CYP24A1 Overexpression Research Articles

VDR, SOD-2, and CYP24A1 Gene Expression in Different Genotypes of BsmI SNP of the Vitamin D Receptor Gene in Individuals with Hypovitaminosis.

Hypovitaminosis D is a public health problem due to its implications for various diseases. Vitamin D has numerous functions, such as modulating the metabolism of cellular tissues, and it is expressed through the vitamin D receptor (VDR) gene that may influence gene expression modulation, which plays an important role in vitamin D metabolism. To evaluate the effect of the genotypes of BsmI single nucleotide polymorphism (SNP) of the VDR gene on VDR, SOD2, and CYP24A1 gene expression in individuals with low serum vitamin D levels. This was a cross-sectional analytical study. After signing the informed consent form, individuals were invited to participate and answered a structured questionnaire with identification data. Blood was collected for biochemical analysis, and vitamin D was measured by chemiluminescence; BsmI polymorphism was determined using real-time polymerase chain reaction (PCR) assays with TaqMan allelic discrimination, and gene expression was conducted by qRT-PCR using QuantiFast SYBR® Green PCR Master Mix. Data were analyzed using the SPSS 20.0 software, and differences were considered significant at p < 0.05. 98 individuals with vitamin D ≤ 20 ng/dL were evaluated, and the BsmI SNP of the VDR gene showed CYP24A1 overexpression and low SOD2 expression. BsmI SNP of the VDR gene can modulate the expression of the genes evaluated without interfering with serum levels.

MiR-30b-5p attenuates neuropathic pain by the CYP24A1-Wnt/β-catenin signaling in CCI rats.

MicroRNAs (miRNAs) has been reported to act as key regulators of neuronal function. Increasing evidence has showed that miRNAs exert significant effects in neuropathic pain. We explored the role of miR-30b-5p in neuropathic pain by establishing a rat model of chronic constrictive injury (CCI). The sciatic nerve of CCI rats was used to induce chronic neuropathic pain. The expression and cellular distribution of miR-30b-5p were determined by RT-qPCR and FISH. The mRNA level, protein level, and cellular distribution of CYP24A1 were detected by RT-qPCR, western blot, and immunofluorescence staining assays, respectively. The interaction between miR-30b-5p and CYP24A1 was examined by a luciferase reporter assay. The behavioral effects of miR-30b-5p were assessed after intrathecal administration. Mechanical stimuli and radiant heat were applied to assess mechanical allodynia and thermal hyperalgesia of rats. ELISA was performed to measure the concentration of inflammatory cytokines. MiR-30b-5p expression was significantly downregulated in the spinal cord tissues and of CCI rats. Overexpression of miR-30b-5p attenuated symptoms of neuropathic pain, including mechanical allodynia and thermal hyperalgesia. Additionally, miR-30b-5p overexpression suppressed neuroinflammation by reducing the levels of IL-6, TNF-α and COX2 and elevating the levels of IL-10 in CCI rats. Mechanistically, CYP24A1 was a target of miR-30b-5p, and its expression was negatively regulated by miR-30b-5p. Moreover, CYP24A1 expression was upregulated in CCI rats and knockdown of CYP24A1 attenuated neuropathic pain and neuroinflammation. Furthermore, miR-30b-5p reduced the levels of the Wnt pathway-related genes in CCI rats by downregulating CYP24A1. Rescue assays showed that overexpression of CYP24A1 or activation of Wnt pathway reduced the alleviative effects of miR-30b-5p overexpression on neuropathic pain in CCI rats. Overall,miR-30b-5p inhibits neuropathic pain progression in CCI rats by inhibiting the CYP24A1-Wnt/β-catenin pathway.

Could CYP24A1 promoter methylation status affect the gene expression in the colorectal cancer patients?

CYP24A1 up-regulation has been reported in different cancers, including colorectal cancer (CRC). CYP24A1 initiates the degradation of 1, 25-dihydroxyvitamin D3 and is a candidate biomarker for the progression and prognosis of CRC. Despite many studies for discovering the exact mechanism underlying the CYP24A1 overexpression, the results are inconsistent. The present study aimed to determine whether the promoter methylation status of the CYP24A1 gene is the cause of CYP24A1 overexpression in CRC patients. In this regards, after quantification of the CYP24A1 expression by quantitative real-time polymerase chain reaction (qRT-PCR), we measured the methylation status of the CYP24A1 promoter region by methylation-specific PCR (MSP) in 50 cases of fresh human colorectal cancer and 34 paired adjacent tissues. CYP24A1 was overexpressed in the CRC tissues (Fold change = 17.2, P=0.001); however, we didn't find any significant differences regarding the CYP24A1 promoter methylation status between the CRC samples and the paired adjacent tissues. We concluded that the CYP24A1 promoter hypomethylation is not the reason for the gene up-regulation in our CRC patients; therefore, other mechanisms involved in the CYP24A1 overexpression should be investigated.

In silico identification and screening of CYP24A1 inhibitors: 3D QSAR pharmacophore mapping and molecular dynamics analysis

Vitamin D is a key signalling molecule that plays a vital role in the regulation of calcium phosphate homeostasis and bone remodelling. The circulating biologically active form of vitamin D is regulated by the catabolic mechanism of cytochrome P450 24-hydroxylase (CYP24A1) enzyme. The over-expression of CYP24A1 negatively regulates the vitamin D level, which is the causative agent of chronic kidney disease, osteoporosis and several types of cancers. In this study, we found three potential lead molecules adverse to CYP24A1 through structure-based, atom-based pharmacophore and e-pharmacophore-based screening methods. Analysis was done by bioinformatics methods and tools like binding affinity (binding free energy), chemical reactivity (DFT studies) and molecular dynamics simulation (protein–ligand stability). Combined computational investigation showed that the compounds NCI_95001, NCI_382818 and UNPD_141613 may have inhibitory effects against the CYP24A1 protein.

Oncogenic Potential of CYP24A1 in Lung Adenocarcinoma

IntroductionWe have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. MethodsTwo low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. ResultsOver-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. ConclusionsOur data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.

Progesterone potentiates the growth inhibitory effects of calcitriol in endometrial cancer via suppression of CYP24A1.

Here, we evaluated the expression of CYP24A1, a protein that inactivates vitamin D in tissues. CYP24A1 expression was increased in advanced-stage endometrial tumors compared to normal tissues. Similarly, endometrial cancer cells expressed higher levels of CYP24A1 than immortalized endometrial epithelial cells. RT-PCR and Western blotting were used to examine CYP24A1 mRNA and protein levels in endometrial cancer cells after 8, 24, 72, and 120 h of exposure to progesterone, progestin derivatives and calcitriol, either alone or in combination. Progestins inhibited calcitriol-induced expression of CYP24A1 and splice variant CYP24SV mRNA and protein in cancer cells. Furthermore, actinomycin D, but not cycloheximide, blocked calcitriol-induced CYP24A1 splicing. siRNA-induced knockdown of CYP24A1 expression sensitized endometrial cancer cells to calcitriol-induced growth inhibition. These data suggest that CYP24A1 overexpression reduces the antitumor effects of calcitriol in cancer cells and that progestins may be beneficial for maintaining calcitriol's anti-endometrial cancer activity.

Abstract 866: Delineation of novel CYP24A1 transcriptional regulators

Abstract The anti-proliferative and pro-apoptotic effects of 1á,25 dihydroxycholecalciferol (1,25D3) are well-documented in various tumor model systems in vitro and in vivo. However, limited anti-tumor effects of 1,25D3 have been observed in clinical trials. One potential reason may be the over-expression of CYP24A1, the primary 1,25D3 degrading enzyme, in tumors, and the subsequent rapid local inactivation of 1,25D3. A CYP24A1 inhibitor might be added to improve the anti-tumor activity of 1,25D3. However, most CYP24A1 inhibitors are non-specific, and their use is accompanied by a striking increase in the CYP24A1 expression level. In this study, we screened a small molecule library to identify novel CYP24A1 transcriptional inhibitors using a luciferase reporter assay in a human prostate cancer PC3 cell line stably expressing CYP24A1 promoter-driving luciferase reporter. Screening of DIVERSet™, a diverse library of 55,230 compounds, resulted in the identification of 150 hits, each of which had over 50% inhibition of 1,25D3-induced CYP24A1 promoter activity. After confirming initial observations, four hits (CPI3, CPI8, CPI11 and CPI17) displaying the strongest inhibitory effect were chosen to further examine their effects on endogenous and 1,25D3-regulated CYP24A1 expression in cancer cells. Prostate cancer (PC3) cells, bladder cancer (RT112, RT112/D21) cells, kidney cancer (A498, ACHN and 786-O) cells were treated with CPI3, CPI8, CPI11 and CPI17 alone or followed by 100 nM of 1,25D3. qRT-PCR analysis showed that 1,25D3 induced CYP24A1 mRNA expression in these cancer cells and the induction of CYP24A1 was significantly inhibited by the treatment with CPI8 and CPI17 (P &amp;lt; 0.01). Western blot showed the reduction of 1,25D3-induced CYP24A1 protein expression in CPI8 and CPI17 treated cancer cells. These results indicate that CPI8 and CPI17 inhibit CYP24A1 expression at the transcriptional level. To investigate whether these compounds affect the transcriptional expression of vitamin D receptor (VDR) and other vitamin D target genes, the expression of VDR and p21Waf1 were measured by qRT-PCR in kidney cancer A498, ACHN and 786-O cells treated with CPI8, which displayed the strongest inhibitory effect on CYP24A1 expression. Treatment with CPI8 did not affect VDR expression in kidney cancer cells. In contrast with the reduction of CYP24A1 expression by CPI8, treatment with CPI8 markedly increased p21Waf1 mRNA and protein expression compared to 1,25D3 alone. These results indicate that CPI8 differentially affects the expression of vitamin D target genes. In summary, we identified novel CYP24A1 inhibitors, which differentially regulate 1,25D3 target genes. Inhibiting CYP24A1 expression, while increasing the expression of other anti-proliferative 1,25D3-target genes, may be a useful approach to enhance 1,25D3 antitumor activity. Supported by NIH/NCI grant CA67267. Citation Format: Wei Luo, Yingyu Ma, Mikhail Chernov, Donald L. Trump, Candace S. Johnson. Delineation of novel CYP24A1 transcriptional regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 866.

Vitamin D Signaling Modulators in Cancer Therapy.

The antiproliferative and pro-apoptotic effects of 1α,25-dihydroxycholecalciferol (1,25(OH)2D3, 1,25D3, calcitriol) have been demonstrated in various tumor model systems in vitro and in vivo. However, limited antitumor effects of 1,25D3 have been observed in clinical trials. This may be attributed to a variety of factors including overexpression of the primary 1,25D3 degrading enzyme, CYP24A1, in tumors, which would lead to rapid local inactivation of 1,25D3. An alternative strategy for improving the antitumor activity of 1,25D3 involves the combination with a selective CYP24A1 inhibitor. The validity of this approach is supported by numerous preclinical investigations, which demonstrate that CYP24A1 inhibitors suppress 1,25D3 catabolism in tumor cells and increase the effects of 1,25D3 on gene expression and cell growth. Studies are now required to determine whether selective CYP24A1 inhibitors+1,25D3 can be used safely and effectively in patients. CYP24A1 inhibitors plus 1,25D3 can cause dose-limiting toxicity of vitamin D (hypercalcemia) in some patients. Dexamethasone significantly reduces 1,25D3-mediated hypercalcemia and enhances the antitumor activity of 1,25D3, increases VDR-ligand binding, and increases VDR protein expression. Efforts to dissect the mechanisms responsible for CYP24A1 overexpression and combinational effect of 1,25D3/dexamethasone in tumors are underway. Understanding the cross talk between vitamin D receptor (VDR) and glucocorticoid receptor (GR) signaling axes is of crucial importance to the design of new therapies that include 1,25D3 and dexamethasone. Insights gained from these studies are expected to yield novel strategies to improve the efficacy of 1,25D3 treatment.

Impact of CYP24A1 overexpression on growth of colorectal tumour xenografts in mice fed with vitamin D and soy.

Our previous studies showed that the 1,25‐dihydroxyvitamin D (1,25‐D3) catabolizing enzyme, 1,25‐dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumours and its level correlated with increased proliferation. We hypothesised that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the anti‐tumourigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumour growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP‐tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3/kg) or low vitamin D (100 IU D3/kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25‐D3, the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumour volume only in the control xenografts, while the tumours overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumours. Irrespective of the dietary vitamin D3, dietary soy is able to increase tumour volume when tumours overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an anti‐tumourigenic effect only if CYP24A1 levels are normal.

Abstract 37: Role and regulation of CYP24A1 in endometrial cancer

Abstract The cytochrome P450 enzyme, 24-hydroxylase, encoded by CYP24A1 is critical for the catabolism of 1,25(OH)2D3 (calcitriol). The unbalanced high levels of CYP24A1 seem to be a determinant of calcitriol resistance in tumors. We have previously shown that progesterone enhances calcitriol antitumor activity by upregulating vitamin D receptor expression and promoting apoptosis in endometrial cancer cells. In the present study, we evaluated CYP24A1 protein expression in normal and endometrial tumor tissues, assessed the effect of progesterone and calcitriol on CYP24A1 and its spliced variant expression in endometrial cancer cell lines and correlated this with tumor cell growth. Expression of CYP24A1 was assessed in tissue microarrays by immunohistochemistry. A grade-dependent increase of CYP24A1 expression was found in endometrial carcinomas. Endometrial cancer cells expressed high levels of CYP24A1 compared to immortalized endometrial epithelial cells. Furthermore, CYP24A1 is induced in cells in response to calcitriol. Regulation of CYP24A1 by progesterone, progestin derivatives, calcitriol and their combination was examined by RT-PCR and Western blotting following 8, 24, 72 and 120 h exposure of cells to hormones. In all cancer cell lines, progesterone, medroxyprogesterone acetate and norgestrol attenuated calcitriol-induced CYP24A1, spliced variant CYP24SV transcripts and protein expression at 72 and 120 h of treatment. Furthermore, knockdown of CYP24A1 gene expression by siRNA sensitized endometrial cancer cells to the growth suppressive effect of calcitriol. The data suggest that CYP24A1 overexpression limits calcitriol anti-proliferative signaling in cancer cells, and provide evidence that progestins may be beneficial in preserving calcitriol action in endometrial cancer. Citation Format: Amber A. Bokhari, Laura R. Lee, Raboteau Dewayne, Chad A. Hamilton, George L. Maxwell, Gustavo C. Rodriguez, Viqar Syed. Role and regulation of CYP24A1 in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2015-37

Increased CYP24A1 expression is associated with BRAFV600E mutation and advanced stages in papillary thyroid carcinoma

1α, 25(OH)2 D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1α, 25(OH)2 D3. This study investigates the expression of CYP24A1 and the effect of BRAF(V600E) on its expression in thyroid cancer. We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAF(V600E) mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAF(V600E) and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR. CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAF(V600E) mutation (P < 0·01). In thyroid cancer cell lines expressing BRAF(V600E) , CYP24A1 expression was significantly higher when compared to those without BRAF(V600E) expression. BRAF(V600E) transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAF(V600E) inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines. CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAF(V600E) mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAF(V600E) inhibitor PLX4720.

Abstract 5383: Overexpression of the vitamin D catabolizing enzyme CYP24A1 is caused by gene amplification and results in highly proliferative colorectal tumors.

Abstract Vitamin D insufficiency increases risk of colorectal cancer. Vitamin D is produced photochemically in the skin, thus, low sunlight exposure results in vitamin D insufficiency. Vitamin D is further processed in the liver to its storage form calcidiol (25-OH vitamin D3). Calcidiol can be activated by 1α-hydroxylation to the secosteroid hormone calcitriol (1α,25-OH vitamin D3). Although systemic levels of bioactive calcitriol are regulated by the kidneys, almost every tissue can synthesize and degrade calcitriol. Tissue calcitriol acts in an autocrine/paracrine manner and controls proliferation, apoptosis, and differentiation. In colorectal cancer, the calcidiol and calcitriol-degrading enzyme CYP24A1 is substantially overexpressed both on mRNA and protein level. High CYP24A1 levels markedly reduce the half-life of vitamin D metabolites, likely reducing the anti-tumorigenic effects of calcitriol in the tumor. The causes and consequences of this overexpression are not fully understood. Here, we investigated gene amplification of the CYP24A1 locus (20q13.2) as a possible cause of CYP24A1 overexpression and increased proliferation as a consequence thereof. Quantitative real time PCR assays showed that approximately 60% of colorectal tumors carry CYP24A1 gene amplification (n=127). This gene amplification correlated with increased mRNA expression (ρ=0.38, p&amp;lt;0.001). Aberrantly high CYP24A1 expression may reduce the anti-proliferative actions of calcitriol and lead to increased proliferation. In our colorectal cancer cohort, CYP24A1 mRNA expression correlated with expression of several proliferation markers (e.g. CDC6 ρ=0.60, p&amp;lt;0.001). In conclusion, our data suggest that CYP24A1 gene amplification results in increased mRNA expression in colorectal tumors. Further, high CYP24A1 expression correlates with increased proliferation, possibly caused by an inhibition of the anti-proliferative effects of calcitriol. Tumor specific inhibition of CYP24A1 may provide a future strategy to restore local vitamin D levels and its anti-tumorigenic activities. Citation Format: Julia Höbaus, Abhishek Aggarwal, Doris M. Hummel, Ursula Thiem, Irfete Fetahu, Ildiko Mesteri, Enikö Kallay. Overexpression of the vitamin D catabolizing enzyme CYP24A1 is caused by gene amplification and results in highly proliferative colorectal tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5383. doi:10.1158/1538-7445.AM2013-5383

Increased copy‐number and not DNA hypomethylation causes overexpression of the candidate proto‐oncogene CYP24A1 in colorectal cancer

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.What’s new?Recently, it has been suggested that the association between colorectal cancer and reduced levels of circulating vitamin D may be related to overexpression of the vitamin D-catabolizing enzyme, CYP24A1 in the tumor. In this search for a mechanistic explanation, increased CYP24A1 gene copy number was associated with the enzyme’s overexpression in 60 percent of colorectal tumors, and expression was correlated strongly with proliferation markers. The findings suggest that CYP24A1 overexpression is likely to deplete tumor calcitriol (1,25-dihydroxyvitamin D3) levels, possibly increasing the proliferative potential of the tumors.

Abstract 1136: Gene amplification and epigenetic modifications: Mechanisms behind unbalanced expression of the proto-oncogene CYP24A1 in colorectal tumors

Abstract There is an inverse association between colorectal cancer and pre-diagnostic Vitamin D levels (25-OHD3), underlining the importance of Vitamin D in the prevention of colon cancer. 1α,25(OH)2D3 (1,25D3) is the most active form of Vitamin D and exhibits anti-proliferative, pro-differentiating, anti-angiogenic functions. Both, calcitriol and the calcitriol precursor 25(OH)D3 can be catabolized by the 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1). CYP24A1 is expressed at very low basal levels in the colon but is strongly induced by 1,25D3. This induction is due to a complex formation of the 1,25D3 and the Vitamin D Receptor and binding of this complex to Vitamin D Responsive Elements (VDRE) located within the CYP24A1 promoter region. This feedback loop shortens the biological half-life of 1,25D3. A high expression of the Vitamin D catabolizing enzyme CYP24A1 implies faster degradation of 1,25D3, thereby restricting its anti-tumorigenic functions. A genomic copy number gain and an overexpression of CYP24A1 on mRNA level have been observed in different malignancies, such as colon and breast. Our aim is to test whether the observed overexpression of CYP24A1 is due to gene amplification and/or epigenetic modulations. In human colon cancer cell lines we found an increase in genomic copy number of CYP24A1 compared to human placenta using real time-PCR. In human colorectal cancer patients (n=89) we observed a gain of CYP24A1 copy number in approximately 40% of the tumors compared to their respective adjacent mucosa indicating a role of CYP24A1 gene amplification in tumorigenesis. Further, on mRNA level we could show a trend toward increased CYP24A1 expression in tumors compared with their adjacent mucosa using reverse transcription qRT-PCR. However, the effect of copy number gain can be impaired by epigenetic regulation. Aberrant DNA methylation is a characteristic of various tumors. The promoter region of the CYP24A1 gene contains several CpG islands. Therefore we have examined if there are any differences in the methylation status of the CYP24A1 promoter between the tumor tissue and the adjacent “normal” mucosa. First we have compared the methylation status in 4 cell lines. Bisulfit-sequencing of the genomic DNA of the highly differentiated colon cancer cell line Caco2Aq showed heavy methylation of the CYP24A1 promoter including the VDREs located within this promoter area. In comparison, the colon cell lines Coga1A, LS174, and Coga13 (derived from less differentiated tumors) appeared largely unmethylated. Using methylation specific PCR we found that human colorectal tumors are often less methylated than their adjacent mucosa. In conclusion, our data indicate that both gene amplification and DNA-hypomethylation could be mechanisms leading to the overexpression of CYP24A1 in colorectal tumor patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1136. doi:10.1158/1538-7445.AM2011-1136

CYP24A1Is an Independent Prognostic Marker of Survival in Patients with Lung Adenocarcinoma

The active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25-D(3)), exerts antiproliferative effects in cancers, including lung adenocarcinoma (AC). CYP24A1 is overexpressed in many cancers and encodes the enzyme that catabolizes 1,25-D(3). The purpose of our study was to assess CYP24A1 as a prognostic marker and to study its relevance to antiproliferative activity of 1,25-D(3) in lung AC cells. Tumors and corresponding normal specimens from 86 patients with lung AC (stages I-III) were available. Affymetrix array data and subsequent confirmation by quantitative real time-PCR were used to determine CYP24A1 mRNA expression. A subsequent validation set of 101 lung AC was used to confirm CYP24A1 mRNA expression and its associations with clinical variables. The antiproliferative effects of 1,25-D(3) were examined using lung cancer cell lines with high as well as low expression of CYP24A1 mRNA. CYP24A1 mRNA was elevated 8- to 50-fold in lung AC (compared to normal nonneoplastic lung) and significantly higher in poorly differentiated cancers. At 5 years of follow-up, the probability of survival was 42% (high CYP24A1, n = 29) versus 81% (low CYP24A1, n = 57) (P = 0.007). The validation set of 101 tumors showed that CYP24A1 was independently prognostic of survival (multivariate Cox model adjusted for age, gender, and stage, P = 0.001). A549 cells (high CYP24A1) were more resistant to antiproliferative effects of 1,25-D(3) compared with SKLU-1 cells (low CYP24A1). CYP24A1 overexpression is associated with poorer survival in lung AC. This may relate to abrogation of antiproliferative effects of 1,25-D(3) in high CYP24A1 expressing lung AC.

Abstract 4944: Regulation of vitamin D 24-hydroxylase gene expression in human prostate cancer by epigenetic mechanisms

Abstract 1,25-dihydroxycholecalciferol (calcitriol), the active form of vitamin D3, plays a major role in regulating calcium homeostasis and bone mineralization. Calcitriol also modulates cellular proliferation and differentiation in a variety of cell types. Furthermore, calcitriol has an anti-tumor effect in several tumor models. 1,25-(OH)2D-hydroxylase (24-hydroxylase), encoded by the CYP24A1 gene, plays a key role in attenuation of calcitriol effects by degrading calcitriol to a much less active vitamin D metabolite. CYP24A1 is dysregulated in wide range of tumors. The over-expression of CYP24A1 is associated with poor prognosis of some human cancers. Interestingly, CYP24A1 is down-regulated in human prostate cancer. Little is known about the mechanism(s) underlying the regulation of CYP24A1 expression in human prostate cancer. We have analyzed 3 prostate cancer cell lines (LNCaP, DU-145 and PC3) which express variable levels of CYP24A1. We have found that CYP24A1 gene expression is inversely correlated with promoter DNA methylation in human prostate cell lines. Treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (DAC), activates CYP24A1 gene expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter by methylases SssI, HpaII and HhaI represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases acetylated H3K9 and dimethylated H3K4 and simultaneously decreases dimethylated H3K9 at the CYP24A1 promoter. ChIP-qPCR assay also shows that treatment of prostate cancer cells with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. We have also collected paired samples of benign and malignant tissue from 30 human prostatectomy samples. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to matched benign lesions (p&amp;lt;0.001). Bisulfite DNA pyrosequencing reveals that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Taken together, these data indicate that the repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. Epigenetic repression of CYP24A1 may involve blocked recruitment of VDR to CYP24A1 regulatory regions, and nucleation of repressive chromatin structures at the CYP24A1 promoter. This study was supported by NIH/NCI grants CA067267, CA095045 and CA085142. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4944.

CYP24A1 and the Metabolism of Vitamin D

CYP24A1 belongs to a class of enzymes called mitochondrial P450s, which catalyze specific reactions in the biosynthesis and degradation of hormones. CYP24A1 metabolizes vitamin D, which functions in calcium homeostasis and cellular growth processes. An overexpression of CYP24A1 in disease states may lead to bone disorders, kidney disease, and breast or prostate cancer. A potential treatment could be an inhibitor of CYP24A1. CYP24A1’s mechanism of action is poorly understood as it has been relatively difficult to crystallize. As a membrane protein, CYP24A1 has extensive hydrophobic regions, making this process more difficult. The Stout Lab recently overcame this challenge and solved the first crystal structure for CYP24A1, but without vitamin D bound. Now that a structure of CYP24A1 has been determined, further understanding of enzyme function can be gained by solving structures with vitamin D and designed inhibitors bound in the active site. To assist in this effort the Lutheran High School of San Diego SMART Team (Students Modeling A Research Topic) used data from the Stout Lab to create a physical model of CYP24A1 using 3D printing technology. The model will be very useful in understanding the conformational changes that occur in this large complex protein structure upon vitamin D binding. Supported by grants from the HHMI Pre‐College Program, and Cytochroma, Inc. for research at TSRI.

The Candidate Oncogene CYP24A1: A Potential Biomarker for Colorectal Tumorigenesis

The main autocrine/paracrine role of the active metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D(3) 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D(3), we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D(3) availability, decreasing its antiproliferative effect.