Targeting the HGF/Met signalling pathway in cancer

Under normal conditions, hepatocyte growth factor (HGF)-induced Met tyrosine kinase (TK) activation is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand activated receptor internalisation and degradation. Despite these controls, HGF/Met signalling contributes to oncogenesis and tumour progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumour metastasis. The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programmes. Pathway inhibitors can be divided broadly into biologicals and low molecular weight synthetic TK inhibitors; of these, the latter now outnumber all other inhibitor types. We review here the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development as well as the latest clinical trial results. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents and model systems available concerning HGF/Met oncogenic signalling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.     

Mass spectrometric identification of serine hydrolase OVCA2 in the medulloblastoma cell line DAOY

OVCA2 is a putative serine-hydrolase. Performing protein profiling in human tumour cell lines, OVCA2 was detected in DAOY medulloblastoma cells as a high abundance protein. The protein was unambiguously identified by 2D gel-electrophoresis and MALDI-MS and MS/MS, its presence was confirmed by western blotting. Immunohistochemistry revealed expression in medulloblastoma and predominantly in oligodendrocytes. Computational approaches predicted functional motifs and domains, interaction with apoptosis-related protein BAG and 3D structure. In addition to the presence of OVCA2 in medulloblastoma, it was furthermore detectable in three out of 10 human tumour cell-lines as a high abundance protein probably suggesting a role in the tumour biology.     

Roles of hepatocyte growth factor (HGF) activator and HGF activator inhibitor in the pericellular activation of HGF/scatter factor

The activation of hepatocyte growth factor (HGF)/ scatter factor (SF) in an extracellular milieu is a critical limiting step in HGF/SF-induced signaling that is believed to have important roles in invasive growth of tumor cells and regeneration of injured tissue. This activation is caused by a proteolytic cleavage at the bond between Arg⁴⁹⁴-Val⁴⁹⁵ in the single-chain HGF/SF precursor, generating an active two-chain heterodimeric form. The HGF activator (HGFA) is a coagulation factor XII-like serine proteinase critically involved in this process in injured tissues including tumor tissues. In the past several years, the identification of endogenous HGFA inhibitors (HAIs) has provided detailed knowledge of the regulation of HGFA activity. Currently, two types of HAIs, namely HAI-1 and HAI-2, have been reported. Both are Kunitz-type serine proteinase inhibitors and inhibit not only HGFA but also other serine proteinases, such as membrane-type serine protease 1 (matriptase), plasmin, trypsin and kallikreins. HAIs are of particular interest because they are synthesized as type-I transmembrane proteins. Therefore, HAIs must have important regulatory roles in a cell surface proteolytic reaction, which has emerged as an important mechanism for the generation of biologically active proteins mediating a diverse range of cellular functions. This review is a summary and interpretation of recent data regarding the regulation of pericellular HGF/SF activation mediated by HGFA and HAIs and includes a discussion of the possible role of the type I transmembrane Kunitz-type inhibitor in pericellular proteolysis.     

Dlk/ZIP kinase-induced apoptosis in human medulloblastoma cells: requirement of the mitochondrial apoptosis pathway.

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.     

Effects of hepatocyte growth factor (HGF) on human glioma cells in vitro: HGF acts as a motility factor in glioma cells

Expression of c-Met, the receptor for hepatocyte growth factor (HGF), and the biological roles of HGF were examined in cultured human glioma cells. All of the 5 glioma cell lines examined expressed c-Met protein as well as the c-met gene. Expression of the c-met gene was also confirmed in a glioblastoma tissue. Three cell lines (MGM-3, U251, KG-I-C) demonstrated chemotactic response to HGF in a dose-dependent manner. The response was not only chemotactic but also chemokinetic as judged by a checkerboard analysis. The amounts of c-Met mRNA and protein were abundant in the cell lines which showed a migratory response to HGF. Moreover, c-Met protein expression was highest in U251 with the highest migratory response to HGF. Among the cell lines, KG-I-C produced notable amounts of HGF protein as well as of c-Met, suggesting that HGF may act in an autocrine fashion in this case. HGF did not act as an apparent growth factor in the glioma cell lines examined. Furthermore, HGF stimulated the production of metalloproteinase, probably gelatinase A, in U251 cells. © 1996 Wiley-Liss, Inc.     

Curcumin inhibits the Sonic Hedgehog signaling pathway and triggers apoptosis in medulloblastoma cells

Medulloblastoma is an aggressive primary brain tumor that arises in the cerebellum of children and young adults. The Sonic Hedgehog (Shh) signaling pathway that plays important roles in the pathology of this aggressive disease is a promising therapeutic target. In the present report we have shown that curcumin has cytotoxic effects on medulloblastoma cells. Curcumin suppressed also cell proliferation and triggered cell‐cycle arrest at G₂/M phase. Moreover, curcumin inhibited the Shh–Gli1 signaling pathway by downregulating the Shh protein and its most important downstream targets GLI1 and PTCH1. Furthermore, curcumin reduced the levels of β‐catenin, the activate/phosphorylated form of Akt and NF‐κB, which led to downregulating the three common key effectors, namely C‐myc, N‐myc, and Cyclin D1. Consequently, apoptosis was triggered by curcumin through the mitochondrial pathway via downregulation of Bcl‐2, a downstream anti‐apoptotic effector of the Shh signaling. Importantly, the resistant cells that exhibited no decrease in the levels of Shh and Bcl‐2, were sensitized to curcumin by the addition of the Shh antogonist, cyclopamine. Furthermore, we have shown that curcumin enhances the killing efficiency of nontoxic doses of cisplatin and γ‐rays. In addition, we present clear evidence that piperine, an enhancer of curcumin bioavailability in humans, potentiates the apoptotic effect of curcumin against medulloblastoma cells. This effect was mediated through strong downregulation of Bcl‐2. These results indicate that curcumin, a natural nontoxic compound, represents great promise as Shh‐targeted therapy for medulloblastomas. © 2009 Wiley‐Liss, Inc.     

Mutational analysis of PDGFR-RAS/MAPK pathway activation in childhood medulloblastoma

Aberrant signalling via platelet derived growth factor receptors (PDGFRs) and the RAS/MAPK pathway has been implicated in the development of medulloblastoma, the most common malignant brain tumour in childhood. To determine whether genetic mechanisms play a role in the activation of PDGFR-RAS/MAPK signalling in medulloblastoma, we performed a direct sequence analysis of the established mutational "hotspots" of known targets of activating mutations within the pathway (PDGFRA, NRAS, KRAS, HRAS and BRAF) and PDFRFB, in a cohort of 28 primary tumours. A synonymous sequence variation in PDGFRA (CCG to CCA; PRO 567 PRO) was detected in two cases (approximately 7%), but not in 150 normal chromosomes assessed, suggesting that the PDGFRA locus may be associated with medulloblastoma development in certain cases. No evidence for oncogenic mutations affecting NRAS, KRAS, HRAS, BRAF or PDFRFB was found in any case. These data demonstrate that activating mutations in established mutational hotspots within the PDGFR-RAS/MAPK pathway are rare events in medulloblastoma development, and suggest that alternative mechanisms are responsible for RAS/MAPK pathway activation in this disease.     

NDRG2调控HGF/c-MET通路抑制结肠癌细胞的增殖

目的:探讨抑癌基因NDRG2通过调节HGF/c-MET信号通路,抑制结肠癌HT29细胞增殖的机制。方法:建立NDRG2过表达细胞株HT29-NDRG2及对照组HT29-Cherry,并于第1、2、3、4、5天以MTT法检测细胞增殖;以不同浓度的HGF（0ng/ml、1ng/ml、2ng/ml、4ng/ml、8ng/ml、10ng/ml）刺激HT29亲本细胞,并于第1、2、3天以MTT法检测HT29细胞的增殖;采用qPCR法检测NDRG2过表达细胞株HT29-NDRG2与对照组HT29-Cherry两组细胞中HGF的表达水平;Western blot检测HT29-Cherry和HT29-NDRG2两组细胞以及添加HGF（10ng/ml）刺激HT29亲本细胞后各组细胞中HGF/c-MET通路中关键分子的表达。结果:过表达NDRG2以及HGF刺激均可以显著抑制HT29细胞的增殖能力;NDRG2的高表达可以上调HGF的转录水平;HGF可以刺激c-MET和p-ERK1/2,并上调p 21和p 27,抑制HT29细胞的增殖;过表达NDRG2明显上调c-MET、p-ERK1/2,并诱导p 21和p 27的高表达,从而抑制HT29细胞的增殖。结论:NDRG2可以通过调节HGF/c-MET信号通路从而抑制结肠癌细胞HT29细胞的增殖能力。     

HGF/c-MET信号通路抑制剂在抗胰腺癌中的研究进展

胰腺癌是恶性程度较高的肿瘤之一.靶向治疗日渐成为胰腺癌治疗的重要组成部分.研究证实肝细胞生长因子及其受体(HGF/c-MET)信号通路在胰腺癌发生、发展过程中起重要作用,通过抑制该条信号通路能够起到显著的抗胰腺癌作用.因此,HGF/c-MET靶向抑制剂的研究为胰腺癌的治疗开辟了一条新的途径.     

HGF/c-Met acts as an alternative angiogenic pathway in sunitinib-resistant tumors

Molecular and cellular mechanisms underlying resistance/low responsiveness to antiangiogenic compounds are under extensive investigations. Both populations of tumor and stroma (nontumor compartment) seem to contribute in inherent/acquired resistance to antiangiogenic therapy. Here, investigating in vivo efficacy of sunitinib in experimental models resulted in the identification of tumors that were resistant/sensitive to the therapy. Analysis of tumor protein lysates indicated a greater concentration of hepatocyte growth factor (HGF) in resistant tumors than in sensitive ones. In addition, using flow cytometry, c-Met expression was found to be significantly higher in endothelial cells than in tumor cells, suggesting that HGF might target the vascular endothelial cells in resistant tumors. Combination of sunitinib and a selective c-Met inhibitor significantly inhibited tumor growth compared with sunitinib or c-Met inhibitor alone in resistant tumors. Histology and in vitro analyses suggested that combination treatment mainly targeted the vasculature in the resistant tumors. Conversely, systemic injection of HGF in the sensitive tumor models conferred resistance to sunitinib through maintenance of tumor angiogenesis. In conclusion, our study indicates a role for HGF/c-Met pathway in development of resistance to antiangiogenic therapy and suggests a potential strategy to circumvent resistance to vascular endothelial growth factor receptor tyrosine kinase inhibitor in the clinic.     

Protein chemical identification and characterization of the human variants of far upstream element binding protein in medulloblastoma DAOY cell line

The assembly of trans-acting proteins on sequence-specific DNA cis-elements is crucial in the regulation of eukaryotic gene expression. Far upstream element binding proteins (FAB) are proteins that regulate the expression of the c-myc oncogene by binding to the far upstream element of the c-myc gene. The present study unambiguously identified the two human variants of FAB (FAB1, FAB2) in the medulloblastoma DAOY cell line and characterized their structure for the first time by tandem mass spectrometry independent of antibody availability and specificity. The study also tentatively assigned the third variant (FAB3) at the level of mass spectrometry, although tandem mass spectrometric analysis failed to corroborate the result. These findings open up an exciting possibility for discerning the cellular roles of FAB in tumor biology.     

熊果酸通过HGF/c-met通路逆转肺癌A549细胞上皮间质转化及血管生成

目的:探索熊果酸抑制由肝细胞生长因子(HGF)诱导肺癌细胞上皮间质转化(epithelial mesenchymal transition, EMT)及血管生成的作用机制研究。方法:利用CCK-8、侵袭实验、Western blot实验和小管形成实验研究熊果酸对HGF诱导的肺癌细胞增殖、细胞侵袭、EMT、管腔形成能力和相关信号通路分子表达的影响。结果:熊果酸能显著抑制肺癌A549细胞增殖,其作用肺癌A549细胞24 h的IC₅₀值为31.75μmol/L; HGF能使肺癌细胞穿透小室滤膜的数量显著增多(P<0.01),加入熊果酸作用后,肺癌细胞穿透小室滤膜的数量又显著降低(P<0.01);HGF能诱导肺癌EMT的形成(P<0.01),熊果酸则能抑制肺癌EMT形成(P<0.01);HGF能使小管形成明显增加(P<0.01),而熊果酸则能明显抑制小管的形成(P<0.01);此外,我们还研究了熊果酸对相关信号通路的影响,发现熊果酸能显著抑制HGF激活的HGF/c-met信号通路。同时,我们使用c-met抑制剂SU11274进一步研究其...     

Role of Wnt pathway in medulloblastoma oncogenesis

To clarify the roles of Wnt pathway in medulloblastoma oncogenesis, immunohistochemical staining of beta-catenin and Wnt-1 and genomic analyses of CTNNB1 (beta-catenin) and AXIN1 (axin 1) were examined in 23 sporadic cases. Accumulation of beta-catenin in tumor cells was immunohistochemically proven in 5 cases; 2 cases showed positive immunoreactivity for Wnt-1 and another 2 showed mutation of either CTNNB1 or AXIN1. AXIN1 mutation was in exon 3, corresponding to GSK-3beta binding site and CTNNB1 mutation was in exon 3, corresponding to its phosphorylation site. Disruption of these proteins could result in upregulation of the Wnt signaling and accumulation of beta-catenin, followed by cell proliferation and medulloblastoma oncogenesis.