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Proc Natl Acad Sci U S A. Sep 9, 2008; 105(36): 13544–13549.
Published online Sep 3, 2008. doi:  10.1073/pnas.0800041105
PMCID: PMC2533226
Medical Sciences

Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcΔ716 mice


The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth via mTOR complex 1 (mTORC1), whose activation has been implicated in many human cancers. However, mTORC1's status in gastrointestinal tumors has not been characterized thoroughly. We have found that the mTORC1 pathway is activated with increased expression of the mTOR protein in intestinal polyps of the ApcΔ716 heterozygous mutant mouse, a model for human familial adenomatous polyposis. An 8-week treatment with RAD001 (everolimus) suppressed the mTORC1 activity in these polyps and inhibited proliferation of the adenoma cells as well as tumor angiogenesis, which significantly reduced not only the number of polyps but also their size. β-Catenin knockdown in the colon cancer cell lines reduced the mTOR level and thereby inhibited the mTORC1 signaling. These results suggest that the Wnt signaling contributes to mTORC1 activation through the increased level of mTOR and that the activation plays important roles in the intestinal polyp formation and growth. Indeed, long-term RAD001 treatment significantly reduced mortality of the ApcΔ716 mice. Thus, we propose that the mTOR inhibitors may be efficacious for therapy and prevention of colonic adenomas and cancers with Wnt signaling activation.

Keywords: adenoma, colon cancer, mammalian target of rapamycin, RAD001, rapamycin

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that belongs to the phosphoinositide 3-kinase related kinase family. mTOR plays essential roles in regulation of cell growth, proliferation, and motility as a component of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of mTOR, raptor, and mLST8, and Rheb-induced activation of mTORC1 enhances translation of a subset of mRNA through phosphorylation of S6 kinase and 4E-BP1, which induces cell growth (1). mTORC1 is regulated by various upstream signals including those emanated by growth factors, nutrients, energy, and stress. mTORC1 is activated in various types of neoplastic diseases, especially in those with constitutive activation of the PI3K–Akt pathway. On the other hand, mTORC2 is composed of mTOR, rictor, and mLST8 and plays important roles in Akt phosphorylation at Ser-473 (2) and in the regulation of actin cytoskeleton (3).

Rapamycin, a natural product derived from a bacterial species (Streptomyces hygroscopicus), is currently used for prevention of allograft rejection in organ transplants. It has been shown that rapamycin first binds to FKBP12, and the FKBP/rapamycin complex then binds and inhibits mTORC1, but not mTORC2. In vitro studies have shown that mTORC1 inhibitors induce cell-cycle arrest in various cell types, including many cancer cell lines and endothelial cells. Rapamycin-induced apoptosis has also been demonstrated for several cancer cell lines (4). In addition, anticancer activity of mTORC1 inhibitors has been established in in vivo studies using xenograft models in mice and gene-targeted or transgenic mice that spontaneously develop tumors caused by activation of the PI3K/Akt pathway (5, 6). Based on these results, many clinical trials with these drugs aimed at treatment of various malignancies including lymphoma, sarcoma, and glioblastoma (7) are in progress.

Colorectal cancer is one of the leading causes of cancer deaths. Most human colorectal cancers suffer somatic mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, which leads to activation of the Wnt signaling via β-catenin stabilization. Accumulated β-catenin then translocates to the nucleus where it binds and activates TCF/LEF transcription factors (8). Mutation of the APC gene appears to be the triggering event in colorectal tumorigenesis, and its germ-line mutations cause intestinal polyposis in both humans and mice (9).

In the present study, we have demonstrated that the mTORC1 pathway is activated in intestinal polyps of ApcΔ716 mice, a mouse model of familial adenomatous polyposis (FAP). A novel mTOR inhibitor RAD001 (everolimus) showed marked antitumor effects in these mice, targeting both polyp epithelial cells and vascular endothelial cells. We further show that the mTOR protein level is regulated by β-catenin, which may account for the mTORC1 activation in colon polyps and cancers with β-catenin stabilization.


The mTOR Signaling Pathway Is Activated in the ApcΔ716 Intestinal Polyps.

To investigate the activation status of the mTOR signaling pathway in intestinal polyps induced by Wnt signaling activation, we examined phosphorylation of S6, which is catalyzed by S6 kinase in an mTOR-dependent manner (10), in the intestinal polyps and the normal ileum in ApcΔ716 mice. Western blot analysis showed that the S6 phosphorylation was elevated in the ileal polyps as compared with the normal ileum (Fig. 1A). Immunostaining revealed that phospho-S6 (p-S6) was expressed predominantly in adenoma epithelial cells of the polyps (Fig. 1B). In the normal ileum, S6 phosphorylation was found mainly in the crypt epithelial cells, with occasional signals in the villus epithelial cells (Fig. 1C). To test whether the increased S6 phosphorylation in the intestinal polyps depends on the mTOR signaling pathway, we treated ApcΔ716 mice with RAD001 (10 mg/kg) for 3 days. Phosphorylation of S6 in the normal ileum and adjacent polyps of ApcΔ716 mice was strongly inhibited by administration of RAD001 (Fig. 1 D–F). These results indicate that the mTORC1 pathway is strongly activated in the intestinal polyps compared with the normal mucosa in the ApcΔ716 mouse.

Fig. 1.
Activation of the mTOR signaling pathway in ApcΔ716 mouse polyps. (A) Western blot analysis of S6 phosphorylated at Ser-235/236 (p-S6) and total S6 in the normal ileal mucosa and polyps of the ApcΔ716 mouse. COX-2 is known to be expressed ...

Treatment with RAD001 Inhibits Polyp Formation and Reduces Mortality in the ApcΔ716 Mouse.

The activation of the mTORC1 pathway in the ApcΔ716 intestinal polyps suggested that mTORC1 inhibitors might suppress intestinal polyp formation. To investigate the role of the mTORC1 pathway activation in polyp formation, we treated ApcΔ716 mice with RAD001 for 8 weeks from 6 to 14 weeks of age (Fig. 2A). Treatment with RAD001 significantly inhibited both polyp formation [number (Fig. 2B1)] and polyp expansion [size (Fig. 2 B2 and B3)] in ApcΔ716 mice in a dose-dependent manner (Fig. 2 B and C). Large-size polyps, particularly those >1.5 mm in diameter, were not found in the RAD001-treated ApcΔ716 mice (Fig. 2 B and C). Interestingly, RAD001-treated mouse polyps showed a collapsed morphology at the top (Fig. 2 D and E), reminiscent of that of COX-2 inhibitor-treated polyps (11). These results suggest that mTORC1 pathway activation is essential for tumor expansion in the ApcΔ716 intestinal polyps. To investigate whether RAD001 treatment could cause regression of already-formed polyps, we also treated older ApcΔ716 mice from 13 to 21 weeks of age that had already developed large polyps. RAD001 treatment in these ApcΔ716 mice also reduced the number of large-size polyps (> 1.5 mm) [supporting information (SI) Fig. S1]. To test whether RAD001-treatment would improve mortality of ApcΔ716 mouse, we examined the effect of long-term treatment of the ApcΔ716 mouse with RAD001. Although all of the placebo-treated mice became moribund within 30 weeks of age, 50% of the mice treated with RAD001 at 10 mg/kg lived >1.5 years (Fig. 2F). These results indicate that activation of the mTORC1 pathway plays important roles in polyp formation at both the initiation and expansion steps, and that inhibition of the mTORC1 pathway may be an effective treatment for FAP patients.

Fig. 2.
RAD001 suppresses polyp formation with significant effects on survival in the ApcΔ716 mouse. (A) Schematic diagram of the RAD001 treatment schedule. Mice were treated with RAD001 once a day for 8 weeks. (B) Number of polyps per mouse scored ( ...

Inhibitory Effect of RAD001 on Polyp Formation Is Attributable to Inhibition of Tumor Cell Proliferation.

Effects of mTORC1 inhibitors on cell growth are known to differ among cancer cell lines (4). To gain insights into the mechanism of the polyp inhibition by RAD001, we evaluated in vivo cell proliferation and apoptosis in RAD001-treated polyps by BrdU incorporation and TUNEL assay, respectively. Treatment with RAD001 reduced the BrdU labeling index of the adenoma cells by 60% (Fig. 3 A and B). In contrast, TUNEL assay did not show any significant difference in the apoptotic frequency of the polyps between the RAD001-treated and placebo-treated mice (Fig. 3 C and D).

Fig. 3.
RAD001 attenuates adenoma cell growth but does not induce apoptosis. (A) Photographs of the small intestinal adenoma epithelium labeled with BrdU in placebo-treated (Left) and RAD001-treated (Right) ApcΔ716 mouse. (B) BrdU labeling indices of ...

Treatment with rapamycin can reduce expression of cyclin D, cyclin E, and cyclin A in NIH 3T3 and human B cells (12, 13). Consistently, Aoki et al. (14) reported that activation of the mTOR pathway accelerated cell-cycle progression from G1 to S in DLD-1 cells. Because these results suggested that RAD001 affected cell-cycle progression in adenoma cells, we then examined expression of cyclins in the polyps of RAD001-treated ApcΔ716 mice. Although the level of cyclin D protein in the polyps was not affected by treatment with RAD001 (data not shown), those of cyclin A and cyclin E were elevated 3.2 and 4.0 times, respectively, in the polyps of placebo-treated mice, whereas expression of cyclin E in the polyps was reduced to 33% of the placebo control, even only after 3 days of treatment (Fig. 3E). Cyclin A expression was reduced by 45% in the polyps of ApcΔ716 mice treated with RAD001 for 8 weeks (Fig. 3E). These results show that inhibition of polyp formation by RAD001 is associated with inhibition of adenoma cell proliferation in vivo without affecting their apoptosis.

Treatment with RAD001 Inhibits Tumor Angiogenesis.

Treatment with RAD001 caused regression of the already-formed polyps (Fig. S1). Furthermore, some large polyps in the ApcΔ716 mice treated with RAD001 showed a collapsed morphology at the top (Fig. 2 D and E). These results suggest that RAD001 may possess other effects than inhibition of adenoma cell proliferation, by which it causes regression of the preexisting polyps in ApcΔ716 mice. Guba et al. (15) reported that rapamycin treatment caused regression of transplanted CT-26, a mouse colon cancer cell line, through inhibition of tumor cell-induced angiogenesis. Thus, we examined angiogenesis in RAD001-treated ApcΔ716 mice. Treatment for 4 weeks significantly reduced the number of microvessels in the polyps (placebo, 23 ± 3.0 vs. RAD001, 17 ± 3.1) without affecting their numbers in the normal intestine (placebo, 16 ± 1.4 vs. RAD001, 15 ± 1.2) (Fig. 4 A and B). Several studies showed that mTOR inhibitors could reduce not only tumor cell growth but also angiogenesis through suppression of vascular endothelial growth factor (VEGF) expression. Because treatment with anti-VEGF-A mAb inhibited adenoma cell growth in Apcmin/+ mice (16), treatment with RAD001 might inhibit polyp formation in ApcΔ716 mice also through suppression of VEGF expression. However, there was no significant difference in the VEGF expression levels in polyps between placebo- and RAD001-treated ApcΔ716 mice (Fig. 4C). In addition, preliminary determination of the expression levels of various angiogenesis-related factors, including bFGF and insulin-like growth factor (IGF) using an antibody array, revealed no significant difference in the levels of such factors in the polyps between placebo-treated and RAD001-treated ApcΔ716 mice (data not shown). These results suggest that the intestinal polyp inhibition by RAD001 was independent of the suppression of angiogenesis-related factors such as VEGF in ApcΔ716 mice. It is also reported that rapamycin directly inhibits endothelial cell growth (17, 18). Accordingly, we examined p-S6-positive endothelial cells in adenoma blood vessels by double immunostaining for p-S6, and CD31, a marker of endothelial cells. About 10% of the angiogenic vessels in adenomas were positively stained for p-S6 (Fig. 4D). However, no endothelial cells in the normal villi or crypts showed S6 phosphorylation [normal intestine 0% vs. adenoma 10 ± 2.8% (Fig. 4D)]. Phosphorylation of S6 in the angiogenic vessels of the polyps disappeared after the RAD001 treatment (Fig. 4D). These results suggest that RAD001 may directly inhibit angiogenic vessels through suppression of the mTOR pathway and thereby reduce blood vessel formation, leading to regression of the already-formed polyps.

Fig. 4.
Treatment with RAD001 inhibits angiogenesis in the polyps of ApcΔ716 mouse. (A) Immunostaining of von Willebrand Factor (vWF) in the luminal side of a small intestinal polyp of placebo-treated (Left) and RAD001-treated (Right) ApcΔ716 ...

The Wnt Signaling Stimulates the mTOR Pathway.

mTORC1 is stimulated by various upstream signals, including those emanated by growth factors, nutrients, and energy, among which the PI3K–Akt signaling pathway is the most prominent (1). To investigate the mechanism of the mTOR pathway activation in the polyps of ApcΔ716 mice, we first examined whether PI3K pathway inhibition would affect the mTOR pathway activation status in these polyps by treating ApcΔ716 mice with wortmannin, a potent PI3K inhibitor. Although treatment with RAD001 for 3 days could strongly inhibit S6 phosphorylation, wortmannin failed to suppress S6 phosphorylation in the ApcΔ716 mouse, even at a dose sufficient to inhibit Akt phosphorylation (Fig. S2A). These results indicate that pathways other than the PI3K–Akt pathway activate the mTORC1 signaling in the intestine of the ApcΔ716 mice. In addition to PI3K–Akt, the Raf-Mek1/2-Erk1/2 activation or AMP-activated protein kinase (AMPK) inhibition can activate the mTORC1 signaling (19, 20). However, phosphorylation of Erk1/2 at Thr-202/Tyr-204 was reduced to 33% in the polyps as compared with the normal tissue, suggesting that the Erk pathway was not activated in the polyps (Fig. S2B). On the other hand, AMPK phosphorylation at Thr-172 was elevated 3.3 times in the polyps, suggesting that the AMPK pathway was not suppressed (Fig. S2C). These results indicate that mTORC1 pathway activation in the ApcΔ716 intestinal polyps is independent of Erk and AMPK signaling. Nutrients such as leucine can also activate the mTORC1 pathway (21). Starved WT mice showed reduction in the S6 phosphorylation level in the normal intestinal epithelium compared with free-feeding WT mice. In contrast, starved ApcΔ716 mice did not exhibit any reduction in the S6 phosphorylation level in the polyps compared with the normal tissues, suggesting that the mTORC1 pathway in the polyps is independent of nutrient status (Fig. S2D). These results indicate that mTORC1 is constitutively activated in the polyps of ApcΔ716 mice.

To explore the activation mechanism of the mTORC1 pathway, we determined the mTOR expression levels in the polyps and normal intestine of ApcΔ716 mice by Western blotting and immunostaining. Expression of mTOR protein was higher in the polyps than in the normal ileum (Fig. 5A). An immunohistochemical analysis showed that mTOR protein was expressed strongly in the adenoma epithelium and in the proliferative zone of crypts where Wnt signaling was activated (Fig. S3 A–D). Increased expression of mTOR protein has been reported for several types of human tumors, including colon cancer (22, 23), and reduced expression of mTOR protein impairs the mTORC1 signaling (2).

Fig. 5.
Wnt signaling positively regulates mTOR protein expression. (A) Western blot analysis of mTOR, p-S6, and S6 in the normal ileal mucosa (N) and polyps (P) of the ApcΔ716 mouse. β-Actin is shown as a loading control. (B) Western blot analysis ...

The intestinal polyps of ApcΔ716 mice are caused by the canonical Wnt signaling activation through loss of the heterozygosity of the Apc gene (24), which leads to β-catenin stabilization. The stabilized β-catenin moves into the nucleus where it binds to TCF/LEF transcription factors and thereby increases expression of the Wnt-target genes. To elucidate the roles of Wnt signaling activation in the mTOR signaling regulation, we examined the effects of β-catenin knockdown on mTOR pathway in SW480, a colon cancer cell line with APC mutations. Transfection of siRNA for the CTNNB1 gene encoding β-catenin markedly reduced the β-catenin protein level in SW480 (Fig. 5B). Consistently, the β-catenin knockdown also suppressed the TCF-dependent transcription in TOPflash reporter gene assays in SW480 (data not shown). Notably, the Wnt signaling inhibition by CTNNB1 knockdown markedly reduced S6 phosphorylation at Ser-240/244 in SW480 cells (Fig. 5B). We then examined mTOR expression level in SW480 cells treated with two different siRNAs for CTNNB1. Interestingly, not only the mTOR phosphorylation at Ser-2448, a PI3K-Akt pathway dependent phosphorylation site (25), but also the total mTOR level was reduced in the CTNNB1 siRNA-transfected SW480 (Fig. 5 C and D). Reduction of the total mTOR protein by CTNNB1 siRNA was also observed in another colon cancer cell line, DLD-1, in which APC is mutated (data not shown). These results suggest that the Wnt signaling activation may increase the mTOR expression level itself. We confirmed that the mTOR mRNA level was significantly reduced to ≈60% in the CTNNB1 siRNA-transfected SW480 (Fig. S3 E and F). Consistent with this result, expression of the mTOR mRNA was higher in the polyps than in the normal ileum (Fig. S3G). These results indicate that Wnt signaling regulates mTOR expression at the mRNA levels. To investigate whether fluctuation in the mTOR protein level could affect the mTORC1 pathway signaling in colon cancer cells, we constructed mTOR-knockdown SW480 cells by using a retroviral shRNA. Phosphorylation of S6 kinase was reduced in the mTOR knockdown cells as compared with the controls (Fig. S3H). These results strongly suggest that the elevated level of the mTOR protein leads to the activation of the mTORC1 signaling in intestinal tumors.


We have shown that the mTORC1 pathway is strongly activated in the adenoma epithelium of ApcΔ716 mice as compared with neighboring normal intestinal mucosa (Fig. 1), and that mTORC1 inhibitor RAD001 significantly suppresses polyp formation in these mice and prolonged their survival (Fig. 2).

Although APC gene mutations are found in most cases of colorectal cancer, CTNNB1 gene mutations, that facilitate Wnt signaling via β-catenin stabilization, have also been reported (26). We confirmed that mTORC1 was activated in the intestinal polyps of CtnbΔex3 mice [carrying stabilized β-catenin and developing intestinal polyps (27)] (data not shown). These results suggest that activation of mTORC1 depends on the β-catenin stabilization, rather than mutations in Apc itself. These results suggest a different mechanism from that reported by Inoki et al. (28).

We have also found that RAD001 affects both proliferation of polyp epithelial cells in vivo and tumor angiogenesis (Figs. 3 and and4).4). Although RAD001 treatment was shown to reduce the level of VEGF in melanoma allograft models,§ the strong antiangiogenic effect of RAD001 was not accompanied by down-regulation of VEGF in the intestinal polyps of ApcΔ716 mice (Fig. 4C). On the other hand, mTORC1 inhibitors have been shown to inhibit proliferation of vascular endothelial cells (29). Riesterer et al. (17) reported that inhibition of mTOR by rapamycin induced endothelial cell death through caspase 3 activation and treatment-dependent degradation of Akt protein. Some angiogenic vessels in adenomas showed the mTORC1 signal activation (Fig. 4D). These results suggest that RAD001 directly targets vascular endothelial cells, which results in endothelial cell death and growth suppression by abrogating survival signals such as through Akt, rather than indirectly inhibiting angiogenesis through the VEGF–HIF-1α pathway (30). COX-2 has been shown to play important roles in intestinal polyp expansion of ApcΔ716 mice through induction of tumor angiogenesis associated with an increase level of VEGF (11, 31, 32). However, our preliminary experiments showed that COX-2 expression was unaffected by RAD001 in the ApcΔ716 mouse (data not shown), which is consistent with the above data of the unchanged VEGF level. Thus, inhibition of angiogenesis by RAD001 is probably independent of COX-2 expression in these polyps.

Clinical trials using mTORC1 inhibitors for caner therapy are underway in glioblastoma, lung cancer, and renal cell carcinoma (7). So far, however, colon cancer is not among the major targets for mTORC1 inhibitor trials. mTORC1 inhibitors have been shown to inhibit the proliferation of colon cancer cells in vitro, although their effects vary among the cell lines (33, 34). Our present results demonstrate that the RAD001 may be effective against spontaneous intestinal tumors with mTORC1 signaling activation. In addition, RAD001 treatment dramatically improved the survival of ApcΔ716 mice (Fig. 2F). These results suggest a possibility for clinical trials using mTORC1 inhibitors in early human colon polyps. We noted that healthy ApcΔ716 mice treated for >1 year with RAD001 had a significant number of large polyps without malignant progression [diameter >2 mm: 54 ± 28, with total polyp number: 140 ± 60 (mean ± SD (n = 4) (data not shown)]. These results suggest that the inhibitory effect of RAD001 on intestinal polyp formation may be somewhat attenuated in a long-term treatment. However, phosphorylation of S6 and eIF4G was reduced in the polyps of such ApcΔ716 mice (data not shown), indicating that the inhibitory effect of RAD001 on the mTORC1 pathway itself was not compromised in the polyps of these mice. Thus, other mechanisms may be activated in such polyps to make the polyps resistant to RAD001. Recent reports showed that prolonged treatment of rapamycin altered the phosphorylation status of Akt at Ser-473 in several tissues and cell lines (35). Consistently, phosphorylation of Akt and its substrate Foxo1 was markedly increased in the samples of the long-term-treated ApcΔ716 mice compared with the short-term samples (data not shown). Because activation of Akt pathway is involved in cell survival, these results suggest that Akt phosphorylation and activation induced by long-term treatment with RAD001 may contribute to development of large polyps in the ApcΔ716 mice. Thus it may be more beneficial to patients if mTORC1 inhibitor is combined with Akt inhibitors for treatment and prevention of adenomas.

What activates the mTORC1 pathway in ApcΔ716 intestinal polyps? We have excluded the involvement of PI3K-Akt, Erk1/2, and AMPK signaling and the nutrient status that are frequently involved in mTORC1 activation (Fig. S2 A–D). Furthermore, treatment with meloxicam, a COX-2 selective inhibitor, did not alter the S6 phosphorylation level in the ApcΔ716 polyps (data not shown). Recently, Inoki et al. (28) reported that inhibition of GSK3β induced by Wnt signaling drove the mTORC1 signaling through TSC2 inhibition. Therefore, it was conceivable that mTORC1 signaling in ApcΔ716 intestinal polyps was activated by the inhibition of GSK3β. However, the kinase activity of GSK3β was still retained in the polyps and in the normal tissue of ApcΔ716 intestine (data not shown). Interestingly, the mTOR protein and mRNA expression level were markedly increased in the polyps as compared with the normal tissue (Fig. 5A and Fig. S3G). In addition, siRNA-mediated knockdown of β-catenin in the SW480 colon cancer cell line decreased the mTOR mRNA and protein levels (Fig. 5 C and D and Fig. S3 E and F) and S6 phosphorylation (Fig. 5B). The reduced level of mTOR caused by shRNA suppressed the mTORC1 signaling in SW480 cells (Fig. S3H). To test the possibility that the level of mTOR mRNA may be affected by proliferation rate, we have examined the effect of cell-cycle arrest in SW480 cells. The level of mTOR mRNA was not affected by the double thymidine block, suggesting that the reduced expression of mTOR by β-catenin knockdown was not caused by the reduced rate of proliferation (data not shown). These results suggest that the regulation of the mTOR level through Wnt signaling plays a crucial role in the activation of mTORC1. Thus, we propose that the Wnt signaling contributes to the up-regulation of mTOR, leading to the mTORC1 activation (Fig. S4).

In conclusion, we have demonstrated that the mTORC1 pathway activation plays important roles in intestinal polyp formation of ApcΔ716 mice, and that RAD001 potently suppresses polyp formation with significant effects on survival. These results suggest that RAD001 and other mTORC1 inhibitors may be useful agents for therapy and prevention of colon polyps and cancers.

Materials and Methods


The construction of ApcΔ716 knockout mice has been described (24). Male and female ApcΔ716 mice aged 6 and 14 weeks were used for each treatment group. All animal experiments were approved by the Animal Care and Use Committee of Kyoto University.

Drug Administration.

RAD001 (everolimus) was provided by the Novartis Institutes for BioMedical Research. Three and 10 mg/kg per day of RAD001 was administrated (five times a week) as a micro emulsion containing 2% (wt/wt) RAD001 diluted in distilled deionized water by oral gavage. Wortmannin (5 mg/kg per day) (Sigma) was i.p. administrated for 3 consecutive days.

For further details on polyp scoring, animal survival assay, histological analysis, immunostaining, Western blot analysis, TUNEL analysis, BrdU staining, microvessel density, antibody array, cells culture, transfection of RNA oligonucleotides, retroviral shRNA infection, RT-PCR analysis, and quantitative RT-PCR analysis see SI Text.

Supplementary Material

Supporting Information:


We thank Drs. M. Oshima, M. Sonoshita, and A. Matsunaga for scientific discussion and Dr. M. Tsujii (Osaka University, Osaka) for the SW480 cells. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. T.F. and K.A. are Japan Society for the Promotion of Science research fellows.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0800041105/DCSupplemental.

§O'Reilly TM, et al. American Association for Cancer Research Meeting, April 16–20, 2005, Anaheim, CA (abstr).


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