Down-Regulation of the Met Receptor Tyrosine Kinase by Presenilin-dependent Regulated Intramembrane Proteolysis

Hepatocyte growth factor/scatter factor (HGF/SF) acts through the membrane-anchored Met receptor tyrosine kinase to induce invasive growth. Deregulation of this signaling is associated with tumorigenesis and involves, in most cases, overexpression of the receptor. We demonstrate that Met is processed in epithelial cells by presenilin-dependent regulated intramembrane proteolysis (PS-RIP) independently of ligand stimulation. The proteolytic process involves sequential cleavage by metalloproteases and the γ-secretase complex, leading to generation of labile fragments. In normal epithelial cells, although expression of cleavable Met by PS-RIP is down-regulated, uncleavable Met displayed membrane accumulation and induced ligand-independent motility and morphogenesis. Inversely, in transformed cells, the Met inhibitory antibody DN30 is able to promote Met PS-RIP, resulting in down-regulation of the receptor and inhibition of the Met-dependent invasive growth. This demonstrates the original involvement of a proteolytic process in degradation of the Met receptor implicated in negative regulation of invasive growth.     

Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor.

Scatter Factor (SF) is a fibroblast-secreted protein which promotes motility and matrix invasion of epithelial cells. Hepatocyte Growth Factor (HGF) is a powerful mitogen for hepatocytes and other epithelial tissues. SF and HGF, purified according to their respective biological activities, were interchangeable and equally effective in assays for cell growth, motility and invasion. Both bound with identical affinities to the same sites in target cells. The receptor for SF and HGF was identified as the product of the MET oncogene by: (i) ligand binding and coprecipitation in immunocomplexes; (ii) chemical crosslinking to the Met beta subunit; (iii) transfer of binding activity in insect cells by a baculovirus carrying the MET cDNA; (iv) ligand-induced tyrosine phosphorylation of the Met beta subunit. SF and HGF cDNA clones from human fibroblasts, placenta and liver had virtually identical sequences. We conclude that the same molecule (SF/HGF) acts as a growth or motility factor through a single receptor in different target cells.     

How to make tubes: signaling by the Met receptor tyrosine kinase

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the receptor tyrosine kinase Met, stimulates cells derived from a variety of different organs to form elongated hollow tubules when grown in three-dimensional gels. In vivo data also indicate a role for HGF/SF and Met in tubule formation during liver and kidney regeneration and mammary gland formation. Activation of Met results in the recruitment of a myriad of signal transducers that regulate dissociation of adherens junctions and the stimulation of cellular motility, survival, proliferation and morphogenesis during tubule formation. Among these many signal transducers, the Gab1 adaptor protein and its effector, the SHP2 tyrosine phosphatase, have been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAP kinase pathway. Here, we discuss the contribution of these and other signaling pathways and the role of HGF/SF and Met in the formation of epithelial cell tubules both in vitro in branching-morphogenesis assays and in vivo during organogenesis.     

The Gab1 PH domain is required for localization of Gab1 at sites of cell-cell contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase

Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.     

The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells

Depending on the target cells and culture conditions, scatter factor/hepatocyte growth factor (SF/HGF) mediates several distinct activities, i.e., cell motility, proliferation, invasiveness, tubular morphogenesis, angiogenesis, or cytotoxicity. A small isoform of SF/HGF encoded by a natural splice variant, which consists of the NH2-terminal hairpin structure and the first two kringle domains but not the protease homology region, induces cell motility but not mitogenesis. Two types of SF/HGF receptors have recently been discovered in epithelial cells, the high affinity c-Met receptor tyrosine kinase, and low affinity/high capacity binding sites, which are probably located on heparan sulfate proteoglycans. In the present study, we have addressed the question whether the various biological activities of SF/HGF are transduced into cells by a single type of receptor. We have here examined MDCK epithelial cells transfected with a hybrid cDNA encoding the ligand binding domain of the nerve growth factor (NGF) receptor and the membrane-spanning and tyrosine kinase domains of the Met receptor. We demonstrate that all biological effects of SF/HGF upon epithelial cells such as the induction of cell motility, proliferation, invasiveness, and tubular morphogenesis can now be triggered by the addition of NGF. Thus, it is likely that all known biological signals of SF/HGF are transduced through the receptor tyrosine kinase encoded by the c-Met protooncogene.     

Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.     

Constitutive activation of met kinase in non-small-cell lung carcinomas correlates with anchorage-independent cell survival

Lung cancer is currently the most frequent cause of cancer death in North America. Hepatocyte growth factor (HGF) and its receptor Met are frequently over-expressed in non-small-cell lung carcinomas (NSCLC), but their potential role in tumor progression is not clearly known. To assess the role of HGF/Met signaling in lung carcinomas, we have examined the expression, activation status, and function of Met in NSCLC cell lines (n = 7), established from primary tumors or pleural fluids of cancer patients. We observed Met expression in three NSCLC cell lines, two of which exhibited constitutive tyrosine-phosphorylation of Met, and Met kinase activity. In addition, the observed constitutive activation of Met was sustained under anchorage-independent conditions, and correlated with phosphatidyl inositol 3-kinase-dependent cell survival. Immunoreactive HGF-like protein was secreted by two Met-positive and two Met-negative NSCLC cell lines. However HGF activity, as determined by the ability to induce cell scattering and tyrosine-phosphorylation of Met in reporter cell lines, was detected in conditioned medium from only one Met-negative NSCLC cell line: none of the conditioned media from Met-expressing NSCLC cell lines showed detectable HGF activity. Thus, constitutive activation of Met in NSCLC cell lines may occur at least in part through intracrine, or HGF-independent mechanisms. Interestingly, additional paracrine stimulation with exogenous recombinant HGF was required for DNA synthesis and correlated with increased activation of ERK1/2 in all Met-positive NSCLC cell lines, regardless of the basal activation status of Met. These findings indicate that a medium level of constitutive activation of Met occurs in some NSCLC cell lines, and correlates with survival of detached carcinoma cells; whereas additional paracrine stimulation by recombinant HGF is required for DNA synthesis. Thus constitutive and paracrine activation of Met may provide complementary signals that promote survival and proliferation, respectively, during tumor progression of NSCLC.     

Receptor-type protein tyrosine phosphatase beta (RPTP-beta) directly dephosphorylates and regulates hepatocyte growth factor receptor (HGFR/Met) function

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.     

c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination

Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.     

NK1, a Natural Splice Variant of Hepatocyte Growth Factor/Scatter Factor, Is a Partial Agonist In Vivo

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen, motogen, and morphogen for epithelial cells expressing its tyrosine kinase receptor, the c-met proto-oncogene product, and is required for normal development in the mouse. Inappropriate stimulation of Met signal transduction induces aberrant morphogenesis and oncogenesis in mice and has been implicated in human cancer. NK1 is a naturally occurring HGF/SF splice variant composed of only the amino terminus and first kringle domain. While the biological activities of NK1 have been controversial, in vitro data suggest that it may have therapeutic value as an HGF/SF antagonist. Here, we directly test this hypothesis in vivo by expressing mouse NK1 in transgenic mice and comparing the consequent effects with those observed for mice carrying an HGF/SF transgene. Despite robust expression, NK1 did not behave as an HGF/SF antagonist in vivo. Instead, NK1-transgenic mice displayed most of the phenotypic characteristics associated with HGF/SF-transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, progressive renal disease, aberrant pigment cell localization, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors. And like HGF/SF-transgenic livers, NK1 livers had higher levels of tyrosine-phosphorylated complexes associated with Met, suggesting that the mechanistic basis for the effects of NK1 overexpression in vivo was autocrine activation of Met. We conclude that NK1 acts in vivo as a partial agonist. As such, the efficacy of NK1 as a therapeutic HGF/SF antagonist must be seriously questioned.     

Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial-mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes. We provide evidence that signals involving CrkII and CrkL are required for the breakdown of adherens junctions, the spreading of epithelial colonies, and the formation of lamellipodia in response to HGF. The overexpression of a CrkI SH3 domain mutant blocks these HGF-dependent events. In addition, the overexpression of CrkII or CrkL promotes lamellipodia formation, loss of adherens junctions, cell spreading, and dispersal of colonies of breast cancer epithelial cells in the absence of HGF. Stable lines of epithelial cells overexpressing CrkII show enhanced activation of Rac1 and Rap1. The Crk-dependent breakdown of adherens junctions and cell spreading is inhibited by the expression of a dominant negative mutant of Rac1 but not Rap1. These findings provide evidence that Crk adapter proteins play a critical role in the breakdown of adherens junctions and the spreading of sheets of epithelial cells.     

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.     

Expression of functional tyrosine kinases on immortalized Kaposi's sarcoma cells

Kaposi's sarcoma (KS) is the most frequent malignant lesion in patients with AIDS and is characterized by spindle cell proliferation, inflammatory cell infiltration, angiogenesis, edema, and invasiveness. KS origin is still debated. The complex aspect of this disease is probably supported by multiple concomitant pathogenetic factors, among which growth factors and their cognate tyrosine kinase receptors are deeply involved. Here we have investigated the expression status and functional integrity of KDR and Met receptors, as well as of their ligands, in an immortalized KS cell line (KS-IMM). The MET and KDR genes encode the tyrosine kinase receptors for Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth Factor (VEGF) respectively. Both factors are pleiotropic cytokines controlling growth, survival, motility, invasive migration and differentiation of endothelial cells. We have found that KS-IMM cells, which retain most of the features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice, express the Met receptor, but not its ligand. The receptor, which is basally inactive, is functional, being tyrosine phosphorylated in response to ligand stimulation and mediating the expected HGF-dependent biological responses of motility, invasion and proliferation. Moreover, we report that KS-IMM cells coexpress VEGF and KDR and that KDR is constitutively tyrosine phosphorylated, possibly as a consequence of the establishment of an autocrine loop. The receptor, however, maintains responsiveness to exogenously added ligand, by increasing the level of tyrosine phosphorylation and by responding in biological assays of motility, invasion and proliferation. Finally, we have found that the two growth factors synergize in a motility assay. These data show that HGF and VEGF are growth factors active on KS-IMM cells.     

Isoquercitrin,ingredients in Tetrastigma hemsleyanum Diels et Gilg,inhibits hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

ABSTRACT

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.     

InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase

The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.     

Autocrine activation of the hepatocyte growth factor receptor/met tyrosine kinase induces tumor cell motility by regulating pseudopodial protrusion

The multiple beta-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFalpha antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFalpha antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.     

Decreased Expression of Met During Differentiation in Rat Lung

Organ-specific stem cells play key roles in maintaining the epithelial cell layers of lung. Bronchioalveolar stem cells (BASCs) are distal lung epithelial stem cells of adult mice. Alveolar type 2 (AT2) cells have important functions and serve as progenitor cells of alveolar type 1 (AT1) cells to repair the epithelium when they are injured. Hepatocyte growth factor (HGF) elicits mitogenic, morphogenic, and anti-apoptotic effects on lung epithelial cells through tyrosine phosphorylation of Met receptor, and thus is recognized as a pulmotrophic factor. To understand which cells HGF targets in lung, we identified the cells expressing Met by immunofluorescence assay. Met was strongly expressed in BASCs, which expressed an AT2 cell marker, pro-SP-C, and a club cell marker, CCSP. In alveoli, we found higher expression of Met in primary AT2 than in AT1 cells, which was confirmed using primary AT2 cells. We further examined the mitogenic activity of HGF in AT2-cell-derived alveolar-like cysts (ALCs) in 3D culture. Multicellular ALCs expressed Met, and HGF enhanced the ALC production. Taking these findings together, BASCs could also be an important target for HGF, and HGF-Met signaling could function more potent on cells that have greater multipotency in adult lung.Key words: HGF, Met, BASC, alveolus, ALC     

Hepatocyte Growth Factor-Induced Expression of Ornithine Decarboxylase, c-met,and c-mycIs Differently Affected by Protein Kinase Inhibitors in Human Hepatoma Cells HepG2

Binding of hepatocyte growth factor (HGF) to its receptor Met induces autophosphorylation and activation of the tyrosine kinase activity. In HGF-treated HepG2 cells, we studied: (i) the expression patterns of early(c-myc,c-jun,and c-fos) and delayed-early (ornithinedecarboxylase and c-met) response genes and (ii) thepossible involvement of protein kinase transducersin the control of the expression of c-metand of other genes eventually induced downstream. c-metand c-mycmRNAs peaked 1–2 h after HGF, while c-junandc-fosmRNAs slightly increased at 1 h. Ornithinedecarboxylase activity was induced earlier (4 h) thanthe mRNA (8–10 h). The transducers involved in HGF-triggered gene inductions were investigated using different protein kinase inhibitors: genistein for the receptor tyrosine kinase, herbimycin A for the nonreceptor tyrosine kinase (pp60^c-src), wortmannin for phosphatidylinositol 3-kinase (PI3K) and H7 for protein kinase C (PKC). The similarity of responses to PKC inhibition led to suppose that c-mycand ornithinedecarboxylase mRNAs were induced sequentially along the same transduction pathway triggered by HGF. Ornithine decarboxylase activity seemed to be largely regulated by phosphorylation(s). The mRNA expression of c-junwas likely to undergo a negative regulation through a mechanism involving PI3K, while that ofc-metseemed to be almost independent from various protein kinases (PI3K, pp60^c-src, and PKC).     

Cooperative effect of hepatocyte growth factor and fibronectin in anchorage-independent survival of mammary carcinoma cells: requirement for phosphatidylinositol 3-kinase activity.

Anchorage-independent survival and growth are critical characteristics of malignant cells. We showed previously that the addition of exogenous hepatocyte growth factor (HGF) and the presence of fibronectin fibrils stimulate anchorage-independent colony growth of a murine mammary carcinoma, SP1, which expresses both HGF and HGF receptor (Met; R. Saulnier et al., Exp. Cell Res., 222: 360-369, 1996). We now show that tyrosine phosphorylation of Met in carcinoma cells is augmented by cell adhesion and spreading on fibronectin substratum. In contrast, detached serum-starved cells exhibit reduced tyrosine phosphorylation of Met and undergo apoptotic cell death within 18-24 h. Under these conditions, the addition of HGF stimulates tyrosine phosphorylation of Met and restores survival of carcinoma cells. Soluble fibronectin also stimulates cell survival and shows a cooperative survival response with HGF but does not affect tyrosine phosphorylation of Met; these results indicate that fibronectin acts via a pathway independent of Met in detached cells. We demonstrated previously that inhibition of phosphatidylinositol (PI) 3-kinase activity blocks HGF-induced DNA synthesis of carcinoma cells (N. Rahimi et al., J. Biol. Chem., 271: 24850-24855, 1996). We now show in detached cells a cooperative effect of HGF and FN in the activation of PI 3-kinase and on the phosphorylation of PKB/Akt at serine 473. PI 3-kinase activity is also required for the HGF- and fibronectin-induced survival responses, as well as anchorage-independent colony growth. However, c-Src kinase or MEK1/2 activities are not required for the cell survival effect. Together, these results demonstrate that the PI 3-kinase/Akt pathway is a key effector of the HGF- and fibronectin-induced survival response of breast carcinoma cells under detached conditions and corroborate an interaction between integrin and HGF/ Met signalling pathways in the development of invasive breast cancer.