Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

Promise and challenges on the horizon of MET-targeted cancer therapeutics

MET(MNNG HOS transforming gene) is one of the receptor tyrosine kinases whose activities are frequently altered in human cancers, and it is a promising therapeutic target. MET is normally activated by its lone ligand, hepatocyte growth factor(HGF), eliciting its diverse biological activities that are crucial for development and physiology. Alteration of the HGF-MET axis results in inappropriate activation of a cascade of intracellular signaling pathways that contributes to hallmark cancer events including deregulated cell proliferation and survival, angiogenesis, invasion, andmetastasis. Aberrant MET activation results from autocrine or paracrine mechanisms due to overexpression of HGF and/or MET or from a ligand-independent mechanism caused by activating mutations or amplification of MET. The literature provides compelling evidence for the role of MET signaling in cancer development and progression. The finding that cancer cells often use MET activation to escape therapies targeting other pathways strengthens the argument for MET-targeted therapeutics. Diverse strategies have been explored to deactivate MET signaling, and compounds and biologics targeting the MET pathway are in clinical development. Despite promising results from various clinical trials, we are still waiting for true MET-targeted therapeutics in the clinic. This review will explore recent progress and hurdles in the pursuit of METtargeted cancer drugs and discuss the challenges in such development.     

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling.The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor.Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.     

HGF/MET signaling in ovarian cancer

Ovarian cancer is the leading cause of death from gynecological cancers in North America and Europe. Despite its clinical significance, the factors that regulate the development and progression of ovarian cancer are among the least understood of all major human malignancies. A growth factor with pleiotropic effects, which has attracted increasing attention in recent years, is the hepatocyte growth factor (HGF) and its receptor MET. While deregulated HGF/MET signaling is observed in many tumors, the consequences of MET activation are complex and context dependent. Recent observations have demonstrated a cross-talk of other signaling pathways with MET signaling. This review summarizes the key findings and recent advances in our understanding of HGF and MET in the transformation and progression of ovarian cancer. We will begin with a brief discussion on the role of HGF and MET in the physiology of normal ovarian surface epithelium (OSE) and ovarian cancer development. In particular, the coexpression of HGF and MET in OSE of women with hereditary ovarian cancer syndromes emphasizes their importance in neoplastic transformation of OSE. The involvement of HGF in other aspects of tumor progression, such as invasion and metastasis, and novel downstream target genes activated by HGF is summarized next. The therapeutic potential of HGF to treat ovarian cancer and to improve response to conventional chemotherapy is also described. Finally, the most recent progress in drug development and future areas of research in terms of their potential clinical implications are discussed.     

Inhibition of the MET Kinase Activity and Cell Growth in MET-Addicted Cancer Cells by Bi-Paratopic Linking

MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.     

The hepatocyte growth factor (HGF)–MET receptor tyrosine kinase signaling pathway: Diverse roles in modulating immune cell functions

Hepatocyte growth factor (HGF) signaling via the MET receptor is essential for embryonic development and tissue repair. On the other hand, deregulated MET signaling promotes tumor progression in diverse types of cancers. Even though oncogenic MET signaling remains the major research focus, the HGF–MET axis has also been implicated in diverse aspects of immune cell development and functions. In the presence of other hematopoietic growth factors, HGF promotes the development of erythroid, myeloid and lymphoid lineage cells and thrombocytes. In monocytes and macrophages responding to inflammatory stimuli, induction of autocrine HGF–MET signaling can contribute to tissue repair via stimulating anti-inflammatory cytokine production. HGF–MET signaling can also modulate adaptive immune response by facilitating the migration of Langerhans cells and dendritic cells to draining lymph nodes. However, MET signaling has also been shown to induce tolerogenic dendritic cells in mouse models of graft-versus-host disease and experimental autoimmune encephalomyelitis. HGF–MET axis is also implicated in promoting thymopoiesis and the survival and migration of B lymphocytes. Recent studies have shown that MET signaling induces cardiotropism in activated T lymphocytes. Further understanding of the HGF–MET axis in the immune system would allow its therapeutic manipulation to improve immune cell reconstitution, restore immune homeostasis and to treat immuno-inflammatory diseases.     

Activation of Ras/Erk pathway by a novel MET-interacting protein RanBPM

MET is a receptor protein-tyrosine kinase (RPTK) for hepatocyte growth factor (HGF), which is a multifunctional cytokine controlling cell growth, morphogenesis, and motility. MET overexpression has been identified in a variety of human cancers. Oncogenic missense mutations of the tyrosine kinase domain of the MET gene have been identified in human papillary renal cell carcinomas. In this study, RanBPM, also known as RanBP9, is identified as a novel interacting protein of MET through yeast two-hybrid screen. RanBPM contains a conserved SPRY (repeats in splA and RyR) domain. We demonstrate that RanBPM can interact with MET in vitro and in vivo, and the interaction can be strengthened by HGF stimulation. RanBPM interacts with the tyrosine kinase domain of MET through its SPRY domain. We show that RanBPM can induce GTP-Ras association and Erk phosphorylation and elevate serum response element-luciferase (SRE-LUC) expression, indicating that RanBPM can activate the Ras-Erk-SRE pathway. We demonstrate that RanBPM, which itself is not a guanine exchange protein, stimulates Ras activation by recruiting Sos. On the cellular level, A704 cells, a human renal carcinoma cell line, transfected with RanBPM exhibit increased migration ability. Our data suggest that RanBPM, functioning as an adaptor protein for the MET tyrosine kinase domain, can augment the HGF-MET signaling pathway and that RanBPM overexpression may cause constitutive activation of the Ras signaling pathway.     

The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.     

MET molecular mechanisms and therapies in lung cancer

The MET tyrosine kinase signaling pathway is upregulated in many cancers, including lung cancer. The pathway normally promotes mitosis, cell motility and cell survival; but in cancer it can also promote cell proliferation, invasion, metastasis, and angiogenesis. The activating ligand, hepatocyte growth factor, is normally secreted by fibroblasts and smooth muscle cells, but can also be produced by tumor cells. MET upregulation in lung cancer is caused by overexpression and mutation. These mutations can vary with ethnicity. MET signaling affects cytoskeletal proteins such as paxillin, which participates in cell adhesion, growth and motility. Therapeutic approaches that block MET signaling are being studied, and include the use of: small interference RNA, Geldanamycin, competitive HGF homologues, decoy receptors, and direct MET inhibitors such as K252a, SU11274, PHA665752 and PF2341066. It is hoped that blocking MET signaling may one day become an effective treatment for some lung cancers.     

Anti-apoptotic Role of Caspase-cleaved GAB1 Adaptor Protein in Hepatocyte Growth Factor/Scatter Factor-MET Receptor Protein Signaling

The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

The GRB2 associated binder 1 (GAB1) is an essential docking/adaptor protein for transmitting intracellular signals of the MET tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). We found that in response to hours of HGF/SF treatment, the GAB1 protein level is degraded by a mechanism involving MET activity and the proteasomal machinery. We also showed that GAB1 is both multi- and poly-ubiquitinated in a CBL-dependent manner. A long term exposure to HGF/SF caused a more sustained down-regulation of GAB1 than of MET, associated with a loss of reactivation of the ERK MAP kinases to subsequent acute ligand treatment. These data demonstrate that GAB1 is ubiquitinated by CBL and degraded by the proteasome, and plays a role in negative-feedback regulation of HGF/SF–MET signaling.     

The HGF/MET pathway as target for the treatment of multiple myeloma and B-cell lymphomas

Hepatocyte growth factor (HGF) and its receptor MET are essential during embryonic development and throughout postnatal life. However, aberrant MET activation, due to overexpression, mutations, or autocrine ligand production, contributes to the development and progression of a variety of human cancers, often being associated with poor clinical outcome and drug resistance. B cell malignancies arise from B cells that are clonally expanded at different stages of differentiation. Despite major therapeutic advances, most mature B cell malignancies remain incurable and biologically-oriented therapeutic strategies are urgently needed. This review addresses the role of the HGF/MET pathway during B cell development and discusses how its aberrant activation contributes to the development of B cell lymphoproliferative disorders, with particular emphasis on multiple myeloma and diffuse large B cell lymphoma. These insights, combined with the recent development of clinical-grade agents targeting the MET pathway, provide the rationale to envision the HGF/MET pathway as a new promising target for the treatment of B cell malignancies.     

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.     

Quantitative phospho-proteomic profiling of hepatocyte growth factor (HGF)-MET signaling in colorectal cancer

Colorectal cancer (CRC) is the second leading cause of death from cancer. The MET receptor tyrosine kinase and/or its ligand HGF are frequently amplified or overexpressed in CRC. It is known that tyrosine phosphorylated proteins are involved in progression and metastasis of colorectal cancer; however, little is known about the MET phospho-proteome in CRC. High resolution mass spectrometry was used to characterize immunoaffinity-purified, phosphotyrosine (pY)-containing tryptic peptides of the MET-expressing CRC cell model, DLD1. A total of 266 unambiguously identified pY sites spanning 168 proteins were identified. Quantification of mass spectrometry ion currents identified 161 pY sites, including many not previously linked to MET signaling, that were modulated in abundance by HGF stimulation. Overlay of these data with protein-protein interaction data sets suggested that many of the identified HGF-modulated phospho-proteins may be directly or indirectly associated with MET. Analysis of pY sequence motifs indicated a prevalence of Src family kinase consensus sequences, and reciprocal signaling between Src and MET was confirmed by using selective small molecule inhibitors of these kinases. Therefore, using quantitative phospho-proteomics profiling, kinase modulation by ligand and inhibitors, and data integration, an outline of the MET signaling network was generated for the CRC model.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

HGF regulates the development of cortical pyramidal dendrites

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.     

Distinct intracellular signaling mediates C‐MET regulation of dendritic growth and synaptogenesis

Hepatocyte growth factor (HGF) activation of the MET receptor tyrosine kinase influences multiple neurodevelopmental processes. Evidence from human imaging and mouse models shows that, in the forebrain, disruptions in MET signaling alter circuit formation and function. One likely means of modulation is by controlling neuron maturation. Here, we examined the signaling mechanisms through which MET exerts developmental effects in the neocortex. In situ hybridization revealed that hgf is located near MET‐expressing neurons, including deep neocortical layers and periventricular zones. Western blot analyses of neocortical crude membranes demonstrated that HGF‐induced MET autophosphorylation peaks during synaptogenesis, with a striking reduction in activation between P14 and P17 just before pruning. In vitro analysis of postnatal neocortical neurons assessed the roles of intracellular signaling following MET activation. There is rapid, HGF‐induced phosphorylation of MET, ERK1/2, and Akt that is accompanied by two major morphological changes: increases in total dendritic growth and synapse density. Selective inhibition of each signaling pathway altered only one of the two distinct events. MAPK/ERK pathway inhibition significantly reduced the HGF‐induced increase in dendritic length, but had no effect on synapse density. In contrast, inhibition of the PI3K/Akt pathway reduced HGF‐induced increases in synapse density, with no effect on dendritic length. The data reveal a key role for MET activation during the period of neocortical neuron growth and synaptogenesis, with distinct biological outcomes mediated via discrete MET‐linked intracellular signaling pathways in the same neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1160–1181, 2016     

The role of HGF/c-MET signaling pathway in lymphoma

Inappropriate activation of c-mesenchymal-epithelial transition (MET), the receptor tyrosine kinase (RTK) for hepatocyte growth factor (HGF), has been implicated in tumorigenesis and represented a promising therapeutic target for developing anticancer agents. In contrast to other solid tumors, there are limited data describing the functional role of HGF/c-MET signaling pathway in lymphoma. In the current review, we summarize recent findings about the expression, cellular mechanisms/functions, and therapeutic application of HGF/c-MET in different types of lymphoma, especially B cell lymphoma, T and NK cell lymphoma, and Hodgkin lymphoma. We also discuss the existing problems and future directions about studying the HGF/c-MET pathway in lymphoma cells.