Kinetics of Wnt-Driven β-Catenin Stabilization Revealed by Quantitative and Temporal Imaging

The Wnt/β-catenin signal transduction pathway regulates a broad range of developmental processes. Aberrant activation of the Wnt pathway leads to cancer and degenerative diseases. β-catenin is a key signaling molecule that is frequently used as a direct monitor of Wnt pathway activation. This paper describes a multi-parametric method for quantitative analysis of cellular β-catenin protein levels in a rapid and high-throughput manner. The assay offers temporally resolved detection of Wnt-stimulated accumulation of β-catenin, simultaneously detecting cell number, and it sheds light onto the kinetics of posttranslational stabilization of β-catenin

Fate specification and tissue-specific cell cycle control of the <i>Caenorhabditis elegans</i> intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant

Regulation of Epithelial Branching Morphogenesis and Cancer Cell Growth of the Prostate by Wnt Signaling

Although Wnt signaling has been shown to be important for embryonic morphogenesis and cancer pathogenesis of several tissues, its role in prostatic development and tumorigenesis is not well understood. Here we show that Wnt signaling regulated prostatic epithelial branching morphogenesis and luminal epithelial cell differentiation in developing rat prostate organ cultures. Specifically, Wnt signaling regulated the proliferation of prostate epithelial progenitor cells. Assessment of the expression levels of a Wnt pathway transcriptional target gene, Axin2, showed that the Wnt pathway was activated in the developing prostate, but was down-regulated in the adult. Castration resulted in an upregulation of Axin2 whereas androgen replacement resulted in a down regulation of Axin2. Such dynamic changes of Wnt activity was also confirmed in a BAT-gal transgenic mouse line in which β-galactosidase reporter is expressed under the control of β-catenin/T cell factor responsive elements. Furthermore, we evaluated the role of Wnt signaling in prostate tumorigenesis. Axin2 expression was found upregulated in the majority of human prostate cancer cell lines examined. Moreover, addition of a Wnt pathway inhibitor, Dickkopf 1 (DKK1), into the culture medium significantly inhibited prostate cancer cell growth and migration. These findings suggest that Wnt signaling regulates prostatic epithelial ductal branching morphogenesis by influencing cell proliferation, and highlights a role for Wnt pathway activation in prostatic cancer progression

Mathematical and Statistical Techniques for Systems Medicine: The Wnt Signaling Pathway as a Case Study

The last decade has seen an explosion in models that describe phenomena in systems medicine. Such models are especially useful for studying signaling pathways, such as the Wnt pathway. In this chapter we use the Wnt pathway to showcase current mathematical and statistical techniques that enable modelers to gain insight into (models of) gene regulation, and generate testable predictions. We introduce a range of modeling frameworks, but focus on ordinary differential equation (ODE) models since they remain the most widely used approach in systems biology and medicine and continue to offer great potential. We present methods for the analysis of a single model, comprising applications of standard dynamical systems approaches such as nondimensionalization, steady state, asymptotic and sensitivity analysis, and more recent statistical and algebraic approaches to compare models with data. We present parameter estimation and model comparison techniques, focusing on Bayesian analysis and coplanarity via algebraic geometry. Our intention is that this (non exhaustive) review may serve as a useful starting point for the analysis of models in systems medicine.Comment: Submitted to 'Systems Medicine' as a book chapte

WNT signalling in prostate cancer

Genome sequencing and gene expression analyses of prostate tumours have highlighted the potential importance of genetic and epigenetic changes observed in WNT signalling pathway components in prostate tumours-particularly in the development of castration-resistant prostate cancer. WNT signalling is also important in the prostate tumour microenvironment, in which WNT proteins secreted by the tumour stroma promote resistance to therapy, and in prostate cancer stem or progenitor cells, in which WNT-β-catenin signals promote self-renewal or expansion. Preclinical studies have demonstrated the potential of inhibitors that target WNT receptor complexes at the cell membrane or that block the interaction of β-catenin with lymphoid enhancer-binding factor 1 and the androgen receptor, in preventing prostate cancer progression. Some WNT signalling inhibitors are in phase I trials, but they have yet to be tested in patients with prostate cancer

Rho GTPase Cdc42 Is a Direct Interacting Partner of Adenomatous Polyposis Coli Protein and Can Alter Its Cellular Localization

Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC1–1638 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC1–1338 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins

SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Background Transforming growth factor (TGF)-β plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/β-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics associated with breast tumor initiating cells. The phenotypic and genotypic changes that occur in response to SFRP1 loss are consistent with EMT, including a substantial increase in the expression of ZEB2. Considering that ZEB2 has been shown to interact with mediators of TGF-β signaling, we sought to determine whether TGF-β signaling is altered in TERT-siSFRP1 cells. Methods Luciferase reporter assays and real-time PCR analysis were employed to measure TGF-β transcriptional targets. Western blot analysis was used to evaluate TGF-β-mediated ERK1/2 phosphorylation. Migration chamber assays were utilized to quantify cellular migration. TERT-siSFRP1 cells were transfected with Stealth RNAi™ siRNA in order to knock-down the expression of ZEB2. Results TERT-siSFRP1 cells exhibit a significant increase in both TGF-β-mediated luciferase activity as well as TGF-β transcriptional targets, including Integrin β3 and PAI-1. Phosphorylation of ERK1/2 is increased in TERT-siSFRP1 cells in response to enhanced TGF-β signaling. Furthermore, when the TGF-β pathway is blocked with a TGF-βR antagonist (LY364947), cellular migration is significantly hindered. Finally, we found that when ZEB2 is knocked-down, there is a significant reduction in the expression of exogeneous and endogenous TGF-β transcriptional targets and cellular migration is impeded. Conclusions We demonstrate that down-regulation of SFRP1 renders mammary epithelial cells more sensitive to TGF-β signaling which can be partially ameliorated by blocking the expression of ZEB2

Uncovering packaging features of co-regulated modules based on human protein interaction and transcriptional regulatory networks

<p>Abstract</p> <p>Background</p> <p>Network co-regulated modules are believed to have the functionality of packaging multiple biological entities, and can thus be assumed to coordinate many biological functions in their network neighbouring regions.</p> <p>Results</p> <p>Here, we weighted edges of a human protein interaction network and a transcriptional regulatory network to construct an integrated network, and introduce a probabilistic model and a bipartite graph framework to exploit human co-regulated modules and uncover their specific features in packaging different biological entities (genes, protein complexes or metabolic pathways). Finally, we identified 96 human co-regulated modules based on this method, and evaluate its effectiveness by comparing it with four other methods.</p> <p>Conclusions</p> <p>Dysfunctions in co-regulated interactions often occur in the development of cancer. Therefore, we focussed on an example co-regulated module and found that it could integrate a number of cancer-related genes. This was extended to causal dysfunctions of some complexes maintained by several physically interacting proteins, thus coordinating several metabolic pathways that directly underlie cancer.</p

AV-65, a novel Wnt/β-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model

Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that β-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/β-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished β-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery

Voluntary exercise inhibits intestinal tumorigenesis in ApcMin/+ mice and azoxymethane/dextran sulfate sodium-treated mice

<p>Abstract</p> <p>Background</p> <p>Epidemiological studies suggest that physical activity reduces the risk of colon cancer in humans. Results from animal studies, however, are inconclusive. The present study investigated the effects of voluntary exercise on intestinal tumor formation in two different animal models, <it>Apc</it>^Min/+mice and azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice.</p> <p>Methods</p> <p>In Experiments 1 and 2, five-week old female <it>Apc</it>^Min/+mice were either housed in regular cages or cages equipped with a running wheel for 6 weeks (for mice maintained on the AIN93G diet; Experiment 1) or 9 weeks (for mice on a high-fat diet; Experiment 2). In Experiment 3, male CF-1 mice at 6 weeks of age were given a dose of AOM (10 mg/kg body weight, i.p.) and, 12 days later, 1.5% DSS in drinking fluid for 1 week. The mice were then maintained on a high-fat diet and housed in regular cages or cages equipped with a running wheel for 16 weeks.</p> <p>Results</p> <p>In the <it>Apc</it>^Min/+mice maintained on either the AIN93G or the high-fat diet, voluntary exercise decreased the number of small intestinal tumors. In the AOM/DSS-treated mice maintained on a high-fat diet, voluntary exercise also decreased the number of colon tumors. In <it>Apc</it>^Min/+mice, voluntary exercise decreased the ratio of serum insulin like growth factor (IGF)-1 to IGF binding protein (BP)-3 levels. It also decreased prostaglandin E₂and nuclear ��-catenin levels, but increased E-cadherin levels in the tumors.</p> <p>Conclusion</p> <p>These results indicate hat voluntary exercise inhibited intestinal tumorigenesis in <it>Apc</it>^Min/+mice and AOM/DSS-treated mice, and the inhibitory effect is associated with decreased IGF-1/IGFBP-3 ratio, aberrant β-catenin signaling, and arachidonic acid metabolism.</p