Primary Investigator Texas A&M University, United States
Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high probability of reoccurrence and limited targeted treatment options. Within the tumor microenvironment, it is known that TNBC cells have the ability to reprogram their metabolism to become more efficient for tumor proliferation, such as the Warburg Effect. However, although many tumor cells prefer to be glycolytic, new research suggests there is a balance between glycolytic and oxidative cancer cells, working together to contribute to their rapid growth in a process known as metabolic symbiosis [1]. The key player in this process is lactose dehydrogenase B (LDH-B), the enzyme responsible for the conversion of lactate to pyruvate. Lactate is produced as a byproduct of glycolysis and exits glycolytic cancer cells through the MCT4 transporter. Lactate then enters oxidative cancer cells through the MCT1 transporter and LDH-B converts this lactate back into pyruvate. This allows oxidative cancer cells to immediately begin the TCA reactions without initially completing glycolysis. Therefore, therapies that target or interrupt this metabolic symbiosis are imperative for treating cancers that are dependent on this process, such as TNBC. The first known selective uncompetitive inhibitor of LDH-B is AXKO-0046, an indole derivative discovered by Shibata et al [2]. This research aims to quantify the metabolic changes that occur in TNBC populations when introduced to AKXO-0046. This will happen via metabolic assays and fluorescence lifetime imaging microscopy (FLIM), a technique dependent on the fluorescent lifetimes of two metabolic coenzymes, NADH and FAD.
Materials and
Methods: For all experiments, MDA-MB-231 cells, a TNBC cell line, are used. The cells are cultured according to ATCC’s instructions. First, an XTT cell viability assay (CyQUANT Ca. No.: X12223) was performed with varying concentrations of AXKO-0046 (MedChem Express Cat. No.: HY-147216), a highly selective and uncompetitive LDH-B inhibitor. MDA-MB-231 cells were plated and grown for 24 hours in a 96-well plate with growth media (ThermoFisher Cat. No.: 11875085). After 24 hours, varying concentrations (0.1 μM, 1.0 μM, 5.0 μM, and 10 μM) of AXKO-0046 were added to the wells. The cell viability assay was performed according to the manufacturer's instructions 24 hours after AXKO-0046 introduction to measure changes in the cell population. After confirming that AXKO-0046 decreases TNBC cell numbers, preliminary imaging experiments using fluorescence lifetime imaging microscopy (FLIM) on a multiphoton microscope commenced. Cells were plated in imaging dishes and left to grow in growth media for 48 hours. Similar to the cell viability assay, varying concentrations (0.1 μM, 1.0 μM, 5.0 μM, and 10 μM) of AXKO-0046 were added to the imaging dishes. FLIM was performed 24 hours after AXKO-0046 introduction to qualitatively and quantitatively characterize changes in metabolism. The cell’s morphology and confluency were examined as well as the redox ratio (NADH/(NADH+FAD) to establish how much the cellular metabolism is affected.
Results, Conclusions, and Discussions: AXKO-0046 significantly decreased the TNBC cell population viability, especially at higher concentrations such as 10 μM after 24 hours. Imaging experiments revealed that the morphology of the cells changed, with a greater proportion of round cells, indicative of unhealthy metabolism in TNBC cells. Additionally, the confluency decreased as LDH-B inhibition leads to cell death due to perturbed metabolism. The redox ratio of cell populations affected by AXKO-0046 decreased due to the inability to effectively execute metabolic symbiosis. These findings support a metabolic effect of LDH-B on TNBC cells and the possibility of using an LDH-B inhibitor as a TNBC treatment.
Acknowledgements (Optional): I would like to thank Dr. Linghao Hu for his assistance during the assay experiments and Blanche Ter Hofstede for her assistance during imaging experiments. This project is funded by the NIH R35 GM142990 and CZI 2022-251380 grants received by Dr. Walsh’s lab.
References: [1] A. S. Dias, C. R. Almeida, L. A. Helguero, and I. F. Duarte, “Metabolic crosstalk in the breast cancer microenvironment,” European Journal of Cancer, vol. 121, pp. 154–171, Nov. 2019, doi: https://doi.org/10.1016/j.ejca.2019.09.002. [2] S. Shibata et al., “Identification of the first highly selective inhibitor of human lactate dehydrogenase B,” Scientific Reports, vol. 11, no. 1, p. 21353, Nov. 2021, doi: https://doi.org/10.1038/s41598-021-00820-7.
