Aryl hydrocarbon receptor (AHR) is a potential tumour suppressor in pituitary adenomas

Pituitary adenomas (PA) represent the largest group of intracranial neoplasms and yet the molecular mechanisms driving this disease remain largely unknown. The aim of this study was to use a high-throughput screening method to identify molecular pathways that may be playing a significant and consistent role in PA. RNA profiling using microarrays on eight local PAs identified the aryl hydrocarbon receptor (AHR) signalling pathway as a key canonical pathway downregulated in all PA types. This was confirmed by real-time PCR in 31 tumours. The AHR has been shown to regulate cell cycle progression in various cell types; however, its role in pituitary tissue has never been investigated. In order to validate the role of AHR in PA behaviour, further functional studies were undertaken. Over-expression of AHR in GH3 cells revealed a tumour suppressor potential independent of exogenous ligand activation by benzo α-pyrene (BαP). Cell cycle analysis and quantitative PCR of cell cycle regulator genes revealed that both unstimulated and BαP-stimulated AHR reduced E2F-driven transcription and altered expression of cell cycle regulator genes, thus increasing the percentage of cells in G0/G1 phase and slowing the proliferation rate of GH3 cells. Co-immunoprecipitation confirmed the interaction between AHR and retinoblastoma (Rb1) protein supporting this as a functional mechanism for the observed reduction. Endogenous Ahr reduction using silencing RNA confirmed the tumour suppressive function of the Ahr. These data support a mechanistic pathway for the putative tumour suppressive role of AHR specifically in PA, possibly through its role as a cell cycle co-regulator, even in the absence of exogenous ligands.peer-reviewe

RNASeq of pituitary adenomas reveals dysfunctional metabolic, secretory and differentiation molecular pathways

Pituitary adenomas consist of a group of highly heterogeneous intracranial tumours with variable presentation, clinical prognosis and management. High throughput sequencing was used in order to try and identify common de-regulated pathways and characterize tumours according to specific molecular behaviour. RNA sequencing (RNAseq) was chosen since it provides not only information regarding the expression profile but also the mutational load of each specific tumour analysed. 58 locally resected tumours (36 non-functional tumours; 17 growth hormone-secreting; 3 prolactin-secreting; 2 Cushing’s) were stored in RNAlater (Qiagen, US) and RNA was extracted to purified. RNAseq was performed on all samples plus a control on the BGI-Seq500 platform (Beijing Genomics Ind., China). Bioinformatic analyses was also performed by BGI with additional analyses still being carried out. Preliminary data reveals a number of known and novel de-regulated pathways which characteristically differentiate between different tumour types such as hormone signalling and production pathways. However, novel metabolic pathways also appear to differ significantly, not only between controls and tumours but also between different tumour types with changes in lipid transport and glucose metabolism being observed. Additionally various hormone receptor signalling pathways were also found to be altered. Verification and additional bioinformatic analyses will be required to further delve into the vast data that is generated by this technique which has provided a wealth of information.peer-reviewe

The down-regulated tumour suppressor Wnt inhibitory factor 1 (WIF1) regulates non-canonical Wnt signalling in pituitary adenomas (PA)

The Wnt developmental pathway has been implicated in tumour growth and development in a number of tissues. Most frequently, the canonical Wnt pathway acting through the nuclear translocation of B-catenin has been the key player in driving tumour growth. However, non-canonical Wnt pathways, namely the Wnt-Calcium signalling and the Wnt-planar polarity pathways have also been found de-regulated in a number of cancers. In this study, microarray analysis on locally resected PA revealed strong down-regulation Wnt pathway antagonists, namely the Wnt inhibitory factor 1 (WIF1) and the secreted frizzled-related proteins 2 and 4 (SFRP2 and 4). These results have been confirmed by qPCR and shown that WIF1 is under – expressed in all tumour types while SFRP’s tend to be repressed in functional PAs. The aim of this study was to functionally assess the role of WIF1 in PA in relation to the different Wnt signalling pathways using two established cell lines, the rat sommatotroph/lactrotroph GH3 and prolactinoma MMQ cell lines in the presence of known canonical and non-canonical Wnt ligands, Wnt3, Wnt4 and Wnt5a. WIF1 over-expression reduced significantly GH3 and MMQ cell proliferation using a fluorescent-based Alamar Blue assay, both in the absence and presence of Wnt ligands. However, both Wnt ligands and lithium chloride, an established canonical Wnt pathway activator, failed to activate β-catenin driven transcription using the TOP/FOP flash luciferase system. In fact, canonical Wnt signalling appears to be completely absent in GH3 and MMQ cells. In order to study the influence of Wnt ligands and their inhibitor, WIF1, on other non-canonical Wnt pathways, the Wnt-Calcium signalling pathway was chosen, owing to the important role that calcium signalling plays in regulating hormone release from these cells. Using the FluoForte Calcium assay (Enzo Biologicals, US) to assess free calcium in real-time in the chosen cell lines, we studied the effect of the Wnt ligands in the absence and presence of the WIF1 inhibitor. Wnt ligands activated calcium release with variable potentials with WIF1 displaying an inhibitory but selective role on this effect. Real-time PCR of targets of the canonical and non-canonical Wnt pathways is also being undertaken together with analysis of growth hormone and prolactin secretion from both cell lines. Preliminary data reveals that the Wnt agonists may activate the Wnt-Calcium signalling pathway and WIF1 could play a role in PA by inhibiting specific aspects of this pathway.peer-reviewe

Novel and classical molecular pathways identified in pituitary tumorigenesis using mRNA profiling

Pituitary tumorigenesis has been analysed from multiple perspectives, yet mRNA expression profiling studies are limited. In this study, microarray analysis was used to identify pathways and networks related to pituitary tumour physiology using key de-regulated genes and bioinformatics analysis. Eight pituitary adenomas (five non-functional tumours, two GH-secreting tumours and a TSH/prolactin-secreting tumour) and a pool of random normal control RNA were profiled for RNA expression using the 29 kb Affymetrix HuGene 1.0 ST chip. Microarray data was analysed using GeneSpring GX 11.0 and network analysis was done using the Ingenuity Pathway Analysis (IPA) software. Data obtained from the microarray was verified on 30 tumours (20 non-functional tumours, six GH-secreting, two prolactinomas and two Cushing’s) using quantitative PCR of key genes involved in the networks identified by the IPA. Different analyses between controls and tumour types were carried out. Among the classical networks discovered known to be involved in pituitary tumorigenesis were the cAMP signalling pathway and the Wnt developmental pathway although both networks were driven by genes not previously described in any other study. Different tumour types were also found to be characterised by variable novel de-regulated molecular pathways, such as the GABA signalling and aryl hydrocarbon receptor-signalling pathways in GH-secreting tumours, and the p53 signalling de-regulation in the non-functional tumours. Cluster analysis from microarray data was also able to distinguish between tumour types, identifying one non-functional tumour as belonging to the functional tumours by its mRNA expression profile. This study validates the use for gene expression profiling for the correct characterisation of pituitary adenoma sub-types and identification of key pathways involved in pituitary tumorigenesis thereby proposing possible therapeutic targets.peer-reviewe

Functional analysis of aryl hydrocarbon receptor (AHR) polymorphisms in pituitary adenomas (PAs) in the presence of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

BACKGROUND: PAs are the most frequent pituitary neoplasms, however molecular pathogenesis is largely unknown. The AHR is a ligand-activated transcription factor that regulates expression of various genes that mediate cellular response to xenobiotics. The exact functional role of two AHR single nucleotide polymorphisms (SNPs); Arginine554Lysine (Arg554Lys) and Valine570Isoleucine (Val570Ile) has not yet been established, however studies suggest that these mutations might increase risk of developing PAs. To date, functional analysis of regarding the significance of these AHR SNPs in pituitary pathophysiology has never been analysed.AIMS: • Elucidate the effect of wildtype and polymorphic AHR on GH3 cell proliferation and on AHR-transcriptional response in the presence and absence of TCDD. • Determine the allele frequency of the most common AHR SNP; the Arg554Lys in PA patients and in a small cohort of the Maltese population.METHOD: The two missense mutations were introduced within the AHR-expressing vector and transfected in GH3 cells by magnetofaction, followed by the exposure to TCDD. Cell viability of GH3 transfected cells was measured using the MTT assay. Functional analysis of GH3 transfected cells treated with TCDD was carried out using luciferase assay and real-time PCR to detect and quantify the AHR-transcriptional activity. Genotyping of the Arg554Lys was performed on PA patients and neonatal controls using allele specific PCR. The Mann-Whitney test was used to compare two groups and Kruskall-Wallis test was used to compare three groups or more.RESULTS: In the absence and presence of low TCDD concentrations (1 and 10 nM), over-expression of wildtype AHR (wtAHR) did not affect GH3 cell proliferation. GH3 cells transfected with the AHR mutants did not exhibit any significant differences in their proliferative ability when compared with the wtAHR, both in the presence and absence of TCDD. Luciferase reporter analysis showed that there was a significant difference between the treated and untreated wtAHR (P=0.016), however this difference was not observed between the treated and untreated AHR mutants. Statistically significant difference in Cyp1a1 gene expression analysis was detected between the treated and untreated wtAHR (P=0.021), Arg554Lys (P=0.005) and Val570Ile (P=0.054). Genotyping of the Arg554Lys in patients with PA gave a minor allele frequency (MAF) of 3% vs 0% in neonatal controls.CONCLUSION: Gene expression and quantification analyses of AHR-target genes suggests that these AHR mutants might interfere with AHR target gene expression. Genotyping results suggested that this mutation is quite rare and may be similar to the frequencies of other European populations.peer-reviewe

Over-expression of AIP protein in GH3 cells reduces cAMP signalling and growth hormone secretion

Mutations in the AIP gene have been linked to familial cases of pituitary adenomas (Vierimaa et al, 2006). Analysis of the protein support its role as a tumour suppressor since mutations cause a loss-of-function with reduced protein interactions and over-expression of wild-type (WT) AIP reduces cell proliferation (Leontiou et al, 2008). AIP interacts with a number of interesting proteins, among them are the phosphodiesterases, PDE4A5 and PDE2A, the G proteins, Gαq and Gα13, survivin, RET, nuclear receptors and others (Trivellin & Korbonits, 2011). However, the mechanism by which AIP dysfunction causes increased susceptibility to pituitary adenomas remains unknown. Owing to AIP’s interaction with the phosphodiesterases and G proteins, we investigated the effect of WT and mutant AIP proteins on cAMP signalling and its downstream effectors in cell cultures. WT AIP, R304X-AIP mutant and empty vector (EV) were transfected into GH3 cells. Basal and forskolin – induced cAMP signalling was analyzed using cAMP assays, CRE-promoter luciferase assays, real-time PCR and finally growth hormone (GH) assays. WT AIP was able to reduce forskolin-induced, but not basal, cAMP signaling. Total cAMP, the luciferase activity of cAMP-driven promoter and target gene expression were reduced when compared to EV and R304X mutant. Additionally, analysis of GH secretion which occurs after cAMP cascade activation, was slightly but significantly reduced in WT over-expressing GH3 cells treated with forskolin. Addition of IBMX, a phosphodiesterase inhibitor, did not reverse the effect of AIP on cAMP signalling or GH secretion, indicating that this effect occurs independently of AIP-phosphodiesterase interaction. AIP protein appears inhibit pituitary cells from proliferation by suppressing cAMP production, activation of which is known to cause tumour formation (Lania et al, 2003) and thereby also influencing GH secretion. However, this effect appears not to be mediated by the AIP-phosphodiesterase interaction, suggesting G protein involvement in mediating this outcome.DECLARATION OF INTEREST: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.FUNDING: This work was supported, however funding details unavailable.peer-reviewe

OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.Peer reviewe

Spatial data infrastructures in Malta : state of play 2006

Based on limited information, we have found that in Malta, production, management and dissemination of (rather large scale) spatial reference and core thematic data is almost the exclusive responsibility of the Malta Environmental and Planning Authority (MEPA) in general and the subordinate National Mapping Agency (NMA) in particular. Other organizations which are producing spatial information and conducting spatial information system projects include the Land Registry, the National Statistics Office and the Local councils. The private sector seems to play an increasing role in data production, besides systems development and consultancies. Utilities are also increasingly looking at GIS as a means to develop and manage their business.peer-reviewe

Biomarkers of pituitary adenoma behaviour

The pathological behaviour of pituitary adenomas (PAs) is complex and difficult to predict. In this study, the proliferation marker, Ki-67, pituitary tumour transforming gene (PTTG), vascular endothelial growth factor (VEGF), cyclin D1, c-MYC and pituitary adenylate cyclase-activating peptide (PACAP) protein expression were analyzed using immunohistochemistry in 74 PA samples (48 non-functional PAs, 26 functional PAs) and correlated with tumour characteristics including size, extension and tumour behaviour patterns. Correlation of protein marker expression with clinical characteristics yielded significant results. A correlation between PTTG expression and age at diagnosis, tumour size, tumour regrowth and Ki-67 was observed. Cyclin D1 and c-MYC also showed significant correlations with gender, tumour size, age at diagnosis and other protein markers. Significant differences in protein expression in the chosen markers were also observed between different tumour types, between patients treated pre-operatively with somatostatin analogues and in tumours with different intensity on MR imaging). Significant correlations were also observed between the markers themselves, with a possible direct link between two of the studied markers which substantiate data from other in vitro studies. Differences in protein localization were also analyzed to identify possible differences in biological behaviour arising in relation to nuclear vs cytoplasmic localization of the studied biomarkers. VEGF and PACAP similarly appeared interesting but exhibited few statistically significant correlations on detailed analysis. In conclusion, interesting and novel observations on the differences in expression of tumour markers studied are reported. Specifically, Ki-67 and PTTG appear to be very strongly correlated to tumour regrowth/recurrence and may be considered useful tools in predicting the proliferative potential of the resected tumours. Further data on the differential role of Cyclin D1 and cMYC in pituitary tumorigenesis and possibly tumour prognosis are presented.Principal author (MG, JV) was funded by the University of Malta Research funds (MEDRP02-05) and the Faculty of Medicine and Surgery (MDSIN08-22).RF is funded by the REACH HIGH Scholars Programme – Post-Doctoral Grant. The Research work disclosed in this publication is partially funded by the REACH HIGH Scholars Programme – Post Doctoral Grants. The grant is part-financed by the European Union, Operational Programme II – Cohesion Policy 2014 – 2020 “Investing in human capital to create more opportunities and promote the wellbeing of society” – European Social Fund.peer-reviewe

The kinematics of fixed-seat rowing : a structured synthesis

Olympic-style sliding-seat rowing is a sport that has been extensively researched, with studies investigating aspects related to the physiology, biomechanics, kinematics, and the performance of rowers. In contrast, studies on the more classic form of fixed-seat rowing are sparse. The aim of this study is to address this lacuna by analysing for the first time the specific kinematics of fixed-seat rowing as practised by able-bodied athletes, thus (i) documenting how this technique is performed in a manner that is replicable by others and (ii) showing how this technique compares and contrasts with the more standard sliding-seat technique. Fixed-seat rowing was replicated in a biomechanics laboratory where experienced fixed-seat rowers, marked with reflective markers following the modified Helen–Hayes model, were asked to row in a manner that mimics rowing on a fixed-seat boat. The findings from this study, complimented with data gathered through the observation of athletes rowing on water, were compared to sliding-seat ergometer rowing and other control experiments. The results show that, in fixed-seat rowing, there is more forward and backward thoracic movement than in sliding-seat rowing (75–77° vs. 44–52°, p < 0.0005). Tilting of the upper body stems was noted to result from rotations around the pelvis, as in sliding-seat rowing, rather than from spinal movements. The results also confirmed knee flexion in fixed-seat rowing with a range of motion of 30–35°. This is less pronounced than in standard-seat rowing, but not insignificant. These findings provide a biomechanical explanation as to why fixed-seat rowers do not have an increased risk of back injuries when compared with their sliding-seat counterparts. They also provide athletes, coaches, and related personnel with precise and detailed information of how fixed-seat rowing is performed so that they may formulate better and more specific evidence-based training programs to meliorate technique and performance.peer-reviewe