carcinoma (Fig. 1A and D), with the majority of carcinomas showing significantly increased nuclear β-catenin compared with normal tissue (Wilcoxon rank sum, P < 2.2e-16). Nuclear β-catenin was also significantly increased within hyperplastic polyps compared with normal tissue (Wilcoxon rank sum, P = 5.817e-06), and even more so in adenomas (Wilcoxon rank sum, P = 8.774e-05; Fig. 1A), indicative of their high β-catenin–mediated transcriptional activity, due to their APC mutations typically observed in >80% of adenomas (29). These results support the widely held view that APC mutation alone can cause nuclear accumulation of β-catenin, and argue against the notion that the latter requires, in addition, an activating KRAS mutation (30). As expected from the nuclear β-catenin, Axin2 has a highly significant tendency to be overexpressed in hyperplastic polyps and adenoma compared with normal tissue (Wilcoxon rank sum, P = 6.333e-07 and 3.655e-05, respectively), which increases even further in carcinomas (Wilcoxon rank sum, P = 7.444e-05; Fig. 1B and D). In turn, the pattern of increasing Axin2 expression through the tumor progression from benign to malignant is closely mirrored by Dvl2, whose levels also increase significantly (Wilcoxon rank sum, P = 3.953e-12) from moderate in hyperplastic polyps and adenomas to high in carcinomas, in which it exhibits a punctate cytoplasmic staining pattern (Supplementary Fig. S2; Fig. 1C and D). Indeed, there is a remarkable correlation between the Dvl2 and Axin2 expression levels in the different tumor stages (Pearson's correlation, r = 0.5394386; P < 2.2e-16), indicating that Dvl2 may be upregulated, along with Axin2, in response to APC loss from the onset of colorectal tumorigenesis. Consistent with this observation, we found that stimulation of HEK293 cells by Wnt3a causes an increase of endogenous Dvl2 protein levels, although its transcript levels remain unchanged (Supplementary Fig. S3). Thus, Dvl2 can be upregulated posttranscriptionally upon Wnt stimulation, providing a possible explanation of why this protein accumulates in cancer cells whose Wnt/β-catenin pathway is hyperactive.

Next, we tested whether Dvl2 contributes to the β-catenin–dependent intestinal tumorigenesis in the ApcMin model, that is, whether Dvl2 loss would suppress the intestinal tumor load in these mutants. ApcMin/+ (called Min/+) mice develop numerous intestinal tumors over the course of 3 to 4 months (5), likely reflecting a β-catenin–dependent transcriptional switch in the intestinal epithelium (22, 31). Dvl2 homozygosity causes various embryonic and perinatal defects; however, 50% of these Dvl2−/− mice survive and develop into apparently normal healthy adults (19). We thus generated Min/+ Dvl2+/− and Min/+ Dvl2−/− compound mutant mice, and found that the adenoma numbers of 120-day-old mice were reduced significantly in a Dvl2 dose–dependent manner, that is, noticeably in Dvl2+/− (P = 0.038, two-tailed t test; Fig. 2A), and even more so in Dvl2−/−, on average to ∼55% of their matched Dvl2+/+ controls (P = 0.0065, two-tailed t test; Fig. 2A). The disease onset in Dvl2−/− may also be slightly delayed compared with the other two experimental cohorts, as revealed by Kaplan-Meier survival plots (Fig. 2B); although this delay is not statistically significant, due to the high intrinsic variation of disease onset, the observed delay may nevertheless be indicative of the reduced tumor numbers in some of the animals. Our results identify Dvl2 as a contributor to the intestinal tumor incidence in this mouse model. Notably, the Dvl2−/− mice retain the function of two Dvl paralogs, Dvl1 and Dvl3, each of which shares overlapping redundant functions with Dvl2 (double mutants being embryonic lethal; refs. 19, 20). Therefore, the functional contribution of Dvl2 to intestinal neoplasia (Fig. 2) is likely an underestimate of the overall Dvl function in this process. Indeed, we detect transcripts of both paralogs, Dvl1 and Dvl3, in lysates of wt and Dvl2−/− mutant intestinal epithelia (Supplementary Fig. S4). Technical difficulties with the available antibodies prevented us from assessing the Dvl protein levels in intestinal lysates, but our transcript data suggest that the total Dvl function may be reduced by approximately half in the Dvl2−/− mutant intestine.

Figure 2.

Dvl2 deficiency reduces the intestinal tumor numbers in ApcMin mice. A, adenoma counts in 120-d-old ApcMin Dvl2+/+ (n = 16), ApcMin Dvl2−/+ (n = 23), and ApcMin Dvl2−/− (n = 9) mice; each square represents one mouse; horizontal lines, SEM; *, statistical significance. B, Kaplan-Meier survival plots of all three cohorts.

Figure 2.

Dvl2 deficiency reduces the intestinal tumor numbers in ApcMin mice. A, adenoma counts in 120-d-old ApcMin Dvl2+/+ (n = 16), ApcMin Dvl2−/+ (n = 23), and ApcMin Dvl2−/− (n = 9) mice; each square represents one mouse; horizontal lines, SEM; *, statistical significance. B, Kaplan-Meier survival plots of all three cohorts.

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While scoring tumors, we noticed that the small intestines of the Dvl2−/− mice were significantly shorter than those of their controls (P < 0.0001, t test; Fig. 3A). This gut shortening to 75% ± 2% (mean ± SD) of normal length is fully penetrant and highly consistent between individuals. It is also observed in a normal Min+/+ background and is already manifest at 8 days of age (Fig. 3B). The circumference of the Dvl2−/− mutant intestines appears normal (6–7 mm in wt and mutants, upon opening up and flattening out), although the accuracy of these measurements is limited to ±0.5 mm. The body weights of the mutants are also normal and so are their organ weights (Table 1), possibly because each of the organs assessed expresses at least one of the two Dvl2 paralogs at high levels (32). Thus, the small intestine appears to be particularly vulnerable to the loss of Dvl2. Evidently, intestinal length and tumor numbers represent functional readouts that are sensitive to partial Dvl loss.

Figure 3.

Dvl2 mutants have short guts. Length measurements of the small intestines from Dvl2−/− and littermate controls (n > 3) at (A) 120 d and (B) 8 or 30 d after birth; statistical significance and symbols as in Fig. 2 (see also text).

Figure 3.

Dvl2 mutants have short guts. Length measurements of the small intestines from Dvl2−/− and littermate controls (n > 3) at (A) 120 d and (B) 8 or 30 d after birth; statistical significance and symbols as in Fig. 2 (see also text).

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Table 1.

Intestine lengths, crypt diameters, and organ weights of wt and Dvl2 mutant mice

Dvl2 (+/+)Dvl2 (+/−)Dvl2 (−/−)Dvl2 (−/−)/Dvl2 (+/+) (percent)P (t test)
Small intestine (cm) 34.1 ± 0.70 34.9 ± 0.55 25.5 ± 0. 26 75 P < 0.005 
Crypt width (μm) 41.63 ± 0.42 — 38.63 ± 0.53 93 P < 0.005 
Spleen (g) 0.09 ± 0.00 — 0.09 ± 0.01 100 — 
Kidneys (g) 0.26 ± 0.01 — 0.29 ± 0.03 111 — 
Liver (g) 1.11 ± 0.12 — 1.10 ± 0.27 99 — 
Heart (g) 0.12 ± 0.00 — 0.12 ± 0.00 100 — 
Dvl2 (+/+)Dvl2 (+/−)Dvl2 (−/−)Dvl2 (−/−)/Dvl2 (+/+) (percent)P (t test)
Small intestine (cm) 34.1 ± 0.70 34.9 ± 0.55 25.5 ± 0. 26 75 P < 0.005 
Crypt width (μm) 41.63 ± 0.42 — 38.63 ± 0.53 93 P < 0.005 
Spleen (g) 0.09 ± 0.00 — 0.09 ± 0.01 100 — 
Kidneys (g) 0.26 ± 0.01 — 0.29 ± 0.03 111 — 
Liver (g) 1.11 ± 0.12 — 1.10 ± 0.27 99 — 
Heart (g) 0.12 ± 0.00 — 0.12 ± 0.00 100 — 

NOTE: Measurements of small intestines (n > 10 per genotype) and organ weights (n = 3 per genotype) from 120 day-old mice. Crypt diameters (n = 132, 3 mice per genotype) were calculated with AxioVision software (Zeiss); ± represents standard error.

To examine the underlying defect of the “short gut” phenotype, we examined longitudinal sections through wt and Dvl2−/− mutant small intestines, both of which revealed a normal overall tissue architecture (Fig. 4A–C). However, the diameters of the mutant intestinal crypts appeared slightly reduced (Fig. 4A, arrows; also compare B and C). To quantify this effect, we measured the diameters of individual crypts (n = 111, 4 mice per genotype) selected on the basis of their orientation parallel to the sectional plane, but blinded to the genotype, and found that the crypt diameters are reduced in the Dvl2−/− samples, on average to 93% of their matched wt samples (Table 1; Fig. 4D). Although relatively small, this reduction is highly statistically significant (P < 0.0001, t test). It likely contributes to the shortened intestines of the Dvl2−/− mutants, but does not fully account for this phenotype. Indeed, we estimate that the reduced crypt diameters could account for ∼30% of the total length reduction (2.6 of 9 cm) seen in 4-month-old mice. The remainder is most likely due to fewer crypts; based on our measurements of gut length, circumference, and crypt diameter, we estimate that the total numbers of crypts in the small intestine are reduced to between 93% and 75% of the wt. Notably, each crypt contains a few long-lived stem cells with tumor-forming potential (33), so lower crypt numbers in the Dvl2−/− mutants could explain at least partly why they develop fewer tumors (although we could not detect a significant reduction of bromodeoxyuridine-incorporating cells in the mutants; Supplementary Fig. S5).

Figure 4.

Dvl2 mutants have reduced crypt diameters. A to C, longitudinal sections through the small intestine of a 2-mo-old Dvl2−/− and littermate controls (A) after H&E staining; arrows, individual narrow crypts in the mutant. Scale bars, 100 μm; B and C, immunofluorescence images, revealing decreased crypt diameters in Dvl2−/− compared with littermate controls, as measured along apicobasal axis of intestinal crypt cells (between asterisks); blue, 4′,6-diamidino-2-phenylindole staining; green, α-β-catenin staining; scale bars, 25 μm. D, measurements of crypt diameters (n = 111) from sections as shown in A (four mice per genotype); small squares, mean; bars, SEM; *, statistical significance.

Figure 4.

Dvl2 mutants have reduced crypt diameters. A to C, longitudinal sections through the small intestine of a 2-mo-old Dvl2−/− and littermate controls (A) after H&E staining; arrows, individual narrow crypts in the mutant. Scale bars, 100 μm; B and C, immunofluorescence images, revealing decreased crypt diameters in Dvl2−/− compared with littermate controls, as measured along apicobasal axis of intestinal crypt cells (between asterisks); blue, 4′,6-diamidino-2-phenylindole staining; green, α-β-catenin staining; scale bars, 25 μm. D, measurements of crypt diameters (n = 111) from sections as shown in A (four mice per genotype); small squares, mean; bars, SEM; *, statistical significance.

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The crypt diameter can be taken as a measure of cell size, specifically of the apicobasal axis of individual crypt cells, visualized by staining of the membrane-associated β-catenin (Fig. 4B and C), whose length seemed reduced in Dvl2−/− mutant crypts (data not shown), suggesting that Dvl2 may promote cell size (or growth) in intestinal crypts. Cell size is controlled primarily by the mTOR signaling pathway and its well-established S6 kinase effector arm that results in phosphorylation of ribosomal protein S6 (pS6; ref. 17). mTOR can be activated by several growth factors and kinases (17), for example, by Ras signaling (34), but also by Wnt/Dvl signaling, which was reported to affect cell size in tissue culture (35). Interestingly, high levels of pS6 staining have been observed in normal murine intestinal crypts and in Apc mutant intestinal tumors; furthermore, mTORC1 transcription depends on β-catenin in APC mutant colorectal cancer cells (18).

We thus asked whether the reduced crypt diameters in the Dvl2 mutants might be due to reduced mTOR signaling, by staining histologic sections of intestinal preparations with antibodies against pS6. We confirmed that the crypts and adenomas are generally positive for this mTOR signaling readout (Supplementary Fig. S6; ref. 18), although the staining was somewhat variable, and depended on the type of fixation. We chose to examine the phosphorylation of 4E-BP1 (p4E-BP1), an equally well-established readout of mTOR signaling that controls translational initiation through eIF-4E (17) and is thought to be important in oncogenesis (34). These p4E-BP1 stainings turned out to be far more robust; we observe highly restricted p4E-BP1 staining throughout normal crypts, apparently in every cell (Fig. 5A). Likewise, every single adenoma shows p4E-BP1 staining in most if not all cells (Fig. 5B). Indeed, we used p4E-BP1 staining to identify nascent polyps, appearing as tubes within a single villus as previously described (Fig. 5C; ref. 36). To confirm their identification, we stained adjacent sections for β-catenin, which was nuclear throughout the polyp, in every cell (Fig. 5D), again, arguing against the notion that APC loss is insufficient to cause nuclear accumulation of β-catenin (30). Thus, mTOR signaling is activated with full penetrance throughout normal crypts and in every adenoma. Indeed, the p4E-BP1 staining is a diagnostic marker for adenomas in the ApcMin model. Given that virtually all adenomas in Apc mutant mice show Apc inactivation (36), this strongly supports the notion that the activation of mTOR signaling in adenomas is a direct consequence of β-catenin–dependent transcription due to Apc loss (18). Notwithstanding this, we were unable to detect a consistent reduction of pS6 or p4E-BP1 staining in normal crypts or adenomas of Dvl2−/− mice compared with their controls (Supplementary Fig. S7A and B; data not shown), although we found a slight reduction of pS6 levels in crypt-enriched intestinal lysates from Dvl2−/− versus Dvl2+/− littermate controls by Western blot analysis (Supplementary Fig. S7C). Given the redundancy problem with Dvl2 paralogs, this is perhaps not surprising; indeed, Wnt/β-catenin signaling was not detectably reduced in embryos even upon simultaneous knockout of two Dvl paralogs (19, 20). In addition, a subtle attenuation of mTOR signaling in Dvl2−/− mutants would be difficult to detect by immunohistochemistry.

Figure 5.

mTOR signaling is induced in murine intestinal crypts and in nascent polyps. A and B, longitudinal sections through (A) normal crypts and (B) a small adenoma in the small intestine of 120-d-old ApcMin mice, stained for p4E-BP1; strong signals are characteristic for normal crypts and adenomas. C and D, high magnification views of adjacent sections through a nascent polyp in a 120-d-old ApcMin mouse, stained for (C) p4E-BP1 and (D) β-catenin, revealing nuclear β-catenin in every cell (arrowheads). Scale bars, 100 μm.

Figure 5.

mTOR signaling is induced in murine intestinal crypts and in nascent polyps. A and B, longitudinal sections through (A) normal crypts and (B) a small adenoma in the small intestine of 120-d-old ApcMin mice, stained for p4E-BP1; strong signals are characteristic for normal crypts and adenomas. C and D, high magnification views of adjacent sections through a nascent polyp in a 120-d-old ApcMin mouse, stained for (C) p4E-BP1 and (D) β-catenin, revealing nuclear β-catenin in every cell (arrowheads). Scale bars, 100 μm.

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Notably, both Dvl2 loss and mTOR inhibition have comparable tumor-suppressive effects in the ApcMin model; oral administration of the mTOR inhibitor RAD001 to ApcMin mice reduces their intestinal tumor numbers by ∼50% (Supplementary Fig. S8A), similar to Dvl2 homozygosity (Fig. 2A), although again, we cannot detect a robust reduction of mTOR signaling in adenomas of treated mice compared with their controls, by staining these with p4E-BP1 or pS6 antibodies (Supplementary Fig. S8B and C; data not shown). Our findings with RAD001 confirm earlier results from this mTOR inhibitor in a different Apc mutant model (18) and reinforce the conclusion that the high mTOR signaling levels observed in crypts or adenomas promote the intestinal tumorigenesis driven by Apc loss.

Given the fully penetrant activation of mTOR signaling in murine adenomas, we also screened our TMA of human colorectal tumors with pS6 antibody (and with p4E-BP1 antibody, although this did not produce reliable staining of these samples). Although we observe very low pS6 signals in normal intestinal mucosa (Fig. 6A, left), hyperplastic polyps consistently show high levels of pS6 staining, apparently in every single cell (Fig. 6A, right), thus mirroring the murine adenomas. mTOR signaling is therefore a hallmark of these polyps and may be a direct consequence of activating mutations in their KRAS/BRAF signaling pathway, as often found in these polyps (37). Indeed, the cells in the hyperplastic polyps are visibly larger than those in the adjacent normal epithelium (Fig. 6A and B), suggesting that their growth is stimulated by their mTOR signaling. Adenomas and carcinomas also have a high tendency to show strong pS6 staining (Fig. 6C), although on average, their probability of elevated mTOR activity is lower than that of the hyperplastic polyps, with approximately one and two thirds of all adenomas and carcinomas, respectively, showing robust pS6 staining (Fig. 6D). Basically the same was found with cyclin D1 (data not shown), another mTOR signaling target whose translational stimulation requires phosphorylated 4E-BP1 (38). Thus, the mTOR signaling pathway has a significant tendency to remain active throughout the progression of colorectal cancer.

Figure 6.

mTOR signaling is elevated in human colorectal tumors. A to C, TMA cores of (A) normal mucosa (left) and matched hyperplastic polyp (right), and (B) adenoma (left) compared with adenocarcinoma (right), stained for pS6; C, high magnification views, as in A, of a different matched sample pair. D, boxplots of the α-pS6 TMA scoring results, as in Fig. 1D.

Figure 6.

mTOR signaling is elevated in human colorectal tumors. A to C, TMA cores of (A) normal mucosa (left) and matched hyperplastic polyp (right), and (B) adenoma (left) compared with adenocarcinoma (right), stained for pS6; C, high magnification views, as in A, of a different matched sample pair. D, boxplots of the α-pS6 TMA scoring results, as in Fig. 1D.

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Our work provides two lines of evidence for a tumor-promoting role of Dvl2 in colorectal cancer. First, the Dvl2 protein levels are elevated through the cancer progression, closely correlating with Axin2 protein levels that increase in parallel (Fig. 1). Therefore, Dvl2 may be upregulated, in parallel to Axin2, as a direct result of APC loss. However, whereas the upregulation of Axin2 is likely to be due to transcriptional stimulation by β-catenin (28), that of Dvl2 may occur at the posttranscriptional level (Supplementary Fig. S3), although we note that the transcript levels of Dvl2 are also elevated >2× in response to Apc inactivation throughout the intestinal epithelium (6). Importantly, given that Dvl2 causes β-catenin accumulation upon overexpression, in the absence of a Wnt signal (9), this implies that the high Dvl2 levels in colorectal carcinomas contribute to, or maintain, the high levels of (nuclear) β-catenin at advanced stages. Second, Dvl2 deficiency reduced the tumor load in ApcMin mutant mice (Fig. 2), providing experimental evidence for its tumor-promoting role in this mouse model for colorectal cancer. The reduced tumor numbers in the Dvl2−/− mutants could be partly due to reduced crypt numbers and partly to reduced crypt cell growth (see below). Notably, overexpression of Dvl paralogs has been observed in cervical carcinomas (39) and seems to contribute to the pathogenesis of mesothelioma and small cell lung cancer (40, 41). Furthermore, Dvl proteins seem to become hyperactive in colorectal cancer cells due to transcriptional silencing of their inhibitor DACT3 (42). These results, together with our own work, highlight the potential of Dvl2 as a therapeutic target in cancers driven by hyperactive Wnt/β-catenin signaling.

Perhaps our most interesting result was that Dvl2 deficiency reduces the length of the small intestine. This mutant phenotype results in part from a reduction of the crypt numbers, but also from a reduced crypt diameter, itself a measure of crypt cell size. Given the normal role of Dvl in transducing Wnt signals to β-catenin, it is possible that some or all aspects of this mutant phenotype are a consequence of attenuated β-catenin–mediated transcription. In particular, the narrowed crypts may reflect attenuated mTOR signaling, given that (a) mTOR signaling commonly regulates cell size (17); (b) mTOR signaling is high in normal crypts; and (c) signaling through this pathway is stimulated by Dvl and β-catenin, through transcriptional stimulation of mTORC1 (18, 35). Our inability to detect a robust reduction of mTOR signaling in the Dvl2−/− mutant crypts could be due to the above-mentioned redundancy problem; also, we may not have analyzed mTOR signaling during the critical time window, or in the critical subset of cells, responsible for the narrow crypt and/or short gut phenotype. Interestingly, a small cell phenotype was also observed upon conditional deletion of c-myc (43), a key transcriptional target of hyperactive Wnt/β-catenin signaling in murine and human intestinal epithelial cells (21, 44), indicating that Wnt/β-catenin signaling could affect cell size. Indeed, Wnt/Dvl and mTOR signaling might act synergistically on common targets (sequentially, or in parallel): intriguingly, many of the reported transcriptional targets of Wnt/β-catenin signaling (including c-myc, cyclin D1, vascular endothelial growth factor, survivin, and matrix metalloproteinase 9) have independently been identified as translational targets of the mTOR/eIF-4E pathway (e.g., refs. 34, 45). Furthermore, although cyclin D1 is thought to be a transcriptional target of β-catenin (2), cyclin D protein rather than transcript levels are upregulated in the murine intestine upon Apc inactivation (46) and in Wnt-stimulated tissue culture cells (35). Thus, the transcriptional changes induced by Wnt/Dvl signaling may generally be accompanied by mTOR-dependent translational changes.

Alternatively, it is also possible that the shortened intestines in the Dvl2−/− mutants reflect one of the β-catenin–independent (“noncanonical”) Dvl functions (7). We note that gut elongation is compromised in Wnt5a and Ror2 knockout mice, along with other noncanonical Wnt signaling defects, although in both mutants, the short gut phenotypes result from gross morphologic abnormalities in the early embryonic midgut primordium, such as convergent extension defects (47, 48).

It is striking that the mTOR signaling pathway is upregulated not only in normal murine intestinal crypts and in all intestinal adenomas (Supplementary Figs. S6–8; Fig. 5B and C; ref. 18), but also in human hyperplastic polyps, with a significant tendency of being active also in adenomas and colorectal carcinomas (Fig. 6C and D), strongly supporting the notion that mTOR has potential as a therapeutic target in colorectal cancer (18). Indeed, we extended the results of these authors, showing efficacy of the mTOR inhibitor RAD001 in reducing the intestinal tumor load in a different Apc model. mTOR inhibitors have been in use clinically as immunosuppressants for many years and have begun to show great promise in cancer treatment, in particular of renal cell carcinomas, but also in other types of solid tumors (49). Our findings, together with previous results (18), provide evidence for a future application of these inhibitors in colorectal cancers, at least in those prescreened for mTOR signaling readouts, and possibly also in young familial adenomatous polyposis patients, carriers of APC germ line mutations, which develop thousands of adenomas before reaching adulthood (50).

No potential conflicts of interest were disclosed.

We thank Tony Wynshaw-Boris for the Dvl2 knockout strain; Owen Samson for the technical advice regarding the analysis of mouse intestines and for countless stimulating discussions; Tracey Butcher and her animal staff; and Paul Sylvester, Rob Longman, and Paul Durdey for their assistance.

Grant Support: Cancer Research UK (grant no C7379/A8709 M. Fiedler and M. de la Roche, and M. Bienz) and a Clinician Scientist Fellowship from Cancer Research UK (A.E.K. Ibrahim). Core support was provided by the Medical Research Council (U1051030200000101).

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.

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