Trafficking and signalling of oncogenic met

PhDThe Receptor Tyrosine Kinase (RTK) Met influences behaviour of several cancers by controlling growth, survival and metastasis. Recently, compartmentalisation of signals generated by RTKs, due to their endocytosis / trafficking, has emerged as a major determinant of various cell functions. The aim of my project was to study oncogenic Met signalling in relation to endosomal trafficking and to determine the consequences of such spatial changes on tumour cell growth and migration in vitro and in vivo. The model studied was NIH3T3 cells stably transfected with Wild type (Wt) Met or with three distinct mutants reported in human cancers. I found that two activating mutations in the kinase domain are highly tumorigenic in vivo. Using functional assays and tumour growth experiments, I demonstrated that one mutant is highly sensitive to Met specific tyrosine kinase inhibitors (TKI) while another is resistant. Such results suggested that therapeutical approaches to these mutants should be different. Furthermore, I demonstrated a direct link between endocytosis and tumorigenicity, suggesting a major role for Met endosomal signalling in cancer progression. Using confocal microscopy and quantitative biochemical assays, I demonstrated that Met mutants displayed an increased endocytosis and recycling and a decreased degradation profile. This led to an accumulation of phosphorylated Met on endosomes that induced activation of the GTPase Rac1, loss of stress fibres and increased cell migration. Blocking endocytosis by pharmacological and genetic 4 means inhibited mutants’ anchorage independent growth and, strikingly, tumorigenesis and experimental metastasis. Interestingly, the mutant resistant to TKI inhibition was sensitive to endocytosis inhibition. Taken together, these results suggest that Met localisation constitutes a major determinant in neoplastic development, while Met activation alone is insufficient to effect this change

L’autophagie : le yin et le yang des cancers

International audienceAutophagy is a self-cannibalism process essential for tissue homeostasis, which can be activated following different environmental stressful conditions. In normal cells, autophagy could act as a brake to prevent tumorigenesis, but cancer cells are able to hijack this process to their own benefit, to promote tumor growth and/or tumor resistance to anti-cancer therapies. Scientists and clinicians attempt to modulate this process to improve therapies, using autophagy inhibitors or activators, some of them being tested currently in clinical trials against several types of tumors. Thus, it appears that autophagy is at the center of a showdown between cancer cells and anti-cancer therapies. In this review, we focus on the mechanisms by which autophagy could be either the yin or the yang of cancers

STK38 at the crossroad between autophagy and apoptosis

We describe the STK38 protein kinase as a conserved regulator of autophagy. We discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Furthermore, we also provide evidence demonstrating that STK38 with the small GTPase RalB, assist the co-ordination between autophagic and apoptotic events upon autophagy induction, hence proposing a role for STK38 in determining cellular fate in response to autophagic conditions

Localization of RalB signaling at endomembrane compartments and its modulation by autophagy

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The pro-apoptotic STK38 kinase is a new Beclin1 Partner positively regulating autophagy

SummaryAutophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions

Adipose-derived cardiomyogenic cells: in vitro expansion and functional improvement in a mouse model of myocardial infarction

1755-3245 (Electronic) Journal Article Research Support, Non-U.S. Gov'tAIMS: Cells derived from the stroma vascular fraction (SVF) of mouse adipose tissue can spontaneously give rise to rare, functional, cardiac-like cells in vitro. This study aimed to improve the production of adipose-derived cardiomyogenic cells (AD-CMG), to characterize them and to assess their cardiac fate and functional outcomes after their administration in a mouse model of acute myocardial infarction. METHODS AND RESULTS: The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD81(+), CD90(-), CD117(-), and Flk-1(-) and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by immunohistochemistry as well as quantitative polymerase chain reaction. Echocardiography showed a significant reduction of remodelling and stability of left ventricle ejection fraction in the AD-CMG cell-treated group vs. controls. Vascular density analysis revealed that AD-CMG administration was also associated with stimulation of angiogenesis in peri-infarct areas. CONCLUSION: Cardiomyogenic cells can be selected and expanded in large amounts from mouse adipose tissue. They can survive and differentiate in an acute myocardial infarction model, avoiding remodelling and impairment of cardiac function, and can promote neo-vascularization in the ischaemic heart

Preconditioning by mitochondrial reactive oxygen species improves the proangiogenic potential of adipose-derived cells-based therapy

1524-4636 (Electronic) Journal Article Research Support, Non-U.S. Gov'tOBJECTIVE: Transplantation of adipose-derived stroma cells (ADSCs) stimulates neovascularization after experimental ischemic injury. ADSC proangiogenic potential is likely mediated by their ability to differentiate into endothelial cells and produce a wide array of angiogenic and antiapoptotic factors. Mitochondrial reactive oxygen species (ROS) have been shown to control ADSC differentiation. We therefore hypothesized that mitochondrial ROS production may change the ADSC proangiogenic properties. METHODS AND RESULTS: The use of pharmacological strategies (mitochondrial inhibitors, antimycin, and rotenone, with or without antioxidants) allowed us to specifically and precisely modulate mitochondrial ROS generation in ADSCs. We showed that transient stimulation of mitochondrial ROS generation in ADSCs before their injection in ischemic hindlimb strongly improved revascularization and the number of ADSC-derived CD31-positive cells in ischemic area. Mitochondrial ROS generation increased the secretion of the proangiogenic and antiapoptotic factors, VEGF and HGF, but did not affect ADSC ability to differentiate into endothelial cells, in vitro. Moreover, mitochondrial ROS-induced ADSC preconditioning greatly protect ADSCs against oxidative stress-induced cell death. CONCLUSIONS: Our study demonstrates that in vitro preconditioning by moderate mitochondrial ROS generation strongly increases in vivo ADSC proangiogenic properties and emphasizes the crucial role of mitochondrial ROS in ADSC fate