Abstract 3696: L-arginine depletion causes different proliferation responses in breast cancer subtypes - Relation to gene expression profiles of L-arginine metabolic pathways

Metabolic fingerprinting provides insight into cancer biology, improving personalized therapeutic strategies. We sought to unravel responses of the molecular subtypes of breast cancer to L-arginine depletion in vitro. Availability of the semi-essential amino acid L-arginine limits protein synthesis and cell proliferation. L-Arginine is the substrate for nitric oxide synthases (NOS) and arginases (ARG), enzymes also involved in the regulation of cell proliferation. Clinical studies have provided contrasting results regarding the role of L-arginine in breast cancer.We studied the response of different breast cancer cell lines to L-arginine depletion and the molecular mechanisms underlying this effect. MCF-7, SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 cells were cultured under standard conditions and compared to MCF-12A normal breast epithelial cells. Cells were maintained in culture media with 200 mg/L (control) or 0 mg/L of L-arginine (depletion) for up to 240 h. Cell proliferation was analyzed and intracellular concentrations of L-arginine, L-ornithine, and L-citrulline were measured by UPLC-MS/MS every 24 h. mRNA expression of genes involved in L-arginine metabolism was quantified by qRT-PCR. MCF-12A control cells did not proliferate in the absence of extracellular L-arginine. Their intracellular L-arginine content dropped sharply, whilst L-ornithine and L-citrulline were only slightly reduced. By contrast, proliferation of BT-474, SK-BR-3, and MDA-MB-468 cells was unaffected by L-arginine depletion (p<0.05), whilst MCF-7 and MDA-MB-231 cells showed significantly higher proliferation rates in the absence of L-arginine (p<0.05). L-arginine concentration dropped sharply within 24 h in MCF-7 cells, but only by 25-50% in the other cell lines (p<0.01). L-ornithine dropped to almost zero within 24 h in both estrogen receptor positive cell lines (MCF-7 and BT-474), whilst being only slightly reduced in SK-BR-3 cells (p<0.01). L-ornithine was not significantly reduced in MDA-MB-231, but strongly reduced in MDA-MB-468 cells (p<0.05). L-citrulline showed no significant change in any of the cell lines. mRNA expression of NOS, ARG, and the L-arginine salvage pathway (ASS1 and ASL) was differentially expressed amongst the cell lines. SK-BR-3 showed high expression of the L-arginine salvage pathway; BT-474 had the highest expression levels of NOS and ARG2, and MDA-MB had high ARG2 but very low NOS expression. Gene expression patterns of MCF-12A and MCF-7 were very similar. In conclusion, metabolic fingerprinting reveals complex and differential regulation of L-arginine pathways in breast cancer cell lines. These analyses may help to understand differences in proliferation during L-arginine depletion, thus offering a perspective to further personalize treatment options within classical molecular breast cancer subtypes. Citation Format: Juliane Hannemann, Fiona Franke, Mariola Kastner, Rainer Böger. L-arginine depletion causes different proliferation responses in breast cancer subtypes - Relation to gene expression profiles of L-arginine metabolic pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3696.