Intracellular Expression of the T-Cell Factor-1 RNA Aptamer As an Intramer

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Intracellular expression of the T-cell factor-1 RNA aptamer as an intramer

Kang Hyun Choi,1 Min Woo Park,1 and tumorigenic potential of HCT116 colon cancer cells. Seung Yeon Lee,1 Mi-Ya Jeon,1 Mee Young Kim,1 Such RNA intramer could lead to valuable gene therapeu- Hee Kyu Lee,1 Jaehoon Yu,2 Hong-Jin Kim,3 tics for TCF/B-catenin-mediated carcinogenesis. [Mol Kyungsook Han,4 Heviran Lee,5 Keerang Park,6 Cancer Ther 2006;5(9):2428–34] Woong June Park,1 and Sunjoo Jeong1 Introduction 1Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, T-cell factor (TCF)-1 is a transcription factor that binds to Dankook University; 2Department of Chemistry & Education, specific DNA through its high mobility group-1 domain and Seoul National University; 3College of Pharmacy, Chung-Ang is likely to be involved in the expansion of T lymphocytes 4 University, Seoul, Korea; School of Computer Science (1–8). TCF family proteins bind to DNA in a sequence- and Engineering, Inha University, Incheon, Korea; 5Department of Microbiology, University of Ulsan College of Medicine, Seoul, specific manner and they seem to act as architectural Korea; and 6JS Gene Therapy R&D Center, Jooseong College, proteins for the assembly of other transcription factors (9). Chung-buk, Korea h-Catenin is a potent transcriptional coactivator of TCF family proteins and the formation of a transcriptional complexby an oncogenic h-catenin with TCF might be a Abstract central event in cancer cell development (10, 11). The TCF/ T-cell factor (TCF)-1 protein forms the transcriptional h-catenin complex–mediated Wnt signaling is also a critical complex with B-catenin and regulates the expression of regulator of immature thymocyte development as well as of diverse target genes during early development and other early development (12–16). It is likely that TCF family carcinogenesis. We have selected previously an RNA proteins are critical modulators of the expression of genes aptamer that binds to the DNA-binding domain of TCF-1 that control the decision between proliferation and apopto- and have shown that it interfered with binding of TCF-1 to sis in the cells, such as cyclin D1 and c-myc,aswellas its specific DNA recognition sequences in vitro.Asan for cancer cell metastasis by matrixmetalloproteinase-7 approach to modulate the transcription by TCF/B-catenin (MMP-7) expression (17, 18). complex in the cells, we have developed the RNA High-affinity molecules, such as nucleic acid ligands, expression vector for stable expression of RNA aptamer can modulate the transcriptional activity of transcription inside of the mammalian cells. High level of RNA was factors. Reiterated in vitro selection procedures are able to expressed as an intramer in the fusion with the stable RNA select specific RNA molecules from random RNA library, transcript. The RNA intramer inhibited TCF/B-catenin and nucleic acids selected by this procedure are generally transcription activity as shown by luciferase assay. It referred to as ‘‘aptamers’’ (19–21). We have reported also modulated the expression of TCF/B-catenin target previously the in vitro selection of RNA aptamers that genes, such as cyclin D1 and matrix metalloproteinase-7, bind to TCF-1 and characterized the one of the selected as predicted to be as an effective inhibitor of the TCF RNA aptamers (#10 RNA aptamer) in vitro. It binds to the function. In addition, it efficiently reduced the growth rate TCF-1 protein containing high mobility group-1 domain, thereby interfering with its binding to DNA (22). Here, we have developed an RNA expression vector for this aptamer and overexpressed it in mammalian cells (23). Intracellularly expressed aptamers, ‘‘intramers’’, have Received 6/20/05; revised 5/25/06; accepted 6/29/06. advantages of high-level cellular expression and excep- Grant support: Korea Science and Engineering Foundation grants tional specificity, which make it possible to distinguish R01-2003-000-10461-0,2005-01131,and R01-2006-000-10194-0, Korea Research Foundation grant KRF-2004-015-C00378, between similar cellular proteins and to differentially and Department of Education of Korean Government BK21 Graduate modulate their functions (24, 25). We designed the RNA Program for RNA Biology (H.K. Lee). intramer to accumulate large amount of RNA transcript The costs of publication of this article were defrayed in part by the in the fusion with aberrant spliced mRNA. Most impor- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to tantly, we showed that #10 RNA intramer inhibited the indicate this fact. transcriptional activity of TCF-1 in the cells and down- Note: K.H. Choi and M.W. Park contributed equally to this work. regulated the TCF/h-catenin-mediated expression of Requests for reprints: Sunjoo Jeong,Department of Molecular Biology, cyclin D1 and MMP-7. Cell growth rate as well as BK21 Graduate Program for RNA Biology,Institute of Nanosensor and tumorigenic potential of colon cancer cells was also Biotechnology,Dankook University,Hannam-dong san 8,Yongsan-ku, Seoul 140-714,Korea. Phone: 82-2-709-2819; Fax: 82-2-793-0176. reduced by stably expressed RNA aptamer. Further E-mail: [email protected] refinement of this intramer could provide a way for Copyright C 2006 American Association for Cancer Research. modulating TCF-1-mediated T-cell development as well as doi:10.1158/1535-7163.MCT-05-0204 cancer cell development.

Mol Cancer Ther 2006;5(9). September 2006

Materials and Methods pDHFR/Aptamer #10 (in sense orientation) and pDHFR/ Plasmids, Proteins, and Reagents Inverse #10 (in inverse orientation) clones. The level of FLAG-tagged TCF-1 expression clone was generated by expression of the RNA aptamer in 293T cells was estimated introducing mouse TCF-1 into the EcoRI site of the vector by reverse transcription-PCR (RT-PCR) using the above pcDNA3.1. luciferase reporter plasmids, wild-type TCF aptamer-specific primer sets (DHFR-F1 and DHFR-R1). reporter pGL3-OT and mutant pGL3-OF, were kindly PCR (22–30 cycles) was done with each transfected sample. provided by Dr. Shivdasani (Dana-Farber Cancer Institute, RT-PCR Analysis Boston, MA). Nuclear factor-nB reporter (3Â nB-Luc) and Total cellular RNA was isolated from cells by the acid p65 expression clones were from Dr. Bill Sugden (University guanidium thiocyanate/phenol-chloroform extraction of Wisconsin, Madison WI). pCAN-h-catenin was kindly method. Isolated RNA (2 Ag) was reverse transcribed with provided by Dr. McCrea (The University of Texas M.D. M-MuLV reverse transcriptase (Roche, Indianapolis, IN), 25 Anderson Cancer Center, Houston, TX). The control vector Amol/L random hexamer (Promega), 500 Amol/L each of for normalizing transfection frequencies, pCMV-h,was deoxynucleotide triphosphates, and 20 units RNase inhib- purchased from Clontech (Mountain View, CA). Lipofect- itor. One fifth of the resulting cDNA was used in the AMINE was purchased from Invitrogen (Carlsbad, CA) PCR. PCR primers specific for cyclin D1 were 5¶-CTGG- and DMRIE-C was from Invitrogen. o-Nitrophenyl-h-D- CCATGAACTACCTGGA-3¶ (forward primer; corres- galactopyranoside was from Sigma-Aldrich (St. Louis, ponding to 385–404 bp) and 5¶-GTCACACTTGATCACT- MO). Anti-FLAG M2 monoclonal antibody and anti- CTGG-3¶ (reverse primer; corresponding to 848–867 bp), a-tubulin antibody were purchased from Sigma-Aldrich which produces a 483-bp PCR product. PCR primers for h- and anti-p65 C20 antibody and anti-cyclin D1 (sc-246) were catenin were 5¶-CGGGATCCACAAGAAACGGCTTTCA-3¶ from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- (forward) and 5¶-GAGAATTCCAGGTCAGTATCAAA- h-catenin antibody was from Transduction Laboratories CCA-3¶ (reverse), which generates a 334-bp PCR product. (San Jose, CA). PCR primers for MMP-7 were 5¶-ATGTTAAACT- Cell Culture,Transfection, and Luciferase Assay CCCGCGTCATA-3¶ (forward) and 5¶-CAGCATACAG- Human embryonic kidney 293T cells, human colorectal GAAGTTAATCC-3¶ (reverse), which generates a 481-bp carcinoma HCT116 cells and the murine immature thy- PCR product. Each primer set was amplified at 95jC for 30 moma S49.1 cells (American Type Culture Collection, seconds, 50jC (cyclin D1) for 2 minutes, 58jC (MMP-7) Manassas, VA) were cultured in DMEM with 10% fetal for 2 minutes, or 54jC(h-catenin) for 30 seconds and 72jC bovine serum and antibiotics. 293T and HCT116 cells were for 30 seconds for the indicated number of cycles. PCR for transfected with LipofectAMINE and S49.1 immature T-cell aptamer RNA #10 was done with DHFR-F1 and DHFR-R1 line was transfected by electroporation. Stable transfectants primers as described above. An 18S rRNA control primer of #10 RNA aptamer and #10 inverse RNA were selected by set (Ambion, Austin, TX) was used as control. zeocin. For luciferase assays, the luciferase reporter, an Western Blot Analysis aptamer expressing DNA or RNA, and pCMV-h were Cells were harvested and lysed with 2Â sample buffer cotransfected into the cells. Twenty-four hours later, cells [125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 40% glycerol]. were harvested, washed, and lysed in cell lysis buffer. Whole-cell extracts (20 Ag) were subjected to 12% SDS- Luciferase activity was determined with a Luciferase assay PAGE and the proteins were transferred to nitrocellulose system (Promega, Madison, WI). Readings were made with membranes. The membranes were incubated with blocking a Turner luminometer TD-20/20, and relative luciferase buffer (5% skim milk, 0.1% Tween 20 in TBS) for 1 hour at activities were obtained by normalizing for h-galactosidase room temperature and with anti-cyclin D1, anti-FLAG, activity. Transfection efficiencies were determined by anti-h-catenin, or anti-p65 antibodies for 1 hour at room counting green fluorescent protein (GFP)–expressed cells temperature. The membranes were washed with washing following cotransfection of cells with pEGFP vector. buffer (0.1% Tween 20 in TBS) thrice and incubated for Construction and Expression of the RNA Intramer 1 hour at room temperature with horseradish peroxidase– The expression vector for the RNA aptamer, pDHFR, was conjugated anti-mouse antibody. Anti-a-tubulin antibody constructed using the fact that a splice variant of was used for the controls. dihydrofolate reductase (DHFR) RNA can be highly Cell Growth Assay and Soft Agar Colony Formation expressed in cells (26). Fragment containing human DHFR Cell growth curve was generated by crystal violet splicing variant cDNA (residue 31 mutation in 831 bp) was staining. Cells were seeded with low cell density (5,000 cut from the retroviral vector DC/SV/R/DHFR and ligated per 24 wells) in triplicates and allowed to grow for 6 days. into the pcDNA vector with BamHI and HindIII sites to In each day, cells were washed with PBS, fixed with 1% generate the pDHFR vector (23). To insert the aptamer into glutaraldehyde, and stained with 0.1% crystal violet. After pDHFR, the DNA sequence of the aptamer was amplified destaining, crystal violet was solved with 1 mol/L acetic ¶ A from the pUC19-aptamer clone with DHFR-F1 (5 -TCA- acid and 595 was measured. For soft agar colony CCGCGGGAGCTCGGTACC-3¶) and DHFR-R1 (5¶-CCCC- formation assays, 5,000 cells were seeded in six-well plates GCGGATCCTCCTCTGCAAAGCTT-3¶) primers. The with 0.7% agar. After 10 to 20 days, colonies formed were amplified fragment was digested with SacII and cloned fixed in 70% ethanol, washed with water, and stained with into the same site of the pDHFR vector, generating 0.005% crystal violet for 20 minutes.

Mol Cancer Ther 2006;5(9). September 2006

Results In vitro Characterization of #10 RNA Aptamer We have described previously the selection and characterization of TCF-1 binding RNA aptamers in vitro (22). The #10 RNA aptamer was shown to specifically bind to COOH-terminal 200 amino acids of TCF-1 protein, which contains the DNA-binding high mobility group-1 domain. Secondary structure of #10 RNA aptamer was also determined by the RNase mapping (Fig. 1). Because #10 RNA aptamer bound to the DNA-binding domain of TCF-1, we also confirmed that the RNA aptamer interfered with DNA binding by TCF-1 proteins (22). Intracellular Expression of #10 RNA Aptamer as an Intramer As a way of overcoming instability and low efficiency of RNA transfection, we have developed a vector expressing stabilized RNA aptamer transcripts in mammalian cells. As shown in Fig. 2A, the expression vector contains DHFR cDNA (831 bp) harboring a cryptic splice site that can accumulate high levels of aberrantly spliced DHFR transcripts (26). The DNA sequence encoding #10 aptamer was inserted at the 5¶ end of the DHFR cDNA (pDHFR/ Aptamer) and was thus expressed as chimeric aptamer- DHFR-RNA (931 nt). The secondary structure of this chimeric RNA was predicted by mfold to retain the specific stem-loop structure of #10 aptamer due to the stable and extensive secondary structure of the aberrant DHFR transcript (23, 26). Inverse orientation of #10 aptamer was also inserted to DHFR vector, generating pDHFR/Inverse. To evaluate the expression level of #10 RNA intramer in mammalian cells, pDHFR/Aptamer DNA (200 ng) was transfected into 293T cells (2 Â 106) and the level of

Figure 2. Expression of RNA aptamer #10 as an intramer. A, design of the RNA aptamer expression vector,pDHFR/Aptamer. The vector was generated by cloning the DHFR cDNA containing cryptic splice sites into pcDNA. The DNA sequence encoding RNA aptamer #10 (100 nt) was inserted into the 5¶ end of DHFR cDNA (831 nt),generating pDHFR/ Aptamer #10. B, expression of RNA intramer in 293T cells as shown by RT-PCR. Cells (2 Â 106) were transfected with 200 ng pDHFR/Aptamer #10 DNA and harvested after 24 h. Level of the expressed RNA aptamer #10 was compared with that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA by various cycles of RT-PCR. Total RNA (5.7 Ag) was prepared from the cells and equal amounts of the RNAs (2 Ag) were reverse transcribed into cDNA. One of 20th amount of cDNA was used for each PCR. Lanes 1 and 5, 24 cycles; lanes 2 and 6, 26 cycles; lanes 3 and 7, 28 cycles; lanes 4 and 8, 30 cycles of PCR; lane 9, sample not treated with reverse transcriptase; lane 10, PCR control with the aptamer plasmid DNA. C, estimation of the amount of the RNA intramer molecules in 293T cells. The amount of RNA aptamer #10 expressed in the cells (1:100 dilutions of expressed cDNA) was estimated by comparison with in vitro – transcribed (IVT) RNA (0.1 and 0.5 ng in vitro – transcribed cDNA) by RT- PCR. Lanes 1, 5, and 9, 22 cycles; lanes 2, 6, and 10, 24 cycles; lanes 3, 7, and 11, 26 cycles; lanes 4, 8, and 12, 28 cycles of PCR. D, stability of the transfected RNA aptamer measured by RT-PCR. Lane 1, marker; lane 2, no reverse transcriptase,negative control; lane 3, PCR-positive control; lane 4, RNA from nontransfected cells; lane 5, RNA isolated 0 h after transfection; lane 6, RNA isolated 24 h after transfection.

RNA transcripts was measured by RT-PCR analysis. As shown in Fig. 2B, expression of the RNA intramer (#10 Figure 1. Structure of the RNA aptamer #10 as determined by the aptamer) was higher than that of abundant transcripts, RNase mapping (22). Shades, nucleotides from the selected sequence. such as glyceraldehyde-3-phosphate dehydrogenase mRNA.

Mol Cancer Ther 2006;5(9). September 2006

In addition, the intracellular level of expressed RNA Transcriptional Modulation of theTCF-1Protein by the intramer (expressed, diluted at 1:100) was compared with RNA Intramer known amounts (0.1 and 0. 5 ng) of in vitro –transcribed To see if the RNA intramer inhibited the transcriptional RNA (Fig. 2C). Because the amount of the diluted RNA activity of TCF-1, 293T cells were cotransfected with the intramer was estimated to be 0.2 ng by densitometry, at #10 aptamer or #10 inverse along with FLAG-tagged TCF-1 least 1.08 Ag RNA was expressed by the transfected cells and the TCF-responsive luciferase reporter, pGL3-OT (2 Â 106). Based on the molecular weight of #10 RNA (Fig. 3A). Overexpression of TCF-1 cDNA induced lucifer- 4 aptamer (3.1 Â 10 Da) and Avogadro’s number, we ase activity as expected (27). RNA intramer (#10 aptamer) 7 calculated that at least 1 Â 10 RNA intramer molecules reduced TCF-1-mediated transcription in a dose-depen- were expressed per cell. Because we obtained 50% dent manner. This inhibitory effect was sequence specific transfection efficiency for 293T cells as determined by because RNA intramer #10 in reverse orientation (#10 the expression of cotransfected green fluorescent protein inverse) did not affect transcriptional activity. Moderate 7 (data not shown), we expected that 2 Â 10 RNA inhibitory effect of RNA intramer could be partially intramer molecules were expressed in each transfected explained by 50% transfection efficiency as determined by cell. We were concerned that the #10 aptamer transfected green fluorescent protein expression. However, the expres- into the cells might be subjected to cellular RNase sion of FLAG-tagged TCF-1 protein was detected by activities, thus reducing its effect in the cells. Therefore, Western blot analysis with anti-FLAG antibody and was expression of #10 aptamer was also monitored to confirm not significantly reduced by cotransfected #10 aptamer or the stability of the RNA (Fig. 2D). We usually detect the #10 inverse (Fig. 3B). In addition, the expression of the RNA expression of the RNA intramer for several days, which intramers did not alter the morphology of the cells (data not may provide enough time to be an effective inhibitor in shown). We also tested whether the RNA intramer could the cancer cells. modulate the endogenous TCF-1 in immature T cells. As

Figure 3. Modulation of TCF transcriptional activity by intracellularly expressed aptamer DNA. A, 293T cells were transfected with FLAG-tagged TCF-1 cDNA and luciferase reporter (pGL3-OT) together with pDHFR (vector),pDHFR/Aptamer #10 (#10 aptamer) or pDHFR/Inverse #10 (#10 inverse). Luciferase activities were measured 24 h after transfection. They were normalized using the h-galactosidase activity from the cotransfected pCMV-h plasmid and are presented as relative fold activities compared with vector-transfected cells. Columns, mean of five independent experiments; bars, SD. *, P < 0.005. B, Western blot analysis was done with anti-FLAG antibody to detect TCF-1 protein expression in transfected cells as shown in (A). C, S49.1 immature T cells were cotransfected with the luciferase reporters and RNA aptamer #10 by electroporation. Luciferase activities of wild-type TCF luciferase reporter (pGL3-OT; OT) were measured and are presented as relative fold activities compared with that the activity obtained with the mutant TCF luciferase reporter (pGL3-OF; OF). Three independent experiments were done. D, 293T cells were transfected with p65-expressing cDNA and nuclear factor-nB(NF-jB) luciferase reporter (3Â nB-Luc) together with pDHFR/Aptamer #10 (#10 aptamer). Luciferase activity was measured 24 h after transfection. Relative fold activity was presented compared with nuclear factor-nB reporter – transfected cells. Three independent experiments were done. Expression level of p65 protein was shown by Western blot analysis with anti-p65 antibody.

Mol Cancer Ther 2006;5(9). September 2006

shown in Fig. 3C, #10 RNA inhibited the TCF-1 transcrip- h-catenin target gene, such as MMP-7, in HCT116 colon tional activity in cells of the immature T-cell line S49.1. The cancer cells (28). As shown in Fig. 4B, expression of MMP-7 specificity of the #10 aptamer on TCF-1-activated transcrip- mRNA was also reduced by the #10 aptamer but not by the tion was also confirmed by the luciferase assay with the #10 inverse (Fig. 4C). Interestingly, transfection of #10 reporter activated by nonrelated transcription factors nuclear aptamer did not affect the expression of c-myc protein in factor-nBin293Tcells(Fig.3D). HCT116 cells (Fig. 4A), suggesting differential effects on Specific Inhibition of Target Gene Expression and Cell different TCF/h-catenin target genes. These results showed Growth that the intracellular expressed TCF-1 binding RNA TCF family proteins are known to interact with the aptamer could be an effective and selective inhibitor of transcriptional activator h-catenin and activate expression TCF/h-catenin target genes in colon cancer cells. of diverse target genes, such as cyclin D1, c-myc, and Because #10 RNA intramer suppressed the expression of MMP-7 (17, 18, 28). Because the high-level expression of cyclin D1, we tested the effect of the RNA aptamer on the h-catenin is related to the high level of target gene cell growth of colon cancer cell line, HCT116. As shown in expression in colon cancer cells, we used human colon Fig. 5A, cell growth was significantly reduced by the #10 carcinoma HCT116 as a test colon cancer cell line. As RNA intramer (aptamer) compared with that by #10 shown in Fig. 4A, endogenous protein levels of h-catenin inverse RNA (inverse). We also examined whether the and cyclin D1 are high in HCT116 cells as expected. tumorigenic potential of HCT116 colon cancer cell was However, transfection of #10 RNA aptamer greatly reduced suppressed by the stably expressed #10 RNA intramer. As the cyclin D1 protein level but not by #10 inverse. We also shown in Fig. 5B by colony-forming assay, size of colonies tested the effect of #10 RNA aptamer on the other was significantly reduced by the expressed #10 RNA aptamer (Fig. 5B, bottom) compared with #10 inverse (Fig. 5B, top). These results suggested that #10 RNA aptamer can be an effective inhibitor of cell growth as well as tumorigenesis in colon cancer cells.

Discussion Because TCF/h-catenin-mediated transcription is impor- tant regulator for carcinogenesis of diverse cells, notably for colon cancer development, a molecule that could modulate this process would be a useful agent for antitumor therapy (29, 30). Some conventional drugs have been shown to reduce the TCF/h-catenin-mediated transcription in colon cancer cells (31–33) and small-molecule antagonists have been selected from the diverse chemical library to inhibit TCF/h-catenin protein complex(34). We report here the first characterization of an RNA aptamer that inhibits the binding of the transcription factor TCF-1 to DNA, thus modulating the expression of various target genes. Cell growth rate and tumorigenic potential of colon cancer cell line HCT116 were also seemed to be reduced by TCF-1 binding RNA aptamer. RNA aptamer #10 efficiently reduced the expressions of TCF/h-catenin target genes, such as cyclin D1 and MMP-7, as shown by luciferase assay, RT-PCR, and Western blot analysis (Fig. 4). However, more significantly, we observed no obvious alteration on the protein level of c-myc, another notable TCF/h-catenin target gene. Such a selective regulation of TCF/h-catenin signaling by the RNA aptamer might be of greatest utility if it could modulate the expres- Figure 4. Inhibition of the TCF target genes by pDHFR/Aptamer #10. sion of a subset of genes and regulate specific subpathway A, Western blot analysis of HCT116 colon cancer cells with anti-cyclin D1 of Wnt signaling. Because it was proposed recently that antibody. HCT116 cells were transfected with pDHFR/Aptamer #10 cyclin D1 may not be an immediate target of TCF/h-catenin (aptamer) or pDHFR/Inverse #10 (inverse). Level of h-catenin protein was confirmed and the protein level of a-tubulin served as the loading following Apc loss in the intestine (35), it will be interesting control. B, RT-PCR analysis of MMP-7 expression. HCT116 cells were to study the mechanism of selective inhibition of transcrip- transfected with either pDHFR vector (À),pDHFR/Aptamer #10 (aptamer), tion by RNA aptamer #10. We observed more significant or pDHFR/Inverse #10 (inverse). Total RNA was prepared and the reduction of transcriptional activity for the endogenously expression of MMP-7 mRNA was analyzed. Expression of RNA aptamer was also confirmed and glyceraldehyde-3-phosphate dehydrogenase expressed TCF-1 in T-cell lines than for the exogenously mRNA served as the loading control. overexpressed protein in 293T cells.

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Kang Hyun Choi, Min Woo Park, Seung Yeon Lee, et al.

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