Methylation status of Vitamin D receptor gene promoter in benign and malignant adrenal tumors

We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5' regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesi

Vitamin D receptor gene polymorphisms and prognosis of breast cancer among African-American and Hispanic women.

BackgroundVitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). Although African-Americans have the lowest levels of serum vitamin D, there is a dearth of information on VDR gene polymorphisms and breast cancer among African-Americans and Hispanics. This study examines whether VDR gene polymorphisms are associated with breast cancer in these cohorts.MethodsBlood was collected from 232 breast cancer patients (Cases) and 349 non-cancer subjects (Controls). Genotyping for four polymorphic variants of VDR (FokI, BsmI, TaqI and ApaI) was performed using the PCR-RFLP method.ResultsAn increased association of the VDR-Fok1 f allele with breast cancer was observed in African-Americans (OR = 1.9, p = 0.07). Furthermore, the FbTA, FbtA and fbtA haplotypes were associated with breast cancer among African-Americans (p<0.05). Latinas were more likely to have the VDR-ApaI alleles (Aa or aa) (p = 0.008). The VDR-ApaI aa genotype was significantly associated with poorly-differentiated breast tumors (p = 0.04) in combined Cases. Kaplan-Meier survival analysis showed decreased 5-year disease-free-survival (DFS) in breast cancer patients who had the VDR-Fok1 FF genotype (p<0.05). The Cox regression with multivariate analysis revealed the independent predictor value of the VDR-FokI polymorphism for DFS. The other three variants of VDR (BsmI, TaqI and ApaI) were not associated with disease outcome.ConclusionsVDR haplotypes are associated with breast cancer in African-Americans, but not in Hispanic/Latinas. The VDR-FokI FF genotype is linked with poor prognosis in African-American women with breast cancer

Deletion of vitamin D receptor leads to premature emphysema/COPD by increased matrix metalloproteinases and lymphoid aggregates formation

Deficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. The level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR(−/−)) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-κB) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction

Post-Translational Nuclear Protein Modification and High Vitamin-D Receptor Levels in Genetic Hypercalciuric Stone-Forming Rats

Studies conducted on Genetic Hypercalciuric-Stone Forming (GHS) rats have shown that their excess calcium urine excretion might be caused by significantly high vitamin D receptor (VDR) levels found in key calcium-transporting tissues, since high VDR raises physiological response to 1,25(OH)2D3, the hormonally active metabolite of vitamin D that regulates calcium levels in the body. We suggest that the high VDR levels in GHS tissue are a result of altered post-translational modification by ubiquitin and Small Ubiquitin-like Modifier (SUMO), which regulate the degradation of nuclear proteins substrates such as VDR. Our inquiry begins by overexpressing ubiquitin, SUMO-1, SUMO-2, and SUMO-3 in rat tissue to perform targeted gene expression of VDR. Western blot will be used to analyze resulting changes in the VDR signaling pathway and the degree to which the GHS rat phenotype is emulated with the added ubiquitin or SUMO. Our proceeding step will be to measure changes in VDR levels after performing an siRNA-facilitated knockdown of the E1, E2, and E3 enzymes that facilitate ubiquitination of nuclear proteins. Our final experiment will consist of isolating proteins from GHS bone marrow, intestinal, and renal cells and recording ubiquitin levels to compare with control tissue. If there is a consistent trend in the ubiquitin levels of GHS cells compared to control cells, and the western blot and enzyme knockdown procedures reveal that altered ubiquitin/SUMO presence affects VDR levels, then there will be evidence to show that the general hypercalciuiric phenotype could be an eventual result of insufficient VDR degradation due to abnormal post-translational protein modification

Molecular Static and Dynamic Analyses reveal Flaw in Murine Model used by US FDA to Detect Drug Carcinogenicity

The US FDA currently accepts carcinogenicity studies of pharmaceutical drugs based on murine models. In addition to 6 month studies with p53(+/-) and ras.H2 transgenic mice, lifetime studies (typically 2 years) in WT mice or rats are also considered as evidence that a drug lacks carcinogenic activity. This model is not always exhaustive. For example, during the acceptance testing of the ARB Olmesartan[1], possible carcinogenicity observed in hamsters was not able to be duplicated in rats, or in transgenic mice. We have previously used the static molecular modeling of AutoDock to demonstrate that Olmesartan has agonostic activity in the PDB:1DB1 model of the human VDR Nuclear Receptor[2], while it has antagonistic activity in the PDB:1RK3 model of the rat VDR. This agonism has now been confirmed with Molecular Dynamics, using GROMACS. The murine VDR indeed lost its ability to bind the DRIP-205 co-activator when Olmesartan was the ligand, while the human VDR was activated by Olmesartan similarly to its native ligand (1,25-dihydroxyvitamin-D). Since the VDR is believed to express 913 genes[3], many of which are known to be associated with cancer pathogenesis, good homology between human VDR, and the animal model VDR, is exceedingly important. CONCLUSION: The murine environment is inadequate to accurately model drug carcinogenic activity in humans. A species should be chosen which has a VDR LBP homology closer to that of man. AutoDock and GROMACS molecular analyses are useful in resolving any remaining anomalies in the observed data.

References:
1. FDA CDER: NDA-21-286, Sankyo Pharma Inc Available from URL http://www.fda.gov/cder/foi/nda/2002/21-286_Benicar.htm
2. Marshall TG: VDR Nuclear Receptor Competence is the Key to Recovery from Chronic Inflammatory and Autoimmune Disease. Abstract presentation, DMM2006.
Available from URL http://AutoimmunityResearch.org/karolinska-handout.pdf
3. Wang TT, et al: Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin-D3 target genes. Mol Endocrinol. 2005 Nov;19(11):2685-95.
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The Association between Vitamin D Receptor Expression and Prolonged Overall Survival in Breast Cancer.

Summary In this study, we analyzed vitamin D receptor (VDR) expression and survival in a breast cancer patient cohort of 82 breast cancer patients. Immunohistochemical analysis was possible in 91.5% of the patients (75/82). Staining was evaluated using the semi-quantitative assay according to Remmele and Stegner (immunoreactivity score [IRS]). IRS 0–1 was negative/very low, IRS 2–4 was moderate to high, and IRS 6–12 was high. Statistical analysis was performed by Spearman’s correlation test (p<0.05 significant). Overall survival was analyzed using Kaplan-Meier estimations. Only 6 patients had a negative IRS. Moderate IRS values were present in 20 patients. Most of the patients had a high IRS (49). For survival analysis, data were dichotomized (IRS 0–4: negative to moderate and IRS 6–12: high VDR expression). In univariate analysis, VDR expression showed significant differences in progression-free survival (PFS) and overall survival (OS). Patients with high IRS scores showed significantly better PFS and OS than patients with moderate/negative IRS scores for VDR expression. Tumor size was significantly correlated to PFS. When analyzed separately, the three different IRS groups showed significant differences in VDR expression. The present data suggest that VDR expression in breast cancer tissue may be of clinical significance, and the results provide evidence that VDR may be a factor with prognostic relevance. (J Histochem Cytochem 60:121–129, 2012). Keywords: breast cancer, vitamin D receptor, immunohistochemistry, prognosi

Disruption of Vitamin D and Calcium Signaling in Keratinocytes Predisposes to Skin Cancer.

1,25 dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, and calcium regulate epidermal differentiation. 1,25(OH)2D exerts its effects through the vitamin D receptor (VDR), a transcription factor in the nuclear hormone receptor family, whereas calcium acts through the calcium sensing receptor (Casr), a membrane bound member of the G protein coupled receptor family. We have developed mouse models in which the Vdr and Casr have been deleted in the epidermis ((epid) Vdr (-∕-) and (epid) Casr (-∕-)). Both genotypes show abnormalities in calcium induced epidermal differentiation in vivo and in vitro, associated with altered hedgehog (HH) and β-catenin signaling that when abnormally expressed lead to basal cell carcinomas (BCC) and trichofolliculomas, respectively. The Vdr (-∕-) mice are susceptible to tumor formation following UVB or chemical carcinogen exposure. More recently we found that the keratinocytes from these mice over express long non-coding RNA (lncRNA) oncogenes such as H19 and under express lncRNA tumor suppressors such as lincRNA-21. Spontaneous tumors have not been observed in either the (epid) Vdr (-∕-) or (epid) Casr (-∕-). But in mice with epidermal specific deletion of both Vdr and Casr ((epid) Vdr (-∕-)/(epid) Casr (-∕-) [DKO]) tumor formation occurs spontaneously when the DKO mice are placed on a low calcium diet. These results demonstrate important interactions between vitamin D and calcium signaling through their respective receptors that lead to cancer when these signals are disrupted. The roles of the β-catenin, hedgehog, and lncRNA pathways in predisposing the epidermis to tumor formation when vitamin D and calcium signaling are disrupted will be discussed

Overexpression of hedgehog signaling is associated with epidermal tumor formation in vitamin D receptor-null mice.

The vitamin D receptor (VDR) ligand, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), reduces proliferation and enhances differentiation, and thus has been investigated for a role in preventing or treating cancer. Mice deficient for the VDR display a hyperproliferative response in the hair follicle and epidermis and decreased epidermal differentiation. Unlike their wild-type littermates, when treated with 7,12 dimethylbenzanthracene (DMBA) or UVB, they develop skin tumors, including some characteristic of overexpression of the hedgehog (Hh) pathway. Both the epidermis and utricles of the VDR-null animals overexpress elements of the Hh pathway (sonic hedgehog (Shh) 2.02-fold, patched1 1.58-fold, smoothened 3.54-fold, glioma-associated oncogene homolog (Gli)1 1.17-fold, and Gli2 1.66-fold). This overexpression occurs at an age (11 weeks) at which epidermal hyperproliferation is most visible and is spatially controlled in the epidermis. DMBA- or UVB-induced tumors in the VDR-null mice also overexpress elements of this pathway. Moreover, 1,25(OH)(2)D(3) downregulates the expression of some members of the Hh pathway in an epidermal explants culture system, suggesting a direct regulation by 1,25(OH)(2)D(3). Our results suggest that increased expression of Shh in the keratinocytes of the VDR-null animal activates the Hh pathway, predisposing the skin to the development of both malignant and benign epidermal neoplasms

Hypothalamic Vitamin D Improves Glucose Homeostasis and Reduces Weight

Despite clear associations between vitamin D deficiency and obesity and/or type 2 diabetes, a causal relationship is not established. Vitamin D receptors (VDRs) are found within multiple tissues, including the brain. Given the importance of the brain in controlling both glucose levels and body weight, we hypothesized that activation of central VDR links vitamin D to the regulation of glucose and energy homeostasis. Indeed, we found that small doses of active vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D3) (calcitriol), into the third ventricle of the brain improved glucose tolerance and markedly increased hepatic insulin sensitivity, an effect that is dependent upon VDR within the paraventricular nucleus of the hypothalamus. In addition, chronic central administration of 1,25D3 dramatically decreased body weight by lowering food intake in obese rodents. Our data indicate that 1,25D3-mediated changes in food intake occur through action within the arcuate nucleus. We found that VDR colocalized with and activated key appetite-regulating neurons in the arcuate, namely proopiomelanocortin neurons. Together, these findings define a novel pathway for vitamin D regulation of metabolism with unique and divergent roles for central nervous system VDR signaling. Specifically, our data suggest that vitamin D regulates glucose homeostasis via the paraventricular nuclei and energy homeostasis via the arcuate nuclei