1,25 dihydroxyvitamin D regulation of glucose metabolism in breast cancer prevention.

摘要

Breast cancer is the second leading cancer among women in the US. Substantial evidence from epidemiological, clinical, animal and cell-based studies supports that vitamin D may be an effective agent for breast cancer prevention, but the mechanism is not clear. Metabolic reprogramming that alters the cellular utilization of glucose including the "Warburg effect" is an important characteristic which allows rapid cell proliferation in more adverse conditions, and promotes the progression of cells to the development of a tumorigenic phenotype. The current research focused on the effects and the underlying molecular mechanisms of 1,25 dihydroxyvitamin D (1,25(OH)2D), the bioactive form of vitamin D, in regulating the metabolic reprogramming in breast epithelial cells during cancer development. The untransformed MCF10A and Harvey- ras oncogene transfected (MCF10A-ras) human breast epithelial cells were employed as a multistage carcinogenesis model for studying early breast cancer progression. The major approaches to study cellular metabolism included the measurement of metabolite fluxes at the cell membrane by a selective micro-biosensor, [13C6]glucose flux by 13 C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of glucose metabolism enzymes by quantitative PCR, etc. Results indicated that the MCF10A-ras cells exhibited enhanced glycolytic activity and lactate production, decreased glucose flux through the tricarboxylic acid cycle, as well as an increase in the utilization of glucose in the pentose phosphate pathway, suggesting the role of H-ras oncogene in the metabolic reprogramming of MCF10A cells during early mammary carcinogenesis. In MCF10A-ras cells, 1,25(OH)2D treatment for four days inhibited these alterations of glucose metabolism which promotes breast cancer progression, by reducing glycolysis and lactate production and reducing glucose flux through the tricarboxylic acid (TCA) cycle. In addition, 1,25(OH)2D reduced cell "glucose addiction", an important biological consequence of metabolic reprogramming, by preventing the increase in G1 cell cycle arrest in response to glucose restriction noted in MCF10A-ras cells. These results suggest a novel mechanism involving the regulation of glucose metabolism by which 1,25(OH)2D may prevent breast cancer progression. Further, 1,25(OH)2D treatment in MCF10A-ras cells reduced both the mRNA and protein expression of pyruvate carboxylase (PC), a key enzyme involved in glucose metabolism which has recently gained recognition for its anaplerotic role in cancer metabolism. shRNA induced knockdown of PC had a similar effect as 1,25(OH)2D in preventing the increase in cell cycle arrest at glucose restriction, suggesting that the effects of 1,25(OH) 2D in reducing glucose addiction may be mediated by the inhibition of PC expression. Study of human PC gene promoter using the reporter gene assay revealed the presence of a negative regulatory element by 1,25(OH)2D on the PC distal promoter (-575 to +23), which is a putative vitamin D responsive element (VDRE), supporting that 1,25(OH)2D inhibited the expression of PC potentially through a VDR mediated transcriptional regulation in MCF10A- ras cells. Therefore, negative regulation of the PC gene may be part of the mechanism by which 1,25(OH)2D regulates glucose metabolism and prevents early breast cancer progression. On the other hand, preliminary data on dietary vitamin D and the risk of mammary tumorigenesis in vivo showed that long-term exposure to high level of dietary vitamin D (10,000 IU/kg diet) beginning at pre-puberty (immediately post-weaning) led to a 40% reduction in incidence of MNU-induced mammary tumor in Sprague-Dawley rats. However, vitamin D did not alter the tumor latency, tumor frequency or tumor volume in rats that developed tumor, and did not inhibit the mammary expression of PC gene. This data provided evidence that high dietary vitamin D may modulate early life events during mammary tissue development to provide protection against breast cancer even following powerful carcinogenic exposure. In conclusion, these results demonstrate that 1,25(OH) 2D inhibits the alterations of glucose metabolism in H-ras oncogene transformed human breast epithelial cells during early progression to cancer, potentially through the VDR mediated transcriptional inhibition of PC gene expression, which may be a novel mechanism by which vitamin D prevents breast cancer progression. Additionally, data from the animal study suggests early exposure to high dietary vitamin D may be protective against breast cancer. Overall, findings from these exploratory studies consistently support that vitamin D may be an effective agent for breast cancer prevention.