Cancer deaths account for 23% of all deaths in Western Europe and the United States ranking second only to deaths from heart disease. When deaths are categorized by age, sex, and cause, cancer is by far the main cause of deaths among men and women between 40 and 79 years of age [Jemal, 03a]. Among can­cers, malignancies of the prostate are the most commonly diagnosed tumors in European and American males [Howe, 01; Kieschke, 02]. In 2003 malignancies of the prostate will be the second most common fatal can­cer in men (10%) after lung cancer (31%), followed by colorectal cancer (10%) [Jemal, 03b], as estimated by the American Cancer Society.

In Germany approximately 700,000 men suffer from prostate cancer. It is the most frequently (18,7%) diagnosed tumor with an incidence of 31.500 cases displacing 1998 lung cancer as the most frequently diagnosed carcinoma [Kieschke, 02]. Prostate cancer afflicts men at an average age of 72 years, 6 years above that of cancer in general. The five-year relative survival rate of prostate cancer in Germany is 70%. In general, the number of diagnosed prostatic malignancies has increased, but the numbers of deaths per year have decreased in the last years. The reasons for this increase in incidence is the aging population and most importantly, the improved early detection methods such as serum testing of prostate specific antigen (PSA) and the digital rectal examination (DRE). Early detection as well as improved surgical intervention and radiation therapy have reduced the number of deaths significantly. But prostate cancer still ranks third of all lethal cancers causing about 18,000 deaths per year in Germany [Kieschke, 02].

Although more men die with prostate cancer than of prostate cancer, there is still no effective cure for many patients suffering especially from aggressive and advanced forms of prostate cancers. PSA screening is one of very few preoperative parameters of prognostic relevance. So far it is not possible to distinguish between aggressive and minor severe forms at an early stage of the disease.

The prostate is a walnut-sized gland surrounding the urethra at the base of the bladder. It is surrounded by a fibroelastic capsule that penetrates the gland to divide it into lobes. The prostate contributes to the seminal fluid an alkaline liquid which is rich in spermine, phosphlipids, cholesterol, fibrinogenase, cit­ric acid, fibrinolysin, zinc and acid phosphatase and other proteins. The seminal fluid consists further of the fluid produced in the seminal vesicles and the sperm. The sperm, produced in the testis, enters the upper portion of the prostate through the vas deferens. Sperm and fluid from the seminal vesicles then mix with secretions emitted from the prostate to form the seminal fluid that is expelled at the time of ejaculation.

Interestingly, the prostate is neither required for viability nor for basal levels of fertility. It is widely discussed that this might be the reason for its high incidence of cancer as other vitally important organs of the urogenital system, such as the seminal vesicles and bulbourethral glands, are nearly immune to neoplasias [Abate-Shen, 00a].

The cause of prostate cancer is still not very well understood. A distinguishing feature of this cancer is its intimate association with aging [Abate-Shen, 00d]. Usually clinically detectable prostate cancer is not manifest until the age of 60 or 70. Hereditary factors account for about 10% of prostate cancers and are generally associated with an early onset of the disease [Carter, 92]. To date, two family susceptility loci have been mapped to X chromosome and to a region of chromosome 1q, although no candidate gene has been found so far [Smith, 96; Xu, 98]. Another feature is that African American men have a higher incidence and more aggressive forms of prostate cancer than white men who in turn have a higher incidence than men of Asian origin. Additionally, androgens play a povital role in all stages of the disease. High fat diets are also suspected to increase the risk of prostate cancer while a diet rich in soy may be protective. These observations have been proposed as reasons for the low incidence of this cancer in Asia [Kristal, 02].

Prostate cancers are generally of multifocal nature and belong the most heterogeneous tumors in humans [Macintosh, 98]. 70% of the tumors arise in the peripheral zone, whereas 15-20% arise in the central zone, and 10 -15% arise in the transitional zone.

Most of the prostate tumors are adenocarcinomas (95%), only about 4% of cases have transitional cell morphology and are thought to arise from the uroehelial lining of the prostatic urethra. Few cases have neuroendcrine morpholgy. These cells are believed to arise from the neuroendocrine stem cells normally present in the prostate (Fig. 1).

Fig. 1 Schematic view of the cell types within a human prostatic duct. Neuroendocrine cells are morphological indistinguishable from basal cells. Taken from [Abate-Shen, 00b].

Prostate cancer progresses from an enlargement (benign prostatic hyperplasia [BPH]) to precursor lesions (prostate intraepithelial neoplasias [PIN]) on to invasive carcinomas and ultimately to metastases. BPH is an abnormal growth of prostate cells occurring in nearly all men over the age of 70. Cells from a BPH are larger in shape but they do not spread to other tissues. Thus benign tumors are not treated unless symptoms such as pain and/or difficulties in the urine flow require treatment. The PIN is a precursor of carcinoma. It is recognized as a continuum between low-grade and high-grade forms with high-grade PIN thought to represent the immediate precursor of early invasive carcinoma [Abate-Shen, 00e].

Nowadays there are two major systems used for the pathological graduation of malignant prostate cancers: The Tumor Node Metastasis (TNM), which evaluates the location and size of a tumor and the Gleason grading system, which describes the tumor’s degree of differentiation and cell anaplasia. For better understanding of the clinical terminology used in this study a brief description of the main characteristics and pathological classifications of prostate cancer is given in the next paragraphs.

Although especially in older patients with early stage cancers it is enough to carefully watch the tumor growth as these cancers usually grow at a very slow rate and the possible risks and side effects of therapy may outweigh the possible benefits, many prostate tumors need treated through surgery, radiation or hormones.

The importance of androgens in prostate cancer was first described by Huggins and Hodges in the early 1940s [Huggins, 02]. Since then, significant research has shown that the interrelationship between hormone and cancer is very complex and is best exemplified by the recurrence and progression of prostate cancer after hormonal therapy to a lethally resistant phenotype [So, 03].

Androgens, principally testosterone, play a critical role in the development and growth of the male reproductive system. Their biological actions are mediated by the androgen receptor (AR), a ligand-dependent transcription factor, belonging to the nuclear receptor superfamily. These androgen-AR complexes interact with various transcription activators or repressors in order to modulate transcription of androgen target genes via specific DNA sequences [Lee, 03]. The AR is composed of an N-terminal domain, a DNA binding domain, a hinge region and a ligand binding domain. In its inactive form the AR is complexed to heat-shock proteins in the cytoplasm. After binding a specific ligand (i.e. 5alpha-dihydrotestosterone) the ligand-receptor-complex translocates to the nucleus and binds a specific androgen responsive element (ARE) within the promoter of various genes. Genes affected by the AR are for example the KLK3 (Kallikrein3) gene which codes for PSA and the homeobox gene NKX3-1 [Gregory, 98].

Androgen ablation and anti-androgen therapy has become the cornerstone of treatment for patients with locally advanced or metastatic prostate cancer. Among the earliest detected effects of androgen withdrawal are decreases in the intranuclear concentration of androgens and the AR as well as decreased PSA levels in the blood. Although 80 – 90% of patients respond initially to this therapy the majority gradually develops resistance [Laufer, 00]. The mechanism of change from tumors being androgen-responsive to being androgen-unresponsive is poorly understood [Suzuki, 03]. Interestingly, clinical findings indicated that in androgen ablation therapy-resistant prostate cancer PSA and other genes regulated by androgens as well as the AR by itself are still expressed. This led to the assumption that a ligand-independent activation of the androgen receptor may be the underlying mechanism of androgen independence. In fact, multiple signaling pathways have been implicated in AR non-steroidal activation including estrogen, progesterone, peptide growth factors and cytokines [Debes, 02]. These factors are able to induce transactivation of the AR under androgen-depleted conditions reviewed in[Huang, 02]. Dysregulation of the AR in prostate cancer further results in an abnormal profile of AR-regulated genes which include cell cycle regulators, transcription factors and proteins important for cell survival, lipogenesis, and secretion. Additionally, this receptor is a target for somatic mutation and deregulated androgen signaling is a potential consequence of such mutations reviewed in [Bentel, 96]. Not only is this androgen independence a sign of an emerging disease, it is also associated with a poor prognosis [Sadar, 99b].

Digital rectal examination (DRE), measurement of the prostate specific antigen (PSA) in the blood and the transrectal ultrasonography (TRUS) are the main parameter used in prostate cancer diagnosis. Nowadays prostate cancer is not diagnosed by symptoms, but because of increased levels of PSA in the blood and abnormal findings in the DRE. Thus it was possible to diagnose more and more patients at earlier stages of the disease, hoping to increase the probability of a cure.

The main goal of metaGen Pharmaceuticals GmbH was the identification of novel target genes for the development of therapeutic antibodies or small-molecule drugs in different tumor entities. One of the main questions was to find the best method for the identification of new genes. The first description of a “high-capacity system to monitor the expression of many genes in parallel” was published in 1995 [Schena, 95]. They showed that it was possible to detect the expression of 45 Arabidopsis genes simultaneously by spotting the complementary DNAs of these genes on a glass slide and hybridizing samples of RNA to this chip. The development in this field has been more than dramatic in the last years. Today it is possible to detect the expression of the complete human genome, represented by approximately 47,000 transcripts on only one DNA chip (Affymetrix, Santa Clara, CA, USA).

Nowadays two major groups of DNA microarrays are available: First, cDNA microarrays where oligonucleotides or cDNAs are spotted to a glass microscope slide, second, high density microarrays where nucleotides are synthesized to a specific matrix (Affymetrix). The advantage of the first method is the high flexibility of genes spotted to the slide and the possibility of hybridizing two different samples simultaneously to the chip at relatively low cost. Affymetrix chips only provide the possibility of hybridizing one pool of mRNA at a time at relatively high cost, but have the considerable advantage of synthesizing more than 40,000 genes to one chip. Probe preparation and analysis procedures are quite the same for both chips: The isolated RNA from tissues or cell lines is labeled with fluorochromes before hybridization. A scanner records the intensity of fluorescence per probeset and different bioinformatics tools are used to interpret the huge amount of data sets.

In order to detect genes relevant in different cancer entities metaGen decided to use the Affymetrix technology. At that time it was not possible to synthesize more than approximately 10,000 genes on one chip. Thus 5 chips would have been necessary to analyze the whole genome for differentially expressed genes. First of all this approach was much too expensive and on the other hand, most of the genes present on these chips are not relevant in prostate cancers.

Consequently, a customized Cancer-Chip was designed at metaGen for the identification of tumor specific genes (Fig. 4). The chip design based on a bioinformatic attempt mining systematically expressed sequence tag (EST) libraries [Schmitt, 99c]. Briefly, about 4 million ESTs of public

http://www.ncbi.nlm.nih.gov/dbEST/

Fig. 4 The metaGen Affymetrix Cancer-Chip (metg001A). This chip contains about 6200 probe sets which represent roughly 3,000 genes. Nearly half of the sequences represent genes which have been shown to be overexpressed in various tumor entities.

These sequences and most of the known tumor associated genes made up the main part of the newly developed metg001A Cancer-Chip. This concentration of cancer associated genes on one chip made it possible to screen for relevant tumor markers in different entities at relatively low cost and in dramatically reduced time. By the use of this proprietary chip it was strongly expected to find overexpressed genes in cancers which have not yet been discovered by other groups and by other methods.

Hybridization of the metg001A chip with 52 matched prostate normal and tumor tissues revealed a number of genes differentially expressed in prostate cancer patients. One of these genes, the Transient Receptor Potential Protein 8 (TRPM8), was selected for further evaluation. At the time of identification TRPM8 was a completely unknown gene, not described in the literature.

Up to now, some very interesting features of this gene and especially of this whole protein family have come up. A short overview will be given in the next chapters.

Calcium signals control a wide range of cellular events, ranging from secretion and contraction to gene expression. Calcium can control cell growth and cell differentiation but also induce apoptosis. The concentration of free calcium ions in the cytosol is generally less than 10 µM, a thousand times less than that from the extra cellular space. In general, there are two possibilities for increasing intracellular free calcium ([Ca2+]i) levels: 1. opening of channels located in the plasma membrane allowing calcium ions to move along the electrophysiological gradient to the interior of the cell. 2. Internal stores as the endoplasmatic reticulum and the sarcoplasmatic reticulum release Ca2+ into the cytoplasm. These stores have a limited capacity and must be refilled from the external environment. This process of replenishing is accomplished by store operated Ca2+ channels (SOC) which are located in the plasma membrane. They trigger calcium from the external environment through processes known as capacitive Ca2+ entry to cell intracellular stores [Hardie, 92; Montell, 97]. This group of calcium channels is named non-voltage-gated channels, because their activation is independent of changes in voltage. Consequently the second large group of Ca2+ permeable channels is called voltage-gated channels.

The hypothesis that multiple mutations have to occur before progression from normal to invasive carcinoma can occur has been proven by the identification of multiple chromosomal abnormalities in cancers, including prostate cancer reviewed in [Bova, 96]. In prostate cancer one of the most frequent ly observed chromosomal aberrations is the loss of chromosomal region 8p12-22 [Bova, 93; Macoska, 95; Vocke, 96]. A loss of heterozygosity in this region occurs in 63% of prostate intraepithelial neoplasias (PIN). Although no bona fide tumor suppressor has been mapped to this region, the most probable candidate at this loci is the NKX3-1 gene, which maps to 8p21 [Abate-Shen, 00c; He, 97]. NKX3-1 belongs to the large group of homeobox genes. The homeobox sequence encodes a 60-amino acid domain called the homeodomain responsible for DNA binding. These genes are mainly specific nuclear proteins which act as transcription factors. They are the master developmental control genes which regulate cell differentiation and other morphogenic processes [Nunes, 03]. Recent research has demonstrated that deregulation of developemental genes cause cancer. In many cancers such as leukemia, colon, skin, breast, ovary and prostate alterations of gene expression of these genes have been described. For example the PRX homeobox family is strongly connected with human diseases, especially cancer [Silberstein, 02].

The ability of cells to determine which gene needs to be expressed at a given time and to coordinate transcription is a complex process. Various elements like transcription factors, matrix attachment regions, locus control regions, promoters, gene methylation, enhancers and silencers control the complex transcription machinery [Werner, 03b]. First, the chromatin structure of the DNA displays a physical barrier for transcription factors and polymerases to bind to their target DNA sequences [Emerson, 02]. Diverse enzymes modulate the accessibility of DNA by changing the structure of the histones and by modeling the nucleosomes in an ATP-dependent manner. Second and most important for gene expression is the transcription initiation. Transcription requires binding of transcriptional regulatory proteins, RNA polymerases and proteins called mediators to the promoter region of a gene. A promoter is a region of DNA extending 150-300 base pairs upstream from the transcription start site andrepresents the central processor of the transcription control. The sites where the transcription factors bind are generally composed of 10 to 30 nucleotides, but usually only a small number of core nucleotides are necessary for binding [Werner, 03a].

The complexity and precision of intron removal during mRNA splicing is still an amazing process, although it is known since 1977 [Berget, 77; Chow, 77]. In recent years it has become clear that most human genes express more than one mRNA by alternative splicing [Faustino, 03b]. Human genes contain on average 8 exons with an average length of 145 nucleotides. Introns are usually more than 10 times of this size, some are much larger [Lander, 01b]. The classical splicing signals at the intron/exons borders are present in 99% of all human introns (Fig. 7). They are necessary for the recognition of exons by the spliceosome, which catalyses the cut-and-paste reactions that removes introns and joins exons [Faustino, 03a]. Surrounding these major splicing signals different auxiliary elements such as exonic splicing enhancers or silencers (ESE and ESS) and intronic enhancers of silencers (ISE and ISS) are commonly found. Together with the binding factors these elements are required for efficient splicing of constitutive and alternative exons.

Fig. 7 Classical and auxiliary splicing sites and binding factors taken from [Faustino, 03c]. A) Classical and auxiliary splicing site. These sites are found in >99% of the human introns necessary for exon recognition (n = G; A; U, or C; y = pyrimidine; r = purine). B) Classical and auxiliary binding factors. (ISE/ISS = Intronic Splicing Enhancer/Silencer; ESE, ESS = Exonic Splicing Enhancer /Silencer)

Alternative splicing is characterized by the connection of different 5’ and 3’ splice sites within a gene, resulting in multiple mRNAs expressed by one gene. This process leads to transcripts with one or more skipped exons, variable positions of exons and additions of alternative exons, either within a gene or at its 5’ or 3’ end. This effect may lead to an expanded protein repertoire which could explain the apparent discrepancy between gene number and the complexity of higher eukaryotes [Mercatante, 02b]. Up to 59% of human genes were found to be spliced alternatively [Lander, 01a] and ~ 80% of these splicing results in an altered protein [Modrek, 02]. In most cases regulation of alternative gene expression is cell type specific. The regulation is mediated by intronic repressors or activator elements distinct from the classical splicing sequences. Expression of these alternative mRNA forms was seen during specific stages of development, in specific cells or tissues as well as in numerous diseases including cancer [Mercatante, 02a].

Until now four classes of mRNA splicing (two cis acting and two trans acting) leading to a disease have been described reviewed in [Faustino, 03d]: First and most common are mutations in the constitutive splice sites. These can lead to unnatural mRNAs which are deleted by a nonsense-mediated decay or to the loss of function of the resulting protein (protein instability, truncations). Second the disruption of alternative splice sites which have been described for 4 diseases (Familial isolated growth hormone deficiency type II- caused by mutations in the growth hormone gene; Frasier syndrome - caused by mutations in the WT-1 gene; Frontotemporal dementia and Parkinsonism linked to Chromosome 17 - caused by mutations in the MAPT gene and the atypical cystic fibrosis - caused by polymorphisms of the CFTR gene) The two trans-acting classes are characterized by mutations either in the basal splicing machinery or mutation in factors regulating the alternative splicing machinery. The major group of splicing factors are the serine/arginine-rich (SR) proteins which are members of a conserved family of proteins that bind to the active sites of RNA polymerase II and thus function as key regulators of alternative RNA splicing [Zahler, 92a; Fu, 95b; Graveley, 00a]. They have dual functions and serve as splicing enhancer or splicing repressor proteins, depending on where in the pre-mRNA they bind reviewed in [Akusjarvi, 03].

Aberrations in alternative splicing were found as a contributing factor or cause to the development, progression or maintenance of cancer. Up to now there are only models of how this process is regulated. Most likely specific repression of activation complexes surrounding the regulated splice sites serve to enhance or inhibit the recognition of the classical splice sites by the basic splicing machinery [Charlet, 02]. But it remains unclear why the expression of different transcripts is enhanced in one tissue, whereas it is repressed in others. One interesting question is, if the altered expression of splice variants in cancer is a cause for the disease (i.e. due to mutations within the auxiliary elements within exons or introns) or the effect from other disordered genes which might be involved in the splicing machinery.