Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis
Introduction
Lynch syndrome (LS) is a hereditary cancer syndrome with a 60% lifetime risk of endometrial cancer (EC). LS is defined by mutations in DNA mismatch repair genes, including MSH2, but molecular determinants of LS-EC development are not well-defined. Identifying mechanisms influencing EC development could offer new opportunities for prevention. Our recent studies in Msh2-deficient mice revealed mitochondrial dysfunction in EC pathogenesis, despite MSH2 not directly participating in mitochondrial DNA (mtDNA) repair. We aimed to define the mechanism of mitochondrial dysfunction and its impact on metabolic reprogramming to identify targets for EC prevention.
Methods
We assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines from this mouse model (21B, 369, 1386), and human EC cells and isogenic MSH2-knockdown controls. mtDNA damage was measured using a RT-PCR. Mitochondrial content was quantified in vitro by immunofluorescent mitochondrial staining (TOM20) using confocal microscopy and in vivo by anti-TOM20 immunohistochemical staining (IHC) of Msh2KO and wildtype (WT) endometrium. TOM20 IHC was performed on human ECs from patients with and without LS. Mitochondrial stress tests (MSTs) assessed mitochondrial function. Polar metabolite profiling via ultra-high resolution ion chromatography mass spectrometry was conducted on Msh2KO EC and MSH2-intact EC from the Pten+/- mouse model. Glycolysis was suppressed in vitro by replacing glucose with galactose in culture media and viability measured using CellTiter Glo.
Results
mtDNA damage was elevated in MSH2-deficient MFE280 (4.8-fold) and RL95-2 (7.9-fold) cell lines compared to MSH2-intact KLE and Hec50 cell lines (p<0.001). MSH2 knockdown increased mtDNA damage in KLE and Hec50 cells by 2.5- to 5.7-fold vs control (p<0.05 for all). MSH2-deficient mouse and human cell lines exhibited significant reductions in mitochondrial content compared to MSH2-intact counterparts by immunofluorescence, with Msh2KO cell lines exhibiting over 60% reductions in mitochondrial volume compared to MecPK and MSH2-deficient human EC lines exhibiting over 95% reductions in mitochondrial volume compared to KLE cells (p<0.0001 for all). IHC also demonstrated reduced mitochondrial content during EC development in Msh2KO mice and in LS-related human ECs. MSTs revealed decreased baseline (0.17, 0.23, and 0.29 pmol/min relative to MecPK in 21B, 369, and 1386 cells, respectively, p<0.0001) and induced mitochondrial function (0.18, 0.26, and 0.39 pmol/min relative to MecPK, respectively, p<0.0001) and in a panel of MSH2-deficient human cells (MFE280, RL95-2, and Hec59) compared to MSH2-intact human cells (KLE, Hec1a, Hec50), with p<0.0001 for each comparison. Within the top 10 differentially modulated metabolites in Msh2KO EC compared to Pten+/- EC were glucose 6-phosphate (log2 fold-change (FC) -1.79), mannose 6-phosphate (log2FC -1.77), fructose 6-phosphate (log2FC -1.54), and lactate (log2FC 0.68) with padj<0.05. Galactose reduced viability in MSH2-deficient mouse cells (0.21, 0.10, and 0.09 surviving fraction in 21B, 369, and 1386 cells, respectively) more than in MSH2-intact cells (surviving fraction 0.56, p<0.0001). MSH2-deficient RL95-2 cells had lower viability following galactose (surviving fraction 0.21) compared to MSH2-intact KLE cells (surviving fraction 0.60, p<0.01).
Conclusion
Mitochondrial DNA damage in MSH2-deficient cells suggests an indirect role of MSH2 in mtDNA repair. Msh2-related mitochondrial depletion leads to reduced mitochondrial function and metabolic reprogramming toward glycolysis, as signified by increased usage of glycolytic intermediates and increased dependence on glycolysis for survival. Mitochondrial and metabolic aberrations could be further evaluated as novel biomarkers for EC prevention in women with LS.