Abstract A036: Macropinocytosis mediates metabolic resistance to loss of glutamine transport in triple-negative breast cancer

Abstract

Abstract Introduction Although a nonessential amino acid in normal cells, the demand for glutamine is dramatically increased throughout malignant transformation, supporting a range of metabolic processes including mitochondrial ATP production, protein synthesis, purine and pyrimidine biosynthesis. We previously shown that triple-negative breast cancer (TNBC) cells rely on glutamine uptake by the amino acid transporter ASCT2 to sustain their unique glutamine metabolism, thereby supporting in vitro growth and in vivo tumour formation. However, it is known that TNBC cells can also utilise non-transporter mediated nutrient uptake facilitated by processes such as macropinocytosis. Methods We examined proliferation of human breast cancer cell lines after ASCT2 CRISPR/Cas9 knockout (clonal and polyclonal populations) and shRNA knockdown. Proteomics and mRNAseq analysis further examined cellular and adaptive changes to ASCT2 knockout. Cellular changes were further analysed by western blotting, with macropinocytosis examined using 70kDa dextran-FITC uptake. Metabolic changes were assessed using targeted metabolomics approaches including 13C-labelled substrate tracing and liquid chromatography coupled tandem-mass spectrometry (LC-MS/MS) to determine intracellular levels of key tricarboxylic acid (TCA) cycle intermediates, glycolytic metabolites, fatty acid precursors, nucleotides, and amino acids in human TNBC cell lines in vitro. Results Despite our previous data showing a significant reduction in cell growth after ASCT2 knockdown, ASCT2 knockout was well-tolerated by both TNBC and Luminal A breast cancer cell lines, with proliferation rates similar to non-targeted CRISPR/Cas9 control cells. This adaptation to ASCT2 knockout was not due to the high glutamine levels present in culture media, as the knockout cells could be cloned in media containing either standard 2 mM glutamine or physiological 0.5 mM glutamine. Previous data have shown that TNBC cell lines can undergo constitutive macropinocytosis, and that this can be enhanced when cells are cultured in low nutrient conditions. Indeed, not only did the TNBC cell line HCC1806 undergo constitutive macropinocytosis, the amount of macropinocytosis was significantly increased (5-10 fold) in 5 separate ASCT2 knockout clones tested. By comparison, the ASCT2 shRNA knockdown cell line, which are published to have a significant proliferation deficit, showed a modest 2-fold increase in macropinocytosis, which may explain why they maintain a growth phenotype compared to the knockout lines. Despite in-depth analysis of gene and protein levels by mRNAseq and proteomics, ASCT2 knockout cells showed only a few significant changes in known macropinocytotic genes. There was, however, a substantial upregulation of Ser473-Akt phosphorylation which may drive the adaptive macropinocytosis in TNBC. Conclusion These data suggest that the constitutive macropinocytosis present in TNBC cell lines provides a novel resistance mechanism to strategies targeting glutamine uptake alone. Citation Format: Kanu Wahi, Natasha Freidman, Qian Wang, Yi Fang Guan, Jeff Holst. Macropinocytosis mediates metabolic resistance to loss of glutamine transport in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A036.