METHODS OF DIAGNOSING CANCER AND OF PREDICTING RESPONSE OF CANCER TO DENDROGENIN A TREATMENT

Abstract

The present invention relates to methods for the diagnosis and the treatment of cancer, in particular breast cancer. In particular, the present invention relates to a method of diagnosing cancer in a subject comprising the steps of i) determining the expression level of hGSTA1 in a tumor sample obtained from the subject, ii) comparing the expression level determined at step i) with its predetermined reference value and ii) concluding that the subject suffers from a cancer when the expression level of hGSTA1 is lower than its predetermined reference value.

Claims

1. A method of diagnosing cancer in a subject comprising the steps of i) determining an expression level of hGSTA1 in a tumor sample obtained from the subject, ii) comparing the expression level determined at step i) with its predetermined reference value and iii) concluding that the subject suffers from a cancer when the expression level of hGSTA1 is lower than its predetermined reference value.

2. A method for determining the survival time of subject suffering from a cancer comprising the steps of i) determining an expression level of hGSTA1 in a tumor sample obtained from the subject, ii) comparing the expression level determined at step i) with its predetermined reference value and iii) concluding that the subject will have a long survival time when the expression level of hGSTA1 is higher than its predetermined reference value or concluding that the subject will have a short survival time when the expression level of hGSTA1 is lower than its predetermined reference value.

3. A method for determining whether a subject suffering from a cancer will achieve a response with dendrogenin A of comprising i) determining an expression level of hGSTA1 in a tumor sample obtained from the subject, ii) comparing the expression level determined at step i) with its predetermined reference value and iii) concluding that the subject will achieve a response with dendrogenin A when the expression level of hGSTA1 is lower than its predetermined reference value.

4. The method of claim 1, wherein the cancer is selected from the group consisting of bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

5. The method of claim 4 wherein the cancer is breast cancer.

6. The method of claim 1 wherein when it is determined that the subject suffers from cancer, a nucleic acid encoding for hGSTA1, a chemotherapeutic agent, a radiotherapeutic agent or an immunotherapeutic agent is administered to the subject.

7. The method of claim 3 wherein when it is determined that the subject will achieve a response with dendrogenin A, the subject is then administered with said dendrogenin A.

8. The method of claim 2, wherein the cancer is selected from the group consisting of bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

9. The method of claim 3, wherein the cancer is selected from the group consisting of bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

Description

FIGURES

[0086] FIG. 1: a) Dendrogenin A is the product of the enzymatic conjugation of 5,6α-epoxy-cholesterol (5,6α-EC) with histamine (HA). The reaction is an SN2 reaction resulting from the attack of the primary amine (a) of histamine (HA) on the C6 through the β side of the steroid backbone giving a 6β substitute product. b) The rat Glutathione S-transferase B (GST-B) catalyzed the stereoselective glutathionylation of 5,6α-EC to give 5α-dihydroxycholestan-6β-yl-S-glutathione (CDO-SG), a cholesterol metabolite with the precise stereochemistry of DDA at C6.

[0087] FIG. 2: Measurement of glutathionylation activity of hGSTA1. Glutathionylation of CDNB was done with recombinant hGSTA1 in the presence of (a) increasing concentrations of CDNB and a single GSH concentration or (b) increasing concentrations of GSH and a single CDNB concentration. c) The glutathionylation of CDNB was done in the presence of 1 and 10 μM 5,6α-EC or Histamine (HA). The glutathionylation of 5,6α-EC was done in the presence of (d) increasing hGSTA1 concentrations, (e) increasing GSH concentrations or (f) increasing 5,6α-EC concentrations. The reaction of CDO-S-G biosynthesis by hGSTA1 was carried out in the presence of (g) 1 and 10 mM CDNB or 1 and 10 μM HA, (h) or 1 μM 5,6β-EC or 10 μM CT, OCDO, 27-HC or 22(R)-HC.

[0088] FIG. 3: Measurement of the histaminylation of 5,6α-EC by hGSTA1. Histaminylation of 5,6α-EC was measured using recombinant hGSTA1 in the presence of (a) increasing concentrations of HA and a single 5,6α-EC concentration or (b) increasing concentrations of 5,6α-EC and a single HA concentration. (c) The product of this reaction was characterized by mass spectrometry and gave the expected mass of 514.4371 confirming the production of DDA by GSTA1. (d) The reaction of DDA biosynthesis by hGSTA1 was carried out in the presence of 10 mM CDNB with increasing concentrations of GSH ranging from 1 μM to 1 mM, (e) or 1 μM 5,6β-EC or 10 μM CT, OCDO, 27-HC or 22(R)-HC.

[0089] FIG. 4: Expression of GSTA1 in HEK293 induced DDAS activity. The expression of hGSTA1 by transfection of HEK293T cells with a plasmid encoding hGSTA1 (p-hGSTA1) was confirmed by Western blot (a) and immunocytochemistry (b). The functionality of hGSTA1 was validated by measurement of DCNB glutathionylation (c). (d) Transfected cells activated the biosynthesis of DDA and thus DDAS activity.

[0090] FIG. 5: Expression of DDAS and quantification of endogenous DDA in human breast tumors and normal tissues. Photomicrographs of immunohistochemical staining of DDAS expression in biopsies from patients. The stain is diaminobenzidine counterstained with hematoxylin. (a) DDAS is specifically expressed in (1) lactating duct terminal units (LTDU) and (3) in lactating ducts (LD), but not in tumor cells (1,3). DDAS is expressed in the cytoplasm of epithelial cells LD and LDTU (2, 4). (b) Quantification of DDAS expression in normal breast and in tumors after immunostaining was done as described in the “Methods” section (0 is no expression and 3 is high expression). DDA was quantified in tumors and adjacent normal tissues from 50 patients as described in the “Methods” section (c). Each symbol represents the mean concentration of DDA in each normal or tumor sample that was analyzed twice. The black line indicates the group sample mean±S.E., *P<0.001. (d) Paired comparison of the level of DDA between normal tissue and the corresponding tumor (p=0.024, n=50, paired Wilcoxon signed rank test).

EXAMPLE

[0091] Material & Methods

[0092] Chemicals.

[0093] [.sup.14C]cholesterol (58 mCi/mmol) and [.sup.3H]cholesterol (53 Ci/mmol) were purchased from Perkin Elmer. Sterols, oxysterols and drugs were from Steraloids or Sigma-Aldrich. DDA and 5,6α-EC and 5,6β-EC were synthesized in our laboratory according to published procedures.sup.10,27. 3β,5α-dihydroxycholestan-6β-yl-S-glutathione (CDO-SG) was synthesized as previously described.sup.14 and was 99% pure by TLC. Solvents were from Sigma, Fischer, Scharlau or VWR. TLC plates were from Macherey Nagel. C18 Sep-Pack cartridges were from Waters.

[0094] Measurement of GSTA1 Activity.

[0095] Glutathione S-transferase activity was measured according to the method of Habig et al.sup.28. Briefly, 2 μg of hGSTA1 (NBP1-30307, Novus company) were incubated in the presence of 1 mM glutathione in 0.1 M potassium phosphate buffer (pH 7.0) and 100 μM 1-chloro-2,4-dinitrobenzene (CDNB, Sigma) in ethanol (2.85% vol/vol). The mixture was incubated for 30 min at 30° C. and the reaction was stopped with 200 μl MeOH. Measurements were carried out spectrophotometrically at 345 nm (A.sub.m=8.5 mM.sup.−1.Math.cm.sup.−1). The negative control corresponds to the same test performed in absence of hGSTA1.

[0096] Measurement of CDO-SG and DDA Biosynthesis.

[0097] The final assay volume of 100 μl with 0.1 M potassium phosphate buffer (pH 7.5), 2 μg hGSTA1 (1 μg/μ1), was incubated with increasing concentrations of [.sup.3H]5,6α-EC (50 nM to 500 nM), GSH (1 μM to 1 mM) or HA (0.1 to 100 μM). The mixture was incubated for 30 minutes at 30° C., and the reaction was stopped by addition of 200 μl MeOH. After centrifugation 10 min at 18,000 g (4° C.), the supernatant was evaporated to dryness using a speed vac concentrator (Savant SP01010, Thermo scientific) and the residue was resuspended in 20 μl MeOH. Samples were spotted on HPTLC NanoSilgur 10×10 cm plates (Macherey Nagel, ref 811042) and developed using using ethyl acetate/butanol/acetic acid/water (6:2:3:2) for CDO-SG and CH.sub.2Cl/MeOH/NH.sub.4OH 84:15:1 for DDA. Standards were co-spotted with samples. Plates were revealed by sulfuric acid impregnation and heating. The radioactivity was quantified by liquid scintillation counting of the ethanol extract of the silica scraped at the retention factor (Rf) corresponding to authentic 5,6α-EC, CDO-SG or DDA standards.

[0098] Cell Culture.

[0099] HEK293T cells were from the ATCC. HEK293T cells were grown in RPMI 1640 medium supplemented with 1.2 mM glutamine, 5% fetal bovine serum, penicillin, and streptomycin (50 units/mL) in a humidified atmosphere with 5% CO.sub.2 at 37° C. Expression of hGSTA1 in HEK293T. Cells were transfected using the Neon™ transfection system following the instructions of the Invitrogen Company (program 13, 5.10.sup.6 cells). Cells were transfected with 4 μg of a mock plasmid (pCMV6-AC, PS100020, Origene Company) or a plasmid encoding hGSTA1 (pCMV6-hGSTA1, SC321900, Origene Company). Proteins were separated on 4-12% SDS-PAGE gels (NuPAGE Novex Invitrogen 4-12% Bis-Tris protein gels, NP0335BOX), electro-transferred onto PVDF membranes and incubated overnight at 4° C. with rabbit anti-GSTA1 (PA5-29811, Thermo Scientific) or anti-Actin (C4, MAB1501, Millipore). Visualization was carried out using an ECL plus kit (Pierce), and revealed with the ChemiDoc™ MP Imaging System (Biorad). Immunocytochemistry was carried as previously described.sup.10 using an anti-hGSTA1 (PA5-29811, Thermo Scientific, dilution1/500) and revealed with an anti-HRP secondary antibody (Abeam, Anti-Rabbit IgG, 1/500) after Giemsa staining.

[0100] Measure of DDA Biosynthesis in Cells Transfected with hGSTA1.

[0101] Transfected cells were seeded in 6-well plates at 100 000 cells per well in triplicate. After 24 h, the cells were incubated with [14C]-5,6αEC (0,072 μCi) in EtOH 0.1%. After 48 h, the cells were incubated with HA at 100 μM for 96 h. Cells were then scraped and pelleted by centrifugation for 5 min at 1,400 rpm, and then extracted, developed and analyzed as described with recombinant hGSTA1.

[0102] MS Experiments.

[0103] Mass spectrometry experiments were performed on an Exactive mass spectrometer (Thermo Scientific) equipped with a HESi-II probe electrospray ionization source operating in positive mode. Samples were introduced using a syringe pump and a 500 μL syringe (Thermo scientific) at a flow rate of 10 μL/min. The Exactive MS was tuned using a 100 fmol/μL Dendrogenin A solution to optimize parameters. Optimized parameters were as follows: spray voltage, 3.8 kV; heated capillary temperature, 150° C.; capillary voltage, 82.50 V; tube lens voltage, 185 V; skimmer voltage, 34 V, sheath gas (nitrogen) flow rate, 20 (arbitrary units); auxiliary gas flow rate, 10 (arbitrary units). The instrument was set to a maximum injection time of 50 ms with 2 microscans per spectrum. The data were acquired via the Thermo Exactive Tune software.

[0104] Preparation of Tissues.

[0105] All human samples were collected with the approval of institutional review board of the Purpan University Hospital and of the Claudius Regaud Institute. Normal and tumor human tissues obtained by surgical resection were stored frozen at −80° C. and thawed in ice before use. Patients' clinical characteristics and tumor pathological features were obtained from the medical reports and followed the standard procedures in our institution. Tissues were homogenized at 4° C. in buffer A (50 mM Tris, 0.5% butylated hydroxytoluene (5 mg ml.sup.−1), 150 mM KCl, pH 7.4) (5 vol g.sup.−1 of tissue) with a Polytron B homogenizer and centrifuged 5 min at 2,500 r.p.m. Homogenates and sera were diluted with buffer A to obtain samples at 10 mg protein per ml. Quantification of DDA from tumors and normal matching tissues was done exactly as previously described.sup.10.

[0106] Immunohistochemistry.

[0107] Human tissues were analysed for hGSTA1 expression. Immunohistochemical staining was done on paraffin-embedded tissue sections, using a specific anti-human hGSTA1 antibody (PA5-29811, Thermo Scientific). Immunostaining of paraffin sections was preceded by an antigen retrieval technique by heating in 10 mM citrate buffer, pH 6, with a microwave oven twice for 10 min each time. After incubation with the antibody for 1 h at room temperature, sections were incubated with biotin-conjugated polyclonal anti-rabbit immunoglobulin antibody followed by the streptavidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Laboratories, CA) and were then counterstained with hematoxylin. Negative controls were incubated in buffered solution without primary antibody. Immunohistochemistry was performed on 3-μm-thick representative whole tissue sections from 50 cases, which were mounted on polylysine-coated slides. hGSTA1 antibody (PA5-29811, ThermoScientific) was used at 1:100. Heat-induced antigen retrieval was employed: 30 min in buffer pH 6.0 in a pre-treatment module (Labvision, Fremont, Calif., USA) for hGSTA1 antibody. Sections were blocked with 1.5% H.sub.2O.sub.2 in methanol for 10 min and incubated with hGSTA1 antibodies for 60 min at room temperature. Detection was achieved with the Vector avidin-biotin complex (ABC) system (Vector Laboratories, Burlingame, Calif., USA) according to the manufacturer's recommendations, using 3,3′-diaminobenzidine (Dako, Glostrup, Denmark) as a chromogenic substrate. Slides were lightly counterstained with haematoxylin. Positive controls (normal breast section) and negative controls (omission of the primary antibody and substitution of the primary antibody by IgG-matched control) were included in each experiment. hGSTA1 immunostains obtained with the two antibodies were analyzed independently by five of the authors (MV, SSP, MP, FD and MLT) using the Allred scoring system that combines the staining intensity and the percentage of stained cells (intensity score 0-3+% score 0-5).sup.29. For each case, the score was assessed separately for the cytoplasmic, nuclear and membrane reactivity. An Allred score of >2 was considered as positive. Immunohistochemical analysis with the hGSTA1 clone was carried out with the observers blinded to the results of the analysis of the HEK23T-hGSTA1 clone. For a comparison with fibromatosis, we considered the expression patterns and levels of β-catenin in the stromal cells of phyllodes tumors and in the most abundant component of metaplastic carcinomas. Data analysis was performed with the results obtained with each antibody and also with combined results (ie, for nuclear/cytoplasmic staining, positivity was defined as positive for at least one antibody; for membrane staining, the highest Allred score was taken into account).

[0108] Statistical Analysis.

[0109] Values are the mean±S.E. of three independent experiments each carried out in duplicate. Statistical analysis was carried out using a Student's t-test for unpaired variables. * and ** in the figures refer to statistical probabilities (P) of <0.001 and <0.0001, respectively, compared with control cells that received solvent vehicle alone. Tissues from patients were analyzed for significance and pairing with Wilcoxon signed rank tests. In the figures, *, ** and *** refer to P<0.05, P<0.01 and P<0.001, respectively, compared with controls (vehicle) unless otherwise specified. Prism software was used for all the analyses.

[0110] Results

[0111] hGSTA1 Catalyzed DDA Biosynthesis

[0112] Commercially available hGSTA1 was found to be functionally active and catalyzed the conjugation of the xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) with GSH to give S-(2,4-dinitrophenyl)-glutathione (CDNB-SG) with K.sub.m.sup.CDNB=32.2 μM, V.sub.m.sup.CDNB=274 μmol.Math.min.sup.−1.Math.mg prot for CDNB and K.sub.m.sup.GSH=35.1 μM, V.sub.m.sup.GSH=8.12 μmol.Math.min.sup.−1.Math.mg prot for GSH (FIG. 2a-b and table 1). 5,6-EC and HA were not inhibitors of CDNB-SG biosynthesis (FIG. 2c). We therefore tested whether hGSTA1 could catalyze the formation of CDO-SG from [.sup.14C]-5,6-EC and GSH as reported for rat GST B.sup.14,15. We showed that hGSTA1 catalyzed the biosynthesis of CDO-SG with a K.sub.m.sup.5,6EC=0.44 μM and K.sub.m.sup.GSH=47 μM, while no reaction occurred in the absence of the enzyme establishing its obligatory requirement for CDO-SG biosynthesis (FIG. 2d-f, and table 1). We found that hGSTA1 had a 73-fold higher affinity for 5,6-EC than for CDNB while the K.sub.m.sup.GSH was in the same range for both catalytic activities. The maximum velocities were V.sub.m.sup.5,6-EC=1.33 pmol.Math.min.sup.−1.Math.mg prot V.sub.m.sup.GSH=1.50 pmol.Math.min.sup.−1.Math.mg prot (Table 1) showing a 24.10.sup.6 fold weaker capacity to produce CDO-SG than to produce CDNB-SG. CDNB did not inhibit CDO-SG biosynthesis while HA was a potent inhibitor of the reaction (FIG. 2g) suggesting that HA was a possible substrate of hGSTA1. We next found that ring B oxysterols such as 5,6β-EC, CT and OCDO were potent inhibitors of CDO-SG biosynthesis while side chain oxysterols were not inhibitors up to 10 μM (FIG. 2h).

[0113] We next showed that hGSTA1 catalyzed the biosynthesis of DDA from [.sup.14C]-5,6-EC and HA with a K.sub.m.sup.5,6EC=0.28 μM and K.sub.m.sup.HA=0.35 μM and maximum velocities: V.sub.m.sup.5,6EC=0.81 pmol.Math.min.sup.−1.Math.mg prot and V.sub.m.sup.HA=0.66 pmol.Math.min.sup.−1.Math.mg prot (FIG. 3a-b, Table 1). No reaction occurred in the absence of enzyme as opposed to what was found for xenobiotic conjugation that occurred spontaneously. While hGSTA1 is known to facilitate or to accelerate the conjugation reaction of the CDNB with GSH, we showed here that DDAS is absolutely required for DDA biosynthesis. The production of DDA by hGSTA1 was confirmed by mass spectrometry giving a MS1 parent peak with a mass of 514 that gave a fragment of 496 in MS2 (FIG. 3c), which is characteristic of DDA.sup.10. hGSTA1 had a comparable affinity for 5,6α-EC and HA, in the range of their reported endogenous concentrations ([5,6-EC]=0.5 μM.sup.6, [HA]=12 μM.sup.17). Next we showed that GSH inhibited the biosynthesis of DDA dose-dependently giving an IC.sub.50 of 52 μM while CDNB did not inhibit this reaction and that ring B oxysterols but not side chain oxysterols were inhibitors of DDAS (FIG. 3d). 5,6β-EC was found to be the most potent inhibitor of the oxysterol series we tested (FIG. 3e). To determine whether hGSTA1 catalyzed DDAS in cells, a plasmid encoding hGSTA1 was expressed in HEK293 cells. The expression of the enzyme was confirmed by Western blotting (FIG. 4a) and immunocytochemistry showed that the enzyme was mainly cytoplasmic and nuclear (FIG. 4c). We found that hGSTA1-transfected cells glutathionylated CDNB (FIG. 4A) and, importantly, showed DDAS activity, establishing that hGSTA1 catalyzed DDA biosynthesis in intact cells (FIG. 4d). Altogether, these data established that hGSTA1 catalyzed DDA biosynthesis.

[0114] DDAS is Selectively Expressed in the Epithelial Cells of Lactating Duct and Lobular Terminal Units.

[0115] We previously found DDA in whole extracts of normal breast.sup.10 but the nature of the cells producing DDA remained to be established. We then analysed DDAS immuno-histochemically in normal breast tissues from patients. Fifty normal breast tissue samples were found to be 97.9% positive for DDAS, which was selectively expressed in the epithelial cells of lactating ducts and lobular terminal units from all samples. DDAS was not found in adipocytes, endothelial cells from vessels, fibroblasts or myo-epithelial cells. The cellular localization of DDAS in epithelial cells was mainly cytoplasmic (FIG. 5a) and in 4% of the samples was also found in the nuclei. These data established that DDAS is selectively expressed in the epithelial cells of lactating ducts and lobular terminal units thus identifying the source of DDA production in the breast.

[0116] DDAS is not Expressed in Breast Cancer (BC) Cells and Tumors.

[0117] We previously showed that BC cell lines did not produce DDA.sup.10 and consistently we found that these cells do not express DDAS (supplementary Table 1). We next analyzed 50 tumors from patients with BC by IHC and showed that DDAS expression was considerably decreased in the tumors of patients (FIG. 5b) compared to normal matched tissues. Analysis of the DDA levels in tumors showed a mean 8-fold decrease in DDA from 425±288 to 49.9±10.7 ng/mg tissue (FIG. 3c), importantly, the measured decreased in DDA between the normal tissue and the matched tumor tissues was paired (Wilcoxon test, p<0.005) (FIG. 3c). These data established that DDAS was not expressed in BC cells and considerably decreased in BC tumors and this decrease paralleled the decrease in DDA we measured.

[0118] Discussion

[0119] DDA is a steroidal alkaloid recently discovered in mammals as the product of an enzyme-catalyzed conjugation of 5,6α-EC with the α-amino group of HA.sup.10. The discovery of DDA was important because DDA posesses TS properties, which has never been reported for a cholesterol metabolite. Moreover, DDA is the first mammalian steroidal alkaloid found to date′° defining a new class of steroids in mammals and a new class of LXR endogenous ligands with sub-μM affinity.sup.8,9.

[0120] DDA was not found in BC cell lines and found in lower amounts in BC biopsies establishing a deregulation of DDA metabolism in BC. The identification of the DDA synthase (DDAS) was thus a crucial challenge to understand this deregulation.

[0121] We hypothesized that hGSTA1 was a possible candidate for DDAS activity, because its rat orthologue GST-B was reported to catalyze a similar reaction using 5,6α-EC and GSH as a nucleophile. Two different groups showed that GST-B catalyzed the stereoselective biosynhesis 5α-dihydroxycholestan-6β-yl-S-glutathione (CDO-SG), which displays the same C5αOH-C6β-substituted stereochemistry than DDA.sup.14,15 We report in the present study its human ortholog (hGSTA1) catalyzed also CDO-SG biosynthesis, establishing that human enzyme catalyzed the same reaction than the rat enzyme and used 5,6α-EC and GSH as endogenous substrates. The K.sub.m obtained for 5,6α-EC transformation was 0.44 μM which is consistent with an endogenous concentration of 0.5 μM in 5,6α-EC measured in mammalian tissues.sup.6. Replacing of GSH by HA, we found that hGSTA1 catalyzed the biosynthesis of DDA from 5,6α-EC with a similar Km for 5,6α-EC and a 135 fold weaker K.sub.m for HA than for GSH, establishing a preference of hGSTA1 for HA over GSH as substrate. Again, the K.sub.m obtained for HA transformation was 0.35 μM which is consistent with the endogenous concentration found for HA in human breast.sup.17. We found that the xenosubstrate of hGSTA1 CDNB did not inhibit CDO-SG and DDA biosynthesis, strongly suggesting that the binding sites for CDNB and 5,6α-EC were different. The physiological function previously attributed to hGSTA1 was xenobiotic detoxification, steroidogenesis.sup.18 and elimination of 5,6α-EC by conjugation with GSH.sup.14. GSTA1 has a catalytic efficiency 60 fold higher to transform 5,6α-EC compared to CDNB, and 60 fold higher to transform HA compared to GSH. We next established that the expression of GSTA1 in cells induced DDAS activity. Altogether these data showed that GSTA1 catalyzed DDAS activity, which maybe the primary function of GSTA1.

[0122] The fact that 5,6β-EC, the diasteroisomer of 5.6α-EC, was a potent inhibitor of DDAS is interesting, because it can potentially impact on DDA biosynthesis. 5,6β-EC is the major product of ROS activated lipoperoxidation.sup.6,19 suggesting that inflammation may be detrimental to the production of the tumor suppressor DDA and thus can contribute to carcinogenicity. In the late seventies, Watabe's group reported the existence of a cholesterol 5,6α epoxidase.sup.20, which supports the existence of a metabolic pathways contributing to DDA biosynthesis from cholesterol. They showed that a yet unidentified cytochrome P450 catalyzed the stereoselective biosynthesis 5.6α-EC.sup.20. Further studies are now required to identify this cytochrome p450.

[0123] Normal breast is a heterogeneous tissue containing different types of cells. HIC studies revealed that DDAS is selectively expressed in the cytoplasm of epithelial cells from lactiferous ducts and lobules giving the cellular origin of DDA production in the breast. The absence of DDAS in BC tumors explains the decrease in the DDA content found in tumors. The absence of DDA in BC tissue due to a lack of DDAS expression is consistent with the increased amount of HA.sup.17,21 and 5,6-EC.sup.22 measured in ductal breast cancer and breast epithelial hyperplasic tissues respectively. Statins are broadly used anti-cholesterol drugs prescribed for the prevention of cardiovascular diseases.sup.23, but their benefit in the chemoprevention of breast cancer is still a matter of debate.sup.24. Since long term statin use was recently reported to increase the risk of both invasive ductal carcinoma and invasive lobular carcinoma.sup.25, in which we found that DDAS expression was reduced, it raises the important question of the impact of statins on the DDA content in the breast and deserves further investigations.

[0124] We found that DDAS expression reflected DDA levels in breast cancer tissues. The accuracy of the quantification of DDA levels by LC/MS in BC tumors is limited by possible contamination with normal tissues; in addition this method is laborious and expensive. The identification of DDAS enabled the measurement of its expression in BC by immunohistochemistry. This approach will be cheaper and more precise than the quantification of DDA and useful for histopathology platforms.

[0125] DDA has been detected in different mammalian organs such as lung, brain and spleen but found to be absent from their corresponding tumor cell lines.sup.10. Further studies will be required to determine if this correlates with the loss of DDAS activity.

[0126] DDA was active on tumor cell lines of different origins that do not contain measurable amounts of DDA.sup.8-11 and that do not or only weakly express GSTA1.sup.26. Thus the absence of GSTA1 and the expression of LXRβ, the molecular target of DDA, may be predictive of a response to DDA.

[0127] In summary, we report in the present study the first identification and molecular identification of DDAS: the enzyme that catalyzes the biosynthesis of the tumor suppressor dendrogenin A.

TABLE-US-00002 TABLE 1 Enzymatic parameters of hGSTA1 for exogenous and endogenous substrates. Substrates Product K.sub.m V.sub.m K.sub.m.sup.CDNB K.sub.m.sup.GSH V.sub.m.sup.CDNB V.sub.m.sup.GSH (μM) (μM) (μmol .Math. min.sup.−1 .Math. mg.sup.−1) (μmol .Math. min.sup.−1 .Math. mg.sup.−1) CDNB GSH CDNB-S-G 32.2 35.1 274 8.12 K.sub.m.sup.5,6-EC K.sub.m.sup.GSH V.sub.m.sup.5,6-EC V.sub.m.sup.GSH (μM) (μM) (pmol .Math. min.sup.−1 .Math. mg.pr) (pmol .Math. min.sup.−1 .Math. mg .Math. pr) 5,6α-EC GSH CDO-SG 0.44 47 1.33 1.50 5,6β-EC GSH — — — — — K.sub.m.sup.5,6-EC K.sub.m.sup.His V.sub.m.sup.5,6-EC V.sub.m.sup.His (μM) (μM) (pmol .Math. min.sup.−1 .Math. mg.pr) (pmol .Math. min.sup.−1 .Math. mg .Math. pr) 5,6α-EC His DDA 0.28 0.35 0.81 0.66 5,6β-EC His — — — — — CDNB: 1,chloro-2,4-dinitrobenzene; GSH: glutathione; CDNB-S-G: S-(2,4-dinitrophenyl)-glutathione 5,6α-EC: 5,6α-epoxy-cholesterol; 5,6βEC: 5,6β-epoxy-cholesterol; His: histamine; CDO-SG: 5α-dihydroxycholestan-6β-yl-S-glutathione; dendrogenin A (DDA): 5α-hydroxy-6β-[2-(1H-imidazol-4-yl) ethylamin] cholestan-3β-ol; 5α-hydroxy-6β-[S-glutathione] cholestan-3β-ol,3β,

TABLE-US-00003 SUPPLEMENTARY TABLE 1 Quantification of GSTA1 in normal and tumor cells. GSTA1 Cell line Cell type expression activity MCF-7 Breast carcinoma, human — N.M. TS/A Breast carcinoma, mouse — N.M. SKBr3 Breast carcinoma, human — N.M. ZR75-1 Breast carcinoma, human — N.M. BT474 Breast carcinoma, human — N.M. HCC 1937 Breast carcinoma, human — N.M. MDA-MB-231 Breast carcinoma, human — N.M. *Mean ± S.E.M. values (see Methods) N.M., not measurable.

REFERENCES

[0128] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0129] 1. Wu, Q. et al. 27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth. Cell Rep 5, 637-45 (2013). [0130] 2. Nelson, E. R. et al. 27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology. Science 342, 1094-8 (2013). [0131] 3. Yue, S. et al. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. Cell Metab 19, 393-406 (2014). [0132] 4. de Medina, P. et al. Auraptene is an inhibitor of cholesterol esterification and a modulator of estrogen receptors. Mol Pharmacol 78, 827-36 (2010). [0133] 5. Paillasse, M. R. et al. Signaling through cholesterol esterification: a new pathway for the cholecystokinin 2 receptor involved in cell growth and invasion. J Lipid Res 50, 2203-11 (2009). [0134] 6. Poirot, M. & Silvente-Poirot, S. Cholesterol-5,6-epoxides: chemistry, biochemistry, metabolic fate and cancer. Biochimie 95, 622-31 (2013). [0135] 7. Silvente-Poirot, S. & Poirot, M. Cholesterol epoxide hydrolase and cancer. Curr Opin Pharmacol 12, 696-703 (2012). [0136] 8. Segala, G. et al. Dendrogenin A links Liver X Receptor to autophagic cell death in melanoma. submitted (2014). [0137] 9. Segala, G. et al. Abstract 1662: Dendrogenin A is a newly identified mammalian steroidal alkaloid that induced autophagic cell death in melanoma cells through an LXRbeta-, Nur77- and Nor1-dependent way. Cancer Research 73, 1662 (2013). [0138] 10. de Medina, P. et al. Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties. Nat Commun 4, 1840 (2013). [0139] 11. de Medina, P., Paillasse, M. R., Payre, B., Silvente-Poirot, S. & Poirot, M. Synthesis of new alkylaminooxysterols with potent cell differentiating activities: identification of leads for the treatment of cancer and neurodegenerative diseases. J Med Chem 52, 7765-77 (2009). [0140] 12. Khalifa, S. A. et al. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Biochem Biophys Res Commun 446, 681-6 (2014). [0141] 13. Paillasse, M. R. et al. Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles. J Lipid Res 53, 718-25 (2012). [0142] 14. Watabe, T., Sawahata, T. & Horie, J. Evidence for the formation of a steroid S-glutathione conjugate from an epoxysteroid precursor. Biochem Biophys Res Commun 87, 469-75 (1979). [0143] 15. Meyer, D. J. & Ketterer, B. 5 alpha,6 alpha-Epoxy-cholestan-3 beta-ol (cholesterol alpha-oxide): A specific substrate for rat liver glutathione transferase B. FEBS Lett 150, 499-502 (1982). [0144] 16. Mannervik, B., Board, P. G., Hayes, J. D., Listowsky, I. & Pearson, W. R. Nomenclature for mammalian soluble glutathione transferases. Methods Enzymol 401, 1-8 (2005). [0145] 17. Reynolds, J. L. et al. Histamine in human breast cancer. Br J Surg 85, 538-41 (1998). [0146] 18. Mannervik, B. Five decades with glutathione and the GSTome. J Biol Chem 287, 6072-83 (2012). [0147] 19. Segala, G. et al. 5,6-Epoxy-cholesterols contribute to the anticancer pharmacology of tamoxifen in breast cancer cells. Biochem Pharmacol 86, 175-89 (2013). [0148] 20. Watabe, T. & Sawahata, T. Biotransformation of cholesterol to cholestane-3beta,5alpha,6beta-triol via cholesterol alpha-epoxide (5 alpha,6alpha-epoxycholestan-3beta-ol) in bovine adrenal cortex. J Biol Chem 254, 3854-60 (1979). [0149] 21. von Mach-Szczypinski, J., Stanosz, S., Sieja, K. & Stanosz, M. Metabolism of histamine in tissues of primary ductal breast cancer. Metabolism 58, 867-70 (2009). [0150] 22. Wrensch, M. R. et al. Breast fluid cholesterol and cholesterol beta-epoxide concentrations in women with benign breast disease. Cancer Res 49, 2168-74 (1989). [0151] 23. LaRosa, J. C., He, J. & Vupputuri, S. Effect of statins on risk of coronary disease—A meta-analysis of randomized controlled trials. Jama-Journal of the American Medical Association 282, 2340-2346 (1999). [0152] 24. Silvente-Poirot, S. & Poirot, M. Cancer. Cholesterol and cancer, in the balance. Science 343, 1445-6 (2014). [0153] 25. McDougall, J. A. et al. Long-term statin use and risk of ductal and lobular breast cancer among women 55 to 74 years of age. Cancer Epidemiol Biomarkers Prev 22, 1529-37 (2013). [0154] 26. Tew, K. D. et al. Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program. Mol Pharmacol 50, 149-59 (1996). [0155] 27. Voisin, M., Silvente-Poirot, S. & Poirot, M. One step synthesis of 6-oxo-cholestan-3beta,5alpha-diol. Biochem Biophys Res Commun 446, 782-5 (2014). [0156] 28. Habig, W. H. et al. The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc Natl Acad Sci USA 71, 3879-82 (1974). [0157] 29. Harvey, J. M., Clark, G. M., Osborne, C. K. & Allred, D. C. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 17, 1474-81 (1999).