Sunjoo Jeong

β-Catenin stabilizes Cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3′-UTR

Cyclooxygenase-2 (COX-2) mRNA is induced in the majority of human colorectal carcinomas. Transcriptional regulation plays a key role in COX-2 expression in human colon carcinoma cells, but post-transcriptional regulation of its mRNA is also critical for tumorigenesis. Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. β-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2. Here we found that COX-2 mRNA was also substantially stabilized by activating β-catenin in NIH3T3 and 293T cells. We identified the β-catenin-responsive element in the proximal region of the COX-2 3′-untranslated region (3′-UTR) and showed that β-catenin interacted with AU-rich elements (ARE) of 3′-UTR in vitro and in vivo. Interestingly, β-catenin induced the cytoplasmic localization of the RNA stabilizing factor, HuR, which may bind to β-catenin in an RNA-mediated complex and facilitate β-catenin-dependent stabilization of COX-2 mRNA. Taken together, we provided evidences for β-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

Modulation of Oncogenic Transcription and Alternative Splicing by β-Catenin and an RNA Aptamer in Colon Cancer Cells

Activated beta-catenin regulates the transcription of oncogenic target genes and is critical for tumorigenesis. Because nuclear functions are frequently coupled, we investigated whether it also has a role in alternative splicing of oncogenic genes. We showed that stabilized beta-catenin caused alternative splicing of estrogen receptor-beta pre-mRNA in colon cancer cells. To establish a direct role of beta-catenin in regulated splicing, we selected a high-affinity RNA aptamer that associated with beta-catenin in vivo. Nuclear localized aptamer inhibited beta-catenin-dependent transcription of cyclin D1 and c-myc in colon cancer cells; thus, cells stably expressing the aptamer exhibited cell cycle arrest and reduced tumor forming potential. Most significantly, the aptamer prevented the alternative splicing induced by stabilized beta-catenin. Taken together, our results establish that beta-catenin has an important role in both transcription and splicing, and that its action can be modulated by a high-affinity RNA aptamer. The RNA aptamer could be further developed as a specific inhibitor for cancer therapeutics.

SR Proteins Induce Alternative Exon Skipping through Their Activities on the Flanking Constitutive Exons

ABSTRACT SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.

β-Catenin Regulates Multiple Steps of RNA Metabolism as Revealed by the RNA Aptamer in Colon Cancer Cells

Nuclear β-catenin forms a transcription complex with TCF-4, which is implicated in colon cancer development and progression. Recently, we and others have shown that β-catenin could be a regulator of RNA splicing and it also stabilizes the cyclooxygenase-2 (COX-2) mRNA. Here, we further explored the role of β-catenin in the RNA metabolism in colon cancer cells. To specifically modulate the subcellular functions of β-catenin, we expressed the RNA aptamer in the form of RNA intramers with unique cellular localizations. The nucleus-expressed RNA intramer proved to be effective in reducing the protein-protein interaction between β-catenin and TCF-4, thus shown to be a specific regulator of β-catenin–activated transcription. It could also regulate the alternative splicing of E1A minigene in diverse colon cancer cell lines. In addition, we tested whether β-catenin could stabilize any other mRNAs and found that cyclin D1 mRNA was also bound and stabilized by β-catenin. Significantly, the cytoplasm-expressed RNA intramer reverted the β-catenin–induced COX-2 and cyclin D1 mRNA stabilization. We show here that β-catenin regulated multiple steps of RNA metabolism in colon cancer cells and might be the protein factor coordinating RNA metabolism. We suggest that the RNA intramers could provide useful ways for inhibiting β-catenin–mediated transcription and RNA metabolism, which might further enhance the antitumorigenic effects of these molecules in colon cancer cells. [Cancer Res 2007;67(19):9315–20]

Inhibition of the angiogenesis by the MCP-1 (monocyte chemoattractant protein-1) binding peptide

The CC chemokine, monocyte chemoattractant protein‐1 (MCP‐1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP‐1‐induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP‐1 induced angiogenesis. Surface plasmon resonance measurements demonstrated specific binding of the peptide to MCP‐1 but not to the other homologous protein, MCP‐3. Flow cytometry revealed that the peptide inhibited the MCP‐1 binding to THP‐1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP‐1 induced angiogenesis. Our observations indicate that the MCP‐1‐binding peptide exerts its anti‐angiogenic effect by interfering with the interaction between MCP‐1 and its receptor.

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

T-cell factor (TCF)-1 protein forms the transcriptional complex with β-catenin and regulates the expression of diverse target genes during early development and carcinogenesis. We have selected previously an RNA aptamer that binds to the DNA-binding domain of TCF-1 and have shown that it interfered with binding of TCF-1 to its specific DNA recognition sequences in vitro. As an approach to modulate the transcription by TCF/β-catenin complex in the cells, we have developed the RNA expression vector for stable expression of RNA aptamer inside of the mammalian cells. High level of RNA was expressed as an intramer in the fusion with the stable RNA transcript. The RNA intramer inhibited TCF/β-catenin transcription activity as shown by luciferase assay. It also modulated the expression of TCF/β-catenin target genes, such as cyclin D1 and matrix metalloproteinase-7, as predicted to be as an effective inhibitor of the TCF function. In addition, it efficiently reduced the growth rate and tumorigenic potential of HCT116 colon cancer cells. Such RNA intramer could lead to valuable gene therapeutics for TCF/β-catenin-mediated carcinogenesis. [Mol Cancer Ther 2006;5(9):2428–34]

β-Catenin binds to the downstream region and regulates the expression C-reactive protein gene

C-Reactive protein (CRP) is a major acute-phase response protein, which is activated by various cytokines. We investigated the mechanism of TNF-α-induced CRP expression and found that the p50 subunit of NF-κB was responsible for the transcriptional activation of CRP. Since the p50 protein acts as a positive regulator of CRP expression without an inherent transactivation domain, we looked for an interaction partner that could provide p50 with such a domain. We found that β-catenin enhanced the expression of a CRP mRNA in concert with p50 subunit. Protein–protein interaction between p50 and β-catenin was important for CRP expression and their interactions to CRP promoter were induced after TNF-a treatment. Since gene expression depends upon the proximity of promoters and distal regulatory sites, we explored the long-range genomic interaction at the CRP locus by chromosome conformation capture (3C). We identified a binding site for β-catenin in the downstream of CRP gene by 3C and confirmed TNF-α-induced association of β-catenin and p50 by chromatin immunoprecipitation and co-immunoprecipitation assays. Our findings provide evidence that transcription of the CRP gene depends upon p50 and β-catenin proteins, which is accompanied by close proximity between promoter and the downstream region of CRP gene.

Modulation of transcription by the peroxisome proliferator-activated receptor δ–binding RNA aptamer in colon cancer cells

Peroxisome proliferator-activated receptor δ (PPAR-δ), one of three PPAR subtypes, is a lipid-sensing nuclear receptor that has been implicated in multiple processes, including inflammation and cancer. To directly establish the role of PPAR-δ in colon cancer development and progression, we selected high-affinity RNA aptamers and expressed them in several colon cancer cell lines. Nuclear-expressed aptamers efficiently inhibited PPAR-δ–dependent transcription from a synthetic peroxisome proliferator response element–driven luciferase reporter. PPAR-δ–specific aptamers suppressed transcription from natural promoters of vascular endothelial cell growth factor-A and cyclooxygenase-2. Moreover, vascular endothelial cell growth factor-A and cyclooxygenase-2 mRNA levels were significantly reduced by the PPAR-δ–specific aptamers in colon cancer cells. Most significantly, HCT116 colon cancer cells with high-level expression of PPAR-δ–specific aptamers exhibited a striking loss of tumorigenic potential. Further study on these RNA aptamers could provide an opportunity to modulate PPAR-δ–mediated colon cancer development and progression. Taken together, our results establish an important role for PPAR-δ in transcription of tumor-promoting genes, which can be specifically modulated by high-affinity RNA intramers in colon cancer cells. The RNA intramers may be further developed as specific inhibitors for cancer therapeutic strategies. [Mol Cancer Ther 2009;8(9):2664–73]

Annexin A2 binds RNA and reduces the frameshifting efficiency of infectious bronchitis virus.

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.

Selection and characterization of tenascin C targeting peptide.

Since tenascin C is a factor expressed highly in the tumor-associated matrix, it would be a desirable first step for targeting the tumor-specific microenvironment. In fact, a high level of tenascin C expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. Therefore, the targeted binding of tenascin C in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. We isolated a peptide that bound to tenascin C by phage display peptide library selection, and the selected peptide specifically recognized tenascin C protein in xenograft mouse tissue. We also observed exclusive staining of tenascin C by the selected peptide in tumor patient tissues. Moreover, the peptide reduced tenascin C-induced cell rounding and migration. We propose that the tenascin C targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors.