Loss Of Β-catenin Research Articles

Loss of β-catenin promotes chondrogenic differentiation of aortic valve interstitial cells.

The Wnt/β-catenin signaling pathway has been implicated in human heart valve disease and is required for early heart valve formation in mouse and zebrafish. However, the specific functions of Wnt/β-catenin signaling activity in heart valve maturation and maintenance in adults have not been determined previously. Here, we show that Wnt/β-catenin signaling inhibits Sox9 nuclear localization and proteoglycan expression in cultured chicken embryo aortic valves. Loss of β-catenin in vivo in mice, using Periostin(Postn)Cre-mediated tissue-restricted loss of β-catenin (Ctnnb1) in valvular interstitial cells, leads to the formation of aberrant chondrogenic nodules and induction of chondrogenic gene expression in adult aortic valves. These nodular cells strongly express nuclear Sox9 and Sox9 downstream chondrogenic extracellular matrix genes, including Aggrecan, Col2a1, and Col10a1. Excessive chondrogenic proteoglycan accumulation and disruption of stratified extracellular matrix maintenance in the aortic valve leaflets are characteristics of myxomatous valve disease. Both in vitro and in vivo data demonstrate that the loss of Wnt/β-catenin signaling leads to increased nuclear expression of Sox9 concomitant with induced expression of chondrogenic extracellular matrix proteins. β-Catenin limits Sox9 nuclear localization and inhibits chondrogenic differentiation during valve development and in adult aortic valve homeostasis.

Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis.

Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

PARP-1 regulates epithelial-mesenchymal transition (EMT) in prostate tumorigenesis

Poly (ADP-ribose) polymerase (PARP) is involved in key cellular processes such as DNA replication and repair, gene transcription, cell proliferation and apoptosis. The role of PARP-1 in prostate cancer development and progression is not fully understood. The present study investigated the function of PARP-1 in prostate growth and tumorigenesis in vivo. Functional inactivation of PARP-1 by gene-targeted deletion led to a significant reduction in the prostate gland size in young PARP-1-/- mice (6 weeks) compared with wild-type (WT) littermates. To determine the effect of PARP-1 functional loss on prostate cancer onset, PARP-1-/- mice were crossed with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Pathological assessment of prostate tumors revealed that TRAMP+/-, PARP-1-/- mice exhibited higher grade prostate tumors compared with TRAMP+/- PARP-1+/+ (16-28 weeks) that was associated with a significantly increased proliferative index and decreased apoptosis among the epithelial cells in TRAMP+/- PARP-1-/- prostate tumors. Furthermore tumors harboring PARP-1 loss, exhibited a downregulation of nuclear androgen receptor. Impairing PARP-1 led to increased levels of transforming growth factor-β (TGF-β) and Smads that correlated with induction of epithelial-mesenchymal transition (EMT), as established by loss of E-cadherin and β-catenin and upregulation of N-cadherin and ZEB-1. Our findings suggest that impaired PARP-1 function promotes prostate tumorigenesis in vivo via TGF-β-induced EMT. Defining the EMT control by PARP-1 during prostate cancer progression is of translational significance for optimizing PARP-1 therapeutic targeting and predicting response in metastatic castration-resistant prostate cancer.

Cooperation of structurally different aryl hydrocarbon receptor agonists and β-catenin in the regulation of CYP1A expression

The ligand-activated nuclear receptor AhR (aryl hydrocarbon receptor) mediates the response of hepatocytes to various exogenous compounds. AhR is classically activated by planar, aromatic hydrocarbons, but also by other, structurally rather unrelated compounds. Recent data show that the canonical Wnt/β-catenin signaling pathway is also involved in the regulation of hepatic zonal gene expression and drug metabolism in mammalian liver. Previous studies indicate that the loss of β-catenin in hepatocytes diminishes the response to the AhR agonists 3-methylcholanthrene (3MC) in vivo and to 2,3,7,8-tetrachlorodibenzo-[p]-dioxin in vitro. The knockout of β-catenin also impairs the zonal pattern of AhR target gene induction by 3MC. However, it is presently unknown whether the chemical nature of the AhR agonist influences the AhR/β-catenin interaction. Moreover, no information is available about the dose-response curves of AhR activation in the absence or presence of Wnt/β-catenin signaling. In the present study, we have analyzed AhR-dependent responses to different concentrations of structurally unrelated AhR agonists in vivo and in vitro. The results demonstrate that β-catenin is essential to obtain the maximum AhR response. Moreover, using transgenic mouse models which allow for the ablation of β-catenin at different age of mice, we demonstrate that the presence of β-catenin, not postnatal developmental effects in β-catenin-deficient livers, is responsible for the observed interplay of β-catenin and the AhR.

Signet ring cell carcinoma of the ampulla of Vater: Immunophenotype and differentiation

Signet ring cell carcinoma (SRC) of the ampulla of Vater is extremely rare and the histogenesis remains unknown. In the present study, to investigate the immunohistochemical phenotypes, discuss the histological origin and evaluate the correlation between the immunohistochemical phenotypes and survival of ampullary SRC patients, a retrospective review was conducted. This included all ampullary carcinoma patients treated at The First Affiliated Hospital, College of Medicine, Zhejiang University, and was performed over a five-year period between 2008 and 2012. Eight resected ampullary SRC specimens were examined histopathologically and immunohistochemically, using cytokeratin (CK) and mucin (MUC) immunohistochemical phenotypes. Of all 162 patients with ampullary lesions, eight cases (4.9%) of ampullary SRC were identified. Immunohistochemical analyses of the eight cases revealed the positive expression of CK7 in five, CK19 in seven, CK20 in one, MUC1 in five, MUC2 in three, caudal-related homeobox transcription factor 2 in one, MUC5AC in seven and MUC6 in four of the eight cases, while loss of E-cadherin and β-catenin was observed in four of the eight cases. According to immunohistochemical classification, ampullary SRC can be classified into four subtypes: Intestinal (I), pancreatobiliary (PB), gastric and mixed types (composed of I mucosa lining and PB epithelium). Patients with the I-type ampullary SRC demonstrated a more favorable prognosis than that of patients with the PB-type ampullary SRC. Additionally, patients with ampullary SRC of I or PB type with gastric differentiation may have a worse prognosis than others. The coexpression of the E-cadherin/β-catenin complex may also indicate poor prognosis in PB-type ampullary SRC. In conclusion, the clinical five-year follow-up of the patients with pure SRC was more positive than that of those with I-, PB-, gastric- or mixed-type ampullary SRC. The coexpression of the E-cadherin/β-catenin complex may present a poor prognosis in the PB type of ampullary SRC.

β-Catenin inactivation is a pre-requisite for chick retina regeneration.

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.

Curcumin suppresses cell proliferation through inhibition of the Wnt/β-catenin signaling pathway in medulloblastoma.

Recently, the survival rate of medulloblastoma patients has greatly improved; yet, patients undergoing current treatment regimes suffer from serious therapy-related side-effects. The aim of the present study was to investigate the anticancer effects of curcumin on medulloblastoma cells by testing its capacity to suppress proliferation and regulate the Wnt/β-catenin pathway. In the present study, cell proliferation was determined by MTT assay. Cell cycle was observed by flow cytometry. The changes in the Wnt/β-catenin pathway were analyzed by immunofluorescence, western blot analysis and RT-PCR. Curcumin treatment resulted in a dose- and time-dependent inhibition of proliferation in the medulloblastoma cell line. Curcumin treatment arrested the cell-cycle at the G2/M phase. Furthermore, curcumin treatment led to activation of GSK-3β, reduced expression of β-catenin and its downstream target cyclin D1. The attenuation of the Wnt/β‑catenin pathway was due to the loss of nuclear β-catenin. In conclusion, curcumin can inhibit cell growth by suppressing the Wnt/β-catenin signaling pathway, and it has the potential to be developed as a therapeutic agent for medulloblastoma.

Prognostic Significance of β-Catenin, E-Cadherin, and SOX9 in Colorectal Cancer: Results from a Large Population-Representative Series.

Robust biomarkers that can precisely stratify patients according to treatment needs are in great demand. The literature is inconclusive for most reported prognostic markers for colorectal cancer (CRC). Hence, adequately reported studies in large representative series are necessary to determine their clinical potential. We investigated the prognostic value of three Wnt signaling-associated proteins, β-catenin, E-cadherin, and SOX9, in a population-representative single-hospital series of 1290 Norwegian CRC patients by performing immunohistochemical analyses of each marker using the tissue microarray technology. Loss of membranous or cytosolic β-catenin and loss of cytosolic E-cadherin protein expression were significantly associated with reduced 5-year survival in 903 patients who underwent major resection (722 evaluable tissue cores) independently of standard clinicopathological high-risk parameters. Pre-specified subgroup analyses demonstrated particular effect for stage IV patients for β-catenin membrane staining (P = 0.018; formal interaction test P = 0.025). Among those who underwent complete resection (714 patients, 568 evaluable), 5-year time-to-recurrence analyses were performed, and stage II patients with loss of cytosolic E-cadherin were identified as an independent high-risk subgroup (P = 0.020, formal interaction test was not significant). Nuclear β-catenin and SOX9 protein, regardless of intracellular location, were not associated with prognosis. In conclusion, the protein expression level of membranous or cytosolic β-catenin and E-cadherin predicts CRC patient subgroups with inferior prognosis.

Loss of β-catenin triggers oxidative stress and impairs hematopoietic regeneration

Accidental or deliberate ionizing radiation exposure can be fatal due to widespread hematopoietic destruction. However, little is known about either the course of injury or the molecular pathways that regulate the subsequent regenerative response. Here we show that the Wnt signaling pathway is critically important for regeneration after radiation-induced injury. Using Wnt reporter mice, we show that radiation triggers activation of Wnt signaling in hematopoietic stem and progenitor cells. β-Catenin-deficient mice, which lack the ability to activate canonical Wnt signaling, exhibited impaired hematopoietic stem cell regeneration and bone marrow recovery after radiation. We found that, as part of the mechanism, hematopoietic stem cells lacking β-catenin fail to suppress the generation of reactive oxygen species and cannot resolve DNA double-strand breaks after radiation. Consistent with the impaired response to radiation, β-catenin-deficient mice are also unable to recover effectively after chemotherapy. Collectively, these data indicate that regenerative responses to distinct hematopoietic injuries share a genetic dependence on β-catenin and raise the possibility that modulation of Wnt signaling may be a path to improving bone marrow recovery after damage.

Distinct requirements for beta-catenin in pancreatic epithelial growth and patterning

Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate β-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component β-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of β-catenin function to β-cell development remains controversial. To resolve the potential roles of β-catenin in development of MPCs and β-cells, we generated pancreas- and pre-endocrine-specific β-catenin knockout mice. Pancreas-specific loss of β-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in β-cell mass. The loss of β-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for β-catenin in maintaining proximal–distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent “trunk” fate when β-catenin is deleted. Moreover, β-catenin maintains proximal–distal patterning, in part, by inhibiting Notch signaling. Subsequently, β-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for β-catenin along the route of β-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.

Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (ShhCreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The ShhCreERT2/+; β-cateninflox(ex3)/+; BmprIAflox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.

High concordance of molecular tumor alterations between pre-operative curettage and hysterectomy specimens in patients with endometrial carcinoma

ObjectiveMolecular alterations in endometrial cancer have been shown to be prognostically significant but have not yet been implemented in the current clinical risk assessment. Few studies have investigated the reliability of molecular alterations in pre-operative specimens. Therefore, the objective was to determine whether molecular analysis of pre-operative endometrial cancer samples accurately reflects those alterations in the subsequent hysterectomy specimens. MethodsPaired pre-operative and hysterectomy specimens of 48 patients diagnosed with endometrial carcinoma, 42 endometrioid (EEC) and 6 non-endometrioid (NEEC) carcinomas, were analyzed for immunohistochemical expression of p53, PTEN and β-catenin. Tumor DNA was isolated and analyzed for microsatellite instability (MSI), TP53 mutations and somatic hot spot mutations in 13 genes. ResultsIn EEC patients, loss of PTEN, nuclear β-catenin and p53-mutant expression was found in 43%, 7% and 12%, respectively. No nuclear β-catenin was found in 5 of 6 NEEC patients, all serous cancers, whereas a p53-mutant expression was present in all serous cases. MSI was found in 19.5%, all EEC. Concordance for PTEN, β-catenin, p53 expression and MSI status was found in 79%, 92%, 79% and 93.5%, respectively. We detected 65 hot spot mutations in 39/48 (81%) tumors. Overall concordance of the GynCarta multigene analysis was 99.8%. ConclusionsThe results confirm the reliability of immunohistochemical and DNA-based techniques in the evaluation of molecular alterations in pre-operative endometrial specimens and high concordance rates with the definitive hysterectomy specimens. The resulting molecular signature provides initial pre-operative diagnostic information on the status of oncogenic pathways, which may contribute to individualized treatment strategies.

WNT-pathway components as predictive markers useful for diagnosis, prevention and therapy in inflammatory bowel disease and sporadic colorectal cancer

The key role of the Wnt/β-catenin signaling in colorectal cancer (CRC) insurgence and progression is now recognized and several therapeutic strategies targeting this pathway are currently in developing. Wnt/β-catenin signaling not only dominates the early stages of sporadic colorectal cancer (SCC), but could also represent the connection between inflammatory bowel diseases (IBD) and increased risk of developing SCC. The knowledge on the sequential molecular events of Wnt-signaling cascade in IBD and during colorectal carcinogenesis, might provide new diagnostic/prognostic markers and could be helpful for optimizing the treatment protocols, thus improving the efficacy of Wnt-targeting therapies. We performed a comparative evaluation of the expression of some crucial molecules participating to Wnt signaling in an animal model of chemically-induced CRC and in human tissues obtained from patients suffering from IBD or at sequential stages of SCC. Specifically, we analyzed upstream events of Wnt signaling including β-catenin nuclear translocation and loss of E-cadherin and APC functions, and downstream events including c-Myc and Cyclin-D1 expression. We demonstrated that these crucial components of the Wnt/β-catenin pathway, when evaluated by immunohistochemistry using a multiparametric approach that includes the analyses of both expression and localization, could be potent markers for diagnosis, prevention and therapy in IBD and SCC, also possessing a predictive value for responsiveness to Wnt-targeting therapies. Furthermore, we showed that the animal model of chemically-induced CRC mimics the molecular events of Wnt signaling during IBD and SCC development in humans and may therefore be suitable for testing chemopreventive or therapeutic drugs targeting this pathway.

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the β-catenin pathway to skeletal development. Inactivation of β-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of β-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear β-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1−/− embryos. Inactivating β-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→β-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating β-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-β-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation, Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.

The Role for Dickkopf-Homolog-1 in the Pathogenesis of Crohn’s Disease-Associated Fistulae

BackgroundOne of the most challenging conditions in Crohn’s disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae.MethodsDkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-β or IL-13. Knock-down of DKK-1 or β-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR.ResultsDkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-β induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-β-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of β-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-β. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF.DiscussionWe demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.

Differential Expression of E-Cadherin, β-Catenin, and S100A4 in Intestinal Type and Nonintestinal Type Ampulla of Vater Cancers

Background/AimsEpithelial-mesenchymal transition (EMT)-related proteins may exhibit differential expression in intestinal type or pancreatobiliary type ampulla of Vater carcinomas (AVCs). We evaluated the expression of E-cadherin, β-catenin, and S100A4 in intestinal and nonintestinal type AVCs and analyzed their relationships with clinicopathological variables and survival.MethodsA clinicopathological review of 105 patients with AVCs and immunohistochemical staining for E-cadherin, β-catenin, and S100A4 were performed. The association between clinicopathological parameters, histological type, and expression of EMT proteins and their effects on survival were analyzed.ResultsSixty-five intestinal type, 35 pancreatobiliary type, and five other types of AVCs were identified. The severity of EMT changes differed between the AVC types; membranous loss of E-cadherin and β-catenin was observed in nonintestinal type tumors, whereas aberrant nonmembranous β-catenin expression was observed in intestinal type tumors. EMT-related changes were more pronounced in the invasive tumor margin than in the tumor center, and these EMT-related changes were related to tumor aggressiveness. Among the clinicopathological parameters, a desmoplastic reaction was related to overall survival, and the reaction was more severe in nonintestinal type than in intestinal type AVCs.ConclusionsDysregulation of E-cadherin, β-cadherin, and S100A4 expression may play a role in the carcinogenesis and tumor progression of AVCs.

Abstract C12: Beta-catenin enhances synovial sarcomagenesis and provides a therapeutic target.

Abstract β-catenin is a master regulator in the cellular biology of development and neoplasia. Its dysregulation is implicated as a driver of colorectal carcinogenesis and the epithelial-mesenchymal transition in other cancers. Nuclear β-catenin is known to be a poor prognostic sign in synovial sarcoma, the most common soft-tissue sarcoma in adolescents and young adults. Beyond this prognostic correlation, the biology of β-catenin's role in synovial sarcoma has not been explored. We show through genetic experiments in mice demonstrate that loss of β-catenin hampers SS18-SSX2-driven synovial sarcomagenesis. Expression of a stabilized form of β-catenin greatly enhances synovial sarcomagenesis. Stabilization of β-catenin specifically enables transformation under the influence of the SS18-SSX2 fusion oncogene by blocking differentiation and apoptosis. As part of the mechanism of this impact, β-catenin recruits BRG1-containing SWI/SNF chromatin remodeling complexes to activated target genes. Compounds disrupting the transcriptional activity of β-catenin are toxic to human and mouse synovial sarcoma cell lines. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C12. Citation Format: Kevin B. Jones, Benjamin Illum, Huifeng Jin, Julie Zhu Mu, Michael Monument, Allie Grossmann, Mario R. Capecchi. Beta-catenin enhances synovial sarcomagenesis and provides a therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C12.

Abstract A30: Epigenetic regulation of Wnt/β-catenin signaling in vivo by PRC2 protein EZH2

Abstract Introduction: Colon cancer results from the accumulation of both acquired genetic and epigenetic changes that transform normal glandular epithelium into invasive adenocarcinomas. The Enhancer of Zeste Homolog 2 (EZH2) protein is a core component of the Polycomb Repressive Complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). EZH2 is overexpressed in several malignancies including colon cancer; EZH2's role in tumor initiation however, is less clear. Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) that utilizes a type-three secretion system (T3SS) to activate Wnt/β-catenin signaling, represents the earliest molecular and functional changes associated with colon carcinogenesis; the increases in β-catenin recorded during TMCH however, are neither due to mutations in Adenomatous polyposis coli (Apc) or β-catenin (CTNNB1) gene. Aim: To investigate if EZH2 is critical in epigenetically regulating Wnt/β-catenin signaling in response to bacterial infection. Experimental Procedures: TMCH or tumorigenesis were induced by CR infection (108cfu) in wild type and ApcMin/+ mice, respectively. AOM (@10mg/Kg body wt.)/DSS (2.5%; 3 cycles) model of colitis-associated cancer and de-identified human adenoma and adenocarcinoma samples were used to further examine evidence of epigenetically-regulated Wnt/β-catenin signaling. Results: Following CR infection, EZH2 protein increased significantly in the crypt at day 6 and particularly at day 12 (peak hyperplasia) and coincided with expression of EZH2 target protein H3K27me3. The changes accrued were specific to CR as T3SS mutant escV failed to elicit alterations in either EZH2, H3K27me3 or crypt hyperplasia. Mechanistically, siRNA-mediated knockdown of EZH2 led to: i) attenuated cell migration, ii) reduced colonosphere formation and, iii) decreased β-catenin/TCF4-dependent TOPflash reporter activity due to loss of β-catenin. Chromatin immunoprecipitation revealed EZH2's occupancy on WIF1 (Wnt Inhibitory Factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown either via siRNA or EZH2-inhibitor DZNep (5μM), relative levels of H3K27me3 decreased which led to the transcriptional upregulation of WIF1 as was evidenced by WIF1 promoter activity. Besides EZH2, we also discovered increases in miR-203 expression in the crypts at days-6 and 12 post-infection that correlated with reduced levels of its target WIF1, at these time points; overexpression of miR-203 in Young Adult Mouse Colon primary cells resulted in decreased WIF1 transcription. Tumors in the distal colons of ApcMin/+ mice following CR infection expressed high levels of EZH2 and β-catenin and a concomitant decrease in WIF1. In response to AOM/DSS, colonic tumors at 24 weeks expressed high levels of EZH2 and β-catenin and a concomitant decrease in WIF1. Pharmacological intervention with honokiol (HNK; @5mg/Kg body wt. p.o.) a week prior to and during AOM/DSS-induced colon carcinogenesis reduced both tumor burden and tumor size. Immunohistochemical analysis of HNK-treated tissues revealed significant reduction in EZH2 and β-catenin staining while WIF1 expression was dramatically upregulated. In human adenomas/adenocarcinomas, simultaneous increases in EZH2 and β-catenin staining coincided with a corresponding decrease in WIF1 positivity. Conclusions: 1. CR infection induces EZH2 overexpression that correlates with aberrant Wnt/β-catenin signaling in the colonic crypts.2. EZH2 downregulates WIF1 expression which may correlate with earliest stages of colon tumorigenesis. 3. Honokiol-induced upregulation of WIF1 to block aberrant Wnt/β-catenin signaling may be a novel strategy to target epigenetic abnormalities for colon cancer prevention and therapeutics. Citation Format: Badal C. Roy, Dharmalingam Subramaniam, Ishfaq Ahmed, Shrikant Anant, Shahid Umar. Epigenetic regulation of Wnt/β-catenin signaling in vivo by PRC2 protein EZH2. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A30.

Targeting the Wnt Pathway in Synovial Sarcoma Models

Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In an SYT-SSX2 transgenic model, we show that a constitutive Wnt/β-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of β-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/β-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/β-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents. Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT–SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans.