Vitamin D, obesity and breast cancer metastasis

Breast cancer is the second leading cause of cancer death among women in the United States. Many epidemiological studies have demonstrated that obesity is a risk factor for breast cancer. As leptin is an adipokine released from adipose tissue in proportion to the adipose tissue size, obese people generally have higher serum leptin levels than non-obese individuals. Many researchers have shown that leptin exerts migratory and invasive effects through leptin receptors in cancer cells, including breast cancer, and studies on cell models were conducted in conditions with medium containing high glucose concentrations. However, the mechanisms by which leptin induces breast cancer cells migration at physiological glucose levels are not fully elucidated. In the present studies, the effect of leptin on migratory capability was investigated focusing on leptin-mediated altered energy metabolism, as migration is an energy-requiring process. Two murine metastatic 4T1 and metM-wnt^lung cell lines are employed in this study. Leptin treatment for 4 days increased migration in 4T1 and metM-wnt^lung murine breast cancer cells when cultured in 5 mM glucose medium. We determined, using stably labeled glucose, that leptin did not alter glucose incorporation into palmitate in 4T1 cells, but leptin increased palmitate synthesis from glucose in metM-wnt^lung cells. Additionally, in 4T1 cells, leptin treatment increased Glut1 mRNA level and decreased Fasn mRNA level. However, both Glut1 and Fasn mRNA level were increased with leptin treatment in metM-wnt^lung cells. These different effects of leptin on glucose and fatty acid metabolism may lead to different energy status regulated through AMP-activated protein kinase (AMPK), a master sensor of energy status. While AMPK was activated with leptin treatment in 4T1 cells, it was inactivated with leptin treatment in metM-wnt^lung cells. We also determined that glycolysis is necessary for leptin-mediated increased migratory capability in metM-wnt^lung cells, but not in 4T1 cells. However, fatty acid metabolism is not required in leptin-induced migration in either 4T1 or metM-wnt^lung cells. Furthermore, glutamine metabolism is also not involved in increased migration with leptin treatment in metM-wnt^lung. Thus, leptin-mediated alteration in energy metabolism is differentially regulated in 4T1 and metM-wnt^lung cells during leptin-induced migration. Another factor that may regulate breast cancer migration potentially through leptin is vitamin D, as it has been shown to inhibit breast cancer metastasis and vitamin D impacts adipocytes including adipogenesis and inflammation. However, the link between vitamin D’s regulation of adipocytes and the effect on breast cancer metastasis is not understood. Here, we demonstrated that migration of MDA-MB-231 cells was reduced when exposed to conditioned media from differentiated mature 3T3-L1 adipocytes treated with the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) compared to vehicle-treated controls. In addition, 1,25(OH)₂D decreased mRNA level of leptin (Lep), adiponectin (Adipoq), IGF-1, IL-6, and MCP-1. Consistent with the change in mRNA level, concentrations of leptin, adiponectin, IGF-1, and IL-6, but not MCP-1, in adipocyte conditioned media were decreased with 1,25(OH)₂D treatment. Although adipocyte leptin mRNA level and release were decreased with 1,25(OH)₂D, leptin alone did not induce migration as a chemoattractant, suggesting other factors underly the impact of 1,25(OH)₂D on adipocytes to decrease migration of breast cancer cells. In addition to the impact of 1,25(OH)₂D on adipocytes, 1,25(OH)₂D exerts its effect in breast cancer cells since breast tissue expresses vitamin D receptor (VDR). However, loss of VDR is reported during tumor progression in cancer cells, implicating that vitamin D may not exert its protective effect on more advanced stages of cancer. In this study, MCF-10A series, a series of human cells representing different stages of breast cancer, were employed, including untransformed MCF10A, Harvey-ras oncogene transfected early progression of breast cancer cells model (MCF10A-ras), and metastatic MCF10CA1a cells. Consistent with previous literature, we demonstrated that the basal VDR mRNA level was reduced in metastatic MCF10CA1a compared to untransformed MCF10A cells, however, treatment with 1,25(OH)₂D reduced mRNA level of VDR in MCF10A and MCF10A-ras cell but increased VDR mRNA level and induced a trend toward an increase in VDR transcriptional specific activity in MCF10CA1a cells with 1,25(OH)₂D treatment compared to MCF10A. Together, these results indicate that vitamin D may induce inhibitory effects on metastatic breast cancer cells through upregulation of VDR level by 1,25(OH)₂D. Collectively, the present studies provide a novel insight into leptin-mediated changes in energy metabolisms during leptin-induced migration with two murine breast cancer cells. In addition, these studies demonstrate the importance of 1,25(OH)₂D regulation of adipocytes in breast cancer cell migration as well as 1,25(OH)₂D regulation of VDR level and activity in metastatic breast cancer cells.