WO2014128127A1

WO2014128127A1 - Methods and pharmaceutical compositions for treatment of prostate cancer

Info

Publication number: WO2014128127A1
Application number: PCT/EP2014/053144
Authority: WO
Inventors: Michael PRIMIG; Frédéric CHALMEL; Romain Mathieu
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2013-02-19
Filing date: 2014-02-18
Publication date: 2014-08-28

Abstract

The present invention relates methods and compositions for the treatment of prostate cancer.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR TREATMENT OF

PROSTATE CANCER

FIELD OF THE INVENTION:

The present invention relates to methods and compositions for the treatment of prostate cancer.

BACKGROUND OF THE INVENTION:

Prostate cancer (PCa) is the most commonly diagnosed noncutaneous malignancy among men and the second most common cause of cancer-associated mortality in western countries [1, 2]. Up to a third of localized PCa patients develop a locally advanced cancer or metastases within 15 years [3, 4]. Hormone (androgen deprivation) therapy is an established treatment to delay the progression of metastatic tumors. However, within two years most patients develop the hormonally insensitive disease form called castration-resistant prostate cancer (CRPC) [5]. The high morbidity and mortality of CRPC remains a major public health challenge in part due to the unsatisfactory and ineffective treatment options available. The molecular mechanisms and their interplay governing the initiation and progression to this refractory status are still poorly understood in spite of the intensive research [6]. Therefore improved biomarkers as well as new gene-based therapeutic strategies focusing on PCa progression are eagerly awaited.

The idea to direct the immune system toward malign tumors stems from the initial observation that a patient suffering from skin cancer had developed antibodies against a protein called melanoma antigen (MAGEAl). This protein was present in the tumor and in testis but not in healthy non-testicular tissues [7]. Ever since this discovery was made 254 additional Cancer/Testis (CT) genes have been identified using molecular biological methods and genome biological approaches based on microarrays or serial analysis of gene expression. Following up on this work, genes such as MAGE A3 are being investigated in clinical trials [10, 11]. Apart from their potential usefulness as targets for immunotherapy [12], CT genes are also interesting from the perspective of the etiology and the progression of cancer because gametogenesis and oncogenesis share features including immortality, genetic instability and invasiveness; reviewed in [13]. Consistently, CT gene expression levels are often correlated with tumor progression and clinical outcomes in various types of somatic malignancies [14- 19]. This is notably the case for PCa where expression screens of known CT genes yielded information on their stage-specific induction as the disease unfolds [20]; reviewed in [21, 22]. It is noteworthy that a genome-wide comparison of gene expression patterns across a large number of healthy and pathological human tissues marked out colon-, and hormone-sensitive prostate cancers as failing to express testis-specific (or testis-selective) genes [23]. In contrast to this finding, three recent landmark studies have associated well-known CT genes with tumor aggressiveness and poor outcomes [20, 22, 24]. However, to the best of our knowledge, a systematic screening of CT genes showing altered expression in PCa has never been carried out.

SUMMARY OF THE INVENTION:

The present invention relates to a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors for use in the treatment of prostate cancer in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors analyzed a large gene expression dataset assembled from normal testicular sample data (total testis, seminiferous tubules, spermatocytes and spermatids) [25] with the output of published studies covering nine normal testis, sixteen healthy prostates, 68 HSPC, four CRPC and 44 other non-testicular and non-prostatic healthy tissues [26, 27]. This led to the identification of 98 genes including known CT genes and novel candidates which discriminate between CRPC and HSPC. Of particular interest are the roles of germline- associated sequence-specific transcription factors in controlling CT gene expression in cancer. Therefore, among the 98 candidates genes, the inventors further validated the orphan nuclear receptor NR6A1 (also called GCNF for germ cell nuclear factor) - a DNA binding repressor essential for embryogenesis, neurogenesis, normal female fertility and testicular gene expression in the mouse [28-32]- at the protein level. Using tissue microarrays prepared from a large cohort of patients suffering from prostate cancers with clinicopathologic information, the inventors subsequently observed NR6A1 's cellular presence to be significantly associated with prostate cancer progression. These results demonstrate that NR6Al is a novel biomarker for aggressive PCa.

The inventors surprisingly found that NR6A1 transcript levels were significantly increased in healthy testis and in CRPCs compared to healthy non-testicular tissues. The inventors also demonstrated that NR6A1 protein expression was significantly higher in primary tumors from patients with metastatic compared to localized PCa and further increased in CRPCs, indicating that NR6A1 upregulation correlates with disease progression and aggressiveness. The inventors also demonstrated that increased NR6A1 immunoreactivity was significantly associated with high Gleason score, advanced T stage and proliferation in both HSPCs and CRPCs.

Therapeutic methods and uses

Accordingly, the present invention relates to a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors for use in the treatment of prostate cancer in a subject in need thereof.

As used herein, the term "subject" denotes a mammal. In a preferred embodiment of the invention, a subject according to the invention refers to any subject (preferably human) afflicted with prostate cancer.

The method of the invention may be performed for any type of prostate cancer such as revised in the World Health Organisation Classification of prostate cancer and selected from the group: Malignant neoplasm of prostate (C61); low and high grade dysplasia of prostate (N42.3, D07.5); Benign neoplasm of prostate (D21.1); Neoplasm of uncertain or unknown behavior of prostate (D40); localized prostate cancer; advanced prostate cancer; locally advanced prostate cancer; metastatic prostate cancer; hormone-sensitive prostate cancer (HSPCs); castration-resistant (CRPCs) prostate cancer.

As used herein, the term "NR6A1" has its general meaning in the art and refers to nuclear receptor subfamily 6 group A member 1. NR6A1 is also known as germ cell nuclear factor (GCNF) [29-32]. The term "NR6A1" refers to the orphan nuclear that is a transcriptional repressor that plays a critical role during embryogenesis, neurogenesis and gametogenesis. NR6A1 is characterized such as known nuclear receptor by a modular structure composed of a N-terminal regulatory domain (activation function 1 (AFl), followed by a DNA-binding domain (DBD), a ligand-binding domain (LBD) and a C-terminal domain (activation function 2 (AF2)). NR6A1 repress transcription through nuclear translocation, homodimerization or heterodimerization, and binding to specific DNA sequences. NR6A1 as a transcriptional repressor form a homodimeric form and bind to unique DRO (direct-repeat spaced by 0 nucleotides) response elements that control the post-meiotic expression of protamines replacing histones and packaging DNA into the sperm head. NR6A1 may regulate hormonal signaling pathways by modulating the expression of DRO response element- regulated genes (Schweitzer et al, 2008. Expert Opin. Ther. Patents (2008) 18(8)). The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., NR6A1 or GCNF) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.

An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.

The term "NR6A1 antagonist" refers to a compound that selectively blocks or inactivates NR6A1. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates NR6A1 with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the nuclear receptor family. Compounds that prefer NR6A1, but that may also block or inactivate other nuclear receptor sub-types, as partial or full antagonists, are contemplated. The "NR6A1 antagonist" refers to compounds that blocks NR6A1 nuclear translocation, homodimerization or heterodimerization, and binding to specific DNA sequences. The "NR6A1 antagonist" may also consist in compounds that inhibit the binding of the ligand to NR6A1 such as compounds having the ability to bind NR6A1 ligand with high affinity and specificity or compounds that compete with NR6A1 ligand. Typically, a NR6A1 antagonist is a small organic molecule, a peptide, a polypeptide, an aptamer or an intra-antibody. In another embodiment, the NR6A1 antagonist of the invention is an aptamer.

Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then after raising aptamers directed against NR6A1 of the invention as above described, the skilled man in the art can easily select those inhibiting NR6A1.

In one embodiment, the compound of the invention is an inhibitor of NR6A1 expression.

Inhibitors of NR6A1 expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of NR6A1 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of NR6A1 proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding NR6A1 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of NR6A1 expression for use in the present invention. NR6A1 gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that NR6A1 expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of NR6A1 expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of R A. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of NR6A1 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of NR6A1 expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing NR6A1. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation. In one embodiment, the present invention relates to a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors for use in the prevention or treatment of castration-resistant prostate cancer in a subject in need thereof.

In one embodiment, the present invention relates to a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors for use in the prevention or treatment of advanced prostate cancer and metastatic prostate cancer in a subject in need thereof.

In another embodiment, the present invention relates to a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors for use in the inhibition of the progression of the localized prostate cancer to locally advanced prostate cancer, metastatic prostate cancer or castration resistance prostate cancer. In one embodiment, the present invention relates to a method of treating prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors.

In one embodiment, the present invention relates to a method of preventing or treating castration-resistant prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6Al expression inhibitors.

In one embodiment, the present invention relates to a method of preventing or treating advanced prostate cancer and metastatic prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting ofNR6Al antagonists or NR6A1 expression inhibitors.

In another embodiment, the present invention relates to a method of inhibiting the progression of the localized prostate cancer to locally advanced prostate cancer, metastatic prostate cancer or castration resistance prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6 A 1 expression inhibitors.

Pharmaceutical composition

The compound of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the treatment of prostate cancer in a subject in need thereof.

Typically, the compound of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The compound of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used. In one embodiment, the present invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the prevention or treatment of castration-resistant prostate cancer in a subject in need thereof.

In one embodiment, the present invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the prevention or treatment of advanced prostate cancer and metastatic prostate cancer in a subject in need thereof.

In another embodiment, the present invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the inhibition of the progression of the localized prostate cancer to locally advanced prostate cancer, metastatic prostate cancer or castration resistance prostate cancer in a subject in need thereof. Screening method

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the prevention or treatment of prostate cancer in a subject in need thereof, wherein the method comprises the steps of: i) providing candidate compounds and ii) selecting candidate compounds that blocks the action of NR6A1.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the prevention or treatment of prostate cancer in a subject in need thereof, wherein the method comprises the steps of: - providing a NR6A1, providing a cell, tissue sample or organism expressing the NR6A1,

- providing a candidate compound such as small organic molecule, intra-antibodies, peptide or polypeptide,

measuring the activity of the NR6A1 ,

and selecting positively candidate compounds that blocks the action of NR6A1, inhibits NR6A1 expression, or blocks NR6A1 homodimerization or heterodimerization.

Methods for measuring the activity of the NR6A1 are well known in the art. For example, measuring the NR6A1 activity involves determining a Ki on the NR6A1 cloned and transfected in a stable manner into a CHO cell line or measuring NR6A1 homodimerization or heterodimerization level, or measuring NR6A1 nuclear translocation in the presence or absence of the candidate compound.

Tests and assays for screening and determining whether a candidate compound is a NR6A1 antagonist are well known in the art. In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to reduce NR6A1 activity.

Activities of the candidate compounds, their ability to bind NR6A1 and their ability to inhibit NR6A1 activity may be tested using isolated germ cells expressing NR6A1, CHO cell line cloned and transfected in a stable manner by the human NR6A1.

Cells expressing another receptor than NR6A1 may be used to assess selectivity of the candidate compounds.

Prognostics and diagnostics methods

A further aspect of the invention relates to a method of identifying a subject having a prostate cancer which comprises the step of analyzing a biological sample from said subject for:

(i) determining the NR6A1 expression level,

(ii) comparing the NR6A1 expression level in the sample with a reference value,

(iii) detecting differential in the NR6A1 expression level between the sample and the reference value is indicative of a subject having a prostate cancer. Analyzing the NR6A1 expression level may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.

In a preferred embodiment, the NR6A1 expression level is assessed by analyzing the expression of mRNA transcript or mRNA precursors, such as nascent RNA, of NR6A1 gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a biological sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays (AFFYMETRIX).

Advantageously, the analysis of the expression level of mRNA transcribed from the gene encoding for NR6A1 involves the process of nucleic acid amplification, e. g., by RT- PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991), self sustained sequence replication (Guatelli et al, 1990), transcriptional amplification system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi et al, 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

In another preferred embodiment, the NR6A1 expression level is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin- streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for NR6A1.

Said analysis can be assessed by a variety of techniques well known from one of skill in the art including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (RIA).

A reference value can be a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the NR6A1 expression levels (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the NR6A1 expression level (or ratio, or score) determined in a biological sample derived from one or more subjects having or at risk of having or developing a prostate cancer. In one embodiment of the present invention, the threshold value may also be derived from NR6A1 expression level (or ratio, or score) determined in a biological sample derived from one or more subjects having or at risk of having or developing a prostate cancer. Furthermore, retrospective measurement of the NR6A1 expression levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

In one embodiment of the invention, the reference value may consist in expression level measured in a biological sample associated with a healthy subject not afflicted with prostate cancer or in a biological sample associated with a subject afflicted with prostate cancer.

According to the invention, low NR6A1 expression level is indicative of subject not having a prostate cancer and high NR6A1 expression level is indicative of subject having a prostate cancer. In another embodiment, the present invention relates to a method of identifying if a subject afflicted with prostate cancer is at risk of having or developing a locally advanced prostate cancer, a metastatic prostate cancer or a castration-resistant prostate cancer which comprises the step of analyzing a biological sample from said subject for:

(i) determining the NR6A1 expression level,

(ii) comparing the NR6A1 expression level in the sample with a reference value,

(iii) detecting differential in the NR6A1 expression level between the sample and the reference value is indicative of a subject at risk of having or developing a locally advanced prostate cancer, a metastatic prostate cancer or a castration-resistant prostate cancer.

A further aspect of the invention relates to a method of monitoring prostate cancer progression by performing the method of the invention.

In one embodiment, the present invention relates to a method of treating prostate cancer in a subject in need thereof comprising the steps of:

(i) identifying a subject having a prostate cancer by performing the method according to the invention, and

(ii) administering to said subject a compound which is selected from the group consisting of NR6 A 1 antagonists or NR6Al expression inhibitors.

In another embodiment, the present invention relates to a method of preventing the progression of the prostate cancer to locally advanced prostate cancer, metastatic prostate cancer or castration resistance prostate cancer in a subject in need thereof comprising the steps of:

(i) identifying a subject afflicted with prostate cancer at risk of having or developing a locally advanced prostate cancer, a metastatic prostate cancer or a castration-resistant prostate cancer by performing the method according to the invention,

(ii) administering to said subject a compound which is selected from the group consisting of NR6 A 1 antagonists or NR6A1 expression inhibitors.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: Resulting Cancer/Testis signatures and NR6A1 transcript expression in prostate cancers. Box plots displaying log2 expression signals (y-axis) of NR6A1 transcript in healthy prostate, HSPC, CRPC and 39 other cancer types (x-axis) based on the assembled GeneChip expression data. The total numbers of samples are indicated. The large horizontal red line represents the Background Expression Cutoff (BEC = 5.5, corresponding to the overall median log2 -transformed intensity). CRPC and acute myeloid leukemia (AML) samples are highlighted with a grey box and red bold fonts.

EXAMPLE:

Material & Methods

Gene-based expression screening

Gene expression dataset. We assembled a large gene expression dataset from several independent microarray studies generated on Affymetrix GeneChip U133 Plus 2.0 microarray consisting of 21 '248 coding genes publicly available at the NCBFs GEO (dataset IDs: GSE2109, GSE3325, GSE6565, GSE7307, GSE11839) [26]and ArrayExpress (dataset ID: E- TABM-130) [27]. It included 613 samples (Supplemental file 1) covering eight previously published testicular samples (total testis, seminiferous tubules as well as enriched populations of pachytene spermatocytes and round spermatids samples) [25] together with nine additional healthy testes (hT), sixteen healthy prostates (hP), 508 samples corresponding to 44 different non-testicular and non-prostatic healthy tissues (nTnP), 4 localized CRPC [33] and 68 localized HSPC (ExpO). Prostate cancer samples were from radical prostatectomy series or rapid autopsy program (ExpO).

Raw data pre-processing. The microarray data were pre-processed using AMEN [34]. GeneChip data were quality controlled and normalized using the Robust Multi- Array Average (RMA) method as published [25].

Statistical filtration of transcripts specifically expressed in HSPC or CRPC. Probesets were selected when their intensity values were above the Background Expression Cutoff (BEC = 5.5, corresponding to the overall median log2 -transformed intensity) in their corresponding PCa category and below the BEC in the other category as well as in hP (Fig. 1 A). To avoid inclusion of transcripts with signal values close to the BEC, only those whose signals were at least two-fold higher were retained. A LIMMA statistical test (F-value adjusted with the False Discovery Rate method: p < 0.01) was employed to discriminate probesets with statistically significant changes across individual samples. Finally, remaining transcripts were categorized into transcripts specifically expressed in HSPC or in CRPC.

Statistical filtration of transcripts preferentially expressed in testis. Probesets were selected if at least one intensity value across hT was above the BEC and below the BEC in hP and nTnP (with a maximum of 3 nTnP exceptions) (Fig. 1). Only those whose signals were at least two-fold higher in at least one hT than in nTnP were retained. Finally, a LIMMA statistical test [p(FDR) < 0.01] was employed to identify probesets with significant changes.

Statistical filtration of novel Cancer/Testis genes (CT genes) associated with PCa. The novel CT genes associated with HSPC or CRPC were identified by intersecting the transcripts specifically expressed in HSPC or CRPC with the transcripts preferentially expressed in testis, respectively (Fig. 1).

Functional data mining. Enrichment of GO terms was estimated using AMEN [34] with the Fisher exact probability (Gaussian Hypergeometric test). A GO term was considered to be significantly enriched when the FDR-corrected p-value was < 0.05 and the number of gene bearing this annotation was > 3.

Patients and tissues - Tissue microarrays (TMA)

224 cases of HSPC were obtained from patients treated by radical prostatectomy (with negative surgical margins) at the Montsouris Institute of Paris. Patients' characteristics are the following: median age: 63 years (46-77), median preoperative PSA: 8.9 ng/ml (1.5-20), 139 pT2 cases and 85 pT3, 51 Gleason score 6 or less, 161 Gleason score 7, and 12 Gleason score 8 or more (Table I). 57 Normal prostate samples were obtained from patients with urinary bladder cancer without prostate cancer treated by radical prostatocystectomy [64 years (48- 81)]. From the same institution, 111 prostate cancers with biochemical relapse, defined as 2 consecutive increases in serum PSA 0.2 ng/ml or greater, were matched with 111 tumors without recurrence after at least 4 year follow up. The date of PSA recurrence was defined as the date of the first increase of serum PSA after surgery. Each of these patients was matched with 1 patient that presented identical age group (ie, under 50 years old, 50-60, 60-70, and over 70 years old), preoperative PSA rate group (ie under lOng/ml, 10-15 ng/ml, 15-20 ng/ml), Gleason score, and pathological stage, but free of recurrence after at least the same follow up. Patients' characteristics and follow-up are summarized in Table II.

Fifty cases of CRPC were selected from 323 patients treated with exclusive androgen deprivation therapy (ADT), between 1988 and 2008. Patients were selected if they initially responded to exclusive ADT (decrease in PSA level without clinical or radiological progression), and had post hormonal relapse tissue sample suitable for analysis. Hormonal therapies were LHRH agonist, steroidal or non-steroidal antiandrogen or complete androgen blockage. No patient received chemotherapy, radiation therapy, prostatectomy or 5-alpha reductase inhibitors. Hormonal relapse was defined as 2 consecutive rises in PSA with at least 1 week interval, and with serum testosterone level under castration level (50 ng/dl). Tissues were collected by trans-uretral resection (TUR), performed in all cases because of lower urinary tract symptoms associated with local tumor progression (Table I).

Prostate cancer metastases were obtained from 20 patients treated in the University Hospital of Poitiers, by either radical prostatectomy or radiotherapy. No hormonal deprivation had been performed before metastases resection. Metastatic tissues were either lymph nodes (n=10) or bone (n= 10).

Tissue microarrays were constructed as previously described [35] with prostate cancer tissues obtained from the above mentioned normal prostate, HSPC, bone and lymph node metastatic lesions, and CRPC specimens, including 4 cores (0.6 mm diameter) per cancer.

Immunohistochemistry

Immunohistochemistry (IHC) was performed on normal prostate and on prostate cancer Tissue Microarrays (TMA). IHC was performed on de-paraffinized sections and samples were treated with citrate buffer pH 6 for 30 minutes at 80°C and then kept 20 minutes at room temperature. The slides were rinsed in IX PBS (phosphate buffered saline, pH 7.4), treated with 3 % hydrogen peroxide in IX PBS for 5 minutes. Sections were pre-incubated twice in 5% human serum albumin (Sigma, Saint-Quentin Fallavier, France) for 20 min. Next, the slides were incubated overnight at 4°C with rabbit polyclonal antibodies against NR6A1 (Abeam ab38816) at 1 :200, in 5% human serum albumin and 0.1% Tween. IHC staining was performed at room temperature with biotinylated goat anti-rabbit IgG (Dako) and streptavidin-biotin peroxidase (Dako) for 1 hour each at a dilution of 1/500. Slides were then stained for three minutes with 3,3'-diaminobenzidine Enhanced System (Sigma). Finally, the sections were counterstained with 0.2 % Hematoxylin. The fraction of anti-NR6Al stained nuclei/cells was determined on the entire section area. Negative controls included incubation without the primary antibody. IHC staining was also performed on TMA slides using antibodies directed against the proliferation marker Ki-67 (DakoCytomation, dilution 1/50, incubation 30 min). Tissue microarrays were reviewed by two experienced uropathologists (N.R-L., G.F.) in a blinded fashion. In case of inter-observer variability (different categories in the case of categorical data or variability more than 10% in the case of continuous data), TMA were rescored by both uropathologists until a consensus was reached. For the NR6A1 and Ki-67 nuclear epithelial staining, positive cells were expressed as a percentage of total epithelial cells.

Statistical analysis

Survival analyses were conducted using Kaplan-Meier method, and curves were compared with the long-rank test. Hazard ratios were calculated using Cox regression for multivariate analysis. To evaluate the association between markers' expression and both Gleason score and pathological stage, either Chi-square, nonparametric Mann-Whitney or Kruskall-Wallis tests were used for categorical and continuous variables, respectively. The association between 2 continuous variables was tested using the non parametric Spearmen test. Results

Initial genome-wide screening and functional analysis

By comparing CRPC, HSPC and healthy prostate (hP) samples through a gene expression screening, we selected 1046 and 35 transcripts (or probeset IDs) with a restricted expression in CRPC and in HSPC (Fig. 1 and Supplemental file 2). In addition, we integrated data obtained with healthy testis (hT) samples and 45 non-testicular normal tissues (including hP samples) we identified 2668 transcripts classified as Preferentially Expressed in Testis (PET). Finally, the intersection of both filtration strategies yielded 98 potential CT genes (corresponding to 111 transcripts) detected in testicular samples of which 95 (108 transcripts) were detected only in CRPC and three (three transcripts) exclusively in HSPC (Fig. 1, 2A and Supplemental file 2).

We next explored the biological processes associated with the 95 target genes detected only in CRPC by searching for enriched Gene Ontology (GO). We found significant over- representation of terms associated with genes related to cell cycle progression and checkpoint control (CCNE2, CDC25A, CDC25C, CDC6, CEP78, DDX11, MNS1, NEDD1, SESN3, TIPIN) such as regulation of cell cycle arrest (GO:0071156, 5 observed/~l expected, p- value=0.0469), DNA replication (GO:0006260, 5/~l, 0.0426), cell cycle process (GO:0022402, 10/-3, 0.0426) and more specifically M phase (GO:0000279, 7/~2, 0.0427) and interphase (GO:0051325, 7/~l, 0.0186). We also found genes demonstrated or proposed to be associated with spermatogenesis (SPAGl, MICALC, NR6A1) and more precisely meiosis (MNS1), the acrosome (SPACA3, SPESP1) and sperm motility (DNAH14, DNAH2) as well as loci related to spermatogenesis failures (CNE2, MYBLl) but without significant enrichment for none of these terms. NR6A1 transcript is upregulated in castration-resistant prostate cancers

Among the 98 CT gene candidates, 15 encode for DNA binding proteins annotated as known or putative transcription factors (Supplemental file 2). We decided to further investigate a ligand-activated transcriptional repressor, NR6A1 [36]. Its transcript (Affymetrix probeset ID:211402_x_at) was consistently above the threshold of detection in all CRPC (4 out of 4) and testicular samples, and at the lowest concentration in HSPC (64/68) and the other healthy tissues (including normal prostate) (Fig. 2B). In addition, we found that NPv6Al 's transcript accumulation in CRPC, with the exception of acute myeloid leukemia (AML), did not occur in 38 other cancer types sampled by the Expression Project for Oncology (expO, International Genomics Consortium, http://www.intgen.org) (R. Mathieu, M. Primig and F. Chalmel, unpublished observation) (Fig. 2B). These results lead us to further examine NR6A1 at the protein level by immunocytochemistry (IHC) analyses.

NR6A1 protein accumulates in castrate-refractory disease

We monitored NR6A1 protein accumulation in prostate cancer by IHC using a polyclonal antibody on tissue microarrays (TMA). First, we observed that the protein indeed accumulated in the nuclei of cancer cells in high grade HSPC and CRPC sections (Fig. 3C-D) as compared to normal prostate and low grade tumor samples (Fig. 3A-B). Moreover, the fraction of anti-NR6Al stained nuclei/cells was found to be significantly higher in HSPC sections (median=~5%, range=[0%-95%]) than in normal prostate (median=0%, range=[0%- 90%]) and further increased in CRPC (median=~70%, range=[20%-95%]) (p<0.0001, Kruskall-Wallis nonparametric test).

NR6A1 is highly expressed in cases of metastatic prostate cancers

Next, we asked if NR6 A 1 -positive staining was associated with PCa metastasis as well. Using a TMA containing bone- and lymph node metastatic lesions (n=20), we demonstrated substantially increased NR6A1 staining in all metastatic sites (Fig. 3E-F). The positive nuclear fraction of NR6A1 -stained cells was found to be significantly higher in metastatic lesions (median=80%, range=[0%-100%]) than in normal prostate and localized HSPC samples (p<0.0001, Kruskall-Wallis nonparametric test) (Table I). NR6A1 protein expression is associated with tumor progression and high grade prostate cancers

To further refine the picture of NR6A1 expression in PCa we examined the correlation of its protein level with traditional clinicopathological indicators (Table I). In the group of HSPC, the positive nuclear fraction of NR6A1 -stained tumor cells was significantly lower in localized pT2 tumors (median=0%, range=[0%-95%]) than in locally advanced pT3 prostate cancer (25%, [0%-90%]) (p = 0.02, Mann- Whitney nonparametric test) (Table I). Moreover, the percentage of NR6A1 positive cells was significantly increased in high grade tumors (Gleason 8-10; 60%, [5%-90%]) when compared to low (Gleason 5-6; median=0%, range=[0%-95%]) or intermediate (Gleason 7; 5%, [0%-95%]) grades (p=0.02, Kruskall- Wallis nonparametric test) (Table I). Finally, no significant association was found between NR6A1 staining and age at diagnosis (p=0.5, Spearman nonparametric test), or preoperative PSA level (p=0.6) in HSPC.

NR6A1 protein is associated with tumor cell proliferation

We subsequently examined correlations of the NR6A1 -positive staining in HSPC and CRPC with proliferation as determined by Ki-67 proliferative marker analysis. The median percentage of Ki-67 positive cells was 15% in CRPC (range=[3%-90%]) and 1% in HSPC (range=[0%-20%]). In the whole patient population, a statistically significant level of co- immunostaining was observed for NR6A1 and Ki-67 (p=0.002, Spearman test).

NR6A1 protein is not associated with biochemical relapse after radical prostatectomy

Finally, we examined if NR6A1 protein expression could help predict cancer recurrence, independently from the above-mentioned clinicopathological indicators. To address this issue, we conducted an individually matched nested case-control study: 11 1 HSPC with biochemical relapse after radical prostatectomy were matched with 111 HSPC with identical age, Gleason score, pathological stage and preoperative PSA but without recurrence (Table II). After adjusting for Gleason score, stage and PSA level, we concluded that NR6A1 status was not associated with biochemical relapse in HSPC (p=0.5, log-rank test) (Supplemental file 3).

Discussion Genome-wide expression screening strategies using GeneChips are powerful methods for pinpointing transcriptional events of interest despite a number of issues that complicate the interpretation of the data output. For example, in the case of heterogeneous samples it must be kept in mind that the resulting signal is an average of the transcripts present in the different cell types. Furthermore, changes in R A concentrations across samples may be due to transcriptional effects, varying transcript stability or altered DNA copy numbers (or a combination of all of them). Finally, another critical issue especially pertinent for human samples is the extent to which transcript levels are reproducible between individuals. This problem can usually be overcome by analyzing large numbers of samples. This is, however, difficult when they are rare like in the case for CRPC samples. Gene-based screening strategies based on RNA profiling experiments are in any case a prelude for follow-up experimental validation by, for example immunohistochemical (IHC) analysis using tissue microarrays (TMA) [37]. The sequential combination of both array technologies has become a widely used approach to identify and validate candidate biomarkers at the transcript and protein level prior large-scale and expensive clinical trials [38].

In the present study we report the outcome of a genome-wide expression screening that aimed at the identification of novel Cancer/Testis genes relevant for prostate cancer (PCa). To this end, we assembled a large GeneChip expression data set from normal testicular samples obtained in our own laboratory [25] combined with data from HSPC, CRPC and a range of healthy tissues (including normal prostate) available via public repositories [27]. We were able to identify 98 genes for which transcripts were reliably detectable in healthy testicular samples and in either CRPC (95 genes) or HSPC (3) and undetectable in 45 non- testicular healthy tissue controls (including normal prostate). This low yield regarding the three candidates associated with HSPC is not really surprising given a previous observation that this type of prostate cancer does not appear to express testis-selective genes [9]. To internally validate and support the validity of our screening approach, we observed among our candidates, sixteen known CT genes which included five genes belonging to the synovial sarcoma X breakpoint (SSX) family and four genes from the melanoma antigen gene (MAGE) family (Supplemental file 2). Members of SSX family have been recently related with PCa progression and proposed as targets for new therapeutic approaches [39]. Upregulation of MageA subfamily in CRPC has been previously reported [20]. Moreover, MAGE-C2 protein has been already associated with metastatic PCa and CRPC, and identified as an independent predictor of biochemical recurrence after radical prostatectomy [40]. As expected (since tumors and testes both contain dividing cells), we found that one-tenth of the 98 selected genes was implicated in the control of the cell cycle progression by using functional enrichment analysis. Furthermore, we found a number of poorly characterized genes that thus constitute novel targets potentially relevant for spermatogenesis or prostate cancer (or both); see Fig. 2A and Supplemental file 2 and neXtProt for annotation and references [48].

Bearing in mind that transcriptional regulators critical for cell differentiation such as meiosis and gametogenesis may contribute to the etiology and the development of somatic cancer [13], we therefore focused our analysis on genes proposed or known to encode transcription factors. As one of such candidates, one regulator appeared particularly interesting: the orphan nuclear receptor NR6A1, a transcriptional repressor that plays a critical role during embryogenesis, neurogenesis and gametogenesis in mouse models [28-32] for which no immunohistochemical (IHC) data are currently available in the HPA (http://www.proteinatlas.org). We decided to further investigate this gene, since its transcript was homogeneously and significantly detected across all four CRPC and healthy testicular samples and repressed in HSPC samples and all healthy non-testicular controls including normal prostate. It is important to notice that, in the mouse, it was previously observed that Nr6al is expressed in maturing male gem cells in the mouse [41-43], in the developing nervous system during embryogenesis [44] as well as in a variety of somatic tissues, including epididymis, oviduct, brain, and pituitary using a very sensitive lacZ reporter gene assay [45]. One obvious explanation is that CT gene screens do not include samples from the entire set of human tissues harvested at all stages of pre-, and post-natal development, making it very difficult to categorize a gene as unambiguously testis-specific.

It was previously shown that rodent and human NR6A1 proteins are present in the nuclei of testicular germ cells [46, 47], and our validation experiments suggest the same to be true for prostate cancer cells. To further investigate this question and to ask if the protein's accumulation was significantly linked to cancer progression we monitored it by IHC analysis on TMA (see patients characteristics in Material and Methods) (Table I). Consistently NR6A1 expression at the protein level indeed mirrors mRNA concentrations obtained by GeneChips. First, we observed that the protein mostly localized to the nuclei of prostate cancer cells which is consistent with the idea that it might unfold its biological activity as a transcriptional regulator during prostate cancer development. Furthermore, we observed that increased NR6A1 expression at the protein level is associated with progression to locally advanced HSPC, CRPC and metastatic lesions. To further refine the picture we examined the correlation of its protein level with traditional clinicopathological indicators (Table I). We showed that the NR6A1 's protein accumulation was positively correlated with aggressive PCa phenotypes as defined by traditional indicators such as advanced pT stage and high Gleason score. These results are in keeping with the significant association observed between NR6A1 and tumor cell proliferation determined by Ki-67 marker analysis. Taken together, these results strongly suggest that elevation of NR6A1 (from organ confined to locally advanced tumors, metastatic lesions and CRPC) is a relatively late event in PCa. Focusing on biochemical recurrence after radical prostatectomy, NR6A1 staining has no independent prognostic value in multivariate analysis when adjusting for the validated clinicopathological indicators (including pT stage, Gleason score and preoperative PSA level). This observation may be due to its close association with the established prognostic factors. However, our findings strongly suggest that NR6A1 is a novel biomarker for aggressiveness of PCa.

In the light of our results regarding the positive correlation between NR6A1 staining and PCa aggressiveness, one can raise the question whether it could play a role in the progression of PCa to an advanced stage disease. In addition to its nuclear accumulation in certain forms of HSPC and CRPC, NR6A1 has several key characteristics that support this hypothesis. First, the strong RNA signals we observed in PCa are not simply a consequence of tumor progression towards metastasis in general because the transcript levels were not found to be significantly elevated in 38 other types of cancer (with the exception of acute myelogenous leukemia). This may mean that the gene is not ubiquitously up-regulated in dividing and transformed cells marking out the restricted de-repression of NR6A1 in CRPC and AML as a special property of these malignancies. NR6A1 might therefore at least in part be responsible for a number of specific features observed in both pathologies. Second, this ligand-activated transcriptional repressor is an orphan member of the nuclear receptor gene superfamily that binds its cognate DNA response elements as a homodimer [48-52]. NR6A1 is unable to form heterodimer with other nuclear receptors [48] but it can recruit nuclear corepressors [36, 51, 53-56] or bind essential transcriptional activators [31, 32, 36, 48, 51, 52, 55, 56]. Thus, NR6A1 regulates the temporal and spatial gene expression of several processes related to stem cell growth and differentiation, neurogenesis and germ cell differentiation - that is to say it can alter cell-fate [41, 49, 57-59]. Third, neuroendocrine differentiation is known to be involved in prostate cancer proliferation and invasion [60], and several studies have reported that neuroendocrine cell number increases in high grade and high stage tumors, particularly in HSPC and CPRC [61-63]. Since these cells are likely androgen- independent, it is conceivable that hormonal therapy does not eliminate neuroendocrine cancer cells and thus contributes to tumor recurrence [60]. Vitamin A-derived retinoic acid (RA) influences the differentiation of neural stem cells from neural induction through adulthood during the development of the vertebrate nervous system [64]. It was hypothesized that RA-regulated neurogenesis might involve NR6A1 -signaling [58] as its transcript was up-regulated during RA-induced neural differentiation of certain embryonal carcinoma (EC) cell lines [53, 54]. We speculate that NR6A1 might contribute the molecular pathways leading to neuroendocrine differentiation in prostate cancer. If this theory is proven, blocking NR6A1- signaling should likely extend the therapeutic window of androgen deprivation therapy. Experimental work addressing these issues is under way.

In conclusion, the inventors discovered that the human orphan nuclear receptor gene, NR6A1, is elevated both at the transcript and protein levels in high grade HSPC, CRPC and metastatic lesions, using a gene-based expression screening of Cancer/Testis genes relevant for prostate cancer combined with a TMA-based analysis. To the best of our knowledge, this is the first study to report the association between NR6A1 and prostate cancer progression. Future in vitro and in vivo studies will investigate whether the pleiotropic function of this transcriptional repressor plays a critical role in the tumor progression and/or aggressiveness or if the observed expression signature is simply a consequence of the disease. These future insights may provide rationale for its use as reliable biomarker of aggressiveness and as a promising target for therapeutic intervention in the clinic. REFERENCES:

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Claims

CLAIMS:

1. A method of treating prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6 A 1 expression inhibitors.

2. A method of preventing or treating castration-resistant prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors.

3. A method of preventing or treating advanced prostate cancer and metastatic prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors.

4. A method of inhibiting the progression of the localized prostate cancer to locally advanced prostate cancer, metastatic prostate cancer or castration resistance prostate cancer in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of NR6A1 antagonists or NR6A1 expression inhibitors.

5. A method of screening a candidate compound for use as a drug for the prevention or treatment of prostate cancer in a subject in need thereof, wherein the method comprises the steps of:

- providing a NR6A1, providing a cell, tissue sample or organism expressing the

NR6A1,

measuring the activity of the NR6A1 ,

- and selecting positively candidate compounds that blocks the action of NR6A1, inhibits NR6A1 expression, or blocks NR6A1 homodimerization or heterodimerization.

6. A method of identifying a subject having a prostate cancer which comprises the step of analyzing a biological sample from said subject for:

(i) determining the NR6A1 expression level,

(ii) comparing the NR6A1 expression level in the sample with a reference value,

(iii) detecting differential in the NR6A1 expression level between the sample and the reference value is indicative of a subject having a prostate cancer.

7. A method of identifying if a subject afflicted with prostate cancer is at risk of having or developing a locally advanced prostate cancer, a metastatic prostate cancer or a castration- resistant prostate cancer which comprises the step of analyzing a biological sample from said subject for:

(i) determining the NR6A1 expression level,

(ii) comparing the NR6A1 expression level in the sample with a reference value,