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1.1 Incidence of prostate cancer

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.

1.2 Biological function of the prostate

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. [Seite 2↓]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].

1.3 Development of Prostate cancer

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].

1.4 Pathological classifications of prostate cancer

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).

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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.

1.4.1 The TNM staging system for prostate cancer

The TNM staging system was already developed in 1977 by Ammon et al., but it was not before 1997 that it was predominantly used for evaluating prostate cancer. The TNM staging is based on the location and size of the tumor. It evaluates the local tumor growth (T), the lymph nodes (N) and the distant metastases (M) (Fig. 2). Tumors staged as T1 (a-c) are small and unapparent, they cannot be felt during rectal examination. It may be found by chance when surgery is done for other reasons, usually for BPH (Benign Prostate Hyperplasia). There is no sign that the tumor has spread outside the prostate. A stage 2 tumor is locally restricted tumor, yet lager in size and which can be detected during rectal examination or through biopsy. Tumors stage 3, have spread outside the prostate to nearby tissues such as the seminal vesicles. The last T stage of the TNM system is characterized by tumors which have spread or [Seite 4↓]are attached to organs near the prostate, such as the bladder. The affection of the lymph nodes is described by pathologist as N status (N0-N3). The M status characterizes the existence of metastasis in organs such as lungs, liver or brain.

Fig. 2 Anatomical staging of prostate cancer. The TNM system evaluates the location and size of a tumor in the prostate. T = local tumor growth, N = the lymph nodes, M = distant metastases.

1.4.2 The Gleason grading system

In contrast to the TNM system, which evaluates the localization and size of the tumor, the Gleason grading system published by Gleason and Mellinger in 1974 evaluates the tumor’s degree of differentiation and cell anaplasia [Gleason, 74b] (Fig. 3). Thereby the variation in cell size, shape and staining properties are taken into account. It distinguishes between well differentiated cells, moderately and poorly differentiated cells (cells which are distorted and irregular). The Gleason grading is obtained by summing the degree of cellular differentiation found on the two predominant patterns in a pathological specimen. Well differentiated cells which look closest to normal cells receive the Gleason score 1, poorly differentiated cells get score 5. These two grades are referred to as the Gleason grade. Score 2- 4 is considered as low grade, score 5-7 is considered as moderate, and score 8-10 is considered as a high grade tumor which is poorly differentiated.

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Fig. 3 Gleason Grading of the prostate [Gleason, 74a].

1.5 Treatment of prostate cancer

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.

1.5.1 Classical treatment

The radical prostatectomy and the radiation therapy are the most commonly used treatment forms for clinically localized prostate cancer (T1 and T2). The surgery involves removal of the entire prostate and in some cases of the surrounding tissues as part of the urethra and the seminal vesicles. Radiation may be used to destroy cancer cells that may have remained in the area after surgery, but it is also used as a stand alone therapy1.

Tumors which have spread out of the prostate gland (T3) and are thus beyond the reach of a local treatment by surgery or radiation, are treated by hormonal therapy. Although hormonal therapy cannot cure, it usually shrinks or stops the advance of the disease. Drugs which are used nowadays for treatment are either antiandrogens, which block the action of the androgens (for example flutamides and bicalutamide) or drugs which block the testicals from producing testosterone (e.g. luteinizing hormone-releasing hormone (LH-RH) agonists as leuprolide and goserelin). Finally aminoglutethimide and ketoconazole are used to prevent the adrenal glands from producing androgens.

Chemotherapy is seldom used for prostate cancer treatment as the response rate is very low. Usually these unspecific systemic drugs are given when hormone therapy has failed. Today drugs such as Docetaxel, Doxorubicin or Estramustine phosphate are used for treatment.

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1.5.2  New treatment forms

In the last years the development of target drugs for the treatment of cancers has dramatically increased, a progress that is likely to continue in the future. This approach is based on the targeting of genes found to be overexpressed in tumors or other disease by monoclonal antibodies, small-molecules, immunotoxins and antisense oligonucleotides. This form of therapy has considerable advantage over unspecific systemic drugs such as the chemotherapy. They are more specific, thus less toxic, and more effective in the treatment of cancer [Stockwin, 03b]. Antibodies (150 kD) are used to target the extracellular portion of membrane proteins, whereas small-molecules can also inhibit the function of intracellular localized proteins as they can penetrate through the membrane (smaller than 1 kD) [Seemann, 90a].

For example antibodies are used to treat indications as diverse as cancer, inflammation and infectious disease. They can be used as cell targeting reagents and thus tag specific cells for complement- or effector-mediated lysis. Antibodies can further be modified to deliver toxic or modulatory payloads (radionuclides or enzymes) [Stockwin, 03c].

Up to now several monoclonal antibodies have been developed, especially those gainst the extracellular portion of receptor tyrosine kinases [Seemann, 90c]. Herceptin, a humanized monoclonal antibody against the Her2/neu receptor tyrosine kinase, was shown to prolong the survival of women with Her-2/neu positive metastatic breast cancer, when combined with chemotherapy [Seemann, 90b].

As an example for a small-molecule the STI-571 has to be named, it was shown to inhibit the Bcr-Abl, c-kit and platelet derived growth factor receptor tyrosine kinases, and thus produced dramatic clinical responses in patients with Bcr-Abl positive chronic myeloid leukemia and c-kit positive gastrointestinal stromal tumors [von Bubnoff, 03].

It seems possible that targeted drugs will be used in association with existing medical, surgical, and radiotherapeutic therapies and will play an important role in the aim of curing cancer.

1.6 androgens in prostate cancer

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 [Seite 7↓]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].

1.7 Diagnosis of prostate cancer

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.

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1.7.1  Prostate Specific Antigen

Prostate cancer antigen (PSA) is a tissue specific tumor marker routinely used to diagnose prostate cancer and to monitor treatment response, prognosis and progression of prostate cancer [Sadar, 99a]. It is a single-chain glycoprotein with a molecular mass of about 33 kD which functions in the liquefaction of seminal coagulum. Serum levels of PSA of healthy patients are between 0 – 4 ng/ml. In prostate tumor patients the PSA levels can raise up to 100 ng/ml. Generally, PSA levels rise with tumor volume, but it is expressed in all stages of cancer [Caplan, 02b]. Although PSA is the best marker for prostate cancer existing today, it is still far from being perfect. For example, PSA tends to increase with age and rises in men with evidence of benign prostatic hyperthrophy. Thus many men are diagnosed falsely positive for prostate cancer. On the other hand PSA levels do not increase in some patients with prostate cancer which leads to a false negative diagnosis. Additionally, preoperative PSA cannot be used to predict capsular penetration or seminal vesicle invasion. Further, PSA is not able to predict progression in adenocarcinomas of the prostate following radical prostatectomy [Sauvageot, 98b].

1.7.2 Regulation of PSA

The Kallikrein 3 (KLK3) gene which codes for PSA is primarily regulated by androgens. In the proximal promoter of the KLK3 gene are two functional androgen-response elements (AREs) located [Riegman, 91; Cleutjens, 96]. The core region of the enhancer could be mapped within a 440-bp fragment. A functionally active, high-affinity androgen receptor binding site (GGAACATATTGTATC) was identified in the center of this fragment. Mutation of this element almost completely abolished PSA promoter activity. Therefore PSA levels undergo a sharp decline following an anti-androgen therapy or surgical castration. However, when in the absence of androgens the tumors change to an androgen-independent state, PSA levels increase due to an alternative activation mode. At this stage tumor progression is mostly inexorably and untreatable.

1.8 Microarray analysis in cancer research

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 [Seite 9↓]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 public2 and proprietary databases were sorted for tissues specificity and into pairs of benign and cancer tissues. The numbers of ESTs matching to a specific sequence were counted for each pool (normal, tumor and tissue). The sequences which exhibited significant differential expression between normal and cancer tissue were selected and added to the metg001A chip (The procedure is described in detail in “Methods”).

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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.

1.9 Transient Receptor Potential Channels

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.

1.9.1 The TRP superfamily

The outstanding feature of the TRP superfamily is its considerable diversity in modes of activation and function. They are involved in processes ranging from sensory physiology of cold and heat to vasorelaxation and male fertility [Montell, 02d]. The discovery that TRP channels are able to sense temperatures and flavors was honoured as one of the top ten scientific achievements in the year 2002 by Sience, showing the increasing importance of TRPs [02].

Biochemically the TRPs belong to the group of non-voltage gated ion channels - the so called capacitive calcium entry (CCE) channels [Nilius, 03c]. They are activated by various chemical and physical stimuli and also by depletion of [Seite 11↓]intracellular Ca2+ stores, which is followed by a cation influx to the cytosol. TRPs consist of six transmembrane spanning helices, a pore region between Transmembrane (TM) 5 and TM 6 and cytoplasmatic N- and C-termini [Clapham, 01a] (Fig. 5).

Fig. 5 Architecture of TRP channels. A) TRP channels consist of six transmembrane spanning helices and a pore region between TM5 and TM6 where different mono- and divalent cations can pass through the pore [Clapham, 01b]. B) Top view of the TRPV5/6 heterotetrameric channel. The complex is formed by four momomeric subunits of TRPV5/6. The calcium binding site within the pore is formed by 4 aspartate residues [den Dekker, 03].

The first member of the TRP family was identified as a Drosophila gene responsible for visual transduction [Lo, 81]. Because of its transient rather than sustained response to light in mutant flies it was named transient receptor potential (trp). Up to now more than 20 mammalian TRP members are known. They are classified into three subfamilies according to their structural and sequence similarities [Nilius, 03d; Grimm, 03b]. The first group are the TRPC (C stands for canonical subfamily) which have a high homology to Drosophila TRP channels. Second, the TRPV subfamily (V stands for vanilloid) which are closely related to vanilloid receptor 1 (TRPV1), and third the TRPM family members which are highly homologue to the tumor suppressor melastatin (TRPM1).

The phylogenetic tree of all mammalian TRPs as known today is shown in Fig. 6.

Fig. 6 The phylogentic tree of the mammaliean TRP channels based on their homology [Nilius, 03a].

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TRPs are activated mainly through the phospholipase C (PLC) and G-protein coupled receptors (GPCR) which in turn generate inositol (1,4,5) trisphosphate and diacylglycerol (DAG) [Bakowski, 02; Putney, Jr., 97; Putney, Jr., 99]. But also DAG by itself has proved to activate TRP channels [Hofmann, 99b; Chyb, 99]. Surprisingly during the last years new TRPs have been indentified which can be activated by chemical and physical stimuli such as heat, cold, mechanical stress, bitter-sweet compounds, reactive oxygen species, pH, pheromones, phorbolesters and vanilloid compounds. For example TRPV1 responds to capsaicin and temperatures over 43°C by generating inward membrane currents, suggesting that it functions as a transducer of painful thermal stimuli [Caterina, 97a].These findings are remarkable as these functions give insight into new modes of channel regulation [Nilius, 03b].

1.9.2 TRPM 1 to 7

Less research has been done on members of the TRPM family. Until now 8 homologs of TRPMs are known, counting form TRPM1 to TRPM8. The name (TRPM) has been chosen because the first described member of the group was melastatin (MLSN) [Montell, 02a]. Prior to the implementation of a unified nomenclature by Montell at al. in 2002 this subfamily was also known as LTRPC. Members are characterized by relatively long N- and C-termini with some of them having entire enzyme domains linked to their C-terminus. For example TRPM2 has an ADP-ribose pyrophosphatase [Perraud, 01b] and TRPM6 and TRPM7 have an atypical α-kinase domain in TRP [Perraud, 01a; Runnels, 01; Schlingmann, 02]. Although most of the functions of TRPMs are not known, some TRPM appear to play an important role in cancer and cell proliferation. For example TRPM1 was described as a putative tumor suppressor gene expressed in melanocytes correlating inversely with tumor aggressiveness and the potential for melanoma metastasis [Montell, 02b; Duncan, 98a]. Another channel, TRPM2, was shown to mediate apoptotic cell death when activated by H2O2 in HEK293 cells. This process was accompanied by an increase in intracellular calcium levels ([Ca2+]i) [Duncan, 98b; Zhang, 03a]. TRPM3 is expressed in human brain and kidney. It is supposed to play a role in the renal homeostasis as it increases Ca2+ entry during reduction of extracellular osmolarity [Grimm, 03a].

TRPM4 is a remarkable example of the great functional diversity of the TRP protein family. It was shown to be directly activated by cytoplasmatic calcium but the following large inward current is carried primarily by monovalent cations such as Na+ [Launay, 02]. This shows that TRPM4 is most likely impermeable for calcium. Another striking feature of this protein is that it is the only voltage dependant channel of this group [Nilius, 03]. TRPM6 was shown to be involved in familial hypomagnesemia being responsible for renal excretion of calcium and magnesium [Walder, 02]. TRPM7 was described as a channel and a protein kinase as well.

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1.9.3  TRPM8

TRPM8, formerly known as Trp-p8, is the latest identified gene of all TRP channels. First mentioned in 2001 as a gene upregulated in prostate cancer and other malignancies [Tsavaler, 01c] it was shown to be expressed also in a large spectrum of nonprostatic primary cancers such as melanoma, colorectal carcinomas and breast carcinomas. In normal human tissues expression was found mainly in the prostate with trace expressions in testis, breast, thymus and lung.

TRPM8 is closest related to TRPM2 followed by TRPM1 with which it shares 34% sequence identity. Tsavaler et al. suggested that TRPM8 could be an oncogen or tumor promoter gene. It was assumed to belong to the 7-transmembrane proteins.

To describe the importance of TRPM8 for the development of a small-molecule or antibody based therapy is a deal between Dendreon and Genentech from 2002. The agreement “provides for upfront and milestone payments totaling over US $ 110 million for the resulting development of TRPM8 products”3 .The deal concerns the development of monoclonal antibodies, small molecules and other products derived from Dendreon's TRPM8 gene platform.

In 2002 two manuscripts were published showing the identification of the mouse and rat ortholog of TRPM8 [McKemy, 02a; Peier, 02e]. Interestingly, both genes were identified in cells from neuronal origin. The mouse ortholog was isolated from newborn dorsal root ganglia and the rats from trigeminal neurons of newborn rats. The striking new discovery was that these channels could be activated by cold and different cooling agents such as menthol, icilin and eucalyptol when overexpressed in cells. Following activation an increase in intracellular calcium was observed which could be suppressed by removal of extracelluar calcium. The authors suggest that TRP channels are the primary transducers of thermal stimuli. Until now 4 heat activated TRP channels and two cold sensing channels have been described: TRPV4 (27-34°C) [Guler, 02], TRPV3 (20-40°C) [Xu, 02; Smith, 02], TRPV1 (>43°C) [Caterina, 97b] and TRPV2 (>53°C) [Caterina, 99] TRPM8 (8-28°C) [McKemy, 02b] and ANKTM1(12-24°C)[Story, 03].

1.10 Calcium signaling

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 [Seite 14↓]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.

1.11 Genetic alterations of prostate cancer

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].

1.12 Control of gene expression

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 [Seite 15↓]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 [Seite 16↓]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.

[Seite 17↓]

1.13  Aim of the Thesis

The aim of this thesis was the identification of new genes differentially expressed in prostate tumors. Therefore microdissected matched prostate cancer and benign tissues of 52 prostate cancer patients were hybridized to a proprietary high density Cancer-Chip based on Affymetrix GeneChip technology. The intention was to identify genes differentially expressed in prostate cancers which had not yet been discovered by other groups. One of these genes was then selected for a more detailed analysis.

In order to answer, whether the selected gene could be used as a target for a small-molecule or antibody based therapy the following questions were aimed to be answered.

  1. Is the gene expressed in other cancer entities than the prostate?
  2. In which normal tissues is it expressed?
  3. Does it correlate with prostate cancer progression?
  4. In which cell compartiment(s) is the gene expressed?
  5. Is the gene an oncogene?
  6. How is it regulated?
  7. Is the gene also useful for diagnostics or other possible cancer therapies?

The answers should provide evidence whether it is worth to develope a specific drug against this gene or not.

Fußnoten und Endnoten

1 http://www.cancer.gov/cancerinfo/wyntk/prostate

2  http://www.ncbi.nlm.nih.gov/dbEST/

3 http://www.pharmaventures.com/ag_demo/pr_11144.html

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