Patent Application
20110197293
The present invention relates to the identification of two tumor enriched transcripts and their shared CpG island located within the body of human TRPM2 gene on chromosome 21. The first of these transcripts is named TRPM2-AS (transient receptor potential cation channel, subfamily M, member 2-antisense). The second transcript, located across the shared CpG island, is named TRPM2-TE (tumor-enriched transient receptor potential cation channel, subfamily M, member 2). Such transcripts and their shared CpG island are useful as diagnostic, prognostic and therapeutic agents in cancer and in other diseases in which cell death by oxidative stress plays a relevant role such as neurodegenerative diseases, diabetes and stroke.
There is overwhelming evidence indicating that human cancer is a genetic disease caused by sequential accumulation of mutations in oncogenes and tumor suppressor genes [1-3]. However, it is also increasingly apparent that cancer not only depends on genetic alterations, but also on epigenetic changes that collaborate with genetic traits to drive progressive stages of tumor evolution [4-6]. One of the main epigenetic cancer features is an altered DNA methylation pattern, composed of global demethylation and promoter-localized hypermethylation. While the latter phenomenon is more understood, a certain number of speculative hypotheses have been formulated about the former. For example, global hypomethylation of cancer cells could result in a de-repression of short interspersed repeated sequences (SINEs), long interspersed repeated sequences (LINEs) and retroviral sequences [7,8]. Thereby, aberrant antisense RNA transcripts would be yielded, some of which could interfere with key tumor suppressors [7]. This would then inhibit their expression and promote the evolution of malignant clones. Indeed, it would be interesting to systematically identify antisense transcripts whose expression is enriched in cancer cells, in order to study their possible role in cancerogenesis.
Over the past few years, the authors of the present invention have developed a software program called AntiHunter, aimed at identifying antisense transcripts in the Expressed Sequence Tag (EST) database [9,10]. The authors were able to apply AntiHunter to the genome-wide identification of antisense transcripts in melanoma. Several antisense transcripts showing enrichment in melanoma were identified by the program. Here, the authors report one of them, TRPM2-AS, mapped within the locus of TRPM2, an ion channel described as a member of the transient receptor potential (TRP) superfamily [11], which is a diverse group of voltage-independent calcium-permeable cation channels [12]. TRPM2 is activated by several second messengers [13-15], oxidative stress and TNF-α and is capable of mediating susceptibility to cell death [16, 17, 15, 18-20]. Moreover, a specific role for TRPM2 in cancer cell death has been recently proposed [21].
Intriguingly, graphical viewing of the AntiHunter results in a genomic browser indicated the presence of another tumor-enriched TRPM2 transcript in the same region, TRPM2-TE, located across a CpG island shared with TRPM2-AS. CpG islands are genomic regions that contain a high frequency of CG dinucleotides. In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length. The “p” in CpG notation refers to the phosphodiester bond between the cytidine and the guanosine. Expression analysis of TRPM2-AS and TRPM2-TE in malignant melanoma indicated that they were consistently up-regulated. It was also determined that TRPM2-TE could contribute to the down-regulation of TRPM2 function in cancer cells and that increased expression of wild type TRPM2 in melanoma cells lead to increased susceptibility to cell death. Finally, expression analysis in other cancer types indicated that TRPM2 silencing in cancer might have an even wider role than anticipated, reinforcing the relevance of TRPM2-AS and TRPM2-TE transcripts and the shared CpG island as diagnostic, prognostic and therapeutic tools in cancer and in other diseases in which cell death by oxidative stress plays a relevant role such as neurodegenerative diseases, diabetes and stroke.
In the present invention, the authors describe the computational identification of a melanoma-enriched antisense transcript, TRPM2-AS (accession number GenBank EU362988), mapped within the locus of TRPM2, an ion channel capable of mediating susceptibility to cell death. Analysis of the TRPM2-AS genomic region indicated its presence in the same region of another tumor-enriched TRPM2 transcript, TRPM2-TE (accession number GenBank EU362987), located across a CpG island shared with TRPM2-AS. Quantitative PCR experiments confirmed that TRPM2-AS and TRPM2-TE transcripts were upregulated in melanoma, and their activation was consistent with the methylation status of the shared CpG island. Functional knock-out of TRPM2-AS and TRPM2-TE, as well as over-expression of wild-type TRPM2, increased melanoma susceptibility to apoptosis and necrosis. Finally, further expression analysis in other cancer types indicated that TRPM2-AS and TRPM2-TE over-expression might have an even wider role than anticipated, reinforcing the relevance of the authors' computational approach to identifying new diagnostic/prognostic tools and therapeutic targets.
It is therefore an object of the present invention a nucleic acid molecule being able to modulate the expression of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene and to modulate apoptosis.
In one aspect of the invention the nucleic acid molecule is an antisense in respect to the TRPM2 gene. Preferably it has substantially the sequence of SEQ ID No. 1.
In another aspect the nucleic acid molecule is sense in respect to the TRPM2 gene. Preferably it has substantially the sequence of SEQ ID No. 2.
It is within the scope of the invention an expression vector comprising the nucleic acid molecule as above described.
It is within the scope of the invention a host cell transformed with the expression vector as above described.
It is within the scope of the invention a non human transgenic animal bearing the nucleic acid molecule as above described.
It is within the scope of the invention the nucleic acid molecule as above described for medical use, preferably for use as anti-neurodegenerative disease, more preferably if neurodegenerative disease is selected from the group of Alzheimer's or Parkinson's disease.
It is within the scope of the invention the nucleic acid molecule as above described for use as anti-apoptotic agent in ischemic cells. Preferably ischemic cells are selected from the group of: neurons, cardiomyocites, kidney cells, lung cells, pancreas beta-cells.
It is within the scope of the invention the nucleic acid molecule as above described for use as CpG island methylation agent. Preferably the nucleic acid molecule belongs to the group of methylated oligonucleotides MO1, MO2 and MO3, for use as de-novo methylation agents of the CpG island located between TRPM2-AS and TRPM2-TE transcripts.
It is within the scope of the invention the nucleic acid molecule as above described for the diagnosis and prognosis of cancer.
The nucleic acid molecule, or the vector or of the host cell are advantageously used to modulate the expression of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene in vivo or in vitro.
It is within the scope of the invention a molecule able to down regulate the nucleic acid molecule as above described, for use as a pro-apoptotic and/or pro-necrosis therapy agent in cancer cells.
It is within the scope of the invention a method to down-regulate TRPM2-TE and/or TRPM2-AS transcripts in melanoma cells characterized by methylating the CpG island located between TRPM2-AS and TRPM2-TE transcripts.
It is within the scope of the invention a method to induce apoptosis in melanoma cells characterized by down regulating TRPM2-TE and/or TRPM2-AS transcripts in said melanoma cells.
The invention will be now described by non limiting examples referring to the following figures:
) and closed circles (), indicate methylation in 0-29%, 30-79%, 80-100% of cases respectively. The ‘AS fold’ and ‘TE fold’ columns represent the fold induction of TRPM2-AS and TRPM2-TE transcripts from
Computational identification of antisense transcripts in melanoma. The AntiHunter software [9,10], capable of performing genome wide searches for antisense transcripts, was used for the identification of antisense ESTs from melanoma. Antisense ESTs identified by the program were visualized in their genomic context using the UCSC genome browser (http://genome.ucsc.edu/), in order to prioritize for further analysis those showing expression enriched in cancer tissues.
The melanoma cell lines Mel Ho, Mel Juso, Mel Ju, Mel Im and HMB2 were described previously [36], whereas the cell lines DettMel, GR4, MaL, MR255, MR299, MR304 and MSR3 were from Hospital San Raffaele melanoma patient's repository. The cell lines Mel Ho and Mel Juso were derived from primary cutaneous melanomas; Mel Im, Mel Ju, HMB2, DettMel, GR4, MaL, MR255, MR299, MR304 and MSR3 were derived from metastases of malignant melanomas. Cells and normal human epidermal melanocytes (NHEM) were cultured as previously described [37].
Tissue samples from primary human melanoma and melanoma metastasis obtained from patients undergoing surgical treatment were immediately snap frozen and stored at −80° C. Informed consent was obtained from all patients and investigations were according to institutional guidelines and to the Declaration of Helsinki principles.
Confluent cells (1-5×106) were trypsinised, pelleted and RNA extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. On column DNase digestion was performed with 80 μl of DNaseI (Invitrogen) for 40 minutes at room temperature. The eluted RNAs were quantified with a Nanodrop while their integrity was controlled on a 1% agarose gel. Subsequently cDNAs were generated by a reverse-transcriptase reaction performed using the ThermoScript RT-PCR System (Invitrogen). Briefly, 1 μg of total cellular RNA, 1 μl of dNTPs (10 mmol/L) and 1 μl of dN6-primer (50 ng/μl), were mixed and incubated for 5 minutes at 65° C. Then, 1 μl of Thermoscript reverse transcriptase, 4 μl of 5× first strand buffer, 1 μl of 0.1 mol/L DTT, 1 μl of RNaseOUT (40 U/μl), and sterile RNase-free water, were added to a 20 μl total reaction volume. In order to estimate the amount of genomic DNA contamination in our preparations, each RNA was used to produce a parallel RT-minus reaction where the RT-enzyme was substituted with water. Reactions were incubated for 10 minutes at 25° C., and the RNAs were then transcribed for 1 hour at 55° C. Subsequently, reverse transcriptase was inactivated at 85° C. for 5 minutes and RNA was degraded by digestion with 1 μl of RNase H (2 U/μl) at 37° C. for 20 minutes. cDNAs were controlled by PCR amplification of Beta-actin.
5′- and 3′-Rapid Amplification of cDNA Ends (RACE).
Human XG Malignant Melanoma (A375) marathon-ready cDNA (Clontech, Mountain View, Calif.) was used as template for PCR according to the manufacturer's instructions. Primer sequences are available on request. The largest-size PCR products were gel purified by the Illustra™ DNA and gel band purification kit (GE Healthcare, Buckinghamshire, UK) and cloned into a pGEM®-T Easy Vector System (Promega). Individual clones were sequenced using the DYEnamic ET Dye Terminator kit (Amersham Biosciences).
Aliquots of 3×106 cells were lysed in 200 μl RIPA-buffer (Roche) and incubated for 15 min at 4° C. Insoluble fragments were removed by centrifugation at 13,000 rpm for 10 minutes and the supernatant lysate was stored at −20° C. For western blotting, 40 μg protein lysates were loaded and separated on 12.75% SDS-PAGE gels and subsequently blotted onto a PVDF membrane (Biorad, Hercules, Calif., USA). After blocking for 1 hour with 3% BSA/TBST (0.05% Tween) the membrane was incubated for 16 hours at 4° C. with the primary antibodies [polyclonal anti-TRPM2 antibody generated by Biogenes (Berlin, Germany), 1:2,000] and anti-beta-actin (Sigma, Deisenhofen, Germany, 1:2,500)). The peptide sequence recognized by the TRPM2 antibody reads as follows: MEVYKGYMDDPRNT (SEQ ID No. 24). Subsequently, the membrane was washed three times in TBST, incubated for 1 hour with alkaline phosphate-coupled secondary antibody (Chemicon; 1:2,000) and then washed again. Finally, immunoreactions were visualized by NBT/BCIP (Sigma) staining
A panel of Mel Im cell clones with reduced TRPM2-TE expression were established by stable transfection with an antisense expression plasmid (base −312 to +18 relative to the ATG codon cloned in antisense orientation into pCMX-PL1). Mel Im cell clones with induced TRPM2-FL expression were generated by stable transfection with a sense expression plasmid containing the coding sequence of TRPM2-FL from the translation start to the stop codon. Plasmids were cotransfected with pcDNA3 (Invitrogen), containing the selectable marker for neomycin resistance. Controls received pcDNA3 alone. Transfections were performed using Lipofectamin plus (Invitrogen) according to the manufacturer's instructions. One day after transfection, cells were placed in selection medium containing 50 μg/ml G418 (Sigma). After 25 days of selection, individual G418-resistant colonies were subcloned. TRPM2-FL expression levels of these clones were checked in western blot and quantitative real-time PCR. Cell proliferation was determined using the XTT assay (Roche, Mannheim, Germany). Apoptotic cells were detected by staining with AnnexinV-FITS and propidium iodide using the AnnexinV Kit (Caltag Laboratories/Invitrogen, Karlsruhe, Germany) according to the manufacturer's manual, stained cells were measured by flow cytometry with the BD FACS Calibur System (BD Biosciences, San Jose, Calif., USA). Data were analyzed and histograms generated using the Cellquest™ software (BD Biosciences). Measurement of caspase activity was another assay used to detect rates of apoptosis. Therefore cells were stained with CaspACE™ FITC-VAD-FMK In Situ Marker (Promega, USA). 5 μM of CaspACE™ FITC-VAD-FMK In Situ Marker were added to the cell medium and cells were incubated for 20 minutes protected from light. After incubation, cells were washed with PBS twice and cell pellets were resuspended in 400 μl PBS before analyzing by flow cytometry. To enhance apoptosis, 2×104 cells were seeded into each well of a six-well plate and stimulated with 2 mM H2O2 for 30 minutes before staining. To analyze the influence of the TRPM2-TE iso form on apoptosis, the TRPM2-FL expressing cell clones were transiently transfected with an expression plasmid for TRPM2-TE. For transient transfections, 2×104 cells were seeded into each well of a six-well plate and transfected with 0.5 μg TRPM2-TE plasmid DNA using the lipofectamine plus method (Invitrogen) according to the manufacturer instructions. 24 h after transfection, cells were stimulated with 2 mM H2O2 for 30 minutes and caspase activity was measured as described above. All experiments were performed at least three times.
Genomic DNA was isolated and purified with TRIzol reagent (Invitrogen) and quantified with a Nanodrop. Average 100 ng of each DNA sample were subjected to bisulphite modification with the MethylEasy Kit (Human Genetic Signatures) allowing the sulphonation reaction to proceed for no less than 8 hours at 55° C. Following conversion, 3 μl of the modified DNAs were PCR amplified with the primers CpG-8U 5′-TTGATTTGATTTGGTTTTTGGA-3′ (SEQ ID No. 3) and CpG-8L 5′-CAAAACAAAAACTTCCTCTATA-3′ (SEQ ID No. 4) (1 ng/μl each) in the presence of buffer 10× (Qiagen), dNTPs (2 mmol/L), 5× Solution Q (Qiagen), Taq DNA Polymerase (3U), with the reaction conditions being: 1 minute of pre-incubation at 95° C., then amplification reactions were performed by 35 repetitive cycles of denaturing for 1 minute at 95° C., annealing for 2 minutes at 55° C. and extension for 2 minutes at 72° C. A semi-nested PCR reaction was performed in same conditions except for the forward primer changed to CpG-1U 5′-TGTAGATTGTGTTTGAATTTGGTTA-3′ (SEQ ID No. 5), using 4 μl of the previous 472 bp per product as template. The resulting 370 bp product was then gel-purified, cloned and sequenced using standard procedures.
Beta-actin (Exons 5-6) was amplified from cDNAs using the specific primers: LC+hBactin-U 5′-TCCTCCTGGAGAAGAGCTA-3′ (SEQ ID No. 6) and LC+hBactin-L 5′-GGATGCCACAGGACTCCAT-3′ (SEQ ID No. 7) coupled to human Universal Probe Library (UPL) probe number 11 (Roche) resulting in a 124 bp fragment. TRPM2-FL (Exons 20-21) was amplified by RT-PCR from cDNAs using the specific primers: RCH_LONG_TRPM2_U 5′-ACCTCCTCATCGCCATGTT-3′ (SEQ ID No. 8) and RCH_LONG_TRPM2_L 5′-CTTCCAAATCTGGTCCGTGT-3′ (SEQ ID No. 9) coupled to human UPL probe number 11, resulting in a 68 bp fragment. TRPM2-AS (Exons 1-2) was amplified by RT-PCR from cDNAs using the specific primers: RCH-asmel-1F 5′-CCAGGAACCAGAACCAAACT-3′ (SEQ ID No. 10) and RCH-asmel-1R 5′-TGTCCGTCTGCTGAGACATC-3′ (SEQ ID No. 11) coupled to human UPL probe number 57, resulting in a 61 bp fragment. TRPM2-TE was amplified from cDNAs using the primers: UPL-TSS-U 5′-GATGTTTTGGCGGAAGGAC-3′ (SEQ ID No. 12) and UPL-TSS-Rev 5′-CAGGAAGACGTGACGCAAG-3′ (SEQ ID No. 13) coupled to human UPL probe number 6, resulting in a 88 bp intronless fragment. The contribution of genomic DNA contamination to the detected signal was subtracted by running a parallel real-time quantification from a RT-minus reaction, where the RT-enzyme was substituted with water. When RNA was not available to perform the RT-minus control, the contribution of DNA contamination to the detected signal was subtracted by performing a real-time quantification on the same samples, using a couple of primers specific for the Beta-actin promoter: LC-ProBACT-U1 5′-TCTGCAGGAGCGTACAGAAC-3′ (SEQ ID No. 14) and LC-ProBACT-L1 5′-ACATCTCTTGGGCACTGAGC-3′ (SEQ ID No. 15) coupled to human UPL probe number 81 (Roche) resulting in a 85 bp fragment. Real time PCR reactions were performed on a LightCycler 480 instrument (Roche), on 96 multiwell plates (Roche) using LightCycler 480 ProbeMaster Mix (Roche) following the manufacturer instructions. Briefly, all reactions were performed in a final 30 μl volume containing 15 μl of 2× MasterMix, 0.6 μl of each primer (20 μmol/L), 0.3 μl of the corresponding UPL probe (10 μmol/L), and 10 μl of template cDNA. Only for TRPM2-AS the reaction was supplemented with 6 μl of 5× Solution-Q (Qiagen) and the cDNA template lowered to 7 μl. The resulting mix was pre-incubated for 10 minutes at 95° C., then amplification reactions were performed by 45 repetitive cycles of denaturing for 10 seconds at 95° C., annealing for 15 seconds at 58° C. and a final extension plus single acquisition step at 72° C. for 1 second. Levels of target gene transcripts were normalized to transcript levels of a reference gene (Beta-actin) and calculated using a relative quantification model with efficiency correction. Amplification efficiency of primer pairs was calculated from serial dilutions of a representative cDNA template over a concentration range of 3 log orders.
siRNA Experiments.
Two siRNAs designed to target the TRPM2-AS transcript (TRPM2-AS—657 and TRPM2-AS—734), one siRNA designed to target the TRPM2-TE transcript (TRPM2-TE—55) and a control (non-specific) siRNA were synthesized by MWG biotech (Germany) as follows:
1) TRPM2-AS—657, 5′-CCAGUAACUCCGCCCAAAU(dTdT)-3′, (SEQ ID No. 16) target sequence starting at nt 657 of TRPM2-AS.
2) TRPM2-AS—734, 5′-CCACUUACUCAUCCAAGAA(dTdT)-3′, (SEQ ID No. 17) target sequence starting at nt 734 of TRPM2-AS.
3) TRPM2-TE—55, 5′-GAAGGACCACAGAGGAAGU(dTdT)-3′, (SEQ ID No. 18) target sequence starting at nt 55 of TRPM2-TE.
4) Non-specific, 5′-AGGUAGUGUAAUCGCCUUG(dTdT)-3′, (SEQ ID No. 19) no known target in the human transcriptome.
All siRNA sequences were subjected to BLAST search to confirm the absence of similarity to any known additional transcript in the human genome. siRNAs were transfected in the human melanoma cell line Mel Im using the transfection reagent Lipofectamine RNAiMax from Invitrogen. Final siRNA concentration in the medium was 10 nM. Cell were harvested for RNA extraction at 24 hours. qPCR were performed to measure the relative amount of TRPM2-AS, TRPM2-TE and TRPM2-FL transcripts.
Apoptosis was measured after a 72 h transfection with any of the following siRNA: TRPM2-AS—657, TRPM2-AS—734, TRPM2-TE—55 or the non-specific siRNA. Early and late apoptotic cells were assessed by flow cytometry using the Annexin V-Fitc Apoptosis Detection kit (Immunostep, Salamanca, Spain), following manufacturer's instructions. Data acquisition and analysis were done in a FACSort Cytometer (Becton Dickinson, Franklin Lakes, N.J.) using CellQuest and FCS Express software.
Three phosphorothioate oligonucleotides were designed in which the cytosines in CpG dinucleotides were replaced by m5 CpG. These oligonucleotides are all from the CpG island within the TRPM2 gene (genomic coordinates: chromosome 21:44669421-44670121, NCBI build 35) and were meant to induce methylation within this targeted region using the metod developed by Yao et al. [34].
1) Methylated oligonucleotide 1 (MO1): mCGGmCGGGGAmCGCTGCCTGAGCTCCmCG (SEQ ID No. 20) at genomic coordinates chr 21: 44669769-44669794, NCBI build 35).
2) Methylated oligonucleotide 2 (MO2): CCCTmCGTAACmCGCACTGmCGAGTTC (SEQ ID No. 21) at genomic coordinates chr21: 44669697-44669720, NCBI build 35).
3) Methylated oligonucleotide 3 (M03): TGCmCGGGCTGCTGAGTTTmCGCmCGGC (SEQ ID No. 22) at genomic coordinates chr21: 44669657-44669678, NCBI build 35).
Two control oligonucleotides were designed as well:
4) S01 is identical at MO1 except that it doesn't contain m5C residues as MO1.
5) MOS is random, i.e., it is not significantly related to the any sequence in the human genome, but contains 3 m5 CpG residues and has a base composition similar to MO1, MO2 and MO3: TCCTmCGGGCTGCTGAGTTTmCGCmCGGCCC (SEQ ID No. 23).
The oligonucleotides were purchased from MWG (Germany). The oligonucleotides were purified by high-performance liquid chromatography yielding a purity of more than 95% of full-length oligonucleotides.
Tumor cells were seeded in six-well plates at a density of 2×105 cells/ml and were treated with the following liposome-encapsulated oligonucleotide mixes at a final concentration of 3 μM:
Cells were harvested after 24 hours of transfection. DNA was extracted and the methylation status of the CpG island was checked after 24 hours as previously described.
Results are expressed as mean±SD (range) or percent. Comparison between groups was made using the Student's unpaired t-test. A p value <0.05 was considered statistically significant. All calculations were performed using the GraphPad Prism software (GraphPad software Inc, San Diego, USA). For real time PCR, data analysis was performed using the relative expression software tool (REST 2005 BETA V1.9.12) [38]. *, p<0.05; **, p<0.01.
Using a newly developed version of our AntiHunter software, the authors performed a genome-wide search for antisense transcripts expressed in human ESTs (Expressed Sequence Tags) from melanoma. Several antisense transcripts scattered throughout the genome were identified (data not shown) and here the authors describe one of them, named TRPM2-AS, mapped on chromosome 21. TRPM2-AS is antisense with respect to TRPM2, an ion channel capable of conferring cell-death upon oxidative stress. AntiHunter identified two melanoma antisense ESTs, BF689755 and BF690298, located in the body of the TRPM2 gene and both corresponding to TRPM2-AS. The TRPM2 locus and the relevant molecules herein described are schematically represented in
A 701 bp CpG island, partially superimposed to the TRPM2-AS transcript, was mapped at coordinates 44,669,421-44,670,121 by the “CpGplot” program from the EMBOSS package (www.emboss.org). A prediction for a 570 bp DNA segment containing a PolII promoter was made within the CpG island (coordinates 44,669,484-44,670,053) by “First-EF”, a first-exon and promoter prediction program for human DNA [22], suggesting the CpG island could be involved in the transcriptional regulation of TRPM2-AS (
The transcript spans a genomic region of 17,553 nucleotides from position 44,669,840 to position 44,687,392. Sequencing of the TRPM2-TE transcript revealed other interesting features. It lacked part of exon 26 and the entire exon 27. Removal of part of exon 26 does not affect the putative protein product, as the first methionine is positioned downstream (see below), whereas the ablation of exon 27 corresponds to the already described TRPM2-AC splice variant [23]. The resulting deletion of 34 amino acids removes part of the Nudix domain and it has been reported to impair the capability of TRPM2-FL to be activated by ADP-ribose (ADPR, [23]). However, it leaves intact the segment showing similarity to the CORE region of the NUDT9 domain, containing all the structure required for ADPRase activity [24]. The predicted TRPM2-TE-AC open reading frame ranged from nucleotide 320 to nucleotide 874, encoding for a 184 amino acid protein, whose predicted molecular size is 21,083 Da. The full length TRPM2-TE isoform without the ΔC deletion encoded for a protein of 218 amino acids, with a predicted molecular mass of 25,012 Da. A schematic representation of TRPM2-TE protein products, compared to TRPM2-FL, is shown in
The authors analyzed TRPM2-AS and TRPM2-TE expression levels in 12 melanoma cell lines and five tumor tissues, with the cell line MaL being the immortalized counterpart of the fresh tumor #3 (FT3). As a control, the authors used normal melanocytes from two independent healthy donors, NHEM1 and NHEM2. The expression level of these two transcripts was also measured in eight normal tissues: heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Results are shown in
Methylation Status of the Shared CpG Island Correlates with the Expression Levels of TRPM2-AS and TRPM2-TE Transcripts.
The authors tried to ascertain if the degree of methylation of the CpG island shared by TRPM2-AS and TRPM2-TE was correlated with the transcriptional activation of these two molecules in melanoma. For this purpose, seven DNAs from the samples analyzed in
To analyze the functional role of TRPM2-TE in melanoma cells, the authors down-regulated its expression in melanoma cell line Mel Im by stable transfection with a TRPM2-TE antisense expression construct. Successful down-regulation of TRPM2-TE in cell clones 1 and 2 (TRPM2-TE-AS 1, TRPM2-TE-AS 2) was verified by quantitative RT-PCR (
The authors generated melanoma cell clones which expressed higher TRPM2-FL levels than parental cells by stable transfection of Mel Im cells with an expression construct for TRPM2-FL. Over-expression was tested by quantitative RT-PCR revealing that the cell clones 3 and 7 had a strongly increased expression of TRPM2-FL when compared to mock-transfected cell clones mock 1 and mock 2 (
In order to investigate the possibility that overexpression of TRPM2-AS and TRPM2-TE transcripts is a wider phenomenon in cancer, quantitative RT-PCR experiments were performed on other tumor types. In
Expression analysis of TRPM2-AS, TRPM2-TE and TRPM2-FL transcripts in resected non-small cell lung cancer patients revealed that TRPM2-AS was over-expressed in 17 samples (63%), under-expressed in 9 samples (33%) and equally expressed in one sample (4%). TRPM2-TE was over-expressed in 24 samples (89%), under-expressed in 2 samples (7%) and equally expressed in one sample (4%). TRPM2-FL was over-expressed in 9 samples (33%), under-expressed in 13 (48%) samples and equally expressed in 5 samples (18%) (
It is conceivable that TRPM2-TE and TRPM2-AS exert their function by counteracting the action of TRPM2-FL within the cell. Therefore, since the overall capability of TRPM2-FL to induce cell death could be affected by the amounts of these two molecules, the authors calculated the following value for each sample from the fold induction data: TRPM2-FL-TRPM2-AS-TRPM2-TE. This means that this new value, named TRPM2-FL Relative Fold Induction (TRFI), is calculated by subtracting the figure of TRPM2-AS and TRPM2-TE fold induction from the TRPM2-FL one. In order to keep in account saturating concentration of these molecules in the cell, the fold induction values used for calculation were kept within the −/+10-fold range. For example, from
The average TRFI of short term survival patients (0-30 months, dark grey boxes) differs from the one of long term survival patients (31-88 months, light grey boxes): −17.41 vs. −8.77. A t-test applied to the TRFI values of both distributions determined that this difference was significant (p-value: 6.57E-08). No significant difference was found in stage 3 samples (data not shown). Tumor stage (TS), overall surviving (OS) after surgery and whether patient was still living after the end of the clinical follow-up is also shown in the table. This data demonstrate that the TRFI value is a significant prognostic marker in lung cancer. The month survival of patients from stage I and stage II having a TRFI<−10 (dotted line; n=11; average survival: 29 months) and a TRFI>−10 (solid line; n=7; average survival: 67 months) was plotted in a Kaplan-Meyer survival plot in
siRNAs Targeting TRPM2-AS and TRPM2-TE Transcripts Down-Regulate their Expression in Melanoma Cells
siRNAs experiments, shown in
Following the protocol of Yao et al [34], the authors designed three methylated oligonucleotides aimed at inducing new methylation in melanoma cells of the CpG island located in between TRPM2-AS and TRPM2-TE transcripts. Three methylated oligonucleotides, MON1, MON2 and MON3, targeting the CpG island were designed. Two negative control oligonucleotides, SO1 and MOS, were designed as well. The first of these two control oligonucleotides, SO1, had the same sequence of MO1, but it was completely not-methylated. The second control oligo, MOS, was not significantly related to any sequence in the human genome. Oligonucleotides were transfected in melanoma Mel Im cells. After 24 hours, DNA was extracted and treated with sodium bisulphite to convert cytosine residues to uracil in single-stranded DNA, under conditions whereby methylated cytosines remain essentially non-reactive. As previously described, primers were used to PCR-amplify the region at coordinates 74-369 of the predicted CpG island, containing 19 CpG dinucleotides, whose methylation status was determined after cloning and sequencing of 5 clones. The results are shown in
In the study presented here, the authors mined the EST melanoma database with their recently developed software AntiHunter and identified a new antisense transcript, TRPM2-AS. Natural Antisense Transcripts (NATs) are supposed to negatively regulate the conjugated sense transcript by means of a variety of regulatory mechanisms [9]. The sense partner of TRPM2-AS is TRPM2, a gene whose protein product encodes for an ion channel capable of conferring cell-death upon oxidative stress. Although the direct involvement of TRPM2 in tumor cell death has been shown only very recently [21], the authors contemplated that activation of TRPM2-AS could interfere with the proapoptotic role of TRPM2 during the process of cancerogenesis.
Quantitative RT-PCR experiments revealed transcriptional activation of TRPM2-AS in about 80% of melanoma cell lines and tumor tissues vs. controls. Visualization of the AntiHunter output, using a genome browser, allowed the authors to hint at other interesting features of the investigated region. First, a CpG island/Pol II promoter, mapped within intron 24 of TRPM2, appeared to be closely positioned to the TRPM2-AS transcription start site. Second, five ESTs from prostate carcinoma appeared to have a transcription start site located within the same intron 24, suggesting the CpG island could serve also as a promoter for a new Tumor Enriched TRPM2 transcript (TRPM2-TE). Quantitative RT-PCR experiments showed that TRPM2-TE expression was activated in melanoma and coupled to the expression of TRPM2-AS suggesting a bidirectional role for the shared promoter region. Sequencing of bisulphite treated DNAs of several melanoma samples indicated the methylation status of the CpG island was at least partially correlated with the expression levels of TRPM2-AS and TRPM2-TE transcripts. Since this CpG island appeared to be mostly hypermethylated in normal melanocytes, it is conceivable that its demethylation could activate TRPM2-AS and TRPM2-TE expression in melanoma, contributing to the functional downregulation of TRPM2-FL.
No obvious indication on the regulatory role of TRPM2-AS over FL-TRPM2 could be determined by the qPCR data shown in
Experimental evidence for the dominant-negative role of TRPM2-TE was determined by the present invention experiments. Knock-down of this transcript significantly increased the susceptibility of melanoma cells to apoptosis and necrosis. Since the present experimental approach to TRPM2-TE depletion also removed a significant fraction of TRPM2-FL from the cells (see
Quantitative RT-PCR experiments performed in lung and breast cancer (
A large sequencing effort directed toward human colorectal and breast cancer DNA gene coding regions and splicing consensi failed to find any mutation within TRPM2-FL [2]. Therefore, it is conceivable that TRPM2-FL inactivation in cancer is mainly obtained by epigenetic means, such as those described in the present invention. A possible explanation is that TRPM2-FL function could be needed in the initial phases of tumor development, while becoming harmful at later stages. Accordingly, since metastasis is a cellular heterogeneous process, it is unlikely to be mediated by permanent genetic mutations. However, epigenetically mediated gene silencing is an excellent candidate for supporting such cellular dynamics [33]. The kind of epigenetic regulation is shown here to allow the fine tuning of the TRPM2-FL requirements of the tumor by acting on the methylation status of the inner CpG island. Another important player in this scenario should be the promoter of the TRPM2-FL gene, whose activation strength should also contribute to the functional activity of TRPM2-FL.
From the data reported in the present invention, restoration of TRPM2-FL activity in cancer cells is an attractive therapeutic opportunity. Since inactivation of TRPM2-FL is likely to correlate with the hypomethylation of the CpG island shared by TRPM2-AS and TRPM2-TE transcripts, a simple way to restore its activity is to artificially methylate it de novo. Experimental evidence indicates the feasibility of this approach, obtained by delivering a methylated oligonucleotide complementary to the targeted CpG island to the nucleus [34]. The authors delivered a methylated oligonucleotide to the shared CpG island with positive results, as shown in
The systematic identification of antisense transcripts in the EST database holds great promise for the identification of new and interesting biological and pathological phenomena. With the advent of new sequencing technologies that generate order-of-magnitudes larger sequencing data at fractional costs [35], it is conceivable that the identification of regulatory antisense transcripts obtained by mining the EST database will gain even more ground in the future.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2009/054173 | 9/23/2009 | WO | 00 | 4/21/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61101123 | Sep 2008 | US |
