[MET exon 14 splicing sites mutations: A new therapeutic opportunity in lung cancer]. / Les mutations des sites d'épissage de l'exon 14 de MET. Une nouvelle opportunité thérapeutique dans le cancer du poumon.

The mutations leading to MET exon 14 skipping represent a new class of molecular alterations described in various cancers. These alterations are observed in 2 to 3 % of cases of non-small cell lung cancer (NSCLC). Several cases of NSCLC carrying such alterations and achieving objective response to MET tyrosine kinase inhibitorshave recently been published. This review summarizes the molecular mechanisms responsible for MET exon 14 skipping as well as the consequences of the loss of this exon on receptor activity. We also describe the clinical characteristics of patients with METΔ14 mutations. Finally, we address the issues related to the detection of these mutations in lung cancer, and the need to anticipate resistance to MET inhibitors.

Necrosis- and apoptosis-related Met cleavages have divergent functional consequences.

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.

Caspases shutdown nonsense-mediated mRNA decay during apoptosis.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that plays integral roles in eliminating mRNAs with premature termination codons to prevent the synthesis of truncated proteins that could be pathogenic. One response to the accumulation of detrimental proteins is apoptosis, which involves the activation of enzymatic pathways leading to protein and nucleic acid cleavage and culminating in cell death. It is not clear whether NMD is required to ensure the accurate expression of apoptosis genes or is no longer necessary since cytotoxic proteins are not an issue during cell death. The present study shows that caspases cleave the two NMD factors UPF1 and UPF2 during apoptosis impairing NMD. Our results demonstrate a new regulatory pathway for NMD that occurs during apoptosis and provide evidence for role of the UPF cleaved fragments in apoptosis and NMD inhibition.

Caspase-generated fragment of the Met receptor favors apoptosis via the intrinsic pathway independently of its tyrosine kinase activity.

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas the deregulation of Met signaling is associated with tumorigenesis. While ligand-activated Met promotes survival, caspase-dependent generation of the p40 Met fragment leads to apoptosis induction - hallmark of the dependence receptor. Although the survival signaling pathways induced by Met are well described, the pro-apoptotic signaling pathways are unknown. We show that, although p40 Met contains the entire kinase domain, it accelerates apoptosis independently of kinase activity. In cell cultures undergoing apoptosis, the fragment shows a mitochondrial localization, required for p40 Met-induced cell death. Fulminant hepatic failure induced in mice leads to the generation of p40 Met localized also in the mitochondria, demonstrating caspase cleavage of Met in vivo. According to its localization, the fragment induces mitochondrial permeabilization, which is inhibited by Bak silencing and Bcl-xL overexpression. Moreover, Met silencing delays mitochondrial permeabilization induced by an apoptotic treatment. Thus, the Met-dependence receptor in addition to its well-known role in survival signaling mediated by its kinase activity, also participates in the intrinsic apoptosis pathway through the generation of p40 Met - a caspase-dependent fragment of Met implicated in the mitochondrial permeabilization process.

Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis.

Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle.

The shadow of death on the MET tyrosine kinase receptor.

The MET tyrosine kinase receptor is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF-MET system is necessary for embryonic development, and aberrant MET signalling favours tumorigenesis and metastasis. MET is a prototype of tyrosine kinase receptor, which is able to counteract apoptosis through the initiation of a survival signal involving notably the PI3K-Akt pathway. Paradoxically, the MET receptor is also able to promote apoptosis when activated by HGF/SF or independently of ligand stimulation. The molecular mechanisms underlying this uncommon response have been recently investigated and revealed dual antiapoptotic or proapoptotic property of MET according to the cell type or stress conditions. Although the involvement of MET in the regulation of integrated biological responses mostly took into account its efficient antiapoptotic function, its proapoptotic responses could also be important for regulation of the survival/apoptosis balance and play a role during the development or tumour progression.

Regulation of the Ets-1 transcription factor by sumoylation and ubiquitinylation.

Sumoylation and ubiquitinylation reversibly regulate the activity of transcription factors through covalent attachment to lysine residues of target proteins. We examined whether the Ets-1 transcription factor is modified by sumoylation and/or ubiquitinylation. Among four potential SUMO motifs in Ets-1, we identified lysines 15 and 227 within the LK(15)YE and IK(227)QE motifs, as being the sumoylation acceptor sites. Using transfection of Ets-1 wildtype (WT) or its sumoylation deficient version (Ets-1 K15R/K227R), as well as WT or mutant proteins of the SUMO pathway, we further demonstrated that the E2 SUMO-conjugating enzyme Ubc9 and a E3 SUMO ligase, PIASy, can enhance Ets-1 sumoylation, while a SUMO protease, SENP1, can desumoylate Ets-1. We also found that Ets-1 is modified by K48-linked polyubiquitinylation independently of the sumoylation acceptor sites and is degraded through the 26S proteasome pathway, while sumoylation of Ets-1 does not affect its stability. Finally, sumoylation of Ets-1 leads to reduced transactivation and we demonstrated that previously identified critical lysine residues in Synergistic Control motifs are the sumoylation acceptor sites of Ets-1. These data show that Ets-1 can be modified by sumoylation and/or ubiquitinylation, with sumoylation repressing transcriptional activity of Ets-1 and having no clear antagonistic action on the ubiquitin-proteasome degradation pathway.

Amplification of apoptosis through sequential caspase cleavage of the MET tyrosine kinase receptor.

Activation of the MET tyrosine kinase receptor by hepatocyte growth factor/scatter factor is classically associated with cell survival. Nonetheless, stress stimuli can lead to a caspase-dependent cleavage of MET within its juxtamembrane region, which generate a proapoptotic 40 kDa fragment (p40 MET). We report here that p40 MET is in fact generated through an additional caspase cleavage of MET within its extreme C-terminal region, which removes only few amino acids. We evidenced a hierarchical organization of these cleavages, with the C-terminal cleavage favoring the juxtamembrane one. As a functional consequence, the removal of the last amino acids of p40 MET increases its apoptotic capacity. Finally, cells expressing a MET receptor mutated at the C-terminal caspase site are unable to generate p40 MET and are resistant to apoptosis, indicating that generation of p40 MET amplifies apoptosis. These results revealed a two-step caspase cleavage of MET resulting in the reshaping of this survival receptor to a proapoptotic factor.

C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor gamma-chain-associated signaling pathway and by agglutination.

A peptide from the C-terminal domain of thrombospondin-1 (Arg-Phe-Tyr-Val-Val-Met-Trp-Lys; known as 4N1-1) has been reported to induce platelet aggregation and to bind to the integrin-associated protein (IAP), which is also known as CD47. In this study, it was discovered that 4N1-1 or its derivative peptide, 4N1K, induces rapid phosphorylation of the Fc receptor (FcR) gamma chain, Syk, SLP-76, and phospholipase C gamma2 in human platelets. A specific inhibitor of Src family kinases, 4-amino-4-(4-methylphenyl)-7-(t-butyl) pyrazola[3,4-d]pyrimidine, prevented phosphorylation of these proteins, abolished platelet secretion, and reduced aggregation by approximately 50%. A similar inhibition of aggregation to 4N1-1 was obtained in the presence of Arg-Gly-Asp-Ser in mouse platelets deficient in FcR gamma chain or SLP-76 and in patients with type I Glanzmann thrombasthenia. These results show that 4N1-1 signals through a pathway similar to that used by the collagen receptor glycoprotein (GP) VI. The alphaIIbbeta3-independent aggregation induced by 4N1-1 was also observed in fixed platelets and platelets from patients with Bernard-Soulier syndrome, which are deficient in GPIbalpha. Surprisingly, the ability of 4N1-1 to stimulate aggregation and tyrosine phosphorylation was not altered in platelets pretreated with anti-IAP antibodies and in IAP-deficient mice. These results show that the C-terminal peptide of thrombospondin induces platelet aggregation through the FcR gamma-chain signaling pathway and through agglutination. The latter pathway is independent of signaling events and does not use GPIbalpha or alphaIIbbeta3. Neither of these pathways is mediated by IAP.

The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen.

The major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor gamma-chain (FcR gamma-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.

A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI.

The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.

Sequential activation of ERK and repression of JNK by scatter factor/hepatocyte growth factor in madin-darby canine kidney epithelial cells.

The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.

The multisubstrate docking site of the MET receptor is dispensable for MET-mediated RAS signaling and cell scattering.

The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association-kinase assay we found that the mutated receptor can still associate and phosphorylate a approximately 250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering.

The ETS1 transcription factor is expressed during epithelial-mesenchymal transitions in the chick embryo and is activated in scatter factor-stimulated MDCK epithelial cells.

In embryos and in human tumors, the expression of the ETS1 transcription factor correlates with the occurrence of invasive processes. Although this was demonstrated in cells of mesodermal origin, the expression of ETS1 was not detected in epithelial cells. In the present study, we show that during early organogenesis in the chick embryo, ETS1 mRNA expression was transiently induced in epithelial structures, during emigration of neural crest cells and dispersion of somites into the mesenchymal sclerotome. In contrast, the expression of ETS1 was not detected in situations where epithelial layers stayed cohesive while forming a new structure, such as the dermomyotome forming the myotome. The involvement of ETS1 in epithelial cell dissociation was examined in MDCK epithelial cells stimulated by scatter factor/hepatocyte growth factor (SF/HGF), a potent inducer of cell dissociation and motility. SF/HGF was found to stimulate ETS1 mRNA and protein expressions, and these increases coincided with the dispersion of cells and the expression of protease mRNAs, such as urokinase-type plasminogen activator and collagenase, but not with the protease inhibitor, plasminogen activator inhibitor type 1. Furthermore, we showed that SF/HGF was able to induce a transcriptional response involving ETS1 by using artificial as well as cellular promoters, such as the urokinase-type plasminogen activator and collagenase 1 promoters, containing RAS-responsive elements with essential ETS-binding sites. These data demonstrate expression of ETS1 during epithelial-mesenchymal transitions in the developing embryo and show that ETS1 can act as a downstream effector of SF/HGF in MDCK epithelial cells. Taken together, these data identify ETS1 as a molecular actor of epithelia cell dissociation.