Linderapyrone: A Wnt signal inhibitor isolated from Lindera umbellata.

Linderapyrone, a Wnt signal inhibitor was isolated from the methanolic extract of the stems and twigs of Lindera umbellata together with epi-(-)-linderol A. Linderapyrone inhibited TCF/ß-catenin transcriptional activity that was evaluated using cell-based TOPFlash luciferase assay system. To evaluate the structure-activity relationship and mechanism, we synthesized linderapyrone and its derivatives from piperitone. As the results of further bioassay for synthesized compounds, we found both of pyrone and monoterpene moieties were necessary for inhibitory effect. cDNA microarray analysis in a linderapyrone derivative treated human colorectal cancer cells showed that this compound downregulates Wnt signaling pathway. Moreover, we successes to synthesize the derivative of linderapyrone that has stronger inhibitory effect than linderapyrone and ICG-001 (positive control).

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Salinomycin exerts anti-colorectal cancer activity by targeting the ß-catenin/T-cell factor complex.

BACKGROUND AND PURPOSE: Salinomycin is a well-known inhibitor of human cancer stem cells (CSCs). However, the molecular mechanism(s) by which salinomycin targets colorectal CSCs is poorly understood. Here, we have investigated underlying antitumour mechanisms of salinomycin in colorectal cancer cells and three tumour models. EXPERIMENTAL APPROACH: The inhibitory effect of salinomycin on the Wnt/ß-catenin pathway was analysed with the SuperTopFlash reporter system. The mRNA expression of Wnt target genes was evaluated with real-time PCR. Effects of salinomycin on ß-catenin/TCF4E interaction were examined using co-immunoprecipitation and an in vitro GST pull-down assay. Cell proliferation was determined by BrdU incorporation and soft agar colony formation assay. The stemness of the cells was assessed by sphere formation assay. Antitumour effects of salinomycin on colorectal cancers was evaluated with colorectal CSC xenografts, APCmin/+ transgenic mice, and patient-derived colorectal tumour xenografts. KEY RESULTS: Salinomycin blocked ß-catenin/TCF4E complex formation in colorectal cancer cells and in an in vitro GST pull-down assay, thus decreasing expression of Wnt target genes. Salinomycin also suppressed the transcriptional activity mediated by ß-catenin/LEF1 or ß-catenin/TCF4E complex and exhibited an inhibitory effect on the sphere formation, proliferation, and anchorage-independent growth of colorectal cancer cells. In colorectal tumour xenografts and APCmin/+ transgenic mice, administration of salinomycin significantly reduced tumour growth and the expression of CSC-related Wnt target genes including LGR5. CONCLUSIONS AND IMPLICATIONS: Our study suggested that salinomycin could suppress the growth of colorectal cancer by disrupting the ß-catenin/TCF complex and thus may be a promising agent for colorectal cancer treatment.

Novel screening system revealed that intracellular cholesterol trafficking can be a good target for colon cancer prevention.

In conventional research methods for cancer prevention, cell proliferation and apoptosis have been intensively targeted rather than the protection of normal or benign tumor cells from malignant transformation. In this study, we aimed to identify candidate colon cancer chemopreventive drugs based on the transcriptional activities of TCF/LEF, NF-κB and NRF2, that play important roles in the process of malignant transformation. We screened a "validated library" consisting of 1280 approved drugs to identify hit compounds that decreased TCF/LEF and NF-κB transcriptional activity and increased NRF2 transcriptional activity. Based on the evaluation of these 3 transcriptional activities, 8 compounds were identified as candidate chemopreventive drugs for colorectal cancer. One of those, itraconazole, is a clinically used anti-fungal drug and was examined in the Min mouse model of familial adenomatous polyposis. Treatment with itraconazole significantly suppressed intestinal polyp formation and the effects of itraconazole on transcriptional activities may be exerted partly through inhibition of intracellular cholesterol trafficking. This screen represents one of the first attempts to identify chemopreventive agents using integrated criteria consisting of the inhibition of TCF/LEF, NF-κB and induction of NRF2 transcriptional activity.

Optimization of Peptidomimetics as Selective Inhibitors for the ß-Catenin/T-Cell Factor Protein-Protein Interaction.

The ß-catenin/T-cell factor (Tcf) protein-protein interaction (PPI) plays a critical role in the ß-catenin signaling pathway which is hyperactivated in many cancers and fibroses. Based on compound 1, which was designed to target the Tcf4 G13ANDE17 binding site of ß-catenin, extensive structure-activity relationship studies have been conducted. As a result, compounds 53 and 57 were found to disrupt the ß-catenin/Tcf PPI with the Ki values of 0.64 and 0.44 µM, respectively, and exhibit good selectivity for ß-catenin/Tcf over ß-catenin/E-cadherin and ß-catenin/adenomatous polyposis coli (APC) PPIs. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) cell viability assays revealed that 56, the ethyl ester of 53, was more potent than 53 in inhibiting viability of most of the Wnt/ß-catenin hyperactive cancer cells. Further cell-based studies indicated that 56 disrupted the ß-catenin/Tcf PPI without affecting the ß-catenin/E-cadherin and ß-catenin/APC PPIs, suppressed transactivation of Wnt/ß-catenin signaling in dose-dependent manners, and inhibited migration and invasiveness of Wnt/ß-catenin-dependent cancer cells.

vIRF3 encoded by Kaposi's sarcoma-associated herpesvirus inhibits T-cell factor-dependent transcription via a CREB-binding protein-interaction motif.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Like other herpesviruses, KSHV has two distinct life cycles: latent and lytic. Among KSHV latent genes, viral interferon regulatory factor 3 (vIRF3), which shares homology with cellular IRFs, is a multifunctional protein. To identify unknown functions of vIRF3, we performed luciferase-reporter assays in the presence of vIRF3. These analyses revealed that overexpression of vIRF3 inhibited T-cell factor (TCF)-dependent transcriptional activity. This TCF-dependent transcription was associated with the Wnt signaling pathway, which normally regulates embryonic development, but contributes to oncogenesis under dysregulated conditions. Using a mutagenesis analysis, we identified a CREB-binding protein-interaction motif (LXXLL) in vIRF3 as an important region for its inhibitory activity. Collectively, our findings provide insight into the dysregulation of host signaling pathways in KSHV-infected cells.

Discovery of Selective Small-Molecule Inhibitors for the ß-Catenin/T-Cell Factor Protein-Protein Interaction through the Optimization of the Acyl Hydrazone Moiety.

Acyl hydrazone is an important functional group for the discovery of bioactive small molecules. This functional group is also recognized as a pan assay interference structure. In this study, a new small-molecule inhibitor for the ß-catenin/Tcf protein-protein interaction (PPI), ZINC02092166, was identified through AlphaScreen and FP assays. This compound contains an acyl hydrazone group and exhibits higher inhibitory activities in cell-based assays than biochemical assays. Inhibitor optimization resulted in chemically stable derivatives that disrupt the ß-catenin/Tcf PPI. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure-activity relationship studies. This series of inhibitors with a new scaffold exhibits dual selectivity for ß-catenin/Tcf over ß-catenin/cadherin and ß-catenin/APC PPIs. One derivative of this series suppresses canonical Wnt signaling, downregulates the expression of Wnt target genes, and inhibits the growth of cancer cells. This compound represents a solid starting point for the development of potent and selective ß-catenin/Tcf inhibitors.

Salinomycin decreases doxorubicin resistance in hepatocellular carcinoma cells by inhibiting the ß-catenin/TCF complex association via FOXO3a activation.

Doxorubicin is a conventional and effective chemotherapy drug against hepatocellular carcinoma (HCC). However, during long-term doxorubicin monotherapy, HCC cells may eventually develop acquired-resistance to doxorubicin which results in recurrence and a poor prognosis. Salinomycin, an ionophore antibiotic, was recently reported to selectively kill human cancer stem cells (CSCs) which were response for chemoresistance. In this study, salinomycin was found to exert synergistic cytotoxicity with doxorubicin in HCC cells and be capable of inhibiting doxorubicin-induced epithelial-mesenchymal transition (EMT), an important cellular process involved in the acquired chemoresistance of tumors. Further experiments revealed that FOXO3a, a multifunctional transcription factor that can be activated by salinomycin, was vital in mediating doxorubicin-induced EMT. In addition, activated FOXO3a disturbed the interaction between ß-catenin and TCF and inhibited the expression of ß-catenin/TCF target genes (ZEB1, c-Myc and CyclinD1), which played important roles in doxorubicin-induced EMT in HCC cells. Finally, the enhanced curative efficacy of combination treatment of doxorubicin and salinomycin for HCC was confirmed in established xenograft models. In summary, the present study identifies a new doxorubicin-based chemotherapy for advanced HCC and provides a potential anti-cancer strategy targeting FOXO3a and related cell pathway molecules.

Endostar, a modified recombinant human endostatin, suppresses angiogenesis through inhibition of Wnt/ß-catenin signaling pathway.

Endostar, a novel modified recombinant human endostatin, is now widely studied for the treatment of diseases that are characterized or caused by pathological angiogenesis. However, its molecular mechanism remains unclear. In this study, we investigated the effects of Endostar on the Wnt/ß-catenin signaling pathway, which has emerged as an important aspect of angiogenesis. We showed that Endostar significantly inhibited the proliferation, migration, invasion, and capillary-like tube formation of human umbilical vascular endothelial cells in a dose-dependent manner. Using a luciferase reporter assay, we also demonstrated that Endostar suppressed ß-catenin-dependent T cell factor transcriptional activity in increasing doses. Moreover, we found that Endostar treatment also restricted the stabilized mutant ß-catenin-mediated increase in transcriptional activity, suggesting that Endostar exerts its inhibitory influence on Wnt/ß-catenin signaling by targeting ß-catenin or its downstream molecules. Western blot and immunofluorescence results revealed that Endostar significantly decreased nuclear and total ß-catenin levels. Finally, we discovered that Endostar down-regulated cyclin D1 and VEGF, two proteins that are known as the downstream targets of Wnt/ß-catenin signaling and that also play important roles in angiogenesis. Our findings suggested that Endostar inhibits angiogenesis and that the downregulation of the Wnt/ß-catenin signaling pathway may be involved in the inhibition of angiogenesis by Endostar. These results support the use of Endostar in further clinical applications.

Investigation of 3-aryl-pyrimido[5,4-e][1,2,4]triazine-5,7-diones as small molecule antagonists of ß-catenin/TCF transcription.

Nearly all colorectal cancers (CRCs) and varied subsets of other cancers have somatic mutations leading to ß-catenin stabilization and increased ß-catenin/TCF transcriptional activity. Inhibition of stabilized ß-catenin in CRC cell lines arrests their growth and highlights the potential of this mechanism for novel cancer therapeutics. We have pursued efforts to develop small molecules that inhibit ß-catenin/TCF transcriptional activity. We used xanthothricin, a known ß-catenin/TCF antagonist of microbial origin, as a lead compound to synthesize related analogues with drug-like features such as low molecular weight and good metabolic stability. We studied a panel of six candidate Wnt/ß-catenin/Tcf-regulated genes and found that two of them (Axin2, Lgr5) were reproducibly activated (9-10 fold) in rat intestinal epithelial cells (IEC-6) following ß-catenin stabilization by Wnt-3a ligand treatment. Two previously reported ß-catenin/TCF antagonists (calphostin C, xanthothricin) and XAV939 (tankyrase antagonist) inhibited Wnt-activated genes in a dose-dependent fashion. We found that four of our compounds also potently inhibited Wnt-mediated activation in the panel of target genes. We investigated the mechanism of action for one of these (8c) and demonstrated these novel small molecules inhibit ß-catenin transcriptional activity by degrading ß-catenin via a proteasome-dependent, but GSK3ß-, APC-, AXIN2- and ßTrCP-independent, pathway. The data indicate the compounds act at the level of ß-catenin to inhibit Wnt/ß-catenin/TCF function and highlight a robust strategy for assessing the activity of ß-catenin/TCF antagonists.

The role of CDX2 in Caco-2 cell differentiation.

BACKGROUND: CDX2 plays a key part in the differentiation of Caco-2 cells, a colon carcinoma derived cell line that undergoes spontaneous differentiation. The effect of CDX2 expression in Caco-2 cells over time in culture has not been studied yet on a genome-wide level. METHODS: The impact of CDX2 expression on the genomic profile of Caco-2 cells was studied by transducing cells with CDX2 targeting shRNAs. Knockdown efficiency was assessed on mRNA level and protein level by RTPCR, microarrays, and Western blots. Gene set enrichment analysis was performed to assess regulation of specific gene sets. RESULTS: CDX2 expression had an inhibitory effect on the transcriptional activity of ß-catenin/TCF at early stages of culturing, while at later stages, its role in the trans-activation of target genes specific for small intestinal enterocytes seemed more dominant. CONCLUSIONS: The unique induction of a small intestinal signature upon differentiation in Caco-2 cells seems to be at least partially under the control of CDX2.

In vitro effects of a small-molecule antagonist of the Tcf/ß-catenin complex on endometrial and endometriotic cells of patients with endometriosis.

BACKGROUND: Our previous studies suggested that aberrant activation of Wnt/ß-catenin signaling might be involved in the pathophysiology of endometriosis. We hypothesized that inhibition of Wnt/ß-catenin signaling might result in inhibition of cell proliferation, migration, and/or invasion of endometrial and endometriotic epithelial and stromal cells of patients with endometriosis. OBJECTIVES: The aim of the present study was to evaluate the effects of a small-molecule antagonist of the Tcf/ß-catenin complex (PKF 115-584) on cell proliferation, migration, and invasion of endometrial and endometriotic epithelial and stromal cells. METHODS: One hundred twenty-six patients (78 with and 48 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of PKF 115-584 on cell proliferation, migration, and invasion and on the Tcf/ß-catenin target genes were evaluated in endometrial epithelial and stromal cells of patients with and without endometriosis, and in endometrial and endometriotic epithelial and stromal cells of the same patients. RESULTS: The inhibitory effects of PKF 115-584 on cell migration and invasion in endometrial epithelial and stromal cells of patients with endometriosis prepared from the menstrual phase were significantly higher than those of patients without endometriosis. Levels of total and active forms of MMP-9 were significantly higher in epithelial and stromal cells prepared from menstrual endometrium in patients with endometriosis compared to patients without endometriosis. Treatment with PKF 115-584 inhibited MMP-9 activity to undetectable levels in both menstrual endometrial epithelial and stromal cells of patients with endometriosis. The number of invasive cells was significantly higher in epithelial and stromal cells of endometriotic tissue compared with matched eutopic endometrium of the same patients. Treatment with PKF 115-584 decreased the number of invasive endometriotic epithelial cells by 73% and stromal cells by 75%. CONCLUSIONS: The present findings demonstrated that cellular mechanisms known to be involved in endometriotic lesion development are inhibited by targeting the Wnt/ß-catenin pathway.

Rational design of small-molecule inhibitors for ß-catenin/T-cell factor protein-protein interactions by bioisostere replacement.

A new hot spot-based design strategy using bioisostere replacement is reported to rationally design nonpeptidic small-molecule inhibitors for protein-protein interactions. This method is applied to design new potent inhibitors for ß-catenin/T-cell factor (Tcf) interactions. Three hot spot regions of Tcf for binding to ß-catenin were quantitatively evaluated; the key binding elements around K435 and K508 of ß-catenin were derived; a bioisostere library was used to generate new fragments that can match the proposed critical binding elements. The most potent inhibitor, with a molecular weight of 230, has a Kd of 0.531 µM for binding to ß-catenin and a Ki of 3.14 µM to completely disrupt ß-catenin/Tcf interactions. The binding mode of the designed inhibitors was validated by the site-directed mutagenesis and structure-activity relationship (SAR) studies. This study provides a new approach to design new small-molecule inhibitors that bind to ß-catenin and effectively disrupt ß-catenin/Tcf interactions specific for canonical Wnt signaling.

IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of ß-catenin/TCF signaling.

Proinflammatory cytokines induce guanylate-binding protein 1 (GBP-1) protein expression in intestinal epithelial tissues. GBP-1 has been described as influencing a number of cellular processes important for epithelial homeostasis, including cell proliferation. However, many questions remain as to the role of GBP-1 in intestinal mucosal homeostasis. We therefore sought to investigate the function of proinflammatory cytokine-induced GBP-1 during intestinal epithelial cell proliferation. Through the use of complementary GBP-1 overexpression and small interfering RNA-mediated knockdown studies, we now show that GBP-1 acts to inhibit pro-mitogenic ß-catenin/T cell factor (TCF) signaling. Interestingly, proinflammatory cytokine-induced GBP-1 was found to be a potent suppressor of ß-catenin protein levels and ß-catenin serine 552 phosphorylation. Neither glycogen synthase kinase 3ß nor proteasomal inhibition alleviated GBP-1-mediated suppression of cell proliferation or ß-catenin/TCF signaling, indicating a non-canonical mechanism of ß-catenin inhibition. Together, these data show that cytokine-induced GBP-1 retards cell proliferation by forming a negative feedback loop that suppresses ß-catenin/TCF signaling.

Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

Activation of the sonic hedgehog (SHh) pathway is required for the growth of numerous tissues and organs and recent evidence indicates that this pathway is often recruited to stimulate growth of cancer stem cells (CSCs) and to orchestrate the reprogramming of cancer cells via epithelial mesenchymal transition (EMT). The objectives of this study were to examine the molecular mechanisms by which (-)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, inhibits self-renewal capacity of pancreatic CSCs and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that EGCG inhibited the expression of pluripotency maintaining transcription factors (Nanog, c-Myc and Oct-4) and self-renewal capacity of pancreatic CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of EGCG on self-renewal capacity of CSCs. EGCG inhibited cell proliferation and induced apoptosis by inhibiting the expression of Bcl-2 and XIAP and activating caspase-3. Interestingly, EGCG also inhibited the components of SHh pathway (smoothened, patched, Gli1 and Gli2) and Gli transcriptional activity. Furthermore, EGCG inhibited EMT by inhibiting the expression of Snail, Slug and ZEB1, and TCF/LEF transcriptional activity, which correlated with significantly reduced CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Furthermore, combination of quercetin with EGCG had synergistic inhibitory effects on self-renewal capacity of CSCs through attenuation of TCF/LEF and Gli activities. Since aberrant SHh signaling occurs in pancreatic tumorigenesis, therapeutics that target SHh pathway may improve the outcomes of patients with pancreatic cancer by targeting CSCs.

Inhibition of Wnt signaling by cucurbitacin B in breast cancer cells: reduction of Wnt-associated proteins and reduced translocation of galectin-3-mediated ß-catenin to the nucleus.

The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anti-cancer and anti-inflamatory activities. We investigated the anti-cancer activity of cucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina Linn. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that cucurbitacin B from T. cucumerina Linn. has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 with an IC50 of 4.60 and 88.75 µg/ml, respectively. Growth inhibition was attributed to G2/M phase arrest and apoptosis. Cyclin D1, c-Myc, and ß-catenin expression levels were reduced. Western blot analysis showed increased PARP cleavage and decreased Wnt-associated signaling molecules ß-catenin, galectin-3, cyclin D1 and c-Myc, and corresponding changes in phosphorylated GSK-3ß levels. Cucurbitacin B treatment inhibited translocation to the nucleus of ß-catenin and galectin-3. The depletion of ß-catenin and galectin-3 in the nucleus was confirmed by cellular protein fractionation. T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcriptional activity was disrupted in cucurbitacin B treated cells as tested by a TCF reporter assay. The relative luciferase activity was reduced when we treated cells with cucurbitacin B compound for 24 h. Our data suggest that cucurbitacin B may in part induce apoptosis and exert growth inhibitory effect via interruption the Wnt signaling.

ß-Catenin signaling increases during melanoma progression and promotes tumor cell survival and chemoresistance.

Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy.

Regulation of TCF3 by Wnt-dependent phosphorylation during vertebrate axis specification.

A commonly accepted model of Wnt/ß-catenin signaling involves target gene activation by a complex of ß-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves ß-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative in vivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.

PKG inhibits TCF signaling in colon cancer cells by blocking beta-catenin expression and activating FOXO4.

Activation of cGMP-dependent protein kinase (PKG) has anti-tumor effects in colon cancer cells but the mechanisms are not fully understood. This study has examined the regulation of beta-catenin/TCF signaling, as this pathway has been highlighted as central to the anti-tumor effects of PKG. We show that PKG activation in SW620 cells results in reduced beta-catenin expression and a dramatic inhibition of TCF-dependent transcription. PKG did not affect protein stability, nor did it increase phosphorylation of the amino-terminal Ser33/37/Thr41 residues that are known to target beta-catenin for degradation. However, we found that PKG potently inhibited transcription from a luciferase reporter driven by the human CTNNB1 promoter, and this corresponded to reduced beta-catenin mRNA levels. Although PKG was able to inhibit transcription from both the CTNNB1 and TCF reporters, the effect on protein levels was less consistent. Ectopic PKG had a marginal effect on beta-catenin protein levels in SW480 and HCT116 but was able to inhibit TCF-reporter activity by over 80%. Investigation of alternative mechanisms revealed that cJun-N-terminal kinase (JNK) activation was required for the PKG-dependent regulation of TCF activity. PKG activation caused beta-catenin to bind to FOXO4 in colon cancer cells, and this required JNK. Activation of PKG was also found to increase the nuclear content of FOXO4 and increase the expression of the FOXO target genes MnSOD and catalase. FOXO4 activation was required for the inhibition of TCF activity as FOXO4-specific short-interfering RNA completely blocked the inhibitory effect of PKG. These data illustrate a dual-inhibitory effect of PKG on TCF activity in colon cancer cells that involves reduced expression of beta-catenin at the transcriptional level, and also beta-catenin sequestration by FOXO4 activation.

Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis.

E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect.