PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration.

A fundamental property of neural crest (NC) migration is contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibians and zebrafish by controlling cell polarity in a cell contact-dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin, which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial-to-mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. Here, we show that platelet-derived growth factor receptor alpha (PDGFRα) and its ligand platelet-derived growth factor A (PDGF-A) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently, impairing CIL. Moreover, we identify phosphatidylinositol-3-kinase (PI3K)/AKT as a downstream effector of the PDGFRα cellular response during CIL. Our results lead us to propose PDGF-A/PDGFRα signalling as a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, we show that once NC cells have undergone EMT, the same PDGF-A/PDGFRα works as an NC chemoattractant, guiding their directional migration.

A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling.

DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected]

N-terminal modification of VEGF-A C terminus-derived peptides delineates structural features involved in neuropilin-1 binding and functional activity.

The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.

Interstrain differences in chronic hepatitis and tumor development in a murine model of inflammation-mediated hepatocarcinogenesis.

UNLABELLED: Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4(-/-) ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1(-/-) ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background. CONCLUSION: Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain.

Imatinib and Nilotinib increase glioblastoma cell invasion via Abl-independent stimulation of p130Cas and FAK signalling.

Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRß) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility.

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.

C9ORF72 expansions, parkinsonism, and Parkinson disease: a clinicopathologic study.

OBJECTIVE: To determine the histopathologic bases for the observed incidence of parkinsonism in families with C9ORF72 expansions, which typically cause amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia. METHODS: DNA was extracted from 377 brains with the histopathologic diagnosis of idiopathic Parkinson disease or related disorders and analyzed for C9ORF72 expansions. α-Synuclein and p62 immunohistochemistry of the substantia nigra (SN) was undertaken in brains of 17 ALS cases with (C9ORF72+) and 51 without (C9ORF72-) the C9ORF72 expansion. RESULTS: Only 1 of 338 cases with pathologically confirmed idiopathic Parkinson disease had a C9ORF72 expansion. Similarly, only 1 of 17 C9ORF72+ brains displayed features suggestive of α-synucleinopathy. In contrast, p62-positive, TDP-43-negative neuronal cytoplasmic inclusions within the SN were considerably more frequent in C9ORF72+ brain tissue than in the C9ORF72- brains (p = 0.005). Furthermore, there was a more marked loss of dopaminergic neurons in the SN of C9ORF72+ ALS brains than C9ORF72- ALS brains (p = 0.029). CONCLUSIONS: SN involvement is common in C9ORF72+ ALS but can be clearly distinguished from Parkinson disease-related mechanisms by the presence of p62-positive inclusions and the absence of α-synuclein-positive Lewy bodies or Lewy neurites.