Addition Of HGF Research Articles

MACC1 Promotes the Progression and Is a Novel Biomarker for Predicting Immunotherapy Response in Colorectal Cancer.

Aims As one of the most prevalent malignant diseases in the world, the mechanisms of metastasis in colon cancer are poorly understood. The aim of this study was to investigate the role of the HGF/c-MET axis in the proliferation and metastasis in colon cancer. Methods The effect of MACC1 on cell proliferation and metastasis was analyzed through a series of in vitro experiments. The role of MACC1 in cancer cells was demonstrated by overexpression and silencing of MACC1 in gain or loss function experiments. To investigate the relationship between MACC1 and c-MET/HGF, we detected c-MET protein expression by disrupting with or overexpressing MACC1. The bioinformatics analysis was used to investigate the correlation between MACC1 and c-MET, and the c-MET expression after the interference of HGF with MACC1 was determined. Subsequently, the function of c-MET was verified in colon cancer cells by a series of experiments. The mouse tumor transplantation model experiment is most suitable in vivo. Results The results indicated that the overexpression of MACC1 could accelerate proliferation and facilitate metastasis in colon cancer cell lines. Furthermore, c-MET was determined to be the downstream regulator of MACC1. The addition of HGF could stimulate the expression of MACC1. With further exploration, we proved that c-MET is downstream of MACC1 in colon cancer and that overexpression of c-MET in colon cancer enhances cell proliferation and migration capability. At last, MACC1 expression level negatively correlates with the infiltration levels and several immune checkpoint biomarkers. High MACC1 expression has a lower response rate with ICIs in COAD. Conclusions We found that, under the regulation of the MACC1/HGF/c-MET axis, the proliferation and metastasis of colorectal cancer are increased by MACC1, which can be a novel biomarker for predicting ICIs response in colorectal cancer. Our findings provide a new idea for the targeted treatment of colorectal cancer.

Prognostic value of hepatocyte growth factor for muscle-invasive bladder cancer

PurposeThe HGF/MET pathway is involved in cell motility, angiogenesis, proliferation, and cancer invasion. We assessed the clinical utility of plasma HGF level as a prognostic biomarker in patients with MIBC.MethodsWe retrospectively analyzed 565 patients with MIBC who underwent radical cystectomy. Logistic regression and Cox regression models were used, and predictive accuracies were estimated using the area under the curve and concordance index. To estimate the clinical utility of HGF, DCA and MCID were applied.ResultsPlasma HGF level was significantly higher in patients with advanced pathologic stage and LN metastasis (p = 0.01 and p < 0.001, respectively). Higher HGF levels were associated with an increased risk of harboring LN metastasis and non-organ-confined disease (OR1.21, 95%CI 1.12–1.32, p < 0.001, and OR1.35, 95%CI 1.23–1.48, p < 0.001, respectively) on multivariable analyses; the addition of HGF improved the predictive accuracies of a standard preoperative model (+ 7%, p < 0.001 and + 8%, p < 0.001, respectively). According to the DCA and MCID, half of the patients had a net benefit by including HGF, but the absolute magnitude remained limited. In pre- and postoperative predictive models, a higher HGF level was significant prognosticator of worse RFS, OS, and CSS; in the preoperative model, the addition of HGF improved accuracies by 6% and 5% for RFS and CSS, respectively.ConclusionPreoperative HGF identified MIBC patients who harbored features of clinically and biologically aggressive disease. Plasma HGF could serve, as part of a panel, as a biomarker to aid in preoperative treatment planning regarding intensity of treatment in patients with clinical MIBC.

Abstract 1343: Preclinical evaluation of a PKC and MET inhibitor combination in metastatic uveal melanoma

Abstract Background: Uveal melanoma (UM) is a common intraocular tumor that primarily metastasizes to the liver. Patients with metastatic uveal melanoma (MUM) have a poor prognosis with a median overall survival of 10 months. The liver microenvironment has high levels of HGF, and high MET receptor expression in UM is associated with metastatic progression. HGF induces cell proliferation and survival through MAPK and PI3Ksignaling and may elicit resistance to MEK inhibitors in MUM patients. IDE196is a potent and selective PKC inhibitor currently in clinical trials for MUM(NCT03947385). In order to identify combination partners for IDE196, we hypothesized that the rich growth factor microenvironment in the liver may prevent MUM tumor cells from responding to IDE196 and that a combination of IDE196 with a MET inhibitor may synergistically inhibit tumor cell proliferation. Methods: Combinations were assessed in 92.1 and MEL-202 (primary) and also in MM28(metastatic). Cells were treated for 3-5 days with increasing doses of IDE196 (0-10uM) in combination with either increasing doses of HGF (0-100ng/ml), or with increasing doses of crizotinib (0-10uM) in the presence of HGF, to form a matrix of doses. Cell viability was assessed using cell-titer glo. To assess PD modulation, cells were treated for 2hrs with IDE196, HGF or crizotinib alone or in combination and relevant phospho MAPK and PI3K effectors were assessed by western blotting. Results: Addition of HGF alone significantly antagonized IDE196 activity at doses ≥1.23ng/ml. In 92.1 cells, HGF modestly antagonized IDE196, which could be attributed to a lower expression level of MET receptor in this cell line. Crizotinib synergized strongly with IDE196 at clinically relevant doses (80nM) in MEL202 and MM28 in contrast to the 92.1 where mild or no synergy was observed. PD analysis showed that in the absence of HGF, IDE196 dose-dependently inhibited pERK, pMARCKS and pPKC-delta in all three cell lines. Addition of HGF dose-dependently induced pMET, pERK levels, pAkt and pPRAS40 levels in all cell lines which was not inhibited by IDE196. Addition of crizotinib alone or in combination with IDE196 strongly inhibited the activation of these pathways in these cell lines. Our results demonstrate that high HGF signaling, as observed in the liver, may activate MAPK and PI3K signaling to antagonizeIDE196 activity and that this antagonism may be overcome by combining IDE196 with a MET inhibitor. Independently, a retrospective analysis of human clinical samples from a Phase 1IDE196 monotherapy trial (NCT02601378) identified cMET expression/activation as a prognostic biomarker. IDEAYA plans to evaluate the combination of IDE196 and crizotinib in a Phase 1/2clinical trial. Citation Format: Marie-Claire Wagle, Nandini Ravindran, Divya Pankajakshan, Mark Lackner, Zineb Mounir. Preclinical evaluation of a PKC and MET inhibitor combination in metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1343.

Chidamide increases the sensitivity of Non-small Cell Lung Cancer to Crizotinib by decreasing c-MET mRNA methylation.

Introduction: Crizotinib is a kinase inhibitor targeting c-MET/ALK/ROS1 used as the first-line chemical for the treatment of non-small cell lung cancer (NSCLC) with ALK mutations. Although c-MET is frequently overexpressed in 35-72% of NSCLC, most NSCLCs are primarily resistant to crizotinib treatment.Method: A set of NSCLC cell lines were used to test the effect of chidamide on the primary crizotinib resistance in vitro and in vivo. Relationships between the synergistic effect of chidamide and c-MET expression and RNA methylation were systemically studied with a battery of molecular biological assays.Results: We found for the first time that chidamide could sensitize the effect of crizotinib in a set of ALK mutation-free NSCLC cell lines, especially those with high levels of c-MET expression. Notably, chidamide could not increase the sensitivity of NSCLC cells to crizotinib cultured in serum-free medium without hepatocyte growth factor (HGF; a c-MET ligand). In contrast, the addition of HGF into the serum-/HGF-free medium could restore the synergistic effect of chidamide. Moreover, the synergistic effect of chidamide could also be abolished either by treatment with c-MET antibody or siRNA-knockdown of c-MET expression. While cells with low or no c-MET expression were primarily resistant to chidamide-crizotinib cotreatment, enforced c-MET overexpression could increase the sensitivity of these cells to chidamide-crizotinib cotreatment. Furthermore, chidamide could decrease c-MET expression by inhibiting mRNA N6-methyladenosine (m6A) modification through the downregulation of METTL3 and WTAP expression. Chidamide-crizotinib cotreatment significantly suppressed the activity of c-MET downstream molecules.Conclusion: Chidamide downregulated c-MET expression by decreasing its mRNA m6A methylation, subsequently increasing the crizotinib sensitivity of NSCLC cells in a c-MET-/HGF-dependent manner.

Abstract LB-C07: Preclinical evaluation of MCLA-129: A bispecific antibody targeting c-MET and EGFR

Abstract NSCLC is the most common form of cancer. Approximately 20% of NSCLC patients have mutations in the growth factor (GF) receptor EGFR. These patients respond to tyrosine kinase inhibitors (TKIs) that target mutant EGFR; however relapse eventually occurs. c-MET is one of the most common GF pathways upregulated in resistant patients. Met amplification is found in 5-10% of tumors that acquire resistance to EGFR TKIs in the first line of treatment and this frequency becomes higher in later lines of treatment. Preclinical studies have shown that co-inhibition of c-MET and EGFR inhibits growth and survival in these resistant tumors. We describe here an unbiased functional screen of bispecific antibodies (bAbs) targeting EGFR and c-MET to select MCLA-129 and its evaluation in an in vivo xenograft NSCLC model. Large collections of common light chain Fab binding ‘arms’ against EGFR and c-MET were derived from immunized MeMo® mice and used in different combinations (based on epitope and sequence diversity) to generate IgG1 Biclonics® bAbs. The panel of &amp;gt; 400 bAbs was screened for proliferation inhibition of the N87 cell line stimulated with EGF and/or HGF. Five bAbs inhibited N87 growth comparable to the combination of cetuximab and a MetMab analog (C+M). Two of the most potent Biclonics® bAbs were selected for further characterization and screened for inhibitory activity in NSCLC cell lines PC-9 and HCC827 that harbour an EGFR exon 19 deletion. The addition of HGF reversed the inhibitory effect of the EGFR TKI erlotinib and gefitinib in both cell lines. Combination of bAbs or C+M with erlotinib or gefinitinib resulted in inhibition of growth and downstream phosphorylation similar to erlotinib treatment without GF addition in both HGF alone or HGF + EGF culture conditions. The best bAb was as potent (PC-9) or more potent (HCC827) than C+M. The lead bAb, MCLA-129 was ADCC enhanced and shown to effectively mediate ADCC against NSCLC cell lines with both high and low affinity FcR bearing effector cells. MCLA-129 was then tested in an genetically engineered immunodeficient xenograft mouse model where endogenous mouse HGF promoter drives the expression of human HGF bypassing the problem of the low affinity binding of mouse HGF for human c-MET. Mice engrafted with HCC827 cells and treated with MCLA-129 (25 mg/kg) displayed significant tumor regression (below baseline) and a much slower tumor regrowth rate upon drug cessation and this activity was enhanced when combined with erlotinib (6mg/kg). This potent therapeutic activity was achieved without the contribution of ADCC and was not observed in mice that received only erlotinib, a bivalent c-MET antagonizing antibody or their combination. To model acquired resistance to EGFR TKI treatment, animals were treated with erlotinib until tumors were &amp;gt;500mm3, MCLA-129 was added to erlotinib in one randomized group. In this group, immediate tumor inhibition was observed, which persisted over the treatment period. In summary, MCLA-129 was synergistic with EGFR targeting TKI’s to reverse c-MET mediated resistance in NSCLC cell lines both in vitro and in vivo. The activity of MCLA-129 was superior to relevant comparator biologics that were combined to inhibit EGFR and c-MET signaling in the absence of functional immunity. These preclinical data suggest MCLA-129 could benefit NSCLC patients that become resistant to EGFR targeted therapies and warrants clinical evaluation. Citation Format: Cecile Geuijen, Mario di Matteo, Tristan Gallenne, Sarah Trusso Cafarello, Roy Nijhuis, Therese Visser, Willem Bartelink, Carina Bartelink-Clements, Berina Eppink, Rinse Klooster, John dekruif, Massimiliano Mazzone, Mark Throsby. Preclinical evaluation of MCLA-129: A bispecific antibody targeting c-MET and EGFR [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C07. doi:10.1158/1535-7163.TARG-19-LB-C07

Activation of MET promotes resistance to sorafenib in hepatocellular carcinoma cells via the AKT/ERK1/2-EGR1 pathway

Sorafenib is an oral multikinase inhibitor that has become an established therapeutic approach in advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib in clinical therapy is often affected by drug resistance. Therefore, it is important to explore the mechanisms underlying sorafenib resistance and to develop individualized therapeutic strategies for coping with this problem. In this study, we found that addition of HGF to sorafenib-treated HCC cells activated MET and re-stimulated the downstream AKT and ERK1/2 pathways. Thereby, restored sorafenib-treated HCC cells proliferation, migration and invasion ability, and rescued cells from apoptosis. In addition, we found that HGF treatment of HCC cells induced early growth response protein (EGR1) expression, which is involved in sorafenib resistance. Importantly, the HGF rescued effect in sorafenib-treated HCC cells could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating EGR1 expression with small interfering RNA (siRNA). Therefore, inhibition of the HGF/MET pathway may improve response to sorafenib in HCC, and combination therapy should be further investigated.

Exogenous HGF Bypasses the Effects of ErbB Inhibition on Tumor Cell Viability in Medulloblastoma Cell Lines.

Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed a limited clinical response in medulloblastoma. The present study investigated the role of micro-environmental growth factors expressed in the brain, such as HGF and EGF, in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (ErbB1-4) inhibition in medulloblastoma cell lines. Medulloblastoma cell lines were treated with tyrosine kinase inhibitors crizotinib or canertinib, targeting MET and ErbB1-4, respectively. Upon treatment, cells were stimulated with VEGF-A, PDGF-AB, HGF, FGF-2 or EGF. Subsequently, we measured cell viability and expression levels of growth factors and downstream signaling proteins. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and demonstrated phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Crizotinib and canertinib both inhibited cell viability and phosphorylation of Akt and ERK1/2. Specifically targeting MET using shRNA’s resulted in decreased cell viability. Interestingly, addition of HGF to canertinib significantly enhanced cell viability as well as phosphorylation of Akt and ERK1/2. The HGF-induced bypass of canertinib was reversed by addition of crizotinib. HGF protein was hardly released by medulloblastoma cells itself. Addition of canertinib did not affect RTK cell surface or growth factor expression levels. This manuscript points to the bypassing capacity of exogenous HGF in medulloblastoma cell lines. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of MET and EGFR inhibitors in medulloblastoma.

Abstract 4025: Exogenous HGF bypasses ErbB inhibition on tumor cell viability in medulloblastoma cell lines

Abstract Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed limited clinical responses in medulloblastoma. An emerging concept is the role of the tumor microenvironment in bypassing targeted therapies by producing ligands that can compensate for the drug-inhibited RTK via alternative routes of pathway activation. The aim of this study is to investigate the role of well-known CNS expressed growth factors in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (EGFR, ErbB2-4) inhibition on tumor cell viability and downstream signaling in medulloblastoma cell lines. Five different medulloblastoma cell lines (DAOY, RES256, UW402, UW426, and UW473) were treated with crizotinib or canertinib, targeting MET or EGFR, ErbB2-4, respectively. Upon treatment, cells were stimulated with the CNS expressed growth factor VEGF-A PDGF-AB, HGF, FGF-2 or EGF basic. Subsequently, a cell viability assay was used to measure cell viability upon growth factor stimulation, compared to non-growth factor stimulated cells. Expression levels of respective receptor tyrosine kinases (RTK’s) and growth factors were analyzed using flow cytometry and human growth factor antibody arrays. Phosphorylation status of RTK's and downstream signaling effectors was visualized using western blot analysis and human phospho-kinase antibody arrays. We observed high MET and EGFR cell surface expression levels. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and resulted in downstream phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Whereas crizotinib and canertinib inhibited cell viability and phosphorylation of Akt and ERK1/2, addition of HGF to canertinib significantly enhanced cell viability and phosphorylation of Akt and ERK1/2. The HGF induced bypass of canertinib was reversed by the addition of crizotinib. HGF protein was hardly released by medulloblastoma cell lines. This suggests that MET activation is mainly dependent on paracrine HGF contribution. In conclusion, these data point to the bypassing capacity of well-known CNS expressed paracrine growth factors (e.g. HGF) in medulloblastoma cell lines and provides a potential explanation for the limited clinical response of single RTK inhibitors in medulloblastoma clinical trials. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of tyrosine kinase inhibitors, targeting for example MET and EGFR in medulloblastoma. Citation Format: Walderik W. Zomerman, Sabine LA Plasschaert, Sander H. Diks, Harm-Jan Lourens, Tiny Meeuwsen-de Boer, Eelco W. Hoving, Wilfred FA den Dunnen, Eveline SJM de Bont. Exogenous HGF bypasses ErbB inhibition on tumor cell viability in medulloblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2015-4025

Knockdown of WAVE3 impairs HGF induced migration and invasion of prostate cancer cells.

BackgroundThe WASP (Wiskott-Aldrich syndrome protein) and WAVE (WASP Verpolin homologous) family of proteins are structurally related and responsible for regulation of actin polymerization through their interaction with actin related proteins 2&3 (ARP 2/3). WAVE-3 has exhibited an association with disease progression and poorer prognosis of certain malignancies. In the current study, we determined the role of WAVE-3 in hepatocyte growth factor induced cellular changes including cell matrix interaction, invasion and cellular motility, and pathways that may be responsible for the changes in prostate cancer cells.MethodsWe used hammer head ribozymes to knock down the expression of WAVE-3 in PC-3 prostate cancer cell line. In vitro cellular functional assays including growth, invasion, adhesion, motility and invasion, were performed to assess the effects of WAVE-3 knock down. Further experimentation was performed to investigate the role of different pathway through expression and phosphorylation status of various intermediate proteins.ResultsWAVE-3 knockdown reduced invasive potential and motility of prostate cancer cells. Following addition of HGF, control cells showed significantly increased invasion and motility (p value <0.5) and marked increase in cellular growth. However, WAVE-3 knockdown cell line failed to show any increase in these trends (p value <0.5) except increased growth compared with control cells. Further experiments revealed that HGF-induced activation of Paxillin was weakened by the knockdown of WAVE-3. Our study also indicated that reduced invasiveness following WAVE-3 knockdown, may be related to reduce activity of MMP-2.ConclusionsOur studies suggest a vital role of WAVE-3 in HGF induced invasion and migration in which Paxillin and MMP-2 are involved. Further study will shed light on its potential as therapeutic target to suppress local invasion and metastasis of prostate cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0203-3) contains supplementary material, which is available to authorized users.

Abstract 4513: IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors

Abstract EGFR is one of the oncogenic drivers in non-small-cell lung adenocarcinoma (NSCLC AC). EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib and afatinib, show remarkable single agent activity in NSCLC AC with EGFR mutations. However, a majority of the tumors develop resistance within twelve months. T790M secondary EGFR mutation and activation of alternative signaling pathways, such as MET, are the two most common resistance mechanisms. To address this clinical challenge, we developed IMGN289, an EGFR antibody-drug conjugate consisting of the novel J2898A antibody (Ab), which selectively binds to and inhibits EGFR-driven tumor cell growth, conjugated to the maytansinoid DM1, a potent anti-microtubule agent, via the SMCC thioether linker. IMGN289 showed significantly enhanced cytotoxic activity, relative to cetuximab or unconjugated J2898A, against tumor cell line models that are dependent on EGFR signaling (Chittenden et al., AACR 2013). To evaluate the ability of IMGN289 to overcome mechanisms of EGFR TKI resistance, IMGN289 activity was tested in erlotinib-resistant EGFR-mutant cell lines which overexpressed MET (HCC827-ER) or carry the T790M EGFR mutation (HCC827-EPR) (Suda et al., Clin Cancer Res 2010). Despite variable levels of EGFR expression in these cell lines, (193,000, 32,500 and 422,000 antigens per cell in HCC827, HCC827-ER and HCC827-EPR, respectively), IMGN289 showed comparable cytotoxic activities against these cell lines in vitro. In addition to MET overexpression, the MET pathway can also be activated by HGF, the MET ligand. While addition of HGF made the HCC827 cell line resistant to EGFR TKIs (Yano et al., Cancer Res 2008) and decreased growth inhibition by cetuximab, IMGN289 activity was unaffected. Additionally, we generated an erlotinib-resistant HCC827-ER-E4 cell line by exposing the EGFR-mutant HCC827 cells to increasing concentrations of erlotinib for ∼6 months, followed by subcloning. The HCC827-ER-E4 cell line expressed mesenchymal markers and acquired a mesenchymal phenotype. It expressed lower levels of EGFR, HER3 and MET compared to the parental cell line and showed no loss of PTEN. In the presence of erlotinib, HCC827-ER-E4 cells retained high levels of pAKT and pERK, suggesting that activation of alternative signaling pathways may be responsible for the erlotinib resistance. In vitro cytotoxic assays showed that IMGN289 was equally cytotoxic to the parental and the HCC827-ER-E4 cell line. In conclusion, IMGN289 demonstrated EGFR-targeted cytotoxicity against NSCLC cell lines that harbor some of the most common mechanisms of resistance to EGFR TKIs. IMGN289 represents a promising novel candidate for treatment of EGFR-expressing NSCLC AC that has acquired resistance to EGFR TKIs. Citation Format: Yulius Y. Setiady, Ling Dong, Anna Skaletskaya, Jan Pinkas, Robert J. Lutz, John M. Lambert, Thomas Chittenden. IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4513. doi:10.1158/1538-7445.AM2014-4513

Maturation defects in NOD macrophages impacts beta cell regeneration (HUM7P.286)

Abstract In Type 1 Diabetes (T1D), CD4+ T cells drive autoimmune destruction of pancreatic β cells by activating M1 macrophages which hinders β cell repair. Endogenous β cell growth can occur if inflammation is tempered. A more controlled inflammatory response is necessary to induce the regenerative process, characterized by a transition of macrophage phenotypes from M1 to M2. Macrophages from NOD mice have several maturation defects. We hypothesize that upon β cell destruction in autoimmunity defective macrophages fail to transition from the M1 to M2 phenotype, rendering β cells unable to initiate the repair program. We adoptively transferred NOD.scid mice with BDC-2.5-TCR-Tg splenocytes to cause T1D. Mice receiving a CD4+ T cell-depleting antibody (GK1.5) had a restricted inflammatory response and delayed T1D onset. We observed low levels of M1 macrophage genes in pancreatic tissue after GK1.5, while M2-macrophage gene expression was maintained. Control mice demonstrated a persistent M1 phenotype overwhelming the M2 macrophage lineage. β cell maintenance is achieved though β cell expressed growth factors, facilitated through M2 macrophages. In pancreatic injury we observed that autoimmune mice have decreased growth factor expression, e.g. HGF, compared to non-autoimmune mice. These macrophage defects may render β cells unable to initiate replicative programs. We propose that addition of HGF to the islet will provide the necessary cues from macrophages for β cell growth and function.

Immunomagnetic exclusion of PECAM-1-positive endothelial cells in fetal mouse liver cell cultures causes impaired growth and gene expression of hepatoblasts and stellate cells.

Previous studies using mice having defective VEGF signaling have demonstrated that vascular development is indispensable for early hepatic organogenesis. However, not only whether its action lasts during later hepatic development, but also what molecules are involved in that action remains to be determined. The present study was undertaken to examine the effects of primitive sinusoidal endothelial cells on hepatic growth and maturation in primary culture of fetal mouse liver cells, and to determine their molecular mechanisms. When endothelial cells were excluded from E12.5 liver cell cultures by using PECAM-1-antibody-coated magnetic beads, the growth of hepatoblasts and stellate cells was conspicuously reduced and hepatic maturation was also suppressed. Conditioned medium prepared from fetal liver cell cultures containing almost all hepatic cell types stimulated the growth and gene expression of hepatoblasts and stellate cells similarly to the cultures in the presence of endothelial cells. HGF mRNA expression was downregulated in endothelial cellfree cultures of fetal liver cells, and the addition of HGF to the culture medium rescued the cells from the effects of endothelial cell depletion. These data suggest that humoral factors, including HGF, which are produced by endothelial cells or stellate cells, are involved in fetal hepatocyte growth and maturation.

α-Lipoic acid-induced inhibition of proliferation and met phosphorylation in human non-small cell lung cancer cells

α-Lipoic acid (α-LA), a naturally occurring anti-oxidant and co-factor for metabolic enzymes, suppresses the growth of different types of tumor cells. The mechanisms that are responsible for these results, however, remain to be elucidated. In the present study, we investigated the effects of α-LA on the proliferation and activation status of definitive receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and Met/hepatocyte growth factor (HGF) receptor, in gefitinib-sensitive human non-small cell lung cancer cells harboring EGFRs with an activating mutation. The enantiomers R-α-LA and S-α-LA suppressed cell proliferation and increased the level of reactive oxygen species in HCC-827 and PC-9 human non-small cell lung cancer cells in an indistinguishable dose-dependent fashion. A phospho-receptor tyrosine kinase array and cell cycle analysis indicated that α-LA decreased tyrosine phosphorylation levels of EGFR, ErbB2, and Met, and this was associated with an inhibition in the cell-cycle transition from the G1 phase to the S phase without inducing apoptosis. Gefitinib, an inhibitor for EGFR tyrosine kinase, inhibited EGFR tyrosine phosphorylation/activation and proliferation of the cells. Instead, the addition of HGF induced Met tyrosine phosphorylation, and this was associated with a resistance to gefitinib-induced growth inhibition, which meant a gain in proliferative ability. In the presence of gefitinib and HGF, the addition of α-LA suppressed Met tyrosine phosphorylation, and this was associated with an inhibition in cell growth. These results suggest that the suppression of tyrosine phosphorylation/activation of growth factor receptors that is critical for the proliferation of human non-small cell lung cancer cells is a mechanism by which α-LA exerts growth inhibition for cancer cells.

The polyphenol epigallocatechin-3-gallate affects lipid rafts to block activation of the c-Met receptor in prostate cancer cells

The HGF/c-Met pathway is an important regulator of signaling pathways responsible for invasion and metastasis of most human cancers, including prostate cancer. Exposure of DU145 prostate tumor cells to HGF stimulates the PI3-kinase and MAPK pathways, leading to increased scattering, motility, and invasion, which was prevented by the addition of EGCG. EGCG acted at the level of preventing phosphorylation of tyrosines 1234/1235 in the kinase domain of the c-Met receptor without effecting dimerization. HGF-induced changes were independent of the formation of reactive oxygen species, suggesting that EGCG functioned independent of its antioxidant ability. ECG, another tea polyphenol, was as effective as EGCG, while EGC and EC were less effective. EGCG added up to 4 h after the addition of HGF still blocked cell scattering and reduced the HGF-induced phosphorylation of c-Met, Akt, and Erk, suggesting that EGCG could act both by preventing activation of c-Met by HGF and by attenuating the activity of pathways already induced by HGF. HGF did not activate the MAPK and PI3-K pathways in cells treated with methyl-beta-cyclodextrin (mCD) to remove cholesterol. Furthermore, subcellular fractionation approaches demonstrated that only phosphorylated c-Met accumulated in Triton X-100 membrane insoluble fractions, supporting a role for lipid rafts in regulating c-Met signaling. Finally, EGCG treatment inhibited DiIC16 incorporation into membrane lipid ordered domains, and cholesterol partially inhibited the EGCG effects on signaling. Together, these results suggest that green tea polyphenols with the R1 galloyl group prevent activation of the c-Met receptor by altering the structure or function of lipid rafts.

Guiding ES cell differentiation into the definitive endoderm lineages

The generation of specific lineages of the definitive endoderm from embryonic stem (ES) cells is an important issue in developmental biology, as well as in regenerative medicine. We have established a procedure, in which M15 cells, derived from a mesonephros, are used as supporting cell line for ES cell differentiation. Under selective culture conditions, by addition of Activin and bFGF, definitive endoderm are generated from the ES cells at a high efficiency. Then, under the presence of Activin and bFGF, the differentiation into pancreatic fates is further promoted, upon the expense of the hepatic fates. On the other hand, removal of Activin, following by the addition of HGF and Dexamethasone potentiated the differentiation of ES cells into hepatic lineages. Further maturation of the pancreatic precursor could be achieved by in vivo differentiation of the differentiated ES cells. Engraftment of the cells under kidney capsule further permit the differentiation of ES cells into mature cell types containing the three pancreatic cell lineages.

HGF suppresses the production of collagen type III and α-SMA induced by TGF-β1 in healing fibroblasts

The aim of this study was to examine the effectiveness of HGF in blocking TGF-beta1-induced collagen III and alpha-smooth muscle actin (alpha-SMA) production in rat healing fibroblasts, fibroblasts were obtained from healing medial collateral ligament (MCL) injury. Cell culture was supplemented with 5 ng/ml of TGF-beta1 along with increasing doses of HGF (10-40 ng/ml). The productions of collagen III in supernatants culture were assayed by enzyme-linked immunosorbent assay. Expression of alpha-SMA was assessed by Western blot. Treatment with TGF-beta1 significantly stimulated collagen III and alpha-SMA production in healing fibroblasts. Remarkably, the addition of HGF reduced productions of all components induced by TGF-beta1 in a dose-dependent manner. This study shows that HGF antagonizes the action of TGF-beta1 effectively in cultured healing MCL injury fibroblasts. The results provide a cellular and molecular basis for HGF's acting as a therapeutic agent for MCL scar formation and poor healing.

Regulation of HGF and SDF-1 expression by oral fibroblasts – Implications for invasion of oral cancer

Invasion and metastasis of oral squamous cell carcinoma (OSCC) is dependent on signals received from stromal fibroblasts present in the surrounding connective tissue. The aim of this study was to investigate the regulation of expression of two important signaling molecules--HGF and SDF-1--by both stromal fibroblasts and their 'activated' form, myofibroblasts, and to determine the role of these two factors in stimulating OSCC cell invasion in vitro. Fibroblasts and myofibroblasts produced similar levels of HGF and SDF-1. IL-1alpha and OSCC cell conditioned medium both stimulated HGF and SDF-1 expression, while TGF-beta(1) inhibited production of each factor. Myofibroblast-derived conditioned medium stimulated OSCC cell invasion through matrigel. Blocking antibodies to both HGF and SDF-1 reduced the level of invasion. In fibroblast-free organotypic raft cultures, addition of HGF and SDF-1 stimulated OSCC cell invasion into the underlying collagen gel, although the pattern of invasion differed from that induced by fibroblasts. Fibroblast-derived HGF and SDF-1 appear to play central roles in the reciprocal interactions between OSCC cells and underlying stromal fibroblasts leading to the local invasion of oral cancer.

Transduction of Exogenous Constitutively Activated Stat3 into Dispersed Islets Induces Proliferation of Rat Pancreatic β-cells

In vitro proliferation of functional islet mass/beta-cells by transduction of cell cycle regulatory genes has not been well documented due to the lack of either an effective method for gene transduction to islets or effective genes for beta-cell proliferation. Signal transducers and activators of transcription- 3 (Stat3) are among the most important molecules for cell proliferation and for cell antiapoptosis. In this study, adenovirus-mediated gene transduction was performed on dispersed islet cells, and reaggregated islet mass functions, such as capabilities for reaggregation, proliferation, and glucose- stimulated insulin secretion (GSIS), were evaluated. The constitutively activated form of Stat3 (Stat3-C) was effectively transduced to most of the islet cells, even at a low density of adenovirus vector. There was no difference in the spontaneous reaggregation capability between Stat3-C transduced and control islet cells. BrdU incorporation in Stat3-C-transduced islet cells was significantly higher (2.8-fold) than that in the control cells, and it was significantly elevated (4-fold) by the addition of HGF in culture media. GSIS in Stat3-C-transduced islet cells was preserved to the level in controls by 5 days in culture. The results indicated that gene transduction into islet cells could be enhanced by first dispersing the cells. Stat3-C induced cell proliferation of beta-cells without loss of insulin secretion activity at the glucose challenge, and HGF enhanced the beta-cell proliferative activity of Stat3-C.

Malaria sporozoite: migrating for a living – a response

In a recent paper, Carrolo et al. reported experiments performed using Plasmodium yoelii and Hepa 1–6 cells showing that cells wounded mechanically or by migrating sporozoites release an infection susceptibility inducing factor (ISIF) into the medium, which enhances infection [1xHepatocyte growth factor and its receptor are required for malaria infection. Carrolo, M. et al. Nat. Med. 2003; 9: 1363–1369Crossref | PubMed | Scopus (84)See all References][1]. Using Plasmodium berghei sporozoites, the authors then identified HGF as the ISIF, based on the observation that addition of HGF to the cultures has the same effects on infection as conditioned-medium ISIF, and, more importantly, that ISIF activity is neutralized by anti-HGF antibodies. Mota argues that it is difficult to envisage that, although P. yoelii and P. berghei infections can be increased by the same factor, and that primary hepatocytes produce this factor and respond to it, the system will be different with respect to this factor between the two species.However, we consider that it is too premature to conclude that HGF/Met signalling is a general feature of Plasmodium sporozoite infection for three reasons. First, P. yoelii and P. berghei display major differences with regard to sporozoite infection, including a differential CD81 requirement and a better infectivity for mice by P. yoelii. Therefore, interpreting results from one species to the other requires great caution. Second, although both recombinant HGF and conditioned culture medium were shown to have similar effects on P. berghei infection, one can hardly conclude whether HGF mediates all ISIF activity, or if other factors in the conditioned medium also account for the ISIF activity. In the absence of experimental confirmation that HGF has an ISIF activity on P. yoelii, we think it is too early to ascertain that the same factor mediates ISIF activity for both species. Finally, Mota and colleagues found that HGF/Met signalling induces a host cytoskeleton remodelling that is required for P. berghei infection, based on the observation that cytochalasin D inhibits early steps of the parasite development. Our observation that cytochalasin D does not inhibit P. yoelii development clearly indicates that the system is different between the two species, at least in the mechanisms involved (Silvie et al., unpublished results). Interestingly, Mota reports a background activation of Met in Hepa 1–6 cells. In this regard, it can be expected that interfering with Met signalling probably has detrimental effects on survival and proliferation of hepatoma cells, thus supporting our hypothesis that the role of HGF/Met signalling during Plasmodium infection might be to promote the survival of infected cells.

In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis.

To investigate the function of Bcl-xL in the skin, we established keratinocyte-specific Bcl-x gene-targeted mice under the keratin 5 promoter (K5). K5.Bcl-xL-/- mice were viable, devoid of alteration in the development of skin or appendages. However, they harbored spontaneous apoptotic keratinocytes in the epidermis. Bcl-xL-deficient keratinocytes cultured in vitro readily underwent apoptosis in the absence of growth factors, but the addition of HGF or EGF resulted in restoration of cell survival, which was reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K). Topical treatment of K5.Bcl-xL-/- mice with wortmannin sensitized the skin for apoptosis induced by UV (UV) B, although wild-type epidermis was only marginally affected by this treatment, suggesting that the resistance to UVB largely depended on PI3K-Akt signaling in Bcl-xL-deficient mice but not in wild-type mice. Furthermore, UVB irradiation resulted in redistribution of phosphorylated Akt from the basal layer to the suprabasal layer, indicating that Akt could spatially cooperate with Bcl-xL upon UVB exposure in the upper epidermis where Bcl-xL is normally localized. These results suggest that Bcl-xL and the PI3K-Akt pathway form a cooperative, intercompensatory axis for the protection of epidermal keratinocytes from apoptosis in vivo.