Methods and Pharmaceutical Compositions Using Orexins (OXA, OXB) for the Treatment of Prostate Cancers

Abstract

The present disclosure relates to methods and pharmaceutical compositions for the treatment of prostate cancers. In particular, the present invention relates to an OX1R agonist for use in the treatment of prostate cancer in a subject in need thereof.

Claims

1. A method for the treatment of prostate cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a OX1R agonist.

2. The method of claim 1 wherein the OX1R agonist is a small organic molecule.

3. The method of claim 1 wherein the OX1R agonist is an antibody.

4. The method of claim 1 wherein the OX1R agonist is selected from the group consisting of chimeric antibodies, humanized antibodies or full human monoclonal antibodies.

5. The method of claim 1 wherein the OX1R agonist is a polypeptide.

6. The method of claim 1 wherein the OX1R agonist is a functional equivalent of Orexin-A or Orexin-B.

7. The method of claim 1 wherein the OX1R agonist is a polypeptide having at least 80% of identity with SEQ ID NO:2 or 3.

8. The method of claim 1 wherein the OX1R agonist is an immunoadhesin.

9. The method of claim 1 wherein the OX1R is an aptamer.

10. The method of claim 1 wherein the prostate cancer is selected from the group consisting of prostate adenocarcinoma, prostate neuroendocrine tumors, advanced prostate cancer and androgen-independent prostate cancer.

11. The method of claim 1 wherein the prostate cancer is an andregeno-independent prostate cancer.

12. The method of claim 1 wherein the subject is further administered with a chemotherapeutic agent.

13. A method for treating a prostate cancer in a subject in need thereof comprising the steps consisting of i) determining the expression level of OX1R in a tumour tissue sample obtained from the subject, ii) comparing the expression level determined at step i) with a reference value and iii) administering the subject with a therapeutically effective amount of an OX1R agonist when the level determined at step i) is higher than the reference value.

14. A method for screening a drug for the treatment of prostate cancer comprising the steps of i) providing a plurality of test substances ii) determining whether the test substances are OX1R agonists and iii) positively selecting the test substances that are OX1R agonists.

Description

FIGURES

[0072] FIG. 1. Representative microscopic fields of prostate tumors at various grades showing the immunocytochemical distribution of the orexin type 1 (OX1R) receptor, and the neuroendocrine marker EM66. (A) Photomicrograph of an adenocarcinomatous formation in a Gleason's score (4+3) CaP showing OX1R-like immunoreactivity (LI). (B) Cancerous mass in a Gleason's score (3+3) CaP where malignant cells infiltrate the acinar lumen. Some of the invasive cells show OX1R-LI while others are totally unstained. (C) Section of a Gleason's score (4+5) CaP showing a well-defined cancerous formation strongly labelled with the OX1R antibodies. (D) Higher magnification of C reveals that most of the cancerous cells display OX1R-LI. (E and F) Consecutive sections of a Gleason's score (4+5) CaP showing that OX1R (E) and EM66 (F) antibodies label the same adenocarcinomatous structures. Scale bars: 100 μm (A, C, E, F); 50 μm (B and D).

[0073] FIG. 2. Representative microscopic fields of prostate tumors at various grades showing the immunocytochemical distribution of orexin A (OxA), α-actin and PGP 9.5. (A) Section of a Gleason's score (4+3) CaP treated with OxA antibodies showing a cancerous formation totally unstained. (B) Photomicrogaph of a Gleason's score (3+3) CaP showing the presence of OxA-immunoreactive “fiber like” structures in the stroma of the gland. (C and D) Consecutive sections of a Gleason's score (3+3) CaP showing that OxA antibodies (C) and α-actin antibodies (D; a specific marker of smooth muscle fibers) do not label the same structures. (E and F) Consecutive sections of a Gleason's score (3+3) CaP showing that OxA antibodies (E) and PGP 9.5 antibodies (F; a specific marker of nerve fibers) do not label the same structures. Scale bars: 100 μm.

[0074] FIG. 3. Quantification of OX1R-immunoreactive cells in benign prostate hyperplasia and prostate tumor sections at various stages. The number of OX1R-immunoreactive cells is significantly higher in the CaP sections as compared to the BPH sections whatever the grade of the cancer. In addition, the percentage of cancer cells labelled with the OX1R antibodies increases with the grade of the CaP. Values are mean±SEM of 5 determinations performed on 20 distinct sections of BPH and CaP at various stages. Data were analyzed by using the Mann-Whitney U test. *, p<0.05; ***, p<0.001; ns, not significant.

[0075] FIG. 4. Expression of OX1R mRNAs in prostate cancer cell lines. OX1R mRNA levels were determined by quantitative PCR and adjusted to the signal intensity of HPRT1 in two distinct prostate cancer cell lines, i.e. LNCaP cells which are androgeno-dependent, and DU145 cells which are androgeno-independent. (A) The OX1R gene is expressed in the androgeno-independent DU145 cell line, but not in the androgeno-dependent LNCaP cells. (B) Induction of a neuroendocrine differentiation, by addition of db-cAMP (1 mM)/IBMX (0.1 mM) in the culture medium, induces a significant increase of OX1R mRNA expression in the DU145 cells, but does not promote the expression of the OX1R gene in the LNCaP cells. (C) Protein expression assessed by Western blot on whole lysates of DU145 cells confirms the up-regulation of OX1R in DU145 cells exhibiting a neuroendocrine phenotype. Equal protein loading was determined by reprobing with antibodies to α-tubulin. Values are mean±SEM of 5 determinations in four independent experiments for each cell line. Data were analyzed by using the Mann-Whitney U test. ***, p<0.001.

[0076] FIG. 5. Effects of orexin A and B on the apoptosis of native and trans-differentiated DU145 cells. Impact of orexin A and B (OxA and OxB; 10-6 M) on the apoptosis level of the androgeno-independent DU145 cells, submitted or not to a neuroendocrine differentiation (by addition of db-cAMP (1 mM)/IBMX (0.1 mM)), was assessed by using an Apo-ONE Homogeneous Caspase3/7 assay. (A and B) Addition of OxA or OxB in the culture medium has no effect on the apoptosis rate of native DU145 cells. (C and D) When the DU145 cells are submitted to a neuroendocrine differentiation, orexins induce a significant increase of the cell apoptosis. (E and F) In the same set of experiments, the effects of OxA and OxB on the HT-29 cell line, used as a positive control, were investigated. As previously described, orexins strongly promote the apoptosis of the HT-29 cells. Values are mean±SEM of 4 determinations in 10 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ns, non significant.

[0077] FIG. 6 Effect of inoculation of orexin-A on the growth of tumors developed by xenografting human DU-145 cells in nude mice—DU-145 cells were inoculated in the flank of nude mice at day 0. Left, Mice were injected (2 injections/week) intraperitoneally with 100 μl of orexin-A solutions (0.22 μmoles of orexin-A/Kg) starting at day 1 (white circles) or day 26 (white triangles) or with 100 μl of PBS (black circles) for controls. Right, after 45 days of treatment, mice were sacrificed and tumor volume and weight were then challenged. The development of tumors was followed by calliper measurement. Data are means±SE of 8 tumors in each group. *** p<0.01 versus control.

EXAMPLE

Example 1. Material & Methods

[0078] Immunohistochemical Procedure

[0079] Deparaffinized sections (15-μm thick) from 5 BPH and 15 prostate tumors at various stages (Gleason scores: 3+3, 4+3 and 4+5) were obtained from the Department of Pathology of the University Hospital of Rouen. CaP sections were incubated for 1 h at room temperature with rabbit polyclonal antibodies against orexin A (#AB3704, Millipore, Billerica, Mass.) diluted 1:1000, or to OX1R (#PAB8017, Abnova, Taipan, Taiwan) diluted 1:250, or to OX2R (#OX2R22-A, Alpha Diagnostic International Inc, San Antonio, Tex.) diluted 1:250, or to EM66 19 diluted 1:600, or to α-actin (#AB5694, Abcam, Paris, France) diluted 1:500, or to protein gene product PGP9.5 (# AB1761, Chemicon International, Temecula, Calif.) diluted 1:250. The sections were incubated with a streptavidin-biotin-peroxydase complex (Dako Corporation, Carpinteria, Calif.), and the enzymatic activity was revealed with diaminobenzidine. The slices were then counterstained with hematoxylin. Observations were made under a Nikon E 600 light microscope.

[0080] The specificity of the immunoreactions was controlled by (1) substitution of the primary antibodies with Tris buffer saline (TBS; pH 7.4) and (2) preincubation of the orexin A antiserum (diluted 1:1000) with synthetic human orexin A (10-6 M; Tocris Bioscience, Bristol, UK), or preincubation of the EM66 antiserum (diluted 1:600) with recombinant human EM66 (10-6 M).

[0081] OX1R-immunoreactive cells present in the adenocarcinomatous masses (for the CaP sections) or the acini (for the BPH sections) were quantified. For this, 5 independent fields of 20 distinct sections of BPH or prostate tumors were photographed at a 20× magnification. The number of immunostained cells present in each image was evaluated by using the cell counter plugin of the image analysis software Image J, and expressed as a percentage of the total number of cells present on the photomicrograph.

[0082] Cancer Cell Lines

[0083] Two human-derived CaP cell lines were used, i.e. the androgen-responsive cell line LNCaP, and the androgen-unresponsive cell line DU145 as well as the colorectal adenocarcinoma HT-29 cell line. The three cell lines were purchased from American Type Culture Collection (ATCC, Rockville, Md.). The LNCaP cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% glutamine (complete medium). The DU145 cells were maintained in Dulbecco modified Eagle's minimal essential medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin (complete medium). The HT-29 cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 1% glutamine. The cells were grown at 37° C. in a humidified 95% air/5% CO2 atmosphere and the culture medium was replaced every 3 days. When the cells reached 70-80% confluence, they were washed with phosphate buffer saline (PBS; pH 7.4), harvested by a brief incubation with 0.25% trypsin-EDTA solution, and seeded as suggested by ATCC.

[0084] RNA Extraction, Reverse Transcription and Quantitative PCR

[0085] Total RNA from cell lines was extracted with the Tri-reagent (Sigma-Aldrich, Lyon, France), purified by using a Nucleospin kit (Macherey-Nagel, Hoerdt, France), and quantified with a Nanodrop spectrometer (Nanodrop Technologies, Wilmington, Del.). Contaminating genomic DNA was removed by treatment with deoxyribonuclease I, and cDNAs were synthesized from 1 to 5 μg RNA using the ImProm II Reverse Transcriptase (Promega Corp., Madison, Wis.). Quantitative PCR was performed by using the 7900 HT Fast Real-time PCR System and Gene Expression Master Mix 2× assay (Applied Biosystems, Courtaboeuf, France). OX1R and hypoxanthine ribosyltransferase (HPRT1) primers were designed by using Primer Express software version 3.0: HPRT1 forward primer 5′-GACTTTGCTTTCCTTGGTCAGGCA-3′(SEQ ID NO:4); HPRT1 reverse primer 5′-ACAATCCGCCCAAAGGGAACTGA-3′(SEQ ID NO:5); OX1R forward primer 5′-GTGGGCAACACGCTGGTCTG-3′(SEQ ID NO:6); OX1R reverse primer 5′-GGCCCCAGAGCTTGCGGAAT-3′(SEQ ID NO:7). The purity of the PCR products was assessed by dissociation curves. The amount of target cDNA was calculated by the comparative threshold (Ct) method and expressed by means of the 2-ΔΔCt method according to Applied Biosystems instructions using HPRT1 as an internal control. Expression of HPRT1 mRNA was not affected by treatments or the nature of the tissues, and the ratio of ΔCt value did not vary with the amount of cDNA.

[0086] Neuroendocrine Differentiation of DU145 Cells

[0087] DU145 cells were seeded at a density of 5×104 cells/well in 12-well plates and starved 24 h in minimum medium (complete medium in which 10% FBS was replaced by 1% FBS). Neuroendocrine differentiation of the cells was carried out by adding to the culture medium 1 mM dituryl-cyclic adenosine monophosphate (db-cAMP)/0.1 mM 3-isobutyl-1-methylxanthine (IBMX). Medium was changed every day until day 5.

[0088] Ability of db-cAMP/IBMX to induce a neuroendocrine differentiation of the DU145 cells was assessed by quantifying the increase of expression of three markers of neuroendocrine differentiation, i.e. chromograninA, secretogranin II and neurone specific enolase, and the occurrence of neurite-like extensions as previously described14.

[0089] Viability and Apoptosis of DU145 Cells

[0090] Cell viability was determined by using the CellTiter-Blue® assay (Promega, Charbonnières, France). Cell apoptosis was quantified by evaluating the enzymatic activities of Caspase 3/7 by using the Apo-ONE® Homogeneous Caspase-3/7 Assay (promega). Briefly, the enzymes were measured in intact and differentiated DU145 culture cells after a 3-days incubation with OxA or OxB (10-6M) in 96-well plates. The cells were incubated with fluorogenic peptide specific to caspase 3 and 7 enzymes, and detection of fluorescent product over time was monitored in a spectrofluorometer Flex Station 3 (Molecular Devices, St. Grégoire, France) at 485 nm excitation and 527 nm emission. Data were normalized to equivalent cell numbers per treatment group.

[0091] Statistical Analysis

[0092] All of the experiments were performed in triplicate and repeated at least three times. Results are expressed as mean±SEM. All statistical analyses were performed with GraphPad Prism 4 data analysis software. The Mann-Whitney U test was used for comparison of the mean values between two groups. Differences were considered statistically significant at *: p<0.05, **: p<0.01, ***: p<0.001.

Example 2. Results

[0093] 2.1. Immunohistochemical Distribution of Orexin Type 1 Receptor and Orexin A in Prostate Tumors

[0094] The distribution and localization of OX1R and OxA was investigated on BPH and CaP sections at various stages (Gleason's score 3+3=low grade, 4+3=medium grade and 4+5=high grade). In BPH, OX1R-like immunoreactivity (LI) was confined to scattered cells observed just in a few acini (data not shown). In low and medium grade CaP, some cancerous foci contained cells positive for OX1R (FIG. 1A, B). In high grade CaP, the adenocarcinomatous formations were heavily labelled with the OX1R antibodies (FIG. 1C), and a vast majority of the malignant cells exhibited OX1R-LI (FIG. 1D). In contrast, OX2R was only detected in a few cancer cells only in high grade CaP. Treatment of consecutive sections with either OX1R antibodies, or antibodies directed against the peptide EM66 (which is a marker of neuroendocrine differentiation) revealed that OX1R and EM66 were present in the same carcinomatous formations, whatever the grade of the tumor (FIGS. 1E and F).

[0095] OxA-LI was never observed in cancerous foci whatever the grade of the CaP (FIG. 2A). In contrast, “fiber-like” structures of the stroma were immunostained with the OxA antibodies (FIG. 2B, C, E). Treatment of consecutive sections with the OxA or α-actin (a specific marker of smooth muscle fibers) antibodies revealed that the two antibodies did not label the same structures in the stroma (FIG. 2C, D). Similarly, FIGS. 2E and F show that OxA and PGP 9.5 (a specific marker of nerve fibers) antibodies did not label the same structures.

[0096] Quantification of immunoreactive cells revealed that the number of OX1R-expressing cells present in the CaP sections was significantly higher (p<0.001) compared to BPH sections (FIG. 3). In addition, the percentage of OX1R-positive cancer cells increased significantly (p<0.05) with the severity of the CaP (FIG. 3).

[0097] 2.2. Expression of OX1R mRNA in Prostate Cancer Cell Lines

[0098] Quantitative RT-PCR performed in two distinct prostate cancer cell lines, i.e. the AD LNCaP and the AI DU145 cells, revealed that the OX1R gene was only expressed in the AI cell line (FIG. 4A). DU145 cells exhibiting a neuroendocrine phenotype showed a highly significant increase of OX1R mRNA expression as compared to the native cells (p<0.001; FIG. 4B), that was confirmed by a western-blot analysis (FIG. 4C). In contrast, induction of a neuroendocrine differentiation in LNCaP cells did not promote the expression of the OX1R gene (FIG. 4B).

[0099] 2.3. Effects of Orexins on the Apoptosis and Viability of DU145 Cells

[0100] Treatment of native DU145 cells with OxA or OxB (10-6 M each) for 3 days did not alter apoptosis (FIG. 5A, B) but increased cell growth (p<0.05). When the cells were submitted to a neuroendocrine differentiation, incubation with Orexins induced a significant apoptosis (p<0.05; FIG. 5C, D) without altering the number of viable cells. In the same set of experiments, HT-29 cells, used as a positive control, were treated with orexins. As previously described16, OxA and OxB promoted significantly the apoptosis of HT-29 cells (p<0.001 and p<0.01 respectively; FIG. 5E, F) and inhibited cell growth (p<0.05).

[0101] 2.4. Discussion

[0102] This is the first report investigating the presence and function of orexins and their receptors in CaP. The immunohistochemical experiments indicate that the type 1 orexin receptor (OX1R) is present in abundance in carcimatous foci of CaP sections whereas it is virtually absent of non-cancerous prostate tissues. We have performed the same experiments for OX2R and we have found that the orexin type 2 receptor is present in a few cancer cells only in high grade CaP. Such an observation has already been made in colorectal cancer that shows an ectopic expression of the OX1R but not of the OX2R16. We have thus decided to focus our study on OX1R. The quantitative analysis reveals that the number of OX1R-stained cells in the adenocarcinomatous masses increases with the grade of the cancer, and that the orexin receptor is exclusively present in cancerous structures labeled with EM66, a fragment of the granin secretogranin II which has been previously identified as a marker of neuroendocrine differentiation19. Altogether, these findings support the view that the expression of OX1R is closely associated with advanced CaP and the acquisition of a neuroendocrine phenotype by CaP cells. Consistent with this hypothesis, our in vitro data reveal that OX1R is expressed in the AI cell line DU145, but not in the AD cell line LNCaP, and that induction of a neuroendocrine differentiation in the DU145 cells results in an important increase of OX1R expression and production.

[0103] The presence of the endogenous ligand of OX1R was also investigated in CaP sections. OxA-LI was detected in “fiber-like” structures of the stroma that do not correspond to smooth muscle fibers or nerve fibers. These OxA-stained structures are probably fibroblasts but this cannot definitely be confirmed as no specific markers of fibroblasts are available. Anyway, in all of the CaP sections treated, we have never observed OxA-labeling in the cancerous foci whatever the grade of the CaP. This observation strongly suggests that OX1Rs expressed by prostate cancer cells might not be activated by a paracrine loop as the stroma structures exhibiting OxA-LI were always located at distance from the cancerous foci. Activation of OX1R in CaP by circulating orexins may be a possibility. However, levels of orexins in human plasma are very low (between 2 and 40 pmol/L) 20 in comparison of the 7 nmol/L Kd of the human OX1R21, making unlikely the activation of OX1R in CaP by circulating orexins. Altogether, our data indicate that OX1R ectopically expressed by prostate cancer cells is probably not activated by endogenous orexins in vivo.

[0104] The in vitro experiments reveal that orexins have no effect on the apoptosis of native DU145 cells but stimulate their growth. Consistent with this finding, it has been recently shown that orexin A stimulates INS-1 rat insulinoma cell proliferation via interaction with the OX1R22. In contrast, when the DU145 cells are submitted to a neuroendocrine differentiation, OxA and OxB promote cell death. As trans-differentiated DU145 cells overexpress OX1R, this supports the idea that OX1R level is determinant for the induction of apoptosis as previously suggested for colon cancer cells16. Such an observation has already been reported for the angiotensin II type 2 receptor (AT2R) in hepatocellular carcinoma, in which high levels of AT2R trigger apoptosis while low levels of the receptor do not impact cell death23. The mechanisms underlying OX1R-driven apoptosis in DU145 cells are still unknown. However, it has been shown that transfection of OX1R in CHO cells (devoid of endogenous receptors) is sufficient to confer the ability of orexins to promote apoptosis, and that, in this model as in colon cancer cells, activation of OX1R results in the recruitment and activation of the phosphotyrosine phosphatase SHP-2, and subsequent cytochrome C-mediated mitochondrial apoptosis24. Whether such an intracellular pathway is activated in DU145 cells showing a neuroendocrine phenotype deserves further investigation. The observation that OxA-induced apoptosis increase in DU145 cells with a neuroendocrine phenotype is not associated with a decrease of cell proliferation may be questioning. However, such a phenomenon has already been observed in the same cell line, as it has been reported that inactivation of miR-21 in DU145 cells results in an increase of apoptosis and inhibition of cell motility and invasion, whereas cell proliferation is not affected25.

[0105] In conclusion, the present data show that OX1R-driven apoptosis is strongly expressed in AI CaPs showing a neuroendocrine differentiation, opening new perspectives for the treatment of these advanced CaPs which are the most aggressive and for which no treatment is available until now.

Example 3: In Vivo Results

[0106] Effect of inoculation of orexin-A on the growth of tumors developed by xenografting human DU-145 cells in nude mice is shown in FIGS. 6 A and B

[0107] DU-145 cells were inoculated in the flank of nude mice at day 0. Mice were injected (2 injections/week) intraperitoneally with 100 μl of orexin-A solutions (0.22 μmoles of orexin-A/Kg) starting at day 1 (white circles) or day 26 (white triangles) or with 100 μl of PBS (black circles) for controls (FIG. 6A). After 45 days of treatment, mice were sacrificed and tumor volume and weight were then challenged (FIG. 6B). The development of tumors was followed by calliper measurement. Data are means±SE of 8 tumors in each group. *** p<0.01 versus control.

REFERENCES

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