Introduction
Triple-negative breast cancer (TNBC) is known to be addicted to utilising glutamine to support cell growth. The glutamine transporter, ASCT2, is highly expressed in TNBC and has been shown as a putative therapeutic target in this aggressive breast cancer subset using a range of in vitro and in vivo models. While glutamine targeted therapies including glutaminase inhibitors have gained traction and are being trialled, these have had limited success due to a range of resistance mechanisms.
Methods
We examined proliferation of six human breast cancer cell lines after ASCT2 CRISPR/Cas9 knockout and shRNA knockdown. Metabolic changes were assessed using targeted metabolomics in human TNBC cell lines. Proteomics and mRNAseq analysis further examined cellular and adaptive changes to ASCT2 knockout.
Results
Our study shows that knockout of ASCT2 leads to compensatory upregulation of metabolic pathways including macropinocytosis. This is hardwired into the TNBC subset, with some cell lines undertaking constitutive macropinocytosis, while others upregulate macropinocytosis in response to reduced glutamine transport in the knockout. Macropinocytosis was able to restore proliferation in all TNBC cell lines, with only mild inhibition of colony formation in some cell lines. In addition, HCC1806 TNBC cell line was able to rewire metabolic pathways to make glutamine from alternative sources such as glucose. These data confirm the glutamine addiction of TNBC cells, but show they also have upstream pathways that can compensate for loss of glutamine transport.
Conclusion
We demonstrate that caution should be applied to targeting ASCT2 in TNBC, due to their ability to upregulate compensatory glutamine uptake pathways such as macropinocytosis and glutamine synthesis. TNBC cells continue to rely on glutamine, however therapeutic targeting may need to focus on other unique metabolic pathways such as single-pass glutaminolysis, which couples glutamine and glucose metabolism together.