Vitamin D regulation of HAS2, hyaluronan synthesis and metabolism in triple negative breast cancer cells

The vitamin D receptor (VDR) and its ligand 1,25(OH)(2)D-3 (1,25D) exert anti-tumor effects, but considerable heterogeneity has been reported in different model systems. In general, cell lines derived from aggressive tumor subtypes such as Triple Negative Breast Cancer (TNBC) express low levels of VDR and are less sensitive to 1,25D than those derived from more differentiated tumor types. We have previously reported that 1,25D inhibits hyaluronic acid synthase 2 (HAS2) expression and hyaluronic acid (HA) synthesis in murine TNBC cells. Here we confirmed the inhibitory effect of 1,25D on HA synthesis in human Hs578T cells representative of the mesenchymal/stem-like (MSL) subtype of TNBC. Because HA synthesis requires the production of hexoses for incorporation into HA, we predicted that the high HA production characteristic of Hs578T cells would require sustained metabolic changes through the hexosamine biosynthetic pathway (HBP). We thus examined metabolic gene expression in Hs578T cell variants sorted for High (HA(High)) and Low (HA(Low)) HA production, and the ability of 1,25D to reverse these adaptive changes. HA(High) populations exhibited elevated HA production, smaller size, increased proliferation and higher motility than HA(Low) populations. Despite their more aggressive phenotype, HA(High) populations retained expression of VDR protein at levels comparable to that of parental Hs578T cells and HA(Low) subclones. Treatment with 1,25D decreased production of HA in both HA(High) and HA(Low) populations. We also found that multiple metabolic enzymes were aberrantly expressed in HA(High) cells, especially those involved in glutamine and glucose metabolism. Notably, Glutaminase (GLS), a known oncogene for breast cancer, was strongly upregulated in HA(High) vs. HA(Low) cells and its expression was significantly reduced by 1,25D (100 nM, 24 h). Consistent with this finding, Seahorse extracellular flux analysis indicated that respiration in HA(High) cells was significantly more dependent on exogenous glutamine than HA(Low) cells, however, acute 1,25D exposure did not alter metabolic flux. In contrast to GLS, the glutamate transporter SLC1A7 was significantly reduced in HA(High) cells compared to HA(Low) cells and its expression was enhanced by 1,25D. These findings support the concept that 1,25D can reverse the metabolic gene expression changes associated with HA production in cancer cells with aggressive phenotypes.