Scientific Papers - 2012

Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma: Activating mutations in the gene encoding β-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of β-catenin (β-cat(nc)) and over-activation of the Wnt/β-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the β-cat(nc) cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these β-cat(nc) cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the β-cat(nc) cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours. (Published in Acta Neuropathol, 2012 Feb 18. [Epub ahead of print])

Mitochondrial T3 receptor p43 regulates insulin secretion and glucose homeostasis: Thyroid hormone is a major determinant of energy expenditure and a key regulator of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine receptor (p43) that acts as a mitochondrial transcription factor of the organelle genome, which leads, in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Here we generated mice specifically lacking p43 to address its physiological influence. We found that p43 is required for normal glucose homeostasis. The p43(-/-) mice had a major defect in insulin secretion both in vivo and in isolated pancreatic islets and a loss of glucose-stimulated insulin secretion. Moreover, a high-fat/high-sucrose diet elicited more severe glucose intolerance than that recorded in normal animals. In addition, we observed in p43(-/-) mice both a decrease in pancreatic islet density and in the activity of complexes of the respiratory chain in isolated pancreatic islets. These dysfunctions were associated with a down-regulation of the expression of the glucose transporter Glut2 and of Kir6.2, a key component of the K(ATP) channel. Our findings establish that p43 is an important regulator of glucose homeostasis and pancreatic β-cell function and provide evidence for the first time of a physiological role for a mitochondrial endocrine receptor. (Published in FASEB Journal, Volume 26, Issue 1, pp. 40-50, Jan 2012)

MALDI imaging mass spectrometry reveals COX7A2, TAGLN2 and S100-A10 as novel prognostic markers in Barrett's adenocarcinoma: To characterize proteomic changes found in Barrett's adenocarcinoma and its premalignant stages, the proteomic profiles of histologically defined precursor and invasive carcinoma lesions were analyzed by MALDI imaging MS. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett's adenocarcinoma patient tissue samples was used. The goal was to find proteins that might be used as markers for monitoring cancer development as well as for predicting regional lymph node metastasis and disease outcome. Using mass spectrometry for protein identification and validating the results by immunohistochemistry on an independent validation set, we could identify two of 60 differentially expressed m/z species between Barrett's adenocarcinoma and the precursor lesion: COX7A2 and S100-A10. Furthermore, among 22m/z species that are differentially expressed in Barrett's adenocarcinoma cases with and without regional lymph node metastasis, one was identified as TAGLN2. In the validation set, we found a correlation of the expression levels of COX7A2 and TAGLN2 with a poor prognosis while S100-A10 was confirmed by multivariate analysis as a novel independent prognostic factor in Barrett's adenocarcinoma. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. This article is part of a Special Issue entitled: Translational Proteomics. (Published in J Proteomics [Epub ahead of print], Feb 17, 2012)

Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling: AIMS/HYPOTHESIS: Knowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size.
METHODS: Electron micrographs of rat beta cells fixed either chemically or by high-pressure freezing were compared using a high-content analysis approach. These data were used to develop three-dimensional in silico beta cell models, the slicing of which would reproduce the experimental datasets.
RESULTS: As previously reported, chemically fixed insulin secretory granules appeared as hollow spheres with a mean diameter of ∼350 nm. Remarkably, most granules fixed by high-pressure freezing lacked the characteristic halo between the dense core and the limiting membrane and were smaller than their chemically fixed counterparts. Based on our analyses, we conclude that the mean diameter of rat insulin secretory granules is 243 nm, corresponding to a surface area of 0.19 μm(2). Rat beta cells have a mean volume of 763 μm(3) and contain 5,000-6,000 granules.
CONCLUSIONS/INTERPRETATION: A major reason for the lower mean granule number/rat beta cell relative to previous accounts is a reduced estimation of the mean beta cell volume. These findings imply that each granule contains about twofold more insulin, while its exocytosis increases membrane capacitance about twofold less than assumed previously. Our integrated approach defines new standards for quantitative image analysis of beta cells and could be applied to other cellular systems. (Published in Diabetologia, Jan 18, 2012 [Epub ahead of print])

Bone healing around titanium implants in two rat colitis models: Objective: Crohn's disease is a chronic inflammatory process that has recently been associated with a higher risk of early implant failure. Herein we provide information on the impact of colitis on peri-implant bone formation using preclinical models of chemically induced colitis.
Methods: Colitis was induced by intrarectal instillation of 2,4,6-trinitro-benzene-sulfonic-acid (TNBS). Colitis was also induced by feeding rats dextran-sodium-sulfate (DSS) in drinking water. One week after disease induction, titanium miniscrews were inserted into the tibia. Four weeks after implantation, peri-implant bone volume per tissue volume (BV/TV) and bone-to-implant contacts (BIC) were determined by histomorphometric analysis.
Results: Cortical histomorphometric parameters were similar in the control (n = 10), DSS (n = 10) and TNBS (n = 8) groups. Cortical BV/TV was 92.2 ± 3.7%, 92.0 ± 3.0% and 92.6 ± 2.7%. Cortical BIC was 81.3 ± 8.8%, 83.2 ± 8.4% and 84.0 ± 7.0%, respectively. No significant differences were observed when comparing the medullary BV/TV and BIC (19.5 ± 6.4%, 16.2 ± 5.6% and 15.4 ± 9.0%) and (48.8 ± 12.9%, 49.2 ± 6.2 and 41.9 ± 11.7%), respectively. Successful induction of colitis was confirmed by loss of body weight and colon morphology.
Conclusions: The results suggest bone regeneration around implants is not impaired in chemically induced colitis models. Considering that Crohn's disease can affect any part of the gastrointestinal tract including the mouth, our model only partially reflects the clinical situation. (Published in Clin Oral Implants Res, 2012 Apr 18. [Epub ahead of print])

Late motor decline after accomplished remyelination: Impact for progressive multiple sclerosis: OBJECTIVE: To investigate the impact of single or repeated episodes of reversible demyelination on long-term locomotor performance and neuroaxonal integrity, and to analyze the myelin proteome after remyelination and during aging.
METHODS: Long-term locomotor performance of previously cuprizone-treated animals was monitored using the motor skill sequence (MOSS). Quantitative analysis of myelin proteome and histopathological analysis of neuronal/axonal integrity was performed after successful remyelination. Histopathological findings observed in experimental chronic remyelinated lesions were verified in chronic remyelinated lesions from multiple sclerosis (MS) patients.
RESULTS: Following cessation of cuprizone treatment, animals showed an initial recovery of locomotor performance. However, long after remyelination was completed (approximately 6 months after the last demyelinating episode), locomotor performance again declined in remyelinated animals as compared to age-matched controls. This functional decline was accompanied by brain atrophy and callosal axonal loss. Furthermore, the number of acutely damaged amyloid precursor protein-positive (APP+) axons was still significantly elevated in long-term remyelinated animals as compared to age-matched controls. Confocal analysis revealed that a substantial proportion of these APP+ spheroids were ensheathed by myelin, a finding that was confirmed in the chronic remyelinated lesions of MS patients. Moreover, quantitative analysis of myelin proteome revealed that remyelinated myelin displays alterations in composition that are in some aspects similar to the myelin of older animals.
INTERPRETATION: We propose that even after completed remyelination, axonal degeneration continues to progress at a low level, accumulating over time, and that once a threshold is passed axonal degeneration can become functionally apparent in the long-term. The presented model thus mimics some of the aspects of axonal degeneration in chronic progressive MS. (Published in Ann Neurol, Volume 71, Issue 2, pp. 227-44, Feb 2012)

Erythropoietin Receptor Signaling Is Membrane Raft Dependent: Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. (Published in PLoS One, Volume 7, Issue 4, e34477, Apr 3, 2012)

Measurement of tumor volumes improves RECIST-based response assessments in advanced lung cancer: Objective: This study was designed to (1) estimate the reproducibility of measurement for tumor volumes their longest tumor diameters (LDs); (2) estimate the potential impact of using changes in tumor volumes instead of LDs as the basis for response assessments; and (3) create a data set to help qualify image analysis tools.
Methods: We studied patients with advanced lung cancer who had been followed longitudinally with x-ray computed tomography (CT) in a multi-national trial. A total of 71 time-points from 10 patients with 13 morphologically complex target lesions were analyzed.  A total of 6,461 volume measurements and their corresponding LDs were made by 7 independent teams using their own work flows and image analysis tools.  Inter-team agreement and overall inter-rater concurrence were characterized. 
Results: Inter-team agreement between volume measurements was better than between LD measurements (Iota = 0.945 v. 0.734, p = 0.005).  Variability in determining the nadir was lower for volumes than for LDs (p = 0.005).  Use of standard thresholds for the RECIST-based method and use of experimentally determined cutoffs for categorizing responses showed that volume measurements had a significantly greater sensitivity for detecting partial responses and disease progression. Earlier detection of progression would have led to earlier changes in patient management in a majority of cases.
Conclusions: Our findings indicate that measurement of changes in tumor volumes is adequately reproducible.  Using tumor volumes as the basis for response assessments could have a positive impact on both patient management and clinical trials. The value of volume measurements as criteria for lung tumor response assessments should be further explored.
(Published in Translational Oncology, Volume 5, Issue 1, pp. 19-25, Feb 2012)

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres: Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. (Published in EMBO J [Epub ahead of print], Mar 13, 2012)

Resolving Equivocal HER2 Status in Breast Cancer by Automated and Quantitative RNA Chromogenic In Situ Hybridization (CISH): Background: Fluorescence in situ hybridization (FISH) for HER2 gene amplification and immunohistochemistry (IHC) for HER2 protein overexpression both can generate “equivocal” HER2 results (FISH HER2/CEP17 ratio 1.8-2.2 or IHC score 2+), and some cases generate equivocal results by both methods. This study explores the potential of a novel automated and quantitative HER2 mRNA CISH assay based on the recently developed RNAscope technology for resolving equivocal HER2 status.
Design: Formalin-fixed, paraffin-embedded (FFPE) breast cancer specimens from a non- consecutive series of 73 cases were analyzed for HER2 mRNA using a fully automated RNAscope CISH assay. There were 30 negative and 22 positive cases based on the combined FISH and IHC results, and 21 cases were equivocal by both methods (“dual equivocals”). These cases were preallocated into a training set (n=38) and a validation set (n=35). Automated image analysis of HER2 mRNA staining was used to count
the number of punctate “dots” per cell, each dot corresponding to a single HER2 RNA transcript. and correlated to HER2 FISH, IHC and HER2 mRNA RT-PCR. A probabilistic linear discriminant analysis model for HER2 status based on the training set was built and applied to the validation set.
Results: Evaluable HER2 mRNA CISH results were obtained for 67 cases ( 92%). HER2 mRNA dots per cell correlated strongly to FISH (Spearman r=0.81) and RT-PCR (r=0.86) and definitively separated HER2 positive and negative cases in the training set. Apredictive model based on HER2 mRNA dots/cell in the training set correctly identified 11/12 negative and 5/5 positive cases (concordance=94%) in the validation set. The only discrepant case was reviewed and found to have both amplified ductal carcinoma in situ (DCIS) and Non-amplified invasive components; the HER2 RNA CISH results were from the DCIS. When analyzed on the invasive component only, this case was correctly classified as HER2 negative. The model classified all 16 double equivocal cases in the validation set into positives (n=2) and negatives (n=14).
Conclusions: This quantitative HER2 mRNA CISH assay was highly accurate in assessing HER2 status and may provide an effective means to resolve FISH/IHC dual equivocal cases. The walk-away automation and image analysis-based quantification should minimize both analytical and post-analytical variability in HER2 testing. Quantification of single RNA transcripts in situ in analysis routine clinical specimens demonstrates great potential in predictive biomarker analysis. (Poster presented at the USCAP 2012, Vancouver, Canada)