METHODS OF DIAGNOSING AND TREATING CANCER

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 11βHSD1 and/or 11βHSD2 in a 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 11βHSD1 is lower than its predetermined reference value or when the expression level of 11βHSD2 is higher than its predetermined reference value.

Claims

1. A method of diagnosing cancer in a subject comprising the steps of i) determining the expression level of 11βHSD1 and/or 11 βHSD2 in a 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 11βHSD1 is lower than its predetermined reference value or when the expression level of 11βHSD2 is higher 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 the expression level of 11βHSD1 and/or 11βHSD2 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 will have a long survival time when the expression level of 11βHSD1 is higher than its predetermined reference value or concluding that the subject will have a short survival time when the expression level of 11βHSD2 is lower than its predetermined reference value.

3. A method for determining whether a subject suffering from a cancer will achieve a response with tamoxifen or dendrogenin A comprising the steps of i) determining the expression level of 11βHSD1 and/or 11βHSD2 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 will achieve a response with tamoxifen or dendrogenin A when the expression level of 11βHSD1 is higher than its predetermined reference value or when the expression level of 11βHSD2 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 3 wherein when it is determined that the subject will achieve a response with tamoxifen or dendrogenin A, the method includes a step of administering one or both of tamoxifen and dendrogenin A to the subject.

7. The method of claim 1 wherein when it is determined that the subject suffers from cancer, the method includes a step of administering to the subject at least one of a 11β-HSD2 inhibitor, an inhibitor of 11β-HSD2 expression and a nucleic acid encoding 11β-HSD1.

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.

10. The method of claim 8 wherein the cancer is breast cancer.

11. The method of claim 9 wherein the cancer is breast cancer.

Description

FIGURES

[0112] FIG. 1. OCDO is produced and secreted from MCF7 tumor cells incubated with EC or CT. Representative TLC autoradiograms showing time dependent production of OCDO in MCF7 cells treated with .sup.14C-αEC (A,B) or .sup.14C-βEC (C,D) or .sup.14C-CT (E, F) and quantitative analyses of the metabolites produced in each condition from three separate experiments (±s.e.m). The metabolites extracted from the cells (left panels) or from the medium (right panels) were analyzed by TLC analysis and the region corresponding to radioactive metabolites of interest were recovered and counted using a β-counter.

[0113] FIG. 2. OCDO is a tumor promoter in vitro and in vivo and its inhibition contributes to the anti-tumor effects of Tam and DDA. (A, B) Histograms representing the effect of OCDO or 17β-estrogen (E2) on MCF7 (A) and TS/A (B) cell proliferation after 24 h treatment using a colorimetric immunoassay measuring BrDU incorporation in DNA (C, D) Histogram representing the effect of OCDO on MCF7 (C) and TS/A (D) cell invasion. Data are the mean of three separate experiments (±s.e.m), *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). (E, F) Mice were implanted s.c. with MCF7 (E) or TS/A (F) cells and animals (8 per group) were treated s.c. every day starting on the day of implantation with either the solvent vehicle or OCDO (16 μg/kg for MCF7 or 50 μg/kg for TS/A). Animals were monitored for tumour growth twice a week. The data are representative of three independent experiments. The mean tumor volume±s.e.m is shown, *P<0.05, **P<0.01, ***P<0.001 (analysis of variance (ANOVA), Dunnett's post test). (G) Mean (±s.e.m) of Ki67 positive cell number determined from IHC staining of MCF7 tumor sections from (E), n=8, *P<0.05 (Student's t-test) using HistoQuant, Pannoramic Viewer (3DHistech). (H) Representative Ki67 staining of TS/A tumor sections from (F) showing increased staining in OCDO-treated tumor compared with control-treated tumor. (I, J, K). Murine E0771(I), or human MDA-MB231 (J) or MDA-MB468 (K) cells implanted s.c. into mice (8 per group) and animals were treated s.c. with either the solvent vehicle or OCDO (16 μg/kg for MDA-MB-231 and MDA-MB-468 or 50 μg/kg for E0771). The data are representative of two independent experiments. Statistical analysis was performed as in E and F. (L) Mice were implanted s.c. with TS/A cells and animals (8 per group) were treated s.c. every day with either the solvent vehicle, OCDO (50 μg/kg), Tam (56 mg/kg), DDA (20 mg/kg) or the combination of Tam (56 mg/kg) +OCDO (50 μg/kg) or DDA (20 mg/kg)+OCDO (50 μg/kg). The data are representative of three independent experiments. Statistical analysis was performed as in E and F.

[0114] FIG. 3. We hypothesized that 11β-HSD type 2 (11HSD2) which catalyzes the dehydrogenation of cortisol into cortisone is the enzyme that produces OCDO from CT, and 11β-HSD type 1 (11HSD1) which catalyzes the hydrogenation of cortisone into cortisol is the enzyme that realizes the reverse reaction (CT production from OCDO). We also hypothesized that H6PDH which produces the cofactor NADPH necessary for the 11βHSD1 reductase activity and the production of cortisol is also necessary for the production of CT.

[0115] FIG. 4. 11βHSD2 and 11βHSD1 are the enzymes producing OCDO and CT respectively. HEK-273 cells (5×10.sup.6 cells) were transfected by electroporation with the plasmids coding either the enzymes 11βHSD2 (HSD2), 11βHSD1 (HSD1), H6PDH, the control empty vector (mock) or were co-transfected with a plasmid coding 11βHSD1 or H6PDH, and analyzed as followed: (A) the expression of 11βHSD2 was confirmed by immunoblotting using a specific antibody against 11βHSD2 and normalized with actin; (B, C) the production of cortisone (B) or OCDO (C) was determined by incubating the mock or the HSD2-transfected cells with .sup.3H-cortisol or .sup.14C-CT for 8 h at 37° C. respectively. Lipids extracted from the cell and the media were analyzed by TLC analysis and the region corresponding to radioactive metabolites of interest were recovered and counted using a β-counter; (D) the expression of 11βHSD1 and H6PDH was confirmed by immunoblotting using a specific antibody against 11βHSD1 or H6PDH and normalized with actin; (E, F) HEK-273 cells expressing 11βHSD1, H6PDH, both enzymes or the control empty vector (mock) were incubated either with .sup.3H-cortisone (E) or .sup.14C-OCDO (F) for 72 h and the radioactive metabolites of interest were analyzed as in B and C. The results in B, C, E, F are the mean (±s.e.m) of three experiments, **P<0.01, ***P<0.001 (Student's t-test).

[0116] FIG. 5. Ectopic expression of 11βHSD1 in MCF7 inhibits cell proliferation and OCDO reverses this effect. MCF7 cells were transfected by electroporation with a plasmid coding either the enzymes 11βHSD1 (HSD1) or the control empty vector (mock) and analyzed as followed: (A) the expression of 11βHSD1 was confirmed by immunoblotting using specific antibody against 11βHSD1 and normalized with actin; (B) The production of CT was determined by incubating the mock or the HSD1-transfected cells with .sup.14C-OCDO for 72 h at 37° C. The radioactive metabolites of interest were analyzed as in the legend of FIG. 4B. (C) The proliferation of the mock- or the HSD1-transfected MCF7 cells treated or not with 5 μM OCDO for 24 h were analyzed as in FIG. 2A. The results in B and C are the mean (±s.e.m) of three to five experiments, **P<0.01, ***P<0.001 (Student's t-test), ns: non specific.

[0117] FIG. 6. Knock-down of 11βHSD2 decreases OCDO production, cell proliferation, invasion and survival in MCF7 cells. MCF7 cells (5×10.sup.6 cells) were transfected by electroporation with a plasmid expressing a short-hairpin RNA (shRNA) against 11βHSD2 or a control shRNA, two clones (A and B) were selected and analyzed as followed: (A) the knock down of 11βHSD2 expression in MCF7 was confirmed by immunoblotting as described in FIG. 4 or by qPCR; (B, C) The quantification of cortisone (B) or OCDO (C) produced by the sh-Control (shC A and B) or the shHSD2-transfected cells (shHSD2 A and B) were measured as described in FIG. 4. (D, E) The proliferation of sh-C or shHSD2 was measured using quantification of DNA BrDU incorporation (D) as described in FIG. 2A or by cell counting (E). (F) the formation of colony by sh-C or shHSD2 MCF7 cells was quantified after cell fixing and crystal violet staining. The results are the mean (±s.e.m) of three to five experiments,*P<0.05, **P<0.01 (Student's t-test).

[0118] FIG. 7. Knock-down of 11βHSD2 decreases cell proliferation, invasion and survival in MCF7 cells as well as tumor growth and OCDO reverses these effects. shC or shHSD2 MCF7 cells were analyzed as followed: (A) The proliferation of sh-C or shHSD2 cells treated or not with OCDO 5 μM 24 h was measured using quantification of DNA BrDU incorporation as described in FIG. 2A. (B) The proliferation of sh-C or shHSD2 cells treated or not with increasing concentration of cortisone for 24 h was measured as in (A). (C) The invasiveness of sh-C or shHSD2 cells treated or not with OCDO 5 μM for 72 h was assayed using matrigel-coated filters. (D) the formation of colony by sh-C or shHSD2 cells treated or not with OCDO 1 μM was quantified as described in FIG. 6F. (E) Mice were implanted s.c. with shC or shHSD2 MCF7 cells (5×10.sup.6 cells) and animals (8 per group) were treated s.c. every day starting on the day of implantation with either the solvent vehicle or OCDO (16 μg/kg). Animals were monitored for tumor growth twice a week. The data are representative of three independent experiments. The mean tumor volume±s.e.m is shown, **P<0.01, ***P<0.001 (analysis of variance (ANOVA), Dunnett's post test). (F) Mean (±s.e.m) of Ki67 positive cell number determined from IHC staining of shC or shHSD2 MCF7 tumor sections from (E), n=8, *P<0.05 (Student's t-test) using HistoQuant, Pannoramic Viewer (3DHistech).

EXAMPLE

[0119] Material & Methods

[0120] Materials

[0121] Chemicals [3H]cortisol, [3H]cortisone and [14C]cholesterol were purchased from Perkin Elmer. The radiochemical purity of the compounds was verified by thin-layer chromatography (TLC) and was greater than 98%. Autoradiography experiments were done with GE Healthcare or Kodak phosphor screens. Fulvestrant (ICI 182780) used in vivo was a generous gift from the Institute Claudius Regaud (France). The NEON Transfection system was from Invitrogen, the BrdU cell proliferation elisa was from Roche Diagnosic, all plasmids were from Origene (HSD1 sc109325, HSD2 sc122552, H6PDH sc117481, DHCR7 sc110871, EBP or D8D7I sc116006. Other compounds and chemicals were from Sigma-Aldrich (St. Louis, Mo.), and solvents from VW. The antibodies were from the following company: 11βHSD2 (Santa cruz, H-145), 11βHSD1 (Abcam, EPR9407(2)), H6PDH (Santa Cruz, C-10), EBP (Abgent, RB23728) and DHCR7 (Abcam, ab170388).

[0122] Animals

[0123] Female C57BL/6 Charles River Laboratories (France), Balb/c and NMRI Nude mice (6 weeks old) Janvier (France) were maintained in specific pathogen-free conditions and were included in protocols only following 2 weeks quarantine. All of the animal procedures for the care and use of laboratory animals were conducted according to the ethical guidelines of our institution and followed the general regulations governing animal experimentation.

[0124] Cell Culture

[0125] MCF-7, SKBR3, MDA-MB-231, MDA-MB-468, HEK293T and E0771 cells were from the American Type Culture Collection (ATCC) and cultured until passage 30. TS/A cells were provided by Dr P. L. Lollini (Bologna, Italy) and MELN cells were a generous gift of Dr. G. Freiss (Montpellier, France). MCF-7 cells were grown in RPMI 1640 medium (Lonza) supplemented with 5% fetal bovine serum (FBS) (Dutcher), SKBR3 cells in Mc Coy's medium (invitrogen) 10% SVF, TSA and MDA-MB-468 cells in RPMI 10%, E0771 in RPMI 10% SVF HEPES 10 mM and HEK 293T and MDA-MB-231 in DMEM (Lonza) 10% SVF. All the cells lines were cultured in 1% penicillin and streptomycin (50 U/ml) (invitrogen) in a humidified atmosphere with 5% CO2 at 37° C.

[0126] Cell Transfection

[0127] MCF7 or HEK293T cells (5×10.sup.6 cells) were transfected with 5 μg of the indicated plasmid using the NEON Transfection System and according to the manufacturer's recommendations. Stable clones were established after MCF7 cells were separately transfected with four different shRNA plasmids targeting 11βHSD2 (11βHSD2 shRNA) or with a control shRNA (11βHSD2 SureSilencing ShRNA plasmid, Qiagen). Cells were then cultured for 3 weeks in presence of 0.5 mg/ml puromycin (Life Technologies). Several clones were analyzed by immunoblot analysis and real time RT-qPCR for the knock down of the expression of the protein of interest.

[0128] Analysis of Tumours

[0129] Exponentially growing MCF7, ShMCF7, E0771, MDA-MB231, MDA-MB468 and TS/A cells were collected, washed twice in PBS and resuspended in PBS. TS/A and E9771 tumours were prepared by subcutaneous transplantation of 35×10.sup.3 cells or 3×10.sup.5 cells respectively in 100 μl PBS into the flank of BALB/c or C57B16 mice. For other tumors, 5 to 10×10.sup.6 cells in 200 μl PBS/matrigel (1/1) were injected into the flank of NMRI nude mice. Animals were treated as indicated in the legends. Animals were examined daily, and body weights were measured twice per week. In all the experiments, the tumor volume was determined by direct measurement with a caliper and was calculated using the formula (width.sup.2×length)/2. Tumors were either frozen in liquid nitrogen or fixed in 10% neutral-buffered formalin and embedded in paraffin for immunohistochemical analysis. Paraffin sections were stained with haematoxylin and eosin for histomorphological analyses. Immunohistochemical staining was done on paraffin-embedded tissue sections, using a specific Ki67 antibody (Dako).

[0130] Chemical Synthesis

[0131] 5,6α-EC, 5,6β-EC were synthesized as reported.sup.10, 20. CT and OCDO were synthesized as reported.sup.21.

[0132] Metabolic Activity Assay in Intact Cells

[0133] Cells were plated on six-well plates (1×10.sup.5 cells/well) in the appropriate complete medium. One day after seeding, cells were treated with either .sup.14C-CT (1 μM, 10 μCi/μmol-1 μl/dish) or .sup.14C-OCDO (1 μM, 10 μCi/μmol-1 μL/dish) or .sup.3H-cortisol (200 nM, 89 Ci/mmol-1 μL/dish) or .sup.3H-cortisone (200 nM, 89 Ci/mmol-1 μL/dish) or .sup.14C-αEC or .sup.14C-βEC (600 nM, 20 μCi/μmol-1 μl/dish) at the indicated times. After incubation, cells were washed, scraped, and neutral lipids were extracted with chloroform-methanol as described in.sup.11 and then separated by TLC using Ethyl Acetate as eluant for .sup.14C-CT and .sup.14C-OCDO or chloroform-methanol (87:13, v/v) for .sup.3H-cortisol or .sup.3H-cortisone adapted from.sup.22. The radioactive lipids were detected by autoradiography (KODAK, BioMax MS Film). The positions of the metabolite of interest were determined using purified .sup.14C or .sup.3H standards and the region corresponding of CT, OCDO, cortisol or cortisone was scraped and quantified using a beta counter.

[0134] Cell Proliferation Assay

[0135] Cells, MCF7 (4×10.sup.3), MCF7-sh11bHSD2 (4×10.sup.3), SKBR3 (2.5×10.sup.3), TSA (2.5×10.sup.3), MDA-MB231 (5×10.sup.3) and MDA-MB468 (5×10.sup.3), were seeded in 96-well plates and cultured in complete medium for 24 h. Cells were then treated for 24 h with either the indicated concentration of OCDO, cortisol or cortisone or with 1 μM RU486 or ICI182780 added 30 mn before other treatment. At the end of this time, cells were incubated with BrDU for an additional 8 h and then evaluated for proliferation using the ELISA kit, Roche Diagnostic, as indicated by the manufacturer.

[0136] Cell Invasion Assay

[0137] Invasion assays were carried out using Bio-Coat migration chambers (BD Falcon) with 8 μm filters previously coated with matrigel. Cells, MCF7-sh11βHSD2 or MCF7-shC (1×10.sup.3), were plated in the upper chambers in SVF free medium and the chemoattractant (10% FBS) was added in the lower chambers. After incubating cells in absence or presence of OCDO (5 μM) for 72 h at 37° C. in 5% CO2 incubator, cells that had migrated through the filters were fixed (3.7% PFA) and stained (aqueous crystal violet 0.05%). The entire membranes were mounted on glass slides, and were counted under a microscope. All experiments were performed in duplicate.

[0138] Clonogenic Assay

[0139] Cells, MCF7-sh11bHSD2 (5×10.sup.3), MCF7-shC (5×10.sup.3) or TSA (3×10.sup.3) were seeded in duplicate in 35 cm.sup.2 diameter dish. Twenty four hours after, cells were treated either with OCDO 1 μM or solvent vehicle and the treatment was repeated every 3 days. At day 10, colonies were fixed with 3.7% PFA, stained with an aqueous crystal violet solution (0.05%) and the number of colonies was counted.

[0140] Luciferase Assay

[0141] MELN cells expressing luciferase in an estrogen-dependent manner.sup.23 or MCF7 co-transfected as described above with the plasmid coding the human glucocorticoid receptor hGR and a plasmid GREluc were routinely grown in DMEM or RPMI 1640 respectively supplemented with 5% FBS (Dutcher). Experiments were carried out as described previously .sup.23. Briefly, 50×10.sup.3 cells per well were seeded in 12-well plates and grown for 4 days in phenol red-free medium, containing 5% dextran-coated charcoal-treated FCS. Then, cells were treated for 16 hours with the indicated compounds. At the end of the treatment, cells were washed with PBS and lysed in 250 μL of lysis buffer (Promega). Luciferase activity was measured using the luciferase assay reagent (Promega), according to the manufacturer's instructions. Protein concentrations were measured using the Bradford technique to normalize the luciferase activity data. For each condition, the mean luciferase activity was calculated from the data of three independent wells.

[0142] Immunoblotting

[0143] Cells treated or not as indicated were washed with ice-cold PBS, scraped, and centrifuged at 1200 rpm for 5 min at 4° C. The pellets were resuspended in 100 μL of extraction buffer (50 mM Tris pH 7.4; 5 mM NaCl; 1% tritonX100; 10% glycerol) with 1% protease inhibitor cocktail (Sigma Aldrich), vortexed and centrifuged at 10,000×g for 10 min at 4° C. Whole cell extracts were fractionated by SDS PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer's protocols (Life Technologies). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 1% Tween 20) for 60 min, the membrane was incubated with antibodies against 11βHSD2 (1:1000), 11βHSD1 (1:500), H6PDH (1:500), EBP (1:500) and DHCR7 (1:200) or actin (1:10000, Merck Millipore, C4) at 4° C. overnight. Membranes were washed three times for 10 min and incubated with a 1:10000 dilution of horseradish peroxidase conjugated anti-mouse or anti-rabbit antibodies for 1 h. Blots were washed with TBST three times and developed with the ECL system (Amersham Biosciences) according to the manufacturer's protocols.

[0144] RNA Isolation and qPCR Analysis

[0145] Total RNA from cultured cells were isolated using TRIzol Reagent®(Invitrogen). RNA was quantified using nanodrop (thermofisher). Total RNA (1μg) was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. qRT-PCR was performed with an iCycler iQreal-time PCR detection system (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad) and the indicated primers The threshold cycle (Ct) values of genes of interest were normalized with the Ct values of Cyclophiline A1.

TABLE-US-00002 Primers: forward reverse cycloA1 GCA-TAC-GGG-TCC-TGG-CAT-CTT-GTC-C ATG-GTG-ATC-TTC-TTG-CTG-GTC-TTG-C (SEQ ID NO: 5) (SEQ ID NO: 6) 11βHSD1 GA-CAG-CGA-GGT-CAA-AAG-AAA (SEQ ID GTC-CTC-CCA-TGA-GCT-TTC-CTG (SEQ NO: 7) ID NO: 8) 11βHSD2 CCA-CCG-TAT-TGG-AGT-TGA-ACA (SED ID CGC-GGC-TAA-TGT-CTC-CTG-G (SEQ ID NO: 9) NO: 10) EBP CAC-AGG-GGT-CTT-AGT-CGT-GAC (SEQ ID CCA-GGT-GAA-TGA-ACC-CAC-ACA (SEQ (D8D7I) NO: 11) ID NO: 12) DHCR7 ACT-GGC-GAG-CGT-CAT-CTT-C (SEQ ID TCC-TCG-TTA-TAG-GTG-GAG-TCT-TG NO: 13) (SEQ ID NO: 14) H6PDH GCA-GAG-CAC-AAG-GAT-CAG-TTC (SEQ ID GGC-AGC-TAC-TGT-TGA-TGT-TGC (SEQ NO: 15) ID NO: 16)

[0146] Immunohistochemistry. All samples were collected with the approval of the Institutional Review Board of the Claudius Regaud Institute. Written informed consent was obtained before inclusion in this study. Patients' clinical characteristics and tumour pathological features were obtained from the medical reports and followed the standard procedures in our institution. Immunohistochemistry was performed on formalin-fixed, paraffin embedded sections of the initial tumor biopsies with the following antibodies: DHCR7 1:50, H6PDH 1:100, EBP 1:500, 11β-HSD1 1:50 and 11β-HSD2 1:50. Immunostaining was blindly analyzed by the pathologist (MLT).

[0147] Statistical analyses. Tumour growth curves in animals were analysed for significance by analysis of variance with Dunnett's multiple comparison tests. In other experiments, significant differences in the quantitative data between the control and the treated group were analysed using the Student's t-test for unpaired variables. 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.

[0148] Results

[0149] OCDO is a Metabolite of CT.

[0150] We studied the production of OCDO in breast tumors by incubating MCF7 tumor cells during increasing time with either [.sup.14C]α-EC, [.sup.14 C]β-EC or [.sup.14C]-CT. At the indicated time the cells and the media were collected and analyzed separately. As shown in the TLC autoradiograms of FIGS. 1a and 1c, α-EC and β-EC were converted to CT as a result of ChEH activity however, with prolonged incubation times, OCDO production was observed. The formation of OCDO continued when α-EC or β-EC was totally metabolized to CT at 72 h (FIGS. 1a and 1c), indicating that OCDO is formed from CT. Similar experiment performed with [.sup.14C]-CT confirmed that OCDO is a metabolite of CT (FIG. 1e).

[0151] OCDO Stimulates Tumor Cell Proliferation and Invasion.

[0152] We studied the effects of OCDO on breast tumor cell proliferation and invasion. As shown in FIGS. 2A and 2B, the growth rate of human MCF7 and mouse TS/A cells treated with OCDO for 24 h was increased in a concentration-dependent manner and reached respectively 1.3-fold and 1.7-fold the control. This increased in proliferation was in the same range than with 1 nM estradiol (E2). The invasiveness of MCF7 and TS/A cells treated with OCDO were also increased in a concentration-dependent manner and reached respectively 6-fold and 2.3-fold respectively compared with the control (FIGS. 2C and 2D).

[0153] OCDO Stimulates the Proliferation of Breast Tumors Implanted into Mice.

[0154] We then assayed whether OCDO stimulates the growth of mammary tumors implanted into mice. OCDO treatment significantly increased the growth of human MCF7 (FIG. 2E) and murine TS/A tumors grafted into immunodeficient or immunocompetent mice respectively compared with the control group (FIGS. 2E and 2F). Histological analysis of MCF7 or TS/A tumors indicated that the proliferative marker Ki67 was increased in OCDO-treated tumors compared with control-treated tumors in both tumor models (FIG. 2G and 2H). In addition, OCDO stimulates the growth of other tumor models expressing or not the estrogen receptor such as the mouse E0771 and the human MDA-MB231 and MDA-MB468 cells (FIGS. 2I, 2J and 2K respectively).

[0155] OCDO Reverses the Tumor Growth Inhibition Effect of ChEH Inhibitors in Mice.

[0156] We then assayed the anti-growth effect of Tam or DDA against TS/A tumors in the absence and presence of OCDO. As above, TS/A tumors implanted into immunocompetent mice were treated s.c. every day either with either the solvent vehicle (control), OCDO (50 μg/kg), Tam (56 mg/kg), DDA (20 mg/kg) or the combination of Tam (56 mg/kg)+OCDO (50 μg/kg) or DDA (20 mg/kg) +OCDO (50 μg/kg). As shown in FIG. 2L, after 13 days of treatment, OCDO enhanced TS/A tumor growth by 140% compared with that of the control group (p<0.01). Treatment with Tam or DDA alone significantly inhibited the growth of tumors by 31% (p<0.05) and 33% (p<0.01) respectively compared with the control group. When animals were treated with OCDO and Tam, or OCDO with DDA, the growth of tumors was not statistically different from that of the control group, indicating that the growth inhibitory action of Tam or DDA was reversed by OCDO. These data indicate that the inhibition of OCDO production contributes to the anti-tumor effects of both Tam and DDA.

[0157] Identification of the Enzymes that Regulate the Production of OCDO from CT.

[0158] Since the data we obtained argued for the existence of an enzyme distinct from ChEH that metabolizes CT into OCDO, we hypothesized that a hydroxysteroid dehydrogenases (HSD) would catalyze the dehydrogenation (or oxidation) of the alcohol function in position 6 of CT into a ketone in OCDO. Three main classes of HSD has been described (3β-, 17β- and 11β-hydroxy steroid dehydrogenase). A symmetry axis on the steroid backbone makes equivalent the positions 11βand 7α.sup.15, which suggest us that 11βHSD could be a good candidate for this reaction. 11β-HSD exist as two enzymes, 11β-HSD type 2 (11HSD2) which catalyzes the dehydrogenation of cortisol into cortisone and 11β-HSD type 1 (11HSD1) which realizes the reverse reaction and catalyzes the hydrogenation of cortisone into cortisol.sup.13, 14, 16(FIG. 3A). Interestingly 11βHSD1 accepts also as substrate 7-ketocholesterol which is transformed into 7-hydroxycholesterol.sup.16. Importantly, 11βHSD2 is expressed in MCF7 while 11βHSD1 is not detected.sup.17, suggesting a possible deregulation of the equilibrium between 11βHSD1 and 11βHSD2 expression in tumor cells, that would favor OCDO production. In accordance with this hypothesis, we characterized significant levels of 11βHSD2 at the mRNA and protein level in various human BC cell lines reflecting different BC subtypes while 11βHSD1 expression was not detectable either at the mRNA or protein levels and all the cell lines tested produced OCDO (Table 1).

[0159] To confirm the implication of 11βHSD2 in the production of OCDO from CT, we transfected HEK-273 cells, a cell model previously used to study cortisol/cortisone metabolism.sup.18, with a plasmid coding either the 11βHSD2 (HSD2) or the empty vector (mock). Immunoblot analysis of mock transfected HEK-273 cells did not detect endogenous 11βHSD2 (FIG. 4A). In contrast, in 11βHSD2-transfected HEK-273 cells, 11βHSD2 was well detected migrating (FIG. 4A). We first measured the capacity of the 11βHSD2-transfected HEK-273 cells to produce cortisone when incubated 8 h with .sup.3H-cortisol. As observed in FIG. 4B, 11βHSD2-transfected HEK-273 cells produced 3-fold more cortisone (3.3 pmol/10.sup.6 cells/h) than mock-transfected cells (1.1 pmol/10.sup.6 cells/h), indicating that the encoded enzyme was functional. We then measured the production of OCDO after incubating transfected-HEK-273 cells with [.sup.14C]α-CT for 8 h. As shown in FIG. 4C, 11βHSD2-transfected HEK-273 cells induced a 7-fold increase production of OCDO (195 pmol/10.sup.6 cells/h) compared with mock-transfected HEK-273 cells (29 pmol/10.sup.6 cells/h). Together these data indicate that 11βHSD2 is able to produce significant levels of OCDO in addition to cortisone.

[0160] To study the implication of 11βHSD1 in the transformation of OCDO into CT, HEK293 cells were transfected with a plasmid coding the 11βHSD1 (HSD1) or the empty vector (mock) and with or without a plasmid coding the H6PDH, the enzyme that produces the cofactor NADPH necessary for 11βHSD1 reductase activity as reported in.sup.18 (FIG. 3A). No endogenous expression of 11βHSD1 or H6PDH was detected in HEK293 cells transfected with the empty vector (mock) by western blot analysis (FIG. 4D). In contrast, in 11βHSD1 and H6PDH transfected-HEK293 cells, the proteins were well detected (FIG. 4D). We then measured the capacity of the HEK293 transfected cells to produce cortisol after incubating with .sup.3H-cortisone. As shown in FIG. 4E, low production of cortisol was measured in the mock-transfected cells or in H6PDH-transfected cells (about 0.20 pmol/10.sup.6 cells/h). In contrast, 11βHSD1-transfected cells produced 5-fold more cortisol than mock-transfected cells (1.1 pmol/10.sup.6 cells/h), and this production was increased twice by co-transfecting 11βHSD1 and H6PDH (2 pmol/10.sup.6 cells/h). Together the data indicated that the transfected enzymes 11βHSD1 and H6PDH are functional. We then measured the production of CT after incubating transfected HEK293 cells with [.sup.14C]-OCDO for 24 h. As shown in FIG. 4F, the production of CT was of about 1 pmol/10.sup.6 cells/h in cells transfected with the empty plasmid or with H6PDH while the transfection of the plasmid coding 11βHSD1 induced a 3-fold increased production of CT and the co-tranfection of H6PDH and 11βHSD1 further increased CT production that reached 8-fold (8.5 pmol/10.sup.6 cells/h) the levels of the mock-transfected cells. These data indicate that 11βHSD1 is able to produce significant levels of CT in addition to cortisol.

[0161] Ectopic Expression of 11βHSD1 in MCF-7 Cells Induces CT Production and Decreases Cell Proliferation and OCDO Treatment Reverses this Effect.

[0162] Since MCF7 cells do not express 11βHSD1, we transfected these cells with a plasmid expressing this enzyme (FIG. 5A) and evaluated the impact of its expression on CT production and cell proliferation. As shown in FIG. 5B, the expression of 11βHSD1 in MCF7 cells significantly stimulated OCDO to CT conversion compared with the control (73±12 against 8.5±2.5 pmol/10.sup.6 cells/h). In addition, the expression of 11βHSD1 in MCF7 cells significantly decreased cell proliferation by 45% and OCDO treatment reversed this effect (FIG. 5C), indicating that 11βHSD1 inhibits cell proliferation through transformation of OCDO into CT.

[0163] Knock-Down of 11βHSD2 Decreases Cell Proliferation, Invasion and Survival in MCF7 Cells as Well as Tumor Growth and OCDO Reverses These Effects.

[0164] To study the implication of 11βHSD2 in cell proliferation and survival, we knocked down the expression of 11βHSD2 in MCF7 cells by using shRNA against the enzyme or control shRNA. Two stable clones were selected in which the expression of 11βHSD2 was significantly decreased at both protein and mRNA level (sh11HSD2 A and sh11HSD2 B) and compared with shRNA control clones (shC A and shC B) (FIG. 6A). A significant decrease in cortisone and OCDO production was measured in sh11HSD2 A and B clones compared with shC A and B control clones (FIGS. 6B and 6C respectively). Basal cell proliferation of the two sh11HSD2 clones was significantly decreased (FIG. 6D) and their doubling time was increased by 142% and 150% (FIG. 6E) compared with control clones. Moreover, the knock-down of 11βHSD2 expression also significantly decreased cell survival in a clonogenic assay (FIG. 6F). Importantly, we determined that OCDO was able to reverse the inhibition of cell proliferation induced by decreasing the expression of 11βHSD2 in sh11HSD2 (FIG. 7A) while cortisone even at high concentrations did not (FIG. 7B). Similarly, OCDO reversed the inhibition of cell invasion (FIG. 7C) and cell survival (FIG. 7D) mediated by the knock-down of 11βHSD2. Together these results indicate that 11βHSD2 controls cell proliferation, survival and cell invasion through OCDO production. We then tested the impact of 11βHSD2 knock-down in vivo on ShC or sh11HSD2 cells xenografted in immunodeficiente mice. As shown in FIG. 7E, the basal growth of sh11HSD2 tumors was significantly decreased (by 29%) compared with that of shC tumors. Importantly, subcutaneous treatment with OCDO (15 μg/kg, 5 days/week) reversed the growth inhibition of sh11HSD2 tumors to a level similar to the growth of shC tumors. KI67 staining of the tumors indicated that cell proliferation was increased in ShC tumors through OCDO treatment and decreased in sh11HSD2 tumors, and OCDO reversed the growth inhibition of sh11HSD2 tumors. Together, these date indicate that 11βHSD2 controls tumor growth through OCDO production.

[0165] Expression of the Enzymes Regulating OCDO Production in Breast Cancer Samples and Normal Matched Tissue.

[0166] We then explored the expression of the enzymes regulating OCDO in breast patient samples and normal adjacent tissues. As shown in Table 2, immunohistology analyses showed that 11βHSD2 was mainly expressed in breast tumors (93% of 49 samples) and weakly or not in normal adjacent tissues (8% of 46 samples). 11βHSD2 was also observed in the blood vessels in 43% of breast tumor samples. 11βHSD1 was poorly present either in the tumor samples (25% of 48 samples) and in the normal tissue (38% of 42 samples) and H6PDH showed the same tendency (34% of 32 tumor samples and 57% of the 42 normal cases), however the expression of both enzymes was lower in tumors compared to normal tissue. DHCR7 and D8D7I were found expressed both in tumor and normal tissues. However, for DHCR7 a strong expression was observed in 54% of the 49 tumor samples compared with normal tissue and interestingly the expression of the enzyme was increased in the adipocytes surrounding the tumors (78% of the samples) compared with the adipocytes that were distant. For D8D7I, a strong staining was also observed in 63% of the 50 tumor samples compared with the normal tissues. Together these results indicate that the expressions of the enzymes producing OCDO are increased or high in tumors compared with normal tissue.

[0167] Discussion

[0168] The present study identifies new functions for 11-βHSD2 and 11-βHSD1 as being the enzymes involved in the inter-conversion of OCDO and CT. Thus, several enzymes are involved in the production and regulation of OCDO production. Previously, we showed that the ChEH, that is carried out by D8D7I and DHCR7, mediates the transformation of 5,6-EC into CT that leads to the production of OCDO in tumors.sup.8, 10. The inhibition of ChEH by molecules such Tam or DDA blocks the production of OCDO and its proliferative effect in cancer cells and tumors, while the addition of OCDO reverses these effects.sup.8, 10 and present study. Here, we show that 11-βHSD2 and 11-βHSD1, which are known to regulate the metabolism of the glucocorticoids, cortisol and cortisone in human, are involved in the next step to produce OCDO from CT or to produce CT from OCDO respectively. Importantly, 11-βHSD2 controls both in vitro and in vivo tumor cell proliferation through OCDO production, in add back experiments in which 11-βHSD2 expression has been attenuated. Conversely, 11-βHSD1 re-expression in tumor cells lacking this enzyme inhibits cell proliferation through transformation of OCDO into CT and OCDO addition reverses this effect. Thus, activation of 11-βHSD2 not only promotes inflammation and decreases the inhibition of cell proliferation induced by the inactivation of cortisol into cortisone but also produces an onco-metabolite OCDO that actively participates to cancer proliferation and invasion. Importantly, OCDO increases the proliferation of estrogen-positive or estrogen-negative breast tumors, indicating that OCDO may contribute to stimulate tumor progression even in the absence of estrogens. The 11-βHSD2 enzyme is exclusively oxidative, converting the active cortisol to the inactive cortisone and requiring NAD as cofactor. 11-βHSD1 presents a dual reductase and dehydrogenase activity, depending for the deshydrogenase activity of the presence of H6PDH that produces the co-factor NADP.sup.18. In absence of H6PDH expression, 11-βHSD1 will work as a dehydrogenase as reported in human omental preadipocytes.sup.19. According to our results, the absence or the decrease level of 11-βHSD1 in tissues expressing 11-βHSD2 would favour the production of OCDO in addition to converting cortisol to cortisone. Similarly, the decrease or the absence of H6PDH may favour the dehydrogenase activity of 11-βHSD1 and thus the production of OCDO and cortisone. In the present study, the immunohistology analyses indicate that the expressions of the enzymes producing OCDO, 11βHSD2, D8D7I and DHCR7, are increased or high in tumors compared with normal tissues and that the enzymatic equilibrium between 11βHSD2 and 11βHSD1/H6PDH is shifted toward the production of OCDO in tumors. These results are consistent with the pro-tumor and pro-invasive activity of OCDO that we report in the present study and its secretion by the tumor cells should contribute to tumor proliferation and aggressiveness. 11βHSD2 is also present in cells of the vasculature in 43% of the tumor samples, indicating that OCDO may be secreted in the blood fluid to act at distance of the tumor in addition to an autocrine action and it may actively participate to tumor invasion. An effect of OCDO on the proliferation of blood vessels could be also considered.

[0169] Thus, the discovery of OCDO and its pro-tumor effect as well as the discovery of the enzymes regulating its production are important findings that should have major implications in tumor biology and therapy. Therefore, the activation of OCDO production as well as the expression of the enzymes producing or regulating OCDO could be markers of cancer and of the efficacy of anti-cancer compounds such as Tam or DDA.

TABLE-US-00003 TABLE 1 Expression and activity of 11βHSD1 and 11βHSD2 in BC tumor cells. Different subtypes of breast cancer cells were analyzed for the expression of 11βHSD1 and 11βHSD2 by either qPCR or immunobloting as well as OCDO production by incubating tumor cells with .sup.14C-αEC for 24 h as described in FIG. 1. The amount of OCDO formed per hour was normalized to the number of cells. The results are the mean (±s.e.m) of two to three experiments. 11HSD2 OCDO pro- 11HSD1 duction pmol/10.sup.6 Cells mRNA protein mRNA protein cells/h ± s.e.m MCF-7 >35 − 26.5 + 10.6 ± 2.sup.  BT474 >35 − 26 + 5.76 ± 1.5 SKBr3 >35 − 27.1 +  3.5 ± 0.3 ZR751 >35 − 25.2 + 9.3 ± 1  MDA-MB- >35 − 24.2 + 23.9 ± 3.4 468 MDA231- 28.2 − 28.5 +   2 ± 0.4 MB- HCC1937 33 − 28.0 +  7.3 ± 0.3 LCC1 >35 − 23.7 + 19.5 ± 2.2 LCC2 >35 − 23.6 + 29.3 ± 9.sup.  (TamR) RTx6 >35 − 25.4 +   2 ± 0.1 (TamR) TS/A 35 − 26 ND  9.5 ± 0.1 E0771 35 − 24 ND 22.5 ± 4.sup.  ND: not determined TamR: cells derived from MCF7 resistante to tamoxifen

TABLE-US-00004 TABLE 2 Expression of enzymes regulating OCDO production in breast tumor patient samples and normal matched tissues. Immunohistology analyses using specific antibodies against the enzymes regulating OCDO production were scored as described in the “Materials and Methods” section. Cancer Adjacent normal tissue n % n % 11HSD2 49 93 46 8 11HSD1 48 25 42 38 H6PDH 32 34 42 57 DHCR7 49 83 43 74  54* D8D7I 50 98 43 70  63* *High expression compared with normal tissue

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