In obesity, dysregulation of adipocytokines is involved in several pathological conditions including diabetes and certain cancers. As a member of the adipocytokines, adiponectin plays crucial roles in whole-body energy homeostasis. Recently, it has been reported that the level of plasma adiponectin is reduced in several types of cancer patients. However, it is largely unknown whether and how adiponectin affects colon cancer cell growth. Here, we show that adiponectin suppresses the proliferation of colon cancer cells including HCT116, HT29, and LoVo. In colon cancer cells, adiponectin attenuated cell cycle progression at the G(1)/S boundary and concurrently increased expression of cyclin-dependent kinase inhibitors such as p21 and p27. Adiponectin stimulated AMP-activated protein kinase (AMPK) phosphorylation whereas inhibition of AMPK activity blunted the effect of adiponectin on the proliferation of colon cancer cells. Furthermore, knockdown of adiponectin receptors such as AdipoR1 and AdipoR2 relieved the suppressive effect of adiponectin on the growth of colon cancer cells. In addition, adiponectin repressed the expression of sterol regulatory element binding protein-1c, which is a key lipogenic transcription factor associated with colon cancers. These results suggest that adiponectin could inhibit the growth of colon cancer cells through stimulating AMPK activity.

Activated B-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 B-catenin caused alternative splicing of estrogen receptor-B pre-mRNA in colon cancer cells. To establish a direct role of B-catenin in regulated splicing, we selected a high-affinity RNA aptamer that associated with B-catenin in vivo . Nuclear localized aptamer inhibited B-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 B-catenin. Taken together, our results establish that B-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. (Cancer Res 2006; 66(21): 10560-6)

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.

Nuclear B-catenin forms a transcription complex with TCF-4, which is implicated in colon cancer development and progression. Recently, we and others have shown that B-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 B-catenin in the RNA metabolism in colon cancer cells. To specifically modulate the subcellular functions of B-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 B-catenin and TCF-4, thus shown to be a specific regulator of B-cateninactivated transcription. It could also regulate the alternative splicing of E1A minigene in diverse colon cancer cell lines. In addition, we tested whether B-catenin could stabilize any other mRNAs and found that cyclin D1 mRNA was also bound and stabilized by B-catenin. Significantly, the cytoplasmexpressed RNA intramer reverted the B-catenin-induced COX-2 and cyclin D1 mRNA stabilization. We show here that B-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 B-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):9315-21]

RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, β-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to β-catenin to address RNA recognition by β-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a β-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3′-untranslated region (3′-UTR). More significantly, the C-terminal domain of β-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA–β-catenin–HuR complex in vitro and in cells. Furthermore, the tertiary RNA–protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the β-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA–protein networks in colon cancer cells.

No abstract

T-cell factor (TCF)-1 protein forms the transcriptional complex with B-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/B-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/B-catenin transcription activity as shown by luciferase assay. It also modulated the expression of TCF/B-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/B-catenin-mediated carcinogenesis. [Mol Cancer Ther 2006;5(9):2428 -34]

a b s t r a c tTranscription is activated by signal-induced protein-protein interaction between transcription factors on regulatory elements positioned near their target genes. Here, we tested the utility of the bcatenin binding RNA aptamer as a tool for studying protein-protein interaction within transcription complex and for modulating expression of a target gene. The RNA aptamer bound Armadillo repeats of b-catenin and was effective in disrupting protein-protein interaction between b-catenin and nuclear factor-jB (NF-jB) p50. In addition, the RNA aptamer effectively reduced tumor necrosis factor-a induced transcription from the promoter of C-reactive protein regulated by b-catenin and NFjB p50. Taken together, b-catenin binding RNA aptamer was an effective regulator of b-catenin and NF-jB p50 mediated transcription.