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This article discusses a case-control study that examined the prevalence of zinc hypersensitivity in patients with primary Sjögren syndrome (pSS). The study found that more than half of the patients with pSS were hypersensitive to zinc, compared to none of the healthy controls. The study suggests that zinc allergy may contribute to chronic inflammation in some patients with pSS and supports a connection between pSS and metal allergies. However, further research with larger sample sizes is needed to confirm these findings and understand the mechanisms involved. [Extracted from the article]

Ovarian cancer is the fifth most common cause of cancer‐related death in women. Ovarian clear cell carcinoma (OCCC) is a chemotherapy‐resistant epithelial ovarian cancer with poor prognosis. As a basis for the development of therapeutic agents that could improve the prognosis of OCCC, we performed a screen for proteins critical for the tumorigenicity of OCCC using the CRISPR/Cas9 system. Here we show that knockdown of the phosphate exporter XPR1/SLC53A1 induces the growth arrest and apoptosis of OCCC cells in vitro. Moreover, we show that knockdown of XPR1/SLC53A1 inhibits the proliferation of OCCC cells xenografted into immunocompromised mice. These results suggest that XPR1/SLC53A1 plays a critical role in the tumorigenesis of OCCC cells. We speculate that XPR1/SLC53A1 might be a promising molecular target for the therapeutic treatment of OCCC. [ABSTRACT FROM AUTHOR]

Certain cancers, such as ovarian clear cell carcinoma (OCCC), display high levels of genetic variation between patients, making it difficult to develop effective therapies. In order to identify novel genes critical to OCCC growth, we carried out a comprehensive CRISPR‐Cas9 knockout screen against cell growth using an OCCC cell line and a normal ovarian surface epithelium cell line. We identified the gene encoding DHX38/PRP16, an ATP‐dependent RNA helicase involved in splicing, as critical for the growth and tumorigenesis of OCCC. DHX38/PRP16 knockdown in OCCC cells, but not normal cells, induces apoptosis and impairs OCCC tumorigenesis in a mouse model. Our results suggest that DHX38/PRP16 may play a role in OCCC tumorigenesis and could potentially be a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury. [ABSTRACT FROM AUTHOR]

Background: Dysbiosis of oral microbiota is implicated not only in oral inflammatory lesions, but also in a variety of extraoral diseases. The etiology of palmoplantar pustulosis (PPP) remains unclear; however, it has been suggested that chronic inflammation caused by periodontopathic bacterial infection may play a role. Objectives/Methods: To determine whether patients with PPP have altered diversity and composition of oral microbiota, we conducted the 16S rDNA analysis using saliva samples collected from 21 outpatients with PPP and 10 healthy individuals. Results: We found that the proportion of bacteria in the phylum Proteobacteria was significantly lower in PPP patients (p = 0.025). At the genus level, patients with PPP had a significantly lower abundance of Neisseria (p = 0.014), which best accounted for the observed decrease in Proteobacteria. We also identified multiple minor genera and species that were represented at a significantly higher level in the PPP group, several of which have been associated with periodontal diseases. Conclusion: Our results suggest a possible link between PPP and dysbiosis of oral microbiota, particularly the lower abundance of Neisseria, the most predominant genus of Proteobacteria in healthy oral microbiota. Probiotics that improves oral dysbiosis may be beneficial for patients with PPP as an adjunctive therapy. [ABSTRACT FROM AUTHOR]

Background: The etiology of ulcerative colitis (UC) remains elusive even though many genetic and environmental pathogenic factors have been reported. Aberrant inflammatory responses mediated by specific subsets of T cells have been observed in ulcerative lesions of UC patients. Objectives: To elucidate the involvement of a delayed-type hypersensitivity reaction in UC, we focused on dental metal hypersensitivity, a T cell-mediated, delayed-type allergic reaction that causes oral contact mucositis and systemic cutaneous inflammation. Method: We recruited 65 Japanese UC patients and 22 healthy controls (HC) and used the in vitro lymphocyte stimulation test to quantify their sensitivity to zinc, gold, nickel, and palladium – the metals that have been widely used in dentistry. All subjects were users of metallic dental implants and/or prostheses containing zinc, gold, nickel, and/or palladium as major constituents. Results: Sixty percent of the UC patients were hypersensitive to at least one metal species, whereas 32% of the HC were hypersensitive to only a single metal species. The overall incidence of metal hypersensitivity was significantly higher for UC patients than for HC. Furthermore, a significantly greater proportion of UC patients were hypersensitive to nickel or palladium. The severity of the sensitivity to nickel and palladium was also significantly greater for UC patients than for HC. Conclusions: This pilot study demonstrates that UC patients have a significantly higher incidence of hypersensitivity to nickel and palladium, suggesting the possible involvement of dental metal hypersensitivity in UC pathogenesis. [ABSTRACT FROM AUTHOR]

Introduction: The etiology of irritable bowel syndrome (IBS) remains elusive even though several genetic and environmental pathogenic factors have been reported. IBS is considered to be a functional disorder without any detectable lesions in the patient's bowel. However, many studies have demonstrated that a subset of IBS patients have low‐grade inflammation and aberrant T‐cell activation in their intestinal mucosa. To elucidate the immune mechanism underlying the mucosal inflammation in IBS, we focused on dental metal hypersensitivity, a T cell–mediated, delayed‐type allergic reaction that causes oral contact mucositis and systemic cutaneous inflammation. Methods: We recruited 147 Japanese IBS patients and 22 healthy controls (HCs). The subjects underwent the in vitro lymphocyte stimulation test to quantify their sensitivity to zinc, gold, nickel, and palladium, the metals that have been commonly used in dentistry. Results: A total of 56.5% of the IBS patients were hypersensitive to at least one metal species, whereas 31.8% of HC were hypersensitive to only a single metal species. The overall incidence of metal hypersensitivity was significantly higher for IBS patients than for HC. Furthermore, a significantly higher proportion of IBS patients were hypersensitive to zinc and/or nickel. The severity of the sensitivity to zinc and nickel was also significantly greater for IBS patients than for HC. There was no significant difference in the sensitization rates and the sensitivity among the IBS subtypes. Conclusions: This pilot study demonstrates that IBS patients have a significantly higher prevalence of hypersensitivity to zinc and nickel, suggesting the possible involvement of dental metal hypersensitivity in IBS pathogenesis in a subset of patients. [ABSTRACT FROM AUTHOR]

The Wnt signaling pathway is aberrantly activated in the majority of human colorectal tumors. β‑catenin, a key component of the Wnt signaling pathway, interacts with the T‑cell factor/lymphoid enhancer‑binding factor family of transcription factors and activates transcription of Wnt target genes. Sp5 is one of the Wnt target genes, and its expression is commonly upregulated in colon cancer cells. The present study demonstrates that the expression of Sp5 is not upregulated in the colon cancer cell line HCT116, in which Wnt signaling is constitutively activated. Furthermore, the results demonstrate that Sp5 has the potential to inhibit cell proliferation through upregulation of the cell cycle inhibitor p27. These findings suggest that HCT116 cells downregulate Sp5 to avoid p27‑mediated growth arrest. [ABSTRACT FROM AUTHOR]

Summary Aberrant activation of Wnt/β-catenin signaling is a major driving force in colon cancer. Wnt/β-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc regulates multiple biological processes through its ability to directly modulate gene expression. Here, we identify a direct target of c-Myc, termed MYU , and show that MYU is upregulated in most colon cancers and required for the tumorigenicity of colon cancer cells. Furthermore, we demonstrate that MYU associates with the RNA binding protein hnRNP-K to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. These results suggest that the MYU /hnRNP-K/CDK6 pathway functions downstream of Wnt/c-Myc signaling and plays a critical role in the proliferation and tumorigenicity of colon cancer cells. [ABSTRACT FROM AUTHOR]

Summary Allergic airway inflammation is one of the primary features of allergic asthma. Interleukin-33 (IL-33) is recognized as a key pro-inflammatory cytokine that mediates allergic airway inflammation, and its expression is elevated in this condition, but little is known about the regulatory mechanisms underlying IL-33 induction. Here, we show that the RNA binding protein Mex-3B plays a critical role in the induction of IL-33 in the development of allergic airway inflammation. We generated Mex3b −/− mice and found that they develop significantly less airway inflammation than wild-type mice due to reduced induction of IL-33. Furthermore, we show that Mex-3B directly upregulates IL-33 expression by inhibiting miR-487b-3p-mediated repression of IL-33. Moreover, we show that inhalation of an antisense oligonucleotide targeting Mex-3B suppresses allergic airway inflammation. Our data identify a signaling pathway that post-transcriptionally regulates IL-33 expression and suggest that Mex-3B could be a promising molecular target for the treatment of allergic asthma. [ABSTRACT FROM AUTHOR]

The Dlg1 gene encodes a member of the MAGUK protein family involved in the polarization of epithelial cells. Null mutant mice for the Dlg1 gene (Dlg1-/- mice) exhibit respiratory failure and cyanosis, and die soon after birth. However, the cause of this neonatal lethality has not been determined. In the present study, we further examined Dlg1-/- mice and found severe defects in the cardiovascular system, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle, which would cause the neonatal lethality. These cardiovascular phenotypes resemble those of mutant mice lacking planar cell polarity (PCP) genes and support a recent notion that DLG1 is involved in the PCP pathway. We assessed the degree of involvement of DLG1 in the development of other organs, as the cochlea, intestine, and skeleton, in which PCP signaling has been suggested to play a role. In the organ of Corti, tissue elongation was inhibited accompanied by disorganized arrangement of the hair cell rows, while the orientation of the stereocilia bundle was normal. In the sternum, cleft sternum, abnormal calcification pattern of cartilage, and disorganization of chondrocytes were observed. Furthermore, shortening of the intestine, sternum, and long bones of the limbs was observed. These phenotypes of Dlg1-/- mice involving cellular disorganization and insufficient tissue elongation strongly suggest a defect in the convergent extension movements in these mice. Thus, our present results provide a possibility that DLG1 is particularly required for convergent extension among PCP signaling-dependent processes. [ABSTRACT FROM AUTHOR]

Increased vascular endothelial permeability and inflammation are major pathological mechanisms of pulmonary edema and its life-threatening complication, the acute respiratory distress syndrome (ARDS). We have previously described potent protective effects of hepatocyte growth factor (HGF) against thrombin-induced hyperpermeability and identified the Rac pathway as a key mechanism of HGF-mediated endothelial barrier protection. However, anti-inflammatory effects of HGF are less understood. This study examined effects of HGF on the pulmonary endothelial cell (EC) inflammatory activation and barrier dysfunction caused by the gram-negative bacterial pathogen lipopolysaccharide (LPS). We tested involvement of the novel Rac-specific guanine nucleotide exchange factor Asef in the HGF anti-inflammatory effects. HGF protected the pulmonary EC monolayer against LPS-induced hyperpermeability, disruption of monolayer integrity, activation of NF-kB signaling, expression of adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and production of IL-8. These effects were critically dependent on Asef. Small-interfering RNA-induced downregulation of Asef attenuated HGF protective effects against LPS-induced EC barrier failure. Protective effects of HGF against LPS-induced lung inflammation and vascular leak were also diminished in Asef knockout mice. Taken together, these results demonstrate potent anti-inflammatory effects by HGF and delineate a key role of Asef in the mediation of the HGF barrier protective and anti-inflammatory effects. Modulation of Asef activity may have important implications in therapeutic strategies aimed at the treatment of sepsis and acute lung injury/ARDS-induced gram-negative bacterial pathogens. [ABSTRACT FROM AUTHOR]

Summary The development of cancer is driven not only by genetic mutations but also by epigenetic alterations. Here, we show that TET1-mediated production of 5-hydroxymethylcytosine (5hmC) is required for the tumorigenicity of glioblastoma cells. Furthermore, we demonstrate that chromatin target of PRMT1 (CHTOP) binds to 5hmC. We found that CHTOP is associated with an arginine methyltransferase complex, termed the methylosome, and that this promotes the PRMT1-mediated methylation of arginine 3 of histone H4 (H4R3) in genes involved in glioblastomagenesis, including EGFR , AKT3 , CDK6 , CCND2 , and BRAF . Moreover, we found that CHTOP and PRMT1 are essential for the expression of these genes and that CHTOP is required for the tumorigenicity of glioblastoma cells. These results suggest that 5hmC plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex to selective sites on the chromosome, where it methylates H4R3 and activates the transcription of cancer-related genes. [ABSTRACT FROM AUTHOR]

Intestinal microbiota can indirectly modulate airway physiology and immunity through the gut-lung axis. Recent microbiome studies indicate that patients with coronavirus disease 2019 (COVID-19) exhibit a specific intestinal dysbiosis that is closely associated with the disease pathophysiology. Therefore, rebalancing the intestinal microbiome using probiotics may be effective for controlling COVID-19. However, the rationale for using probiotics in COVID-19 remains unclear. In the present study, an in vitro cytokine response assay was conducted, followed by a single-arm, double-blind, prospective trial to evaluate the immunological efficacy of probiotic lactic acid bacteria against COVID-19. The present study focused on Lactobacillus plantarum (L. plantarum), Bifidobacterium longum and Lactococcus lactis ssp. lactis, which exhibit robust protective effects against infection with respiratory RNA viruses. Considering the feasibility of long-term daily intake for prophylactic purposes, healthy uninfected individuals were enrolled as subjects. Our previous pilot trial demonstrated that oral Qingfei Paidu decoction (QFPD), a Chinese herbal medicine formulated specifically against COVID-19, upregulates plasma TNF-α, IL-1β, IL-18 and IL-8. Therefore, the present study utilized the cytokine changes induced by QFPD to define the innate cytokine index QICI [=(TNF-α) x (IL-1β) x (IL-18) x (IL-8)/(IL-6)] as an indicator of the anti-COVID-19 immunomodulatory potential of the lactic acid bacteria. A total of 20 eligible volunteers were enrolled, 18 of whom completed the intervention. L. plantarum demonstrated a strikingly high innate cytokine index in all subjects in the in vitro cytokine response assay. In the subsequent trial, oral intake of L. plantarum significantly increased the innate cytokine index (mean fold change, 17-fold; P=0.0138) and decreased the plasma level of IL-6 (P=0.0128), a key driver of complex immune dysregulation in COVID-19, as compared with the baseline. The cytokine index increased in 16 of 18 subjects (88.9%) with considerable individual differences in the fold change (1- to 128-fold). In line with these innate cytokine changes, L. plantarum ingestion significantly enhanced the activity of natural killer cells. By contrast, oral B. longum failed to induce a significant increase in the innate cytokine index (mean fold change, 2-fold; P=0.474) as compared with the baseline. In conclusion, L. plantarum demonstrated superior QFPD-like immunomodulatory ability and mimicked the blood cytokine environment produced by early immune responses to viral infection. Daily consumption of L. plantarum as an anti-COVID-19 probiotic may be a possible option for preventing COVID-19 during the pandemic. The present study was prospectively registered in the University Hospital Medical Information Network-Clinical Trials Registry under the trial number UMIN000040479 on 22 May 2020 (https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000046202). [ABSTRACT FROM AUTHOR]

The tumor suppressor adenomatous polyposis coli ( APC) is mutated in familial adenomatous polyposis and in many sporadic colorectal tumors. Adenomatous polyposis coli is known to negatively regulate Wnt signaling by inducing the degradation of β-catenin. Adenomatous polyposis coli also interacts with the guanine nucleotide exchange factors Asef and Asef2 and stimulates their activity, thereby regulating cell adhesion and migration. Here we show that in confluent, non-motile MDCK II cells, Asef/ Asef2 are colocalized with APC at the sites of cell-cell adhesion at the apical and junctional levels. In contrast, in colorectal tumor cells containing mutated APC, significant amounts of Asef/ Asef2 and the truncated mutant APCs are localized mainly in the cytoplasm. These results suggest that localization of the Asef/ Asef2- APC complex at the sites of cell-cell contact is critical for the regulation of cell adhesion, and that the aberrant subcellular localization of these complexes in colorectal tumor cells may contribute to the cell's aberrant adhesive and migratory properties. [ABSTRACT FROM AUTHOR]

Mutant mouse models are indispensable tools for clarifying the functions of genes and elucidating the underlying pathogenic mechanisms of human diseases. We carried out large-scale mutagenesis using the chemical mutagen N-ethyl- N-nitrosourea. One specific aim of our mutagenesis project was to generate novel cancer models. We screened 7012 animals for dominant traits using a necropsy test and thereby established 17 mutant lines predisposed to cancer. Here, we report on a novel cancer model line that developed osteoma, trichogenic tumor, and breast cancer. Using fine mapping and genomic sequencing, we identified a point mutation in the adenomatous polyposis coli ( Apc) gene. The Apc1576 mutants bear a nonsense mutation at codon 1576 in the Apc gene. Although most Apc mutant mice established thus far have multifocal intestinal tumors, mice that are heterozygous for the Apc1576 mutation do not develop intestinal tumors; instead, they develop multifocal breast cancers and trichogenic tumors. Notably, the osteomas that develop in the Apc1576 mutant mice recapitulate the lesion observed in Gardner syndrome, a clinical variant of familial adenomatous polyposis. Our Apc1576 mutant mice will be valuable not only for understanding the function of the Apc gene in detail but also as models of human Gardner syndrome. [ABSTRACT FROM AUTHOR]

The pentaspan membrane glycoprotein CD133 (also known as prominin-1) has been widely used as a marker for both cancer and normal stem cells. However, the function of CD133 has not been elucidated. Here we describe a cancer stem cell line established from clear cell carcinoma of the ovary (CCC) and show that CD133 interacts with plakoglobin (also known as γ-catenin), a desmosomal linker protein. We further demonstrate that knockdown of CD133 by RNA interference (RNAi) results in the downregulation of desmoglein-2, a desmosomal cadherin, and abrogates cell-cell adhesion and tumorigenicity of CCC stem cells. We speculate that CD133 may be a promising target for cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma. Methodology/Principal Findings: In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC- MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells. Conclusions/Significance: Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome. [ABSTRACT FROM AUTHOR]

Tumour cells are known to be dependent on, or 'addicted to', not only oncogenes, but also some non-oncogenes. However, the mechanisms by which tumour cells are addicted to these genes have not been fully explained. Here, we show that overexpression of a member of the ETS family, EHF, is required for the survival of colon tumour cells that contain wild-type p53. We found that EHF directly activates the transcription of RUVBL1, an ATPase associated with chromatin-remodelling complexes. RUVBL1 blocks p53-mediated apoptosis by repressing the expression of p53 and its target genes. Moreover, we found that RUVBL1 represses p53 transcription by binding to the p53 promoter, interfering with RNF20/hBRE1-mediated histone H2B monoubiquitination and promoting PAF1-mediated histone H3K9 trimethylation. These results indicate that EHF-mediated RUVBL1 expression allows colon tumour cells to avoid p53-mediated apoptosis. Thus, EHF and RUVBL1 might be promising molecular targets for the treatment of colon tumours. [ABSTRACT FROM AUTHOR]

Sporadic and familial colorectal tumours usually harbour biallelic adenomatous polyposis coli (APC)-associated mutations that result in constitutive activation of Wnt signalling. Furthermore, APC activates Asef and Asef2, which are guanine-nucleotide exchange factors specific for Rac1 and Cdc42. Here, we show that Asef and Asef2 expression is aberrantly enhanced in intestinal adenomas and tumours. We also show that deficiency of either Asef or Asef2 significantly reduces the number and size of adenomas in ApcMin/+ mice, which are heterozygous for an APC mutation and spontaneously develop adenomas in the intestine. We observed that the APC–Asef/Asef2 complex induces c-Jun amino-terminal kinase-mediated transactivation of matrix metalloproteinase 9, and is required for the invasive activity of colorectal tumour cells. Furthermore, we show that Asef and Asef2 are required for tumour angiogenesis. These results suggest that Asef and Asef2 have a crucial role in intestinal adenoma formation and tumour progression, and might be promising molecular targets for the treatement of colorectal tumours. [ABSTRACT FROM AUTHOR]

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of β-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent β-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in ApcMin/+ mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers. [ABSTRACT FROM AUTHOR]

Background: Although microarray-based studies have revealed global view of gene expression in cancer cells, we still have little knowledge about regulatory mechanisms underlying the transcriptome. Several computational methods applied to yeast data have recently succeeded in identifying expression modules, which is defined as co-expressed gene sets under common regulatory mechanisms. However, such module discovery methods are not applied cancer transcriptome data. Results: In order to decode oncogenic regulatory programs in cancer cells, we developed a novel module discovery method termed EEM by extending a previously reported module discovery method, and applied it to breast cancer expression data. Starting from seed gene sets prepared based on cis-regulatory elements, ChIP-chip data, and gene locus information, EEM identified 10 principal expression modules in breast cancer based on their expression coherence. Moreover, EEM depicted their activity profiles, which predict regulatory programs in each subtypes of breast tumors. For example, our analysis revealed that the expression module regulated by the Polycomb repressive complex 2 (PRC2) is downregulated in triple negative breast cancers, suggesting similarity of transcriptional programs between stem cells and aggressive breast cancer cells. We also found that the activity of the PRC2 expression module is negatively correlated to the expression of EZH2, a component of PRC2 which belongs to the E2F expression module. E2Fdriven EZH2 overexpression may be responsible for the repression of the PRC2 expression modules in triple negative tumors. Furthermore, our network analysis predicts regulatory circuits in breast cancer cells. Conclusion: These results demonstrate that the gene set-based module discovery approach is a powerful tool to decode regulatory programs in cancer cells. [ABSTRACT FROM AUTHOR]

Numerous independent clinical and experimental studies indicate that estrogens confer a protective effect against development of intestinal tumors, however the molecular mechanisms involved remain unclear. Physiological effects of estrogens are predominantly mediated by the action of nuclear estrogen receptors (ERs). A multifunctional protein adenomatous polyposis coli (APC) is a tumor suppressor and thought to act as a gatekeeper in colon tumorigenesis, as loss of function APC mutations trigger the development of colorectal cancer. Here we report that APC physically associates with ERa in the ligand-dependent manner. We have shown in the endogenous setting that the ligand-activated ERa recruits APC to the promoters in ER target genes and that increased levels of ER-dependent recruitment of APC enhances the ER transactivation through stimulation of histone acetylation. Found in majority of human colon tumors APC truncation mutants lost the ability to interact with ER. Thus, here we present the first evidence of a functional interaction between APC and ER that may be accounted for a tumor protective action of estrogens. [ABSTRACT FROM AUTHOR]

The Drosophila Notum gene, which is regulated by the Wingless pathway, encodes a secreted hydrolase that modifies heparan sulfate proteoglycans. In comparative analysis of the gene expression profiles in primary human hepatocellular carcinomas (HCC) and normal organs, we observed that the human ortholog of Drosophila Notum was overexpressed markedly in a subset of HCC, but expressed rarely in adult normal tissues. Immunoblotting confirmed the overexpression of NOTUM protein in 12 of 40 primary HCC cases (30%). High levels of NOTUM protein were significantly associated with intracellular (nuclear or cytoplasmic) accumulation of β-catenin protein: all 10 HCC with high intracellular β-catenin also had high NOTUM expression, whereas only 2 of 30 cases (6.7%) without intracellular β-catenin had high NOTUM expression ( P < 0.00001). NOTUM expression in HepG2 cells was downregulated significantly by induction of a dominant-negative mutant of TCF4, a β-catenin partner. In vivo binding of the β-catenin/TCF complex to the NOTUM promoter was demonstrated by chromatin immunoprecipitation in HepG2 and SW480 cells, where canonical Wnt signaling is activated constitutively. These findings provide evidence that NOTUM is a novel target of β-catenin/TCF4 and is upregulated in Wnt/β-catenin signaling-activated HCC. ( Cancer Sci 2008; 99: 1139–1146) [ABSTRACT FROM AUTHOR]

Abstract: Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies. [Copyright &y& Elsevier]

Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C-terminal region with postsynaptic density (PSD)-95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant-negative construct that disrupts the APC–PSD-95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD-95 and a glutamate receptor subunit, and reduced alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)-induced activity of AMPA receptors; however, the clustering of an N-methyl-d-aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses. [ABSTRACT FROM AUTHOR]

In our previous study, we identified RICS, a novel β-catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits. [ABSTRACT FROM AUTHOR]

B9L/BCL9-2, a novel β-catenin-interacting protein, plays an important role in colorectal carcinogenesis by translocating β-catenin to the nucleus and enhancing β-catenin–T-cell factor-mediated transcription. To elucidate the role of B9L in breast cancers, we studied B9L expression in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast immunohistochemically and compared it to the immunohistochemical expression of known proteins involved in breast carcinogenesis. In breast tissues, B9L immunoreactivity was present exclusively in the nuclei of normal and neoplastic ductal cells. In DCIS, immunohistochemical B9L expression was significantly associated with the tumor nuclear grade, comedo necrosis and the expression of ErbB2/HER-2, c-myc and p53. In IDC, B9L expression was correlated with ErbB2/HER-2 expression and tumor nuclear grade only. In both DCIS and IDC, immunohistochemical B9L expression was not related to the expression of cytoplasmic β-catenin. We demonstrated that nuclear B9L expression was closely associated with the high nuclear grade cancer phenotype and the expression of ErbB2/HER-2 in breast cancers. ( Cancer Sci 2007; 98: 484–490) [ABSTRACT FROM AUTHOR]

Aberrant activation of Wnt signaling is a critical event in the development of human colorectal tumors. The aim of this study was to elucidate the role of B9L and its association with β-catenin in each stage of the adenoma-carcinoma sequence of human colorectal tumorigenesis. We investigated the expression levels of B9L in sporadic colorectal adenomas and carcinomas, categorized according to the Vienna classification, using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemical analysis. B9L was expressed in the nuclei of non-neoplastic colonic mucosa cells and was overexpressed in the nuclei of neoplasias and invasive carcinoma cells. Immunoreactivity to B9L protein was correlated with the progressive grades of colorectal neoplasias, as was the expression of B9L mRNA. A high level of immunoreactivity to nuclear B9L was present in 27% of low-grade neoplasias and in more than 50% of high-grade neoplasias and invasive carcinomas, whereas a high level of nuclear B9L immunoreactivity was not observed in any of the non-neoplastic mucosa samples. The expression of B9L was dramatically increased in low-grade neoplasias compared with that in non-neoplastic mucosa. B9L may play an important role in tumorigenesis induced by aberrant activation of Wnt signaling and may act as a key protein in the progressive dysplasia of adenoma. ( Cancer Sci 2007; 98: 83–87) [ABSTRACT FROM AUTHOR]

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS –/– neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS –/– mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo. [ABSTRACT FROM AUTHOR]

Recently, we have identified human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, as a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP. Human Scribble, classified as a LAP protein containing eucine-rich repeats nd DZ domains, interacts with E6 through its PDZ domains and C-terminal PDZ domain-binding motif of E6 protein. Interaction between human Discs Large (hDlg), which is a substrate of E6 for the ubiquitin-mediated degradation, and adenomatous polyposis coli (APC) has been shown. Here, we investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C-terminal PDZ domain-binding motif of APC. Human Scribble co-localized with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC caused disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg-APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC. [ABSTRACT FROM AUTHOR]

Death receptor-mediated apoptosis is potently inhibited by viral FLIP (FLICE/caspase 8 inhibitory protein), which is composed of two tandemly repeated death effector domains (DEDs), through reduced activation of procaspase 8. Here, we show that equine herpesvirus 2-encoded viral FLIP E8 enhances Wnt/β-catenin signaling in a variety of cell lines. E8 was shown to strikingly augment Wnt3a signaling, as shown both in a luciferase assay for T-cell factor/β-catenin and through induction of endogenous cyclin D1. The effect of E8 was independent of its direct binding activity with DED-containing signaling molecules, including caspase 8 and FADD, in death receptor-mediated apoptosis. E8 enhanced Wnt signaling downstream of stabilized β-catenin, while a long form of cellular FLIP (c-FLIPL) enhanced stabilization of β-catenin in 293T cells. Consequently, coexpression of E8 and c-FLIPL synergistically increased Wnt signaling in 293T cells. Moreover, E8-mediated stimulation of Wnt signaling induced dramatic growth retardation in untransformed cell lines but not in transformed cell lines. Thus, viral FLIP E8 not only inhibits death receptor-mediated apoptosis but also enhances Wnt signaling pathways that are closely related to those of both ontogenesis and oncogenesis. [ABSTRACT FROM AUTHOR]

In the mammalian central nervous system, neurogenesis precedes gliogenesis; neurons are primarily generated at the neural stage, whereas most glial cells are generated perinatally and postnatally. However, the signals that regulate this sequence of events remain unclear. Here we show that Wnt signaling induces neuronal and astroglial differentiation but suppresses oligodendroglial differentiation. We observed that precursor cells infected with a retrovirus encoding β-catenin differentiated into neurons, while astrocytes developed from uninfected precursor cells surrounding infected cells. As neurogenesis proceeded, expression of the bone morphogenetic proteins (BMPs), BMP2, 4 and 7, progressively increased in the cells infected with the retrovirus encoding β-catenin. Furthermore, treatment of cells with Noggin, a BMP antagonist, completely inhibited astroglial differentiation but partially restored oligodendroglial differentiation. These results suggest that Wnt signaling indirectly regulates gliogenesis by inducing BMPs in neuronal cells. Thus, cooperation between Wnt and BMP signaling may play a key role in determining the sequence of neurogenesis and gliogenesis. [ABSTRACT FROM AUTHOR]

The improved IGCR (In–Gel Competitive Reassociation) method was applied to the analysis of human gastric cancer genomic DNA to identify its alterations, and it appeared that the IGCR library contained a fragment of 3′-untranslated region (3′ UTR) of G-protein coupled receptor 30 (GPR30) mRNA. When we searched genomic DNA pairs of gastric cancer patients with this IGCR clone, we found the deletion polymorphism with or without 2 bp (Cytosine and Thymine; CT). We confirmed the existence of a novel mRNA in GPR30 3′UTR by northern blotting, cloned this novel mRNA and named it Leucine Rich Protein in GPR30 3′UTR (LERGU). The EST database search gave one alternative splicing form in this 3′ UTR, which was named as LERGU-1. A novel alternative splicing form of this mRNA was also identified from the stomach total RNA, which was named LERGU-2. The LERGU mRNA was also detected in eight gastric cancer cell lines, but GPR30 mRNA scarcely existed. Furthermore, we detected the 2 bp-deletion form in genomic DNAs and mRNAs derived from gastric cancers, but not in other type cancers. Since the 2 bp-deletion position on LERGU corresponds to its alternative splicing site, this deletion may produce a frame-shifted protein. Overall, our findings suggest that a mutation or disappearance of the normal LERGU protein may have a function in the development of gastric cancer. (Cancer Sci2005; 96: 191–196) [ABSTRACT FROM AUTHOR]

Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of theß-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. TCF-binding elements have been found in the promoter regions of many TCF target genes and characterized by a highly conserved consensus sequence. Utilizing this consensus sequence, we performed an in silico screening for new TCF target genes. Through computational screening and subsequent experimental analysis, we identified a novel TCF target gene, DKK1, which has been shown to be a potent inhibitor of Wnt signaling. Our finding suggests the existence of a novel feedback loop in Wnt signaling.Oncogene (2004) 23, 8520-8526. doi:10.1038/sj.onc.1207892 Published online 20 September 2004 [ABSTRACT FROM AUTHOR]

TGF-β signalling regulates cell growth, differentiation, morphogenesis and apoptosis. MAFbx/Atrogin-1 has been identified as a regulator for skeletal muscle atrophy and encodes an F-box-type E3 ubiquitin ligase. However, little is known about how MAFbx/Atrogin-1 regulates cellular signalling. Here, we identify and genetically characterize MFB-1, a MAFbx/Atrogin-1 homologue fromCaenorhabditis elegans. Themfb-1deletion mutant significantly enhanced the dauer constitutive (Daf-c) phenotype caused by mutations in the DAF-7/TGF-β-like signalling pathway, but not the DAF-2/insulin receptor-like signalling pathway. Conversely, the Daf-c phenotypes of DAF-7 pathway mutants were partially suppressed bymfb-1cDNA transgenes. Therefore, MFB-1 acts genetically downstream in the DAF-7 pathway. A mfb-1::GFP fusion was found to be expressed in the nervous system, hypodermis and intestine and overlapped expression of many DAF-7 pathway genes. We propose that MFB-1 is a novel F-box protein that negatively regulates dauer formation in concert with the DAF-7 signalling pathway inC. elegans. [ABSTRACT FROM AUTHOR]

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of β-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the β-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized β-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development. [ABSTRACT FROM AUTHOR]

We have identified and characterized a novel member of the G protein-coupled receptor (GPCR) family, termed DREG. DREG belongs to the LNB-TM7 subfamily and possesses a long amino-terminus that contains a CUB domain, a PTX domain, a hormone binding domain and a GPCR proteolytic site (GPS) domain. RT-PCR experiments and whole mount in situ hybridization in mice showed that DREG is expressed at high levels in the heart and somite during embryogenesis and in the adult lung. When DREG was transiently expressed in mammalian cultured cells, a 35-kD fragment was generated by endogenous proteolytic processing at the conserved GPS domain. This short fragment was found associated with the cell membrane, typical of GPCRs. DREG was further cleaved in the middle of the extracellular domain, generating a soluble 70-kD fragment containing the CUB and PTX domains. This processing was inhibited by an inhibitor of the endoprotease furin but not of matrix metalloproteinases. These results raise the possibility that DREG plays a role in development, not only as a receptor or an adhesion molecule but also as a secreted ligand. [ABSTRACT FROM AUTHOR]

Inhibitor of β-catenin and T cell factor (ICAT) inhibits Wnt signaling by interfering with the interaction between β-catenin and T cell factor. Here we show that ICAT-/- embryos exhibit malformation of the forebrain and craniofacial bones and lack the kidney. Analysis of the neuronal differentiation of embryonic stem cells revealed that Wnt3a redirects the fate of neural progenitors to a posterior character, whereas ICAT induces forebrain cells by inhibiting Wnt signaling. Furthermore, ICAT-/- embryonic stem cells were found to differentiate into neuronal cells possessing a posterior character. These results suggest that ICAT plays an important role in the anteriorization of neural cells by inhibiting the posteriorizing activity of Wnt signaling. [ABSTRACT FROM AUTHOR]

The tumour suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC binds to β-catenin, a key component of the Wnt signalling pathway, and induces its degradation. APC interacts with microtubules and accumulates at their plus ends in membrane protrusions, and associates with the plasma membrane in an actin-dependent manner. In addition, APC interacts with the Rac-specific guanine nucleotide exchange factor Asef and stimulates its activity, thereby regulating the actin cytoskeletal network and cell morphology. Here we show that overexpression of Asef decreases E-cadherin-mediated cell-cell adhesion and promotes the migration of epithelial Madin-Darby canine kidney cells. Both of these activities are stimulated by truncated APC proteins expressed in colorectal tumour cells. Experiments based on RNA interference and dominant-negative mutants show that both Asef and mutated APC are required for the migration of colorectal tumour cells expressing truncated APC. These results suggest that the APC-Asef complex functions in cell migration as well as in E-cadherin-mediated cell-cell adhesion, and that truncated APC present in colorectal tumour cells contributes to their aberrant migratory properties. [ABSTRACT FROM AUTHOR]

Abstract Background: hDLG, the human homologue of the Drosophila tumour suppressor dlg , functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. hDLG possesses multiple protein-binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain. Results: We studied the significance of the PDZ, SH3, HOOK and GK domains in the cytoplasmic localization of hDLG. We found that mutation of the SH3 or GK domain, but not the PDZ domain, resulted in a re-localization of hDLG to the nucleus. Furthermore, hDLG was found to possess a potential nuclear localization signal in the HOOK domain. Conclusion: These results suggest that the SH3, HOOK and GK domains of hDLG are important for its cytoplasmic localization. [ABSTRACT FROM AUTHOR]

Abstract Background: The PSD-95 family of proteins possesses multiple protein binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain. The PSD-95 proteins function as scaffolding proteins that link ion channels such as the N-methyl-d-aspartate-receptors (NMDA-Rs) with cytoskeletal networks and signalling molecules, thereby controlling synaptic plasticity and learning. Results: We found that the PSD-95 family proteins interact via their GK domains with SPA-1-like protein (SPAL), a GTPase-activating protein (GAP) that is specific for Rap1. SPAL was contained within the NMDA-R-PSD-95 complex, and co-localized with PSD-95 and NMDA-R at the synapses in cultured hippocampal neurones. Furthermore, NMDA stimulation induced the dephosphorylation of SPAL in cultured hippocampal neurones. Conclusion: Our findings suggest that SPAL may be involved in the NMDA-mediated organization of cytoskeletal networks and signal transduction. [ABSTRACT FROM AUTHOR]

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of β-catenin, through its interaction with β-catenin, GSK-3β and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A?KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and β-catenin are transported along microtubules by KAP3?KIF3A?KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration. [ABSTRACT FROM AUTHOR]

Postsynaptic density (PSD)-95 interacts with and mediates clustering of the N-methyl-d-aspartate-receptors (NMDA-R). PSD-95 also interacts with the hDLG-associated protein DAP, which is also called Synapse-associated protein 90-associated protein (SAPAP), and Guanylate kinase-associated protein (GKAP). DAP interacted directly with the dynein light chain (DLC) family of proteins. DLC was contained in the NMDA-R-PSD-95-DAP-neuronal nitric oxide synthase (nNOS) complex. Furthermore, DAP interacted with nNOS and recruited it into the Triton X-100-insoluble fraction of transfected cells. DAP interacts directly with DLC and nNOS, and links these proteins to the NMDA-R-PSD-95 complex. [ABSTRACT FROM AUTHOR]

Background The synaptic protein PSD-95/SAP90 interacts with ion channels such as the N-methyl-D-aspartate-receptor (NMDA-R) via its PDZ domain, and is involved in their clustering. Moreover, it interacts with signalling molecules and plays an important role in coupling NMDA-R to pathways that control synaptic plasticity and learning. Results We report that PSD-95 interacts with the adenomatous polyposis coli (APC) tumour suppressor protein via its PDZ domain. Furthermore, we found that PSD-95, NMDA-R and APC are contained in the same complex in vivo. PSD-95-NMDA-R–APC association was found to require two cysteine residues conserved in the amino-terminus of PSD-95 that are known to be critical for its multimerization. Conclusion Our findings suggest that the PSD-95-NMDA-R-APC complex forms due to the multimerization of PSD-95 monomers, each of which can associate with either NMDA-R or APC. It is possible that APC is involved in the regulation of ion channel clustering and/or organization of signalling molecules. [ABSTRACT FROM AUTHOR]