WO2009016385A1

WO2009016385A1 - Methods of aiding in the diagnosis of prostate cancer

Info

Publication number: WO2009016385A1
Application number: PCT/GB2008/002618
Authority: WO
Inventors: Simak Ali; Laki Buluwela; Jonathan Waxman; Sarah Ngan
Original assignee: Imperial Innovations Limited
Priority date: 2007-07-31
Filing date: 2008-07-31
Publication date: 2009-02-05
Also published as: GB0714842D0

Abstract

A method for aiding in the assessment of prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia in a patient, the method comprising the step of determining the level of CNP nucleic acid and/or protein in a sample from the patient. The patient may be at risk of developing prostate cancer, or have already been diagnosed with prostate cancer, or may have undergone treatment for prostate cancer. The method may be for assessing the likely progression of prostate cancer in the patient, or for assessing prostate function, or for assessing and/or predicting the development of prostate cancer. The method may be for diagnosing prostate cancer in the patient or for assessing the response of the patient to treatment for prostate cancer.

Description

METHODS OF AIDING IN THE DIAGNOSIS OF PROSTATE CANCER

The current invention relates to the diagnosis of prostate disorders including prostate cancer.

Background

Prostate cancer is the most commonly diagnosed cancer in males in the developed world and is the second leading cause of male cancer death.

The growth and development of prostate cancer is stimulated by androgens. Androgens are male sex hormones, such as testosterone. Treatment for prostate cancer is directed towards inhibiting cancer growth by the suppression of endogenous androgen action or the suppression of androgen synthesis. Standard treatment involves androgen ablation therapies, which are mediated either surgically by bilateral orchidectomy, or pharmacologically by the action of anti- androgens (Carter & Coffey (1990) Prostate 16: 39-48; McConnell (1991) Urol. Clin. North Am. 18: 1-13).

Androgen action is mediated by the androgen receptor (AR), which is a transcription factor of the Nuclear Receptor superfamily. Many prostate cancers have AR gene amplification or mutation. The androgen-bound AR stimulates prostate cancer growth through activation of a transcriptional programme that promotes cancer cell proliferation and survival. This is reviewed in Agoulnik & Weigel (2006) J. Cell. Biochem. 99: 362-372. Additionally, other reviews relating to AR and prostate cancer can be found in J Cell Biochem vol 99, issue 2 and vol 91 issue 3.

The use of prostate-specific antigen (PSA) screening over the last 20 years has improved detection of early disease. However, PSA testing has been found to fail in identifying a small but significant proportion of aggressive cancers, while only about 30% of men with a "positive" PSA have a positive biopsy. There is a need for additional, more accurate biomarkers that not only detect prostate cancer but also distinguish indolent from aggressive disease (reviewed in Bradford et al, (2006) Urol. Oncol 24: 538-551 and Steuber et al (2007) World J. Urol. 25: 111- 119).

Description of the invention

Following gene expression microarray analysis and quantitative RT-PCR we have shown that expression of the gene encoding the C- type natriuretic peptide (CNP) is potently regulated by androgens in AR-positive prostate cancer cell lines. We have identified, for example, that the CNP gene shows a 35-fold stimulation of expression within 4 hours of androgen (Rl 881) addition, as shown in Example 1.

Of a 308 up-regulated gene set, CNP gene expression was found to be the most highly induced in the LNCaP prostate cancer cell line with other activators of AR, and was inhibited by the anti-androgen bicalutamide (see Example 1).

C-type natriuretic peptide (CNP) is secreted and can be found at low levels in serum and urine (Clavell et al (1993) Am. J. Physiol. 264: R290-R295) and has a restricted pattern of expression, being mainly found in the Central Nervous System (Komatsu et al. (1991) Endocrinology) 129:1104-1106). Further, CNP can act to stimulate steroidogenesis and testosterone production (El-Gehani et al. (2001) Biol. Reprod. 65: 595-600; Khurana & Pandey (1993) Endocrinology 133: 2141-2149).

The CNP gene name is NPPC; UniGene Hs.247916 [http://wvvw.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=247916]. See, for example, Accession number NM_024409 for the human CNP protein and mRNA sequence; NM_174125 for the bovine sequence and NM_010933 for the mouse sequence. Sequences for CNP from numerous other mammals are available in public databases. The human CNP precursor sequence, as shown in Accession number NM_024409 is:

MHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGQKKGDKAPGGGGANLKG DRSRLLRDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC

1 atgcatctct cccagctgct ggcctgcgcc ctgctgctca cgctgctctc cctccggccc

61 tccgaagcca agcccggggc gccgccgaag gtcccgcgaa ccccgccggc agaggagctg 121 gccgagccgc aggctgcggg cggcggtcag aagaagggcg acaaggctcc cgggggcggg

181 ggcgccaatc tcaagggcga ccggtcgcga ctgctccggg acctgcgcgt ggacaccaag

241 tcgcgggcag cgtgggctcg ccttctgcaa gagcacccca acgcgcgcaa atacaaagga

301 gccaacaaga agggcttgtc caagggctgc ttcggcctca agctggaccg aatcggctcc

361 atgagcggcc tgggatgtta g

CNP is the most highly conserved member of the natriuretic peptide family. It comprises a 17 amino acid ring structure formed by a disulphide bridge, which is important for receptor binding (Scotland et al (2005) Pharmacol. Ther. 105: 85- 93). As with the other natriuretic peptides, CNP encodes a pre-pro-peptide. Following cleavage of the signal peptide, a 126 amino acid residue pre-pro-CNP (in humans, see precursor sequence above) is converted to the pro-peptide and stored. Pro-CNP is subsequently cleaved by furin in the trans-Golgi network to yield a 55 amino acid peptide; CNP53. CNP53 is secreted or further processed to yield a 22 amino acid peptide; CNP22. The CNP22 fragment is the more mature and biologically active form (Scotland et al (2005) Pharmacol. Ther. 105: 85-93; Baxter (2004) Basic Res. Cardiol. 99: 71-75).

In all aspects of the current invention the term "CNP" includes the 126 amino acid CNP precursor (with or without the signal sequence), the shorter pro-peptide, the CNP53 peptide and/or the CNP22 peptide.

The expression and role of CNP in prostate cancer is presently unexplored. The work described in the current invention is the first to identify such a link. The measurement of CNP levels in biological fluids provides quantitative data on disease activity, and provides a new bio marker to monitor patients' response to treatment. It also provides a biomarker to assess effects of new treatments for prostate cancer. Also, the measurement of CNP levels allows investigations into the role of CNP in regulating the growth of prostate cancer cells.

A first aspect of the invention provides a method for aiding in the assessment of prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia in a patient, wherein the method comprises the step of determining the level of CNP nucleic acid and/or protein in a sample from the patient. The CNP nucleic acid or protein may be the full precursor, pre-pro-CNP, pro-CNP or mature CNP (i.e. CNP53 or CNP22) nucleic acid or protein. The CNP may also be measured in conjunction with PSA levels in an embodiment of this aspect of the invention. Combining measuring CNP and PSA levels may provide a more robust and/or definitive assessment of disease state.

The patient may be at risk of developing prostate cancer, for example on the basis of age but may not yet have shown clinical signs of the disease. For example, men older than about 60 years may be at greater risk of prostate cancer than men below the age of 35. Alternatively, the patient may already have been diagnosed with prostate cancer and may or may not have begun treatment therefor. Additionally, the patient may have already undergone treatment for prostate cancer. The patient who has already undergone treatment for prostate cancer may still have signs of the disease or they may have gone into 'remission'. It is envisioned that the method of the current invention will be useful in the assessment of, for example, prostate cancer at all the above mentioned 'stages' of the disease.

It is preferred that the sample is a urine sample but the sample may also be, for example, a blood sample, a blood serum sample, a blood plasma sample, a lymph sample, a sample of seminal fluid or any other sample of body fluid where CNP secretion may be a sign of prostate cancer. The CNP protein or nucleic acid may be contained within cells in these samples or it may be extracellular. The CNP may also be measured in biopsies of suspected cancers. The measurement of mRNA levels in cells in the blood, urine or seminal fluid may provide indications of synthesis of CNP. Alternatively, detection of CNP protein by immunohistochemistry and mRNA by in situ hybridisation and real time RT-PCR from prostate biopsied material (or, for example enriched prostate cells, or cells identified as prostate cells, for example as discussed below) may also be useful. Using urine samples and/or samples of seminal fluid may be more convenient and may also be particularly informative, as the urine and/or seminal fluid may accurately reflect prostate conditions.

It is preferred that if blood, seminal fluid, lymphatic circulation or urine is the source of the said sample containing nucleic acid derived from the patient that the sample is enriched for prostate-derived tissue or cells. Enrichment for prostate cells may be achieved using, for example, cell sorting methods such as fluorescent activated cell sorting (FACS) using a prostate-selective antibody such as one directed to prostate-specific membrane antigen (PSMA) (Silver et ah, (1997) Clinical Cancer Research 3: 81-85). Alternatively, enrichment may be achieved using magnetic beads or other solid support, for example a column, coated with such a pro state- specific antibody, for example an anti-PSMA antibody. Other examples of antigens that may be suitable in methods of enrichment/purification of prostate cells are epithelial cell surface antigens, which would also facilitate the purification of tumour/epithelial cells from fluids such as blood. Alternatively, cells in the sample may be identified as prostate cells, for example on the basis of prostate-selective antibody/antigens as discussed above without necessarily enriching the cells in the sample. The source of the said sample also includes biopsy material and tumour samples, also including fixed paraffin mounted specimens as well as fresh or frozen tissue.

The method may be used for assessing the likely progression of prostate cancer, metastatic prostate cancer and/or benign hyperplasia in the patient. The method may also be useful for aiding in the diagnosis of, or diagnosing, prostate cancer, metastatic prostate cancer and/or benign hyperplasia in the patient. The method may also or alternatively be useful for aiding in the assessment of the likelihood or likely severity or likely progression of prostate cancer in the patient. This may include assessing the likelihood of the development of complications associated with prostate cancer in the patient, for example arising from metastasis of the prostate cancer. The method may also be useful for assessing prostate function. The method may be useful for distinguishing between benign hyperplasia, prostate cancer and metastatic prostate cancer. This in turn may aid in improving the outcome of these conditions by allowing physicians more accurately to assess these conditions and provide the most appropriate treatments.

The method may be useful for assessing and/or predicting the development of prostate cancer in the patient. CNP protein or mRNA levels may also be used as a surrogate marker for the development of prostate cancer.

The screening of prostate cancer patients for changes in CNP protein and/or mRNA levels may be useful for diagnosing those patients that may have or may develop complications associated with prostate cancer.

By "complications associated with prostate cancer" is included such instances as failure to respond to therapy. This may include endocrine therapies (e.g. luteinising hormone-releasing hormone (LHPsH) agonists and anti- androgens such as flutamide) and/or chemotherapy. Also, any other unexpected or undesirable outcome in the patient, which is associated with the prostate cancer, may be termed a 'complication'. Typically, PSA levels are used to monitor the patient's response to treatment and rising PSA levels may be a possible sign of the emergence of resistance to the therapy and/or the re-growth of the tumour.

By "prostate cancer" is included any condition of the cells or tissues of the prostate that has arisen through abnormal cell growth originating from tissues/cells of the prostate. This may include pre-cancerous stages distinguished by abnormal cell growth at one end of the spectrum to metastatic prostate cancer as a more severe form of the disease. Benign hyperplasia may be considered a precancerous stage in some instances. The response of the patient to treatment for prostate cancer may be assessed using the method of the current invention. Such treatment may be for prostate cancer or any prostate related disease with which CNP up-regulation or down-regulation has been associated. Thus, the method may be useful in predicting the future response of the patient to treatment for prostate cancer. The method of the current invention may also be used for assessing the likely progression of response of the patient to treatment for prostate cancer. It may also be useful in prognosis or aiding prognosis.

The normal course of action when deciding whether to treat a patient for prostate cancer, or indeed initially diagnosing the condition, consists of assessing the level and activity of PSA in the patient's urine and assessing the size of the cancer by conducting rectal examinations. This is then followed by a period of "watchful waiting", when surveillance of PSA levels is carried out (Parekh et al (2007) J Natl. Cancer Inst. 99: 496-97; Fall et al. (2007) J. Natl. Cancer Inst. 99: 526-532). When changes occur in these parameters physicians are more likely to take action. The reasons for these delays include the potential of subjecting men who would otherwise live healthy lives with indolent cancers to cancer treatments, which are often accompanied by unpleasant side-effects. By assessing the level of CNP in conjunction with PSA levels, the accuracy of diagnosis and assessment of the need for intervention may be increased. This in turn may lead to a significant improvement in the quality of life of patients. Patients that do not need treatment may be spared the effects of treatment and patients who may not have been considered to be in need of treatment may be provided with life-saving treatments.

The method of the current invention may also be used for choosing patients for treatment for prostate cancer or for monitoring response of patients to treatment or for monitoring relapse in patients. These treatments may include, but are not limited to, endocrine therapies such as anti-androgens such as flutamide, or other endocrine treatments such as luteinising hormone-releasing hormone (LHRH) agonists. On the basis of such selection the patients may be grouped, for example, into particular patient groups for clinical trials. It is envisaged that clinical trial data may be assessed using the method of the current invention. The method may be used, for example, for assessing the progress of patients in clinical trials. The use of CNP as a biomarker in clinical trials of prostate cancer patients may aid in the assessment of, for example, remission of the cancer.

The method of the current invention may comprise the steps of (i) obtaining a sample containing nucleic acid and/or protein from the patient; and (ii) determining whether the sample contains a level of CNP nucleic acid or protein associated with the development, progression or regression (after appropriate treatment) of prostate cancer.

It will be appreciated that determining whether the sample contains a level of CNP nucleic acid or protein associated with prostate cancer may in itself be diagnostic (or prognostic) of prostate cancer or it may be used by the clinician as an aid in reaching a diagnosis or prognosis.

Thus, measurement of CNP levels may be performed or considered alongside other measurements or factors, for example, determining the level of PSA, in the sample from the patient and/or measuring the size of any suspect cancer in the patient through digital rectal examination. Any physical examination may also include taking biopsies of suspected cancerous tissue or monitoring other physical indicators of cancer as appropriate. Simultaneous measurement of other hormones or factors may be helpful, such as for example, blood IGF-I level (Chan et al (1998) Science 279: 563-566). Measurement of CNP levels may provide more detailed information on the severity of individual disease mechanisms.

It will be appreciated that determination of the level of CNP in the sample will be useful to the clinician in determining how to manage prostate cancer in the patient. For example, since our research has indicated that elevated levels of CNP are associated with prostate cancer, the clinician may use the information concerning the levels of CNP to facilitate decision making regarding treatment of the patient. The level of CNP which is indicative of prostate cancer may be defined as the increased level present in samples from patients with prostate cancer relative to levels present in samples from control healthy volunteers. The level of said CNP protein may be, for example, at least 2 standard deviations higher in a sample from a patient with prostate cancer than the control healthy volunteers. The level of mRNA encoding CNP may be, for example, at least 2 standard deviations higher in a sample from a patient with prostate cancer.

The level of CNP in a sample from the patient may be determined using any suitable protein detection or quantitation method, for example using methods employing antibodies specific for CNP. Thus, immunoassay techniques, preferably quantitative techniques, may be used, for example an antibody array or captured ELISA technique, for example as described in the Examples. Preferred embodiments relating to methods for detecting CNP protein include enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), irnmunoradiometric assays (IRJVlA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies.

Exemplary sandwich assays are described by David et al in US Patent Nos.

4,376,110 and 4,486,530, hereby incorporated by reference. Other techniques include: Beads-based immunoassay using Luminex type machine; Antibody arrays

(including membrane based, or glass based); Proteomic analysis (mass spectromotery, antibody coated biochips using SELDI-TOF technique).

It will be appreciated that other antibody- like molecules may be used in the method of the invention including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs.

A suitable ELISA assay for pro-CNP can be obtained from Biomedica. Medizinprodukte GmbH, Germany. Bioassays may alternatively be used for measuring CNP activity, although this may not be preferred, as it may not be convenient to carry out routine assays in this way. CNP related peptide hormones have been shown to inhibit prostate cancer cells (Vesely et al. (2005) Eur. J. CHn. Invest. 35: 700-710) and to affect smooth muscle tone, thereby acting as a vasodilator in blood vessels in smooth muscle component of the prostate. Bioassays may utilise one or more of these properties and give an indication of the activity of CNP in a sample.

As noted above, the level of mature CNP, pro-CNP, pre-pro-CNP or full CNP (unprocessed precursor) nucleic acid and/or protein may be measured. Measurement of full CNP may be most appropriate in cancer biopsies rather than serum or urine (in which the processed forms may be more abundant).

In one preferred embodiment of the invention it is determined whether the level of CNP nucleic acid, in particular mRNA, is a level associated with prostate cancer. Preferably, the sample contains nucleic acid, such as mRNA, and the level of CNP is measured by contacting said nucleic acid with a nucleic acid which hybridises selectively to CNP nucleic acid. This may typically be in the context of a Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) using at least one primer specific to the cnp gene encoding CNP, conducted using standard protocols available in the art. Thus, the PCR primer is an example of a nucleic acid which hybridise selectively to CNP nucleic acid. RT-PCR may be directed towards regions within the coding region of cnp or alternatively to the 5' and/or 3' untranslated regions, as will be well known to those skilled in the art.

By "selectively hybridising" is meant that the nucleic acid has sufficient nucleotide sequence similarity with the said human nucleic acid that it can hybridise under moderately or highly stringent conditions. As is well known in the art, the stringency of nucleic acid hybridisation depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and GC or AT content of the sequence. Thus, any nucleic acid that is capable of selectively hybridising as said is useful in the practice of the invention.

Nucleic acids which can selectively hybridise to the said human nucleic acid include nucleic acids which have > 95 % sequence identity, preferably those with > 98 %, more preferably those with > 99 % sequence identity, over at least a portion of the nucleic acid with the said human nucleic acid. As is well known, human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene would not match perfectly along its entire length with the said human genomic DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said human DNA. Thus, the invention specifically includes nucleic acids which selectively hybridise to CNP mRNA or cDNA but may not hybridise to a CNP gene. For example, nucleic acids which span the intron-exon boundaries of the CNP gene may not be able to selectively hybridise to the CNP mRNA or cDNA.

Typical moderately or highly stringent hybridisation conditions which lead to selective hybridisation are known in the art, for example those described in Molecular Cloning, a laboratory manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. USA, incorporated herein by reference. An example of a typical hybridisation solution and protocol is provided in, for example, WO 02/18637.

By "nucleic acid which selectively hybridises" is also included nucleic acids which will amplify DNA from the said CNP mRNA by any of the well known amplification systems, in particular the polymerase chain reaction (PCR), as noted above.

Although the nucleic acid which is useful in the methods of the invention may be RNA or DNA, DNA is preferred, for example if assessing the patient for CNP polymorphisms. If assessing expression levels then mRNA may be preferred.

Although the nucleic acid that is useful in the methods of the invention may be double-stranded or single-stranded, single- stranded nucleic acid is preferred under some circumstances such as in nucleic acid amplification reactions.

The nucleic acid that is useful in the methods of the invention may be any suitable size. However, for certain diagnostic, probing or amplifying purposes, it is preferred if the nucleic acid has fewer than 10 000, more preferably fewer than

1000, more preferably still from 10 to 100. and in further preference from 15 to 30 base pairs (if the nucleic acid is double-stranded) or bases (if the nucleic acid is single stranded). As is described more fully below, single- stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.

The nucleic acid for use in the methods of the invention is a nucleic acid capable of hybridising to the CNP mRNA. Fragments of the CNP gene and cDNAs derivable from the mRNA encoded by the CNP gene are also preferred nucleic acids for use in the methods of the invention.

It is particularly preferred if the nucleic acid for use in the methods of the invention is an oligonucleotide primer which can be used to amplify a portion of the CNP nucleic acid, particularly CNP mRNA.

Methods and nucleic acids as described in the examples may be used. In particular, a semi-quantitative PCR technique, for example as described in Example 2, may be used.

It is preferred if the nucleic acid is derived from a sample of the tissue in which prostate cancer is suspected or in which prostate cancer may be or has been found. Samples of prostate for example, may be obtained by surgical excision, laproscopy and biopsy, endoscopy and biopsy, and image-guided biopsy. The image for use in obtaining samples using image-guided biopsies of prostate tissue may be generated by ultrasound or by technetium-99-labelled antibodies or antibody fragments which bind or locate selectively at the prostate. Although any sample containing nucleic acid derived from the patient is useful in the methods of the invention, it is preferred if the sample is selected from the group consisting of prostate tissue, blood, urine or semen. Prostate tissue can be obtained from a patient using standard surgical techniques. Cells derived from the prostate are found in small numbers in the urine and in the blood. If necessary these cells can be enriched from the patient sample, as discussed above. Although it is preferred that the sample containing nucleic acid from the patient is, or is derived directly from, a cell of the patient, such as a prostate cell, a sample indirectly derived from a patient, such as a cell grown in culture, is also included within the invention. Equally, although the nucleic acid derived from the patient may have been physically within the patient, it may alternatively have been copied from nucleic acid which was physically within the patient. The tumour tissue may be taken from the primary tumour or from metastases, and particularly may be taken from the margins of the tumour.

A second aspect of the invention provides a method for assessing a prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia treatment regime, the method comprising the step of determining the level of CNP nucleic acid and/or protein in a sample from patients receiving the treatment regime. The sample type is typically of the type discussed above in relation to the first aspect of the invention, for example, a urine sample from the patient. The method may, for example, be used to provide information on the likelihood of the development of complications of prostate cancer in the patient. Thus, levels of CNP may be used as surrogate markers in clinical trials of proposed treatments for prostate cancer. Measurement of CNP may provide an overall assessment of how various factors affect the treatment of and progression of prostate cancer.

A third aspect of the invention provides a method for identifying a compound useful in modulating prostate function, for example in treating or preventing prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia, the method comprising the steps of a) determining whether a test compound is capable of suppressing production of, or activity of, CNP in prostate tissue or a sample from a patient with, for example, prostate cancer and b) selecting a compound which is capable of suppressing production of, or activity of, CNP in prostate tissue or a sample from a patient with, for example, prostate cancer. Other organ tissues may be more accessible for testing than the prostate. For example cells, which may be cancer cells, shed in the urine of the patient may be used. The compound may be administered to the patient or may be applied in vitro to the cells.

The method may comprise the step of determining whether a test compound is capable of suppressing production of, or activity of, CNP in a sample, for example a urine sample from a patient, as discussed hereinbefore.

In all aspects of the invention the patient is typically a human but may alternatively be another mammal.

An in vitro model may be most appropriate for performing the immediately preceding aspect of the invention. Thus, it may be appropriate to test compounds for an effect on production or activity of CNP in an in vitro model system, for example in which the compound is applied in vitro to the cells.

Examples of appropriate in vitro models are:

(i) Primary culture of prostate cells/tissue from a patient with prostate cancer;

(ii) Human prostate cell lines; (iii) Primary culture human cells from an uninvolved part of the prostate removed from patients with prostate cancer.

The test compound may be a small molecule, polypeptide or genetic construct, as will be well known to those skilled in the art. Compounds identified in the methods may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds. The compound may be a drug-like compound or lead compound for the development of a drug-like compound for each of the above methods of identifying a compound. It will be appreciated that the said methods may be useful as screening assays in the development of pharmaceutical compounds or drugs, as well known to those skilled in the art.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, nonselective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

Any documents referred to herein are hereby incorporated by reference. The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention will now be described in more detail by reference to the following, non- limiting, Figures and Examples. Figure legends

Fig. 1. Characterisation of androgen responses in LNCaP cells. (A) LNCaP cell growth is stimulated by the synthetic androgen Rl 881 (1 nM), as measured using the Sulphorhodamine B cell staining assay. (B) Immunoblotting of whole cell lysates following androgen treatment in LNCaP cells. Cells were grown in androgen-depleted medium for 72 hrs, prior to the addition of Rl 881 for 36 hrs. Results of lysates prepared from duplicate cultures for each treatment are shown for the androgen-regulated genes PSA and DRGl.

(C) Secreted PSA levels were measured for culture medium from three replicate cultures grown as in part B, using the Architect chemiluminescence total PSA kit

(Abbott, UK), according to manufacturer's protocols.

(D) RT-PCR analysis of PSA and DRGl in RNAs prepared from LNCaP cells treated with Rl 881 over the time-course shown. From this analysis and subsequent real-time RT-PCR, it was concluded that 24 hours treatment with Rl 881 is optimal for microarray analysis of androgen responses.

(E) Cluster analysis of genes demonstrating androgen regulation by 2-fold or greater in LNCaP cells following 24 hour treatment with Rl 881. For this, bioreplicate cultures were prepared and the extracted RNA subject to quality assays and validated by real-time RT-PCR for two control genes (GAPDH and RPLPO) and the androgen regulated genes PSA, DRG-I and GREB-I. These analyses showed that there was little variation between replicates and that the RNAs were of the highest quality. Three replicates from each treatment set were subsequently used for hybridisation to the Applied Biosystems (ABI) Human Genome Survey Microarray V2.0, which has probes for 29, 098 genes. Raw data was quality assessed and filtered according to the recommendations supplied by the ABIl 700 Data Analysis User Guide and the filtered data "vsn normalised" (12). Differential expression was assessed using linear models and empirical Bayes algorithms as described in (13) and resulted in the identification of 319 androgen up-regulated genes and 300 genes whose expression was reduced upon androgen treatment. Fig. 2. Characterisation of androgen responses in LNCaP cells. (A) Real-time (Taqman) RT-PCR measurement of androgen regulated gene expression in LNCaP cells following 24-hour treatment with Rl 881. Quantitative RT-PCR was performed using Low Density Array (LDA) microfluidic cards from Advanced Biosystems, using Taqman Assay-On-Demand RT-PCR primer sets for 371 andro gen-regulated genes identified from the gene expression microarray analysis described in Figure 1. These 371 androgen-regulated genes consisted of 308 up-regulated genes (there were no available assays for 10 genes) and 63 down-regulated genes, which were those that showed the greatest inhibition by R1881, when ranked according to significance. The expression profiles for these genes are shown in the barchart as fold expression relative to the no ligand control. Rl 881 (1 nM) was added to LNCaP cells for 24 hours at which point the cells were lysed and RNA prepared. As Rl 881 was solubilised in ethanol, an equal volume of ethanol was added to the no ligand control. Each assay was carried out in triplicate, normalised against GAPDH expression and the mean fold induction, relative to the untreated control calculated. This fold induction is presented for all 371 androgen-regulated genes, and for additional control genes and shows that CNP is the most highly androgen-regulated gene identified. (B-C) Quantitative (Taqman) RT-PCR measurement to compare gene expression of PSA (KLK3) and CNP (NPPC) in LNCaP cells over a 24-hour time course with Rl 881. Real-time RT-PCR was used to measure the levels of PSA and CNP mRNA in LNCaP cells following 4, 16 and 24 hours treatment with R1881. Fold expression was determined as for part (A). (D-E) Quantitative (Taqman) RT-PCR measurement to compare gene expression of PSA (KLK3) and CNP (NPPC) in LNCaP cells using RNA prepared 24 hours following the addition of Rl 881 (1 nM), Dihydrotestosterone (DHT, 100 nM), Cyproterone Acetate (CPA, 100 nM), Flutamide (FLUT, 100 nM), Bicalutamide (BIC, 1 μM) or BIC + Rl 881. An equal volume of ethanol was added to the no ligand control. Figure 3. Pro-CNP levels in culture medium shows androgen regulation. (A, B) LNCaP cells were cultured in androgen- free medium for 72 hours prior to the addition of ligands followed by culturing for a further 48 hours prior to collection of culture media. Androgen used were Rl 881 (10 nM), dihydrotestosterone (DHT; 100 nM), Cyproterone acetate (CPA; 100 nM), hydroxyflutamide (FLU: 100 nM), Bicalutamide (BIC; 1000 nM), BIC + 10 nM Rl 881. No ligand refers to the control, where an equal volume of ethanol, the solvent in which the ligands were prepared, was added. The results represent the means of three samples, with standard errors of the mean being represented by the error bars. (C, D) LNCaP cells were cultured in androgen- free medium for 72 hours prior to the addition of R1881 (1 nM) for the time period shown, at which point the culture media were recovered. (A, C) Pro-CNP levels were measured using the Nt-Pro-CNP EIA kit (Biomedica Gruppe, GmbH & Co KG), according to manufacturer's methods. (B, D) Secreted PSA levels were measured for culture medium from three replicate cultures grown as in part B, using the Architect chemiluminescence total PSA kit (Abbott, UK), according to manufacturer's protocols.

EXAMPLE 1 : CNP is highly upregulated upon androgen stimulation of prostate cancer cells.

In order to identify androgen-responsive genes that mediate growth stimulation in prostate cancer cells, we have carried out gene expression microarray analysis. The LNCaP cell line was chosen for these studies because; (i) LNCaP cells express AR; (ii) they grow in an andro gen-regulated manner in cell culture (Figure IA); (iii) they demonstrate androgen-regulated expression of androgen-responsive genes, such as PSA (Figure IB and C); and (iv) they form andro gen-dependent tumours in xenograft models (Sobel & Sadar (2005) J. Urol. 173: 342-359).

Detailed time course studies were undertaken to determine the optimal androgen treatment for expression of androgen-responsive genes (PSA and DRG-I) in the LNCaP cells (Figure ID). On the basis of these studies, RNA from bio-replicate cultures were prepared from LNCaP cells treated with androgen for 24 hours, as well as the no ligand control. Three replicates from each treatment set were subsequently used for hybridisation to the Applied Biosystems (ABI) Human Genome Survey Microarray V2.0, which has probes for 29,098 genes (representing the most comprehensive coverage currently available for any human gene expression microarray platform) and differential gene expression was subsequently analysed.

The analysis has robustly defined 319 genes whose expression was stimulated by androgen by two fold or greater (Figure IE). We have carried out quantitative

RT-PCR (Q-RT-PCR) (Taqman; ABI) analysis for 308 of the up-regulated genes over a time course of androgen treatment. Figure 2A shows the fold induction for all 308 genes following 24 hours androgen treatment. This analysis confirms the two fold or greater induction of the gene set and, more dramatically, highlights CNP as a highly androgen-responsive gene (Figure 2A), showing a 307-fold induction at this time point.

A more detailed analysis shows CNP to be a highly responsive androgen regulated gene, demonstrating a 35-fold stimulation of expression within 4 hours of Rl 881 addition (Figure 2B). Similarly, of a 308 up-regulated gene set, CNP was found to be the most highly induced gene in LNCaP cells in response to 24 hours stimulation with 100 nM Dihydrotestosterone (DHT). where levels were induced to 43.5 fold.

EXAMPLE 2: CNP expression in prostate cancer cell lines

CNP expression in prostate cancer cell lines is measured using real-time PCR. The available prostate cancer cells lines include the AR negative lines DU- 145 and PC3 and the immortalised normal, tumour and metastatic lines, which are AR positive (McConnell (1991) Urol. CHn. North Am. 18: 1-13; Sobel & Sadar (2005) J. Urol. 173: 360-372). These cell lines are used to measure the levels of CNP gene expression.

Claims

1. A method for aiding in the assessment of prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia in a patient, wherein the method comprises the step of determining the level of CNP nucleic acid and/or protein, and/or pro-CNP nucleic acid and/or protein, in a sample from the patient.

2. The method of claim 1 wherein the patient is; (i) at risk of developing prostate cancer; or (ii) has already been diagnosed with prostate cancer; or (iii) has undergone treatment for prostate cancer.

3. The method of claim 1 or 2 wherein the sample is a blood sample, a urine sample, a lymph sample or a sample of seminal fluid.

4. The method of claim 1, 2 or 3 wherein the method is for assessing the likely progression of prostate cancer in the patient.

5. The method of any one of the preceding claims wherein the method is for assessing and/or predicting the development of prostate cancer.

6. The method of claim 1, 2 or 3 wherein the method is for diagnosing prostate cancer in the patient.

7. The method of claim 1, 2 or 3 wherein the method is for assessing the response of the patient to treatment.

8. The method of claim 7 wherein the method is for assessing the likely progression of response of the patient to treatment.

9. The method of claim 1 , 2 or 3 wherein the method is for choosing patients for treatment for prostate cancer.

10. The method of claim 1, 2 or 3 wherein the method is for monitoring response of patients to treatment or for monitoring relapse in patients.

11. The method of claim 1, 2 or 3 wherein the method is for assessing the progress of clinical trials.

12. A method for assessing a prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia treatment regime, the method comprising the step of determining the level of CNP nucleic acid and/or protein in a sample from patients receiving the treatment regime.

13. A method for identifying a compound useful in modulating prostate function, for example in treating or preventing prostate cancer (including metastatic prostate cancer) and/or benign prostate hyperplasia, the method comprising the steps of a) determining whether a test compound is capable of suppressing production of, or activity of, CNP in prostate tissue or a sample from a patient with, for example, prostate cancer and b) selecting a compound which is capable of suppressing production of, or activity of, CNP in prostate tissue or a sample from a patient with, for example, prostate cancer.