Not-so-mighty mitochondria: Mitochondrial dysfunction in mismatch repair deficient endometrial cancer development (234)

Abstract

<b>Objectives:</b> Thirty percent of endometrial cancers exhibit DNA mismatch repair deficiency (MMRd), and inherited or sporadic loss of mismatch repair proteins increases the risk of developing endometrial cancer (EC). Identifying the consequences of MMRd beyond hypermutability could provide novel targetable mechanisms during EC development. Mitochondrial dysfunction has been shown to be a therapeutic vulnerability in other advanced MMRd-related cancers but has yet to be described early in cancer development. The objective of our study was to define the mitochondrial function in MMRd-related EC development. <b>Methods:</b> Our novel mouse model for MMRd-related EC development uses the uterine-targeted loss of <i>Msh2</i> (a commonly mutated gene in MMRd EC), abbreviated Msh2KO. Transcriptomic profiling using the Mouse ClariomD Assay (Affymetrix) was used to compare Msh2KO normal endometrium (<i>n</i>=4) and Msh2KO EC tissue (<i>n</i>=7) versus <i>Msh2</i>-wildtype (WT) normal endometrium (<i>n</i>=5). Differentially expressed genes were further evaluated using Ingenuity Pathway Analysis. Mitochondrial function was evaluated using a panel of five tumor-derived cell lines from Msh2KO mice. Using the Msh2KO cell line panel and a mismatch repair proficient mouse EC line (<i>PTEN</i>-null and <i>KRAS</i>-mutant; abbreviated MecPK), we performed the Seahorse Mitochondrial Stress Test (Agilent) to measure basal and induced mitochondrial function. <b>Results:</b> A total of 1,295 genes were differentially expressed in Msh2KO normal endometrium and 2,938 in Msh2KO EC compared to <i>Msh2</i> WT normal endometrium (90% were downregulated). Fifty-two downregulated genes in Msh2KO EC and 32 in Msh2KO normal endometrium related to mitochondrial electron transport chain function (p<0.05). Pathway analysis showed that the top dysregulated pathway in Msh2KO EC was "Mitochondrial dysfunction" (p=1.5E-7), which was also significantly altered in Msh2KO normal endometrium. Within this pathway, Msh2KO EC and Msh2KO normal endometrium shared significant changes of <i>Casp8</i>, <i>Cpt1a</i>, <i>Gpd2</i>, <i>Ncstn</i>, <i>Ogdh</i>, <i>Ndufa8</i>, and <i>Ndufa9</i> with at least a 2-fold-change decrease in expression compared to Msh2 WT endometrium (p<0.001). Mitochondrial stress tests <i>in vitro</i> revealed that the Msh2KO cell line panel had significantly impaired mitochondrial function compared to MecPK cells. Basal oxygen consumption rate, a measure of baseline mitochondrial function, was decreased by 50-90% in four of five Msh2KO cell lines compared to MecPK (p<0.001). Spare respiratory capacity, a measure of mitochondrial response to induced stress, was decreased by 30-85% in all five Msh2KO cell lines compared to MecPK cells (p<0.001). Because there was no significant difference in extracellular acidification rate between Msh2KO cell lines and MecPK cells during the assay, diminished mitochondrial function was likely due to intrinsic mitochondrial dysfunction rather than an adaptive glycolytic shift. <b>Conclusions:</b> Endometrial cancers from Msh2KO mice exhibit mitochondrial dysfunction at a transcriptomic and functional level. Importantly, this study also revealed molecular changes consistent with mitochondrial dysfunction in histologically-normal endometrium from Msh2KO mice, suggesting intrinsic mitochondrial dysfunction early in MMRd EC development. Further studies are necessary to corroborate findings in human MMRd-related EC. Identifying a unique vulnerability of MMRd endometrium undergoing cancer development would provide a novel target for cancer interception efforts.