Plasticity versus specificity in RTK signalling modalities for distinct biological outcomes in motor neurons.

BACKGROUND: Multiple growth factors are known to control several aspects of neuronal biology, consecutively acting as morphogens to diversify neuronal fates, as guidance cues for axonal growth, and as modulators of survival or death to regulate neuronal numbers. The multiplicity of neuronal types is permitted by the combinatorial usage of growth factor receptors, each of which is expressed in distinct and overlapping subsets of neurons, and by the multitasking role of growth factor receptors, which recruit multiple signalling cascades differentially required for distinct biological outcomes. We have explored signalling robustness in cells where a given receptor tyrosine kinase (RTK) elicits qualitatively distinct outcomes. As the HGF/Met system regulates several biological responses in motor neurons (MN) during neuromuscular development, we have investigated the signalling modalities through which the HGF/Met system impacts on MN biology, and the degree of robustness of each of these functions, when challenged with substitutions of signalling pathways. RESULTS: Using a set of mouse lines carrying signalling mutations that change the Met phosphotyrosine binding preferences, we have asked whether distinct functions of Met in several MN subtypes require specific signalling pathways, and to which extent signalling plasticity allows a pleiotropic system to exert distinct developmental outcomes. The differential ability of signalling mutants to promote muscle migration versus axonal growth allowed us to uncouple an indirect effect of HGF/Met signalling on nerve growth through the regulation of muscle size from a direct regulation of motor growth via the PI3 kinase (PI3K), but not Src kinase, pathway. Furthermore, we found that HGF/Met-triggered expansion of Pea3 expression domain in the spinal cord can be accomplished through several alternative signalling cascades, differentially sensitive to the Pea3 dosage. Finally, we show that the regulation of MN survival by HGF/Met can equally be achieved in vitro and in vivo by alternative signalling cascades involving either PI3K-Akt or Src and Mek pathways. CONCLUSIONS: Our findings distinguish MN survival and fate specification, as RTK-triggered responses allowing substitutions of the downstream signalling routes, from nerve growth patterning, which depends on a selective, non-substitutable pathway.

MET signaling in GABAergic neuronal precursors of the medial ganglionic eminence restricts GDNF activity in cells that express GFRα1 and a new transmembrane receptor partner.

GDNF (glial cell line-derived neurotrophic factor) promotes the differentiation and migration of GABAergic neuronal precursors of the medial ganglionic eminence (MGE). These functions are dependent on the GPI-anchored receptor GFRα1, but independent of its two known transmembrane receptor partners RET and NCAM. Here we show that soluble GFRα1 is also able to promote differentiation and migration of GABAergic MGE neurons. These activities require endogenous production of GDNF. Although GDNF responsiveness is abolished in Gfra1(-/-) neurons, it can be restored upon addition of soluble GFRα1, a result that is only compatible with the existence of a previously unknown transmembrane signaling partner for the GDNF-GFRα1 complex in GABAergic neurons. The roles of two candidate transmembrane receptors previously implicated in GABAergic interneuron development--MET, a receptor for hepatocyte growth factor (HGF), and ErbB4, the neuregulin receptor--were examined. GDNF did not induce the activation of either receptor, nor did inhibition of MET or ErbB4 impair GDNF activity in GABAergic MGE neurons. Unexpectedly, however, inhibition of MET or HGF per se promoted neuronal differentiation and migration and enhanced the activity of GDNF on MGE neurons. These effects were dependent on endogenous GDNF and GFRα1, suggesting that MET signaling negatively regulates GDNF activity in the MGE. In agreement with this, Met mutant MGE neurons showed enhanced responses to GDNF and inhibition of MET or HGF increased Gfra1 mRNA expression in MGE cells. In vivo, expression of MET and GFRα1 overlapped in the MGE, and a loss-of-function mutation in Met increased Gfra1 expression in this region. Together, these observations demonstrate the existence of a novel transmembrane receptor partner for the GDNF-GFRα1 complex and uncover an unexpected interplay between GDNF-GFRα1 and HGF-MET signaling in the early diversification of cortical GABAergic interneuron subtypes.

Pool-specific regulation of motor neuron survival by neurotrophic support.

The precise control of motor neuron (MN) death and survival following initial innervation of skeletal muscle targets is a key step in sculpting a functional motor system, but how this is regulated at the level of individual motor pools remains unclear. Hepatocyte growth factor (HGF) and its receptor Met play key developmental roles in both muscle and MNs. We generated mice (termed "Nes-Met") in which met is inactivated from midembryonic stages onward in the CNS only. Adult animals showed motor behavioral defects suggestive of impaired innervation of pectoral muscles. Correspondingly, in neonatal spinal cords of Nes-Met mutants, we observed death of a discrete population of pea3-expressing MNs at brachial levels. Axonal tracing using pea3 reporter mice revealed a novel target muscle of pea3-expressing MNs: the pectoralis minor muscle. In Nes-Met mice, the pectoralis minor pool initially innervated its target muscle, but required HGF/Met for survival, hence for proper maintenance of muscle innervation. In contrast, HGF/Met was dispensable for the survival of neighboring Met-expressing MN pools, despite its earlier functions for their specification and axon growth. Our results demonstrate the exquisite degree to which outcomes of signaling by receptor tyrosine kinases are regulated on a cell-by-cell basis. They also provide a model for one way in which the multiplicity of neurotrophic factors may allow for regulation of MN numbers in a pool-specific manner.

Met acts through Abl to regulate p53 transcriptional outcomes and cell survival in the developing liver.

BACKGROUND & AIMS: Genetic studies indicate that distinct signaling modulators are each necessary but not individually sufficient for embryonic hepatocyte survival in vivo. Nevertheless, how signaling players are interconnected into functional circuits and how they coordinate the balance of cell survival and death in developing livers are still major unresolved issues. In the present study, we examined the modulation of the p53 pathway by HGF/Met in embryonic livers. METHODS: We combined pharmacological and genetic approaches to biochemically and functionally evaluate p53 pathway modulation in primary embryonic hepatocytes and in developing livers. RT-PCR arrays were applied to investigate the selectivity of p53 transcriptional response triggered by Met. RESULTS: Met recruits p53 to regulate the liver developmental program, by qualitatively modulating its transcriptional properties: turning on the Mdm2 survival gene, while keeping death and cell-cycle arrest genes Pmaip1 and p21 silent. We investigated the mechanism leading to p53 regulation by Met and found that Abl and p38MAPK are required for p53 phosphorylation on S(389), Mdm2 upregulation, and hepatocyte survival. Alteration of this signaling mechanism switches p53 properties, leading to p53-dependent cell death in embryonic livers. RT-PCR array studies affirmed the ability of the Met-Abl-p53 axis to modulate the expression of distinct genes that can be regulated by p53. CONCLUSIONS: A signaling circuit involving Abl and p38MAPK is required downstream of Met for the survival of embryonic hepatocytes, via qualitative regulation of the p53 transcriptional response, by switching its proapoptotic into survival properties.

Enhanced Wild-Type MET Receptor Levels in Mouse Hepatocytes Attenuates Insulin-Mediated Signaling.

Compelling evidence points to the MET receptor tyrosine kinase as a key player during liver development and regeneration. Recently, a role of MET in the pathophysiology of insulin resistance and obesity is emerging. Herein, we aimed to determine whether MET regulates hepatic insulin sensitivity. To achieve this, mice in which the expression of wild-type MET in hepatocytes is slightly enhanced above endogenous levels (Alb-R26Met mice) were analyzed to document glucose homeostasis, energy balance, and insulin signaling in hepatocytes. We found that Alb-R26Met mice exhibited higher body weight and food intake when compared to R26stopMet control mice. Metabolic analyses revealed that Alb-R26Met mice presented age-related glucose and pyruvate intolerance in comparison to R26stopMet controls. Additionally, in Alb-R26Met mice, high MET levels decreased insulin-induced insulin receptor (IR) and AKT phosphorylation compared to control mice. These results were corroborated in vitro by analyzing IR and AKT phosphorylation in primary mouse hepatocytes from Alb-R26Met and R26stopMet mice upon insulin stimulation. Moreover, co-immunoprecipitation assays revealed MET-IR interaction under both basal and insulin stimulation conditions; this effect was enhanced in Alb-R26Met hepatocytes. Altogether, our results indicate that enhanced MET levels alter hepatic glucose homeostasis, which can be an early event for subsequent liver pathologies.

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo.

Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders.

BCL-XL blockage in TNBC models confers vulnerability to inhibition of specific cell cycle regulators.

Cell cycle regulators are frequently altered in Triple-Negative Breast Cancer (TNBC). Emerging agents targeting these signals offer the possibility to design new combinatorial therapies. However, preclinical models that recapitulate TNBC primary resistance and heterogeneity are essential to evaluate the potency of these combined treatments. Methods: Bioinformatic processing of human breast cancer datasets was used to analyse correlations between expression levels of cell cycle regulators and patient survival outcome. The MMTV-R26Met mouse model of TNBC resistance and heterogeneity was employed to analyse expression and targeting vulnerability of cell cycle regulators in the presence of BCL-XL blockage. Robustness of outcomes and selectivity was further explored using a panel of human breast cancer cells. Orthotopic studies in nude mice were applied for preclinical evaluation of efficacy and toxicity. Alterations of protein expression, phosphorylation, and/or cellular localisation were analysed by western blots, reverse phase protein array, and immunocytochemistry. Bioinformatics was performed to highlight drug's mechanisms of action. Results: We report that high expression levels of the BCL2L1 gene encoding BCL-XL and of specific cell cycle regulators correlate with poor survival outcomes of TNBC patients. Blockage of BCL-XL confers vulnerability to drugs targeting CDK1/2/4, but not FOXM1, CDK4/6, Aurora A and Aurora B, to all MMTV-R26Met and human TNBC cell lines tested. Combined blockage of BCL-XL and CDK1/2/4 interfered with tumour growth in vivo. Mechanistically, we show that, co-targeting of BCL-XL and CDK1/2/4 synergistically inhibited cell viability by combinatorial depletion of survival and RTK/AKT signals, and concomitantly restoring FOXO3a tumour suppression actions. This was accompanied by an accumulation of DNA damage and consequently apoptosis. Conclusions: Our studies illustrate the possibility to exploit the vulnerability of TNBC cells to CDK1/2/4 inhibition by targeting BCL-XL. Moreover, they underline that specificity matters in targeting cell cycle regulators for combinatorial anticancer therapies.

Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.

Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth.

Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

Tracking Dynamics of Spontaneous Tumors in Mice Using Photon-Counting Computed Tomography.

Computed tomography is a powerful medical imaging modality for longitudinal studies in cancer to follow neoplasia progression and evaluate anticancer therapies. Here, we report the generation of a photon-counting micro-computed tomography (PC-CT) method based on hybrid pixel detectors with enhanced sensitivity and precision of tumor imaging. We then applied PC-CT for longitudinal imaging in a clinically relevant liver cancer model, the Alb-R26Met mice, and found a remarkable heterogeneity in the dynamics for tumors at the initiation phases. Instead, the growth curve of evolving tumors exhibited a comparable exponential growth, with a constant doubling time. Furthermore, longitudinal PC-CT imaging in mice treated with a combination of MEK and BCL-XL inhibitors revealed a drastic tumor regression accompanied by a striking remodeling of macrophages in the tumor microenvironment. Thus, PC-CT is a powerful system to detect cancer initiation and progression, and to monitor its evolution during treatment.

Proapoptotic function of the MET tyrosine kinase receptor through caspase cleavage.

The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.

Imino-tetrahydro-benzothiazole derivatives as p53 inhibitors: discovery of a highly potent in vivo inhibitor and its action mechanism.

Several neurological disorders manifest symptoms that result from the degeneration and death of specific neurons. p53 is an important modulator of cell death, and its inhibition could be a therapeutic approach to several neuropathologies. Here, we report the design, synthesis, and biological evaluation of novel p53 inhibitors based on the imino-tetrahydrobenzothiazole scaffold. By performing studies on their mechanism of action, we find that cyclic analogue 4b and its open precursor 2b are more potent than pifithrin-alpha (PFT-alpha), which is known to block p53 pro-apoptotic activity in vitro and in vivo without acting on other pro-apoptotic pathways. Using spectroscopic methods, we also demonstrate that open form 2b is more stable than 4b in biological media. Compound 2b is converted into its corresponding active cyclic form through an intramolecular dehydration process and was found two log values more active in vivo than PFT-alpha. Thus, 2b can be considered as a new prodrug prototype that prevents in vivo p53-triggered cell death in several neuropathologies and possibly reduces cancer therapy side effects.

Coordination of signalling networks and tumorigenic properties by ABL in glioblastoma cells.

The cytoplasmic tyrosine kinase ABL exerts positive or negative effects in solid tumours according to the cellular context, thus functioning as a "switch modulator". The therapeutic effects of drugs targeting a set of signals encompassing ABL have been explored in several solid tumours. However, the net contribution of ABL inhibition by these agents remains elusive as these drugs also act on other signalling components. Here, using glioblastoma (GBM) as a cellular paradigm, we report that ABL inhibition exacerbates mesenchymal features as highlighted by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Cells with permanent ABL inhibition exhibit enhanced motility and invasive capabilities, while proliferation and tumorigenic properties are reduced. Intriguingly, permanent ABL inhibition also interferes with GBM neurosphere formation and with expression of stemness markers in sphere-cultured GBM cells. Furthermore, we show that the molecular and biological characteristics of GBM cells with impaired ABL are reversible by restoring ABL levels, thus uncovering a remarkable plasticity of GBM cells to ABL threshold. A phospho-signalling screen revealed that loss of tumorigenic and self-renewal properties in GBM cells under permanent ABL inhibition coincide with drastic changes in the expression and/or phosphorylation levels of multiple signalling components. Our findings identify ABL as a crucial player for migration, invasion, proliferation, tumorigenic, and stem-cell like properties of GBM cells. Taken together, this work supports the notion that the oncogenic role of ABL in GBM cells is associated with its capability to coordinate a signalling setting that determines tumorigenic and stem-cell like properties.

The noncatalytic TrkCNC2 receptor is cleaved by metalloproteases upon neurotrophin-3 stimulation.

The trkC locus encodes catalytic and noncatalytic receptors, generated by alternative splicing. These primary high-affinity neurotrophin-3 (NT-3) receptors may act in concert to modulate responsiveness to NT-3. Signal modulation can also be achieved by receptors that are post-translationally processed. We report that the noncatalytic TrkC receptor, TrkCNC2, is cleaved at the membrane-proximal region of its extracellular domain. This generates a soluble ectodomain (gp90(TrkCNC2)) recovered in the cell culture medium and a membrane-bound fragment (p20(TrkCNC2)), which contains the transmembrane and intracellular regions including the juxtamembrane and the NC2-specific cytoplasmic domains. We also show that this processing, which does not occur in the TrkC catalytic counterpart, is upregulated by NT-3 and upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Moreover, cleavage inhibition after EDTA or 1.10 phenanthroline treatment suggests involvement of a metalloprotease(s). Finally, this post-translational processing was observed not only in TrkCNC2-overexpressing NIH3T3 cells but also in primary cultures of cortical neurons and brain extracts. This study shows that, in addition to alternative splicing, ectodomain shedding represents a novel means of regulating TrkC receptor signaling, and consequently NT-3 biological effects on target cells.

Depleting MET-Expressing Tumor Cells by ADCC Provides a Therapeutic Advantage over Inhibiting HGF/MET Signaling.

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures.

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by "RTK swapping" by interfering with PDGFRß phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.

Combined signaling through ERK, PI3K/AKT, and RAC1/p38 is required for met-triggered cortical neuron migration.

Cell migration is a complex biological process playing a key role in physiological and pathological conditions. During central nervous system development, positioning and function of cortical neurons is tightly regulated by cell migration. Recently, signaling events involving the urokinase-type plasminogen activator receptor, which is a key regulator for the activation of hepatocyte growth factor (HGF), have been implicated in modulating cortical neuron migration. However, the intracellular pathways controlling neuronal migration triggered by the HGF receptor Met have not been elucidated. By combining pharmacological and genetic approaches, we show here that the Ras/ERK pathway and phosphatidylinositol 3-kinase (PI3K) are both required for cortical neuron migration. By dissecting the downstream signals necessary for this event, we found that Rac1/p38 and Akt are required, whereas the c-Jun N-terminal kinase (JNK) and mTOR/p70(s6k) pathways are dispensable. This study demonstrates that concomitant activation of the Ras/ERK, PI3K/Akt, and Rac1/p38 pathways is required to achieve full capacity of cortical neurons to migrate upon HGF stimulation.