Abstract 2376: Pyruvate carboxylase regulates mammary tumor microenvironment composition via central carbon metabolism
Authors: Pfeil Alexander J. , Coleman Michael F. , Kulkoyluoglu-Cotul Eylem , Kiesel Violet , Devericks Emily , Etigunta Suhas K. , Teegarden Dorothy , Wendt Michael K. , Hursting Stephen D. 
Published: June 2022

Introduction: Metastatic triple-negative breast cancer (TNBC) is one of the deadliest cancers amongst women in the US, with a 5-year survival rate of 12%. Due to a lack of targeted therapies, investigation into the metabolic drivers of TNBC progression at both the primary and secondary site is vital to direct future treatment strategies. Pyruvate carboxylase (PC) is an anaplerotic enzyme vital for TNBC metastasis to the lungs, though its expression is commonly suppressed in the primary tumor compared to normal tissue. In this study, we investigated the role of PC expression in the metabolic reprogramming of TNBC cell lines and how its reduced expression in vivo may support primary tumor progression.

Methods: Modulation of PC expression in M-Wnt cell lines was achieved through lentiviral-mediated transduction of PCx-targeting short hairpin RNAs (ShPC). In vitro metabolic alterations to central carbon metabolism following PC suppression were assessed via intracellular lactate and NAD+/NADH assays. Functional shifts in mitochondrial metabolism were assessed via extracellular flux analysis in various substrate conditions. Electron transport chain (ETC)-intrinsic dysfunction was assessed via high-resolution respirometry (HRR) and qPCR analysis of ETC gene expression. C57BL/6J mice were injected with ShPC M-Wnt cells and the resulting tumors were harvested after 4 weeks following injection. Metabolomic analysis, mass cytometry, and HRR were conducted with samples from excised tumors.

Results: PC knockdown in vitro results in reduced oxygen consumption and a gene expression profile indicative of mitochondrial dysfunction. Genes encoding subunits of ETC complexes such as ND4FA10, SDHB, and ATP5c1 were downregulated while markers of mitochondrial biogenesis were upregulated following suppression of PC. Intracellular lactate and cell proliferation assays revealed enhanced flux through lactate dehydrogenase and sensitization of PC knockdown M-Wnt cells to lactate metabolism inhibitors. PC knockdown in vivo resulted in enhanced primary tumor growth and an immunosuppressed tumor microenvironment, as indicated by downregulated cytokine signaling and reduced cell fractions of critical anti-tumor immune cells.

Conclusions: Suppression of PC in vitro results in mitochondrial dysfunction, manifesting in a metabolic phenotype characterized by enhanced production of lactate and reduced oxidative phosphorylation. In vivo, PC knockdown resulted in enhanced primary tumor growth and a tumor immune landscape indicative of immunosuppression. We conclude that PC knockdown promotes distinct metabolic reprogramming of the primary mammary tumor microenvironment associated with immune evasion and enhanced tumor progression.

Funding: This study was supported by grants from the National Cancer Institute (R35 CA197627 to SD Hursting, and RO1 CA232589 to SD Hursting and D. Teegarden)

Citation Format: Alexander J. Pfeil, Michael F. Coleman, Eylem Kulkoyluoglu-Cotul, Violet Kiesel, Emily Devericks, Suhas K. Etigunta, Dorothy Teegarden, Michael K. Wendt, Stephen D. Hursting. Pyruvate carboxylase regulates mammary tumor microenvironment composition via central carbon metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2376.