1,25-dihydroxyvitamin D alters lipid metabolism and epithelial-to-mesenchymal transition in metastatic epithelial breast cancer cells
Evidence suggests that high vitamin D status (marked by serum 25-hydroxyvitamin D, 25(OH)2D) is associated with a decreased risk of breast cancer. It has been established that 1,25-dihydroxyvitamin D (1,25(OH) 2D) can alter glycolysis and the Krebs cycle of breast cancer cells (Jiang et al., 2010; Zheng et al., 2013) but little information is available on 1,25(OH) 2D's alterations of lipid metabolism in breast cancer cells. Thus, the current research investigates if there was an effect of 1,25(OH) 2D on proteins that regulate lipid metabolism in MCF10A, MCF10A-ras, MCF10CA1h, and MCF10CA1a epithelial breast cancer cells. While no significant effects were seen at 24 hours of treatment with 10 nM of 1,25(OH) 2D, several changes were see at 48 hours with the most significant effects in the MCF10CA1a line. With 48 hour treatment of 1,25(OH) 2D there was a significant increase of fatty acid synthase (FAS), acyl-CoA cholesterol acyltransferase 1 (ACAT1), insulin-inducing gene 2 (Insig2), perilipin 2 (PLIN2), and a non-significant trend towards an increase in stearoyl-CoA desaturase 1 (SCD1) in the MCF10CA1a line. Additionally, there was a non-significant trend towards a decrease in carnitine palmitoyl transferase 1a (CPT1a), adipose triglyceride lipase (ATGL), and hormone sensitive lipase (HSL). Lipid accumulation was greater in the MCF10CA1a compared to the other MCF10A cell lines as shown by oil red O staining. 1,25(OH) 2D treatment caused a non-significant trend towards a decrease in total triglyceride concentration in the MCF10CA1a cells. Additionally, a multitude of alterations upon 1,25(OH) 2D treatment were noted in specific phosphotidylcholines (PCs), sphingomyelins, and ceramides using MS-MS. Together, these results suggest that 1,25(OH) 2D may alter lipid metabolism by increasing lipid storage and decreasing lipid degradation in epithelial breast cancer cells. Additionally, while it is known that 1,25(OH) 2D exerts anti-cancer effect on primary breast tumors (Welsh, 2004),the role of 1,25(OH) 2 D in metastasis has yet to be determined. Thus, the current research also investigates if there was an effect of 1,25(OH) 2D on epithelial-to-mesenchymal transition (EMT) protein markers and cell viability in MCF10CA1a metastatic epithelial breast cancer cells. To identify the impact of 1,25(OH) 2D regulation of EMT and metastasis of breast epithelial cells to bone we employed a reconstructed metastasis (rMET) model. Upon 1,25(OH) 2D treatment in the 3D matrix which recapitulates the mammary environment, mammospheres were visually smaller compared to vehicle treated cells though 3-(4,5-dimythylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assays did not show a significant difference in the amount of live cells between treatments in 3D. Additionally, the total number of cells to metastasize and survive in the reconstructed bone matrix (rBM) was significantly reduced in the 1,25(OH) 2D group versus vehicle. To determine the impact of 1,25(OH) 2D regulation of EMT, the mRNA abundance of markers of EMT in the upper well was determined. Treatment with 1,25(OH) 2D induced a significant increase in vimentin and fibronectin mRNA abundance, both markers of EMT, following 48 hours of 10 nM of 1,25(OH) 2D treatment compared to vehicle in 2D cell culture. Additionally, mRNA abundance of epithelial-cadherin (E-cadherin), which is decreased during EMT, had a trend towards an increase while neuronal-cadherin (N-cadherin) was unchanged. Immunofluorescent staining of the upper well mammospheres for E-cadherin, vimentin, perilipin 2 (PLIN2), co-stained with neutral lipid stain, Bodipy, and the nuclear stain, DAPI, were completed. Although a visual difference employing Zeiss microscope, showed little difference between treatments, confocal images are being obtained and analyzed by our laboratory to more carefully assess the differences in protein expression. Together, these results suggest that 1,25(OH) 2D decreases breast to bone metastasis by altering viability in the rBM and alterations in characteristic EMT protein expression in metastatic epithelial breast cancer cells, therefore decreasing cancer aggressiveness. Overall, our findings contribute to the knowledge of vitamin D biology and its function in mammary cancer and advocates that vitamin D is a potentially safe chemopreventative agent.
Teegarden, Purdue University.
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