1α,25‐Dihydroxyvitamin D3 Inhibits De Novo Fatty Acid Synthesis and Neutral Lipid Accumulation in Metastatic Breast Cancer Cells Through Downregulation of Pyruvate Carboxylase
Authors: Wilmanski Tomasz M , Buhman Kimberly , Donkin Shawn S , Burgess John , Teegarden Dorothy 
Published: November 2023

The anaplerotic enzyme pyruvate carboxylase (PC) has recently emerged as a mediator of breast cancer progression. In addition, PC also plays a regulatory role in adipocyte de novo fatty acid synthesis and neutral lipid accumulation, two pathways exploited by metastatic cancer cells. In the present studies, we investigated the effect of the bioactive vitamin D metabolite, 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D), on PC and lipid metabolism in the MCF10 model of breast cancer progression. First, quantification of triacylglycerol (TAG), the most abundant form of neutral lipids, confirmed that neutral lipid accumulation increases with stages of breast cancer progression, with metastatic MCF10CA1a cells demonstrating a 12.46(±1.49) fold higher TAG level than either MCF10A or the Harvey‐ras oncogene transfected MCF10A‐ras cells. In addition, MCF10CA1a cells expressed a 5.14(±0.21) fold higher level of PC mRNA relative to MCF10A and MCF10A‐ras cells. Therefore, our studies focused on the effect of 1,25(OH)2D on lipid metabolism in the MCF10CA1a cell line with highest level of PC and TAG in the MCF10 model. Treatment of MCF10CA1a cells with 1,25(OH)2D decreased both PC mRNA (58%±7 relative to vehicle) and protein (54%±12 relative to vehicle) expression at 5 days, as measured RT‐qPCR and Western blotting respectively. Similarly, treatment with 1,25(OH)2D decreased PC mRNA expression in other breast epithelial cell lines including the MCF10A‐ras (57%±4 relative to vehicle) and murine metastatic 4T1 cells (42%±11 relative to vehicle) at 48 hours. To investigate the effect of 1,25(OH)2D on cancer cell lipid metabolism, we measured glucose incorporation into palmitate, the predominant fatty acid synthesized de novo. MCF10CA1a cells treated with 1,25(OH)2D for five days demonstrated decreased fatty acid synthesis from glucose (48%±5.5 relative to vehicle), as measured by 13C‐[U]‐glucose incorporation into palmitate through high performance liquid chromatography coupled mass spectrometry. Imaging of neutral lipids using boron‐dipyrromethene (BODIPY) as well as TAG quantification additionally confirmed that 1,25(OH)2D inhibits neutral lipid accumulation at 5 (78%±3.9) and 7 (56%±5.4 relative to vehicle) days of treatment. We hypothesized that 1,25(OH)2D regulates lipid metabolism in breast cancer cells through downregulation of PC. To test our hypothesis, MCF10CA1a cells were transiently transfected with a PC containing plasmid under the control of a pCMV6 promoter. PC overexpression inhibited the effect of 1,25(OH)2D on both neutral lipid accumulation and de novo palmitate synthesis from glucose. Together, these results present a novel mechanism through which 1,25(OH)2D regulates breast cancer cell metabolism at late stages of breast cancer progression.Support or Funding InformationThis work was supported by a Project Development Team within the ICTSI NIH/NCRR (GrantNumber UL1TR001108) and the National Institutes of Health, National Cancer Institute(R25CA128770) Cancer Prevention Internship Program. Additional support was received from theIndiana Elks Charities and the SIRG grant, both administered through the Purdue University Centerfor Cancer Research (Purdue University, West Lafayette, IN).