Introduction: Breast cancer (BC) is the second deadliest cancer amongst women in the US, with metastatic breast cancer being particularly deadly. Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate for anaplerotic refilling of TCA intermediates, feeding numerous energetic and biosynthetic pathways. Upregulation of PC is an important contributor to metabolic reprogramming in both primary and metastatic BC. In this study, we investigate whether suppression of PC alters metabolism and drives microenvironmental adaptation in a primary tumor model of BC.
Methods: C57/Bl6 mice were injected with M-Wnt cells transduced with doxycycline-inducible ShRNA targeting PC. Doxycycline treatment began once tumors were palpable. Tumors were harvested 4 weeks following injection. Tumor transcriptomic analysis was conducted via GSEA and enrichment mapping. Digital cytometry using CIBORSORTx was conducted to determine tumor microenvironment immune cell composition. In vitro metabolic adaptation to PC suppression in BC cell lines following knockdown of PC was analyzed. Perturbations of mitochondrial metabolism and respiration were assessed by extracellular flux analysis. Assays of extracellular lactate and glucose concentrations determined changes in the production and utilization of carbon sources in the context of loss of PC
Results: Metabolic assays revealed that cells with PC knockdown export more lactate into their environment and respire less efficiently, without consuming additional glucose. This indicates potential mitochondrial dysfunction with loss of PC-derived anaplerosis. In vivo suppression of PC resulted in increased tumor mass and volume relative to control. Gene expression data from PC knockdown tumors revealed distinct transcriptomic profiles between groups. GSEA analysis further showed profound suppression of immunological pathways following loss of PC, indicating that PC knockdown resulted in a diminished immune response. Digital cytometry supported this finding with PC suppression resulting in decreased proportions of critical innate and adaptive immune cell populations.
Conclusion: Metabolic assays revealed increased flux through lactate production with a decrease in mitochondrial respiration, suggesting that diminished PC-mediated anaplerosis is altering the fate of carbon sources and contributing to metabolic reprogramming. Suppression of PC resulted in tumors with distinct transcriptomic profiles versus control, with immune response signatures being diminished in response to loss of PC. We conclude that PC knockdown promotes a metabolically altered tumor microenvironment associated with immunosuppression, tumor progression, and increased metastatic potential. This work was supported by R35CA197627 to S. Hursting, and R01CA232589 to D. Teegarden and S. Hursting.
Citation Format: Michael F. Coleman, Alexander J. Pfeil, Violet Kiesel, Suhas K. Etigunta, Michael K. Wendt, Dorothy Teegarden, Stephen D. Hursting. Mammary tumor microenvironment reprogramming in response to pyruvate carboxylase modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2323.