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.Met is a receptor tyrosine kinase expressed predominantly by epithelial cells and activated by its stromal ligand, hepatocyte growth factor/scatter factor (HGF/SF). Met activation stimulates a biological program called invasive growth,¹ involving survival, proliferation, invasion, and morphogenesis of epithelial cells. Ligand-stimulated Met acts, furthermore, as an angiogenic and neurotrophic factor.^{2, 3} HGF/SF and Met are essential to several steps of embryogenesis, experiments on transgenic mice having shown that they are necessary for formation of the placenta, liver, limb muscle, neurons, and lung airspace.^{4, 5, 6, 7, 8} In adults, HGF/SF and Met promote regeneration of several organs, including the liver, kidneys, and thymus.^{9, 10, 11, 12, 13}Aberrant Met and HGF/SF signaling contributes to promoting tumorigenesis and metastasis (for review see Furlan et al.).¹⁴ A direct link between Met and cancer has been evidenced by observation of Met germinal mutations linked to hereditary papillary renal carcinoma.¹⁵ Met and/or HGF/SF are/is also overexpressed in several human cancers.¹⁶ Given its important oncogenic activity, Met is the target of many therapeutic agents currently under clinical investigation.¹⁷Downregulation of Met following its activation by HGF/SF is an important negative regulatory mechanism preventing receptor overactivation. We have previously shown that Met expression and activity are also controlled by proteolytic cleavages. Under steady-state conditions, Met is processed by PS-RIP (presenilin-regulated intramembrane proteolysis).^{18, 19} This process involves cleavage of Met within its extracellular juxtamembrane domain by A disintegrin and metalloproteinase (ADAM)-10,²⁰ generating a soluble N-terminal fragment (Met-NTF), which is released into the extracellular space, and a membrane-anchored C-terminal Met fragment (Met-CTF). The latter is in turn efficiently degraded by the lysosome and by further γ-secretase cleavages. Constitutive degradation of the Met receptor by PS-RIP contributes to regulating its half-life.Under apoptotic conditions, Met is cleaved by caspases²¹ within its C-terminal tail and its intracellular juxtamembrane domain. These cleavages remove the C-terminal tail of Met and separate the extracellular ligand-binding domain from the intracellular kinase domain. The generated 40-kDa intracellular fragment, previously called ‘p40Met'' and here called p40Met^caspase, can increase cell death by promoting mitochondrial permeabilization.^{22, 23} Removal of the C-terminal tail of Met is required for the efficient pro-apoptotic action of the fragment. This pro-apoptotic function of Met makes it a member of the dependence receptor family.²⁴ Met cleavages are illustrated in Figure 6a.Although the mechanisms underlying apoptosis have been studied extensively, necrosis has only recently been described as a regulated cell death mechanism.²⁵ Necrosis is an adenosine triphosphate (ATP)-independent cell death mechanism featuring early plasma membrane and organelle disruption. Many pathways can lead to cell necrosis, including calcium overload. This type of cell stress has been amply described in the nervous system, where an increase in intracellular calcium results in neuronal injury and neurodegenerative diseases. In many other cell types, calcium ionophores such as ionomycin can induce rapid necrosis. An increase in intracellular calcium triggers activation of several proteases, including calpains and cathepsins.^{26, 27, 28} Calpains are calcium-dependent proteases capable of cleaving multiple substrates and involved in regulating various cellular processes, including migration, autophagy, apoptosis, and necrosis. Interestingly, the effector role of calpains during necrosis is reminiscent of the function of caspases during apoptosis. Caspases are directly involved in morphological changes observed during apoptosis, while calpains can cleave cytoskeletal proteins such as spectrin and tubulin, thus favoring dismantling of cell structure during necrosis.^{29, 30, 31}Although apoptosis resistance is a hallmark of many cancer cells,³² some such cells remain sensitive to other cell death processes, including necrosis.³³ Thus, a better understanding of the mechanisms underlying necrosis is important, as it could help to elaborate novel therapeutic strategies. Here we show that calcium stress induced by calcium ionophores triggers Met degradation during necrotic cell death. This loss of Met receptor occurs early during the process and is mediated by Met cleavages: by calpains in its intracellular part and by metalloproteases in its extracellular part. These cleavages generate an extracellular fragment and an intracellular fragment with a molecular weight close to that of p40Met^caspase.