WNT Signaling in Drosophila Neuromuscular Junctions

The Wnt -Wingless (Wg) in Drosophila- signaling is an evolutionary conserved, fundamental signal transduction pathway in animals, having a crucial role in early developmental processes. In the adult animal the Wnt cascade is mainly shut off; aberrant activation leads to cancer. One physiological exception in the adult animal is the activation of Wnt signaling in the nervous system. In the present work, we investigated Wg signaling in the Drosophila neuromuscular junctions (NMJs). The fly NMJs closely resemble the glutamatergic synapses in the mammalian central nervous system and serves as a model system to investigate the mechanism of synapse formation and stability. We demonstrate that the trimeric G-protein Go has a fundamental role in the presynaptic cell in the NMJ. It is implicated in the presynaptic Wg pathway, acting downstream of the ligand Wg and its receptor Frizzled2 (Fz2). Furthermore, we prove that the presynaptic Wg-Fz2-Gαo pathway is essential for correct NMJ formation. The neuronal protein Ankyrin2 (Ank2) localizes to the NMJ and has so far been considered to be a static player in NMJ formation, linking the plasma membrane to the cytoskeleton. We identify Ank2 as a direct target of Gαo. The physical and genetic interaction of Gαo with Ank2 represents a novel branch of the presynaptic Wg pathway, regulating the microtubule cytoskeleton in NMJ formation, jointly with the previously established Futsch-dependent branch, which controls microtubule stability downstream of the kinase Sgg (the homolog of GSK3ß). We moreover demonstrate that the Gαo-Ankyrin interaction to regulate the cytoskeleton is conserved in mammalian neuronal cells. Our findings therefore provide a novel, universally valid regulation of the cytoskeleton in the nervous system. Aberrant inactivation of the neuronal Wnt pathway is believed to be involved in the pathogenesis of the Aß peptide in Alzheimer's disease (AD). We modeled AD in Drosophila by expressing Aß42 in the nervous system and in the eye. Neuronal expression drastically shortens the life span of the flies. We prove that this effect depends on the expression specifically in glutamatergic neurons. However, Aß42 does not induce any morphological changes in the NMJ; therefore this synapse is not suitable to study the mechanism of Aß42 induced neurotoxicity. We furthermore demonstrate that genetic activation of the Wnt pathway does not rescue the Aß42 induced phenotypes - in opposition to the dominating view in the field. These results advice caution when interpreting data on the potential interaction of Wnt signaling and AD in other models. -- La voie de signalisation Wnt (Wingless (Wg) chez la drosophile) est conservée dans l'évolution et fondamentale pour le développement des animaux. Cette signalisation est normalement inactive chez l'animal adulte; une activation anormale peut provoquer le cancer. Or, ceci n'est pas le cas dans le système nerveux des adultes. La présente thèse avait pour but d'analyser le rôle de la voie de signalisation Wingless dans la plaque motrice de Drosophila melanogaster. En effet, cette plaque ressemble fortement aux synapses glutaminergiques du système nerveux central des mammifères et procure ainsi un bon modèle pour l'étude des mécanismes impliqués dans la formation et la stabilisation des synapses. Nos résultats montrent que la protéine trimérique Go joue un rôle fondamental dans la fonction de la cellule présynaptique de la plaque motrice. Go est en effet impliqué dans la voie de signalisation Wg, opérant en aval du ligand Wg et de son récepteur Frizzled2. Nous avons pu démontrer que cette voie de signalisation Wg-Fz2-Gαo est essentielle pour le bon développement et le fonctionnement de la plaque motrice. Fait intéressant, nous avons montré que la protéine neuronale Ankyrin2 (Ank2), qui est connue pour jouer un rôle statique en liant la membrane plasmique au cytosquelette dans la plaque motrice, est une cible directe de Gαo. L'interaction physique et génétique entre Gαo et Ank2 constitue ainsi une bifurcation de la voie de signalisation présynaptique Wg. Cette voie régule le cytosquelette des microtubules en coopération avec la branche liée à la protéine Futsch. Cette protéine est l'homologue de la protéine liant les microtubules MAP1B des mammifères et contrôle la stabilité des microtubules opérant en aval de la kinase Sgg (l'homologue de GSK3ß). De plus, la régulation du cytosquelette par l'interaction entre Gαo et Ankyrin est conservée chez les mammifères. Dans leur ensemble, nos résultats ont permis d'identifier un nouveau mode de régulation du cytosquelette dans le système nerveux, probablement valable de manière universelle. La voie de signalisation Wnt est soupçonnée d'être impliquée dans la toxicité provoquée par le peptide Aß dans le cadre de la maladie d'Alzheimer. Nous avons tenté de modéliser la maladie chez la drosophile en exprimant Aß42 spécifiquement dans le cerveau. Cette expérience a montré que l'expression neuronale d'Aß42 réduit la durée de vie des mouches de manière significative par un mécanisme impliquant les cellules glutamatergiques. Par contre, aucune modification morphologique n'est provoquée par Aß42 dans les plaques motrices glutamatergiques. Ces résultats montrent que ce modèle de Drosophile n'est pas adéquat pour l'étude de la maladie d'Alzheimer. De plus, l'activation génétique de la voie de signalisation Wg n'a pas réussi à restaurer les phénotypes de survie ou ceux des yeux causés par Aß42. Ces résultats indiquent que l'implication de la voie de signalisation Wg dans la maladie d'Alzheimer doit être considérée avec prudence

Completeness of case ascertainment and survival time error in English cancer registries: impact on 1-year survival estimates

BACKGROUND: It has been suggested that cancer registries in England are too dependent on processing of information from death certificates, and consequently that cancer survival statistics reported for England are systematically biased and too low. METHODS: We have linked routine cancer registration records for colorectal, lung, and breast cancer patients with information from the Hospital Episode Statistics (HES) database for the period 2001-2007. Based on record linkage with the HES database, records missing in the cancer register were identified, and dates of diagnosis were revised. The effects of those revisions on the estimated survival time and proportion of patients surviving for 1 year or more were studied. Cases that were absent in the cancer register and present in the HES data with a relevant diagnosis code and a relevant surgery code were used to estimate (a) the completeness of the cancer register. Differences in survival times calculated from the two data sources were used to estimate (b) the possible extent of error in the recorded survival time in the cancer register. Finally, we combined (a) and (b) to estimate (c) the resulting differences in 1-year cumulative survival estimates. RESULTS: Completeness of case ascertainment in English cancer registries is high, around 98-99%. Using HES data added 1.9%, 0.4% and 2.0% to the number of colorectal, lung, and breast cancer registrations, respectively. Around 5-6% of rapidly fatal cancer registrations had survival time extended by more than a month, and almost 3% of rapidly fatal breast cancer records were extended by more than a year. The resulting impact on estimates of 1-year survival was small, amounting to 1.0, 0.8, and 0.4 percentage points for colorectal, lung, and breast cancer, respectively. INTERPRETATION: English cancer registration data cannot be dismissed as unfit for the purpose of cancer survival analysis. However, investigators should retain a critical attitude to data quality and sources of error in international cancer survival studies

Predicting death from surgery for lung cancer: a comparison of two scoring systems in two European countries

Objectives: Current British guidelines advocate the use of risk prediction scores such as Thoracoscore to estimate mortality prior to radical surgery for non-small cell lung cancer (NSCLC). A recent publication used the National Lung Cancer Audit (NLCA) to produce a score to predict 90 day mortality (NLCA score). The aim of this study is to validate the NLCA score, and compare its performance with Thoracoscore. Materials and methods: We performed an internal validation using 2858 surgical patients from NLCA and an external validation using 3191 surgical patients from the Danish Lung Cancer Registry (DLCR). We calculated the proportion that died within 90 days of surgery. The discriminatory power of both scores was assessed by a receiver operating characteristic (ROC) and an area under the curve (AUC) calculation. Results: Ninety day mortality was 5% in both groups. AUC values for internal and external validation of NLCA score and validation of Thoracoscore were 0.68 (95% CI 0.63–0.72), 0.60 (95% CI 0.56–0.65) and 0.60 (95% CI 0.54–0.66) respectively. Post-hoc analysis was performed using NLCA records on 15554 surgical patients to derive summary tables for 30 and 90 day mortality, stratified by procedure type, age and performance status. Conclusions: Neither score performs well enough to be advocated for individual risk stratification prior to lung cancer surgery. It may be that additional physiological parameters are required; however this is a further project. In the interim we propose the use of our summary tables that provide the real-life range of mortality for lobectomy and pneumonectomy

Short-term breast cancer survival in relation to ethnicity, stage, grade and receptor status: national cohort study in England.

In the re-organisation of cancer registration in England in 2012, a high priority was given to the recording of cancer stage and other prognostic clinical data items. We extracted 86 852 breast cancer records for women resident in England and diagnosed during 2012-2013. Information on age, ethnicity, socio-economic status, comorbidity, tumour stage, grade, morphology and oestrogen, progesterone and HER2 receptor status was included. The two-year cumulative risk of death from any cause was estimated with the Kaplan-Meier method, and univariate and multivariate Cox proportional hazards regressions were used to estimate hazard ratios (HR) and their 95% confidence intervals (95% CI). The follow-up ended on 31 December 2014. The completeness of registration for prognostic variables was generally high (around 80% or higher), but it was low for progesterone receptor status (41%). Women with negative receptor status for each of the oestrogen, progesterone and HER2 receptors (triple-negative cancers) had an adjusted HR for death of 2.00 (95%CI 1.84-2.17). Black women had an age-adjusted HR of 1.77 (1.48-2.13) compared with White women. The excess mortality of Black women with breast cancer has contributions from socio-economic factors, stage distribution and tumour biology. The study illustrates the richness of detail in the national cancer registration data. This allows for analysis of cancer outcomes at a high level of resolution, and may form the basis for risk stratification.British Journal of Cancer advance online publication, 25 October 2016; doi:10.1038/bjc.2016.335 www.bjcancer.com

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

BACKGROUND: The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. METHODS: In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. RESULTS: Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CONCLUSION: CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis

The evolution of colorectal cancer suggests the involvement of many genes. We performed insertional mutagenesis with the Sleeping Beauty (SB) transposon system in mice carrying germline or somatic Apc mutation. Analysis of common insertion sites (CISs) isolated from 446 tumors revealed many hundreds of candidate cancer drivers. Comparison to human datasets suggested that 234 CIS genes are also deregulated in human colorectal cancers. 183 CIS genes are candidate Wnt targets, and 20 are shown to be novel modifiers of canonical Wnt signaling. We also identified gene mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included members of the FGF signaling cascade. Some 70 genes showed pairwise co-occurrence clustering into 38 sub-networks that may regulate tumor development

Lung adenocarcinoma promotion by air pollutants

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 μm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1β. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for PM2.5 air pollutants and provide impetus for public health policy initiatives to address air pollution to reduce disease burden

Dietary factors, genetic susceptibility and somatic mutations in colorectal cancer: a prospective study.

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