Inactivation of APC is a strongly predisposing event in the development of colorectal cancer1,2, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth3,4,5, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1–S6K–eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Kinzler, K. W. & Vogelstein, B. Lessons from hereditary colorectal cancer. Cell 87, 159–170 (1996)
Korinek, V. et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997)
Ashton, G. H. et al. Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling. Dev. Cell 19, 259–269 (2010)
Fujishita, T., Aoki, K., Lane, H. A., Aoki, M. & Taketo, M. M. Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcΔ716 mice. Proc. Natl Acad. Sci. USA 105, 13544–13549 (2008)
Gulhati, P. et al. Targeted inhibition of mammalian target of rapamycin signaling inhibits tumorigenesis of colorectal cancer. Clin. Cancer Res. 15, 7207–7216 (2009)
Pourdehnad, M. et al. Myc and mTOR converge on a common node in protein synthesis control that confers synthetic lethality in Myc-driven cancers. Proc. Natl Acad. Sci. USA 110, 11988–11993 (2013)
Martineau, Y. et al. Pancreatic tumours escape from translational control through 4E-BP1 loss. Oncogene 33, 1367–1374 (2014)
Jiang, Y. P., Ballou, L. M. & Lin, R. Z. Rapamycin-insensitive regulation of 4E-BP1 in regenerating rat liver. J. Biol. Chem. 276, 10943–10951 (2001)
Bach, S. P., Renehan, A. G. & Potten, C. S. Stem cells: the intestinal stem cell as a paradigm. Carcinogenesis 21, 469–476 (2000)
Bernal, N. P. et al. Evidence for active Wnt signaling during postresection intestinal adaptation. J. Pediatr. Surg. 40, 1025–1029 (2005)
Ireland, H. et al. Inducible Cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of β-catenin. Gastroenterology 126, 1236–1246 (2004)
Muncan, V. et al. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol. Cell. Biol. 26, 8418–8426 (2006)
Zoncu, R., Efeyan, A. & Sabatini, D. M. mTOR: from growth signal integration to cancer, diabetes and ageing. Nature Rev. Mol. Cell Biol. 12, 21–35 (2011)
Yilmaz, Ö. H. et al. mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake. Nature 486, 490–495 (2012)
Farin, H. F., Van Es, J. H. & Clevers, H. Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology 143, 1518–1529 (2012)
She, Q. B. et al. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors. Cancer Cell 18, 39–51 (2010)
Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009)
Fresno, M., Jimenez, A. & Vazquez, D. Inhibition of translation in eukaryotic systems by harringtonine. Eur. J. Biochem. 72, 323–330 (1977)
Schneider-Poetsch, T. et al. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nature Chem. Biol. 6, 209–217 (2010)
Hsieh, A. C. et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485, 55–61 (2012)
Richter, J. D. & Sonenberg, N. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477–480 (2005)
Browne, G. J. & Proud, C. G. A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin. Mol. Cell. Biol. 24, 2986–2997 (2004)
Ryazanov, A. G., Shestakova, E. A. & Natapov, P. G. Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation. Nature 334, 170–173 (1988)
Gorshtein, A. et al. Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro. Endocr. Relat. Cancer 16, 1017–1027 (2009)
Gutzkow, K. B. et al. Cyclic AMP inhibits translation of cyclin D3 in T lymphocytes at the level of elongation by inducing eEF2-phosphorylation. Cell. Signal. 15, 871–881 (2003)
Firczuk, H. et al. An in vivo control map for the eukaryotic mRNA translation machinery. Mol. Syst. Biol. 9, 635 (2013)
Hussey, G. S. et al. Identification of an mRNP complex regulating tumorigenesis at the translational elongation step. Mol. Cell 41, 419–431 (2011)
Nakamura, J. et al. Overexpression of eukaryotic elongation factor eEF2 in gastrointestinal cancers and its involvement in G2/M progression in the cell cycle. Int. J. Oncol. 34, 1181–1189 (2009)
Din, F. V. et al. Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase, and induces autophagy in colorectal cancer cells. Gastroenterology 142, 1504–1515 (2012)
Baan, B. et al. 5-Aminosalicylic acid inhibits cell cycle progression in a phospholipase D dependent manner in colorectal cancer. Gut 61, 1708–1715 (2012)
El Marjou, F. et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 39, 186–193 (2004)
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007)
Shibata, H. et al. Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. Science 278, 120–123 (1997)
Moser, A. R., Pitot, H. C. & Dove, W. F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247, 322–324 (1990)
de Alboran, I. M. et al. Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity 14, 45–55 (2001)
Polak, P. et al. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. Cell Metab. 8, 399–410 (2008)
Luche, H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H. J. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur. J. Immunol. 37, 43–53 (2007)
Tsukiyama-Kohara, K. et al. Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1. Nature Med. 7, 1128–1132 (2001)
Banko, J. L. et al. The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and memory in the hippocampus. J. Neurosci. 25, 9581–9590 (2005)
Shima, H. et al. Disruption of the p70s6k/p85s6k gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 17, 6649–6659 (1998)
Ryazanov, A. G. Elongation factor-2 kinase and its newly discovered relatives. FEBS Lett. 514, 26–29 (2002)
Ruvinsky, I. et al. Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev. 19, 2199–2211 (2005)
Sarbassov, D. D. et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol. Cell 22, 159–168 (2006)
Sengupta, S., Peterson, T. R., Laplante, M., Oh, S. & Sabatini, D. M. mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. Nature 468, 1100–1104 (2010)
W.J.F. is funded by AICR. O.J.S. is funded by Cancer Research UK, European Research Council Investigator Grant (COLONCAN) and the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement number 278568. M.B. is a Medical Research Council Senior Fellow. The authors acknowledge P. Cammareri, J. Morton and C. Murgia for proofreading of the manuscript.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 mTORC1 is activated following Wnt-signal and its inhibition does not affect homeostasis.
a, Representative IHC of phospho-RPS6 (pS6) and phospho-4EBP1 (p4EBP1) show mTORC1 activity during intestinal regeneration, 72 h after 14 Gy γ-irradiation (representative of 5 biological replicates). b, c, Boxplots demonstrating that 72 h of 10 mg kg−1 rapamycin treatment does not alter mitosis or apoptosis in normal intestinal crypts. Whiskers show maximum and minimum, black line shows median (n = 4 per group). NS, not significant, Mann–Whitney U test. d, Intestines imaged on OV100 microscope, 96 h after induction, for red fluorescent protein (RFP). Tissue without the ROSA-tdRFP reporter (Neg control) show no RFP positivity, while the positive control (Pos control) and Rptor-deleted intestines show high RFP positivity (representative of 3 biological replicates). e, f, Boxplot showing that Rptor deletion does not affect mitosis or apoptosis rates in intestinal crypts, 96 h after induction. Whiskers show maximum and minimum, black line shows median (n = 4 per group). NS, not significant, Mann–Whitney U test. Scale bars, 100 µm.
a, Representative IHC of phospho-RPS6 (pS6) and phospho-4EBP1 (p4EBP1) shows maintained loss of mTORC1 signalling 400+ days after Rptor deletion. Arrows indicate unrecombined escaper crypts that still show active mTORC1 signalling (representative of 5 biological replicates). b, c, Boxplots showing that mitosis and apoptosis are unchanged 400+ days after Rptor deletion. Mitosis and apoptosis were counted on H&E sections and are quantified as percent mitosis or apoptosis per crypt. Whiskers show maximum and minimum, black line shows median (n = 5 per group). NS, not significant, Mann–Whitney U test. Scale bars, 100 µm.
Extended Data Figure 3 Wnt signalling is still active after Rptor deletion and rapamycin treatment causes regression of established tumours.
a, b, Representative IHC of MYC and β-catenin showing high MYC levels and nuclear localization of β-catenin 96 h after Apc and Apc/Rptor deletion, demonstrating active Wnt signalling. Nuclear staining (as opposed to membranous staining) of β-catenin is indicative of active Wnt signalling. Scale bar, 100 µm (representative of 3 biological replicates). c, Kaplan–Meyer survival curve of ApcMin/+ mice treated with rapamycin when showing signs of intestinal neoplasia. Rapamycin treatment (10 mg kg−1) started when mice showed signs of intestinal disease, and was withdrawn after 30 days. Animals continued to be observed until signs of intestinal neoplasia. Death of animals in the rapamycin group almost always occurred after rapamycin withdrawal (n = 8 per group). ***P value ≤ 0.001, log-rank test. d, Boxplot showing that 72 h 10 mg kg−1 rapamycin treatment causes an increase in lysozyme-positive cells in tumours. Percentage lysozyme positivity within tumours was calculated using ImageJ software (http://imagej.nih.gov/ij/). Whiskers show maximum and minimum, black line shows median (10 tumours from each of 5 mice per group were measured. **P value ≤ 0.014, Mann–Whitney U test. e, Boxplot showing that 72 h 10 mg kg−1 rapamycin treatment causes a decrease in BrdU positivity within tumours. Percentage BrdU positivity within tumours was calculated using ImageJ software. Whiskers show maximum and minimum, black line shows median (10 tumours from each of 5 mice per group were measured). **P value ≤ 0.021, Mann–Whitney U test. f, Representative IHC of lysozyme, showing a lack of lysozyme-positive paneth cells in remaining cystic tumours after 30 days of 10 mg kg−1 rapamycin treatment. Scale bars, 100 µm (representative of 5 biological replicates).
a, Representative IHC of p21, p16 and p53 after 6 h and 72 h of 10 mg kg−1 rapamycin treatment. Staining shows no induction of these proteins in tumours after rapamycin treatment (representative of 5 biological replicates). b, Representative IHC for LGR5–GFP showing high numbers of LGR5-positive cells after 7 and 30 days of 10 mg kg−1 daily rapamycin treatment (representative of 5 biological replicates). Scale bars, 100 µm.
a, Graph showing that Rptor deletion prevents intestinal crypts from growing ex vivo. Intestinal crypts were isolated and cultured as previously described17, 96 h after Cre induction. Number of viable organoids was counted by eye 72 h after crypt isolation. WT, wild type. Data are average ± standard deviation (n = 3 biological replicates per group).
Extended Data Figure 6 Apc deletion increases translational elongation rates and cycloheximide treatment phenocopies rapamycin treatment.
a, Representative polysome profiles from wild-type ex vivo crypts incubated with harringtonine for 0 s (left) and 180 s (right) before harvest (n = 3 per time point). b, The areas under the sub-polysome (40S, 60S and 80S) and polysome sections as indicated by the dashed lines in a were quantified and expressed as a percentage of their sum. Data in the bar graph are the average ± s.e.m. (n = 3 per time point). c, d, Data are shown for Apc-deleted crypts, as for wild type in b and c (n = 3 biological replicates). e, Representative H&E staining showing that 35 mg kg−1 cycloheximide treatment phenocopies rapamycin treatment 96 h after Apc deletion. Treatment began 24 h after induction (n = 3 biological replicates). f, Representative IHC for BrdU showing a loss of proliferation in tumours after 72 h of 35 mg kg−1 cycloheximide treatment. (n = 3 biological replicates). Arrow highlights normal proliferating crypts. Scale bar, 100 µm.
Graphical representation of findings, and boxplot showing that murine intestinal regeneration after irradiation is dependent on S6K. Animals were exposed to 14 Gy γ-irradiation, and intestinal regeneration was calculated 72 h after exposure by counting the number of viable crypts and multiplying that by the average size of the regenerating crypts. Relative regeneration was calculated by comparing each group to wild-type regeneration. The rapamycin treatment arm is reproduced from Fig. 4 for visual clarity. Whiskers show maximum and minimum, black line shows median (n = 4 per group). *P value = 0.034, Mann–Whitney U test.
a, Representative IHC of phospho-eEF2 and phospho-RPS6 in wild-type (WT), Apc-deficient and Apc- and Eef2k-deficient mice (with or without 72 h 10 mg kg−1 rapamycin (rapa) treatment) shows that rapamycin is unable to induce eEF2 phosphorylation in the absence of eEF2K (n = 6 biological replicates). KO, knockout. Scale bars, 100 µm.
a, Representative IHC of Apc-deleted intestines with or without Eef2k deletion. Antibodies to eEF2K, phospho-RPS6 and cyclin D3 are shown (representative of 3 biological replicates). After Eef2k knockout (KO), cyclin D3 levels are no longer decreased upon 10 mg kg−1 rapamycin (rapa) treatment. b, Boxplot showing the number of cyclin-D3-positive cells per crypt, 96 h after Apc deletion, with and without 10 mg kg−1 rapamycin treatment. Graph shows that in Eef2k knockout animals, rapamycin no longer reduced cyclin D3 levels (n = 3 biological replicates per group). *P value ≤ 0.05, Mann–Whitney U test. c, Western blot analysis of intestinal epithelial cells from Apc-deleted and Apc-deleted Eef2k knockout, with and without 10 mg kg−1 rapamycin. Antibodies to eEF2K, phospho-RPS6, cyclin D3 and β-actin are shown. Each well represents a different mouse from the relevant group. Cyclin D3 levels are no longer reduced after Eef2k deletion. Scale bar, 100 µm.
The ribosome run-off rate of various messages was measured as in Fig. 3. Elongation of Ccnd3 was significantly increased, while Actb, Rps21, Rps6 and Ccnd1 remain unchanged. Data are average ± s.e.m. (n = 3 biological replicates per group). *P value ≤ 0.05, Mann–Whitney U test.
About this article
Cite this article
Faller, W., Jackson, T., Knight, J. et al. mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature 517, 497–500 (2015). https://doi.org/10.1038/nature13896
This article is cited by
Hyperbaric oxygen treatment increases intestinal stem cell proliferation through the mTORC1/S6K1 signaling pathway in Mus musculus
Biological Research (2023)
mTORC1-selective activation of translation elongation promotes disease progression in chronic lymphocytic leukemia
Familial Cancer (2023)
Journal of Gastroenterology (2023)