PXD053894-2

PXD053894 is an original dataset announced via ProteomeXchange.

Title	¬Macropinocytosis mediates metabolic resistance to loss of glutamine transport in triple-negative breast cancer
Description	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 showed 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. We examined proliferation and colony forming ability 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. 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 knockout was not due to the high glutamine levels present in culture media, as the knockout cells could be cloned in media containing physiological 0.5 mM glutamine. Previous data have shown that TNBC cell lines can undergo constitutive macropinocytosis, and that this could 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 enhanced (5-10 fold) in 5 separate ASCT2 knockout clones. By comparison, the ASCT2 knockdown cell line, which have a significant proliferation deficit, showed a modest 2-fold increase in macropinocytosis. Despite in-depth analysis of gene and protein levels by mRNAseq and proteomics, ASCT2 knockout cells did not display a significant alteration in macropinocytic gene expression, but instead showed a substantial upregulation of Ser473-Akt phosphorylation which may drive the adaptive macropinocytosis in TNBC. These data suggest that the constitutive macropinocytosis present in TNBC cell lines provides a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine, however therapeutic targeting may need to focus on other unique TNBC metabolic pathways such as single-pass glutaminolysis, which couples glutamine and glucose metabolism together.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:58:41.520.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Mark Larance
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2024-07-11 17:22:40	ID requested
1	2024-09-16 16:13:46	announced
⏵ 2	2024-10-22 06:58:41	announced	2024-10-22: Updated project metadata.

Dataset with its publication pending

submitter keyword: HCC,cancer, ASCT2

Mark Larance
contact affiliation	Charles Perkins Centre, School of Medical Sciences, University of Sydney, Sydney, Australia.
contact email	mark.larance@sydney.edu.au
lab head
Mark Larance
contact affiliation	The University of Sydney
contact email	mark.larance@sydney.edu.au
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD053894
     1. Label: PRIDE project
     2. Name: ¬Macropinocytosis mediates metabolic resistance to loss of glutamine transport in triple-negative breast cancer