The regulatory role of heparin on c-Met signaling in hepatocellular carcinoma cells

The role of heparin as an anticoagulant is well defined; however, its role in tumorigenesis and tumor progression is not clear yet. Some studies have shown that anticoagulant treatment in cancer patients improve overall survival, however, recent clinical trials have not shown a survival benefit in cancer patients receiving heparin treatment. In our previous studies we have shown the inhibitory effects of heparin on Hepatocyte Growth Factor (HGF)-induced invasion and migration in hepatocellular carcinoma (HCC) cells. In this study, we showed the differential effects of heparin on the behaviors of HCC cells based on the presence or absence of HGF. In the absence of HGF, heparin activated HGF/c-Met signaling and promoted motility and invasion in HCC cells. Heparin treatment led to c-Met receptor dimerization and activated c-Met signaling in an HGF independent manner. Heparin-induced c-Met activation increased migration and invasion through ERK1/2, early growth response factor 1 (EGR1) and Matrix Metalloproteinases (MMP) axis. Interestingly, heparin modestly decreased the proliferation of HCC cells by inhibiting activatory phosphorylation of Akt. The inhibition of c-Met signaling reversed heparin-induced increase in motility and invasion and, proliferation inhibition. Our study provides a new perspective into the role of heparin on c-Met signaling in HCC.     

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.     

The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

Backgroundc-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.MethodsWe utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.ResultsWe have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.Conclusionc-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.     

Inhibition of c-Met activation sensitizes osteosarcoma cells to cisplatin via suppression of the PI3K-Akt signaling

Osteosarcoma is a common malignant bone tumor. Cisplatin (CDDP) achieves a high response rate in osteosarcoma. However, osteosarcoma usually exhibits cisplatin resistance. Many members of receptor tyrosine kinases (RTKs)(1) have been demonstrated to be overexpressed and constitutively activated in various tumors including osteosarcoma, resulting in malignant progression and insensitivity to chemotherapy. Hepatocyte growth factor receptor (HGFR/c-Met) also appears overexpressed and activated in osteosarcoma cells. Nevertheless, which role of c-Met activation in cisplatin efficacy against osteosarcoma cells remains still elusive. This study found that inhibition of c-Met activity by PHA-665752 or blockade of the interaction of autocrined HGF with c-Met with neutralizing anti-HGF antibody promoted cisplatin efficacy in osteosarcoma cells, while addition of recombinant human HGF (rh-HGF) counteracts cisplatin cytotoxicity. Specifically, we demonstrated that inhibition of c-Met activity led to suppression of the PI3K-Akt pathway, thus enhancing cisplatin chemosensitivity. Our study clearly suggests that inhibition of c-Met activity can effectively sensitize osteosarcoma cells to cisplatin via suppression of the PI3K-Akt signaling.     

Matriptase is required for the active form of hepatocyte growth factor induced Met,focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.     

HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.

Although it is established that growth factors and prostaglandins function in the maintenance of gastric mucosal integrity and in the healing of gastric mucosal injury and ulceration, the regulatory relationship between growth factors and prostaglandins in the gastric mucosa is not well characterized. Therefore, we investigated whether hepatocyte growth factor (HGF) affects expression of COX-2 (the inducible form of the prostaglandin synthesizing enzyme, cyclooxygenase) in gastric epithelial cells and whether this action is mediated through the MAP (ERK) kinase signaling pathway. In RGM1 cells (an epithelial cell line derived from normal rat gastric mucosa), HGF caused an increase in COX-2 mRNA and protein by 236% and 175%, respectively (both P<0.05). This induction of COX-2 expression was abolished by pretreatment with the MAPK kinase (MEK) inhibitor PD98059. HGF also triggered a 13-fold increase in c-Met/HGF receptor phosphorylation (P<0.005) and increased ERK2 activity by 684% (P<0.01). Pretreatment with PD98059 abolished the HGF-induced increase in ERK2 activity, but not c-Met/HGF receptor phosphorylation. The specific inhibitor of p38 MAP kinase, SB203580, had no effect on HGF-induced COX-2 expression. Thus, HGF triggers activation of the COX-2 gene in gastric epithelial cells through phosphorylation of c-Met/HGF receptor and activation of the ERK2 signaling pathway.-Jones, M. K., Sasaki, E., Halter, F., Pai, R., Nakamura, T., Arakawa, T., Kuroki, T., Tarnawski, A. S. HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.     

The Multisubstrate Adapter Gab1 Regulates Hepatocyte Growth Factor (Scatter Factor)–c-Met Signaling for Cell Survival and DNA Repair

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.     

Contact inhibition of hepatocyte growth regulated by functional association of the c-Met/hepatocyte growth factor receptor and LAR protein-tyrosine phosphatase

Contact inhibition, the inhibition of cell proliferation by tight cell-cell contact is a fundamental characteristic of normal cells. Using primary cultured hepatocytes, we investigated the mechanisms of contact inhibition that decrease the mitogenic activity of hepatocyte growth factor (HGF), focusing on the regulation of c-Met/HGF-receptor activation. In hepatocytes cultured at a sparse cell density, HGF stimulation induced prolonged c-Met tyrosine phosphorylation for over 5 h and a marked mitogenic response. In contrast, HGF stimulation induced transient c-Met tyrosine phosphorylation in <3 h and failed to induce mitogenic response in hepatocytes cultured at a confluent cell density. Treatment of the confluent cells with HGF plus orthovanadate, a broad spectrum protein-tyrosine phosphatase inhibitor, however, prolonged c-Met tyrosine phosphorylation for over 5 h and permitted the subsequent mitogenic response. The mitogenic response to HGF was associated with the duration of c-Met tyrosine phosphorylation even in the sparse cells. We found that the activity and expression of the protein-tyrosine phosphatase LAR increased following HGF stimulation specifically in confluent hepatocytes and not in sparse hepatocytes. LAR and c-Met were associated, and purified LAR dephosphorylated tyrosine-phosphorylated c-Met in in vitro phosphatase reactions. Furthermore, antisense oligonucleotides specific for LAR mRNA suppressed the expression of LAR, allowed prolonged c-Met tyrosine phosphorylation, and led to acquisition of a mitogenic response in hepatocytes even under the confluent condition. Thus functional association of LAR and c-Met underlies the inhibition of c-Met-mediated mitogenic signaling through the dephosphorylation of c-Met, which specifically occurs under the confluent condition.     

Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines

Activation of the c-Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes mitogenic, motogenic, and morphogenic cellular responses. Aberrant HGF/c-Met signaling has been strongly implicated in tumor cell invasion and metastasis. Both HGF and its receptor c-Met have been shown to be overexpressed in human synovial sarcoma, which often metastasizes to the lung; however, little is known about HGF-mediated biological effects in this sarcoma. Here, we provide evidence that Crk adaptor protein is required for the sustained phosphorylation of c-Met-docking protein Grb2-associated binder 1 (Gab1) in response to HGF, leading to the enhanced cell motility of human synovial sarcoma cell lines SYO-1, HS-SY-II, and Fuji. HGF stimulation induced the sustained phosphorylation on Y307 of Gab1 where Crk was recruited. Crk knockdown by RNA interference disturbed this HGF-induced tyrosine phosphorylation of Gab1. By mutational analysis, we identified that Src homology 2 domain of Crk is indispensable for the induction of the phosphorylation on multiple Tyr-X-X-Pro motifs containing Y307 in Gab1. HGF remarkably stimulated cell motility and scattering of synovial sarcoma cell lines, consistent with the prominent activation of Rac1, extreme filopodia formation, and membrane ruffling. Importantly, the elimination of Crk in these cells induced the disorganization of actin cytoskeleton and complete abolishment of HGF-mediated Rac1 activation and cell motility. Time-lapse microscopic analysis revealed the significant attenuation in scattering of Crk knockdown cells following HGF treatment. Furthermore, the depletion of Crk remarkably inhibited the tumor formation and its invasive growth in vivo. These results suggest that the sustained phosphorylation of Gab1 through Crk in response to HGF contributes to the prominent activation of Rac1 leading to enhanced cell motility, scattering, and cell invasion, which may support the crucial role of Crk in the aggressiveness of human synovial sarcoma.     

c-Met activation promotes extravasation of hepatocellular carcinoma cells into 3D-cultured hepatocyte cells in lab-on-a-chip device

Activation of c-Met signaling is associated with an aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC); however, its contribution to organ preference in metastasis remains unclear. In this study, using a Lab on a Chip device, we defined the role of aberrant c-Met activation in regulating the extravasation and homing capacity of HCC cells. Our studies showed that (i) c-Met overexpression and activation direct HCC cells preferentially towards the hepatocytes-enriched microenvironment, and (ii) blockage of c-Met phosphorylation by a small molecule inhibitor attenuated extravasation and homing capacity of HCC cells. These results, thus, demonstrate the role of c-Met signaling in regulating the colonization of HCC cells preferentially in the liver.     

HGF/c-Met pathway facilitates the perineural invasion of pancreatic cancer by activating the mTOR/NGF axis

Perineural invasion (PNI) is a pathologic feature of pancreatic cancer and is associated with poor outcomes, metastasis, and recurrence in pancreatic cancer patients. However, the molecular mechanism of PNI remains unclear. The present study aimed to investigate the mechanism that HGF/c-Met pathway facilitates the PNI of pancreatic cancer. In this study, we confirmed that c-Met expression was correlated with PNI in pancreatic cancer tissues. Activating the HGF/c-Met signaling pathway potentiated the expression of nerve growth factor (NGF) to recruit nerves and promote the PNI. Activating the HGF/c-Met signaling pathway also enhanced the migration and invasion ability of cancer cells to facilitate cancer cells invading nerves. Mechanistically, HGF/c-Met signaling pathway can active the mTOR/NGF axis to promote the PNI of pancreatic cancer. Additionally, we found that knocking down c-Met expression inhibited cancer cell migration along the nerve, reduced the damage of the sciatic nerve caused by cancer cells and protected the function of the sciatic nerve in vivo. Taken together, our findings suggest a supportive mechanism of the HGF/c-Met signaling pathway in promoting PNI by activating the mTOR/NGF axis in pancreatic cancer. Blocking the HGF/c-Met signaling pathway may be an effective target for the treatment of PNI.Subject terms: Pancreatic cancer, Cancer microenvironment     

Hepatocyte growth factor stimulates the growth and activates mitogen-activated protein kinase in human hepatoma cells

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.     

Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.     

Preclinical Trials for Prevention of Tumor Progression of Hepatocellular Carcinoma by LZ-8 Targeting c-Met Dependent and Independent Pathways

Hepatocellular carcinoma (HCC) is among the most lethal cancers. Mounting studies highlighted the essential role of the HGF/c-MET axis in driving HCC tumor progression. Therefore, c-Met is a potential therapeutic target for HCC. However, several concerns remain unresolved in c-Met targeting. First, the status of active c-Met in HCC must be screened to determine patients suitable for therapy. Second, resistance and side effects have been observed frequently when using conventional c-Met inhibitors. Thus, a preclinical system for screening the status of c-Met signaling and identifying efficient and safe anti-HCC agents is urgently required. In this study, immunohistochemical staining of phosphorylated c-Met (Tyr1234) on tissue sections indicated that HCCs with positive c-Met signaling accounted for approximately 46% in 26 cases. Second, many patient-derived HCC cell lines were established and characterized according to motility and c-Met signaling status. Moreover, LZ8, a medicinal peptide purified from the herb Lingzhi, featuring immunomodulatory and anticancer properties, was capable of suppressing cell migration and slightly reducing the survival rate of both c-Met positive and negative HCCs, HCC372, and HCC329, respectively. LZ8 also suppressed the intrahepatic metastasis of HCC329 in SCID mice. On the molecular level, LZ8 suppressed the expression of c-Met and phosphorylation of c-Met, ERK and AKT in HCC372, and suppressed the phosphorylation of JNK, ERK, and AKT in HCC329. According to receptor array screening, the major receptor tyrosine kinase activated in HCC329 was found to be the epidermal growth factor receptor (EGFR). Moreover, tyrosine-phosphorylated EGFR (the active EGFR) was greatly suppressed in HCC329 by LZ8 treatment. In addition, LZ8 blocked HGF-induced cell migration and c-Met-dependent signaling in HepG2. In summary, we designed a preclinical trial using LZ8 to prevent the tumor progression of patient-derived HCCs with c-Met-positive or -negative signaling.     

Activated c-Met signals through PI3K with dramatic effects on cytoskeletal functions in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive illness with early metastases. There are several receptor tyrosine kinases (RTKs) overexpressed in SCLC, including c-Met. c-Met contains an external semaphorin-like domain, a cytoplasmic juxtamembrane domain, tyrosine kinase domain and multiple tyrosines that bind to adapter molecules. We have previously reported that c-Met is abundantly expressed in the NCI-H69 SCLC cell line and now have determined the downstream effects of stimulating c-Met via its ligand hepatocyte growth factor (HGF). Utilizing unique phospho-specific antibodies generated against various tyrosines of c-Met, we show that Y1003 (binding site for c-Cb1 and a negative regulatory site), Y1313 (binding site for PI3K), Y1230/Y1234/Y1235 (autophosphorylation site), Y1349 (binding site for Grb2), Y1365 (important in cell morphogenesis) are phosphorylated in response to HGF (40 ng/ml, 7.5 min) in H69 cells. Since multiple biological and biochemical effects are transduced through the PI3K pathway, we determine the role of PI3K in the c-Met/HGF stimulation pathway. We initially determined that by inhibiting PI3K with LY294002 (50μM over 72 hours), there was at least a 55% decrease in viability of H69 cells. Since H69 SCLC cells form clusters in cell culture, we determined the effects of HGF and LY294002 on cell motility of the clusters by time-lapse video microscopy. In response to HGF, SCLC moved much faster and formed more clusters, and this was inhibited by LY294002. Finally, we determined the downstream signal transduction of HGF stimulation of c-Met with and without inhibition of c-Met (with geldanamycin, an anisamycin antibiotic that inhibits c-Met in SCLC) or PI3K (with LY294002). We show that association of c-Met with PI3K and GAB2 is diminished by inhibiting c-Met. In summary, activation of the c-Met pathway targets the PI3K pathway in SCLC and this may be an important therapeutic target.