Autophagy and mitophagy in cancer metabolic remodelling.

Metabolic reprogramming in tumours is now recognized as a hallmark of cancer, participating both in tumour growth and cancer progression. Cancer cells develop global metabolic adaptations allowing them to survive in the low oxygen and nutrient tumour microenvironment. Among these metabolic adaptations, cancer cells use glycolysis but also mitochondrial oxidations to produce ATP and building blocks needed for their high proliferation rate. Another particular adaptation of cancer cell metabolism is the use of autophagy and specific forms of autophagy like mitophagy to recycle intracellular components in condition of metabolic stress or during anticancer treatments. The plasticity of cancer cell metabolism is a major limitation of anticancer treatments and could participate to therapy resistances. The aim of this review is to report recent advances in the understanding of the relationship between tumour metabolism and autophagy/mitophagy in order to propose new therapeutic strategies.

Automatic segmentation of deep grey nuclei using a high-resolution 7T magnetic resonance imaging atlas-Quantification of T1 values in healthy volunteers.

We present a new consensus atlas of deep grey nuclei obtained by shape-based averaging of manual segmentation of two experienced neuroradiologists and optimized from 7T MP2RAGE images acquired at (.6 mm)3 in 60 healthy subjects. A group-wise normalization method was used to build a high-contrast and high-resolution T1 -weighted brain template (.5 mm)3 using data from 30 out of the 60 controls. Delineation of 24 deep grey nuclei per hemisphere, including the claustrum and 12 thalamic nuclei, was then performed by two expert neuroradiologists and reviewed by a third neuroradiologist according to tissue contrast and external references based on the Morel atlas. Corresponding deep grey matter structures were also extracted from the Morel and CIT168 atlases. The data-derived, Morel and CIT168 atlases were all applied at the individual level using non-linear registration to fit the subject reference and to extract absolute mean quantitative T1 values derived from the 3D-MP2RAGE volumes, after correction for residual B1+ biases. Three metrics (the Dice and the volumetric similarity coefficients and a novel Hausdorff distance) were used to estimate the inter-rater agreement of manual MRI segmentation and inter-atlas variability, and these metrics were measured to quantify biases due to image registration, and their impact on the measurements of the quantitative T1 values was highlighted. This represents a fully automated segmentation process permitting the extraction of unbiased normative T1 values in a population of young healthy controls as a reference for characterizing subtle structural alterations of deep grey nuclei relevant to a range of neurological diseases.

Structural constraints of functional connectivity drive cognitive impairment in the early stages of multiple sclerosis.

BACKGROUND: The relationship between structural and functional deficits in multiple sclerosis (MS) is unclear. OBJECTIVE: This study explored structure-function relationships during the 5 years following a clinically isolated syndrome and their role in cognitive performance. METHODS: Thirty-two patients were enrolled after their first neurological episode suggestive of MS and followed for 5 years, along with 10 matched healthy controls. We assessed structural (using diffusion tensor imaging) and functional (using resting-state functional magnetic resonance imaging (fMRI)) brain network metrics, clinical and cognitive scores at each follow-up visit. Structural-functional coupling, calculated as the correlation coefficient between strengths of structural and functional networks, was used to assess structure-function relationships. RESULTS: Structural clustering coefficient was significantly increased after 5 years, whereas characteristic path length decreased. Structural connections decreased after 1 year and increased after 5 years. Functional connections and related path lengths were decreased after 5 years. Structural-functional coupling had increased significantly after 5 years. This structural-functional coupling was associated with cognitive and clinical evolution, with stronger coupling associated with a decline in both domains. CONCLUSION: Our findings provide novel biological evidence that MS leads to a more constrained anatomical-dependant functional connectivity. The collapse of this network seems to lead to both cognitive worsening and clinical disability.

Longitudinal study of functional brain network reorganization in clinically isolated syndrome.

BACKGROUND: There is a lack of longitudinal studies exploring the topological organization of functional brain networks at the early stages of multiple sclerosis (MS). OBJECTIVE: This study aims to assess potential brain functional reorganization at rest in patients with CIS (PwCIS) after 1 year of evolution and to characterize the dynamics of functional brain networks at the early stage of the disease. METHODS: We prospectively included 41 PwCIS and 19 matched healthy controls (HCs). They were scanned at baseline and after 1 year. Using graph theory, topological metrics were calculated for each region. Hub disruption index was computed for each metric. RESULTS: Hub disruption indexes of degree and betweenness centrality were negative at baseline in patients (p < 0.05), suggesting brain reorganization. After 1 year, hub disruption indexes for degree and betweenness centrality were still negative (p < 0.00001), but such reorganization appeared more pronounced than at baseline. Different brain regions were driving these alterations. No global efficiency differences were observed between PwCIS and HCs either at baseline or at 1 year. CONCLUSION: Dynamic changes in functional brain networks appear at the early stages of MS and are associated with the maintenance of normal global efficiency in the brain, suggesting a compensatory effect.

Dynamic modular-level alterations of structural-functional coupling in clinically isolated syndrome.

Structural and functional connectivity abnormalities have been reported previously in multiple sclerosis. However, little is known about how each modality evolution relates to the other. Recent studies in other neurological disorders have suggested that structural-functional coupling may be more sensitive in detecting brain alterations than any single modality. Accordingly, this study aimed to investigate the longitudinal evolution of structural-functional coupling, both at the global and modular levels, in the first year following clinically isolated syndrome. We hypothesized that during the course of multiple sclerosis, patients exhibit a decoupling between functional and structural connectivity due to the disruptive nature of the disease. Forty-one consecutive patients with clinically isolated syndrome were prospectively enrolled in this study, along with 19 age-, sex- and educational level-matched healthy control subjects. These participants were followed for 1 year and underwent resting-state functional MRI and diffusion tensor imaging at each time point, along with an extensive neuropsychological assessment. Graph theory analysis revealed structural reorganization at baseline that appeared as an increase in the clustering coefficient in patients compared to controls (P < 0.05), as well as modular-specific alterations. After 1 year of follow-up, both structural and functional reorganization was depicted with abnormal modular-specific connectivity and an increase of the functional betweenness centrality in patients compared to controls (P < 0.01). More importantly, structural-functional decoupling was observed in the salience, visual and somatomotor networks. These alterations were present along with preserved cognitive performance at this stage. These results depict structural damage preceding functional reorganization at a global and modular level during the first year following clinically isolated syndrome along with normal cognitive performance, suggesting a compensation mechanism at this stage of the disease. Principally, structural-functional decoupling observed for the first time in multiple sclerosis suggests that functional reorganization occurs along indirect anatomical pathways.

Usefulness of confocal laser endomicroscopy for predicting postoperative recurrence in patients with Crohn's disease: a pilot study.

BACKGROUND AND AIMS: Confocal laser endomicroscopy (CLE) has been shown to predict relapse in inflammatory bowel disease, but its value in the detection of postoperative recurrence in Crohn's disease (CD) is unknown. The aims of this pilot study performed in patients with CD after ileocolonic resection were to compare the macroscopic appearance of the neoterminal ileum, according to the endoscopic Rutgeerts score, with the microscopic findings provided by CLE 6 to 12 months after surgery and to assess the predictive values of CLE-generated parameters for predicting further recurrence in patients with postoperative endoscopic remission. METHODS: In 25 consecutive patients with CD within 6 to 12 months of surgery, the neoterminal ileum was examined by standard white-light endoscopy (Rutgeerts scale) followed by CLE (Watson grade). Only patients without endoscopic recurrence (Rutgeerts i0 and i1) were then followed endoscopically and clinically (median follow-up 38 months). RESULTS: At the time of the first postoperative colonoscopy, 18 patients (72%) were in endoscopic remission, and 7 (28%) experienced an endoscopic recurrence (Rutgeerts ≥i2). The Rutgeerts score was significantly correlated with the Watson score (ρ = 0 .73; P < .0001). The Watson scores at baseline were significantly higher in patients with further endoscopic recurrence (median 2.0; interquartile range [IQR] 1.5-2.0) than in those with endoscopic remission (median 1.0; IQR 1.0-1.0; P = .032) and were significantly higher in patients with clinical relapse (medium 2.0, IQR 2.0-2.0) compared with those in clinical remission (median 1.0; IQR 1.0-1.0; P = .036). CONCLUSIONS: CLE could be useful in monitoring patients with CD after intestinal resection. Further studies with a larger population are necessary to confirm these preliminary results.

Metabolic reprogramming in cancer cells, consequences on pH and tumour progression: Integrated therapeutic perspectives with dietary lipids as adjuvant to anticancer treatment.

While tumours arise from acquired mutations in oncogenes or tumour-suppressor genes, it is clearly established that cancers are metabolic diseases characterized by metabolic alterations in tumour cells, and also non-tumour cells of the host organism resulting in tumour cachexia and patient weakness. In this review, we aimed at delineating details by which metabolic alterations in cancer cells, characterized by mitochondrial bioenergetics deregulations and the preference for aerobic glycolysis, are critical parameters controlling the aggressive progression of tumours. In particular, metabolic alteration in cancer cells are coupled to the modulation of intracellular and extracellular pH, epithelial-to-mesenchymal transition and associated increased invasiveness, autophagy, and the development of anticancer treatment resistance. Finally, based on mechanistic, pre-clinical and clinical studies, we proposed the adjuvant supplementation of dietary n-3 polyunsaturated fatty acids for a complementary holistic treatment of the cancer disease.

Diffusion Properties and 3D Architecture of Human Lower Leg Muscles Assessed with Ultra-High-Field-Strength Diffusion-Tensor MR Imaging and Tractography: Reproducibility and Sensitivity to Sex Difference and Intramuscular Variability.

Purpose To demonstrate the reproducibility of the diffusion properties and three-dimensional structural organization measurements of the lower leg muscles by using diffusion-tensor imaging (DTI) assessed with ultra-high-field-strength (7.0-T) magnetic resonance (MR) imaging and tractography of skeletal muscle fibers. On the basis of robust statistical mapping analyses, this study also aimed at determining the sensitivity of the measurements to sex difference and intramuscular variability. Materials and Methods All examinations were performed with ethical review board approval; written informed consent was obtained from all volunteers. Reproducibility of diffusion tensor indexes assessment including eigenvalues, mean diffusivity, and fractional anisotropy (FA) as well as muscle volume and architecture (ie, fiber length and pennation angle) were characterized in lower leg muscles (n = 8). Intramuscular variability and sex differences were characterized in young healthy men and women (n = 10 in each group). Student t test, statistical parametric mapping, correlation coefficients (Spearman rho and Pearson product-moment) and coefficient of variation (CV) were used for statistical data analysis. Results High reproducibility of measurements (mean CV ± standard deviation, 4.6% ± 3.8) was determined in diffusion properties and architectural parameters. Significant sex differences were detected in FA (4.2% in women for the entire lower leg; P = .001) and muscle volume (21.7% in men for the entire lower leg; P = .008), whereas architecture parameters were almost identical across sex. Additional differences were found independently of sex in diffusion properties and architecture along several muscles of the lower leg. Conclusion The high-spatial-resolution DTI assessed with 7.0-T MR imaging allows a reproducible assessment of structural organization of superficial and deep muscles, giving indirect information on muscle function. ©RSNA, 2018 Online supplemental material is available for this article.

Anatomic consistencies across epilepsies: a stereotactic-EEG informed high-resolution structural connectivity study.

See Bernasconi (doi:10.1093/brain/awx229) for a scientific commentary on this article. Drug-resistant localization-related epilepsies are now recognized as network diseases. However, the exact relationship between the organization of the epileptogenic network and brain anatomy overall remains incompletely understood. To better understand this relationship, we studied structural connectivity obtained from diffusion weighted imaging in patients with epilepsy using both stereo-electroencephalography (SEEG)-determined epileptic brain regions and whole-brain analysis. High resolution structural connectivity analysis was applied in 15 patients with drug-resistant localization-related epilepsies and 36 healthy control subjects to study structural connectivity changes in epilepsy. Two different methods of structural connectivity analysis were carried out using diffusion weighted imaging, one focusing on the relationship between epileptic regions determined by SEEG investigations and one blinded to epileptic regions looking at whole-brain connectivity. First, we performed zone-based analysis comparing structural connectivity findings in patients and controls within and between SEEG-defined zones of interest. Next, we performed whole-brain structural connectivity analysis in all subjects and compared findings to the same SEEG-defined zones of interest. Finally, structural connectivity findings were correlated against clinical features. Zone-based analysis revealed no significant decreased structural connectivity within nodes of the epilepsy network at the group level, but did demonstrate significant structural connectivity differences between nodes of the epileptogenic network (regions involved in seizures generation and propagation) and the remaining of the brain in patients compared to controls. Whole-brain analyses showed a total of 133 clusters of significantly decreased structural connectivity across all patients. One cluster of significantly increased structural connectivity was identified in a single patient. Clusters of decreased structural connectivity showed topographical preference for both the salience and default mode networks despite clinical heterogeneity within our patient sample. Correlation analysis did not reveal any significant findings regarding either the effect of age at disease onset, disease duration or post-surgical outcome on structural connectivity. Taken together, this work demonstrates that structural connectivity disintegration targets distributed functional networks while sparing the epilepsy network.

Synthesis of Alkyl-Glycerolipids Standards for Gas Chromatography Analysis: Application for Chimera and Shark Liver Oils.

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20-24%), 16:0 (42-54%) and 18:1 (6-16%) and, to a lesser extent, (0.2-2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

Focal Hemodynamic Responses in the Stimulated Hemisphere During High-Definition Transcranial Direct Current Stimulation.

OBJECTIVE: High-definition transcranial direct current stimulation (HD-tDCS) using a 4 × 1 electrode montage has been previously shown using modeling and physiological studies to constrain the electric field within the spatial extent of the electrodes. The aim of this proof-of-concept study was to determine if functional near-infrared spectroscopy (fNIRS) neuroimaging can be used to determine a hemodynamic correlate of this 4 × 1 HD-tDCS electric field on the brain. MATERIALS AND METHODS: In a three session cross-over study design, 13 healthy males received one sham (2 mA, 30 sec) and two real (HD-tDCS-1 and HD-tDCS-2, 2 mA, 10 min) anodal HD-tDCS targeting the left M1 via a 4 × 1 electrode montage (anode on C3 and 4 return electrodes 3.5 cm from anode). The two real HD-tDCS sessions afforded a within-subject replication of the findings. fNIRS was used to measure changes in brain hemodynamics (oxygenated hemoglobin integral-O2 Hbint ) during each 10 min session from two regions of interest (ROIs) in the stimulated left hemisphere that corresponded to "within" (Lin ) and "outside" (Lout ) the spatial extent of the 4 × 1 electrode montage, and two corresponding ROIs (Rin and Rout ) in the right hemisphere. RESULTS: The ANOVA showed that both real anodal HD-tDCS compared to sham induced a significantly greater O2 Hbint in the Lin than Lout ROIs of the stimulated left hemisphere; while there were no significant differences between the real and sham sessions for the right hemisphere ROIs. Intra-class correlation coefficients showed "fair-to-good" reproducibility for the left stimulated hemisphere ROIs. CONCLUSIONS: The greater O2 Hbint "within" than "outside" the spatial extent of the 4 × 1 electrode montage represents a hemodynamic correlate of the electrical field distribution, and thus provides a prospective reliable method to determine the dose of stimulation that is necessary to optimize HD-tDCS parameters in various applications.

Complementary contributions of concurrent EEG and fMRI connectivity for predicting structural connectivity.

While averaged dynamics of brain function are known to estimate the underlying structure, the exact relationship between large-scale function and structure remains an unsolved issue in network neuroscience. These complex functional dynamics, measured by EEG and fMRI, are thought to arise from a shared underlying structural architecture, which can be measured by diffusion MRI (dMRI). While simulation and data transformation (e.g. graph theory measures) have been proposed to refine the understanding of the underlying function-structure relationship, the potential complementary and/or independent contribution of EEG and fMRI to this relationship is still poorly understood. As such, we explored this relationship by analyzing the function-structure correlation in fourteen healthy subjects with simultaneous resting-state EEG-fMRI and dMRI acquisitions. We show that the combination of EEG and fMRI connectivity better explains dMRI connectivity and that this represents a genuine model improvement over fMRI-only models for both group-averaged connectivity matrices and at the individual level. Furthermore, this model improves the prediction within each resting-state network. The best model fit to underlying structure is mediated by fMRI and EEG-δ connectivity in combination with Euclidean distance and interhemispheric connectivity with more local contributions of EEG-γ at the scale of resting-state networks. This highlights that the factors mediating the relationship between functional and structural metrics of connectivity are context and scale dependent, influenced by topological, geometric and architectural features. It also suggests that fMRI studies employing simultaneous EEG measures may characterize additional and essential parts of the underlying neuronal activity of the resting-state, which might be of special interest for both clinical studies and the investigation of resting-state dynamics.

Whole-Brain High-Resolution Structural Connectome: Inter-Subject Validation and Application to the Anatomical Segmentation of the Striatum.

The present study describes extraction of high-resolution structural connectome (HRSC) in 99 healthy subjects, acquired and made available by the Human Connectome Project. Single subject connectomes were then registered to the common surface space to allow assessment of inter-individual reproducibility of this novel technique using a leave-one-out approach. The anatomic relevance of the surface-based connectome was examined via a clustering algorithm, which identified anatomic subdivisions within the striatum. The connectivity of these striatal subdivisions were then mapped on the cortical and other subcortical surfaces. Findings demonstrate that HRSC analysis is robust across individuals and accurately models the actual underlying brain networks related to the striatum. This suggests that this method has the potential to model and characterize the healthy whole-brain structural network at high anatomic resolution.

Understanding the roles of the P2X7 receptor in solid tumour progression and therapeutic perspectives.

P2X7 is an intriguing ionotropic receptor for which the activation by extracellular ATP induces rapid inward cationic currents and intracellular signalling pathways associated with numerous physiological processes such as the induction of the inflammatory cascade, the survival and proliferation of cells. In contrast, long-term stimulation of P2X7 is generally associated with membrane permeabilisation and cell death. Recently, P2X7 has attracted great attention in the cancer field, and particularly in the neoplastic transformation and the progression of solid tumours. A growing number of studies were published; however they often appeared contradictory in their results and conclusions. As such, the involvement of P2X7 in the oncogenic process remains unclear so far. The present review aims to discuss the current knowledge and hypotheses on the involvement of the P2X7 receptor in the development and progression of solid tumours, and highlight the different aspects that require further clarification in order to decipher whether P2X7 could be considered as a cancer biomarker or as a target for pharmacological intervention. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

How do voltage-gated sodium channels enhance migration and invasiveness in cancer cells?

Voltage-gated sodium channels are abnormally expressed in tumors, often as neonatal isoforms, while they are not expressed, or only at a low level, in the matching normal tissue. The level of their expression and their activity is related to the aggressiveness of the disease and to the formation of metastases. A vast knowledge on the regulation of their expression and functioning has been accumulated in normal excitable cells. This helped understand their regulation in cancer cells. However, how voltage-gated sodium channels impose a pro-metastatic behavior to cancer cells is much less documented. This aspect will be addressed in the review. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

High-resolution multi-parametric quantitative magnetic resonance imaging of the human cervical spinal cord at 7T.

Quantitative MRI techniques have the potential to characterize spinal cord tissue impairments occurring in various pathologies, from both microstructural and functional perspectives. By enabling very high image resolution and enhanced tissue contrast, ultra-high field imaging may offer further opportunities for such characterization. In this study, a multi-parametric high-resolution quantitative MRI protocol is proposed to characterize in vivo the human cervical spinal cord at 7T. Multi-parametric quantitative MRI acquizitions including T1, T2* relaxometry mapping and axial diffusion MRI were performed on ten healthy volunteers with a whole-body 7T system using a commercial prototype coil-array dedicated to cervical spinal cord imaging. Automatic cord segmentation and multi-parametric data registration to spinal cord templates enabled robust regional studies within atlas-based WM tracts and GM horns at the C3 cervical level. T1 value, cross-sectional area and GM/WM ratio evolutions along the cervical cord were also reported. An original correction method for B1+-biased T1 mapping sequence was additionally proposed and validated on phantom. As a result, relaxometry and diffusion parameters derived from high-resolution quantitative MRI acquizitions were reported at 7T for the first time. Obtained images, with unmatched resolutions compared to lower field investigations, provided exquisite anatomical details and clear delineation of the spinal cord substructures within an acquisition time of 30min, compatible with clinical investigations. Regional statistically significant differences were highlighted between WM and GM based on T1 and T2* maps (p<10-3), as well as between sensory and motor tracts based on diffusion tensor imaging maps (p<0.05). The proposed protocol demonstrates that ultra-high field spinal cord high-resolution quantitative MRI is feasible and lays the groundwork for future clinical investigations of degenerative spinal cord pathologies.

Effects of Anodal High-Definition Transcranial Direct Current Stimulation on Bilateral Sensorimotor Cortex Activation During Sequential Finger Movements: An fNIRS Study.

Transcranial direct current stimulation (tDCS) is a non-invasive electrical brain stimulation technique that can modulate cortical neuronal excitability and activity. This study utilized functional near infrared spectroscopy (fNIRS) neuroimaging to determine the effects of anodal high-definition (HD)-tDCS on bilateral sensorimotor cortex (SMC) activation. Before (Pre), during (Online), and after (Offline) anodal HD-tDCS (2 mA, 20 min) targeting the left SMC, eight healthy subjects performed a simple finger sequence (SFS) task with their right or left hand in an alternating blocked design (30-s rest and 30-s SFS task, repeated five times). In order to determine the level of bilateral SMC activation during the SFS task, an Oxymon MkIII fNIRS system was used to measure from the left and right SMC, changes in oxygenated (O2Hb) and deoxygenated (HHb) haemoglobin concentration values. The fNIRS data suggests a finding that compared to the Pre condition both the "Online" and "Offline" anodal HD-tDCS conditions induced a significant reduction in bilateral SMC activation (i.e., smaller decrease in HHb) for a similar motor output (i.e., SFS tap rate). These findings could be related to anodal HD-tDCS inducing a greater efficiency of neuronal transmission in the bilateral SMC to perform the same SFS task.

Suppression of PPARß, and DHA treatment, inhibit NaV1.5 and NHE-1 pro-invasive activities.

Peroxisome proliferator-activated receptor ß (PPARß) and NaV1.5 voltage-gated sodium channels have independently been shown to regulate human breast cancer cell invasiveness. The n-3 polyunsaturated docosahexaenoic acid (DHA, 22:6n-3), a natural ligand of PPAR, is effective in increasing survival and chemotherapy efficacy in breast cancer patient with metastasis. DHA reduces breast cancer cell invasiveness and it also inhibits PPARß expression. We have shown previously that NaV1.5 promotes MDA-MB-231 breast cancer cells invasiveness by potentiating the activity of Na(+)/H(+) exchanger type 1 (NHE-1), the major regulator of H(+) efflux in these cells. We report here that DHA inhibited NaV1.5 current and NHE-1 activity in human breast cancer cells, and in turn reduced NaV1.5-dependent cancer cell invasiveness. For the first time, we show that antagonizing PPARß, or inhibiting its expression, reduced NaV1.5 mRNA and protein expression and NaV1.5 current, as well as NHE-1 activity and cell invasiveness. Consistent with these results, the DHA-induced reduction of both NaV1.5 expression and NHE-1 activity was abolished in cancer cells knocked-down for the expression of PPARß (shPPARß). This demonstrates a direct link between the inhibition of PPARß expression and the inhibition of Nav1.5/NHE-1 activities and breast cancer cell invasiveness. This study provides new mechanistic data advocating for the use of natural fatty acids such as DHA to block the development of breast cancer metastases.

NaV1.5 Na⁺ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia.

The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na(+) channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na(+)/H(+) exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

The activation of P2Y2 receptors increases MCF-7 breast cancer cells migration through the MEK-ERK1/2 signalling pathway.

Adenosine 5'-triphosphate (ATP) is found in high concentrations in the extracellular microenvironment of tumours and is postulated to play critical roles in cancer progression. In the present study, we found that stimulation of human MCF-7 breast cancer cells with 30 µM ATP increased their migration by 140 ± 31%, whereas it had minor or no effect on their proliferation. This effect was prevented by the ectonucleotidase apyrase and was antagonized by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, consistently with the participation of P2 receptors. MCF-7 cells expressed messenger RNA for all known P2Y receptors and for P2X2, P2X4, P2X5, P2X6 and P2X7 receptors. Brief applications (20 s) of external ATP resulted in a 50 pA P2X-like inward current. ATP, but not adenosine diphosphate or uridine diphosphate, increased the intracellular calcium concentration in absence of extracellular calcium, and this effect was prevented by the inhibition of phospholipase C. Uridine triphosphate (UTP) (10 µM) and 2-thio-UTP (10 µM) increased intracellular calcium concentration and cell migration to the same extent as ATP. The UTP-dependent increase in cell migration was absent in cells knocked-down for P2Y2. It was inhibited by MEK inhibitor PD98059. UTP induced a time-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which was prevented by the incubation with PD98059. Taken together, these results highlight the importance of the purinergic signalling in cancer cells and indicate that the activation of P2Y2 receptors enhances breast cancer cells migration through the activation of a MEK-ERK1/2-dependent signalling pathway.