Apical-basal polarity and epithelial integrity are maintained in part by the Crumbs (CRB) complex. The C--terminal subunit of MUC1 (MUC1-C) is a transmembrane protein that is expressed at the apical border of normal epithelial cells and aberrantly at high levels over the entire surface of their transformed counterparts. However, it is not known whether MUC1-C contributes to this loss of polarity that is characteristic of carcinoma cells. Here it is demonstrated that MUC1-C downregulates expression of the Crumbs complex CRB3 protein in triple-negative breast cancer (TNBC) cells. MUC1-C associates with ZEB1 on the CRB3 promoter and represses CRB3 transcription. Notably, CRB3 activates the core kinase cassette of the Hippo pathway, which includes LATS1 and LATS2. In this context, targeting MUC1-C was associated with increased phosphorylation of LATS1, consistent with activation of the Hippo pathway, which is critical for regulating cell contact, tissue repair, proliferation, and apoptosis. Also shown is that MUC1-C--mediated suppression of CRB3 and the Hippo pathway is associated with dephosphorylation and activation of the oncogenic YAP protein. In turn, MUC1-C interacts with YAP, promotes formation of YAP/β-catenin complexes, and induces the WNT target gene MYC. These data support a previously unrecognized pathway in which targeting MUC1-C in TNBC cells (i) induces CRB3 expression, (ii) activates the CRB3-driven Hippo pathway, (iii) inactivates YAP, and thereby (iv) suppresses YAP/β-catenin-mediated induction of MYC expression.

These findings demonstrate a previously unrecognized role for the MUC1-C oncoprotein in the regulation of polarity and the Hippo pathway in breast cancer. Mol Cancer Res; 14(12); 1266-76. ©2016 AACR.

Pigment epithelium-derived factor (PEDF) is a secretory protein that inhibits multiple tumor types. PEDF inhibits the Wnt coreceptor, low-density lipoprotein receptor-related protein 6 (LRP6), in the eye, but whether the tumor-suppressive properties of PEDF occur in organs such as the liver is unknown.

Wnt-dependent regulation of PEDF was assessed in the absence and presence of the Wnt coreceptor LRP6. Whole genome expression analysis was performed on PEDF knockout (KO) and control livers (7 months). Interrogation of Wnt/β-catenin signaling was performed in whole livers and human hepatocellular carcinoma (HCC) cell lines after RNA interference of PEDF and restoration of a PEDF-derived peptide. Western diet feeding for 6 to 8 months was used to evaluate whether the absence of PEDF was permissive for HCC formation (n = 12/group).

PEDF levels increased in response to canonical WntWntWnt/β-catenin activation. Enhanced Wnt/β-catenin signaling occurred in KO livers, and PEDF delivery in vivo reduced LRP6 activation. In human HCC cells, RNA interference of PEDF led to increased levels of activated LRP6 and β-catenin, and a PEDF 34-mer peptide decreased LRP6 activation and β-catenin signaling, and reduced Wnt target genes. PEDF KO mice fed a Western diet developed sporadic well-differentiated HCC. Human HCC specimens demonstrated decreased PEDF staining compared with hepatocytes.

PEDF is an endogenous inhibitor of Wnt/β-catenin signaling in the liver.

The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of <em>12</em> glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of <em>Wnt</em> pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma.

OBJECTIVE

To identify molecular markers shared across South African esophageal squamous cell carcinoma (ESCC) cell lines using cytogenetics, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism (SNP) array copy number analysis.

METHODS

We used conventional cytogenetics, FISH, and multicolor FISH to characterize the chromosomal rearrangements of five ESCC cell lines established in South Africa. The whole genome copy number profile was established from 250K SNP arrays, and data was analyzed with the CNAT 4.0 and GISTIC software.

RESULTS

We detected common translocation breakpoints involving chromosomes 1p11-<em>12</em> and 3p11.2, the latter correlated with the deletion, or interruption of the EPHA3 gene. The most significant amplifications involved the following chromosomal regions and genes: 11q13.3 (CCND1, FGF3, FGF4, FGF19, MYEOV), 8q24.21(C-MYC, FAM84B), 11q22.1-q22.3 (BIRC2, BIRC3), 5p15.2 (CTNND2), 3q11.2-q<em>12</em>.2 (MINA) and 18p11.32 (TYMS, YES1). The significant deletions included 1p31.2-p31.1 (CTH, GADD45α, DIRAS3), 2q22.1 (LRP1B), 3p<em>12</em>.1-p14.2 (FHIT), 4q22.1-q32.1 (CASP6, SMAD1), 8p23.2-q11.1 (BNIP3L) and 18q21.1-q21.2 (SMAD4, DCC). The 3p11.2 translocation breakpoint was shared across four cell lines, supporting a role for genes involved at this site, in particular, the EPHA3 gene which has previously been reported to be deleted in ESCC.

CONCLUSIONS

The finding that a significant number of genes that were amplified (FGF3, FGF4, FGF19, CCND1 and C-MYC) or deleted (SFRP2 gene) are involved in the Wnt and fibroblast growth factor signaling pathways, suggests that these pathways may be activated in these cell lines.

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKVBR) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKVBR was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKVBR in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated <em>Wnt</em> signaling were more susceptible to the oncolytic effects of ZIKVBR Furthermore, modulation of <em>Wnt</em> signaling pathway significantly affected ZIKVBR-induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKVBR could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects.Significance: Brazilian Zika virus strain kills aggressive metastatic forms of human CNS tumors and could be a potential oncolytic agent for cancer therapy. Cancer Res; 78(<em>12</em>); 3363-74. ©2018 AACR.

Prior research has shown that in experimental diabetes mellitus, green tea reduces albuminuria by decreasing podocyte apoptosis through activation of the <em>WNT</em> pathway. We investigated the effect of green tea polyphenols (GTP) on residual albuminuria of diabetic subjects with nephropathy. We conducted a randomised, double-blind study in 42 diabetic subjects with a urinary albumin-creatinine ratio (UACR) >30 mg/g, despite administration of the maximum recommended dose of renin-angiotensin (RAS) inhibition. Patients were randomly assigned to two equal groups to receive either GTP (containing 800 mg of epigallocatechin gallate, 17 with type 2 diabetes and 4 with type 1 diabetes) or placebo (21 with type 2 diabetes) for <em>12</em> weeks. Treatment with GTP reduced UACR by 41%, while the placebo group saw a 2% increase in UACR (p = 0.019). Podocyte apoptosis (p = 0.001) and in vitro albumin permeability (p < 0.001) were higher in immortalized human podocytes exposed to plasma from diabetic subjects compared to podocytes treated with plasma from normal individuals. In conclusion, GTP administration reduces albuminuria in diabetic patients receiving the maximum recommended dose of RAS. Reduction in podocyte apoptosis by activation of the <em>WNT</em> pathway may have contributed to this effect.

Background: The incidence of complex atypical hyperplasia and early stage endometrioid endometrial cancer is increasing, in part due to the epidemic of obesity, a risk factor tightly linked to development of endometrial hyperplasia and cancer. The standard upfront treatment for complex atypical hyperplasia and early stage endometrial cancer is hysterectomy. However, non-surgical treatment of early endometrial neoplasia may be necessary due to medical co-morbidities precluding surgery or desired future fertility.

Objective: We sought to evaluate efficacy of the Levonorgestrel Intrauterine Device to treat complex atypical hyperplasia and grade 1 endometrioid endometrial carcinoma.

<strong class="sub-title"> Study design: </strong> A single-institution, single-arm, phase II study of the Levonorgestrel Intrauterine Device (52 mg levonorgestrel, Mirena®) was conducted in patients with complex atypical hyperplasia or grade 1 endometrioid endometrial cancer. The primary endpoint was pathologic response rate at <em>12</em> months, including complete or partial response. Quality of life and toxicity were assessed. Molecular analyses for proliferation markers, hormone-regulated genes, and <em>WNT</em> pathway activation were performed at baseline and 3 months.

<strong class="sub-title"> Results: </strong> Fifty-seven patients were treated (21 endometrial cancer, 36 complex atypical hyperplasia). Median age was 48.0 years, median body mass index was 45.5 kg/m². Of 47 evaluable patients, <em>12</em>-month response rate was 83% (90%CrI 72.7, 90.3); 37 complete responders (8 endometrial cancer; 29 complex atypical hyperplasia), 2 partial responders (2 endometrial cancer), 3 stable disease (2 endometrial cancer, 1 complex atypical hyperplasia), 5 progressive disease (3 endometrial cancer; 2 complex atypical hyperplasia). After stratification for histology, response rate was 90.6% for complex atypical hyperplasia and 66.7% for grade 1 endometrioid endometrial cancer. Four patients (9.5%) had relapse after initial response. Adverse events were mild, primarily irregular bleeding and cramping. Quality of life was not negatively impacted. At 3 months, exogenous progesterone effect was present in 96.9% of responders (31/32) versus 25% (2/8) of non-responders (p=0.001). Non-responders had higher baseline proliferation (Ki67) and lower DKK3 gene expression compared to responders (p=.023 and p=0.030). Non-responders had significantly different changes in sFRP1, FZD8, and RALDH2 compared to responders.

Conclusion: The Levonorgestrel Intrauterine Device has substantial activity in complex atypical hyperplasia and grade 1 endometrioid endometrial cancer, with a modest proportion demonstrating upfront progesterone resistance. Potential biomarkers were identified that may correlate with resistance to therapy, further exploration is warranted.

Keywords: Complex Atypical Hyperplasia; Conservative; Endometrial Cancer; Endometrial Hyperplasia Intrauterine Device; Predictive Biomarkers; Progesterone.

BACKGROUND

Breast cancer metastasis is a highly prevalent cause of death for European females. DNA microarray analysis has established that primary tumors, which remain localized, differ in gene expression from those that metastasize. Crossanalysis of these studies allow to revile the differences that may be used as predictive in the disease prognosis and therapy.

OBJECTIVE

The aim of the project was to validate suggested prognostic and therapeutic markers using meta-analysis of data on gene expression in metastatic and primary breast cancer tumors.

METHODS

Data on relative gene expression values from <em>12</em> studies on primary breast cancer and breast cancer metastasis were retrieved from Genevestigator (Nebion) database. The results of the data meta-analysis were compared with results of literature mining for suggested metastatic breast cancer markers and vectors and consistency of their reported differential expression.

RESULTS

Our analysis suggested that transcriptional expression of the COX2 gene is significantly downregulated in metastatic tissue compared to normal breast tissue, but is not downregulated in primary tumors compared with normal breast tissue and may be used as a differential marker in metastatic breast cancer diagnostics. RRM2 gene expression decreases in metastases when compared to primary breast cancer and could be suggested as a marker to trace breast cancer evolution. Our study also supports MMP1, VCAM1, FZD3, VEGFC, FOXM1 and MUC1 as breast cancer onset markers, as these genes demonstrate significant differential expression in breast neoplasms compared with normal breast tissue.

CONCLUSIONS

COX2 and RRM2 are suggested to be prominent markers for breast cancer metastasis. The crosstalk between upstream regulators of genes differentially expressed in primary breast tumors and metastasis also suggests pathways involving p53, ER1, ERB-B2, TNF and WNT, as the most promising regulators that may be considered for new complex drug therapeutic interventions in breast cancer metastatic progression.

The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating <em>Wnt</em>/β-catenin signaling. We aimed at investigating EZH2's role in epigenetically regulating <em>Wnt</em>/β-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc(Min/+) mice were infected with CR (10(8) CFU); BLT1(-/-)Apc(Min/+) mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and <em>12</em> (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3's occupancy on WIF1 (<em>Wnt</em> inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although β-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and <em>12</em> post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected Apc(Min/+) mice paralleled changes recorded in BLT1(-/-)Apc(Min/+), AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate <em>Wnt</em>/β-catenin-dependent crypt hyperplasia in response to CR infection.

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10-<em>12</em> day incubation. Activation of <em>Wnt</em> signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical <em>Wnt</em> signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.

BACKGROUND

The canonical Wnt signaling is concurrently important for osteoblast differentiation and myeloma cell proliferation. Its activation in myeloma cells and its inhibition in osteoblasts and their progenitors have been identified in the previous studies. Osteoblast progenitors and myeloma cells from a myeloma patient share the same bone marrow (BM) microenvironment, but respond differently to DKK-1 secreted by myeloma cells. The mechanisms remain unclear.

METHODS

Primary multiple myeloma (MM) cells were isolated from BM mononuclear cells of 12 MM patients. Human bone marrow stromal cells (SCs) were obtained from BM adherent cells of these MM patients and 10 healthy donors. The mRNA expression levels of DKK-1 binding receptor LRP5/6 and Kremen1/2 (Krm1/2) were analyzed by Real-time PCR in human myeloma cell line (HMCL) RPMI-8226, NCI-H929, U266, LP-1, CZ-1, KM-3, Sko-007, primary myeloma cells and SCs from 12 MM patients and SCs from 10 healthy donors. The binding capability of DKK-1 binding receptors to DKK-1 on primary myeloma cells and SCs was detected by flow cytometry assay.

RESULTS

The mRNA expression levels of DKK-1 binding receptor LRP5/6 and Krm1/2 in SCs from patients with MM were significantly higher than those in myeloma cells and in SCs from healthy donors. The binding capability to DKK-1of DKK-1 binding receptors on SCs from MM patients was obviously higher than those on myeloma cells and SCs from healthy donors by flow cytometry assay. Similar to the effects of coculture with rhDKK1, coculture of SCs from healthy donors with myeloma cells in the presence or absence of a Transwell insert did up-regulate SCs' mRNA levels of LRP5/6 and Krm1/2, and down-regulate their mRNA levels of β-catenin.

CONCLUSIONS

Compared with myeloma cells, the SCs from MM patients overexpress DKK-1 binding receptors LRP5/6 and Krm1/2 in response to DKK-1 secreted by myeloma cells, which results in intracellular Wnt signaling inhibition. Our study provides a novel insight into mechanisms of myeloma associated osteolytic lesions.

Glioblastoma (GBM) is the most aggressive brain tumor and resistant to current available therapeutics, such as radiation. To improve the clinical efficacy, it is important to understand the cellular mechanisms underlying tumor responses to radiation. Here, we investigated long-term cellular responses of human GBM cells to ionizing radiation. Comparing to the initial response within <em>12</em> hours, gene expression modulation at 7 days after radiation is markedly different. While genes related to cell cycle arrest and DNA damage responses are mostly modulated at the initial stage; immune-related genes are specifically affected as the long-term effect. This later response is associated with increased cellular senescence and inhibition of transcriptional coactivator with PDZ-binding motif (TAZ). Mechanistically, TAZ inhibition does not depend on the canonical Hippo pathway, but relies on enhanced degradation mediated by the β-catenin destruction complex in the <em>Wnt</em> pathway. We further showed that depletion of TAZ by RNAi promotes radiation-induced senescence and growth arrest. Pharmacological activation of the β-catenin destruction complex is able to promote radiation-induced TAZ inhibition and growth arrest in these tumor cells. The correlation between senescence and reduced expression of TAZ as well as β-catenin also occurs in human gliomas treated by radiation. Collectively, these findings suggested that inhibition of TAZ is involved in radiation-induced senescence and might benefit GBM radiotherapy.

Recurrence of Dupuytren's contracture is common yet unpredictable, compromising surgical outcome. The alpha-smooth muscle actin-containing myofibroblast is the active contractile cellular component. Based on recent reports on beta-catenin accumulation in Dupuytren's disease, we investigated a possible relation with disease recurrence. We divided a collection of 143 nodules into those from patients with recurrent or nonrecurrent nodules and with a minimal 3-year followup. We randomly selected <em>12</em> and 11 samples of each group, respectively. We looked at Dupuytren's diathesis, immunohistologic staining for beta-catenin and alpha-smooth muscle actin, and Luck's histologic stages (zones). The expression of selected <em>Wnt</em> genes was examined with TaqMan PCR in separate histologic zones. All samples showed cytoplasmic and nuclear beta-catenin accumulation in myofibroblasts in involutional zones. The risk score of Abe et al. and Dupuytren's diathesis were greater in the recurrent group. Greater <em>Wnt</em>5a expression in the beta-catenin-accumulating involutional zone was seen. We conclude intracellular beta-catenin accumulation, possibly regulated by upstream <em>Wnt</em> signaling pathway activation and confined in myofibroblasts in the involutional zone of Dupuytren's diathesis, is unrelated to disease recurrence. Clinical parameters for Dupuytren's diathesis remain the best way to predict recurrence risk.

Silicosis is a form of occupational lung disease caused by inhalation of crystalline silica dust. While the pathogenesis of silicosis is not clearly understood, the <em>Wnt</em>/β-catenin signaling pathway is thought to play a major role in lung fibrosis. To explore the role of <em>Wnt</em>/β-catenin pathway in silicosis, we blocked <em>Wnt</em>/β-catenin pathway both in silica-treated MLE-<em>12</em> cells (a mouse pulmonary epithelial cell line) and in a mouse silicosis model by using a lentiviral vector expressing a short hairpin RNA silencing β-catenin (Lv-shβ-catenin). In vitro, Lv-shβ-catenin significantly decreased the expression of β-catenin, MMP2 and MMP9, and secretion of TGF-β1. In vivo, intratracheal treatment with Lv-shβ-catenin significantly reduced expression of β-catenin in the lung and levels of TGF-β1 in bronchoalveolar lavage fluid, and notably attenuated pulmonary fibrosis as evidenced by hydroxyproline content and collagen I\III synthesis in silica-administered mice. These results indicate that blockade of the <em>Wnt</em>/β-catenin pathway can prevent the development of silica-induced lung fibrosis. Thus <em>Wnt</em>/β-catenin pathway may be a target in prevention and treatment of silicosis.

Our aim was to test whether pharmacological inhibition of cycloxygenase-2 (COX-2) reverses non-alcoholic steatohepatitis (NASH) in type 2 diabetes mellitus (T2DM) rats via suppression of the non-canonical <em>Wnt</em> signaling pathway expression. Twenty-four male Sprague-Dawley rats were randomly distributed to two groups and were fed with a high fat and sucrose (HF-HS) diet or a normal chow diet, respectively. After four weeks, rats fed with a HF-HS diet were made diabetic with low-dose streptozotocin. At the 9(th) week the diabetic rats fed with a HF-HS diet or the non-diabetic rats fed with a normal chow diet were further divided into two subgroups treated with vehicle or celecoxib (a selective COX-2 inhibitor, 10 mg/Kg/day, gavage) for the last 4 weeks, respectively. At the end of the <em>12</em>(th) week, rats were anesthetized. NASH was assessed by histology. Related cytokine expression was measured at both the protein and gene levels through immunohistochemistry (IHC), Western blot and real-time PCR. T2DM rats fed with a HF-HS diet developed steatohepatitis and insulin resistance associated with elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), insulin levels and the non-alcoholic fatty liver disease (NAFLD) activity score (NAS). The expression of <em>Wnt</em>5a, JNK1, NF-κB p65, and COX-2 were all significantly increased in the T2DM-NASH group compared with the control and control-cele group. Hepatic injury was improved by celecoxib in T2DM-NASH-Cele group indicated by reduced serum ALT and AST levels and hepatic inflammation was reduced by celecoxib showed by histology and the NAFLD activity score (NAS). Serum related metabolic parameters, HOMA-IR and insulin sensitivity index were all improved by celecoxib. The expression of <em>Wnt</em>5a, JNK1, NF-κB p65, and COX-2 expression were all suppressed by celecoxib in T2DM-NASH-Cele group. The results of the present study indicated that celecoxib ameliorated NASH in T2DM rats via suppression of the non-canonical <em>Wnt</em>5a/JNK1 signaling pathway expression.

A critical mediator of cell-cell signaling events during embryogenesis is the highly conserved <em>Wnt</em> family of secreted proteins. Reporter constructs containing multimerized TCF DNA binding sites have been used to detect <em>Wnt</em> beta-catenin dependent activity during animal development. In this report, we have constructed and compared several TCF green fluorescent protein (GFP) reporter constructs. They contained 3, 8, or <em>12</em> TCF binding sites upstream of a minimal promoter driving native or destabilized enhanced GFP (EGFP). We have used the electroporation of somites in the chick embryo as a paradigm to test them in vivo. We have verified that they all respond to <em>Wnt</em> signaling in vivo. We have then assessed their efficiency at reflecting the activity of the <em>Wnt</em> pathway. Using destabilized EGFP reporter constructs, we show that somite cells dynamically regulate <em>Wnt</em>/beta-catenin-dependent signaling, a finding that was confirmed by performing time-lapse video confocal observation of electroporated embryos.

The molecular basis linking folate deficiency to certain health conditions and developmental defects is not fully understood. We examined the consequences of folate deficiency on global gene expression by microarray and compared transcript levels in normal human fibroblast cells (GM03349) grown in folate-deficient and -sufficient medium. The largest represented groups from the selected genes functioned in cell signaling, the cytoskeleton and the extracellular matrix and included the <em>Wnt</em> pathway genes DKK1, WISP1 and WNT5A. Twelve selected genes were further validated by qRT-PCR. Analysis of six genes at 4, 7, 10 and 14 days indicated that the relative differences in transcript levels between folate-sufficient and -deficient cells increases with time. Transcripts for 7 of the <em>12</em> selected genes were detected in the human lymphoblast cell line GM02257, and of these, changes in 4 genes corresponded to the results with fibroblast cells. Fibroblast cells were treated with the compounds homocysteine, methotrexate and the MEK1/2 inhibitor U0<em>12</em>6, and relative transcript levels of six genes were determined. U0<em>12</em>6 caused changes that more closely mimicked those detected in folate-deficient cells. The response of the DKK1 and TAGLN gene promoters to folate deficiency and compounds was examined in NIH3T3 cells using luciferase reporter plasmids. Promoter activity for both genes was decreased by folate deficiency and methotrexate and unaffected by homocysteine. U0<em>12</em>6 caused a decrease in DKK1 promoter activity at 50 microM and had no effect on TAGLN promoter activity. These findings suggest an alternative mechanism for how folate deficiency leads to changes in gene expression and altered cell function.

OBJECTIVE

In this study, we used laser capture microdissection (LCM) and microarray hybridization technology to compare the gene expression profiles of mouse embryonic days 10 and <em>12</em> lenses (E10 and E<em>12</em>).

METHODS

Lens cells of C57/BL6 mouse embryos at E10 and E<em>12</em> were harvested using the PixCell II LCM System. Total RNA was extracted, amplified, labeled, and hybridized to the 430 2.0 mouse chip (Affymetrix) according to the manufacturer's instructions. Data extracted from the images were analyzed using different software programs. Regulated expression of selected genes was confirmed by real-time PCR (RT-PCR).

RESULTS

Analysis of the microarray data from E10 and E<em>12</em> lenses identified 1,573 genes that showed a two fold or greater change in expression level. Among these 1,573 genes, 956 genes were downregulated and 617 were upregulated in E<em>12</em> lenses. In addition to the upregulated expression of beta- and gamma-crystallin genes, genes that regulate the cell cycle showed significant changes of gene expression during the E10 (lens pit) to E<em>12</em> (primary fiber cell induction) time period. Genes involved in insulin-like growth factor (IGF) signaling and Wnt (a family of secreted glycoproteins related to the Drosophila segment polarity gene, wingless, and to the proto-oncogene, int-1) signaling were also differentially regulated. In particular, positive regulators of Wnt signaling were downregulated and negative regulators were upregulated, indicating that modulation of Wnt signaling is important for normal lens morphogenesis.

CONCLUSIONS

Our results provide new information about differential regulation of gene expression during early lens development. Analysis of global gene expression profiles in embryonic mouse lenses has allowed us to identify several molecular pathways that are differentially regulated during early lens development.

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair (MMR) is a characteristic of the majority of tumors from kindreds with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic cancers. To better understand the molecular characteristics of colon cancers with MSI-H, we analyzed these cancers for alterations of genes, such as APC, beta-catenin, and TCF-4 genes, involved in the <em>Wnt</em> signaling pathway. Following the National Cancer Institute (NCI) criteria, 385 unselected colon cancers were classified as follows: 50 (13%) MSI-H tumors, 36 (9%) low-frequency MSI (MSI-L) tumors, and 299 (78%) microsatellite stable (MSS) tumors. The frequency of APC mutations was significantly lower in MSI-H tumors (9 out of 50) than in MSI-L (<em>12</em> out of 20) and MSS (66 out of 100) tumors (P = 0.0005 and P < 0.0001, respectively). In contrast, the frequency of exon 3 mutations in the beta-catenin gene was higher in MSI-H tumors (10 out of 50) than in MSI-L tumors (0 out of 30; P = 0.0110) and MSS tumors (3 out of 100; P = 0.0010). Frameshift mutations in a (A)9 tract of the TCF-4 gene were detected in 44% (22 out of 50) of MSI-H tumors, but not in any of the 20 MSI-L tumors or 40 MSS tumors. In total, 78% of MSI-H tumors and 84% of the remaining tumors had at least one alteration in APC, beta-catenin, or the TCF-4 genes. Although further analysis is needed to functionally characterize the consequences of each of these alterations on beta-catenin/TCF target gene expression, our results suggest that the activation of the <em>Wnt</em> signaling pathway plays a pivotal role in colon tumorigenesis, irrespective of MSI status.

BACKGROUND

Immunohistochemical staining for beta-catenin may be used as an indicator of the integrity of the Wnt signaling and beta-catenin degradation pathways. Among mesenchymal tumors, aberrant nuclear localization of beta-catenin is seen in desmoid-type fibromatoses but has not been described for solitary fibrous tumors that may mimic the former lesions, especially in small biopsy samples.

OBJECTIVE

To study the immunohistochemical expression of beta-catenin in solitary fibrous tumors.

METHODS

We performed immunohistochemical staining for beta-catenin in 12 solitary fibrous tumors, one of which showed histologic features of malignancy.

RESULTS

All the tumors showed strong and diffuse reactivity for beta-catenin. Four tumors (33%) showed nuclear staining for beta-catenin, whereas the remaining tumors showed either a membranous or mixed membranous and cytoplasmic pattern of staining. The only histologically malignant tumor of the group showed a mixed membranous and cytoplasmic pattern of staining for beta-catenin.

CONCLUSIONS

Immunohistochemical staining for beta-catenin in solitary fibrous tumors does not show a consistent pattern, which may be due to differences in tumorigenesis. Larger studies with clinical follow-up are required for estimating the impact of the variable staining pattern on clinical behavior of these tumors.

Endometrial carcinoma, generally, has a good prognosis. However, in some patients, the tumor appears to behave very aggressively, a course that cannot be explained with histopathological characteristics. More insight into the molecular background can be valuable to clarify these differences in tumor behavior. The three components associated with the <em>Wnt</em> pathway--i.e., adenomatous polyposis coli (APC), beta-catenin, and E-cadherin--were evaluated in a case-control study of 28 patients with stage-I endometrial carcinomas to determine their involvement in the development of recurrent disease. Mutation analysis of the mutation cluster region of the APC gene, determination of gene promoter methylation status of the APC-1A and E-cadherin genes, and immunohistochemical analysis of APC, E-cadherin, and beta-catenin were performed using paraffin-embedded tumor tissue. Twenty-one APC gene mutations were detected in <em>12</em> of 28 (43%) patients. Only three mutations would result in a stopcodon in the APC gene. APC gene promoter methylation was assessed in <em>12</em> of 28 (43%) patients. APC immunostaining was absent in two of 24 (8.3%) patients. The occurrence of APC mutations, APC gene promoter methylation, and APC immunostaining were not predictive for recurrence. No E-cadherin expression was observed in four of 24 patients (17%). E-cadherin gene promoter methylation could not be detected in any of the patients. The absence of E-cadherin expression was predictive for distant metastases, but not for local recurrence. Nuclear localization of beta-catenin was present in nine of 24 (38%) patients and was not predictive for recurrent disease. Involvement of epigenetic and genetic aberrations in APC and beta-catenin genes seems to be of minor importance for the development of local recurrences and distant metastases. Although the number of patients is limited, E-cadherin expression appears to be predictive for the development of distant metastases in endometrial carcinoma.

The <em>Wnt</em>/beta-catenin signaling pathway plays an important role in development, tissue homeostasis, and regeneration. Inappropriate activation of the <em>Wnt</em> pathway is linked to a wide range of human cancers. The purpose of this study was to characterize the <em>Wnt</em>/beta-catenin signaling pathway as depicted by the expression of <em>Wnt</em>1, Frizzled-1, <em>Wnt</em>5a, Frizzled-5 and beta-catenin during 4NQO-induced rat tongue carcinogenesis by immunohistochemistry. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4NQO solution at 50 ppm through their drinking water for 4, <em>12</em>, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, an overexpression of <em>Wnt</em>5a was noticed when compared to control group (p<0.05). The <em>Wnt</em>1 showed significant differences (p<0.05) in pre-neoplastic lesions at <em>12</em> weeks following carcinogen exposure. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, <em>Wnt</em>1 was expressed in the majority of the dysplasic cells and tumor cells. This was statistically significant (p<0.05). No significant differences (p>0.05) were found in expression of Frizzled-1, Frizzled-5 or beta-catenin following oral carcinogenesis. Taken together, our results support the belief that expression of <em>Wnt</em>1 and <em>Wnt</em>5a is related to malignant transformation and conversion of oral mucosa.

Recently, we identified WISP-2 (<em>Wnt</em>-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with <em>12</em>-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.

OBJECTIVE

To investigate the effect of CXCL13 (C-X-C motif chemokine 13) on hepatocellular carcinoma and clarify the potential mechanisms.

METHODS

32 patients with hepatocellular carcinoma and <em>12</em> healthy controls were recruited for analyzing the expression of CXCL13 by RT-PCR (reverse transcription-polymerase chain reaction). ELISA (enzyme-linked immune-sorbent assay) was used to test the concentration of serum CXCL13. The interaction between CXCL13 and <em>Wnt</em> signaling was analyzed by western blot. In vitro PBMCs cultured with HepG2 supernatant, the levels of IL-<em>12</em>, IL4, IL-6, and IL-17, and four IgG subclasses were detected by ELISA.

RESULTS

The rate of high expression CXCL13 was 63.4% in advanced HCC patients, and the serum CXCL13 was also at a high level in stage IV HCC patients. Meanwhile CXCL13 level was positively correlated with serum ALT (Alanine Transaminase) and AST (Aspartate Aminotransferase). CXCL13 and <em>Wnt</em>/β-catenin signaling shared a positive feedback loop. Furthermore, CXCL13 could obviously promote the expressions of IL-<em>12</em> and IL-17, and induce IgG4 secreted by B cells.

CONCLUSIONS

The effect of CXCL13 on promoting liver cancer is related to the activation of <em>Wnt</em>/β-catenin pathway and the facilitation of IL-<em>12</em>, IL-17 and IgG4. CXCL13 plays an important role in the progression of HCC, and it may act as a potential target for the diagnosis and treatment of HCC.