Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas.
ABSTRACT: Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.
Project description:Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.
Project description:The receptor tyrosine kinase c-MET and its ligand, hepatocyte growth factor (HGF), regulate multiple cellular processes that stimulate cell proliferation, invasion and angiogenesis. This review provides an overview of the evidence to support c-MET or the HGF/c-MET signaling pathway as relevant targets for personalized cancer treatment based on high frequencies of c-MET and/or HGF overexpression, activation, amplification in non-small cell lung carcinoma (NSCLC), gastric, ovarian, pancreatic, thyroid, breast, head and neck, colon and kidney carcinomas. Additionally, the current knowledge of small molecule inhibitors (tivantinib [ARQ 197]), c-MET/HGF antibodies (rilotumumab and MetMAb) and mechanisms of resistance to c-MET-targeted therapies are discussed.
Project description:Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.
Project description:Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the screening assay of extracts from the root tuber of Tetrastigma hemsleyanum Diels et Gilg, isoquercitrin inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering. Further analysis revealed that isoquercitrin specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met. We also found that isoquercitrin decreased HGF-induced migration and invasion by parental or HGF/SF-transfected bladder carcinoma cell line NBT-II cells. Furthermore, isoquercitrin inhibited HGF/SF-induced epithelial mesenchymal transition in vitro and the invasion/metastasis of HGF/SF-transfected NBT-II cells in vivo. Our data suggest the possible use of isoquercitrin in human cancers associated with dysregulated HGF/SF-Met signaling.
Project description:We recently found that scatter factor (SF), a cell motility factor with a multimodular structure, is identical to hepatocyte growth factor (HGF), a potent mitogen of various cell types. SF/HGF is the ligand of the c-Met receptor tyrosine kinase. Here we used transient expression of naturally occurring and in vitro mutagenized cDNAs of SF/HGF to delineate the protein domains necessary for biological activity and binding to the c-Met receptor. (i) A single-chain SF/HGF resulting from the destruction of the protease cleavage site between heavy and light chain (Arg-494--> Gln) was largely inactive, indicating that proteolytic cleavage is essential for acquisition of the biologically active conformation. (ii) A SF/HGF splice variant encoding a protein with a 5-amino acid deletion in the first kringle domain was as highly active as the wild-type molecule. (iii) The separately expressed light chain (with serine protease homology) was inactive in all assays tested. (iv) The separate heavy chain as well as a naturally occurring splice variant consisting of the N terminus and the first two kringle domains bound the c-Met receptor, stimulated tyrosine auto-phosphorylation, and induced scattering of epithelial cells but not mitogenesis. These data indicate that a functional domain in the N terminus/first two kringle regions of SF/HGF is sufficient for binding to the Met receptor and that this leads to the activation of the downstream signal cascade involved in the motility response. However, the complete SF/HGF protein seems to be required for mitogenic activity.
Project description:Hepatocyte growth factor/scatter factor (HGF/SF), acting through the Met receptor, plays an important role in most human solid tumors, and inappropriate expression of this ligand-receptor pair is often associated with poor prognosis. The molecular basis for the malignant potential of the HGF/SF-Met signal in cancer cells has mostly been attributed to its mitogenic and invasive properties. However, HGF/SF also induces angiogenesis, but the signaling mechanism has not been fully explained, nor has this activity been directly associated with HGF/SF-Met-mediated tumorigenesis. It is known that HGF/SF induces in vitro expression of vascular endothelial growth factor (VEGF), a key agonist of tumor angiogenesis; by contrast, thrombospondin 1 (TSP-1) is a negative regulator of angiogenesis. Here, we show that, in the very same tumor cells, in addition to inducing VEGF expression, HGF/SF dramatically down-regulates TSP-1 expression. We show that TSP-1 shut-off plays an important, extrinsic role in HGF/SF-mediated tumor development, because ectopic expression of TSP-1 markedly inhibits tumor formation through the suppression of angiogenesis. Interestingly, although VEGF-induced expression is sensitive to inhibitors of several pathways, including mitogen-activated protein kinase, phosphoinositide 3-kinase, and signal transducer and activator of transcription 3, TSP-1 shut-off by HGF/SF is prevented solely by inhibiting mitogen-activated protein kinase activation. These studies identify HGF/SF as a key switch for turning on angiogenesis. They suggest that TSP-1 is a useful antagonist to tumor angiogenesis and that it may have therapeutic value when used in conjunction with inhibitors of VEGF.
Project description:MET is located on chromosome 7q31 and is a proto-oncogene that encodes for hepatocyte growth factor (HGF) receptor, a member of the receptor tyrosine kinase (RTK) family. HGF, also known as scatter factor (SF), is the only known ligand for MET. MET is a master regulator of cell growth and division (mitogenesis), mobility (motogenesis), and differentiation (morphogenesis); it plays an important role in normal development and tissue regeneration. The HGF-MET axis is frequently dysregulated in cancer by MET gene amplification, translocation, and mutation, or by MET or HGF protein overexpression. MET dysregulation is associated with an increased propensity for metastatic disease and poor overall prognosis across multiple tumor types. Targeting the dysregulated HGF-MET pathway is an area of active research; a number of monoclonal antibodies to HGF and MET, as well as small molecule inhibitors of MET, are under development. This review summarizes the key biological features of the HGF-MET axis, its dysregulation in cancer, and the therapeutic agents targeting the HGF-MET axis, which are in development.
Project description:The polypeptide growth factor, hepatocyte growth factor/scatter factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade beta-propeller domain of MET harboring the loops connecting the beta-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite "b" face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.
Project description:The receptor tyrosine kinase MET and its ligand, the Hepatocyte Growth Factor/Scattor Factor (HGF/SF), are essential to the migration, morphogenesis, and survival of epithelial cells. In addition, dysregulation of MET signaling has been shown to promote tumor progression and invasion in many cancers. Therefore, HGF/SF and MET are major targets for chemotherapies. Improvement of targeted therapies requires a perfect understanding of tumor microenvironment that strongly modifies half-life, bio-accessibility and thus, efficacy of treatments. In particular, hypoxia is a crucial microenvironmental phenomenon promoting invasion and resistance to treatments. Under hypoxia, MET auto-phosphorylation resulting from ligand stimulation or from receptor overexpression is drastically decreased within minutes of oxygen deprivation but is quickly reversible upon return to normoxia. Besides a decreased phosphorylation of its proximal adaptor GAB1 under hypoxia, activation of the downstream kinases Erk and Akt is maintained, while still being dependent on MET receptor. Consistently, several cellular responses induced by HGF/SF, including motility, morphogenesis, and survival are effectively induced under hypoxia. Interestingly, using a semi-synthetic ligand, we show that HGF/SF binding to MET is strongly impaired during hypoxia but can be quickly restored upon reoxygenation. Finally, we show that two MET-targeting tyrosine kinase inhibitors (TKIs) are less efficient on MET signalling under hypoxia. Like MET loss of phosphorylation, this hypoxia-induced resistance to TKIs is reversible under normoxia. Thus, although hypoxia does not affect downstream signaling or cellular responses induced by MET, it causes immediate resistance to TKIs. These results may prove useful when designing and evaluation of MET-targeted therapies against cancer.
Project description:CrkII, a 40 kDa adaptor possessing a Src homology (SH)2 domain followed by two SH3 domains, although not endowed with catalytic activity, participates in intracellular signalling, presumably by activating the Ras pathway. CrkII was found to be phosphorylated in response to hepatocyte growth factor/scatter factor (HGF/SF) and to associate with the beta-subunit of the HGF receptor (MET). CrkII associated with p(145betaMET) via its SH2 domain. Growth-factor-receptor-bound protein 2 (Grb2) co-immunoprecipitated with CrkII species. By transient transfection of A431 human epidermoid carcinoma cells with wild-type and dominant-negative Grb2 expression constructs lacking either the SH2 or SH3 domains, we have concluded that Grb2 does not contribute to the 'presentation' of CrkII to p(145betaMET). Overexpression of wild-type CrkII in A431 cells enhanced HGF/SF-induced proliferation, while a CrkII dominant-negative mutant lacking the SH2 domain prevented a similar proliferating response to HGF/SF. The effect of CrkII on HGF/SF-induced proliferation was also abolished in cells co-expressing CrkII and Son-of-sevenless lacking the guanine exchange domain, suggesting that CrkII is likely to induce cell proliferation partly via the Ras/mitogen-activated protein kinase route.