Intratumoral Sterol-27-Hydroxylase (CYP27A1) Expression in Relation to Cholesterol Synthesis and Vitamin D Signaling and Its Association with Lethal Prostate Cancer.

BACKGROUND: Higher intratumoral cholesterol synthesis is associated with a worse prognosis in prostate cancer. The vitamin D-regulated enzyme sterol-27-hydroxylase (CYP27A1) converts cholesterol to 27-hydroxycholesterol, potentially lowering intracellular cholesterol levels. We hypothesized that low CYP27A1 expression is associated with high cholesterol synthesis, low vitamin D signaling, and higher risk of lethal prostate cancer. METHODS: In 404 patients from the prospective prostate cancer cohorts within the Health Professionals Follow-up Study (HPFS) and the Physicians' Health Study (PHS), we assessed intratumoral CYP27A1 expression and proxies of cholesterol synthesis using transcriptome profiling, prediagnostic plasma 25-hydroxyvitamin D [25(OH)D; n = 132], and intratumoral vitamin D receptor protein expression (VDR; n = 300). Patients were followed for metastases and prostate cancer mortality (lethal cancer; median follow-up, 15.3 years). RESULTS: CYP27A1 expression was lower in tumors with higher Gleason grade and higher expression of cholesterol synthesis enzymes, including the second rate-limiting enzyme, SQLE. We did not detect consistent associations between CYP27A1 and 25(OH)D, VDR, or CYP24A1 mRNA expression. Lower CYP27A1 was associated with higher risk of lethal cancer in both cohorts, independent of SQLE [adjusted OR for lowest vs. highest quartile of CYP27A1, 2.64; 95% confidence interval (CI), 1.24-5.62]. This association was attenuated when additionally adjusting for Gleason grade (OR, 1.76; 95% CI, 0.75-4.17). CONCLUSIONS: Low CYP27A1 expression was associated with higher cholesterol synthesis and a higher risk of lethal disease. IMPACT: These observations further support the hypothesis that intratumoral cholesterol accumulation through higher synthesis and decreased catabolism is a feature of lethal prostate cancer.

On the Role of Illness Duration and Nutrient Restriction in Cholestatic Alterations that Occur During Critical Illness.

BACKGROUND AND AIMS: Elevated markers of cholestasis are common in response to critical illness, and associated with adverse outcome. The role of illness duration and of nutrient restriction on underlying molecular pathways of such cholestatic responses have not been thoroughly investigated. METHODS: In a mouse model of surgery- and sepsis-induced critical illness, molecular pathways of cholestasis were investigated up to 7 days. To assess which changes are explained by illness-induced lack of feeding, nutrient-restricted healthy mice were studied and compared with ad libitum fed healthy mice. Furthermore, serum bile acid (BA) concentrations were quantified in 1,114 human patients with either short or long intensive care unit (ICU) stay, matched for type and severity of illness, up to ICU-day-7. RESULTS: In critically ill mice, either evoked by surgery or sepsis, circulating and hepatic BA-levels progressively increased with time from day-3 onward, preceded by unsuppressed or upregulated CYP7A1 and CYP27A1 protein expression. From 30 h onward, nuclear farnesoid-X-receptor-retinoid-X-receptor staining was significantly suppressed in both critically ill groups, followed from day-3 onward by decreased gene expression of the apical exporter BA-specific export pump and increased expression of basolateral exporters multidrug resistance-associated protein 3 (MRP3) and MRP4. Nutrient restriction in healthy mice only partly mirrored illness-induced alterations in circulating BA and BA-transporters, without changing nuclear receptors or synthesis markers expression. Also in human critically ill patients, serum BA increased with time in long-stay patients only, similarly for patients with or without sepsis. CONCLUSIONS: Circulating BA concentrations rose days after onset of sepsis- and surgery-induced, critical illness, only partially explained by lack of feeding, preceded by suppressed nuclear feedback-sensors and ongoing BA synthesis. Expression of transporters suggested ongoing reversed BA-flow toward the blood.

Iron depletion induces hepatic secretion of biliary lipids and glutathione in rats.

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

Impact of 27-hydroxylase (CYP27A1) and 27-hydroxycholesterol in breast cancer.

The impact of systemic 27-hydroxycholesterol (27HC) and intratumoral CYP27A1 expression on pathobiology and clinical response to statins in breast cancer needs clarification. 27HC is an oxysterol produced from cholesterol by the monooxygenase CYP27A1, which regulates intracellular cholesterol homeostasis. 27HC also acts as an endogenous selective estrogen receptor (ER) modulator capable of increasing breast cancer growth and metastasis. 27HC levels can be modulated by statins or direct inhibition of CYP27A1, thereby attenuating its pro-tumorigenic activities. Herein, the effect of statins on serum 27HC and tumor-specific CYP27A1 expression was evaluated in 42 breast cancer patients treated with atorvastatin within a phase II clinical trial. Further, the associations between CYP27A1 expression with other primary tumor pathological features and clinical outcomes were studied in two additional independent cohorts. Statin treatment effectively decreased serum 27HC and deregulated CYP27A1 expression in tumors. However, these changes were not associated with anti-proliferative responses to statin treatment. CYP27A1 was heterogeneously expressed among primary tumors, with high expression significantly associated with high tumor grade, ER negativity and basal-like subtype. High CYP27A1 expression was independently prognostic for longer recurrence-free and overall survival. Importantly, the beneficial effect of high CYP27A1 in ER-positive breast cancer seemed limited to women aged ≤50 years. These results establish a link between CYP27A1 and breast cancer pathobiology and prognosis and propose that the efficacy of statins in reducing serum lipids does not directly translate to anti-proliferative effects in tumors. Changes in other undetermined serum or tumor factors suggestively mediate the anti-proliferative effects of statins in breast cancer.

Mast cells express CYP27A1 and CYP27B1 in epithelial skin cancers and psoriasis.

BACKGROUND: Ultraviolet (UV) radiation and the vitamin D system are involved in immunosuppression in the skin. Previous in vitro and animal studies suggest a role for mast cells in these mechanisms. OBJECTIVES: To study vitamin D3 metabolizing enzymes, CYP27A1 and CYP27B1, in mast cells in epithelial skin cancers and psoriasis. MATERIALS AND METHODS: Biopsies were collected from the non-lesional and lesional skin of patients with actinic keratosis (AK), Bowen's disease/squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and psoriasis. CYP27A1 and CYP27B1 in mast cells were analysed using a sequential double-staining method. RESULTS: The percentage of mast cells containing CYP27A1 was significantly higher in lesional than non-lesional skin in all diseases, especially in SCC and BCC. In addition, the percentage of mast cells containing CYP27B1 was significantly increased in BCC, AK, and psoriatic lesions as well. Interestingly, only about 5-6% and 2% of the mast cells expressed CYP27A1 and CYP27B1, respectively, in the non-lesional skin of psoriatic and AK patients. In contrast, 23-38% and 6-9% of the mast cells were immunopositive for CYP27A1 and CYP27B1, respectively, in the non-lesional skin of BCC and SCC patients. In human LAD2 mast cell cultures, about 30% and 15% of the mast cells showed CYP27A1 and CYP27B1, respectively, though the immunostainings of these enzymes were not markedly affected by UVB irradiation. CONCLUSION: Increased proportions of mast cells express vitamin D3 metabolizing enzymes in the lesional skin. Therefore, mast cells may promote an immunosuppressive environment, e.g., in skin carcinoma.

Hematopoietic overexpression of Cyp27a1 reduces hepatic inflammation independently of 27-hydroxycholesterol levels in Ldlr(-/-) mice.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and inflammation. Currently, the underlying mechanisms, leading to hepatic inflammation, are still unknown. The breakdown of free cholesterol inside Kupffer cells (KCs) by the mitochondrial enzyme CYP27A1 produces 27-hydroxycholesterol (27HC). We recently demonstrated that administration of 27HC to hyperlipidemic mice reduced hepatic inflammation. In line, hematopoietic deletion of Cyp27a1 resulted in increased hepatic inflammation. In the current manuscript, the effect of hematopoietic overexpression of Cyp27a1 on the development of NASH and cholesterol trafficking was investigated. We hypothesized that Cyp27a1 overexpression in KCs will lead to reduced hepatic inflammation. METHODS: Irradiated Ldlr(-/-) mice were transplanted (tp) with bone marrow from mice overexpressing Cyp27a1 (Cyp27a1(over)) and wild type (Wt) mice and fed either chow or a high-fat, high-cholesterol (HFC) diet for 3 months. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) from Cyp27a1(over) and Wt mice. RESULTS: In line with our hypothesis, hepatic inflammation in HFC-fed Cyp27a1(over)-tp mice was reduced and KCs were less foamy compared to Wt-tp mice. Remarkably, these changes occurred even though plasma and liver levels of 27HC did not differ between both groups. BMDM from Cyp27a1(over) mice revealed reduced inflammatory gene expression and increased expression of cholesterol transporters compared to Wt BMDM after lipopolysaccharide (LPS) stimulation. CONCLUSIONS: Our data suggest that overexpression of Cyp27a1 in KCs reduces hepatic inflammation independently of 27HC levels in plasma and liver, further pointing towards KCs as specific target for improving the therapy of NASH.

Seasonal variation in expression of markers in the vitamin D pathway in prostate tissue.

PURPOSE: Recent studies suggest variation in genes along the vitamin D pathway, as well as vitamin D receptor (VDR) protein levels, may be associated with prostate cancer. As serum vitamin D levels vary by season, we sought to determine whether the expression of genes on the vitamin D pathway, assessed in prostate tumor tissue, do the same. METHODS: Our study incorporates mRNA expression data from 362 men in the Swedish Watchful Waiting cohort, diagnosed between 1977 and 1999, and 106 men enrolled in the US Physicians' Health Study (PHS) diagnosed between 1983 and 2004. We also assayed for VDR protein expression among 832 men in the PHS and Health Professionals Follow-up Study cohorts. Season was characterized by date of initial tissue specimen collection categorically and by average monthly ultraviolet radiation levels. One-way analysis of variance was used to examine variation in the expression levels of six genes on the vitamin D pathway-VDR, GC, CYP27A1, CYP27B1, RXRα, CYP24A1-and VDR protein by season, adjusted for age at diagnosis and Gleason grade. Variation was also examined separately among lethal and nonlethal cases. RESULTS: Tumor expression levels of the six genes did not vary significantly by season of tissue collection. No consistent patterns emerged from subgroup analyses by lethal versus nonlethal cases. CONCLUSIONS: Unlike circulating levels of 25(OH) vitamin D, expression levels of genes on the vitamin D pathway and VDR protein did not vary overall by season of tissue collection. Epidemiological analyses of vitamin D gene expression may not be biased by seasonality.

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells.

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.

Vitamin D: metabolism.

The biologically active metabolite of vitamin D, 1,25(OH)(2)D(3), affects mineral homeostasis and has numerous other diverse physiologic functions including effects on growth of cancer cells and protection against certain immune disorders. This article reviews the role of vitamin D hydroxylases in providing a tightly regulated supply of 1,25(OH)(2)D(3). The role of extrarenal 1alpha(OH)ase in placenta and macrophages is also discussed, as well as regulation of vitamin D hydroxylases in aging and chronic kidney disease. Understanding specific factors involved in regulating the hydroxylases may lead to the design of drugs that can selectively modulate the hydroxylases. The ability to alter levels of these enzymes would have therapeutic potential for the treatment of various diseases, including bone loss disorders and certain immune diseases.

Regulation of human vitamin D(3) 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells.

In this study, we examined whether 1alpha,25-dihydroxyvitamin D(3) (calcitriol), phenobarbital, and the antiretroviral drug efavirenz, drugs used by patient groups with high incidence of low bone mineral density, could affect the 25-hydroxylase activity or expression of human 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells. Fibroblasts express the 25-hydroxylating enzymes CYP2R1 and CYP27A1. LNCaP cells were found to express two potential vitamin D 25-hydroxylases-CYP2R1 and CYP2J2. The presence in different cells of nuclear receptors vitamin D receptor (VDR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) was also determined. Phenobarbital suppressed the expression of CYP2R1 in fibroblasts and CYP2J2 in LNCaP cells. Efavirenz suppressed the expression of CYP2R1 in fibroblasts but not in LNCaP cells. CYP2J2 was slightly suppressed by efavirenz, whereas CYP27A1 was not affected by any of the two drugs. Calcitriol suppressed the expression of CYP2R1 in both fibroblasts and LNCaP cells but had no clear effect on the expression of either CYP2J2 or CYP27A1. The vitamin D(3) 25-hydroxylase activity in fibroblasts was suppressed by both calcitriol and efavirenz. In LNCaP cells, consumption of substrate (1alpha-hydroxyvitamin D(3)) was used as indicator of metabolism because no 1alpha,25-dihydroxyvitamin D(3) product could be determined. The amount of 1alpha-hydroxyvitamin D(3) remaining in cells treated with calcitriol was significantly increased. Taken together, 25-hydroxylation of vitamin D(3) was suppressed by calcitriol and drugs. The present study provides new information indicating that 25-hydroxylation of vitamin D(3) may be regulated. In addition, the current results may offer a possible explanation for the impaired bone health after treatment with certain drugs.

Relative expression of 1,25-dihydroxyvitamin D3 receptor, vitamin D 1 alpha-hydroxylase, vitamin D 24-hydroxylase, and vitamin D 25-hydroxylase in endometriosis and gynecologic cancers.

The authors demonstrate expression of the vitamin D receptor (VDR) and its hydroxylases in the endometrium and ovaries of women with and without endometriosis and endometrial or ovarian cancer. Immunohistochemistry showed strong staining of the VDR in endometriosis and endometrial cancer, with the most intense staining in epithelial cells. The VDR mRNA was significantly increased in patients with endometrial and ovarian cancer compared to the control group. There was a significantly higher 1 alpha-hydroxylase expression in the endometrium of patients with endometriosis compared to healthy controls. The observed differences in VDR and 1 alpha -hydroxylase mRNA levels were maintained at the protein level. The authors found no differences in 25-OH vitamin D levels between the serum of patients with endometriosis (25.7 +/- 2.1 ng/mL, n = 46) and healthy controls (22.6 +/- 2.0 ng/mL, n = 33, P = .31). They hypothesize that vitamin D might influence the local activity of immune cells and cytokines thought to play important pathogenic roles in the development and maintenance of endometriosis.

CYP27A1 and CYP24 expression as a function of malignant transformation in the colon.

Vitamin D deficiency is strongly associated with the risk of developing colorectal cancer (CRC). Because of the propensity of bioactive 1,25-dihydroxyvitamin D3 to cause toxic hypercalcemia, considerable effort has been directed to identifying safer drugs while retaining the efficacy of the parent compound. However, vitamin D precursors do not present toxicity concerns and may be sufficient for CRC chemoprevention or chemotherapy, providing the appropriate enzymes are present in colonic epithelia. We previously showed that CYP27B1 is present at equally high levels in the colon and CRC irrespective of differentiation but was not present in metastases. In this study we used quantitative immunohistochemistry to show that CYP27A1, converting D3 to 25-hydroxycholecalciferol, is present in increasing concentrations in the nuclei of normal colonic epithelia, aberrant crypt foci (ACF), and adenomatous polyps. Whereas total cellular CYP27A1 remains high in CRC and lymph node metastases, the amount of enzyme present in the nuclei decreases with tumor cell dedifferentiation while rising in the cytoplasm. Similarly, increasing amounts of the deactivating enzyme CYP24 are present in the nuclei of normal colonic epithelia, ACFs, and adenomatous polyps. Although the amount of total CYP24 decreases slightly in CRC as a function of tumor cell dedifferentiation and metastasis, location of this enzyme shifts almost entirely from the nuclear compartment to the cytoplasmic compartment. These data indicate that non-toxic vitamin D precursors should be sufficient for CRC chemoprevention, but that neither vitamin D nor its precursors may be sufficient for CRC chemotherapy.

Regulation of human CYP27A1 by estrogens and androgens in HepG2 and prostate cells.

The regulation of the human CYP27A1 gene by estrogens and androgens was studied in human liver-derived HepG2 and prostate cells. Our results show that the promoter activity, enzymatic activity and mRNA levels of CYP27A1 in HepG2 cells are downregulated by estrogen in presence of ERalpha or ERbeta. Similar effects by estrogen were found in RWPE-1 prostate cells. In contrast, estrogen markedly upregulated the transcriptional activity of CYP27A1 in LNCaP prostate cancer cells. 5alpha-Dihydrotestosterone and androgen receptor upregulated the transcriptional activity of CYP27A1 in HepG2 cells. Progressive deletion experiments indicate that the ERbeta-mediated effects in HepG2 and LNCaP cells are conferred to the same region (-451/+42) whereas ERalpha-mediated effects on this promoter are more complex. The results indicate that the stimulating effect of androgen in HepG2 cells is conferred to a region upstream from -792 in the CYP27A1 promoter. In summary, we have identified the human CYP27A1 gene as a target for estrogens and androgens. The results imply that expression of CYP27A1 may be affected by endogenous sex hormones and pharmacological compounds with estrogenic or androgenic effects.

1alpha,25-dihydroxyvitamin D3 downregulates CYP27B1 and induces CYP24A1 in colon cells.

The antimitotic and prodifferentiating 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), synthesized at various extrarenal sites could potentially prevent sporadic tumor development. Physiological regulation of extrarenal Vitamin D hydroxylases following tissue accumulation of 1alpha,25-(OH)2D3 is unknown. We therefore investigated basal and Vitamin D-regulated expression and activity of the synthesizing (CYP27B1) and metabolizing (CYP24A1) hydroxylase in three cell lines derived from the colon, and compared this to cells from the prostate and mammary gland. Our results show that all cells, irrespective of origin and differentiation, express CYP27B1 mRNA, whereas basal CYP24A1 mRNA is highly expressed only in undifferentiated cells. Treatment with 1alpha,25-(OH)2D3 diminishes CYP27B1 and Vitamin D receptor mRNA expression, but elevates CYP24A1 mRNA to equal levels in all cells. As shown by HPLC, CYP27B1 is active only if basal 24-hydroxylation is not maximally functional. In turn, accumulation of 1alpha,25-(OH)2D3 will induce 24-hydroxylation. We conclude that, although extrarenal and renal metabolic pathways for Vitamin D are similar, malignancy of tumor cells determines extent of Vitamin D catabolism.