Activation of pro-survival metabolic networks by 1,25(OH)2D3 does not hamper the sensitivity of breast cancer cells to chemotherapeutics

Background: We have previously identified 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3], the bioactive form of vitamin D-3, as a potent regulator of energy-utilization and nutrient-sensing pathways in prostate cancer cells. In the current study, we investigated the effects of 1,25(OH)(2)D-3 on breast cancer (BCa) cell metabolism using cell lines representing distinct molecular subtypes, luminal (MCF-7 and T-47D), and triple-negative BCa (MDA-MB-231, MDA-MB-468, and HCC-1143). Methods: 1,25(OH)(2)D-3's effect on BCa cell metabolism was evaluated by employing a combination of real-time measurements of glycolysis/oxygen consumption rates using a biosensor chip system, GC/MS-based metabolomics, gene expression analysis, and assessment of overall energy levels. The influence of treatment on energy-related signaling molecules was investigated by immunoblotting. Results: We show that 1,25(OH)(2)D-3 significantly induces the expression and activity of the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (G6PD) in all BCa cell lines, however differentially influences glycolytic and respiratory rates in the same cells. Although 1,25(OH)(2)D-3 treatment was found to induce seemingly antioxidant responses in MCF-7 cells, such as increased intracellular serine levels, and reduce the expression of its putative target gene thioredoxin-interacting protein (TXNIP), intracellular reactive oxygen species levels were found to be elevated. Serine accumulation in 1,25(OH)(2)D-3-treated cells was not found to hamper the efficacy of chemotherapeutics, including 5-fluorouracil. Detailed analyses of the nature of TXNIP's regulation by 1,25(OH)(2)D-3 included genetic and pharmacological inhibition of signaling molecules and metabolic enzymes including AMP-activated protein kinase and G6PD, as well as by studying the ITCH (E3 ubiquitin ligase)-TXNIP interaction. While these investigations demonstrated minimal involvement of such pathways in the observed non-canonical regulation of TXNIP, inhibition of estrogen receptor (ER) signaling by tamoxifen mirrored the reduction of TXNIP levels by 1,25(OH)(2)D-3, demonstrating that the latter's negative regulation of ER expression is a potential mechanism of TXNIP modulation. Conclusions: Altogether, we propose that regulation of energy metabolism contributes to 1,25(OH)(2)D-3's anticancer effects and that combining 1,25(OH)(2)D-3 with drugs targeting metabolic networks in tumor cells may lead to synergistic effects.