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

Abstract

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

Extended Data Fig. 1. Generation of PolgA^mut/mut;Lgr5-creER;Apc^fl/fl and Lgr5-creER;Apc^fl/fl mice and analysis of colonic adenomas.

a: Breeding scheme. MtDNA mutations can be transmitted down the maternal germline therefore it was essential that only Lgr5-creER;Apc^fl/fl (red) mice from a wild-type PolgA mother used as controls. b: Kaplan-Meier survival curve showing survival time following tamoxifen administration in PolgA^mut/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 PolgA^mut/mut;Apc^fl/fl mice and n=13 Apc^fl/fl mice. Representative images are shown (scale bars 3mm (first column) and 200µm). d: Frequency of adenomas in the colon 23 days post-Apc deletion (unpaired, two tailed, t-test, p=0.7444), n=17 PolgA^mut/mut;Apc^fl/fl mice and n=13 Apc^fl/fl mice, data are mean ±s.d. e: Mean adenoma size in the colon in n=17 PolgA^mut/mut;Apc^fl/fl mice and n=13 Apc^fl/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 PolgA^mut/mut;Apc^fl/fl mice and n=9 Apc^fl/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.

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

a and b: Immunofluorescence was performed to quantify levels of OXPHOS proteins in n=9 PolgA^mut/mut;Apc^fl/fl mice and n=9 Apc^fl/fl mice. Representative images are shown. Scale bars 50µm. 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 b. c and d: dot plots showing Z-scores calculated following quantification of mitochondrial OXPHOS protein levels in adenomas from n=9 PolgA^mut/mut;Apc^fl/fl and n=9 Apc^fl/fl mice with 20 adenomas quantified per mouse. e : Categorical analysis of OXPHOS protein levels in PolgA^mut/mut;Apc^fl/fl (n=9) and Apc^fl/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 PolgA^mut/mut;Apc^fl/fl and n=10 Apc^fl/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 Apc^fl/fl mice and n=7 PolgA^mut/mut;Apc^fl/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: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p<0.0001, NDUFB8: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p=0.9761, UQCRFS1: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p=0.2901, MTCO1: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p=0.0007, ATPB: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p<0.0001. For all panels: * p<0.05, **p<0.01, ***p<0.001

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

a: The frequency of heteroplasmic variants >3% detected in adenomas from PolgA^mut/mut;Apc^fl/fl (n=3 mice per group and n=10 adenomas per mouse) and Apc^fl/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 PolgA^mut/mut;Apc^fl/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).

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 PolgA^mut/mut mice. Immunohistochemistry was performed on n=4 mice per group. Representative images are shown. Scale bars 50µm.

Extended Data Fig. 5. Immunofluorescent images showing the levels of PHGDH, PSAT1 and MTHFD2 in PolgA^+/+ and PolgA^mut/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 50µm.

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 hours. ¹³C labelling is shown as M+6 (glucose) and M+0 denotes no labelling. No significant differences were found between organoids from Apc^fl/fl mice compared with PolgA^mut/mut;Apc^fl/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 Apc^fl/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: 0µM: n=739, 100µM: n=796, 250µM: n=711, 500µM: n=652. On day 5 the numbers of organoids measured were: 0µM: n=1060, 100µM: n=1515, 250µM: n=1088, 500µM: n=1431. Bootstrap estimation of group mean differences (circle) and 95% confidence intervals (vertical bars) are plotted as a sampling distribution.

Figure 1. OXPHOS subunit immunohistochemical and histochemical analysis of human colorectal adenomas and adenocarcinomas.

a: Immunohistochemistry was performed on 26 adenocarcinoma samples and patient matched normal mucosa, and 9 colonic adenoma samples. Representative images showing OXPHOS subunit expression in normal mucosa (CSC024), an adenoma (AD07), and an adenocarcinoma (CSC024). b: Cytochrome c oxidase and succinate dehydrogenase histochemistry was performed on the same samples as in panel a. Representative images of normal colonic mucosa and adenocarcinoma from CRC009 are shown in b. c: Quantification of the mean percentage of normal crypts per subject with defects in the specified OXPHOS subunits (each dot represents the mean percentage of OXPHOS deficient crypts in each subject, n=26 subjects, error bars are s.e.m.), and the percentage of adenomas (n=9) and adenocarcinomas (n=26) analysed with defects in the specified OXPHOS subunits. Scale bars a- 20µm, b 50µm.

Figure 2. Analysis of mitochondrial DNA (mtDNA) mutations detected in 26 colorectal adenocarcinomas compared with normal aged crypts.

a-c: Location, type and consequences of mtDNA mutations detected in colorectal adenocarcinomas in this study (n=41 mutations). d-g: Comparison of the location (d), types (e), and functional consequences (f) of mtDNA mutations in previously published normal crypts (n=129 mutations) and adenocarcinomas^,,, (n=182 mutations). There was a significant difference in the location of the mtDNA mutations in adenocarcinomas compared with normal aged crypts (p=0.0123, Chi-squared analysis (d)), but no significant differences were detected in the types of mutations (p=0.2264, Chi-squared analysis, (e)) or the predicted functional consequences (p=0.1504, Chi-squared analysis (f)). (g) Comparison of MutPred pathogenicity scores for missense mutations in protein encoding genes in normal aging crypts (n=52 mutations) and adenocarcinomas (n=80 mutations) two-tailed, Mann Whitney U Test, p=0.8138, median ±95% confidence intervals are shown. * p<0.05

Figure 3. PolgA^mut/mut;Apc^fl/fl mice have reduced lifespan and enhanced tumour growth due to accelerated cell proliferation and reduced apoptosis compared with Apc^fl/fl mice.

a: Kaplan-Meier survival curve showing survival times post Apc deletion (two-sided Mantel-Cox (Log-Rank) test, p<0.0001, ‘n’ = number of mice) b: β-Catenin immunohistochemistry on small intestinal sections (scale bars 3mm (first column) and 200µm). Immunohistochemistry was performed on PolgA^mut/mut;Apc^fl/fl (n=17) and Apc^fl/fl (n=12) mice 23 days post-Apc deletion, representative images are shown. c: Tumour burden in small intestine of PolgA^mut/mut;Apc^fl/fl (n=17) and Apc^fl/fl (n=12) mice 23 days post-Apc deletion (unpaired, 2 tailed, t-test, error bars show s.d., p=0.0010) d: Immunoflourescent (IF) images showing LGR5+ cells, and cells which have incorporated CldU and IdU. Scale bars 50µm. IF was performed on n=5 mice per group and representative images are shown. e-f: Quantification of the frequency of thymidine analogue incorporation in all cells per adenoma (e), and LGR5+ cells per adenoma per mouse (f). n=5 mice per group with 20 adenomas analysed per mouse (two-sided linear mixed effect regression model with mouse ID as a random effect, p<0.001 in all comparisons). g-h: Apoptotic cells were identified and quantified using cleaved caspase 3 (CC3) immunohistochemistry (g) and TUNEL labelling (h) in mice 23 days post-Apc deletion. n=9 mice per group with a minimum of 10 adenomas analysed per mouse Two-sided linear mixed effect regression model with mouse ID as a random effect, p<0.001 for CC3 and p=0.008 for TUNEL, mean percentage or apoptotic cells per adenoma per mouse are shown ± s.e.m. For all panels: * p<0.05, **p<0.01, ***p<0.001.

Figure 4. Small Intestinal adenomas from PolgA^mut/mut;Apc^fl/fl mice are deficient in mitochondrial Complex I, but the majority retain expression of subunits of complexes III, IV and V.

a and c: Immunofluorescence was performed to quantify levels of OXPHOS proteins on n=9 PolgA^mut/mut;Apc^fl/fl mice and n=10 Apc^fl/fl mice. Representative images are shown. Scale bars 50µm. The white dashed line shows an adenoma region deficient in complex I and III, and the red dashed line highlights deficiency in complex I only in panel a: An adenoma region deficient in complex IV is highlighted by the white dashed line and one with normal complex IV is highlighted by the red dashed line in panel c. b and d: Dot plots showing relative Z-scores calculated following quantification of mitochondrial OXPHOS protein levels in adenomas from PolgA^mut/mut;Apc^fl/fl (n=9) and Apc^fl/fl (n=10) mice using the method in. n=20 adenomas were quantified per mouse. e: Categorical analysis of OXPHOS protein levels PolgA^mut/mut;Apc^fl/fl (n=9 mice) and Apc^fl/fl (n=10 mice) Data points show individual mice ± s.d. f: Dot plots showing raw densitometry values for mitochondrial protein levels. For the adenomas: n=9 PolgA^mut/mut;Apc^fl/fl and n=10 Apc^fl/fl mice with 20 adenomas analysed per mouse. For the normal crypts, n=5 mice were analysed per group with a minimum of 13 crypts quantified per mouse. One-way ANOVA with Tukey’s post-test. P values for within genotype comparisons between normal crypts and adenomas were as follows: TOMM20: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p<0.0001, NDUFB8: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p=0.9995, UQCRFS1: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p=0.1302, MTCO1: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p=0.0001, ATPB: Apc^fl/fl p<0.0001, PolgA^mut/mut;Apc^fl/fl p<0.0001. For all panels: * p<0.05, **p<0.01, ***p<0.001, error bars show s.e.m

Figure 5. Mitochondrial OXPHOS dysfunction causes upregulation of de novo serine synthesis in both non-transformed crypts and adenomas from mice.

a: Heat map showing differential gene expression in non-transformed crypt homogenates from the small intestines of PolgA^+/+ and PolgA^mut/mut mice (n=4 mice per group). b: Mean relative expression of the SSP genes by RT-PCR which were identified to be upregulated by RNASeq analysis in normal crypts. n=7 mice per group, one-way Mann-Whitney U test, p=0.0003 for all genes except Slc1a4 where p=0.0035 c: Mean relative expression of the SSP genes by RT-PCR which were identified to be upregulated by RNASeq analysis in laser-microdissected adenomas. n=6 mice per group, one-way Mann-Whitney U test, p values as follows: Phgdh p=0.0325, Psat1 p=0.066, Psph p=0.0130, Aldh1l2 p=0.1548, Mthfd2 p=0.0043, error bars show s.e.m. d-e: Immunohistochemistry images showing in situ levels of SSP proteins in the non-transformed normal small intestinal mucosa (d), and adenomas (e). Immunohistochemistry was performed on n=4 mice per genotype and representative images are shown. Scale bars 50µm. f: Organoids were generated from n=3 PolgA^mut/mut;Apc^fl/fl and n=3 Apc^fl/fl mice. Representative images of adenoma organoids are shown. Scale bars 100µm. g: Oxygen consumption rates measured by Seahorse analysis in adenoma organoids (n=3 mice per genotype, 8 technical replicates per mouse, mean ±s.e.m per mouse are shown). h: Quantification of major mass isotopomers detected in adenoma organoids following growth in the presence of ¹³C₆-glucose for 24 hours. ¹³C labelling is shown as M+3 (serine) or M+2 (glycine). n=3 mice per group with 3 technical replicates performed per mouse (one-way unpaired t-test, p=0.0143 for labelled serine and p=0.0151 for labelled glycine, data are mean per mouse ±s.e.m). i: Quantification of the growth of adenoma organoids in medium with (+SG) or without (-SG) serine and glycine for 5 days. Data are normalised to organoid area on day 0. Mean organoid size per mouse relative to day 0 ± s.e.m is shown, n=3 mice per group, unpaired, two-tailed t-test, p=0.4140 for Apc^fl/fl and p=0.0021 for PolgA^mut/mut;Apc^fl/fl. For all panels: * p<0.05, **p<0.01, ***p<0.001.

Figure 6. Characterisation of the immune microenvironment in the lamina propria of the small intestine of PolgA^mut/mut and PolgA^+/+ mice at 6 months of age, prior to tumour induction.

a-b: Immune cell infiltration within the distal third of small intestine was analysed by flow cytometry. a shows the relative proportions of each cell type, b shows the absolute numbers. No significant differences were found between the two groups (n=3 mice per group, one-way ANOVA with Tukey’s post-test). c: Mean frequency of B cells, T Cells and Neutrophils/mm² of small intestinal epithelium as quantified by immunohistochemistry. n=5 mice per group, No significant differences were detected by one-way ANOVA with Tukey’s post-test. d-e: Dot plots showing Z-scores calculated following quantification of mitochondrial OXPHOS protein levels in small groups of lamina propria cells in the small intestine (d) and colon (e) of PolgA^mut/mut and PolgA^+/+ mice (n=5 mice per group and a minimum of 50 areas per mouse were analysed). f: Categorical analysis of OXPHOS protein levels (n=5 mice per group). g: Relative expression of the SSP genes in the lamina propria of the small intestine by RT-PCR which had been identified to be upregulated by RNASeq analysis in the crypts (n=6 mice per group, one-way Mann-Whitney U test). P values are as follows: Phgdh p=0.1201, Psat1 p=0.043, Psph p=0.011, Aldh1l2 p=0.0022, Mthfd2 p=0.0043, Slc1a4 p=0.500. Mean values per mouse ± s.e.m are shown. * p<0.05, **p<0.01, ***p<0.001

Figure 7. Mitochondrial OXPHOS dysfunction causes upregulation of de novo serine synthesis in normal ageing human colonic crypts.

Immunoflourescent images showing co-labelling of OXPHOS proteins and SSP enzymes in normal human colonic epithelium. Scale bars; 50µm. Immunofluorescence was performed for each antibody on n=12 human samples. Representative images are shown. b-d: Quantification of the levels of PHGDH, PSAT1 and MTHFD2 in individual human crypts. Every OXPHOS deficient crypt on the section was quantified and OXPHOS normal crypts on the same section were randomly sampled. In b the number of crypts analysed from left to right is: n=45, 46, 40, 62, 43, 50, 28, 29, 21, 16, 16, 17, 33, 31, 41, 17, 20, 8, 32, 27, 15, 17, 24, 24. In c the number of crypts analysed from left to right is: n=47, 57, 58, 70, 44, 56, 54, 39, 73, 11, 21, 20, 33, 33, 39, 16, 30, 8, 61, 45, 31, 22, 51, 49. In d the number of crypts analysed from left to right is: 23, 67, 40, 61, 44, 47, 42, 32, 108, 15, 37, 38, 59, 60, 58, 26, 62, 10, 48, 40, 31, 24, 60, 59. Error bars show mean ±s.d. Data were analysed by two-sided linear mixed effect regression model with mouse ID as a random effect, p<0.0001 in all comparisons. e: Schematic showing the hypothesised mechanism by which mtDNA mutations and OXPHOS defects contribute to tumorigenesis. ***p<0.001