Abstract
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.
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Acknowledgements
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.
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O.J.S., A.E.W. and W.J.F. designed the project. W.J.F., R.A.R., T.J. and S.R. performed breeding and phenotypic analysis of mice; W.J.F., T.J.J. and J.R.P.K. performed translational analysis; M.N.H., A.G.R., N.S., O.M., A.S., J.B.C., M.V., D.J.H., K.B.M., S.A.K., K.M.D., C.J., H.A.C. and M.P. provided advice and material; W.J.F., O.J.S., A.E.W. and M.B. wrote and edited the manuscript.
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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.
Extended Data Figure 2 Rptor deletion is maintained in the small intestine.
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).
Extended Data Figure 4 IHC after rapamycin treatment.
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.
Extended Data Figure 5 Rptor deletion in the intestinal crypt is lethal in vitro.
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.
Extended Data Figure 7 S6k deletion decreases intestinal regeneration.
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.
Extended Data Figure 8 Eef2k deletion drives resistance to rapamycin.
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.
Extended Data Figure 9 Cyclin D3 is regulated at the level of elongation.
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.
Extended Data Figure 10 Ribosomes elongate faster on Ccnd3 after Apc deletion.
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.
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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
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DOI: https://doi.org/10.1038/nature13896
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