1  INTRODUCTION

• 1.1 Incidence of prostate cancer

• 1.2 Biological function of the prostate

• 1.3 Development of Prostate cancer

• 1.4 Pathological classifications of prostate cancer

  • 1.4.1 The TNM staging system for prostate cancer

  • 1.4.2 The Gleason grading system

• 1.5 Treatment of prostate cancer

  • 1.5.1 Classical treatment

  • 1.5.2  New treatment forms

• 1.6 androgens in prostate cancer

• 1.7 Diagnosis of prostate cancer

  • 1.7.1  Prostate Specific Antigen

  • 1.7.2 Regulation of PSA

• 1.8 Microarray analysis in cancer research

• 1.9 Transient Receptor Potential Channels

  • 1.9.1 The TRP superfamily

  • 1.9.2 TRPM 1 to 7

  • 1.9.3  TRPM8

• 1.10 Calcium signaling

• 1.11 Genetic alterations of prostate cancer

• 1.12 Control of gene expression

• 1.13  Aim of the Thesis

2  RESULTS

• 2.1 Gene Chip Expression analysis of prostate cancer patients

  • 2.1.1 Expression of TRPM8 in prostate cancers

  • 2.1.2 Expression of TRPM8 in human cell lines

• 2.2  TRPM8 expression in Human tissues

  • 2.2.1 Electronic Northern of TRPM8

  • 2.2.2 Northern and Dot blot analysis of TRPM8

  • 2.2.3 Real Time PCR of TRPM8 in matched prostate cancer patients

  • 2.2.4 In situ hybridization of TRPM8 in prostate tumors and other entities

• 2.3 FISH experiments of TRPM8 on 2.Q37.2 in LNCaP cells

• 2.4 Homomultimerization of TRPM8 subuntis

• 2.5 activation of TRPM8 by the cooling agent icilin

• 2.6 Gene Chip Analysis of TRPM8 expression in 7 human tissues

• 2.7 TRPM8 expression in neuroendocrine tumors of the lung

• 2.8 Genomic structure of TRPM8

• 2.9 Identification of TRPM8 splice variants

  • 2.9.1 Identification of the TRPM8-regulatory-RNA

  • 2.9.2 Expression of TRPM8 splice variants in prostate tumors

  • 2.9.3 Characterization of TRPM8 Splice variant 16b

  • 2.9.4 Generation of HEK293 cell stable for 16b and TRPM8

  • 2.9.5  Cellular localization of TRPM8 and SV16b in HEK293 cells

  • 2.9.6 Influence of SV 16b on the activation of TRPM8 by icilin

  • 2.9.7 Aberrant splicing of SV 16b

• 2.10  Promotoranalysis of the TRPM8 Gene

  • 2.10.1 Characterization of the TRPM8 Promoter

  • 2.10.2 Genomic structure of the human and mouse TRPM8 promoter

  • 2.10.3 Transcription repression by a highly conserved promoter fragment

  • 2.10.4 Site-directed mutation in the 1.9kb promoter

  • 2.10.5 Androgens enhance transcription of TRPM8

• 2.11 Correlation of TRPM8 to grading and staging of prostate cancer

3  Discussion

• 3.1 Microarray Experiments and Prostate Cancer profiling

• 3.2  overexpression of TRPM8 in Prostate Tumors

• 3.3 TRPM8- a functional calcium Channel

• 3.4  TRPM8 expression is regulated by androgens

• 3.5 TRPM8 Promoter

• 3.6  Expression of TRPM8 in neuroendocrine Tumors

  • 3.6.1 Expression of TRPM8 in neuroendocrine and prostate cells

  • 3.6.2 Expression of TRPM8 in neuroendocrine cells and cells from the nervous system

• 3.7 TRPM8 expression correlates with disease Progression

• 3.8 TRPM8- target For Treatment of prostate cancer

• 3.9 Splice variants of TRPM8

4  Outlook

5  MATERIALS AND METHODS

• 5.1 metg001A Chipdesign

  • 5.1.1 Automated extension of cDNA sequences (AUTEX)

  • 5.1.2 Electronic Northern

• 5.2 gene chip analysis

  • 5.2.1 Tissue Collection

  • 5.2.2 Microdissection

  • 5.2.3  RNA preparation and amplification

  • 5.2.4 Data processing

  • 5.2.5 Prostate Cancer Gene Expression Analysis

  • 5.2.6 probeset comparison of metg001A and U113B

• 5.3 Real-Time PCR

• 5.4 Northern Blot and Dot Blot analysis

• 5.5 In situ Hybridization

• 5.6 Cell culture

  • 5.6.1 Culture of LNCaP cells for androgen activation

• 5.7 Plasmid construction

  • 5.7.1 TRPM8

  • 5.7.2  16b

  • 5.7.3 Promoter constructs

• 5.8 Luciferase Reporter Assay

  • 5.8.1 Site-directed mutations

• 5.9  Intracellular Ca2+ measurements

  • 5.9.1 Fura-2 assay

  • 5.9.2 FLIPR assay

• 5.10  Flow Cytometry Analysis

• 5.11 FISH Analysis

  • 5.11.1 Metaphase preparation

  • 5.11.2  Probe preparation and hybridization

• 5.12 FRET Analysis

• 5.13 Sequencing

• 5.14 Construction of stable cell lines

• 5.15 Western immunoblotting

• 5.16 Immunostaining

6. References

Abbreviations

Attachment

• DANKSAGUNG

• EIDESSTATTLICHE ERKLÄRUNG

• Tab. 1 Upregulated genes in prostate cancer patients identified by Affymetrix GeneChip experiments. Genes were sorted by numbers of patients with a fold change > 2 (quotient of PMQ-values of matched tumor and normal sample for each gene), HGN for HUGO Gene Nomenclature, FC for fold change.

• Tab. 2 Downregulated genes in prostate cancer patients identified by Affymetrix GeneChip experiments. Genes were sorted by numbers of patients with a fold change < 0.5 (quotient of PMQ-values of matched normal and tumor sample for each gene), HGN for HUGO Gene Nomenclature, FC for fold change.

• Tab. 3 Summary of quantitative RT-PCR of splice variants of TRPM8. Expression values were normalized to ß-actin and relative expression was calculated using the ΔΔ CT method.

• Tab. 4 Transcription factor binding sites in the TRPM8 promoter. HGNC stands for the official name given by the “HUGO Gene Nomenclature Committee”. The average frequency/1kb gives an expectation value of matches per 1kb of genomic DNA as given in TRANSFAC and * as blasted against a 200kb region of chromosome 21. Core similarity shows the similarity of the core sequence (usually between 4-6 base pairs) to the database sequences (capital letters). Matrix similarity shows the similarity of a sequence flanking the core region to the databases sequences (lower case letters). A perfect match of a matrix gets 1.0 a "good" match to the matrix usually has a similarity of > 0.80. Both calculation algorithms of the core and matrix are described in Quant et al. [Quandt, 95a].

• Tab.5 Comparison of probesets for TRPM8 on the metg001A chip and the U133B chip. Blue bases indicate incorrect base pairs, red bases show the overlapping sequences in both chips. The grey shaded part points out the false probesets.

• Tab. 6 Oligonucleotid sequences for RealTime PCR

• Tab. 7 Prostate Cancer Patient histopathological and follow up data of samples hybridized to the metg001 Cancer-Chip. R = 1 → Relapse after S; S = Surgery (radical prostatectomy); R = x → patient dead; GG = Gleason Grading,

• 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].

• 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.

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

• 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.

• 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].

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

• 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)

• Fig. 8 Affymetrix microarray analysis of TRPM8 expression in matched tumor and normal prostatic tissues. A) Changefold of PMQ values of 52 matched prostate cancer and normal tissues. B) Boxplots of PMQ-values of TRPM8 expression of prostate tumor patients grouped into normal and tumor. TRPM8 is significantly overexpressed in prostate tumors (p < 0.000001).

• Fig. 9 Affymetrix GeneChip analysis of TRPM8 expression in different cell lines. PMQ values of TRPM8 mRNA expression in 30 cell lines dived from six different tissues including prostate, bladder, colon, mammary gland, lung and pancreas.

• Fig. 10 Electronic Northern analysis of TRPM8 expression in 22 human tissues. FREQ = Frequency of a TRPM8-EST in a pool ESTs derived from either normal or cancer tissues N = Normal tissue, T = Tumor tissue, P-val = p-value, Sig = Significance.

• Fig. 11 Northern blot analysis of TRPM8 expression in various normal human tissues (Clontech, Heidelberg, Germany). The 5‘-probe was 32P-labeled and hybridized to the membrane. TRPM8 is expressed exclusively in prostate with different transcripts sizes of approximately 7.3 kb, 5.6 kb and 4.1kb.

• Fig. 12Cancer Profiling Array representing 241 matched tumor and normal human tissues from 13 cancer entities and several cell lines. A) The TRPM8 specific probe was 32P-labeled and hybridized to the membrane. B) The Ubiquitin specific probe was 32P-labeled and hybridized subsequently to the same blot.

• Fig. 13 Relative expression of TRPM8 mRNA in 42 prostate tumor samples by RT-PCR. Data is shown as relative expression levels from matched tumor and normal prostate tissues. T = tumor, N = normal

• Fig. 14 In situ hybridization of TRPM8 mRNA to a prostate adenocarcinoma. 4 µm sections were hybridized with the TRPM8 probe used in Northern and dot blot experiments. A) and B) hybridization of the antisense TRPM8 probe to an adenocarcinoma of the prostate. C) sense probe of TRPM8 and D) Haematoxilin and Elaun staining of the same patient.

• Fig. 15 FISH mapping of the genomic region of TRPM8 and MYC in normal human XY patients and LNCaP cells. Hybridization signals for TRPM8 (AC005538 on 2q37.2.) are shown in green; hybridization signals for MYC (8q24.12-q24.13) are indicated in red. Picture A1 to C1 show metaphase chromosomes. Pictures A2 to C2 show interphases of the same sample. A) 1 and 2 show hybridization signals specific for TRPM8 in a healthy XY-person. B) 1 and 2 show mapping of TRPM8 in LNCaP cells. C) 1 and 2 show the hybridization signal of MYC to chromosome 8.

• Fig. 16 Spectral karyotyping (SKY) of LNCaP cells taken from [Augustus, 03]. Chromosome 2 and 8 are boxed as these are the chromsomes where TRPM8 and MYC are localized, respectively.

• Fig. 17 FRET analysis of transiently transfected HEK293 with TRPM8-CFP and TRPM8-YFP after 24 h. A) and B) Recovery of fluorescence intensity of the FRET donor (∆FCFP) during disruption of energy transfer by photobleaching of the acceptor (FYFP). The acceptor was selectively bleached at ג = 515 nm. The relative increase of CFP (ΔFCFP (%) intensities compared to initial levels and YFP fluorescence intensity decrease (FYFP (%)) over time. The increase of CFP fluorescence intensity of 15, 3% is a direct evidence of FRET. Data shown are representative for several FRET analyses.

• Fig. 18 Analysis of calcium influx of HEK293 cells expressing TRPM8 using Fura-2. Representative measurement data were taken from calcium experiments of transfected and nontransfected HEK293 cells loaded with Fura-2. WT = wild type

• Fig. 19 Expression of TRPM8 in Affymetrix GeneChip experiments of 7 human tissues. Hybridization experiments were performed with 123 prostate -, 21 ovary -, 90 mammary gland -, 78 bladder -, 102 colon -, 11 pancreas - and 172 lung –specimens of normal and cancer tissue.

• Fig. 20 Affymetrix GeneChip analysis of prostate specific genes in bladder cancer patients. Expression values are shown as PMQ for TRPM8, KLK3, FLOH1 and ACCP.

• Fig. 21 GeneChip analysis of prostate specific genes in lung cancer patients. Expression values are shown as PMQ for TRPM8, KLK3, FLOH1 and ACCP.

• Fig. 22 Strong overexpression of TRPM8 in neuroendocrine tumors. Real-Time PCR of TRPM8 shown as relative expressions to corresponding normal tissue of each patient. A) Pure adenocarcinoma of the lung (panel 1), a lung adenocarcinoma with 10% of neuroendocrine cells (panel 2) and a 100% neuroendocrine tumor also located in the lung (panel 3). B) Expression of TRPM8 in neuroendocrine cell lines. RT-PCR results are shown as relative expression levels to the normal prostate epithelial cell line PrEC.

• Fig. 23 Genomic structure of TRPM8 on Chromosome 2q37.2.

• Fig. 24 Six splice variants of TRPM8. Alternative exons of TRPM8 are indicated as black boxes. Exons of TRPM8 are shown in grey and regions which are not transcribed are shown in stripy.

• Fig. 25 schematic structure of TRPM8 its isoforms 20b and 16b. Number boxes (grey) indicate tranmembrane spanning domains. The blue horizontal beam represents the cell membrane.

• Fig. 26 Genomic localization of the human TRPM8-Regulatory-RNA. The exons of TRPM8 are marked in grey. Black boxes indicate exons of TRPM8 regulatory RNA. Arrows indicate the direction of transcription.

• Fig. 27 Cancer Profiling Array representing 241 matched tumor and normal human tissues from 13 cancer entities and several cell lines. A) The 16b specific probe was 32P-labeled and hybridized to the membrane. B) The Ubiquitin specific probe was 32P-labeled and hybridized subsequently to the same blot.

• Fig. 28 Dot blot of matched prostate cancer and normal tissue hybridized with a SV 16b specific probe. cRNA of samples used for hybridization in Affymetrix microarray analysis were spotted to a nitrocellulose membrane a 16b specific probe was 32P-labeled and hybridized to the membrane.

• Fig. 29 FACS analysis of TRPM8 and 16b stable transfected cells. HEK293 cells stable transfected for TRPM8 and 16b were double stained for V5- and myc-epitope with FITC and PE labeled antibodies respectively. A) and B) show TRPM8-V5 expression of clones 18 and 19 stained with the anti-V5-PE antibody. C) and D) show the same clones, this time stained with anti-myc FITC antibody. M1 represents the mock clone (pcDNA3-1-V5-TOPO (A+B) and pcDNA6-myc-his (C+D). M2 gates the positively stained cells for TRPM8 (A+B) and 16b (C+D).

• Fig. 30 Western Blot of TRPM8-HEK293 cells stable transfected with 16b. Detection was performed with an anti-myc antibody. Lane 1 shows the 16b-protein of an in vitro translation reaction; lane 2 the transient transfected protein; lane 3-5 the empty vector of pcDNA6-myc-his (mock), lane 6-9 shows the 16b stable transfected TRPM8-HEK293 clones (Clone 19, 22, 9 and 16).

• Fig. 31 Co-expression and cellular localization of TRPM8 and SV 16b in HEK293 cells. A) Expression of TRPM8-V5 (red), B) Expression of 16b-myc (green), C) Overlay of pictures (A) and (B). D) Overlay of pictures (A) and (B) plus DAPI staining (blue).

• Fig. 32 Analysis of calcium influx of HEK293 cells co-expressing TRPM8 and 16b using Fura-2. Representative measurement data were taken from calcium experiments of transfected and nontransfected HEK293 cells loaded with Fura-2. WT = wild type.

• Fig. 33 Calcium flux induced by 1 µM Icilin in TRPM8 and 16b transfected HEK293 cells. Free calcium was measured using the FLIPR calcium assay kit and is presented as a change in fluorescence versus time.

• Fig. 34 Aberrant splicing of splice variant 16b. A) Agarose gel of a RT-PCR with 5’ TRPM8 and 3’ 16b specific primers. B) Blue indicates transcribed exons, shaded blue and red indicates untranscribed exons and red marks the alternative exon 16b.

• Fig. 35 Gene therapy approach using the TRPM8 promoter as the prostate specific transcriptional regulator of the Diphteria-Toxin-A expression.

• Fig. 36 Sequence of the promoter region of the human TRPM8 gene on 2q37.2. Grey boxes indicate putative transcription factor binding sites. +1 shows is the transcription start site. Met = methionine, shows the translation start site.

• Fig. 37 Comparison of the genomic structure of the human and mouse TRPM8 promoter region.

• Fig. 38 Homology analysis of the TRPM8 promoter between human and mouse. The 700 bp 5` to the transcription start site of the human TRPM8 were dotted against the mouse genomic region of the TRPM8 gene. Grey lines indicate high homology between the sequence of human and mouse.

• Fig. 39 The TRPM8 promoter revealing a highly conserved region. A) TRPM8 gene promoter. The transcription start is shown as +1, the black box indicates the TRPM8 protein; the grey box indicates a highly conserved sequence among species. B) Alignment of the human, mouse and rat highly conserved 172 bp region in the TRPM8 promoter. DNAs were aligned using CLUSTAL program. Grey boxes indicate the transcription factor binding sites with the 4-6 core base pairs framed in black.

• Fig. 40 Transcriptional activation of the TRPM8 promoter in different cell lines. HEK293, PC3, LNCaP and DU145 were transfected with the 1.9 kb-TRPM8 promoter fragment cloned in front of the luciferase reporter gene (1.9-kb-TRPM8-pGL3). Luciferase activity in the lysates was measured after 24 h. Data were normalized to the PhRL-0 which was used to normalize transfection efficiency. Data is shown relative to the pGL3-Basic vector.

• Fig. 41 Site directed mutated reporter gene constructs of the TRPM8 promoter cloned into the pGL3-basic reporter vector. The deletions in the constructs (SDM 1 to SDM 13) are marked in dark grey. In each mutation at least the core region of the transcription factor binding site was deleted, thus usually 6-8 bases were eliminated (see “Methods” for details).

• Fig. 42 Effect on activation activity of different cis-acting elements in the TRPM8 promoter. The 13 TRPM8 promoter (1.9 kb) constructs were designed each carrying a deletion of 6-9 base pairs specific for one ore more transcription factor binding sites shown in Fig. 41. The mutated constructs were located in front of a luciferase reporter gene in the pGL3-vector. LNCaP cells were transfected for 24 h with the wild-type promoter and the 13 constructs carrying the specific site directed deletions. Activation potential of each deletion construct is shown in relative expression to the wild type promoter (100%).

• Fig. 43 Effects of androgen on the TRPM8 expression. KLK3, NKX3-1 and MYC expression in LNCaP cells. * indicate the significant upregulation in expression compared to ethanol, the solvent of R1881, with p values at least < 0.001 (t-test).

• Fig. 44 Influence of androgens on the promoter activity of TRPM8. LNCaP were cultured in androgen depleted serum before the experiment. The 1.9kb promoter of TRPM8 was cloned in front of the luciferase reporter gene into the pGL3-basic vector (promega). Upon transfection with the TRPM8 promoter, cells were treated with R1881, 10% FCS or ETOH. Analysis was performed after 24h of treatment. Data is shown as relative luciferase activity to the transfection control plasmid phRLTK-null.

• Fig. 45 Microarray results of TRPM8 and KLK3 in correlation to Gleason sum and TNM staging (pT). Data is shown as the median of PMQ for each patient group. At least 3 patients represent one group.

• Fig. 46 TRPM8 Northern blot using 2 different probes for hybridization. A) Northern blot using a 2.7 probe from the 5’-end of the TRPM8 gene. B) Hybridization with a probe from the 3’ end of TRPM8. All other conditions were exactly the same.

• Fig. 47 Schematic drawing of the gene assembly program and the in silico expression profiling in cancers taken from [Schmitt, 99e].

• Fig. 48 Probeset distribution of TRPM8 on metg001A and U133B.

• Fig. 49 Schematic view of site-directed-mutations of the 1.9-TRPM8-pGL3 vector. Red letter in blue boxes indicate the base pairs deleted in Site-directed-mutations (SdM)-pGL3-construct. Light green boxes show the sequence of each transcription factor binding site. Capital letters demonstrate the core binding nucleotides of each transcription factor binding site.

• Fig. 50 Primer for the TRPM8 promoter site-directed mutations. Red letters indicate deleted base-pairs of the 1.9kb-TRPM8-pGL3 contruct.