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Adult tissues set the scene for a continuous battle between cells, where a comparison of cellular fitness results in the elimination of weaker "loser" cells. This phenomenon, named cell competition, is beneficial for tissue integrity and homeostasis. In fact, cell competition plays a crucial role in tumor suppression, through elimination of early malignant cells, as part of Epithelial Defense Against Cancer. However, it is increasingly apparent that cell competition doubles as a tumor-promoting mechanism. The comparative nature of cell competition means that mutational background, proliferation rate and polarity all factor in to determine the outcome of these processes. In this review, we explore the intricate and context-dependent involvement of cell competition in homeostasis and regeneration, as well as during initiation and progression of primary and metastasized colorectal cancer. We provide a comprehensive overview of molecular and cellular mechanisms governing cell competition and its parallels with regeneration.

The influence of the biophysical environment in known models of cell patterning is an area of growing interest to determine how to properly control cell fate and recapitulate tissue organization. This dissertation uses BMP4-induced human embryonic stem cells (hESCs) on gels of defined soft stiffness as a model of gastrulation-stage embryos. Additionally, to probe how mechanical cues affect the regulation of cell fate, traction force microscopy is leveraged to measure stress distributions in tissues. The findings here described indicate that areas of higher tension correlate with early mesoderm differentiation, emphasizing a critical role for tissue architecture in early development. The elevated tension promotes the release of beta-catenin from adhesion junctions and further Wnt signaling through a force-dependent conformational exposure of the tyrosine 654 (Y654) of cadherin-bound beta-catenin. By using a dephosphomimetic mutant for beta-catenin (Y654F), expression of the early mesodermal marker Brachyury (T) is severely affected, indicating a mechanism whereby high cell-cell tension initiates and spatially restrict a release of beta-catenin from junctions to promote mesoderm specification. Furthermore, in order to understand how the mesodermal T signal becomes spatially restricted, this dissertation explored an underappreciated aspect of developmental systems, programmed cell death (PCD). The findings indicate that not only apoptosis may play a mechanical role in equilibrating tissue-level forces, but also that apoptotic cell recognition may play an inhibitory role in the expression of the mesoderm marker T and regulate the boundary of its signal through the phagocytic receptor MERTK. These results provide a framework to understand how mechanical cues and PCD can work in tandem to regulate the self-organization of tissues in developmental processes, such as gastrulation.

ObjectiveThis study aimed to evaluate the Wnt/beta-catenin signaling pathway activity in gingival samples obtained from patients with periodontitis.Materials and methodsFifteen patients with stage III grade B (SIIIGB) and eleven with stage III grade C (SIIIGC) periodontitis were included and compared to 15 control subjects. beta-Catenin, Wnt 3a, Wnt 5a, and Wnt 10b expressions were evaluated by Q-PCR. Topographic localization of tissue beta-catenin, Wnt 5a, and Wnt 10b was measured by immunohistochemical analysis. TNF-alpha was used to assess the inflammatory state of the tissues, while Runx2 was used as a mediator of active destruction.ResultsWnt 3a, Wnt 5a, and Wnt 10b were significantly higher in gingival tissues in both grades of stage 3 periodontitis compared to the control group (p 0.05). beta-Catenin showed intranuclear staining in connective tissue in periodontitis, while it was confined to intracytoplasmic staining in epithelial tissue and the cell walls in the control group. Wnt5a protein expression was elevated in periodontitis, with the most intense staining observed in the connective tissue of SIIIGC samples. Wnt10b showed the highest density in the connective tissue of patients with periodontitis.ConclusionsOur findings suggested that periodontal inflammation disrupts the Wnt/beta-catenin signaling pathway.Clinical RelevancePeriodontitis disrupts Wnt signaling in periodontal tissues in parallel with tissue inflammation and changes in morphology. This change in Wnt-related signaling pathways that regulate tissue homeostasis in the immunoinflammatory response may shed light on host-induced tissue destruction in the pathogenesis of the periodontal disease., Ankara University Scientific Research Projects Office [17H0234001], This research was supported by project 17H0234001 of the Ankara University Scientific Research Projects Office.

ObjectiveThis study aimed to evaluate the Wnt/beta-catenin signaling pathway activity in gingival samples obtained from patients with periodontitis.Materials and methodsFifteen patients with stage III grade B (SIIIGB) and eleven with stage III grade C (SIIIGC) periodontitis were included and compared to 15 control subjects. beta-Catenin, Wnt 3a, Wnt 5a, and Wnt 10b expressions were evaluated by Q-PCR. Topographic localization of tissue beta-catenin, Wnt 5a, and Wnt 10b was measured by immunohistochemical analysis. TNF-alpha was used to assess the inflammatory state of the tissues, while Runx2 was used as a mediator of active destruction.ResultsWnt 3a, Wnt 5a, and Wnt 10b were significantly higher in gingival tissues in both grades of stage 3 periodontitis compared to the control group (p 0.05). beta-Catenin showed intranuclear staining in connective tissue in periodontitis, while it was confined to intracytoplasmic staining in epithelial tissue and the cell walls in the control group. Wnt5a protein expression was elevated in periodontitis, with the most intense staining observed in the connective tissue of SIIIGC samples. Wnt10b showed the highest density in the connective tissue of patients with periodontitis.ConclusionsOur findings suggested that periodontal inflammation disrupts the Wnt/beta-catenin signaling pathway.Clinical RelevancePeriodontitis disrupts Wnt signaling in periodontal tissues in parallel with tissue inflammation and changes in morphology. This change in Wnt-related signaling pathways that regulate tissue homeostasis in the immunoinflammatory response may shed light on host-induced tissue destruction in the pathogenesis of the periodontal disease., Ankara University Scientific Research Projects Office [17H0234001], This research was supported by project 17H0234001 of the Ankara University Scientific Research Projects Office.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3(-/-)), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

The diagnosis of pilomatricoma, the most common matrical tumor, is generally straightforward; however, it exhibits diverse histology associated with various morphological stages and several clinical variants, and matrical differentiation can occur in various neoplastic diseases. A 56-year-old man was admitted to our hospital to resect an 11.0-cm skin tumor on his right shoulder. Because of its large size and surface irregularities, including multiple erosions and ulcers, cutaneous malignancies were clinically suspected. Histologically, the tumor formed numerous nodules with marked matrical differentiation in the superficial to deep dermis. Although the tumor was macroscopically asymmetrical and irregular, each nodule was microscopically round-shaped and consisted of basaloid cells without marked atypia, atypical mitoses, or lymphovascular invasion. Immunohistochemically, the tumor cells were positive for beta-catenin, LEF-1, and PHLDA-1, consistent with their pilomatrical differentiation. We diagnosed the case as a giant pilomatrical tumor with uncertain malignant potential, considering its "contradictory" features, namely, the worrisome histoarchitecture, such as the asymmetrical silhouette, but bland-looking cytological appearance. Unlike typical pilomatrical tumors, this tumor contained numerous epidermal components with features similar to those of the dermal components, resulting in a unique macroscopic and histological appearance. Our case broadens the known histological diversity of pilomatrical tumors.

Desmoid-type fibromatosis (DF) is a locally aggressive but non-metastatic (myo)fibroblastic neoplasm. A hallmark of the tumor is nuclear positivity for beta-catenin in immunohistochemistry due mostly to CTNNB1 mutations. However, a recent study has reported that even beta-catenin ‘nuclear-negative’ DFs can harbor CTNNB1 mutations and that the positive ratio of nuclear beta-catenin in DF is different among antibodies. Here, we reviewed soft tissue lesions for which the possibility of DF was considered and compared the sensitivity and specificity of nuclear beta-catenin for the diagnosis of DF among commonly used anti-beta-catenin antibodies, i.e., clone beta-catenin 1, 17C2 and 14. We analyzed 26 cases of DF, 28 cases of benign fibroblastic lesions, and 27 cases of other soft tissue tumors. The sensitivity and specificity of nuclear beta-catenin for the diagnosis of DF were different among antibodies; 54% and 98% in clone beta-catenin 1, 85% and 84% in 17C2, and 96% and 62% in 14. IHC of LEF1 showed comparable results with IHC of beta-catenin, with a sensitivity of 88% and specificity of 76%. Additionally, when beta-catenin 1 was used, DFs showed characteristic dotted cytoplasmic staining, often appearing as rings. Our results might be helpful for making a correct diagnosis of DF.

Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to β-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased β-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of β-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate β-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating β-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment., Title in Web of Science: Extracellular AGR2 activates neighboring fibroblasts through endocytosis and direct binding to beta-catenin that requires AGR2 dimerization and adhesion domains

Desmoplastic fibroma (DF) is an intraosseous counterpart of desmoid-type soft tissue fibromatosis. It is most frequently seen in the jawbones. The clinical and radiological features of the present cases were nonspecific. The accumulation of beta-catenin in the nuclei of neoplastic cells which is a diagnostic feature of desmoid-type soft tissue fibromatosis could not be detectED in the present DF series. The aim of this study is to report a series of 22 cases of DF involving either mandible or maxilla. A retrospective evaluation of desmoplastic fibroma and beta-catenin, smooth muscle actin, nestin, cyclin D1 immunostaining's patterns. Most of the DF cases expressed only cytoplasmic beta-catenin immunostainings. We suggest that nuclear beta-catenin staining may not be used as a corroborating the diagnosis of DF. Immunohistochemical staining difference of jaw bone desmoplastic fibromas from other soft tissue and bone lesions may be related to the origination of jaw bone from The neural crest. Strong nestin and cyclin D1 positivity in our series supported this. A combined clinical, radiological, and histopathological analysis of the DF cases is essential in the diagnosis and management.

Organogenesis, including renal development, requires an appropriate retinoic acid concentration, which is established by differential expression of aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and cytochrome P450 family 26 subfamily A/B/C member 1 (CYP26A1/B1/C1). In the fetal kidney, ALDH1A2 expresses in the developing stroma and renal vesicle and its derivatives but does not present in the ureteric bud. It remains unclear what may contribute to this expression pattern. Here we show that the glycogen synthase kinase 3 alpha/beta (GSK3A/B) inhibitor CHIR99021 significantly represses ALDH1A2 expression in WiT49, which is a Wilms' tumor cell line that exhibits "triphasic" differential potential and is used as a fetal kidney cell model. CHIR99021 fails to suppress ALDH1A2 as beta-catenin is inhibited, suggesting that the downregulation of ALDH1A2 by CHIR99021 is through Wnt/beta-catenin signaling. Ectopic expression of mouse Wnt1, Wnt3a, Wnt4, and Wnt9b represses ALDH1A2 expression in WiT49 cells. Using immunohistochemistry, we show an inverse correlation of Aldh1a2 expression with beta-catenin in rat E18.5 kidney. ChIP demonstrated that beta-catenin is recruited to the ALDH1A2 promoter, the conserved intron1G, and another site within intron 1 of ALDH1A2. Using a luciferase assay, we further show that the ALDH1A2 promoter and the intron1G element are involved in the repression of ALDH1A2 expression by CHIR99021. Our work demonstrates that ALDH1A2 expression can be directly repressed by the Wnt/beta-catenin signaling in fetal kidney cells, suggesting that Wnt/beta-catenin may play a role in maintaining the expression pattern of ALDH1A2 in the fetal kidney, thus controlling the availability and localization of retinoic acid and regulating aspects of kidney development.

Background: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of β-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling. Methods: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling. Findings: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of β-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes. Interpretation: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression.

The circumventricular organs (CVOs) in the central nervous system (CNS) lack a vascular blood-brain barrier (BBB), creating communication sites for sensory or secretory neurons, involved in body homeostasis. Wnt/β-catenin signaling is essential for BBB development and maintenance in endothelial cells (ECs) in most CNS vessels. Here we show that in mouse development, as well as in adult mouse and zebrafish, CVO ECs rendered Wnt-reporter negative, suggesting low level pathway activity. Characterization of the subfornical organ (SFO) vasculature revealed heterogenous claudin-5 (Cldn5) and Plvap/Meca32 expression indicative for tight and leaky vessels, respectively. Dominant, EC-specific β-catenin transcription in mice, converted phenotypically leaky into BBB-like vessels, by augmenting Cldn5+vessels, stabilizing junctions and by reducing Plvap/Meca32+ and fenestrated vessels, resulting in decreased tracer permeability. Endothelial tightening augmented neuronal activity in the SFO of water restricted mice. Hence, regulating the SFO vessel barrier may influence neuronal function in the context of water homeostasis., SCOPUS: ar.j, info:eu-repo/semantics/published

The pancreas has essential functions. Dysfunction of its cells is associated with pathologies. With the exception of cystic fibrosis, no pancreatic pathology is yet associated with ductal cells dysfunction. The aim of this thesis is to identify the role of ductal cells in the development of pancreatitis and in the appearance of lesions allowing the initiation of pancreatic cancer. We performed a mouse model allowing the ductal inactivation of the LKB1 tumor suppressor, which shows a relationship between a primary cilia defect of the ductal cells and the presence of pancreatitis. By combining this inactivation with the induction of the G12D mutation of the Kras oncogene, we have succeeded in developing a murine model developing IPMN tumors. These results show that disruption of the ductal cells can lead to the development of pancreatitis and pancreatic cancer., Le pancréas présente des fonctions essentielles. Un dysfonctionnement de ces cellules est associé à des pathologies du pancréas. À l’exception de la mucoviscidose, aucune pathologie pancréatique n’est encore associée avec un dysfonctionnement des cellules canalaires. Le but de cette thèse est d’identifier le rôle des cellules canalaires dans le développement d’une pancréatite et dans l’apparition de lésions permettant l’initiation d’un cancer du pancréas. Nous avons réalisé un modèle de souris permettant l’inactivation canalaire du suppresseur de tumeur LKB1 qui montre une relation entre un défaut des cils primaires des cellules canalaires et la présence d’une pancréatite. En combinant cette inactivation avec l’induction de la mutation G12D de l’oncogène Kras, nous sommes parvenus à élaborer un modèle murin développant des tumeurs TIPMP. Ces résultats montrent qu’une perturbation des cellules canalaires peut entraîner le développement d’une pancréatite et d’un cancer du pancréas., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

It is very rewarding for endocrine pathologists to see, in the new book of the World Health Organization (WHO) classification of endocrine organs 1, how the molecular characterization of hyroid tumours has confirmed the types and subtypes of tumours previously recognized by less sophisticated techniques.

Epithelial specific ETS-1 (ESE-1) is one of the E26 transformation-specific transcription factor superfamily and could be considerable interest as a possible target for regulating diverse types of human cancer. Despite its clinical importance, the reported biological role of ESE-1 on cancer development and progression are still controversial and its underlying mechanisms of tumorigenesis remains unclear. The objectives of this dissertation are to elucidate the role of ESE-1 in tumorigenesis. With the evidence in cancer phenotypes, the underlying mechanisms of ESE-1 in colon cancer was also investigated. ESE-1 knockout mice increased azoxymethane (AOM) -induced and dextran sulfate sodium (DSS)-promoted formation of aberrant crypt foci (ACF) compared to wild type mice. Overexpression of ESE-1 suppressed anchorage-independent growth and migration/invasion in human colon cancer cells and while knockdown of ESE-1 reversed anti-cancer activity. Full length ESE-1 was abundantly found in the nucleus, and internal deletion of nuclear localization sequence 2 (NLS2) decreased the amount of nuclear ESE-1. Three lysine residues (318KKK320) in the NLS2 were critical for nuclear localization of ESE-1 and mediates tumor suppressive activity of ESE-1 through reduced beta-catenin transcriptional activity. We identified two anti-cancer natural compounds, epigallocatechin-3-gallate (EGCG) and patchouli alcohol as ESE-1 inducers. Both EGCG and patchouli alcohol increased expression of ESE-1 protein and mRNA in human colon cancer cells. Patchouli alcohol showed reduced the number of tumors and tumor load in Apcmin/+ colon cancer animal model although protein expression level of ESE-1 did not show significant difference. These findings suggest a potential use of ESE-1 as a novel and potential molecular target of natural anti-cancer phytochemicals for colon cancer prevention.

CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Societ

In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: beta-catenin, PPAR-gamma and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of beta-catenin immunoreactive cells was significantly higher in PV then in ET (p lt 0.01) or PMF group of patients (p lt 0.01) and also in ET versus PMF group of patients (p lt 0.01). Erythroid lineage showed absent beta-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-gamma was localized mostly in megakaryocytes and the highest number of PPAR-gamma immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed beta-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-gamma immunopositive megakaryocytes characterized by abnormal maturation.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing -cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat -cell line INS-1E.Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E -cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E -cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal -cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3.These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate -cell maturation. This study indicates a functional role for TFF3 in pancreatic -cell maturation in the perinatal period, which is altered by low protein diet during gestation.

PurposeSporadic desmoid-type fibromatosis (DTF) is a rare, chronic, non-metastasising, disease of the soft tissues. It is characterised by local invasive and unpredictable growth behaviour and a high propensity of local recurrence after surgery thereby often having a great impact on health-related quality of life (HRQL). This study aims to review currently used HRQL measures and to asses HRQL issues among DTF patients.MethodsA mixed methods methodology was used consisting of (1) a systematic literature review, according to the PRISMA guidelines (2009), using search terms related to sporadic DTF and HRQL in commonly used databases (e.g. Embase, Medline Ovid, Web of science, Cochrane Central, Psyc Info, and Google scholar), to provide an overview of measures previously used to evaluate HRQL among DTF patients; (2) focus groups to gain insight into HRQL issues experienced by DTF patients.ResultsThe search strategy identified thirteen articles reporting HRQL measures using a wide variety of cancer-specific HRQL tools, functional scores, symptom scales (e.g. NRS), and single-item outcomes (e.g. pain and functional impairment). No DTF-specific HRQL tool was found. Qualitative analysis of three focus groups (6 males, 9 females) showed that participants emphasised the negative impact of DTF and/or its treatment on several HRQL domains. Six themes were identified: (1) diagnosis, (2) treatment, (3) follow-up and recurrence, (4) physical domain, (5) psychological and emotional domain, and (6) social domain.ConclusionA DTF-specific HRQL tool and consensus regarding the preferred measurement tool among DTF patients is lacking. Our study indicates that HRQL of DTF patients was negatively affected in several domains. A DTF-specific HRQL measure could improve our understanding of short- and long-term effects and, ideally, can be used in both clinic and for research purposes.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry & Biophysics, 2015., This work addresses the central question of how B-CATENIN signaling regulates chondrocyte differentiation during endochondral bone formation and cartilage development through integration with BMP and CCN1 signaling. Even though the functions of B-CATENIN signaling in chondrocytes have been extensively investigated through gain-of-function (GOF) and loss-of-function (LOF) mouse models, the role of B-CATENIN signaling in the committed chondrocyte and its downstream effectors are still unclear. Here we have discovered that cartilage-derived B-CATENIN signaling promotes chondrocyte maturation, angiogenesis, secondary ossification center (SOC) formation and bone ossification. B-CATENIN signaling also induces activation of BMP signaling and mediates primary ossification center (POC) formation through BMP and TAK1 signaling. Next, we identified a matricellular protein, CCN1, induced by B- CATENIN signaling in chondrocytes. In vitro assays demonstrate that CCN1 is required and sufficient to drive chondrocyte maturation. In vivo overexpression of CCN1 in chondrocytes leads to chondrodysplasia with reduced cell number but increased cell size, and results in cartilage degeneration in adult mice. The increase in the expression of CCN1 in injured mouse and human cartilage suggests a correlation between the CCN1 and osteoarthritis progression. Finally, we explored the role of CCN1 in chondrogenesis by overexpressing CCN1 in limb mesenchyme. CCN1 overexpression causes severe chondrodysplasia and reduction in the expression of chondrogenic genes indicating a repressive role of CCN1 in chondrogenesis. Mice with CCN1 overexpression in mesenchyme develop multiple joint defects, which eventually results in cartilage degradation and bone destruction in the knee joints. Taken together, these data have revealed that cartilage-derived B-CATENIN signaling regulates chondrocyte maturation and endochondral bone formation through modulating BMP signaling. Another novel finding is that CCN1 si

Mechanical force is increasingly recognized to be a crucial player in stem cell regulation during normal tissue homeostasis and in cancer development, with Yes-associated protein (YAP) rapidly taking the centre stage as key mechanoprotein. It is critical to unravel the molecular mechanisms underlying the interplay between mechanical force and cell signalling events in normal tissue homeostasis to better understand cancer development, and for the development of therapeutics. My PhD project elucidated that epidermal YAP activity drives β-catenin activation to promote keratinocyte proliferation in mouse skin in vivo (Chapter 3) via mechanotransduction pathways regulated by ROCK, the major mediator of RhoA/C, and elevated collagen-mediated dermal stiffness (Chapter 4). Furthermore, I found evidence that YAP also promotes epidermal Hedgehog signalling via β-catenin activation in mouse skin in vivo (Chapter 5). Interestingly, I observed increased YAP, β-catenin and ROCK activity and dermal fibrosis in mouse and human squamous cell carcinoma (SCC), and in human BCC (Chapter 4, 5). Lastly, I contributed to the development of a simple method to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, Ki67 labelled proliferating cells, Nile Red labelled sebocytes, and DAPI nuclear labelling at the single cell resolution in 3D (Chapter 6).These findings constitute a significant conceptual advance over the established paradigm that YAP purely acts as a mechanosensor in tissue homeostasis, and hence a responder to changes in extracellular matrix stiffness. My work reveals that YAP acts as a central mechanoprotein and also drives tissue mechanics through regulatory interactions with β-catenin, ROCK and Hedgehog signalling pathways. This has important implications for our understanding of normal tissue regeneration and the development of cancers or other regenerative disease displaying increased YAP activity.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. Chemopreventive therapies could be effective way to treat CRC. Tolfenamic acid, one of the NSAIDs, shows anti-cancer activities in several types of cancer. Aberrant Wnt/β-catenin regulation pathway is a major mechanism of colon tumorigenesis. Here, we sought to better define the mechanism by which tolfenamic acid suppresses colorectal tumorigenesis focusing on regulation of β-catenin pathway. Treatment of tolfenamic acid led to a down-regulation of β-catenin expression in dose dependent manner in human colon cancer cell lines without changing mRNA. MG132 inhibited tolfenamic acid-induced downregulation of β-catenin and exogenously overexpression β-catenin was stabilized in the presence of tolfenamic acid. Tolfenamic acid induced an ubiquitin-mediated proteasomal degradation of β-catenin. In addition, tolfenamic acid treatment decreased transcriptional activity of β-catenin and expression of Smad2 and Smad3 while overexpression of Smad 2 inhibited tolfenamic acid-stimulated transcriptional activity of β-catenin. Moreover, tolfenamic acid decreased β-catenin target gene such as vascular endothelial growth factor (VEGF) and cyclin D1. In summary, tolfenamic acid is a promising therapeutic drug targeting Smad 2-mediated downregulation of β-catenin in CRC.

The canonical Wnt/beta-catenin signaling pathway is crucial for blood-brain barrier (BBB) formation in brain endothelial cells. Although glucose transporter 1, claudin-3, and plasmalemma vesicular-associated protein have been identified as Wnt/beta-catenin targets in brain endothelial cells, further downstream targets relevant to BBB formation and function are incompletely explored. By Affymetrix expression analysis, we show that the cytochrome P450 enzyme Cyp1b1 was significantly decreased in beta-catenin-deficient mouse endothelial cells, whereas its close homolog Cyp1a1 was upregulated in an aryl hydrocarbon receptor-dependent manner, hence indicating that beta-catenin is indispensable for Cyp1b1 but not for Cyp1a1 expression. Functionally, Cyp1b1 could generate retinoic acid from retinol leading to cell-autonomous induction of the barrier-related ATP-binding cassette transporter P-glycoprotein. Cyp1b1 could also generate 20-hydroxyeicosatetraenoic acid from arachidonic acid, decreasing endothelial barrier function in vitro. In mice in vivo pharmacological inhibition of Cyp1b1 increased BBB permeability for small molecular tracers, and Cyp1b1 was downregulated in glioma vessels in which BBB function is lost. Hence, we propose Cyp1b1 as a target of beta-catenin indirectly influencing BBB properties via its metabolic activity, and as a potential target for modulating barrier function in endothelial cells.

Primary hyperparathyroidism (pHPT) is a common endocrine disease characterized by excessive secretion of parathyroid hormone and an increased level of serum calcium. Overall, 80-85% of pHPT cases are due to a benign, single parathyroid adenoma (PA), and 15% to multiglandular disease (multiple adenomas/hyperplasia). Parathyroid carcinoma (PC) is rare, accounting for <0.5-1% of pHPT cases. Secondary hyperparathyroidism (sHPT) is a complication of renal failure, with the development of parathyroid tumours and hypercalcaemia. Recurrent mutations in the MEN1 gene have been confirmed by the whole-exome sequencing in 35% of PAs, suggesting that non-protein-coding genes, regulatory elements or epigenetic derangements may also have roles in the majority of PAs. DNA translocations with cyclin D1 overexpression occur in PAs (8%). In PCs, mutations in CDC73/HRPT2 are common. Activation of the WNT/β-catenin signalling pathway (accumulation of nonphosphorylated β-catenin) by an aberrantly truncated LRP5 receptor has been seen for the majority of investigated PAs and sHPT tumours, and possibly by APC inactivation through promoter methylation in PCs. Promoter methylation of several other genes and repressive histone H3 lysine 27 trimethylation by EZH2 of the HIC1 gene may also contribute to parathyroid tumorigenesis. It is possible that a common pathway exists for parathyroid tumour development. CCND1 (cyclin D1) and EZH2 overexpression, accumulation of nonphosphorylated β-catenin and repression of HIC1 have all been observed to occur in PAs, PCs and sHPT tumours. In addition, hypermethylation has been observed for the same genes in PAs and PCs (e.g. SFRP1, CDKN2A and WT1). Whether β-catenin represents a 'hub' in parathyroid tumour development will be discussed.

Research over the last decade recognized the importance of novel molecular pathways in pathogenesis of intracranial meningiomas. In this review, we focus on human brain tumours meningiomas and the involvement of Wnt signalling pathway genes and proteins in this common brain tumour, describing their known functional effects. Meningiomas originate from the meningeal layers of the brain and the spinal cord. Most meningiomas have benign clinical behaviour and are classified as grade I by World Health Organization (WHO). However, up to 20% histologically classified as atypical (grade II) or anaplastic (grade III) are associated with higher recurrent rate and have overall less favourable clinical outcome. Recently, there is emerging evidence that multiple signalling pathways including Wnt pathway contribute to the formation and growth of meningiomas. In the review we present the synopsis on meningioma histopathology and genetics and discuss our research regarding Wnt in meningioma. Epithelial-to-mesenchymal transition, a process in which Wnt signalling plays an important role, is shortly discussed.

Bone is a complex mineralized tissue that functions as a structural support for the body, as well as in mineral storage and homeostasis. Its development is as complex as it’s function, with multiple molecular cues governing several aspects of its development and maintenance. Multiple previous studies over several decades have shown that failure to maintain tight regulation of these cues results in varying degrees of bone malformation, most frequently of the craniofacial variety. Various reports have cited a fundamental importance of both non-canonical and canonical Wnt signaling in regulating osteogenesis. Multiple studies have also revealed the importance of temporal-spatial regulation of their expression in promoting proper bone development.MicroRNAs (miRNAs) are small non-coding RNAs that are able to negatively regulate protein expression, serving as a regulatory mechanism in a variety of developmental processes and diseases. They function by binding to the 3’ Untranslated Region of target mRNAs to induce mRNA degradation or translational inhibition. They are crucial for early embryonic differentiation, as studies have shown that embryonic stem cells that cannot generate miRNA exhibit impaired differentiation.The work presented in this dissertation focuses on two miRNAs that have not been previously characterized in osteogenesis: miR-690 and miR-361. These miRNAs were selected from a screen based on their differential patterns of accumulation in response to osteogenic factors, and their ability to positively regulate osteogenesis. Using embryonic stem cells as a model for early embryonic development, we observed that both miRNAs were capable of affecting Wnt signaling. We also noted a correlation of miRNA expression with accelerated and/or sustained expression of genes that are associated with neural crest specification or differentiation. These studies have established (i) that miR-690 supports osteogenesis by directly regulating Ctnnb1 during differentiation fr

Aim of the study is to determine the possible roles of p53, cyclin D1, B-catenin and Ki-67 in the increase in risk of fractures in patients with giant cell tumor of bone. The study included a total of 164 patients with giant cell tumor of bone (GCTB), 21 (12.8%) with and 143 (87.2%) without fracture. The samples were analyzed immunohistochemically for expression of Ki-67, p53, cyclin D1 and beta-catenin. According to the immunohistochemical expression of p53 and Ki-67 in mononuclear stromal cells, as well as of cyclin D1 in multinuclear giant cells, there was no significant association with immunopositivity and risk of fractures. However, our research revealed that patients with cytoplasmic expression of beta-catenin in stromal cells had three times more frequent occurrence of pathological fractures, which was highly statistically significant (chi(2) = 7.065; p = 0.008). Moreover, a highly statistically significant correlation between the nuclear expression of beta-catenin in giant cells and the incidence of pathological fractures was also found (chi(2) = 8.824; p = 0.003). The study showed that beta-catenin expression highly correlates with the incidence of pathological fractures in patients with GCTB. Taking into account that beta-catenin is closely linked to activation of the Wnt signaling pathway in GCTB pathogenesis, one could postulate that activation of the Wnt pathway is one of the contributing factors to locally destructive behavior of this tumor, as well as to the incidence of pathological fractures.

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.

Wnt/beta-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between beta-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the beta-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3',4,4',5-pentachlorobiphenyl (PCB126). This led us to investigate the AHR interaction with beta-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of p-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a beta-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6-formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryo-toxic, and we found that in the presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholino-oligonudeotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of beta-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of p-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating beta-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes., Correction in: Toxicology and Applied Pharmacology, 2015, Volume: 288, Issue: 2, Pages: 280-280, DOI: 10.1016/j.taap.2015.07.021

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in beta-catenin (CTNNB1). We have compared the dynamics and the potency of beta-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of beta-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of beta-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of beta-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin: beta-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of beta-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of beta-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated beta-catenin.

Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of beta-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a beta-catenin-driven transcription program that leads to endothelial-tomesenchymal transition. TGF-beta/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate beta-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas., This study was supported by grants (to E.D.) from TELETHON-GGP14149, Associazione Italiana per la Ricerca sul Cancro (AIRC) (AIRC IG 14471), "Special Program Molecular Clinical Oncology 5x1000" to AIRC-Gruppo Italiano Malattie Mieloproliferative, Fondazione Cassa di Risparmio delle Provincie Lombarde (CARIPLO) Contract 2012-0678, the European Community (Wnt for Brain Contract 268870; Innovative Training Networks Vessel 317250, Endostem-Health-2009-241440), and Fondazione CARIPLO Contract 2014-1038 (to N.R.). R.C. was supported by the FIRC fellowship 16617. Part of this work was funded by the European consortium European Research Area Network NEURON (to E.T.-L.).Dejana E and Lampugnani MG share the last authorship and corresponding.

Acute myeloid leukaemia (AML) remains the leading cause of leukaemia-related death with a relative five-year survival rate of only 24% in Australia. This poor prognosis is mainly due to disease relapse, which is thought to be attributable to the persistence of leukaemic stem cells (LSC) after chemotherapy. Previous studies have shown that β-catenin is required for the establishment of LSC in mixed lineage leukaemia (MLL)-rearranged AML. Targeted inhibition of β-catenin signalling has been hampered by the lack of pathway components amenable to pharmacoinhibition. In this study we have identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in AML LSC compared to normal HSC (P=0.0114). ShRNA-mediated inhibition of GPR84 impaired cell proliferation (P=0.0009) and AML reconstitution in vivo (P<0.0001). The GPR84- deficient phenotype could be rescued by expression of constitutively active β-catenin in vivo (P=0.0039). Furthermore, GPR84 overexpression accelerated leukaemogenesis in vivo (P=0.0039). These data suggest that inhibiting GPR84/β-catenin signalling may provide a promising novel therapeutic strategy in AML. However, since a GPR84-specific inhibitor has not yet been developed, we aimed to further delineate the GPR84/β- catenin axis in order to pharmacologically inhibit other components of this pathway. Preliminary data from our lab has implicated the G protein GNA13 as a potential downstream target of GPR84. GNA13 overexpression rescued GPR84 deficiency (P=0.0012) through augmentation of cell proliferation in vivo (P<0.0001). In contrast, shRNA-mediated ablation of GNA13 impeded cell proliferation (P=0.0066) and delayed disease onset in vivo (P<0.0001). However, GNA13 may not integrate into a therapeutic regimen since GNA13-deficiency did not sensitize LSC to bortezomib as implicated by previous reports. Hence we conducte

During early vertebrate embryogenesis, the expression of SOXB1 proteins is precisely regulated by a number of different mechanisms, including Wnt/beta-catenin signaling. This is essential for controlling the balance between stemness and differentiation in embryonic stem cells. In the present study, we analyzed the molecular mechanism of LiCl action in NT2/D1 cells and examined the crosstalk between SOXB1 proteins and Wnt signaling in this model system. We have shown that LiCl increases beta-catenin level, induces its translocation to the nucleus and consequently up-regulates beta-catenin/Tcf-dependent transcription in NT2/D1 cells. Our results also suggest that LiCl treatment leads to increased expression of SOX2 and SOX3 proteins in NT2/D1 cells through activation of canonical Wnt signaling. Finally, we have detected a negative feedback loop between beta-catenin and SOX2 expression in NT2/D1 cells. Since beta-catenin and SOX2 have been linked to processes of self-renewal and pluripotency, our results have implications for future research on the maintenance of stemness and lineage commitment of embryonic stem cells.

The Ron receptor tyrosine kinase is over-expressed and over-activated in a cohort of human cancers, with the most compelling data yet found in breast cancer. Specifically, Ron is overexpressed in approximately 50% of human breast cancers, and has been shown to be an independent predictor of both metastases and poor prognosis in women with this disease. While Ron overexpression appears to be an important factor in human breast cancer growth and metastasis, a significant gap exists in our knowledge about the signaling pathways that Ron activates in breast tumors, and about the importance of these pathways with respect to overall tumor growth and metastatic dissemination. Our laboratories have shown that mammary tumors from mice overexpressing Ron selectively in the mammary epithelium exhibit increased levels of the DEK proto-oncogene. In addition, we also show that ligand-induced Ron activation in human and murine breast cancer cell lines induces the accumulation of DEK protein. This accumulation of DEK is significant as DEK overexpression in breast cancer cell lines leads to increases in cell growth and migration while DEK depletion in breast cancer cells leads to dramatic reductions in cell growth and migration. Moreover, we also show that DEK deficient cells are more susceptible to DNA damage. Based on these data, our goal is to test the hypothesis that Ron-mediated DEK upregulation contributes functionally to breast cancer development, dissemination and resistance to clastogenic therapies and that targeting the Ron-DEK signaling axis may represent an important new therapeutic option for the treatment of breast cancer. To test this hypothesis, two Specific Aims were proposed. In Aim 1, we will determine the requirement of DEK in Ron overexpressing breast cancers utilizing a combination of DEK loss of function and Ron transgenic overexpression. Aim 2 will examine the therapeutic utility of targeting Ron and DEK on beta-catenin activation and breast cancer growth., The original document contains color images.

During this funding period, we performed TRAIL-TZD-mediated apoptotic studies in androgen dependent (LNCaP and 22RV1) and androgen independent (DU145 and PC3) prostate cancer cells and the response was maximal in the LNCaP cells with 50 M TZD and 100ng/ml TRAIL combination. This apoptotic response is also associated with increased -catenin cleavage, indicating its potential role. Knockdown of -catenin expression and overexpression of -catenin modulated TRAIL-TZD-induced apoptosis. Studies with the cleaved -catenin protein revealed that this cleaved fragment losses interaction with TCF4, while retaining strong interaction with E-cadherin and -catenin. These indicated (i) a potential mechanism by which TRAIL-TZD antagonizes -catenin/TCF4-induced transcription and (ii) suggested that E-cadherin and -catenin interaction might be critical for apoptosis induction. Based on Mass Spec data various myc -catenin (D/A) mutants were created but they were unable to significantly antagonize -catenin cleavage and apoptosis. Studies also showed that GSK3 inhibition promotes TRAIL-induced apoptosis and TRAIL-TZD significantly reduces expression of GSK3 and increases pGSK3 Ser9 levels. In addition, this apoptosis pathway seems to involve AMPK, since AMPK-dominant negative (DN) overexpression significantly attenuates TRAIL-TZD-induced apoptosis and -catenin cleavage. The C42 and C42B cells showed significant apoptosis with TRAIL-TZD in vitro and C42 cells formed in vivo xenografts in nude mice. Treatment with TRAIL-TZD in vivo however, didn t show any significant effect on prostate tumor regression., The original document contains color images.

Patients with Neurofibromatosis (NF1) exhibit deficient bone healing/ The cause of poor bone healing in NF1 is unclear, and pharmacologic approaches to improve bone repair are lacking. Beta-catenin is a protein that regulates osteoblasts during bone healing. When beta-catenin protein level is high, it prevents osteoblast differentiation, and undifferentiated fibroblast-like cells persist at the fracture site, resulting in a pseudarthrosis. Genetically engineered mice in which the Nf1 gene can be deleted when cells are exposed to Cre-Recombinase were studied. An adenovirus expressing Cre-Recombinase was injected to the fracture site to knock out the gene. Mice in which the beta-catenin gene can be knocked out by exposure to Cre-Recombinase were used to decrease beta-catenin during fracture repair. An open tibial fracture, fixed with an intramedullar pin, was used to study fracture healing. Five to ten mice were studied in each group at each time point, and fracture repair assessed at three and six weeks using radiology and histology. Beta-catenin protein level during fracture repair in mice lacking the Nf1 gene measured five times higher than normal. Mice lacking Nf1 gene showed deficient fracture repair. In contrast, mice lacking Nf1 gene, but that also express a low level of beta-catenin healed their tibia fracture quicker and with more bone as measured using both radiographic and histologic parameters. This work so far shows that beta-catenin protein is elevated during fracture repair in mice lacking Nf1. Inhibition of beta-catenin can improve the quality of the bone repair process., The original document contains color images.

Glycogen synthase kinase 3 beta (GSK3 beta) and cyclin-dependent kinase 5 (CDK5) are tau kinases and have been proposed to contribute to the pathogenesis of Alzheimer's disease. The 3D structures of these kinases are remarkably similar, which led us to hypothesize that both might be capable of binding cyclin proteins-the activating cofactors of all CDKs. CDK5 is normally activated by the cyclin-like proteins p35 and p39. By contrast, we show that GSK3 beta does not bind to p35 but unexpectedly binds to p25, the calpain cleavage product of p35. Indeed, overexpressed GSK3 beta outcompetes CDK5 for p25, whereas CDK5 is the preferred p35 partner. FRET analysis reveals nanometer apposition of GSK3 beta:p25 in cell soma as well as in synaptic regions. Interaction with p25 also alters GSK3 beta substrate specificity. The GSK3 beta:p25 interaction leads to enhanced phosphorylation of tau, but decreased phosphorylation of beta-catenin. A partial explanation for this situation comes from in silico modeling, which predicts that the docking site for p25 on GSK3 beta is the AXIN-binding domain; because of this, p25 inhibits the formation of the GSK3 beta/AXIN/APC destruction complex, thus preventing GSK3 beta from binding to and phosphorylating beta-catenin. Coexpression of GSK3 beta and p25 in cultured neurons results in a neurodegeneration phenotype that exceeds that observed with CDK5 and p25. When p25 is transfected alone, the resulting neuronal damage is blocked more effectively with a specific siRNA against Gsk3 beta than with one against Cdk5. We propose that the effects of p25, although normally attributed to activate CDK5, may be mediated in part by elevated GSK3 beta activity.

Objective It has been proposed that the histamine 1 (H-1) receptor not only promotes allergic reactions but also modulates autoimmune diseases, such as type 1 diabetes. In line with this, it has recently been reported that the H-1-receptor antagonist cetirizine can counteract the activation of signals/factors pertinent to the pathogenesis of type 1 diabetes and cytokine-induced beta-cell destruction. Therefore, the overall aim of this study was to determine whether H-1-receptor antagonists affect cytokine-induced beta-cell death and signaling in vitro. Methods The insulin-producing cell line beta-TC6 was exposed to the proinflammatory cytokines interleukin 1 beta(+) interferon gamma, or hydrogen peroxide. The H-1-receptor antagonists desloratadine and cetirizine were added to the cell cultures and cell viability; macrophage inhibitory factor levels, c-Jun N-terminal kinase phosphorylation, c-Jun expression, and beta-catenin levels were analyzed by flow cytometry, real-time polymerase chain reaction, and immunoblotting. Results Cetirizine protected partially against both cytokine- and hydrogen peroxide-induced cell death. This effect was paralleled by an inhibition of cytokine-induced c-Jun N-terminal kinase phosphorylation, c-Jun induction, and a restoration of macrophage inhibitory factor contents. Cetirizine also increased the beta-TC6 cell contents of beta-catenin at basal conditions. Conclusions Our results indicate a protective effect of a specific H-1-receptor antagonist.

UNLABELLED: Leukocyte cell-derived chemotoxin 2 (LECT2) has been shown to act as a tumor suppressor in hepatocellular carcinoma (HCC). However, the underlying mechanism has not yet been completely defined. Here, we employ a LECT2-affinity column plus liquid chromatography coupled with tandem mass spectrometry to identify LECT2-binding proteins and found that MET receptor strongly interacted with LECT2 protein. Despite the presence of hepatocyte growth factor, the LECT2 binding causes an antagonistic effect to MET receptor activation through recruitment of protein tyrosine phosphatase 1B. The antagonistic effect of LECT2 on MET activation also mainly contributes to the blockage of vascular invasion and metastasis of HCC. Furthermore, serial deletions and mutations of LECT2 showed that the HxGxD motif is primarily responsible for MET receptor binding and its antagonistic effects. CONCLUSION: These findings reveal a novel, specific inhibitory function of LECT2 in HCC by the direct binding and inactivation of MET, opening a potential avenue for treating MET-related liver cancer.

PURPOSE: The extraocular muscles (EOM) and their motor neurons are spared in amyotrophic lateral sclerosis (ALS). In limb muscle axon retraction from the neuromuscular junctions occurs early in the disease. Wnts, a conserved family of secreted signaling molecules, play a critical role in neuromuscular junction formation. This is the first study to examine Wnt signaling for its potential involvement in maintenance of normal morphology in EOMs in ALS. METHODS: EOM and limb muscle axons, neuromuscular junctions, and myofibers from control, aging, and ALS patients and the SOD1G93A mouse model of ALS were quantified for their expression of Wnt1, Wnt3a, Wnt5a, Wnt7a, and beta-catenin. RESULTS: All four Wnt isoforms were expressed in most axon profiles in all human EOMs. Significantly fewer were positive for Wnt1, Wnt3a, and Wnt7a in the human limb muscles. Similar differential patterns in Wnt myofiber expression was also seen, except for Wnt7a, where expression was elevated. In the SOD1G93A mouse, all 4 Wnt isoforms were significantly decreased in the neuromuscular junctions at the terminal stage compared to age matched controls. Beta-catenin was activated in a subset of myofibers in EOM and limb muscle in all patients. CONCLUSIONS: The differences in Wnt expression in EOM and limb muscle, particularly at the neuromuscular junction level, suggest that they play a role in the pathophysiology of ALS. Collectively, the data support a role for Wnt signaling in the preservation of the EOM in ALS and their dysregulation and the subsequent development of pathology in the ALS limb muscles.

BackgroundDeregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells.ResultsWe observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis.ConclusionOur data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

Because of their pluripotent nature, as defined by their self-renewal and differentiation capacity, mouse embryonic stem cells (mESCs) have become an invaluable resource for unraveling the underlying mechanisms that control developmental programs. Understanding the formation and maintenance of bone tissue will undoubtedly facilitate the creation of new therapeutic approaches that address bone-related pathologies. Numerous reports have demonstrated the importance of the Wnt signaling pathway in regulating osteogenesis, however, given the revealed complexity of this regulation further elucidation of the time- and cell-specific response is needed. The in vitro culture of mESCs allows investigators to examine multiple stages of differentiation while simultaneously manipulating this signaling pathway in a time-dependent manner. Through the use of known modulators and activity reporters, studying beta-catenin (CTNNB1) (i.e. the central effector of canonical Wnt signaling) will provide invaluable insights towards a greater understanding of osteogenic processes. For instance, we have found that supplementation of the non-canonical Wnt ligand Wnt5a, during a narrow time window of osteoblast precursor formation, was able to improve osteogenic differentiation at later stages. This supplementation resulted in increased activation of multiple downstream effectors and was also correlated with a decrease in CTNNB1-dependent LEF/TCF transcriptional activity. Interestingly, when studying the impact of high concentrations of glucose within culture media, we found a decrease in osteogenic differentiation, which corresponded to an upregulation of CTNNB1-dependent LEF/TCF reporter activity within the same time period. This change in transcriptional activity was linked to differential activation of the AKT/FOXO signaling pathway. Thus, our work continues to reinforce the dynamic role of CTNNB1 in regulating in vitro osteogenesis. With this knowledge we employed multi-stage supplementatio

Embryonic development and adult homeostasis are dependent upon the coordinated action of signal transduction pathways such as the Wnt signalling pathway which is used iteratively during these processes. In the early post-implantation mouse embryo, Wnt/beta-catenin signalling activity plays a critical role in the formation of the primitive streak, progression of gastrulation and tissue patterning in the anterior posterior axis. The net output of the signalling pathway is influenced by the delivery and post-translational modification of the ligands, the counteracting activities of the activating components and the negative modulators, and the molecular interaction of beta-catenin, TCF and other factors regulating the transcription of downstream target genes.

Colorectal cancer (CRC) is the third most common cancer globally, with approximately 1.2 million new cases every year. The highest incidence rates are seen in developed countries, thereby imposing dietary and lifestyle factors in the etiology of CRC. Accumulating epidemiological evidence suggests that obesity is a risk factor for CRC in particular in men, as weak or no associations are seen in women. The reason for this sex difference is not fully understood, but hormonal factors are suggested to play an important role. CRC is a largely heterogenous disease, and colorectal carcinogenesis can be regarded as a complex process involving multiple genetic and epigenetic alterations engendering tumours with different clinicopathological features. The aims of this thesis were to investigate the associations between obesity, measured as different anthropometric factors, and risk of CRC according to clinocopathological and tumour biological characteristics in men and women, respectively. In addition, the relationship between use of postmenopausal homone therapy (HRT) and oral contraceptives (OC) with CRC risk was examined. All papers are based on tumours from incident CRC in the Malmö Diet and Cancer study, a large prospective population based cohort including 28098 individuals. Baseline examinations comprised anthropometric measurements, questionnaire on medications, socioeconomic- and lifestyle related factors. By the end of follow-up in 2008, 584 cases of incident CRC had been registered. Using the tissue microarray technique (TMA), immunohistochemical (IHC) expression of beta-catenin, cyclin D1, p53 and microsatellite instability (MSI) status of the tumours was investigated. KRAS/BRAF mutation status was assessed by pyrosequencing. Interestingly, we found that obesity was associated with an increased risk of more advanced CRC, i.e. tumour (T)-stage 3 and 4, lymph node positive and metastatic disease, predominantly in men. Further, obesity was associated with an overall i