Multiple genes of the renin-angiotensin system are novel targets of Wnt/?-catenin signaling.
ABSTRACT: Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and ?-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either ?-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule ?-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/?-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms' tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/?-catenin. Our results indicate that blockade of Wnt/?-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury.
Project description:Activation of the renin-angiotensin system (RAS) plays a pivotal role in mediating hypertension, chronic kidney and cardiovascular diseases. As Wnt/?-catenin regulates multiple RAS genes, we speculated that this developmental signaling pathway might also participate in blood pressure (BP) regulation. To test this, we utilized two rat models of experimental hypertension: chronic angiotensin II infusion and remnant kidney after 5/6 nephrectomy. Inhibition of Wnt/?-catenin by ICG-001 blunted angiotensin II-induced hypertension. Interestingly, angiotensin II was able to induce the expression of multiple Wnt genes in vivo and in vitro, thereby creating a vicious cycle between Wnt/?-catenin and RAS activation. In the remnant kidney model, renal ?-catenin was upregulated, and delayed administration of ICG-001 also blunted BP elevation and abolished the induction of angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II type 1 receptor. ICG-001 also reduced albuminuria, serum creatinine and blood urea nitrogen, and inhibited renal expression of fibronectin, collagen I and plasminogen activator inhibitor-1, and suppressed the infiltration of CD3+ T cells and CD68+ monocytes/macrophages. In vitro, incubation with losartan prevented Wnt/?-catenin-mediated fibronectin, ?-smooth muscle actin and Snail1 expression, suggesting that the fibrogenic action of Wnt/?-catenin is dependent on RAS activation. Taken together, these results suggest an intrinsic linkage of Wnt/?-catenin signaling with BP regulation. Our studies also demonstrate that hyperactive Wnt/?-catenin can drive hypertension and kidney damage via RAS activation.
Project description:Activation of the renin-angiotensin system (RAS) is associated with hypertension and heart disease. However, how RAS activation causes cardiac lesions remains elusive. Here we report the involvement of Wnt/?-catenin signaling in this process. In rats with chronic infusion of angiotensin II (Ang II), eight Wnt ligands were induced and ?-catenin activated in both cardiomyocytes and cardiac fibroblasts. Blockade of Wnt/?-catenin signaling by small molecule inhibitor ICG-001 restrained Ang II-induced cardiac hypertrophy by normalizing heart size and inhibiting hypertrophic marker genes. ICG-001 also attenuated myocardial fibrosis and inhibited ?-smooth muscle actin, fibronectin and collagen I expression. These changes were accompanied by a reduced expression of atrial natriuretic peptide and B-type natriuretic peptide. Interestingly, ICG-001 also lowered blood pressure induced by Ang II. In vitro, Ang II induced multiple Wnt ligands and activated ?-catenin in rat primary cardiomyocytes and fibroblasts. ICG-001 inhibited myocyte hypertrophy and Snail1, c-Myc and atrial natriuretic peptide expression, and abolished the fibrogenic effect of Ang II in cardiac fibroblasts. Finally, recombinant Wnt3a was sufficient to induce cardiomyocyte injury and fibroblast activation in vitro. Taken together, these results illustrate an essential role for Wnt/?-catenin in mediating hypertension, cardiac hypertrophy and myocardial fibrosis. Therefore, blockade of this pathway may be a novel strategy for ameliorating hypertensive heart disease.
Project description:Pediatric high-grade gliomas (pedHGG) belong to the most aggressive cancers in children with a poor prognosis due to a lack of efficient therapeutic strategies. The ?-catenin/Wnt-signaling pathway was shown to hold promising potential as a treatment target in adult high-grade gliomas by abrogating tumor cell invasion and the acquisition of stem cell-like characteristics. Since pedHGG differ from their adult counterparts in genetically and biologically we aimed to investigate the effects of ?-catenin/Wnt-signaling pathway-inhibition by the ?-catenin/CBP antagonist ICG-001 in pedHGG cell lines. In contrast to adult HGG, pedHGG cells displayed minimal detectable canonical Wnt-signaling activity. Nevertheless, low doses of ICG-001 inhibited cell migration/invasion, tumorsphere- and colony formation, proliferation in vitro as well as tumor growth in vivo/ovo, suggesting that ICG-001 affects pedHGG tumor cell characteristics independent of ?-catenin/Wnt-signaling. RNA-sequencing analyses support a Wnt/?-catenin-independent effect of ICG-001 on target gene transcription, revealing strong effects on genes involved in cellular metabolic/biosynthetic processes and cell cycle progression. Among these, high mRNA expression of cell cycle regulator JDP2 was found to confer a better prognosis for pedHGG patients. In conclusion, ICG-001 might offer an effective treatment option for pedHGG patients functioning to regulate cell phenotype and gene expression programs in absence of Wnt/?-catenin signaling-activity.
Project description:BACKGROUND:Although canonical Wnt signaling is known to promote tumorigenesis in pancreatic ductal adenocarcinoma (PDAC), a cancer driven principally by mutant K-Ras, the detailed molecular mechanisms by which the Wnt effector ?-catenin regulates such tumorigenesis are largely unknown. We have previously demonstrated that ?-catenin's differential usage of the Kat3 transcriptional coactivator cyclic AMP-response element binding protein-binding protein (CBP) over its highly homologous coactivator p300 increases self-renewal and suppresses differentiation in other types of cancer. AIM/METHODS:To investigate Wnt-mediated carcinogenesis in PDAC, we have used the specific small molecule CBP/?-catenin antagonist, ICG-001, which our lab identified and has extensively characterized, to examine its effects in human pancreatic cancer cells and in both an orthotopic mouse model and a human patient-derived xenograft (PDX) model of PDAC. RESULTS/CONCLUSION:We report for the first time that K-Ras activation increases the CBP/?-catenin interaction in pancreatic cancer; and that ICG-001 specific antagonism of the CBP/?-catenin interaction sensitizes pancreatic cancer cells and tumors to gemcitabine treatment. These effects were associated with increases in the expression of let-7a microRNA; suppression of K-Ras and survivin; and the elimination of drug-resistant cancer stem/tumor-initiating cells.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer due in part to a lack of highly robust cytotoxic or molecular-based therapies. Recent studies investigating ligand-mediated Wnt/?-catenin signaling have highlighted its importance in pancreatic cancer initiation and progression, as well as its potential as a therapeutic target in PDAC. The small-molecule ICG-001 binds cAMP-responsive element binding (CREB)-binding protein (CBP) to disrupt its interaction with ?-catenin and inhibit CBP function as a coactivator of Wnt/?-catenin-mediated transcription. Given its ability to inhibit Wnt/?-catenin-mediated transcription in vitro and in vivo, as well as its efficacy in preclinical models of colorectal cancer and other Wnt-driven diseases, we examined ICG-001 and its potential role as a therapeutic in PDAC. ICG-001 alone significantly inhibited anchorage-dependent and -independent growth of multiple PDAC lines, and augmented in vitro growth inhibition when used in combination with gemcitabine. ICG-001 had only variable modest effects on PDAC apoptosis and instead mediated PDAC growth inhibition primarily through robust induction of G? cell-cycle arrest. These effects, however, seemed decoupled from its inhibition of Wnt/?-catenin-mediated transcription. DNA microarrays performed on PDAC cells in the context of ICG-001 treatment revealed ICG-001 altered the expression of several genes with well-established roles in DNA replication and cell-cycle progression, including direct actions on SKP2 and CDKN1A. ICG-001 also significantly prolonged survival in an in vivo orthotopic xenograft model of PDAC, indicating ICG-001 or derived compounds that disrupt CBP activity are potentially useful small-molecule therapeutics for pancreatic cancer.
Project description:In pediatric glioma cell lines, treatment with ICG-001 had no inhibitory effect on canonical Wnt-target genes but induced significant up regulation of various known β-catenin target genes in both cell lines and top 20 GO-annotations of down-regulated genes by ICG-001 were associated with biosynthetic and metabolic processes and cell cycle division processes. Pediatric cell lines were treated with ICG-001 or DMSO for 48h
Project description:Uremic vascular calcification is a regulated cell-mediated process wherein cells in the arterial wall transdifferentiate to actively calcifying cells resulting in a process resembling bone formation. Wnt signalling is established as a major driver for vessel formation and maturation and for embryonic bone formation, and disturbed Wnt signalling might play a role in vascular calcification. ICG-001 is a small molecule Wnt inhibitor that specifically targets the coactivator CREB binding protein (CBP)/?-catenin-mediated signalling. In the present investigation we examined the effect of ICG-001 on vascular calcification in uremic rats. Uremic vascular calcification was induced in adult male rats by 5/6-nephrectomy, high phosphate diet and alfacalcidol. The presence of uremic vascular calcification in the aorta was associated with induction of gene expression of the Wnt target gene and marker of proliferation, cyclinD1; the mediator of canonical Wnt signalling, ?-catenin and the matricellular proteins, fibronectin and periostin. Furthermore, genes from fibrosis-related pathways, TGF-? and activin A, as well as factors related to epithelial-mesenchymal transition, snail1 and vimentin were induced. ICG-001 treatment had significant effects on gene expression in kidney and aorta from healthy rats. These effects were however limited in uremic rats, and treatment with ICG-001 did not reduce the Ca-content of the uremic vasculature.
Project description:Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/?-catenin-mediated transcriptional program.We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model.ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice.Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.
Project description:Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1-110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/?- and ?-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.
Project description:Interactions between transforming growth factor-? (TGF-?) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated ?-catenin-dependent and transforming growth factor-?1 (TGF-?1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the ?-catenin/CBP (but not ?-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-?1-mediated ?-smooth muscle actin (?-SMA) and collagen induction in AEC. We now demonstrate that TGF-?1 induces LEF/TCF TOPFLASH reporter activation and nuclear ?-catenin accumulation, while LiCl augments TGF-?-induced ?-SMA expression, further confirming co-operation between ?-catenin- and TGF-?-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of ?-catenin and overexpression of ICAT abrogated effects of TGF-?1 on ?-SMA transcription/expression, indicating a requirement for ?-catenin in these Smad3-dependent effects. Following TGF-? treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and ?-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of ?-SMA via complex formation among Smad3, ?-catenin, and CBP. ICG-001 inhibited ?-SMA expression/transcription in response to TGF-? as well as ?-SMA promoter occupancy by ?-catenin and CBP, demonstrating a previously unknown requisite TGF-?1/?-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by ?-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-?.