RESUMEN
BACKGROUND: 27-Hydroxycholesterol (27HC) stimulates estrogen receptor-positive (ER+) breast cancer (BC) progression. Inhibiting the sterol 27-hydroxylase (CYP27A1) abrogates these growth-promoting effects of 27HC in mice. However, the significance of CYP27A1 expression on BC biology and prognosis is unclear. METHODS: Intratumoral CYP27A1 expression in invasive BC was measured by immunohistochemistry in two Swedish population-based cohorts (n = 645 and n = 813, respectively). Cox proportional hazards models were used to evaluate the association between CYP27A1 expression and prognosis. RESULTS: CYP27A1 was highly expressed in less than 1/3 of the tumors. High CYP27A1 expression was more frequent among high-grade tumors lacking hormone receptor expression and with larger tumor sizes. Over a median of 12.2 years follow-up in cohort 1, high CYP27A1 expression was associated with impaired survival, specifically after 5 years from diagnosis among all patients [overall survival (OS), HRadjusted = 1.93, 95%CI = 1.26-2.97, P = 0.003; breast cancer-specific survival (BCSS), HRadjusted = 2.33, 95%CI = 1.28-4.23, P = 0.006] and among patients ≥ 55 years presenting with ER+ tumors [OS, HRadjusted = 1.99, 95%CI = 1.24-3.21, P = 0.004; BCSS, HRadjusted = 2.78, 95%CI = 1.41-5.51, P = 0.003]. Among all patients in cohort 2 (median follow-up of 7.0 years), CYP27A1 expression was significantly associated with shorter OS and RFS in univariable analyses across the full follow-up period. However after adjusting for tumor characteristics and treatments, the association with survival after 5 years from diagnosis was non-significant among all patients [OS, HRadjusted = 1.08, 95%CI = 0.05-2.35, P = 0.83 and RFS, HRadjusted = 1.22, 95%CI = 0.68-2.18, P = 0.50] as well as among patients ≥ 55 years presenting with ER+ tumors [OS, HRadjusted = 0.46 95% CI = 0.11-1.98, P = 0.30 and RFS, HRadjusted = 0.97 95% CI = 0.44-2.10, P = 0.93]. CONCLUSION: CYP27A1 demonstrated great potentials as a biomarker of aggressive tumor biology and late lethal disease in postmenopausal patients with ER+ BC. Future studies should investigate if the benefits of prolonged endocrine therapy and cholesterol-lowering medication in BC are modified by CYP27A1 expression.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/mortalidad , Colestanotriol 26-Monooxigenasa/metabolismo , Recurrencia Local de Neoplasia/epidemiología , Posmenopausia , Anciano , Antineoplásicos Hormonales/uso terapéutico , Biomarcadores de Tumor/análisis , Mama/patología , Mama/cirugía , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Quimioterapia Adyuvante , Colestanotriol 26-Monooxigenasa/análisis , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Hidroxicolesteroles/metabolismo , Inmunohistoquímica , Estimación de Kaplan-Meier , Mastectomía , Persona de Mediana Edad , Clasificación del Tumor , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/prevención & control , Pronóstico , Receptores de Estrógenos/análisis , Receptores de Estrógenos/metabolismo , Factores de TiempoRESUMEN
Self-preparation of nano liquid chromatography (nLC) columns has advantages regarding cost and flexibility. For targeted proteomics, we evaluated several approaches for particle-packing nLC columns and manufacturing fritless silica-based monolithic trap columns (50µm inner diameter). Our preferred approach for nLC column preparation was to magnetically stir Accucore core shell particles (C18 stationary phase) in ACN/water (80/20, v/v) suspensions during pressure-driven filling of polymer-fritted standard fused silica capillaries. The columns were ready for use about one hour after preparation had begun. They had comparable peak capacities (peptides) to commercial columns, and satisfactory within/between-column retention time repeatability, suited for targeted proteomics. Packing with commercial capillary housings/nanospray emitters did not improve performance compared to packing with in-house fritted stock fused silica capillary tubing. For trap columns, several recipes for narrow bore silica-based monolithic columns were evaluated, and we found the recipe by Zou et al. (2005) to be reproducible. Compared to the standard C18 trap column for Accucore nLC columns, monolith trap columns (C8 stationary phase) significantly reduced peak widths. The readily prepared in-house columns were used for targeted detection of the enzyme CYP27A1 in cancer cells, which is associated with proliferation and metastasis of breast cancer.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Nanopartículas/química , Péptidos/análisis , Proteómica/métodos , Dióxido de Silicio/química , Línea Celular Tumoral , Colestanotriol 26-Monooxigenasa/análisis , Colestanotriol 26-Monooxigenasa/aislamiento & purificación , Humanos , Espectrometría de Masas , Microscopía Electrónica de Rastreo , Péptidos/aislamiento & purificación , PresiónRESUMEN
Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis.Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis.Cluster analysis of the whole patient cohort identified a good prognosis group (mean survival=146 months 95% CI 127-165 months) that had a significantly better survival (χ2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group (mean survival=107 months, 95% CI 98-123 months). For the mismatch repair proficient cohort, the good prognosis group had a significantly better survival (χ2=8.985, p=0.003, HR=1.845, 95% CI 1.227-2.774) than the poor prognosis group. Multi-variate analysis showed that cluster group was independently prognostically significant in both the whole patient cohort (p=0.02, HR=1.554, 95% CI 1.072-2.252) and the mismatch repair proficient group (p=0.04, HR=1.530, 95% CI 1.014-2.310).Individual oxysterol metabolising enzymes are overexpressed in colorectal cancer and an oxysterol metabolising enzyme expression profile associated with prognosis has been identified in the whole patient cohort and in mismatch repair proficient colorectal cancers.
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Neoplasias Colorrectales/metabolismo , Reparación de la Incompatibilidad de ADN , Oxiesteroles/metabolismo , Adulto , Anciano , Colestanotriol 26-Monooxigenasa/análisis , Colesterol 24-Hidroxilasa/análisis , Análisis por Conglomerados , Estudios de Cohortes , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Redes y Vías Metabólicas , Persona de Mediana Edad , Pronóstico , Esteroide Hidroxilasas/análisis , Análisis de Matrices TisularesRESUMEN
Vitamin D (VD) deficiency has been suggested as a risk factor for cancer. One recognized mechanism is that the low-serum 25-hydroxyvitamin D (25(OH)D) of VD deficiency reduces intratumoral 25(OH)D conversion to 1α,25-dihydroxyvitamin D (1,25D, the hormonal form of VD), compromising 1,25D-VD receptor (VDR) antitumoral actions. Reduced tumoral VDR and increased CYP24A1, the enzyme that degrades 1,25D and 25(OH)D, further worsen cancer progression. Importantly, in cells expressing CYP27A1 and/or CYP2R1, which convert inert VD into 25(OH)D, low-serum VD may reduce intratumoral 25(OH)D synthesis thereby compromising VDR antitumoral actions because 25(OH)D can activate the VDR directly and enhance 1,25D-VDR action. Therefore, this study examined whether abnormal endometrial expression of CYP27A1 and/or CYP2R1 may impair VDR-antiproliferative properties in endometrial carcinoma (EC). Immunohistochemical analysis of tissue microarrays of normal human endometrium (NE; n=60) and EC (n=157) showed the expected lower VDR expression in EC (P=0.0002). Instead, CYP24A1 expression was lower in EC compared with NE, while CYP27A1 and CYP2R1 expressions were higher (P=0.0002; P=0.03). Furthermore, in NE and EC, CYP2R1 and CYP27A1 expression correlated directly with nuclear VDR levels, an indicator of ligand-induced VDR activation, and inversely with the proliferation marker Ki67. Accordingly, in the endometrioid carcinoma cell lines IK, RL95/2 and HEC1-A, which express VDR, CYP27A1, and CYP2R1, VD efficaciously reduced cell viability and colony number, with a time course that paralleled actual increases in both intracellular 25(OH)D and nuclear VDR levels. Thus, VD may protect from EC progression in part through increased intratumoral 25(OH)D production by CYP27A1 and CYP2R1 for autocrine/paracrine enhancement of 1,25D-VDR-antiproliferative actions.
Asunto(s)
Colecalciferol/farmacología , Colestanotriol 26-Monooxigenasa/metabolismo , Neoplasias Endometriales/química , Endometrio/química , Receptores de Calcitriol/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colestanotriol 26-Monooxigenasa/análisis , Familia 2 del Citocromo P450 , Femenino , Humanos , Persona de Mediana Edad , Receptores de Calcitriol/análisisRESUMEN
Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using ¹5N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.
Asunto(s)
Colestanotriol 26-Monooxigenasa/análisis , Espectrometría de Masas/métodos , Retina/química , Esteroide Hidroxilasas/análisis , Lóbulo Temporal/química , Membrana Celular/química , Colestanotriol 26-Monooxigenasa/metabolismo , Colestenos/análisis , Colestenos/metabolismo , Colesterol/metabolismo , Colesterol 24-Hidroxilasa , Humanos , Hidroxicolesteroles/análisis , Hidroxicolesteroles/metabolismo , Marcaje Isotópico , Isótopos de Nitrógeno , Reproducibilidad de los Resultados , Esteroide Hidroxilasas/metabolismoRESUMEN
UNLABELLED: 25-Hydroxyvitamin D (25[OH]D) can potentially interfere with inflammatory response and fibrogenesis. Its role in disease progression in chronic hepatitis C (CHC) and its relation with histological and sustained virological response (SVR) to therapy are unknown. One hundred ninety-seven patients with biopsy-proven genotype 1 (G1) CHC and 49 healthy subjects matched by age and sex were consecutively evaluated. One hundred sixty-seven patients underwent antiviral therapy with pegylated interferon plus ribavirin. The 25(OH)D serum levels were measured by high-pressure liquid chromatography. Tissue expression of cytochrome (CY) P27A1 and CYP2R1, liver 25-hydroxylating enzymes, were assessed by immunochemistry in 34 patients with CHC, and in eight controls. The 25(OH)D serum levels were significantly lower in CHC than in controls (25.07 +/- 9.92 microg/L versus 43.06 +/- 10.19; P < 0.001). Lower levels of 25(OH)D were independently linked to female sex (P = 0.007) and necroinflammation (P = 0.04) by linear regression analysis. CYP27A1, but not CYP2R1, was directly related to 25(OH)D levels (P = 0.01), and inversely to necroinflammation (P = 0.01). Low 25(OH)D (odds ratio [OR], 0.942; 95% confidence interval [CI], 0.893-0.994) and cholesterol (OR, 0.981; 95%CI, 0.969-0.992) levels, older age (OR, 1.043; 95%CI, 1.002-1.085), high ferritin (OR, 1.003; 95%CI, 1.001-1.005), and necroinflammation (OR, 2.235; 95%CI, 1.014-4.929) were independently associated with severe fibrosis (F3-F4) by multivariate logistic analysis. Seventy patients (41%) achieved SVR. By multivariate analysis, hepatic steatosis (OR, 0.971; 95%CI, 0.944-0.999), lower cholesterol (OR, 1.009; 95% CI, 1.000-1.018), and 25(OH)D levels (OR, 1.039; 95%CI, 1.002-1.077) were independently associated with no SVR. CONCLUSION: G1 CHC patients had low 25(OH)D serum levels, possibly because of reduced CYP27A1 expression. Low vitamin D is linked to severe fibrosis and low SVR on interferon (IFN)-based therapy.
Asunto(s)
Hepatitis C Crónica/tratamiento farmacológico , Interferones/uso terapéutico , Cirrosis Hepática/etiología , Vitamina D/análogos & derivados , Adulto , Anciano , Colestanotriol 26-Monooxigenasa/análisis , Familia 2 del Citocromo P450 , Femenino , Genotipo , Hepacivirus/clasificación , Hepatitis C Crónica/sangre , Hepatitis C Crónica/virología , Humanos , Cirrosis Hepática/sangre , Masculino , Persona de Mediana Edad , Ribavirina/administración & dosificación , Factores de Riesgo , Vitamina D/sangreRESUMEN
Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder believed to be exclusively caused by mutations in the CYP27A1 gene coding for the enzyme sterol 27-hydroxylase. Common findings in CTX are tendon xanthomas, cataracts and progressive neurological dysfunction. Here, we characterize an adult female patient with tendon xanthomas and classic biochemical findings of CTX (i.e. high levels of bile alcohols and cholestanol and extremely low levels of 27-hydroxycholesterol in plasma). Additionally, sterol 27-hydroxylase activity in cultured monocyte-derived macrophages from this patient was <5% of normal. Sequencing the CYP27A1 gene uncovered that the patient is heterozygous for two previously undescribed base substitutions in exon 8, C478A and C479A, which are expected to affect the haeme-binding domain of the enzyme. When expressed in HEK293 cells, the corresponding protein had only 8% of normal enzymatic activity. No other mutation was found in the open reading frame of the CYP27A1 gene, intron-exon boundaries or in the 5'-untranslated region up to 5000 bp distal to the translational start site. Sequencing mRNA isolated from leucocytes from the patient revealed a 1 : 1 ratio of mutated and nonmutated species, with total mRNA levels that were not significantly different from the controls. It is concluded that the patient is heterozygous for two mutations affecting one allele of the CYP27A1 gene and with at least one additional yet undefined gene that is of critical importance for the activity of sterol 27-hydroxylase.