Patent Application
20110229878
This disclosure relates to a consensus nucleotide sequence found within two kilobases of the 5′ end of fifty-six different genes in the human genome and use of the consensus sequence to screen for compounds and other molecules that inhibit transcription or that inhibit or interfere with transcription repressors or repressor complexes.
Human sodium-iodine symporter (hNIS) is a trans-membrane protein enabling thyrocytes, both benign and malignant, to concentrate iodine; permitting radioiodine to be a unique systemic cytotoxic therapy for metastatic tumors. Unfortunately, when hNIS expression is lost in dedifferentiated thyroid carcinomas, there are no effective systemic cytotoxic agents (Ain 2000).
Previous investigations revealed evidence for an alternative mechanism for loss of hNIS transcription, suggesting presence of a trans-acting repressor of hNIS transcription, termed NIS-repressor (Li, et al. 2007).
Multiple cellular and nuclear factors are reported to be important for hNIS transcription, including: TSH (thyrotropin)/receptor (TSHr) (Riedel, et al. 2001), TTF-1 (Schmitt, et al. 2001), and Pax-8 (Pasca di Magliano, et al. 2000), but there are no clear examples of repressing transcription factors in thyroid cells or thyroid carcinomas. In U.S. application 60/907,881, we showed NIS-repressor as a trans-acting protein binding to a specific region of the proximal hNIS promoter, NIS-repressor binding site (NRBS-P); however its composition was not yet known. We also characterized NIS-repressor and investigated the identities of its components and mechanisms of its activity. This involved defining NRBS-P to a narrower region of hNIS promoter and utilizing it to probe nuclear extract, analyzing the probe-bound proteins with liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), to characterize NIS-repressor components. The mass spectrometry analysis data demonstrated human PARP-1 (poly(ADP-ribose)polymerase-I) to be a likely component of the NIS-repressor protein complex. Pharmacological inhibition of PARP-1 activity with PJ34, a PARP-1 inhibitor, stimulated endogenous hNIS mRNA levels, providing evidence that PARP-1 acts as a negative regulatory factor for hNIS transcription and is a likely component of the NIS-repressor complex.
Because of its role in inhibiting the transport of iodine into cells, and in particular, into thyroid cancer cells, there is a need to determine the hNIS repressor binding sites, structure and activities so that anti-thyroid cancer therapies can be maximized. Further, there is a need to determine the general applicability of PARP-1 inhibition to alter transcription regulation and the role of the hNIS repressor binding site in transcription regulation in general.
One aspect of the invention relates to a sodium iodine symporter (NIS)-repressor binding site (NRBS) consisting of a DNA molecule spanning from −1067 to −868 (SEQ ID NO.: 2). Another aspect of the invention relates to a transcription-repressor binding site consisting of a DNA molecule having the sequence 5′-TG(G/A)GCCT(T/C)A(G/A)TTTCCCCA(T/C)CTGT-3′ (SEQ ID NO.: 1) (NRBS consensus sequence) or a nucleotide sequence that hybridizes to the full length of the complement thereof under high stringency conditions. In certain embodiments of this aspect of the invention, there is provided a vector or expression cassette comprising the consensus sequence operably linked to a promoter sequence which is operably linked to a reporter gene, such as a gene encoding a detectable marker, e.g., a luciferase gene. In certain embodiments, the vector is an adenovirus vector.
Yet another aspect of the invention relates to a method of treating thyroid cancer comprising administering to a patient in need thereof a therapeutically effective amount of a PARP-1 inhibitor and a therapeutically effective amount of radiolabeled iodine. In an other aspect of the invention there is provided a method of treating thyroid cancer in a patient comprising contacting thyroid cancer cells in the patient that express and form a NIS repressor protein complex capable of binding to SEQ ID NO.: 1 or SEQ ID NO.: 2 with a PARP-1 inhibitor, and administering to the cells radiolabeled iodine.
Another aspect of the invention relates to a method of screening molecules or compounds that bind to SEQ ID NO. 1 and inhibit or interfere with transcription, said method comprising (1) contacting the test molecule or compound with a nucleotide sequence comprising SEQ ID NO. 1 and (2) determining whether the test molecule or compound binds to SEQ ID NO. 1. Those test compounds or molecules that bind to SEQ ID NO. 1 may be selected for further testing to determine if they modulate target gene expression.
Another aspect of the invention relates to a method for screening molecules or compounds that interfere with NIS repressor binding to the SEQ ID NO. 1, said method comprising (1) contacting the test molecule or compound in the presence of human NIS repressor with a nucleotide sequence comprising SEQ ID NO. 1 and (2) detecting an alteration in binding of the NIS repressor to SEQ ID NO. 1. Those test compounds or molecules that alter NIS repressor binding to that bind to SEQ ID NO. 1 may be selected for further testing to determine if they modulate target gene expression.
Radioiodine therapy remains the only known effective systemic tumoricidal treatment for thyroid carcinoma. Unfortunately, around 10% of such cancers and most dedifferentiated thyroid cancers fail to concentrate radioiodine consequent to loss of sodium-iodine symporter gene (NIS) expression (Ain 2000; Robbins, et al. 1991). For that reason, efforts to understand the mechanisms of this loss may lead to new treatments to restore NIS expression, permitting effective therapy with radioiodine. Our previous study provided evidence of a trans-active protein factor (complex) suppressing NIS transcription under basal conditions, possibly accounting for loss of human NIS expression in some thyroid cancers. This suggested a new target, which we named NIS-repressor, for designing therapies to restore radioiodine uptake in disseminated tumors. We mapped its binding-site in the proximal NIS promoter (NIS-repressor binding site; NRBS-P) (Li et al. 2007). This repressor may function in concert with or independent of epigenetic effects on NIS expression via NIS promoter methylation and histone deacetylation (Venkataraman et al. 1999).
The term “promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, such as a human gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
The term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or repressor is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the nucleotide sequences being linked are typically contiguous. However, some polynucleotide elements may be operably linked, but not directly flanked and may even function in trans from a different allele or chromosome.
The present invention is based, in part, on the identification of a second site in the human sodium-iodine symporter (NIS) promoter region, herein, referred to as NIS-repressor binding site (NRBS-D). We further investigated NIS-repressor by refining NRBS-P, demonstrating sequences at −648 to −620 bp, and an additional NRBS at −987 to −958 bp (NRBS-D; relative to the NIS translation start site) as two core binding sites for NIS-repressor. The homology between NRBS-D and NRBS-P core sequences is 83% in a 23 bp region, with two A/G and two T/C transitions. This constitutes a 23 bp consensus sequence (5′-TG(G/A)GCCT(T/C)A(G/A)TTTCCCCA(T/C)CTGT-3′) (SEQ ID NO. 1) (“consensus NRBS”). NRBS-P and NRBS-D are in opposite orientation in the hNIS promoter and 310 bp apart from each other. A human genome homology search (NCBI/BLAST/blastn suite) shows this consensus sequence to occur (at >90% homology) within two kilobases of the translation start site of 56 different genes, within four kilobases of an additional twenty genes and within seven kilobases of an additional eight genes in the human genome. Among these genes, there are some coding for kinases, receptors, and transporters. A list of genes containing a sequence with >90% homology throughout the entirety of SEQ ID NO. 1 in their promoter regions is shown in Table 1.
EMSA analysis showed proteins in KAK1 nuclear extract that bound to NRBS-P and constitute the NIS-repressor. Electrophoretic analysis of these nuclear extract proteins, UV-crosslinked to the radiolabeled NRBS-P probe, revealed multiple bands, suggesting that NIS-repressor is a protein complex. Several thyroidal transcription factors (Sp1, Ap1, AP2, TTF-1 and Pax8), previously characterized as affecting NIS transcription, were excluded as candidates for NIS-repressor components because double-stranded oligonucleotides containing their respective consensus DNA-binding sites failed to compete against a radiolabeled NRBS-P probe in EMSA analysis.
Unexpectedly, an antibody against human thyroid transcription factor 2 (hTTF-2) (antibody S-18), but not two other anti-TTF-2 antibodies (F-17 or V-20), which recognize different epitopes on TTF-2, altered the migration of the probe-protein complex in supershift assays, demonstrating that human TTF-2 is associated with, or is a part of, the NIS-repressor complex. The three tested antibodies are available from Santa Cruz Biotechnology, Inc. S-18 is an affinity purified goat polyclonal antibody raised against a peptide mapping within an internal region of the human TTF2 polypeptide. The epitope for this antibody is the region from amino acid 100-150 in human TTF2. F-17 is an affinity purified goat polyclonal antibody raised against a peptide mapping within an internal region of human TTF2. The epitope for this antibody is the region from amino acid 140-190 in human TTF2, and S-18 and F-17 do not have competing binding sites. V-20 is an affinity purified goat polyclonal antibody raised against a peptide mapping near the C-terminus of human TTF2.
In one aspect of the invention, an inhibitor of TTF-2 is administered to a patient suffering from thyroid cancer to inhibit the formation of the NIS-repressor complex and/or binding of the NIS repressor to either or both of NRBS-P and NRBS-D and restore iodide uptake in dedifferentiated thyroid carcinoma cells.
Although 5-azacytidine and sodium butyrate have been shown to restore NIS transcription (Venkataraman et al. 1999), these agents did not alter the EMSA pattern using KAK1 nuclear extract, suggesting that NIS-repressor represents a different mechanism of NIS gene regulation. This is consistent with our previous genomic DNase I digestion studies (Li et al. 2007) that failed to demonstrate any effect of these agents on chromatin compaction, suggesting the possibility of non-epigenetic regulatory processes.
The human poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) was identified by proteomic analysis of the nuclear extract from KAK1 cells, as a top candidate for a component of the NIS-repressor complex. PARP-1 was initially known for its role as a DNA-damage sensor, repair and signaling protein. Later studies have shown that PARP-1 also participates in additional critical cellular activities, such as: apoptosis, genetic stability, and gene transcription (Schreiber, et al. 2006). PARP-1 was reported to be able to bind to regulatory sequences by itself (Chiba-Falek, et al. 2005; Zhang, et al. 2002), modify some transcription factors or signal proteins by poly(ADP-ribosyl)ation (Miyamoto, et al. 1999), and influence other protein factors by hetero-complex formation (Simbulan-Rosenthal, et al. 2003). A recent study reveals that PARP-1 has widespread effects upon transcription of diverse genes, either as a positive or negative transcription factor (Krishnakumar, et al. 2008).
ChIP analysis of Cal-62 cells with two commercial anti-PARP-1 antibodies shows that PARP-1 is associated with the NRBS-P region in Cal-62 and KAK1 cells under basal culture conditions without NIS transcription. Furthermore. PJ34, an inhibitor of PARP-1 enzymatic activity (Abdelkarim et al. 2001), effectively stimulated luciferase activity from NIS promoter constructs and also stimulated endogenous hNIS transcription in both KAK1 and Cal-62 cells, confirming that PARP-1 is part of a negative regulatory factor for hNIS gene transcription. Despite the ChIP data indicating that PARP-1 was associated with the hNIS promoter region containing NRBS-P, two different commercial anti-hPARP-1 polyclonal antibodies (that had been effective in the ChIP assay) failed to alter the EMSA pattern on supershift analysis. In addition, two commercial preparations of human PARP-1 failed to produce the same EMSA signals as the nuclear extract from KAK1 cells. It is likely that PARP1 does not directly bind to the NRBS sequence; rather, it is associated with other proteins that contain the critical DNA-binding domain. PJ34 inhibition of PARP1 enzymatic activity may compromise the assembly, stability, or activity of the NIS-repressor protein complex.
In summary, a second core sequence in the human sodium-iodine symporter (hNIS) promoter, NRBS-D, which is a binding site for a trans-active transcriptional repressor, NIS-repressor has been defined. Proteomic analysis revealed PARP-1 as an important constituent of the NIS-repressor protein complex. A known inhibitor of PARP-1 enzymatic activity, PJ34, causes increased endogenous transcription of hNIS in genotypically verified thyroid cancer cells.
In one aspect of the invention there is provided a method of screening for therapeutic agents capable of restoring NIS gene expression and radioiodine uptake in thyroid cancer cells. The method comprises the steps of: i) contacting thyroid cancer cells with a pharmacologic antagonist against one or more components of the NIS repressor protein complex capable of binding to SEQ ID NO. 1, ii) detecting NIS expression or radioiodine uptake by the cell; and iii) selecting the pharmacologic antagonist that results in an increase in NIS expression or radioiodine uptake by the thyroid cancer cells. In certain embodiments the pharmacologic antagonist is an inhibitor of PARP-1 or TTF-2, wherein inhibition thereof comprises inhibition of NIS complex binding to SEQ ID NO. 1 or inhibition of NIS complex formation or function.
The 23 base pair NRBS consensus sequence (SEQ ID NO. 1) may have regulatory importance for multiple diverse human genes. In thyroid oncology, NIS-repressor is a useful target in restoring the effectiveness of radioiodine therapy to dedifferentiated thyroid cancers. In other contexts, the NRBS consensus sequence is a useful target for modifying the expression of one or more of the genes in the human genome to which it is operably linked, some of which appear to play a role in cancer.
In a further aspect of the invention the 23 base pair consensus sequence may be used to screen for compounds or molecules that inhibit or compete with NIS-repressor binding to the consensus sequence (antagonists and agonists of NIS-repressor). In certain embodiments, the consensus sequence is operably linked to a promoter and target gene, e.g., such as a vector which includes the linked elements, and is contacted with a test molecule or compound (or multiple test compounds and/or molecules) under conditions suitable for expression of the target gene. Such assay may also be carried out in the presence of NIS repressor in order to determine those compounds or molecules that may interfere with the activity of the NIS repressor. The effects of such contact on transcription of the target gene are detected via any convenient method, e.g., measurement of a detectable marker encoded by the target gene. The expression of the target gene may be compared to expression of the target gene in the presence of varying amounts of the test compound and/or molecules and/or in the absence of the test compound and/or molecule. Any suitable target gene may be used, such as for example the luciferase gene. The vector may be any suitable plasmid or viral vector, such as an adenovirus vector. One of ordinary skill in the art can construct suitable vectors for use in the present invention.
Alternatively, an electrophoretic mobility shift assay (EMSA) may be used to detect SEQ ID NO. 1-specific binding proteins and/or molecules that bind to SEQ ID NO. 1. Such SEQ ID NO. 1-specific binding proteins may be used to modulate the expression of genes operably linked to SEQ ID NO. 1, such as any of the genes listed in Table 1.
In certain embodiments of the invention, the consensus sequence my be used to select for a “better” NIS-repressor (NIS-repressor agonist) using, for example, the methodology disclosed by Urnov F D, Rebar E J, Biochem Pharmacol. 2002 64(5-6):919-23, which is incorporated herein by reference thereto.
Additional objects and advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure.
The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and H (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.).
As used herein “stringent hybridization conditions” are generally selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. High stringency conditions are selected to be equal to the Tm point for a particular probe. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology, incorporated herein in its entirety.
The following describes materials and methods used in the procedures described in the subsequent Examples.
Five EMSA probes, SHIFT-1, -2, -3, -4, and -5, were prepared with PCR, radiolabeled, and used to probe KAK1 nuclear extract in EMSA. The EMSA results shown in
Seven PCR fragments and three annealed double-strand oligonucleotides were used as unlabeled competitors against the radiolabeled SHIFT-4 probe in EMSA to determine the core sequence for NRBS-D. The seven PCR fragments are: SHIFT-4.1 (150 bp; −1017 to −868), SHIFT-4.2 (100 bp; −967 to −868), SHIFT-4.3 (150 bp; −1067 to −918), SHIFT-4.4 (100 bp; −1017 to −918), SHIFT-4.5 (150 bp; −1017 to −868), SHIFT-4.6 (140 bp; −1007 to −868), and SHIFT-4.7 (130 bp; −997 to −868). The three annealed double-stranded oligonucleotides are: ds-411 (5′-tttattcctctgaggcagggtctattttat-3′, 30 bp; −10.17 to −988) (SEQ ID NO.: 3), ds-412 (5′-tgaggcagggtctattttatccttgttaca-3′, 30 bp; −1007 to −978) (SEQ ID NO.: 4), and ds-413 (5′-tctattttatccttgttacagatggggaaa-3′, 30 bp; −997 to −968) (SEQ ID NO.: 5). Only the sequences of the sense strands are listed. Probe-A and annealed double-stranded Comp-1 were also included as cold competitors, as we considered NRBS-D to be an additional binding site for NIS-repressor, which had already been demonstrated to bind to NRBS-P.
These EMSA results are shown in
Further analysis, using an unlabeled annealed double-stranded oligonucleotide (ds-414; 5′-ccttgttacagatggggaaactaaggccca-3′, 30 bp; −987 to −958) (SEQ ID NO.: 6), sharing a 20 bp sequence with NRBS-D and having an additional unshared 10 bp sequence downstream, revealed strong competition against the radiolabeled Probe-A in EMSA (
In supershift assays, antibodies against human Sp1 (E-3), c-Jun (H-79), c-Fos (H-125), AP-2α (C-18), TTF-1 (F-12), Pax8 (A-15), and PARP-1 failed to alter the EMSA signal mobilities, suggesting that their respective antigens are not associated with the NRBS-P site. This is consistent with other results showing that their respective consensus DNA target sequences are unable to compete against NRBS-P. The anti-TTF-2 antibody (S-18) shifted the EMSA signals, changing the mobility of one of the bands, showing faster migration, and simultaneously changing the single Comp-1 specific signal into multiple constituent bands with faster migration on the gel, as shown in
A human genome homology search (NCBI/BLAST/blastn suite) using the consensus sequence (5′-TG(G/A)GCCT(T/C)A(G/A)TTTCCC-CA(T/C)CTGT-3′ (SEQ ID NO. 1) was undertaken to determine whether the sequence is present in human genes in addition to the hNIS gene. The consensus sequence is shown to occur (at >90% homology throughout the entire sequence of SEQ ID NO. 1) in the human genes listed in Table 1 below.
The electrophoretic mobility shift assay (EMSA) is a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins in crude extracts. Proteins that bind specifically to an end-labeled DNA fragment, (radio-labeled probe) retard the mobility of the fragment during electrophoresis, resulting in discrete band(s) corresponding to the protein-DNA probe complexes. This assay permits the quantitative determination of the affinity, abundance, association and dissociation rate constants, and binding specificity of DNA probe-binding proteins.
Preparation of nuclear extract: Nuclear extracts are prepared from test cells, such as thyroid cells or tumor cells by any acceptable method, such as that encompassed by the NucBuster™ Protein Extraction Kit available from EMD Biosciences Inc./Novagen.
Preparation of Probe: The consensus sequence (SEQ ID NO. 1) is used as probe to detect binding of test molecules/compounds. A polynucleotide comprising the consensus sequence is end-labeled using T4 polynucleotide kinase. Eight pmole annealed double-stranded NRBS probe (consensus sequence), 8 μL γ-P32-ATP (6000 Ci/mmole), 3 μL 10× T4 polynucleotide kinase buffer (New England Biolab) are mixed and distilled water is added to a final reaction volume of 28 μL. Two μL T4 polynucleotide kinase (10000 U/mL (New England BioLab) is added and mixed well. The reaction mixture is incubated at 37° C. for 15 min, unbound label is removed using a QIAquick Nucleotide Removal kit (Qiagen) and the end-labeled probe is eluted with ˜100 μL TE buffer.
EMSA: EMSA is carried out as follows: 3 μL nuclear extract, 1 μL end-labeled probe, 1 μL Poly(dI-dC)-Poly(dI-dC) (0.01 U/μL in 100 mM KCl, 20 mM HEPES, pH 8.0), 1 μL Salmon sperm DNA (500 ng/μL in nuclease-free water), 5 μL 4×EMSA buffer (400 mM KCl, 80 mM HEPES, 0.8 mM EDTA, 80% glycerol, 0.5 mM DTT) are mixed and distilled nuclease-free water is added to bring the reaction mix to 18 μL. The mixture is incubated on ice for 30 minutes, followed by addition of 2 μL of loading buffer (1×EMSA buffer, 0.25% Bromophenol Blue). A 7% non-denaturing PAGE gel is pre-run in 0.5×TBE (5.4 g/L Tris base, 2.75 g/L boric acid, 1 mM EDTA, pH 8.) for 30 minutes at 100V. The entire 20 μL EMSA reaction is loaded into one well of the polyacrylamide gel and run at 100V until the Bromophenol Blue dye has migrated to the end of the gel. The gel is dried on DEAE paper using a standard gel dryer and the dried gel is exposed to X-ray film. A retarded signal relative to free end-labeled probe indicates athe presence of a positive protein-probe interaction complex. When un-labeled DNA probe is added in the EMSA reaction mixture, loss of EMSA signal indicates the signal is probe-specific. When antibody against a specific protein factor is added in the EMSA reaction mixture (Supershift assay), change of EMSA signal indicates that a specific protein factor is involved in the protein-probe complex.
This application is a continuation-in-part of U.S. application Ser. No. 12/724,898, filed Mar. 16, 2010.
This disclosure was made, in part, with support from the Merit Review award program of the U.S. Department of Veterans Affairs and an R01 Grant from the National Cancer Institute of the National Institutes of Health, and the government may have certain rights in this disclosure.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12724898 | Mar 2010 | US |
| Child | 13044120 | US |
