An mTOR feedback loop mediates the 'flare' ('rebound') response to MET tyrosine kinase inhibition.

Targeted therapy significantly impairs tumour growth but suffers from limitations, among which the 'flare' ('rebound') effect. Among cancers driven by tyrosine kinase receptors, those relying on alterations of the MET oncogene benefit from treatment by specific inhibitors. Previously, we reported that discontinuation of MET tyrosine kinase receptor inhibition causes 'rebound' activation of the oncogene, with a post-treatment transient hyperphosphorylation phase that culminates into a dramatic increase in cancer cell proliferation. The molecular mechanisms behind the 'MET burst' after treatment cessation are unknown but critically important for patients. Here we identify a positive feedback loop mediated by the AKT/mTOR pathway leading to (a) enhanced MET translation by activating p70S6K and 4EBP1 and (b) MET hyper-phosphorylation by inactivation of the tyrosine-phosphatase PTP1B. The latter effect is due to m-TOR-driven PTP1B phosphorylation of the inhibitory residues Ser50 and Ser378. These data provide in vitro evidence for the use of mTOR inhibitors to prevent the 'flare effect' in MET targeted therapy, with potential applicative ramifications for patient clinical management.

Dual MET/EGFR therapy leads to complete response and resistance prevention in a MET-amplified gastroesophageal xenopatient cohort.

Amplification of the MET oncogene occurs in 2-4% of gastroesophageal cancers and defines a small and aggressive subset of tumors. Although in vitro studies have given very promising results, clinical trials with MET inhibitors have been disappointing, showing few and short lasting responses. The aim of the work was to exploit a MET-amplified patient-derived xenograft model to optimize anti-MET therapeutic strategies in gastroesophageal cancer. We found that despite the high MET amplification level (26 gene copies), in the absence of qualitative or quantitative alterations of EGFR, MET inhibitors induced only tumor growth inhibition, whereas dual MET/EGFR inhibition led to complete tumor regression. Importantly, the combo treatment completely prevented the onset of resistance, which quite rapidly appeared in tumors treated with MET monotherapy. We found that this secondary resistance was due to EGFR activation and could be overcome by dual MET/EGFR inhibition. Similar results were also obtained in a MET-addicted, established gastric cancer cell line. In vitro experiments performed on tumor-derived primary cells confirmed that MET inhibitors were not able to abrogate the activation of downstream transducers and that only the combined MET/EGFR treatment completely shut off the signaling. Previously reported cases, as well as those described here, showed only partial and transient sensitivity to anti-MET therapy. The finding that combined anti-MET/EGFR therapy-even in the absence of EGFR genetic alterations-induced complete and durable response, represents a proof of concept and guarantees further investigations, opening a new perspective of treatment for these patients.

Targeting the oncogenic Met receptor by antibodies and gene therapy.

The receptor for hepatocyte growth factor (HGF), a tyrosine kinase encoded by the Met oncogene, has a crucial role in cancer growth, invasion and metastasis. It is a validated therapeutic target for 'personalized' treatment of a number of malignancies. Therapeutic tools prompting selective, robust and highly effective Met inhibition potentially represent a major step in the battle against cancer. Antibodies targeting either Met or its ligand HGF, although challenging, demonstrate to be endowed with promising features. Here we briefly review and discuss the state of the art in the field.

Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy.

Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl2), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) - surprisingly - autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl2. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury.

Met signaling regulates growth, repopulating potential and basal cell-fate commitment of mammary luminal progenitors: implications for basal-like breast cancer.

Basal-like breast cancer is an aggressive subtype of mammary carcinoma. Despite expressing basal markers, typical of mammary stem cells, this tumor has been proposed to originate from luminal progenitors, which are downstream of stem cells along the mammary epithelial hierarchy. This suggests that committed luminal progenitors may reacquire basal, stem-like characteristics, but the mechanisms that regulate this transition remain unclear. Using mouse models, we found that luminal progenitors express high levels of the Met receptor for hepatocyte growth factor (HGF), as compared with the other mammary epithelial sub-populations. Constitutive activation of Met led luminal progenitors to attain stem cell properties, including enhanced clonogenic activity in vitro and de novo ability to reconstitute mammary glands in repopulation assays in vivo. Moreover, in response to Met signaling, luminal progenitors gave rise to hyperplastic ductal morphogenesis and preferentially underwent basal lineage commitment at the expense of luminal cell-fate specification. Opposite and symmetric results were produced by systemic pharmacological inhibition of Met. Hence, Met signaling targets luminal progenitors for expansion, impairs their differentiation toward the mature luminal phenotype and enables their commitment toward the basal lineage. These results emphasize a critical role for Met in promoting deregulated proliferation and basal plasticity of normal luminal progenitors in the mammary gland, a complex of events that may be required for sustaining the functional and phenotypic properties of basal-like breast tumors.

Cell delivery of Met docking site peptides inhibit angiogenesis and vascular tumor growth.

Hepatocyte growth factor (HGF) and its receptor Met are responsible for a wide variety of cellular responses, both physiologically during embryo development and tissue homeostasis, and pathologically, particularly during tumor growth and dissemination. In cancer, Met can act as an oncogene on tumor cells, as well as a pro-angiogenic factor activating endothelial cells and inducing new vessel formation. Molecules interfering with Met activity could be valuable therapeutic agents. Here we have investigated the antiangiogenic properties of a synthetic peptide mimicking the docking site of the Met carboxyl-terminal tail, which was delivered into the cells by fusion with the internalization sequences from Antennapedia or HIV-Tat. We showed that these peptides inhibit ligand-dependent endothelial cell proliferation, motility, invasiveness and morphogenesis in vitro to an even greater extent and with much less toxicity than the Met inhibitor PHA-665752, which correlated with interference of HGF-dependent downstream signaling. In vivo, the peptides inhibited HGF-induced angiogenesis in the matrigel sponge assay and impaired xenograft tumor growth and vascularization in Kaposi's sarcoma. These data show that interference with the Met receptor intracellular sequence impairs HGF-induced angiogenesis, suggesting the use of antidocking site compounds as a therapeutic strategy to counteract angiogenesis in cancer as well as in other diseases.

Met-driven invasive growth involves transcriptional regulation of Arhgap12.

Invasive growth is a complex biological program triggered by hepatocyte growth factor (HGF) through its tyrosine kinase receptor encoded by the Met proto-oncogene. The program involves-besides proliferation-cell dissociation, motility and invasiveness, controlled by intracellular signals impinging on PI3K and on the small G-proteins of the Rac/Rho family. The mechanism(s) unbalancing Rac/Rho activation are still not completely clarified. Here, we describe a functional link between HGF and Arhgap12, a gene encoding a previously uncharacterized protein of the RhoGAP family. We identified Arhgap12 as a transcriptional target of HGF, through a novel gene trapping strategy. We found that Arhgap12 mRNA and protein are robustly suppressed by HGF treatment, but not by serum. Arhgap12 displayed GTPase activating protein (GAP) activity towards Rac1 and, upon overexpression, impaired cell scattering, invasion and adhesion to fibronectin in response to HGF. Consistently, Arhgap12 silencing by RNA interference selectively increased the scatter and adhesion responses. These data show that HGF-driven invasive growth involves transcriptional regulation of a Rac1-specific GAP.

Silencing the MET oncogene leads to regression of experimental tumors and metastases.

In spite of the established knowledge of the genetic alterations responsible for cancer onset, the genes promoting and maintaining the invasive/metastatic phenotype are still elusive. The MET proto-oncogene, encoding the tyrosine kinase receptor for hepatocyte growth factor (HGF), senses unfavorable micro-environmental conditions and drives cell invasion and metastasis. MET overexpression, often induced by tumor hypoxia, leads to constitutive activation of the receptor and correlates with poor prognosis. To establish the role of MET in different phases of tumor progression, we developed an inducible lentiviral delivery system of RNA interference. Silencing the endogenous MET gene, overexpressed in tumor cells, resulted in (i) impairment of the execution of the full invasive growth program in vitro, (ii) lack of tumor growth and (iii) decreased generation of experimental metastases in vivo. Notably, silencing MET in already established metastases led to their almost complete regression. This indicates that persistent expression of the MET oncogene is mandatory until the advanced phases of cancer progression.

A positive feedback loop between hepatocyte growth factor receptor and beta-catenin sustains colorectal cancer cell invasive growth.

Overexpressed or activated hepatocyte growth factor receptor, encoded by the MET proto-oncogene, was found in the majority of colorectal carcinomas (CRCs), whose stepwise progression to malignancy requires transcriptional activation of beta-catenin. We here demonstrate that a functional crosstalk between Met and beta-catenin signaling sustains and increases CRC cell invasive properties. Hepatocyte growth factor (HGF) stimulation prompts beta-catenin tyrosine phosphorylation and dissociation from Met, and upregulates beta-catenin expression via the phosphatidylinositol 3-kinase pathway in conditions that mimic those found by the invading and metastasizing cells. Additionally, a transcriptionally active form of beta-catenin, known to be oncogenic, enhances Met expression. Furthermore, HGF treatment increases the activity of the beta-catenin-regulated T-cell factor transcription factor in cells expressing the wild-type or the oncogenic beta-catenin. In the mirror experiments, either Met or beta-catenin knocking down also reduces their protein level. In biological assays, beta-catenin knocking down abrogates the HGF-induced motile phenotype, whereas active beta-catenin fosters ligand-independent cell scattering. Met and beta-catenin also cooperate in promoting entry into the cell cycle and in protecting cells from apoptosis. In conclusion, Met and beta-catenin pathways are mutually activated in CRC cells. This might generate a self-amplifying positive feedback loop resulting in the upregulation of the invasive growth properties of CRC cells.

Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility.

Plexins encode receptors for semaphorins, molecular signals guiding cell migration, and axon pathfinding. The mechanisms mediating plexin function are poorly understood. Plexin activation in adhering cells rapidly leads to retraction of cellular processes and cell rounding "cell collapse"). Here we show that, unexpectedly, this response does not require the activity of Rho-dependent kinase (ROCK) nor the contraction of F-actin cables. Interestingly, integrin-based focal adhesive structures are disassembled within minutes upon plexin activation; this is followed by actin depolymerization and, eventually, by cellular collapse. We also show that plexin activation hinders cell attachment to adhesive substrates, blocks the extension of lamellipodia, and thereby inhibits cell migration. We conclude that plexin signaling uncouples cell substrate-adhesion from cytoskeletal dynamics required for cell migration and axon extension.

A signaling adapter function for alpha6beta4 integrin in the control of HGF-dependent invasive growth.

alpha6beta4 integrin and the Met receptor for HGF have been shown independently to promote invasive growth. We demonstrate here that Met selectively associates with alpha6beta4. In carcinoma cells expressing Met alone, HGF does not exert significant biological effects. Ectopic expression of alpha6beta4 restores HGF-regulated processes. Following Met activation, alpha6beta4 is tyrosine phosphorylated and combines with Shc and PI3K, generating an additional signaling platform that potentiates HGF-triggered activation of Ras- and PI3K-dependent pathways. In the presence of an alpha6beta4 mutant defective for Shc recruitment, Met cannot sustain HGF-mediated responses. Surprisingly, a truncated beta4 unable to bind laminins retains the activity of wild-type alpha6beta4. Such findings invoke an unexpected role for alpha6beta4 in cancer invasion as a functional amplifier of biochemical outputs rather than a mechanical adhesive device.

The transmembrane protein Off-track associates with Plexins and functions downstream of Semaphorin signaling during axon guidance.

The Plexin family of transmembrane proteins appears to function as repulsive receptors for most if not all Semaphorins. Here, we use genetic and biochemical analysis in Drosophila to show that the transmembrane protein Off-track (OTK) associates with Plexin A, the receptor for Sema 1a, and that OTK is a component of the repulsive signaling response to Semaphorin ligands. In vitro, OTK associates with Plexins. In vivo, mutations in the otk gene lead to phenotypes resembling those of loss-of-function mutations of either Sema1a or PlexA. The otk gene displays strong genetic interactions with Sema1a and PlexA, suggesting that OTK and Plexin A function downstream of Sema 1a.

Ligand-regulated binding of FAP68 to the hepatocyte growth factor receptor.

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met). We isolated a human cDNA encoding a novel protein of 68 kDa, which we termed FAP68. This protein is homologous to a previously described FK506-binding protein-associated protein, FAP48, which derives from an alternative spliced form of the same cDNA, lacking an 85-nucleotide exon and leading to an early stop codon. Here we show that epithelial cells, in which the HGF receptor is naturally expressed, contain FAP68 and not FAP48 proteins. FAP68 binding to Met requires the last 30 amino acids of the C-terminal tail, which are unique to the HGF receptor. Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families. FAP68 interacts specifically with the inactive form of HGF receptor, such as a kinase-defective receptor or a dephosphorylated wild type receptor. In vivo, endogenous FAP68 can be coimmunoprecipitated with the HGF receptor in the absence of stimuli and not upon HGF stimulation. Thus, FAP68 represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation. Free FAP68 exerts a specific stimulatory activity toward the downstream target p70 S6 protein kinase (p70S6K). Significantly, nonphosphorylated HGF receptor prevents FAP68 from stimulating p70S6K. These data suggest a role for FAP68 in coupling HGF receptor signaling to the p70S6K pathway.

Differential requirement of the last C-terminal tail of Met receptor for cell transformation and invasiveness.

Biological responses to Hepatocyte Growth Factor are mediated by the tyrosine kinase receptor encoded by the Met oncogene. Under physiological conditions, Met triggers a multi-step genetic program called 'invasive growth' including cell-dissociation, invasion of extracellular matrices and growth. When constitutively activated, Met can induce cell transformation and metastasis. Phosphorylation of two docking tyrosines in the receptor tail is essential for all biological responses. To investigate the role of the C-terminal part of Met, we have generated mutants lacking either the last 26 or 47 amino acids. As expected, mutants lacking the docking sites fail to mediate cell transformation and invasion. Interestingly, while Met Delta26 can mediate invasion, its transforming ability is severely impaired. Moreover, the lack of the last 26 amino acids strongly reduces Met ability to phosphorylate substrates in vitro and in vivo. These data indicate that the last 26 amino acids are required to confer the kinase its full enzymatic activity, which is critical for cell transformation but dispensable for invasive properties. Finally, we also show that up-regulation of Met enzymatic activity by insertion of a point mutation in the kinase domain (M1250T) overcomes the regulatory role played by the last 26 amino acids of the tail. It is concluded that the C-terminal domain of Met is crucial not only for recruitment of transducers but also for regulation of receptor enzymatic activity.

Osteopontin is an autocrine mediator of hepatocyte growth factor-induced invasive growth.

In epithelial cells, hepatocyte growth factor (HGF) activates a genetic program involving cell-cell dissociation ("scattering"), growth and invasiveness. The full program is not elicited by other growth factors like epidermal growth factor, and is aberrantly activated during cancer progression to the invasive-metastatic phenotype. To identify genes involved in the onset of invasive growth, we explored by cDNA microarrays the in vitro transcriptional response to HGF of mouse embryo liver cells. We identified osteopontin (OPN), a secreted matrix protein, as a major HGF transcriptional target. The wave of OPN induction is maximal at 6 h, in concomitance with the initiation of scattering, and is specific, because no other matrix protein among those explored by the microarray is affected. Interestingly, HGF, but not epidermal growth factor, promotes cell adhesion to OPN via the CD44 receptor. Scattering is significantly impaired by antibodies against OPN and CD44; conversely, constitutive OPN overexpression dramatically increases the motile and invasive responses to HGF, leading to disruption of the ordered morphogenetic program triggered by this ligand.

Receptor tyrosine kinases as therapeutic targets: the model of the MET oncogene.

Control of cell growth and differentiation occurs via extracellular signals known as growth factors. Growth factors are high affinity ligands for transmembrane receptors belonging to the family of receptor tyrosine kinases (RTKs). A number of genetic evidences have implicated RTKs in human diseases including developmental disorders and cancer. For instance, germline missense mutations involving the Ret receptor are found in patients affected by multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) or familial medullary thyroid carcinomas. Somatic mutations in the Kit receptor are found in mastocytomas and in gastrointestinal tumors. Germline and sporadic mutations of the Met receptor have been described in kidney and hepatocellular carcinomas. Overexpression of the HER-2/neu receptor in breast cancer has been associated with tumor progression. The enzymatic activity of RTKs is strictly regulated and is usually inhibited under basal conditions. Receptor activation triggers a biochemical signalling cascade inside the cytoplasm, named signal transduction, which is subverted during the malignant transformation of cells. Signal transduction by RTKs is a multistep process which includes: (i) Ligand binding and receptor dimerization, (ii) receptor phosphorylation on tyrosine residues; (iii) recruitment to the receptor and activation of cytoplasmic signaling molecules that transmit signals to the nucleus. Each of the steps involved in this process can potentially be targeted to block the aberrant properties of tyrosine kinase receptors. By using the MET oncogene as a model this review focuses on the strategies that can be applied to therapeutically target RTKs.

A gene trap vector system for identifying transcriptionally responsive genes.

We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.

Macrophage stimulating protein is a novel neurotrophic factor.

Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are alpha/beta heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.

Scatter factors and invasive growth.

Scatter factors are unequivocal signals governing a genetic program that includes cell detachment, repulsion, protection from apoptosis, invasiveness of extracellular matrices and proliferation. This pleiomorphic response is defined as 'invasive growth'. Under physiological conditions, it leads to morphogenic cell movements through the matrix, and--primarily--to ordered building of epithelial tubules. Dysfunctions in invasive growth cause enhanced proliferation, uncontrolled migration into surrounding tissues, and failure to differentiate, events that foster tumour growth and invasiveness. Scatter factors act through tyrosine kinase receptors that belong to the Met oncogene family. Here we discuss how alterations of these receptors or of their signal transduction pathways are responsible for cancer onset and progression towards metastasis.