Overexpressed fibroblast growth factor receptors increase 1,25-dihydroxyvitamin D-dependent differentiation of acute myeloid leukemia cells.

Many malignancies are driven by mutations within the gene for fibroblast growth factor receptor 1 (FGFR1). Previously, we have shown that signal transduction from the FOP2-FGFR1 fusion protein in acute myeloid leukemia KG1 cells is responsible for a low level of expression of the vitamin D receptor gene. In this paper, we address whether other fibroblast growth factor receptors regulate the vitamin D receptor (VDR) gene. We used the human myeloid leukemia U937 and HL60 cells, the bone cancer cell line U2OS, and cell transfection methods to answer the question. For myeloid leukemia cells, overexpression of FGFRs 1-3 genes caused a shift towards monocytic differentiation; this was extracellular regulated kinase (Erk) 1,2-dependent. Overexpression of FGFRs 1-3 genes also upregulated expression of the VDR gene, further sensitizing these cells to 1,25-dihydroxyvitamin D-induced monocyte differentiation. When we increased expression in bone cells, fibroblast growth factor receptors did not upregulate VDR gene expression, nor influence the activity of VDR. Fibroblast growth factor receptors are overexpressed in many neoplasms. Therefore, it may be reasonable to use vitamin D analogs to treat these cancers, to activate VDR and drive cell differentiation.

Investigating the Role of Methylation in Silencing of VDR Gene Expression in Normal Cells during Hematopoiesis and in Their Leukemic Counterparts.

(1) Background: Vitamin D receptor (VDR) is present in multiple types of blood cells, and its ligand, 1,25-dihydroxyvitamin D (1,25D), is important for the proper functioning of the immune system. Activity of VDR is higher in hematopoietic stem and progenitor cells than in fully differentiated blood cells of mice and humans. In some human acute myeloid leukemia (AML) blasts, the expression of the VDR gene is also high. The mechanism of silencing the VDR gene expression during differentiation of blood cells has been addressed in this work. (2) Methods: The cells have been obtained using fluorescence activated sorting from murine tissues and from human umbilical cord blood (UCB). Then, the expression of the VDR gene and transcriptional activity of the VDR protein has been tested in real-time polymerase chain reaction (PCR). Eventually, the methylation of VDR promoter regions was tested using bisulfite sequencing. (3) Results: The CpG islands in VDR promoters were not methylated in the cells studied both in mice and in humans. The use of hypomethylating agents had no effect toward expression of human VDR transcripts, but it increased expression of the VDR-target gene, CYP24A1. (4) Conclusions: The expression of the VDR gene and transcriptional activity of the VDR protein varies at successive stages of hematopoietic differentiation in humans and mice, and in blasts from AML patients. The experiments presented in this case indicate that methylation of the promoter region of the VDR gene is not the major mechanism responsible for these differences.

Investigating the Role of VDR and Megalin in Semi-Selectivity of Side-Chain Modified 19-nor Analogs of Vitamin D.

1,25-dihydroxyvitamin D3 (1,25D3) is implicated in many cellular functions, including cell proliferation and differentiation, thus exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D3 are potent calcemic activities. Therefore, synthetic analogs of 1,25D3 for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. To obtain this goal, the analogs should effectively activate transcription of genes responsible for cell differentiation, leaving the genes responsible for calcium homeostasis less active. In order to better understand this phenomenon, we selected a series of structurally related 19-nor analogs of 1,25D (PRI-5100, PRI-5101, PRI-5105, and PRI-5106) and tested their activities in blood cells and in cells connected to calcium homeostasis. Affinities of analogs to recombinant vitamin D receptor (VDR) protein were not correlated to their pro-differentiating activities. Moreover, the pattern of transcriptional activities of the analogs was different in cell lines originating from various vitamin D-responsive tissues. We thus hypothesized that receptors which participate in transport of the analogs to the cells might contribute to the observed differences. In order to study this hypothesis, we produced renal cells with knock-out of the megalin gene. Our results indicate that megalin has a minor effect on semi-selective activities of vitamin D analogs.