Combining metabolic phenotype determination with metabolomics and transcriptional analyses to reveal pathways regulated by hydroxycarboxylic acid receptor 2

The adaptation of cellular metabolism is considered a hallmark of cancer. Oncogenic signaling pathways support tumorigenesis and cancer progression through the induction of certain metabolic phenotypes associated with altered regulation of key metabolic enzymes. Hydroxycarboxylic acid receptor 2 (HCA2) is a G protein-coupled receptor previously shown to act as a tumor suppressor. Here, we aimed to unveil the connection between cellular metabolism and HCA2 in BT-474 cells. Moreover, we intend to clarify how well this metabolic phenotype is reflected in transcriptional changes and metabolite levels as determined by global metabolomics analyses.

We combined metabolic phenotype determination with metabolomics and transcriptional analyses and identified HCA2 as a regulator of glycolytic flux and fatty acid metabolism in BT-474 breast cancer cells. Thus, HCA2, for which agonists are already widely used to treat diseases such as psoriasis or hyperlipidemia, may prove useful as a target in combination cancer therapy.

We performed both, siRNA mediated knockdown of HCA2 and stimulation with the HCA2-specific agonist monomethyl fumarate. Seahorse technology was used to determine the role of HCA2 in BT-474 breast cancer cell metabolism and its potential to induce a switch in the metabolic phenotype in the presence of different energy substrates. Changes in the mRNA expression of metabolic enzymes were detected with real-time quantitative PCR (RT-qPCR). Untargeted liquid chromatography-mass spectrometry (LC-MS) metabolic profiling was used to determine changes in metabolite levels.

Knockdown or stimulation of HCA2 induced changes in the metabolic phenotype of BT474 cells dependent on the availability of energy substrates. The presence of HCA2 was associated with increased glycolytic flux with no fatty acids available. This was reflected in the increased mRNA expression of the glycolytic enzymes PFKFB4 and PKM2, which are known to promote the Warburg effect and have been described as prognostic markers in different types of cancer. With exogenous palmitate present, HCA2 caused elevated fatty acid oxidation and likely lipolysis. The increase in lipolysis was also detectable at the transcriptional level of ATGL and the metabolite levels of palmitic and stearic acid. Conclusions: We combined metabolic phenotype determination with metabolomics and transcriptional analyses and identified HCA2 as a regulator of glycolytic flux and fatty acid metabolism in BT-474 breast cancer cells. Thus, HCA2, for which agonists are already widely used to treat diseases such as psoriasis or hyperlipidemia, may prove useful as a target in combination cancer therapy.