Age-associated mitochondrial DNA mutations cause metabolic remodeling that contributes to accelerated intestinal tumorigenesis

Abstract

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumors, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mitochondrial DNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting that they may confer a metabolic advantage. To test this, we deleted the tumor suppressor Apc in OXPHOS-deficient intestinal stem cells in mice. The resulting tumors were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumors undergo metabolic remodeling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway. Moreover, normal human colonic crypts upregulate the serine synthesis pathway in response to OXPHOS deficiency before tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodeling that can functionally contribute to accelerated intestinal cancer development.

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Fig. 1: OXPHOS subunit IHC and histochemical analysis of human colorectal adenomas and adenocarcinomas.
Fig. 2: Analysis of mtDNA mutations detected in 26 colorectal adenocarcinomas compared with normal aged crypts.
Fig. 3: PolgAmut/mut;Apcfl/fl mice have a reduced lifespan and enhanced tumor growth due to accelerated cell proliferation and reduced apoptosis compared with Apcfl/fl mice.
Fig. 4: Small intestinal adenomas from PolgAmut/mut;Apcfl/fl mice are deficient in mitochondrial complex I, but the majority retain expression of subunits of complexes III, IV and V.
Fig. 5: Mitochondrial OXPHOS dysfunction causes upregulation of de novo serine synthesis in both non-transformed crypts and adenomas from mice.
Fig. 6: Characterization of the immune microenvironment in the lamina propria of the small intestine of PolgAmut/mut and PolgA+/+ mice at 6 months of age, before tumor induction.
Fig. 7: Mitochondrial OXPHOS dysfunction causes upregulation of de novo serine synthesis in normal aging human colonic crypts.

Data availability

RNA-Seq and DNA next-generation sequencing data have been deposited in the Sequence Read Archive under BioProject accession code PRJNA645504. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

Code used to generate the mitochondrial OXPHOS Z scores and dot pots is freely available at http://mito.ncl.ac.uk/immuno/. The R programming code used in the linear regression mixed-effects modeling is available upon request.

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Acknowledgements

We thank T. Prolla (University of Wisconsin, Washington, United States) for donating the PolgA+/mut mice. We thank C. Alston for assistance with the analysis of mtDNA mutations and staff at the Newcastle University Comparative Biology Centre for animal husbandry. This work was supported by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), Newcastle University Centre for Ageing and Vitality (supported by the Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council and Medical Research Council (MR/L016354/1)), UK NIHR Biomedical Research Centre Age and Age Related Diseases award (to the Newcastle upon Tyne Hospitals NHS Foundation Trust) and NC3Rs (to C.A.R.; NC/K500513/1). O.J.S. is supported by Cancer Research UK grants (A25045, A17196, A12481 and A21139). O.J.S. and D.G. were supported by ERC starting grant 311301 awarded to O.J.S. F.O. is supported by the Medical Research Council (MR/R023026/1). J.L. is supported by Cancer Research UK (C18342/A23390).

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L.C.G., C.B., C.S. and C.A.R. performed the breeding and phenotypic analyses of mice. A.L.M.S., D.H., M.H., J.N.S. and A.B. performed the histology, IHC, immunofluorescence and analysis of mouse and human samples. L.C.G., O.M.R., R.J. and B.G. performed the sequencing and histological analysis of human samples. S.A.C.M., I.M., S.K. and J.C.M. collected and processed the human samples. J.C.W. performed the molecular biology and cell culture experiments. G.H. and A.P. performed the sequencing and bioinformatics analyses of the mouse and human adenomas. Flow cytometric immunophenotyping of the small intestine was carried out by S.A. and G.M. J.L. and F.O. performed the immune cell IHC. L.W. carried out the imaging and analysis of the immune cell IHC. F.R. performed the statistical analysis of the RNA-Seq data. A.P.B. performed the statistical analysis of the experimental data. D.G., J.C.W. and O.J.S. performed the metabolomics analyses and analyzed the data. L.C.G., R.W.T., R.H., D.M.T., N.D.P. and O.J.S. conceived of the ideas, designed the experiments and interpreted the data. All authors contributed to writing and revising the paper.

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Correspondence to Laura C. Greaves.

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F.O. is a director of Fibrofind. J.L. and F.O. are shareholders in Fibrofind. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Generation of PolgAmut/mut;Lgr5-creER;Apcfl/fl and Lgr5-creER;Apcfl/fl mice and analysis of colonic adenomas.

a: Breeding scheme. MtDNA mutations can be transmitted down the maternal germline65 therefore it was essential that only Lgr5-creER;Apcfl/fl (red) mice from a wild-type PolgA mother used as controls. b: Kaplan-Meier survival curve showing survival time following tamoxifen administration in PolgAmut/mut mice. Survival to clinical endpoint or experimental endpoint of 60 days is shown, ‘n’ = number of mice. c: β-Catenin immunohistochemistry was performed on colon sections from n = 17 PolgAmut/mut;Apcfl/fl mice and n = 13 Apcfl/fl mice. Representative images are shown (scale bars 3 mm (first column) and 200 µm). d: Frequency of adenomas in the colon 23 days post-Apc deletion (unpaired, two tailed, t-test, p = 0.7444), n = 17 PolgAmut/mut;Apcfl/fl mice and n = 13 Apcfl/fl mice, data are mean ±s.d. e: Mean adenoma size in the colon in n = 17 PolgAmut/mut;Apcfl/fl mice and n = 13 Apcfl/fl mice 23 days post-Apc deletion. All adenomas on a section were quantified ranging from 5 to 280, mean per mouse ± s.e.m are shown. Two-sided linear mixed effect regression model with mouse ID as a random effect, P < 0.0001. f-g: Quantification of the frequency of thymidine analogue incorporation in all cells per colonic adenoma (f) and LGR5 + cells per colon adenoma per mouse (g). n = 5 mice per group with 18 adenomas analysed per mouse. Mean frequency per adenoma per mouse ± s.e.m is shown. Two-sided linear mixed effect regression model with mouse ID as a random effect, P < 0.001. h, i: Apoptotic cells were quantified using (h) cleaved caspase 3 (CC3) immunohistochemistry n = 7 PolgAmut/mut;Apcfl/fl mice and n = 9 Apcfl/fl mice and (i) TUNEL labelling (n = 9 mice per group) in mice 23 days post-Apc deletion. A minimum of 10 adenomas were analysed per mouse, mean percentage of apoptotic cells per adenoma per mouse ±s.e.m is shown. Two-sided linear mixed effect regression model with mouse ID as a random effect, CC3 P = 0.0092, TUNEL P = 0.002. * P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 2 Colonic adenomas from PolgAmut/mut;Apcfl/fl mice are deficient in mitochondrial complex I, but the majority retain expression of subunits of complexes III, IV and V.

a, b: Immunofluorescence was performed to quantify levels of OXPHOS proteins in n = 9 PolgAmut/mut;Apcfl/fl mice and n = 9 Apcfl/fl mice. Representative images are shown. Scale bars 50 µm. An adenoma deficient in complex I is highlighted by the white dashed line in a. The white dashed line highlights an adenoma deficient in complex IV, and red dashed line shows one with normal complex IV in bd: dot plots showing Z-scores calculated following quantification of mitochondrial OXPHOS protein levels in adenomas from n = 9 PolgAmut/mut;Apcfl/fl and n = 9 Apcfl/fl mice with 20 adenomas quantified per mouse. e: Categorical analysis of OXPHOS protein levels in PolgAmut/mut;Apcfl/fl (n = 9) and Apcfl/fl (n = 9) mice, error bars show mean ±s.d. f, g: dot plots showing Z-scores calculated following quantification of mitochondrial OXPHOS protein levels in normal crypts and adenomas in the small intestine (f) and the colon (g). f: For the adenomas: n = 9 PolgAmut/mut;Apcfl/fl and n = 10 Apcfl/fl mice were analysed with 20 adenomas quantified per mouse. For the normal crypts, n = 5 mice were analysed with a minimum of 13 crypts quantified per mouse. g: For the colonic adenomas: n = 9 mice per group were analysed with a minimum of 20 adenomas quantified per mouse. For the normal crypts, n = 6 Apcfl/fl mice and n = 7 PolgAmut/mut;Apcfl/fl mice were analysed with a minimum of 22 crypts quantified per mouse. h Dot plots showing raw densitometry values for mitochondrial protein levels in the colon (n numbers same as in g, error bars are s.d.). One-way ANOVA with Tukey’s post-test. P values for within genotype comparisons between normal crypts and adenomas were as follows: TOMM20: Apcfl/fl P < 0.0001, PolgAmut/mut;Apcfl/fl P < 0.0001, NDUFB8: Apcfl/fl P < 0.0001, PolgAmut/mut;Apcfl/fl P = 0.9761, UQCRFS1: Apcfl/fl P < 0.0001, PolgAmut/mut;Apcfl/fl P = 0.2901, MTCO1: Apcfl/fl P < 0.0001, PolgAmut/mut;Apcfl/fl P = 0.007, ATPB: Apcfl/fl P < 0.0001, PolgAmut/mut;Apcfl/fl P < 0.0001. For all panels: * P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 3 Analysis of mitochondrial DNA (mtDNA) mutations detected in individual small intestinal adenomas from PolgAmut/mut;Apcfl/fl and Apcfl/fl mice.

a: The frequency of heteroplasmic variants >3% detected in adenomas from PolgAmut/mut;Apcfl/fl (n = 3 mice per group and n = 10 adenomas per mouse) and Apcfl/fl mice (n = 3 mice per group, n = 5 adenomas per mouse), mean ±s.d. are shown. b–d: Analysis of mtDNA variants present at >30% heteroplasmy in individual adenomas from PolgAmut/mut;Apcfl/fl mice (n = 413 mtDNA mutations in total). For location (b), expected values were calculated based on the proportion of the mitochondrial genome taken up by each gene category and observed and expected values compared using Chi-squared analysis. No significant deviation from the expected frequencies was detected (P = 0.4744). Source data

Extended Data Fig. 4 Mitochondrial OXPHOS dysfunction causes upregulation of de novo serine synthesis in vivo in the mouse colon.

Immunohistochemistry images showing in situ levels of SSP proteins in the non-transformed normal colonic mucosa (a) and adenomas (b) of PolgA+/+ and PolgAmut/mut mice. Immunohistochemistry was performed on n = 4 mice per group. Representative images are shown. Scale bars 50 µm.

Extended Data Fig. 5 Immunofluorescent images showing the levels of PHGDH, PSAT1 and MTHFD2 in PolgA+/+ and PolgAmut/mut mice from 1–12 months of age.

Immunofluorescence was performed on n = 3 mice per group at each time point. Representative images are shown. Scale bars 50 µm.

Extended Data Fig. 6 Quantification of major mass isotopomers following growth of adenoma organods in 13C6-glucose and adenoma organoid growth in to the presence of metformin.

a: Quantification of major mass isotopomers following growth in the presence of 13C6-glucose for 24 h. 13C labelling is shown as M + 6 (glucose) and M + 0 denotes no labelling. No significant differences were found between organoids from Apcfl/fl mice compared with PolgAmut/mut;Apcfl/fl mice by one-tailed unpaired t-test. n = 3 mice per group with 3 technical replicates performed per mouse. Error bars show s.e.m. b: A shared group estimation plot comparing the effect of metformin on the volume of individual adenoma organoids generated from Apcfl/fl mice (n = 3) on days 1 and 5 post seeding. Volume data are normalised to day 1. On day 1 the numbers of organoids measured were: 0 µM: n = 739, 100 µM: n = 796, 250 µM: n = 711, 500 µM: n = 652. On day 5 the numbers of organoids measured were: 0 µM: n = 1060, 100 µM: n = 1515, 250 µM: n = 1088, 500 µM: n = 1431. Bootstrap estimation of group mean differences (circle) and 95% confidence intervals (vertical bars) are plotted as a sampling distribution. Source data

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Smith, A.L.M., Whitehall, J.C., Bradshaw, C. et al. Age-associated mitochondrial DNA mutations cause metabolic remodeling that contributes to accelerated intestinal tumorigenesis. Nat Cancer 1, 976–989 (2020). https://doi.org/10.1038/s43018-020-00112-5

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