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.

Identification of targeted therapies for TNBC is an urgent medical need. Using a drug combination screen reliant on synthetic lethal interactions, we identified clinically relevant combination therapies for different TNBC subtypes. Two drug combinations targeting the BET family were further explored. The first, targeting BET and CXCR2, is specific for mesenchymal TNBC and induces apoptosis, whereas the second, targeting BET and the proteasome, is effective for major TNBC subtypes and triggers ferroptosis. Ferroptosis was induced at low drug doses and was associated with increased cellular iron and decreased glutathione levels, concomitant with reduced levels of GPX4 and key glutathione biosynthesis genes. Further functional studies, analysis of clinical datasets and breast cancer specimens revealed a unique vulnerability of TNBC to ferroptosis inducers, enrichment of ferroptosis gene signature, and differential expression of key proteins that increase labile iron and decrease glutathione levels. This study identified potent combination therapies for TNBC and unveiled ferroptosis as a promising therapeutic strategy.

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.

The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26(stopMet) knock-in context (Del-R26(Met)) reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an RTK when occurring in its endogenous domain of activity.

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.

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 PDGFRβ 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.

The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. Deregulation of receptor tyrosine kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis.

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.

Generation of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD.

Enhancing the differentiation potential of human induced pluripotent stem cells (hiPSC) into disease-relevant cell types is instrumental for their widespread application in medicine. Here, we show that hiPSCs downregulated for the signaling modulator GLYPICAN-4 (GPC4) acquire a new biological state characterized by increased hiPSC differentiation capabilities toward ventral midbrain dopaminergic (VMDA) neuron progenitors. This biological trait emerges both in vitro, upon exposing cells to VMDA neuronal differentiation signals, and in vivo, even when transplanting hiPSCs at the extreme conditions of floor-plate stage in rat brains. Moreover, it is compatible with the overall neuronal maturation process toward acquisition of substantia nigra neuron identity. HiPSCs with downregulated GPC4 also retain self-renewal and pluripotency in stemness conditions, in vitro, while losing tumorigenesis in vivo as assessed by flank xenografts. In conclusion, our results highlight GPC4 downregulation as a powerful approach to enhance generation of VMDA neurons. Outcomes may contribute to establish hiPSC lines suitable for translational applications.

Epigenetic modifications such as aberrant DNA methylation reshape the gene expression repertoire in cancer. Here, we used a clinically relevant hepatocellular carcinoma (HCC) mouse model (Alb-R26Met) to explore the impact of DNA methylation on transcriptional switches associated with tumorigenesis. We identified a striking enrichment in genes simultaneously hypermethylated in CpG islands (CGIs) and overexpressed. These hypermethylated CGIs are located either in the 5'-UTR or in the gene body region. Remarkably, such CGI hypermethylation accompanied by gene upregulation also occurs in 56% of HCC patients, which belong to the "HCC proliferative-progenitor" subclass. Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change. Among reprogrammed genes, several are well-known oncogenes. For others not previously linked to cancer, we demonstrate here their action together as an "oncogene module". Thus, hypermethylation of gene body CGIs is predictive of elevated oncogene levels in cancer, offering a novel stratification strategy and perspectives to normalise cancer gene dosages.

The complexity of hepatocellular carcinoma (HCC) challenges the identification of disease-relevant signals. C3G, a guanine nucleotide exchange factor for Rap and other Ras proteins, plays a dual role in cancer acting as either a tumor suppressor or promoter depending on tumor type and stage. The potential relevance of C3G upregulation in HCC patients suggested by database analysis remains unknown. We have explored C3G function in HCC and the underlying mechanisms using public patient data and in vitro and in vivo human and mouse HCC models. We found that C3G is highly expressed in progenitor cells and neonatal hepatocytes, whilst being down-regulated in adult hepatocytes and re-expressed in human HCC patients, mouse HCC models and HCC cell lines. Moreover, high C3G mRNA levels correlate with tumor progression and a lower patient survival rate. C3G expression appears to be tightly modulated within the HCC program, influencing distinct cell biological properties. Hence, high C3G expression levels are necessary for cell tumorigenic properties, as illustrated by reduced colony formation in anchorage-dependent and -independent growth assays induced by permanent C3G silencing using shRNAs. Additionally, we demonstrate that C3G down-regulation interferes with primary HCC tumor formation in xenograft assays, increasing apoptosis and decreasing proliferation. In vitro assays also revealed that C3G down-regulation enhances the pro-migratory, invasive and metastatic properties of HCC cells through an epithelial-mesenchymal switch that favors the acquisition of a more mesenchymal phenotype. Consistently, a low C3G expression in HCC cells correlates with lung metastasis formation in mice. However, the subsequent restoration of C3G levels is associated with metastatic growth. Mechanistically, C3G down-regulation severely impairs HGF/MET signaling activation in HCC cells. Collectively, our results indicate that C3G is a key player in HCC. C3G promotes tumor growth and progression, and the modulation of its levels is essential to ensure distinct biological features of HCC cells throughout the oncogenic program. Furthermore, C3G requirement for HGF/MET signaling full activation provides mechanistic data on how it works, pointing out the relevance of assessing whether high C3G levels could identify HCC responders to MET inhibitors.

The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC).

By using an ensemble-docking strategy, we undertook a large-scale virtual screening campaign in order to identify new putative hits against the MET kinase target. Following a large molecular dynamics sampling of its conformational space, a set of 45 conformers of the kinase was retained as docking targets to take into account the flexibility of the binding site moieties. Our screening funnel started from about 80,000 chemical compounds to be tested in silico for their potential affinities towards the kinase binding site. The top 100 molecules selected-thanks to the molecular docking results-were further analyzed for their interactions, and 25 of the most promising ligands were tested for their ability to inhibit MET activity in cells. F0514-4011 compound was the most efficient and impaired this scattering response to HGF (Hepatocyte Growth Factor) with an IC 50 of 7.2 μ M. Interestingly, careful docking analysis of this molecule with MET suggests a possible conformation halfway between classical type-I and type-II MET inhibitors, with an additional region of interaction. This compound could therefore be an innovative seed to be repositioned from its initial antiviral purpose towards the field of MET inhibitors. Altogether, these results validate our ensemble docking strategy as a cost-effective functional method for drug development.

Cancer stem cells (CSCs) are implicated in tumor initiation, metastasis and drug resistance, and considered as attractive targets for cancer therapy. Here we identified a clinically relevant signaling nexus mediated by AXL receptor, PYK2 and PKCα and show its impact on stemness in TNBC. AXL, PYK2, and PKCα expression correlates with stemness signature in basal-like breast cancer patients, and their depletion in multiple mesenchymal TNBC cell lines markedly reduced the number of mammosphere-forming cells and cells harboring CSCs characteristic markers. Knockdown of PYK2 reduced the levels of AXL, PKCα, FRA1, and PYK2 proteins, and similar trend was obtained upon PKCα depletion. PYK2 depletion decreased AXL transcription through feedback loops mediated by FRA1 and TAZ, whereas PKCα inhibition induced redistribution of AXL to endosomal/lysosomal compartment and enhanced its degradation. PYK2 and PKCα cooperate at a convergence point of multiple stemness-inducing pathways to regulate AXL levels and concomitantly the levels/activation of STAT3, TAZ, FRA1, and SMAD3 as well as the pluripotent transcription factors Nanog and Oct4. Induction of stemness in TNBC sensitized cells to PYK2 and PKCα inhibition suggesting that targeting the AXL-PYK2-PKCα circuit could be an efficient strategy to eliminate CSCs in TNBC.

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.

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.

The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-β activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.

Enhanced activation of the transcription factor MYC and of the receptor tyrosine kinase MET are among the events frequently occurring in hepatocellular carcinoma (HCC). Both genes individually act as drivers of liver cancer initiation and progression. However, their concomitant alteration in HCC has not been explored, nor functionally documented. Here, we analysed databases of five independent human HCC cohorts and found a subset of patients with high levels of MYC and MET (MYChigh/METhigh) characterised by poor prognosis. This clinical observation drove us to explore the functionality of MYC and MET co-occurrence in vivo, combining hydrodynamic tail vein injection for MYC expression in the R26stopMet genetic setting, in which wild-type MET levels are enhanced following the genetic deletion of a stop cassette. Results showed that increased MYC and MET expression in hepatocytes is sufficient to induce liver tumorigenesis even in the absence of pre-existing injuries associated with a chronic disease state. Intriguingly, ectopic MYC in MET tumours increases expression of the Mki67 proliferation marker, and switches them into loss of Afp, Spp1, Gpc3, Epcam accompanied by an increase in Hgma1, Vim, and Hep-Par1 levels. We additionally found a switch in the expression of specific immune checkpoints, with an increase in the Ctla-4 and Lag3 lymphocyte co-inhibitory responses, and in the Icosl co-stimulatory responses of tumour cells. We provide in vitro evidence on the vulnerability of some human HCC cell lines to combined MYC and MET targeting, which are otherwise resistant to single inhibition. Mechanistically, combined blockage of MYC and MET converts a partial cytostatic effect, triggered by individual blockage of MYC or MET, into a cytotoxic effect. Together, these findings highlight a subgroup of HCC characterised by MYChigh/METhigh, and document functional cooperativity between MYC and MET in liver tumorigenesis. Thus, the MYC-R26Met model is a relevant setting for HCC biology, patient classification and treatment.