[Seite 18↓]


2.1 Gene Chip Expression analysis of prostate cancer patients

High throughput gene expression profiling has become an outstanding method for identifying genes differentially expressed in normal and diseased tissue. In order to identify new genes differentially expressed in cancerous tissue, metaGen Pharmaceuticals GmbH designed a customized DNA Cancer-Chip based on Affymetrix technology (Affymetrix, Inc. USA). This oligonucleotide microarray contained 6200 probe sets representing approximately 3,000 genes. The chip design based on a bioinformatic attempt mining approximately 4 million expressed sequence tags (ESTs) of public4 and proprietary databases [Schmitt, 99b]. These ESTs 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. This selection and concentration of genes relevant in tumors on the metg001A chip was expected to increase immensely the percentage of genes differentially expressed in tumors. Additionally, it was expected to identify new genes, which were not yet discovered by other groups and methods.

Therefore 52 prostate tumor samples and its corresponding normal tissues were collected at the time of radical prostatectomy (RP) at the Department of Urology at the University Hospital Charité from 1999 to 2000. All samples were microdissected and hybridized to the metg001A microarray. Microdissection was used in order to specifically select tumor areas and normal glands to further increase the number of identified differentially expressed genes, especially as prostate tumors are among the most heterogeneous of cancers [Singh, 02]. The clinical and pathological features as well as the follow-up data of all patients hybridized to the chip are listed in Tab. 7 of the attachment.

Analysis of hybridization experiments was done according to the metaGen criteria, described in “Methods”. Briefly, a gene was called differentially when it was significantly expressed in both normal and tumor tissue (p-value < 0.05) and the quotient (fold change [FC]) of the normalized perfect match quintile (PMQ) value of each patient and probe set (tumor/normal) was > 2. For genes present only in normal or tumor tissue (p-value > 0.05) no fold change was calculated, but marked as differentially expressed (FC was set to 2). Finally, genes were ranked according to their differentially expression between normal and tumor tissue.

[Seite 19↓]

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.

Microarray analysis of prostate cancer patients revealed that 26% of the probesets (1434 probesets) present on the microarray showed a differential expression in at least one patient. Two criteria were applied for the identification of possible target genes. First, the gene should be overexpressed in tumors in at least 30% of the patients. Second, this gene should not be downregulated in more than 10% of the patients. Following these criteria 38 genes could be identified as overexpressed in prostate tumor patients. From these genes, seven were even upregulated in more than 50% (27 patients) of the tumors. The list of overexpressed genes in prostate tumor patients is shown in Tab. 1.

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.

62 genes could be identified being underexpressed in at least 30% of the patients (FC < 0.5) using the same criteria as for genes downregulated in prostate cancer. [Seite 20↓]From these, 5 genes could be identified to be downregulated in more than 50% of the tumors, listed in Tab. 2.

For a more detailed analysis of this prostate cancer profiling, please refer to the thesis of Christoph Wissmann [Wissmann, 02].

2.1.1 Expression of TRPM8 in prostate cancers

After the identification of genes upregulated in prostate cancer, the next task was to find out which genes would be the best targets for an antibody or small-molecule therapy. The following criteria were set up for genes to be further evaluated: 1. The gene had to be upregulated in at least 30% of all tumors, while downregulation should be less than 10%. 2. The gene had to be new (neither mentioned in the literature nor in patent applications nor filed patents) 3. The gene products should be drugable with preference for proteins located in the plasma membrane. Surprisingly not many genes could be found fulfilling these criteria. One of them was the protein standing on position four of the list of genes upregulated in prostate cancer: TRPM8. It was expressed significantly (p < 0.05) in 103 of the 104 specimens and it was overexpressed in 56% of all tumor patients with a fold change > 2. (p < 0.000001) (Fig. 8). Only in one patient TRPM8 was downregulated in tumors (FC < 0.5).

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).

At the time of data analysis, TRPM8 was completely unknown and database search of the partial cDNA of TRPM8 revealed a high homology to the transient receptor potential (TRP) family of non-voltage-gated cation channels.

2.1.2 Expression of TRPM8 in human cell lines

In order to characterize the expression of TRPM8 among various cell lines 30 established human cell lines were hybridized to the metg001A chip. These cell lines were derived from normal and cancer tissues from the prostate, bladder, colon, mammary gland, lung and pancreas (Fig. 9). Results indicated that [Seite 21↓]TRPM8 is exclusively expressed in prostate tumor cell line LNCaP (p<0.05). No significant TRPM8 expression could be detected in any other cell lines tested.

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.

2.2  TRPM8 expression in Human tissues

Affymetrix gene chip experiments and electronic Northern analysis revealed that TRPM8 expression is restricted to the prostate. To confirm these high-throughput methods and to analyze the distribution of TRPM8 expression in “wet” experiments, classical Northern blots and dot blots as well as Real Time PCR were performed on a large number of patient and tissues.

2.2.1 Electronic Northern of TRPM8

In order to use a gene as a target for a therapeutic approach it has to be shown, that its expression is restricted to the tissue (and disease state) to be targeted, or at least, it should not be expressed in organs essentially for survival such as heart or brain. Ideally, the expression of the therapeutic target gene should be restricted to the tissue of interest. Thus, the drug - its specificity presumed - would not affect any healthy tissue and side effects could be minimized.

[Seite 22↓]

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.

Therefore the TRPM8 expression pattern among diverse normal tissues was examined by electronic Northern (Fig. 10). This in silico approach is a very effective tool to examine expression patterns of genes among divers tissues. Using the proprietary data set of metaGen approximately 4 million ESTs could be analyzed for the expression of TRPM8. Analysis showed impressively that TRPM8 expression among 22 human tissues was significantly restricted to the prostate. (Significance = 100%; p-value < 10-5). This is one of the rare events that a gene is solely expressed in one organ. Further it could be shown that TRPM8 is highly differential expressed between tumor and normal prostate tissues with a significance of 99.8 and a p-value of 0.00158.

2.2.2 Northern and Dot blot analysis of TRPM8

The probe specific for TRPM8 comprised the first 2700 base pairs of the TRPM8 open reading frame (ORF). Hybridizing this probe to a Northern blot from Clontech (Heidelberg, Germany) representing 16 different human normal tissues revealed that TRPM8 expression is restricted completely to the prostate (Fig. 11). Interestingly, besides the expected 5.6 kb fragment two other transcripts of approximately 7.3 and 4.1 kb could be detected.

[Seite 23↓]

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.

Hybridizing the same probe to a Cancer Profiling Array (CPA) (Clontech) which represents cDNA dots of 241 matched normal and tumor tissues of 14 human cancer entities and 9 cancer cell lines confirms the results seen in electronic Northern analysis. TRPM8 is exclusively expressed in the prostate with the exemption of one kidney tumor (Fig. 12).

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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.

2.2.3 Real Time PCR of TRPM8 in matched prostate cancer patients

Quantitative Real Time PCR of TRPM8 expression was done on 42 cDNA from matched tumor and normal prostate cancer patients. Results show that TRPM8 is overexpressed in 64% of prostate tumor patients (Fig. 13). In some patients the relative expression levels of tumor versus normal tissue are very high (20-100 x) which indicates the remarkable overexpression of TRPM8 in prostate tumors. In comparison to microarray experiments Real Time PCR are much more sensitive. [Seite 25↓]These experiments confirm the results gained in gene chip and dot blot experiments.

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

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

In situ hybridization of TRPM8 expression was performed on 60 normal, 23 prostatic intraepithelial neoplasia (PIN) and 91 adenocarcinomas (Gleason Grading 1-10) of the prostate. Additionally, 10 specimens of each tissue from tumors of the mammary gland, ovary, liver, pancreas and bladder were tested for TRPM8 expression. Expression could be detected in 37 of the 60 normal tissues, in 17 of the 23 PINs and in 75 of the 91 adenoncarcinomas of the prostate. All other tissues remained negative. In normal prostate sections, the epithelial cells showed moderately positive hybridizations signals. Strongest signals however were observed in the epithelial cells forming the lumen of the duct (Fig. 14). Smooth muscle cells and connective tissue remained negative. Relative expression levels comparing normal and tumor samples from the prostate samples showed an upregulation in 28 of 56 matched tumor and normal samples. TRPM8 expression correlated positively with disease progression from normal over PIN to low grade tumors, but TRPM8 expression was lost in completely undifferentiated tumor cells (Gleason Grading 9-10). This could be observed [Seite 26↓]especially in cases were high grade tumors and low grade tumors were present in the same section of a specimen.

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.

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

Fluorescence in situ hybridization (FISH) analysis of the genomic region of TRPM8 was performed to gain insight into the molecular mechanism of the overexpression of TRPM8 in prostate tumors and LNCaP cells. As TRPM8 is located at the very end of chromosome 2 (2q37.2), the chromosomal region could possibly by either amplified and/or translocated to other chromosomes. Through translocation, the TRPM8 gene might get under the control of an alternative promoter or enhancer resulting in an altered expression profile. Both chromosomal amplification and translocation are known mechanisms often occurring in tumor tissues [Nowell, 97]. In order to address this question FISH experiments were performed on LNCaP cells. This cell line was chosen, as it is the only cell line expressing TRPM8. The BAC AC005538 (2q37.2) was used as a TRPM8 specific probe for hybridization. As a positive control a commercially available probe for identifying MYC on 8q24.12-q24.13 was used (Vysis Inc., Downers Grove; IL, USA). A comparison of hybridization signals of TRPM8 [Seite 27↓]between LNCaP cells to normal human XY specimens showed that TRPM8 hybridized exclusively to the expected position on chromosome 2, in both the LNCaP cell and the XY patient (Fig. 15). No hybridization signal could be detected anywhere else in the chromosomes.

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.

The finding that chromosome 2 is tripled in LNCaP cells and chromosome 8 (MYC-specific binding) is quadruplicated, is in coherence with the literature [Augustus, 03]. A spectral karyotyping of LNCaP cells shows a predominantly tetraploid karyotype of LNCaP cells. Only chromosome 2 (triploid), 6 (diploid), 19 (triploid) and 21 (triploid) differ from that pattern (Fig. 16).

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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.

Additionally, the signal strength was not enhanced in LNCaP cells compared to normal XY patients. Therefore, it can be concluded that the overexpression of TRPM8 in LNCaP cells and thus most likely also in prostate tumor patients is not due to chromosomal amplifications or rearrangements.

2.4 Homomultimerization of TRPM8 subuntis

TRPs are known to form either homo- or hetero-tetrames. In order to prove direct interactions between TRPM8 subunits, channel multimerization was assessed using the fluorescence resonance energy transport (FRET) technology. Therefore TRPM8 was terminally fused to CFP and YFP vector construct. These plasmids where transiently coexpressed in HEK293 cells and the proximity of the homomerization was measured with FRET. FRET signal was obtained by measuring the increase in fluorescence of donor (CFP) emission during photobleach of the acceptor (YFP). The recovery of donor fluorescence emission was then monitored at 480 nm and was expressed as percentage of CFP emission after acceptor bleach. The FRET analysis showed a strong interaction of TRPM8 homomultimers which is shown in FRET efficiencies of 15.3% (Fig. 17, A).

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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.

2.5 activation of TRPM8 by the cooling agent icilin

It was shown by McKemy et al., that the TRPM8 rat orthologue CMR1 can be activated by cold inducing agents such as icilin, menthol and eucalyptol as well as by temperatures below 28 °C resulting in an increase of intracellular calcium levels. The mouse orthologue of TRPM8 could also be activated by menthol and temperatures ranging from 25°C to 15°C [Peier, 02d]. In order to find out whether human TRPM8 could also be activated with different cooling agents, TRPM8-stable expressing HEK293 cells were loaded with the Ca2+- indicator Fura-2 and exposed to 50 µM of Icilin. These experiments were done in cooperation with Stefan Mergler from the Charité in Berlin.

As displayed in Fig. 18 (A, B) TRPM8 expressing cells showed a threefold increase in intracellular calcium upon stimulation with Icilin. That response was not observed in nontransfected and empty vector transfected cells. The icilin mediated calcium influx occurred within msec. Washout of icilin induced recurrence of [Ca2+]i back to levels of unstimulated cells (recovery effect). The response was dependent on Ca2+ in the buffer, because removal of extracellular calcium suppressed the Icilin response. The result indicated that TRPM8 is localized in the plasma membrane, although transmembrane localization could not be seen in most of the TRPM8-expressing HEK293 cells. This is most likely due to limitations of sensitivity in the cytochemistry procedures.

[Seite 30↓]

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

2.6 Gene Chip Analysis of TRPM8 expression in 7 human tissues

As prostate was the first entity to be hybridized to Affymetrix GeneChips at metaGen, it was interesting to check two years later the expression on TRPM8 in entities other than the prostate. Until today 6 other tissues have been hybridized to several Affymetrix chips (either proprietary or custom designed). Bladder, ovary, mammary gland and pancreas were hybridized to the metg001A Cancer-Chip; lung and colon were hybridized to the U133A and B Affymetrix GeneChip. The probe sets on the U133B were not identical to the ones on the metg001A chip but adequate, as described in detail in “Methods”. Bioinformatic analysis of these 7 tissues revealed that TRPM8 expression was restricted to the prostate with two exceptions. One bladder-tumor sample (out of 79) and one lung cancer (out of 172) expressed TRPM8 (Fig. 19).

[Seite 31↓]
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.

These results were quite unexpected as dot blot and Northern blot experiments showed exclusive expression of TRPM8 in the prostate. The bladder and the lung cancer sample stoud out very clearly from the other samples. Looking closer at the bladder cancer patient, this sample did not only express TRPM8, but also Kallikrein (PSA), Folathydrolase1 (PSMA) and the Acidic Phosphatase Prostate (ACPP) all of which are prostate specific markers (Fig. 20). None of the other 71 bladder samples did express any of these genes. In general these 4 genes were expressed conjointly in nearly 100% of all prostate samples, but very rarely (and never all 4 gene together) in any non-prostatic tissue tested (data not shown). As it was most unlikely that this patient really expressed prostate specific genes, the pathologist at the University of Regensburg (from where the tissue came from) was asked to diagnose this sample again. In response, she told us that this patient was indeed a bladder cancer patient, but that some prostate glands had infiltrated into the bladder. Unfortunately these glands were also microdissected and thus amplified and hybridized on the chip. Consequently this sample was taken out of further analysis of the bladder-experiments at metaGen. The most important aspect of this feature was not the fact that the sample was no pure bladder sample, but the confidence it provided regarding the data gained from the microarray experiments.

[Seite 32↓]

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.

[Seite 33↓]

Strikingly, in lung cancer patients TRPM8 was the only prostate specific gene expressed (Fig. 21). Neither Kallikrein nor Folathydrolase1 nor acidic phosphatase were expressed in this sample. A contamination with prostatic tissue or a prostatic metastasis in the lung could thus be excluded. In order to find an answer to this result the patients data sheets were checked in collaboration with the pathologist and an interesting observation could be made: Lu93 was the only patient run on microarray chips which had a fraction of 10% neuroendocrine tumor cells.

2.7 TRPM8 expression in neuroendocrine tumors of the lung

Real-Time PCR was performed to evaluate the assumed expression of TRPM8 in the patient with a 10% neuroendocrine tumor of the lung (Lu93). Additionally, a 100% neuroendocrine tumor from the lung and a lung adenocarcinoma were analyzed. The mRNA of normal and tumor material of these patients was isolated and quantitative PCR was performed. Results shown in Fig. 22 indicate that TRPM8 is highly overexpressed in both the 10% and the 100% neuroendocrine tumors, but not in the andenocarcinoma of the lung.

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.

2.8 Genomic structure of TRPM8

TRPM8 is located on chromosome 2q37.2 TRPM8 distributed over 100kb at the very end of chromosome 2. The gene consists of 26 exons resulting in 5641 base pairs (Fig. 23). The open reading frame (ORF) has 3312 basepairs resulting in an ORF of 1104 amino acids. The ion pore of TRPM8 consists of 6 transmembrane spanning domains located between exon 16 and 20. Both N and C-termini are located in the cytoplasm.

[Seite 34↓]

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

2.9 Identification of TRPM8 splice variants

TRPM8 splice variants could be identified while cloning the TRPM8 full length gene. The TRPM8 gene was amplified from a normal prostate mRNA pool (Ambion, Huntington, UK). A polyT-T7 primer was used in the reverse transcription RT-reaction which was followed by a PCR reaction using TRPM8 specific primer which bound to the first and last exon of TRPM8. It was interesting to observe that after cloning and sequencing of the constructs a short variant of TRPM8 could be identified. Exons 1-6 of this variant were identical to TRPM8, but exon 6 was elongated by 245 bp ending with a polyadenylation signal (SV 6b). Cloning of the product was possible as the primer used for the cDNA synthesis functioned as the reverse primer in the PCR reaction. This finding was in accordance with the results gained in the Northern blot experiments where two other isoforms of TRPM8 could be detected, although splice variant 6b was smaller than the shortest fragment (4.1kb) seen in the Northern blot experiments (Fig. 11).

In the course of performing genome database search using public5 and proprietary (Incyte Genomics, Palo Alto, CA, USA) cDNA libraries 5 additional alternative transcripts of TRPM8 (Fig. 24) were identified. Using PCR-techniques it was possible to elongate most of the isoforms. The splice variant 6b of the TRPM8 has the SEQ ID NO 1. The splice variant 4a_4b has the SEQ ID NO: 2. It contains of at least 5 exons. Exon 2x is a separate exon (3’end is incomplete) and is located several hundred bases in front of exon 3 of trp-p8. Exon 3 is transcribed as in trp-p8 but exon 4 starts 46 bases earlier compared to trp-p8. The sequence continues from exon 5 to 6a and ends with 6b. Splice variant 16b of TRPM8 has the SEQ ID NO 3. It contains of 16 exons. Exons 1- 15 are identical with TRPM8. Exon 16 has an extention of 104 basepairs immediately beginning after exon 16 and ending in a poly-A signal.

[Seite 35↓]

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.

Splice variant 20b has the SEQ ID NO: 4. The sequence contains of 26 exons. Exons 1-19 and 21-25 are identical to TRPM8. Distinct from TRPM8 splice variant 20b contains an elongated exon 20 with an addition of 127 basepairs immediately beginning after exon 20; exon 26 is truncated: The poly-A signal starts already at basepair 1136. The splice variant avant25 of TRPM8 has the SEQ ID NO: 5. This sequence contains of at least 3 exons with an unknown 3’ end. Avant25 contains an exon of 570 basepairs not found in TRPM8. This exon extends into in exon 25 and 26 but exon 26 is truncated as in splice variant 20b with a poly-A signal beginning at basepair 1136. The splice variant avant13 has the SEQ ID NO: 6. The sequence contains of at least 6 exons with an unknown 3’ end. Avant13 has an exon of 272 basepairs not found in TRPM8. This exon extends into exon 21 but only 20 bases of the 3’end of this exon are within the transcript. The last 4 exons are identical to TRPM8 with a different splice pattern (exons 22, 23, 24 and 26, but exon 26 is truncated with a poly-A signal beginning at basepair 654).

Though it was possible to identify more splice variants, but as they were less abundant and less differentially expressed than those 5 they will not be further discussed here.

The structure of TRPM8 isoforms 16b and 20b is shown in Fig. 25. 16b has one transmembrane spanning domain which results in an extracellular C-terminus. Isoform 20b consists of the same transmembrane spanning domain as TRPM8 but the C-Terminus ends a few amino acids after the last transmembrane spanning domain.

[Seite 36↓]

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.

2.9.1 Identification of the TRPM8-regulatory-RNA

On the basis of in silico analysis of different ESTs in the region of TRPM8 it was possible to identify a gene which is positioned on the opposite strand of the TRPM8 gene on chromosome 2q37.2. (SEQ ID NO: 6) Exon 1 of this RNA lies in intronic regions between exon 12 and 11 of TRPM8; Exon 2 lies in front, over, and behind exon 11. Exon 3 is located between exon 8 and 7 of the TRPM8 gene (Fig. 26). It was named TRPM8-Regulatory-RNA, because it may bind to TRPM8 or its splice variants and thus alter the expression of these genes. Additionally, binding of the mRNA may cause destabilization through activation of mRNA degradation mechanisms or stabilization of the mRNA altering in an elongated translation.

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.

2.9.2 Expression of TRPM8 splice variants in prostate tumors

Real time PCR of the 5 splice variants and the regulatory RNA was performed in order to find out whether these isoforms are as differentially expressed as TRPM8 itself. PCR was performed on samples from prostate cancer tissues used for gene chip experiments and on some additional samples. Tab. 3 shows the results. The splice variants 16b, 20b and avant25 were overexpressed in prostate tumors to 65%, 67% and 60%, respectively, altering an even higher differentially expression than TRPM8 itself. The TRPM8-Regulatory-RNA was overexpressed [Seite 37↓]in 80% of the prostate patients which was the highest differential expression seen. Additionally, relative expression values between a corresponding normal and cancer sample of the isoforms were significantly higher than those of TRPM8 (data not shown). But absolute expression levels of the slice variants were generally lower.

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.

2.9.3 Characterization of TRPM8 Splice variant 16b

The splice variant 16b was chosen for a detailed analysis because a) it is one of the highest differentials expressed splice variants in prostate cancer patients seen in RT-PCR experiments (Tab. 3) and b) most importantly, the N-terminus is probably located in the extracellular space. This extracellular localization makes it an ideal target for the development of a therapeutic antibody, even better than TRPM8 itself as it has a larger epitope for the production of an antibody.

In order to determine the expression profile of splice variant 16b, Northern and dot blot experiments with the 32P-labeled 16b-specific probe were performed on the commercial Cancer Profiling Array (Clonech, Heidelberg, Germany) (Fig. 27).

[Seite 38↓]

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.

Additionally dot blot experiments were carried out on a self spotted blot, to which cRNA from 48 tumor and corresponding normal prostate tissues were spotted to the membrane (Fig. 28). The cRNA spotted was derived from amplified cRNA used for gene chip experiments. Strikingly, the expression pattern of 16b is the same as for TRPM8. The self made dot blots reveal that 16b is overexpressed in 65% of all prostate tumors. These results show that SV 16b would be an even better target for the development of a therapeutic antibody than TRPM8.

[Seite 39↓]

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.

2.9.4 Generation of HEK293 cell stable for 16b and TRPM8

HEK293 cells stable for TRPM8-pcDNA3.1-V5 were transfected with the 16b-pcDNA6-myc constructs for 24h. After antibiotic selection clones were checked for TRPM8 and 16b expression using V5- and myc-specific fluorescent antibodies in the fluorescence activated cell sorter (FACS) (Fig. 29). The percentage of cells which were positive for TRPM8 ranged from approximately 52% to 71% (Fig. 29 A and B). By contrast, cells positive for 16b exhibited only a percentage of 1,3 to 7,6% (Fig. 29 C and D). For control purpose these constructs were checked in Western Blot experiments for the expression of SV 16b (88kD). These experiments showed a strong expression of 16b in all of the six clones tested (Fig. 30).

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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).

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2.9.5  Cellular localization of TRPM8 and SV16b in HEK293 cells

Cellular localization of TRPM8 and its splice variants were analyzed by cloning TRPM8 and 16b into the pcDNA3.1-V5-TOPO and pcDNA6-myc-his vector, respectively. HEK293 cells were used for the genration of stable cell lines expressing TRPM8. SV 16b was transiently transfected into these cells and also into wild type cells for 24 hours, prior fixation and staining with fluorescent antibodies for V5 and myc. Subcellular distribution of TRPM8 and 16b was detected by confocal fluorescence microscopy (Leica Microsystems, Solms, Germany) Fig. 31.

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).

TRPM8 expression could be seen within the cytosol and in the membrane of intracellular compartments, predominantly in the endoplamatic reticulum. The expression exhibited a spotty cluster, which has already been shown for other TRP channels as for example TRPC3 [Hofmann, 99a] (Fig. 31 A). By contrast [Seite 42↓]16b distributed homogenously within the cytoplasm which led to the conclusion that 16b is a soluble protein (Fig. 31 B). In order to examine whether 16b interacts in any form with TRPM8 in vitro, co-transfection of SV 16b in to HEK293 cells stabel transfected for TRPM8 were performed. As shown in Fig. 31 C and D 16b did not alter the localization of TRPM8.

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

In order to examine whether the splice variant 16b had any influence on the activation of TRPM8 by icilin different HEK293 clones expressing TRPM8 and 16b stably, were exposed to 50 µM of Icilin. It could be shown that 16b reduces generally the calcium influx in HEK293 cells. (Fig. 32 D - F), but the results obtained were very unstable. For an unknown reason the empty vector control showed also some inhibiting function (repeated experiments). These findings were supported by FLIPR calcium assay experiments.Fig. 33shows the calcium influx which was induced by 1 µM of Icilin in TRPM8 and 16b transfected HEK293 cells. Again, wild type HEK293 cells show no increase in intracellular calcium when exposed to Icilin. Only when these cells were transfected with TRPM8 a calcium flux could be measured. When cells were co-transfected with TRPM8 and 16b the influx in calcium was quite noticeable reduced, but some clones of 16b transfected cells show an increase in calcium flux (i.e. Clone 19). Additionally, the empty vector (ev) control showed also reduced flux of Ca2+.

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.

[Seite 43↓]

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.

2.9.7 Aberrant splicing of SV 16b

In RT-PCR experiments using TRPM8 and 16b specific primers, it was further possible to identify even more splice variants named 16b -1 to 16b - 4 by RT-PCR using primer specific for TRPM8 (forward primer) and 16b (reverse primer) (Fig. 34). Sequencing of the PCR products revealed the exon scipping shown in Fig. 34 B.

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.

[Seite 44↓]

2.10  Promotoranalysis of the TRPM8 Gene

In the first part of this study it was demonstrated that TRPM8 is an extremely tissue specific gene expressed exclusively in the prostate and neuroencorine tumors. Out of this characteristic arose the question, whether this specificity could be used not only for a small-molecule or antibody based therapy but also for gene therapy. The idea was to clone the TRPM8 promoter in front of the sequence of a certain toxin, such as diphteria toxin A into a viral vector used for gene therapy (Fig. 35) (Li, Diphteria Toxin, cancer research 2002). Expression of the toxin would be restricted to the prostate as the transcription of the toxin would be under the control of the tissue specific TRPM8 promoter. Following that approach several questions need to be answered. 1. How does the TRPM8 promoter look like and which transcription factor (TF) binding sites are present? 2. Is the promoter responsible for the tissue specific expression of TRPM8? 3. And if, is it possible to narrow down a specific part (TF- binding site) responsible for transcriptional activation or repression? 4. As it has been shown that TRPM8 is expressed under the control of androgens, are androgen responsive elements (ARE’s) present? All of these questions will be addressed in the next chapters.

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

2.10.1 Characterization of the TRPM8 Promoter

TRPM8 is a very tissue specific gene of which the expression is restricted to the prostate and neuroencorine tumors. One goal of this study was to identify and characterize the promoter of TRPM8. By blasting the cDNA sequence of TRPM8 gene against the public HTGS (High Throughput Genomic Sequences) database6 a BAC AC005538 was identified which covered the whole genomic region of TRPM8 including 100 MB of the 5’ regions. Fig. 36 shows the 1.9 kb sequence of the TRPM8 promoter including the short 5’ UTR as well as the transcription start site and the first exon of TRPM8. That fragment was subjected to in silico analysis for potential transcription factor binding sites using the commercially available software MatInspector7 [Quandt, 95b].

[Seite 45↓]

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.

[Seite 46↓]

This software utilizes the TRANSFAC8 library, which emphasis on sequences with experimentally verified binding capacity. Tab. 4lists the transcription factors with the highest prediction values. The score for the core sequences listed was in all cases 1.0 which means that the 4-8 basepairs (capital letters) matched 100% to the core sequence of the transcription factor from the database. The matrix similarity comparing the given sequence of the flanking region of the core with the sequence form the database were between 0.90 and 0.99 indicating that even the matrix similarity shows a high identity to known cis-elements (values > 0.8 were designated as good). For the transcription factor NKX3-1 the matrix value was left out as the identification of this transcription factor binding site was based on an own literature [Steadman, 00b]. Steadman identified in gel shift experiments that these hexamers strongly bind NKX3-1. The average frequenz/1kb of these sequences was calculated by blasting each hexamer against a 5 MB sequence of Chromosome 21 which should represent a general distribution of the sequence within the whole genome.

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].

[Seite 47↓]

It was interestingly to note that the predominant transcription factor binding sites identified were from the family of homeobox genes. Cis-elements for the binding of PRX2 (paired related homeobox protein 2) and NKX3-1 was present 5 times each in the 1.9kb promoter sequence of TRPM8. This is a dramatic overrepresentation. The TRPM8 promoter contains a TATA-box and a GC-Box at positions -42 and -67, respectively. Transcription factor binding sites for NKX3-1 (5x), NKX2-5 (5x), USF1 (4x), MYCMAX, LMO2, MYC and ARNT were found in this fragment. Only those binding site were listed which have maximum similarity with described binding sites expressed in core and matrix similarity- values (Tab. 4).

2.10.2 Genomic structure of the human and mouse TRPM8 promoter

Genomic analysis of the promoter region of mouse and human TRPM8 revealed that the mouse orthologue misses the first exon of human TRPM8. But it alters 4 additional exons at the 5’ end, which were so far not seen in humans (Fig. 37). A Dotter analysis of the 700 bp region 5’ to the transcription start site of human TRPM8 to the mouse TRPM8 genomic region is shown in Fig. 38. It demonstrates the high homology between mouse and human of the 172-base pair conserved element. Additionally, it could be illustrated that the GC- and TATA-Box are also conserved between these species.

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

[Seite 48↓]

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.

2.10.3 Transcription repression by a highly conserved promoter fragment

Alignment of the 1.9 fragment of the human TRPM8 promoter sequence with the mouse and rat orthologes revealed an overall identity between human and mouse of 56%. Strikingly a section of 172 bp showed a much higher concordance between the species than the overall sequence. Mouse and human display a similarity of 83% and rat and human of 82% (Fig. 36, Fig. 39). The first assumption, that this could be an alternative exon of TRPM8 was disapproved by repeated RT-PCR demonstrating that it was neither possible to amplify this fragment from any cDNA bank by itself nor with any known exon of TRPM8. To determine if the short part and the 1.9 sequence were able to induce transcription activation these two fragments were cloned each in front of the luciferase reporter vector pGL3-Basic. Different cell lines were transiently transfected with the constructs using PhRL-0 vector for transfection efficiency control. The 1.9 kb TRPM8 promoter showed a more than 13-fold transcriptional activation in DU145 cells (Fig. 40). In LNCaP, PC3 and HEK293 activation ranged from a 9-fold, over 7-fold to nearly 4 fold, respectively. Interestingly, the short element exhibited in all experiments a decrease in activation compared to the pGL3-Basic vector. This indicated that the conserved element might contain a repressor element (Fig. 40).

[Seite 49↓]

The observation that the 172bp and the 1.9kb fragment -although oppositional- both regulate expression identifies this region as promoter site of the human TRPM8 gene. Especially, because a TATA-box with a high significance could be identified at the expected 30-45 base pairs away from the start site. Within the conserved region transcription factor binding site for PRX2 (2x) NKX2-5 and NKX3-1 (2x) were identified. The responding transcription factors all belong to the group of proteins which are characterized by the presence of the homeobox which binds the DNA, indicating that these genes play a predominant role in the activation or repression of TRPM8.

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.

[Seite 50↓]

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.

2.10.4 Site-directed mutation in the 1.9kb promoter

The promoter of TRPM8 exhibits multiple transcription factor binding sites. In order to identify which transcription factor would alter the activity either through activation or repression 13 site-directed deletions of the core 6-8 base pairs of each potential TF-binding site were introduced into the 1.9kb-pGL3-reporter vector using site-directed mutagenesis (Fig. 41). LNCaP cells were transfected for 24 h with these constructs and luminescence was measured. All of the mutations led to a reduction of the activation potential. The strongest inhibition of activation (up to 80%) was achieved when the GC-Box or the TATA-Box were mutated. Neither all NKX3-1 nor PRX2 binding sites showed an agreeing repression pattern.

[Seite 51↓]

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).

[Seite 52↓]

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%).

2.10.5 Androgens enhance transcription of TRPM8

In order to evaluate if TRPM8 is regulated by androgens, LNCaP cells were cultured in steroid reduced medium and subsequently treated with 1nM, 10nM and 100nM of the androgen R1881. After 24 hours of treatment mRNA was isolated and RT-PCR was performed using gene specific primers for TRPM8, KLK3, NKX3-1 and MYC. The results were adjusted to the house keeping gene SDHA (succinate dehydrogenase complex, subunit A) and shown as relative expression to solvent ethanol (Fig. 43). R1881 enhances the transcription of TRPM8 nearly 90 times in LNCaP cells whereas KLK3, the gene which codes for PSA and NKX3-1 are upregulated 30 times and 6 times, respectively. The expression of the transcription factor MYC is not regulated by androgens.

[Seite 53↓]

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).

This was also an interesting finding, especially as the 1.9 kb fragment of the promoter did not show any androgen responsive element (ARE). Additionally, it should be analyzed if androgens alter the activation potential of the 1.9kb-pGL3 promoter construct. Therefore LNCaP cells were treated in the same manner as described above, but additionally when treatment of androgens started, cells were transfected with the 1.9-pGL3-promoter construct and the phRL-TK vector as a transfection efficiency control. After 24 h cells were lysed and luminescence measured. Results are shown in Fig. 44: The activation of the 1.9-pGL3 promoter compared to the pGL3-empthy vector was approximately 8 times, however the addition of R1881 did not activate the promoter significantly when results were adjusted to the empty pGL3 vector (data not separately shown). Thus androgens do not enhance this part of the promoter.

[Seite 54↓]

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.

2.11 Correlation of TRPM8 to grading and staging of prostate cancer

Affymetrix gene chips experiments were analyzed for correlation of TRPM8 expression to the Gleason grading and the TNM staging system. Patients’ PMQ values for TRPM8 and PSA were grouped by staging and grading and the median of each group was calculated. Results showed that TRPM8 mRNA expression increases with Gleason sum from normal tissue to a Gleason sum of 8, but mRNA levels fall to nearly normal levels in high grade tumors (Gleason sum of 9) (Fig. 45 A and B). Correlating TRPM8 expression to the TNM staging system showed similar results. Expression of TRPM8 first rise in early stage tumors compared to normal tissues, but when the tumor has extended through the prostatic capsule into the seminal vesicles (T3) TRPM8 levels fall. Stage 4 cancers, which are characterized by the spreading of the tumor into the bladder neck, show further reduced expression of TRPM8 compared to T3 tumors (Fig. 45 C and D). On the other hand, PSA levels do not show any correlation to either staging or grading.

[Seite 55↓]

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.

Fußnoten und Endnoten

4  http://www.ncbi.nlm.nih.gov/dbEST/

5 http://www.ncbi.nlm.nih.gov/dbEST/

6 http://www.ncbi.nlm.nih.gov/HTGS/

7  http://www.gsf.de/biodv/matinspector.html

8 http://www.gene-regulation.com/pub/databases.html#transfac

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