27-Hydroxylation of oncosterone by CYP27A1 switches its activity from pro-tumor to anti-tumor.

Oncosterone (6-oxo-cholestane-3ß,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or ß- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3ß,5α,6ß-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3ß,26-diol), 27H-CT ((25R)-cholestane-3ß,5α,6ß,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3ß,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.

Chemical synthesis and biochemical properties of cholestane-5α,6ß-diol-3-sulfonate: A non-hydrolysable analogue of cholestane-5α,6ß-diol-3ß-sulfate.

Cholestane-3ß,5α,6ß-triol (CT) is a primary metabolite of 5,6-epoxycholesterols (5,6-EC) that is catalyzed by the cholesterol-5,6-epoxide hydrolase (ChEH). CT is a well-known biomarker for Niemann-Pick disease type C (NP-C), a progressive inherited neurodegenerative disease. On the other hand, CT is known to be metabolized by the 11ß-hydroxysteroid-dehydrogenase of type 2 (11ß-HSD2) into a tumor promoter named oncosterone that stimulates the growth of breast cancer tumors. Sulfation is a major metabolic transformation leading to the production of sulfated oxysterols. The production of cholestane-5α,6ß-diol-3ß-O-sulfate (CDS) has been reported in breast cancer cells. However, no data related to CDS biological properties have been reported so far. These studies have been hampered because sulfate esters of sterols and steroids are rapidly hydrolyzed by steroid sulfatase to give free steroids and sterols. In order to get insight into the biological properties of CDS, we report herein the synthesis and the characterization of cholestane-5α,6ß-diol-3ß-sulfonate (CDSN), a non-hydrolysable analogue of CDS. We show that CDSN is a potent inhibitor of 11ß-HSD2 that blocks oncosterone production on cell lysate. The inhibition of oncosterone biosynthesis of a whole cell assay was observed but results from the blockage by CDSN of the uptake of CT in MCF-7 cells. While CDSN inhibits MCF-7 cell proliferation, we found that it potentiates the cytotoxic activity of post-lanosterol cholesterol biosynthesis inhibitors such as tamoxifen and PBPE. This effect was associated with an increase of free sterols accumulation and the appearance of giant multilamellar bodies, a structural feature reminiscent of Type C Niemann-Pick disease cells and consistent with a possible inhibition by CDSN of NPC1. Altogether, our data showed that CDSN is biologically active and that it is a valuable tool to study the biological properties of CDS and more specifically its impact on immunity and viral infection.

Targeting the liver X receptor with dendrogenin A differentiates tumour cells to secrete immunogenic exosome-enriched vesicles.

Tumour cells are characterized by having lost their differentiation state. They constitutively secrete small extracellular vesicles (sEV) called exosomes when they come from late endosomes. Dendrogenin A (DDA) is an endogenous tumour suppressor cholesterol-derived metabolite. It is a new class of ligand of the nuclear Liver X receptors (LXR) which regulate cholesterol homeostasis and immunity. We hypothesized that DDA, which induces tumour cell differentiation, inhibition of tumour growth and immune cell infiltration into tumours, could functionally modify sEV secreted by tumour cells. Here, we have shown that DDA differentiates tumour cells by acting on the LXRß. This results in an increased production of sEV (DDA-sEV) which includes exosomes. The DDA-sEV secreted from DDA-treated cells were characterized for their content and activity in comparison to sEV secreted from control cells (C-sEV). DDA-sEV were enriched, relatively to C-sEV, in several proteins and lipids such as differentiation antigens, "eat-me" signals, lipidated LC3 and the endosomal phospholipid bis(monoacylglycero)phosphate, which stimulates dendritic cell maturation and a Th1 T lymphocyte polarization. Moreover, DDA-sEV inhibited the growth of tumours implanted into immunocompetent mice compared to control conditions. This study reveals a pharmacological control through a nuclear receptor of exosome-enriched tumour sEV secretion, composition and immune function. Targeting the LXR may be a novel way to reprogram tumour cells and sEV to stimulate immunity against cancer.

The 5,6-epoxycholesterol metabolic pathway in breast cancer: Emergence of new pharmacological targets.

Metabolic pathways have emerged as cornerstones in carcinogenic deregulation providing new therapeutic strategies for cancer management. Recently, a new branch of cholesterol metabolism has been discovered involving the biochemical transformation of 5,6-epoxycholesterols (5,6-ECs). The 5,6-ECs are metabolized in breast cancers to the tumour promoter oncosterone whereas, in normal breast tissue, they are metabolized to the tumour suppressor metabolite, dendrogenin A (DDA). Blocking the mitogenic and invasive potential of oncosterone will present new opportunities for breast cancer treatment. The reactivation of DDA biosynthesis, or its use as a drug, represents promising therapeutic approaches such as DDA-deficiency complementation, activation of breast cancer cell re-differentiation and breast cancer chemoprevention. This review presents current knowledge of the 5,6-EC metabolic pathway in breast cancer, focusing on the 5,6-EC metabolic enzymes ChEH and HSD11B2 and on 5,6-EC metabolite targets, the oxysterol receptor (LXRß) and the glucocorticoid receptor. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

A fast UPLC-HILIC method for an accurate quantification of dendrogenin A in human tissues.

Dendrogenin A (DDA) is a newly-discovered steroidal alkaloid, which remains to date the first ever found in mammals. DDA is a cholesterol metabolites that induces cancer cell differentiation and death in vitro and in vivo, and thus behave like a tumor suppressor metabolite. Preliminary studies performed on 10 patients with estrogen receptor positive breast cancers (ER(+)BC) showed a strong decrease in DDA levels between normal matched tissue and tumors. This suggests that a deregulation on DDA metabolism is associated with breast carcinogenesis. To further investigate DDA metabolism on large cohorts of patients we have developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) procedure for the quantification of DDA in liquid and in solid tissues. This method enabled the identification of DDA analogues such as its geometric isomer C17 and dendrogenin B (C26) in human samples showing that other 5,6α-epoxycholesterol conjugation products with biogenic amines exist as endogenous metabolites . We report here the first complete method of quantification of DDA in liquid and solid tissues using hydrophilic interaction liquid chromatography (HILIC). Two different methods of extraction using either a Bligh and Dyer organic extraction or protein precipitation were successfully applied to quantify DDA in solid and liquid tissues. The protein precipitation method was the fastest. The fact that this method is automatable opens up possibilities to study DDA metabolism in large cohorts of patients.

Chemistry, biochemistry, metabolic fate and mechanism of action of 6-oxo-cholestan-3ß,5α-diol (OCDO), a tumor promoter and cholesterol metabolite.

Oxygenation products of cholesterol, named oxysterols, were suspected since the 20th century to be involved in carcinogenesis. Among the family of oxysterol molecules, cholesterol-5,6-epoxides (5,6-EC) retained the attention of scientists because they contain a putative alkylating epoxide group. However, studies failed into demonstrating that 5,6-EC were direct carcinogens and revealed a surprising chemical stability and unreactivity towards nucleophiles in standard conditions. Analyses of 5,6-EC metabolism in normal cells showed that they were extensively transformed into cholestane-3ß,5α,6ß-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH). Studies performed in cancer cells showed that CT was additionally metabolized into an oxysterol identified as the 6-oxo-cholestan-3ß,5α-diol (OCDO), by the 11ß-hydroxysteroid dehydrogenase of type 2 (HSD2), the enzyme which inactivates cortisol into cortisone. Importantly, OCDO was shown to display tumor promoter properties in breast cancers, by binding to the glucocorticoid receptor, and independently of their estrogen receptor status, revealing the existence of a new tumorigenic pathway centered on 5,6-EC. In breast tumors from patients, OCDO production as well as the expression of the enzymes involved in the pathway producing OCDO, namely ChEH subunits and HSD2, were higher compared to normal tissues, and overexpression of these enzymes correlate with a higher risk of patient death, indicating that this onco-metabolism is of major importance to breast cancer pathology. Herein, we will review the actual knowledge and the future trends in OCDO chemistry, biochemistry, metabolism and mechanism of action and will discuss the impact of OCDO discovery on new anticancer therapeutic strategies.

Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor.

Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3ß,5α,6ß-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) by 11ß-hydroxysteroid-dehydrogenase-type-2 (11ßHSD2). 11ßHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11ßHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11ßHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11ßHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.

Quantitative analysis of the tumor suppressor dendrogenin A using liquid chromatography tandem mass spectrometry.

Dendrogenin A (DDA) was recently identified as a mammalian cholesterol metabolite that displays tumor suppressor and neurostimulating properties at low doses. In breast tumors, DDA levels were found to be decreased compared to normal tissues, evidencing a metabolic deregulation of DDA production in cancers. DDA is an amino-oxysterol that contains three protonatable nitrogen atoms. This makes it physico-chemically different from other oxysterols and it therefore requires specific analytical methods We have previously used a two-step method for the quantification of DDA in biological samples: 1) DDA purification from a Bligh and Dyer extract by RP-HPLC using a 250×4.6mm column, followed by 2) nano-electrospray ionization mass spectrometry (MS) fragmentation to analyze the HPLC fraction of interest. We report here the development a liquid chromatography tandem mass spectrometry method for the analysis of DDA and its analogues. This new method is fast (10min), resolving (peak width <4s) and has a weak carryover (<0.01%). We show that this technique efficiently separates DDA from its C17 isomer and other steroidal alkaloids from the same family establishing a proof of concept for the analysis of this family of amino-oxysterols.

Improvement of 5,6α-epoxycholesterol, 5,6ß-epoxycholesterol, cholestane-3ß,5α,6ß-triol and 6-oxo-cholestan-3ß,5α-diol recovery for quantification by GC/MS.

5,6α-epoxycholesterol (5,6α-EC) and 5,6ß-epoxycholesterol (5,6ß-EC) are oxysterols involved in the anticancer pharmacology of the widely used antitumor drug tamoxifen. They are both metabolized into cholestane-3ß,5α,6ß-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH) enzyme, and CT is metabolized by an as-yet uncharacterized enzyme into 6-oxo-cholestan-3ß,5α-diol (OCDO). A recent feasibility study showed that the 5,6-ECs may represent surrogate markers of tamoxifen activity in breast cancer patients undergoing endocrine therapy, thus there is a growing interest in their accurate quantification. These oxysterols are usually quantified by gas-liquid chromatography coupled to mass spectrometry (GC/MS), using an isotope dilution methodology with the corresponding deuterated oxysterol. This method is considered to be relative quantitative since all of the standards used are deuterated oxysterols, however it is not known whether the preparation of each oxysterol is affected in the same way by the extraction, pre-purification by solid phase extraction (SPE) and trimethylsilylation steps, particularly when using biological samples that contain many other reactive compounds. Thus, in this study we investigated the yield of the 5,6-ECs, CT and OCDO recovery from patient serum samples at different stages of their work-up and trimethylsilylation prior to GC/MS analysis, using [14C]-labeled analogs to follow these oxysterols at each step. We measured a 40 to 60% loss of material for the 5,6-ECs and OCDO, however we also describe the conditions that improved their recovery. Our data also show that the use of deuterated 5,6α-EC, 5,6ß-EC, CT and OCDO is an absolute requirement for their accurate quantification.

Effect of PEG on rheology and stability of nanocrystalline titania hydrosols.

Very stable titania hydrosols were prepared by fast hydrolysis of titanium isopropoxide in a large excess of water. XRD patterns show that these sols contain nanocrystals (5-6 nm) of anatase (70%) and brookite (30%). TEM images indicate that these primary particles form aggregates whose mean hydrodynamic diameter, determined by photon correlation spectroscopy, is in the range of 80-90 nm. The flow curves of these colloids, recorded for several volume fractions of nanoparticles, can be perfectly fitted, in the range 0-100 s(-1), with a power-law model. In this range the behavior is Newtonian but for larger shear rates a shear thinning is observed. The viscosity dependence on particle concentration can be predicted by a Batchelor-type model were the volume fraction of particles is replaced by an effective volume fraction of aggregates, taking into account their fractal dimension. Addition of polyethylene glycol (PEG 2000) induced a marked decrease (more than 50%) of the sol viscosity down to a minimum. This is explained by assuming that PEG adsorbs on the surface of TiO(2) particles producing stabilization by steric effects and leading to formation of more compact aggregates. Without PEG the sol viscosity strongly decreases on aging. This effect is not caused by the growth of primary particles. It is rather interpreted as a progressive reorganization of the aggregates toward a more compact packing.