Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells

Doctor of Philosophy

dissertationThe discovery of the mitochondrial pyruvate carrier (MPC) and the ability to alter the cellular partitioning of pyruvate has allowed for exploration into the consequences for a cell with uncoupled glucose metabolism, unraveling the connections with gene expression to drive cellular proliferation and the biosynthetic requirements of cellular replication. Previous work on the MPC in cancer metabolism tested hypotheses using arguably aggressive and advanced cancer specimens, identifying a growth advantage upon MPC loss through the classic Warburg effect of uncoupled glycolysis and loss of pyruvate oxidation. Now, in the first dysplastic cells in a colon tumor, the directed study of the MPC has demonstrated the importance of metabolic compartmentation in glucose-fueled metabolic pathways in a cell's ability to regulate division autonomously. The cancer metabolism field has only recently begun to use the cell of origin's metabolism as the foundational network for the earliest metabolic aberrations in tumor initiation. In the intestinal and colon epithelium, the resident stem cells, protected at the base of the intestinal or colon crypt, give rise to intestinal and colon tumors, and thus must enact cellular programs to support dysplasia. Both in vivo and ex vivo mouse and human models have demonstrated that the MPC is again a node of regulation, with stem cells expressing lower levels of this transporter and differentiated cells expressing high levels. Interestingly, loss of the MPC throughout the crypt and across the gastrointestinal tract increased the number of tumor foci generated in two models of intestinal carcinogenesis. iv While preserving epithelial function and integrity, as well as organismal parameters of metabolism, MPC loss leading to a significant increase in tumor foci is explained by the subtle yet reproducible hyperproliferation of the stem cell niche. The pro-growth metabolic landscape thus creates a susceptibility towards dysplasia and tumorigenesis. This dissertation identified the gap in knowledge surrounding tumor initiation and addressed the question by linking the metabolic dependencies in adenocarcinoma and intestinal stem cells. The importance of the cell-autonomous approach towards cancer metabolism furthers the understanding of the complexity experienced at the tissue and organism level underlying organismal metabolic aberrations in cancer risk

Oct1/Pou2f1 is selectively required for colon regeneration and regulates colon malignancy.

The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to establish organoids in vitro, but blocked the ability to recover from treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or both Oct1 alleles progressively increased tumor burden in a colon cancer model driven by loss-of-heterozygosity of the tumor suppressor gene Apc. The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with differentially expressed genes between the two models. Oct1 ChIPseq using HCT116 colon carcinoma cells identifies target genes associated with mitotic stability, metabolism, stress response and malignancy. This set of gene targets overlaps significantly with genes differentially expressed in the two tumor models. These results reveal that Oct1 is selectively required for recovery after colon damage, and that Oct1 has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology