High-dose glutamine modulates amino acid transport and mediates pancreatic cancer chemosensitivity
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA884815 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21731933_1.fastq.gz | Fastqsanger.gz | |||
| SRR21731933_2.fastq.gz | Fastqsanger.gz | |||
| SRR21731934_1.fastq.gz | Fastqsanger.gz | |||
| SRR21731934_2.fastq.gz | Fastqsanger.gz | |||
| SRR21731935_1.fastq.gz | Fastqsanger.gz |
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Project 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.
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Project description:Microorganisms can restructure their transcriptional output to adapt to environmental conditions by sensing endogenous metabolite pool. In this study, an Agilent customized microarray representing approximately 4,106 genes was used to study temporal transcript profiles of Bacillus subtilis in response to valine, glutamate and glutamine pulses. Amino-acid-regulated genes were identified having significantly changed expression at one or more time points in response to pulses of valine, glutamate, and glutamine, respectively, and Val-, Glu and Gln-specific genes were further distinguished from them. Different amino acid treatments were compared in terms of both the global temporal profiles and the 5-minute quick regulations, and between-experiment differential genes were identified. The highlighted genes were analyzed based on diverse sources of gene functions using a variety of computational tools, including T-profiler analysis, hierarchical clustering and enrichment of functional categories. The results revealed the common and distinct modes of action of these three amino acids, and should help to elucidate the specific signaling mechanism of each amino acid as an effector. Three amino acids (Glutamate, Glutamine, and Valine) were adopted to perturb the culture of subtilis. Four time-points were investigated for each perturbation. There are two replicates for the first time-point of Valine-treatment experiment.
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