New Insights into the Runt Domain of RUNX2 in Melanoma Cell Proliferation and Migration

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy

Effects of GPR139 agonism on effort expenditure for food reward in rodent models: Evidence for pro-motivational actions

Apathy, deficiency of motivation including willingness to exert effort for reward, is a common symptom in many psychiatric and neurological disorders, including depression and schizophrenia. Despite improved understanding of the neurocircuitry and neurochemistry underlying normal and deficient motivation, there is still no approved pharmacological treatment for such a deficiency. GPR139 is an orphan G protein-coupled receptor expressed in brain regions which contribute to the neural circuitry that controls motivation including effortful responding for reward, typically sweet gustatory reward. The GPR139 agonist TAK-041 is currently under development for treatment of negative symptoms in schizophrenia which include apathy. To date, however, there are no published preclinical data regarding its potential effect on reward motivation or deficiencies thereof. Here we report in vitro evidence confirming that TAK-041 increases intracellular Ca2+ mobilization and has high selectivity for GPR139. In vivo, TAK-041 was brain penetrant and showed a favorable pharmacokinetic profile. It was without effect on extracellular dopamine concentration in the nucleus accumbens. In addition, TAK-041 did not alter the effort exerted to obtain sweet gustatory reward in rats that were moderately food deprived. By contrast, TAK-041 increased the effort exerted to obtain sweet gustatory reward in mice that were only minimally food deprived; furthermore, this effect of TAK-041 occurred both in control mice and in mice in which deficient effortful responding was induced by chronic social stress. Overall, this study provides preclinical evidence in support of GPR139 agonism as a molecular target mechanism for treatment of apathy

A potential role of RUNX2- RUNT domain in modulating the expression of genes involved in bone metastases: an in vitro study with melanoma cells

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling

Runx2 stimulates neoangiogenesis through the Runt domain in melanoma

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma

Animal models for novel drug treatments of tauopathies

A transgenic animal model in which tau protein aggregation is expected to produce behavioural symptoms similar to those observed in certain tauopathies in humans has been examined in this thesis. The transgenic mouse lines, when tested in a heterozygous genetic background, showed no cognitive deficit although non-associative learning was impaired. Fine motor learning and motor coordination, however, were severely compromised suggesting disruption of cerebellar and/or basal ganglia function. This model can be used to test tau aggregation inhibitors, that may be effective in preventing, or even reversing the behavioural symptoms caused by tau-aggregation. Since the cholinergic deficit is a feature of Alzheimer’s disease, the effect of the inhibitors described above was tested on the cholinergic system. This was examined using a pharmacological model in which cholinergic deficit was induced by treatment of mice with scopolamine, a competitive antagonist at M1 muscarinic acetylcholine receptors. For the four tau aggregation inhibitors tested in the present work, there were differing efficacies in reversing the scopolamine-induced cognitive deficit. TRx0014 (methylene blue) was the most effective and more so than rivastigmine, a marketed cholinesterase inhibitor. Co-administration of TRx0014 and rivastigmine at sub-effective doses showed a synergistic effect on the reversal of cholinergic deficits. In conclusion, the present work provided a novel transgenic animal model that closely mimics certain behavioural traits typical of some tauopathies. These animals thus provide a valuable model to test novel tau aggregation inhibitors with potentially disease-modifying consequences. The study also demonstrates that such inhibitors, in addition, are able to reverse the symptoms due to cholinergic disruption in normal mice.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Research data supporting "Hilton et al., Reactivity to conditioned threat cues is distinct from exploratory drive in the elevated plus-maze"

To better characterize potential impacts of prior aversive experience on EPM performance a series of experiments were carried out that systematically varied the time since stimulus presentation and the modality of the conditioning stimulus. Furthermore, we examined whether the EPM measurements taken days before or after threat conditioning measurements held any relationship. MATERIALS AND METHODS: Subjects. Subjects were 104 adult male Lister-Hooded rats (Charles River and Envigo, UK) weighing approx. 250–300g at the start of experiments. All animals were housed in groups of four per cage and kept under a reversed 12 h light/dark cycle (lights off 07:00 h until 19:00 h) and were provided with food and water ad libitum, except for during behavioral procedures, which were conducted during the rats’ dark cycle. The rats were randomly assigned to each group and experimenters were blind to group allocation for subsequent analysis. This research was conducted on Project Licence 70/7548 and has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). See materials and methods for full details.This work was funded by a collaborative research grant with Boehringer Ingelheim Pharma GmbH & Co. awarded to E.N.C