HGF-induced Cell Research Articles

Interruption of Hepatocyte Growth Factor Signaling Augmented Oridonin-Induced Death in Human Non-small Cell Lung Cancer A549 Cells <i>via</i> c-Met-Nuclear Factor-κB-Cyclooxygenase-2 and c-Met-Bcl-2-Caspase-3 Pathways

The aim of this study was to elucidate the molecular mechanisms mediating hepatocyte growth factor (HGF)-induced protection against oridonin-induced apoptosis in A549 cells. Oridonin induced decrease in Bcl-2/Bax ratio and activation of caspase-3, while these processes were reversed by HGF, suggesting that HGF played an anti-apoptotic role in oridonin-induced A549 cell death. HGF-induced protective effect was partially attributed to the activation of nuclear factor (NF)-κB and cyclooxygenase 2 (COX-2), since the protective effect was abolished by inhibition of NF-κB or interruption of COX-2. Then the activated COX-2 could prevent cells from initiating the apoptotic response by promoting prostaglandin E₂ (PGE₂) release. Activation of NF-κB-COX-2 by HGF-treatment triggered the increase in Bcl-2/Bax ratio, inhibition of procaspase-3 cleavage, promotion of Ca²⁺-independent intracellular phospholipase A2 (iPLA2) expression and augmentation of PGE₂ release, leading to antagonizing oridonin-induced cell death in A549 cells. HGF-induced cell survival in response to oridonin administration was associated with the activation of c-Met-NF-κB-COX-2 and c-Met-Bcl-2-caspase-3 signaling pathways. iPLA2, downstream effector of caspase-3, also participated in these processes.

Plasma membrane ion fluxes and NFAT-dependent gene transcription contribute to c-met-induced epithelial scattering

Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and causing them to migrate as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. We have applied a chemical biology approach to identify components of HGF signaling that are required for scattering in an in vitro model system. This approach yields a number of small molecules that block HGF-induced scattering, including a calcium channel blocker. We show that HGF stimulation results in sudden and transient increases in ion channel influxes at the plasma membrane. Although multiple channels occur in the membranes of our model system, we find that TrpC6 is specifically required for HGF-induced scattering. We further demonstrate that HGF-induced ion influxes through TrpC6 channels coincide with a transient increase in nuclear factor of activated T-cells (NFAT)-dependent gene transcription and that NFAT-dependent gene transcription is required for HGF-induced cell scattering.

Foretinib demonstrates anti-tumor activity and improves overall survival in preclinical models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. The purpose of this study is to assess the anti-tumor and anti-angiogenic activities of foretinib, a vascular endothelial growth factor receptor 2 (VEGFR-2) and c-Met inhibitor using mouse models of human HCC. SK-HEP1 and 21-0208 HCC cells as well as patient-derived HCC models were employed to study the anti-tumor and antiangiogenic activities of foretinib. Changes of biomarkers relevant to hepatocyte growth factor (HGF) signaling pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Treatment of SK-HEP1 cells with foretinib resulted in growth inhibition, G2/M cell cycle arrest, reduced colony formation and blockade of HGF-induced cell migration. In both orthotopic and ectopic models of HCC, foretinib potently inhibited tumor growth in a dose-dependent manner. Inhibition of angiogenesis correlated with inactivation of VEGFR-2/c-Met signaling pathways. Foretinib also caused elevation of p27 and Bim but reduced cyclin B1 expression and p-c-Myc, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, foretinib potently inhibited primary tumor growth and significantly prolonged mouse survival. Foretinib demonstrated significant antitumor activities in patient-derived HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.

Osthole Suppresses Hepatocyte Growth Factor (HGF)-Induced Epithelial-Mesenchymal Transition via Repression of the c-Met/Akt/mTOR Pathway in Human Breast Cancer Cells

Substantial activation of the HGF/c-Met signaling pathway is involved in the progression of several types of cancers and associated with increased tumor invasion and metastatic potential. Underlying HGF-induced tumorigenesis, epithelial to mesenchymal transition (EMT) shows a positive correlation with progression in patients. We previously determined that osthole is a potent fatty acid synthase (FASN) inhibitor. FASN is implicated in cancer progression and may regulate lipid raft function. We therefore examined whether osthole could block HGF-induced tumorigenesis by disrupting lipid rafts. Here, we found that osthole could abrogate HGF-induced cell scattering, migration, and invasion in MCF-7 breast cancer cells. Osthole also effectively inhibited the HGF-induced decrease of E-cadherin and increase of vimentin via down-regulation of phosphorylated Akt and mTOR. Interestingly, osthole blocked HGF-induced c-Met phosphorylation and repressed the expression of total c-Met protein in MCF-7 cells. In addition, C75, a pharmacological inhibitor of FASN, repressed the expression of total c-Met protein in MCF-7 cells. Consistent with a role for FASN, loss of c-Met in cells treated with osthole was prevented by the exogenous addition of palmitate. Briefly, our result suggests a connection between FASN activity and c-Met protein expression and that osthole is a potential compound for breast cancer therapy by targeting the major pathway of HGF/c-Met-induced EMT.

Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members

The hepatocyte growth factor (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression, and invasive cancer growth. Similarly, altered expression and splicing (i.e. inclusion of variant exon 5, "v5") of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to further understand how HGF regulates CD44v5 expression. Immortalized nontumorigenic keratinocyte (HaCaT) cells abundantly express both Met receptors and CD44v5 transmembrane glycoproteins. HGF stimulated CD44v5 protein expression and HaCaT cell migration; these events required activation of the ERK1/2 MAPK module and Sam68, a protein involved in RNA processing, splicing, and v5 inclusion. Similar to HaCaT cells, highly migratory MDA-MB-231 breast cancer cells also required Sam68 expression for HGF-induced migration. However, MDA-MB-231 cell migration occurred independently of ERK1/2 and CD44v5 expression and instead required ERK5 signaling to Sam68. Phospho-mutant, but not WT-Sam68, blocked HGF-induced cell migration in both cell types; MDA-MB-435 cells behaved similarly. These results suggest that Sam68 acts as a convergence point for ERK signaling to cell migration; blockade of phospho-Sam68 may provide a new avenue for therapeutic inhibition of metastatic cancers.

Diosgenin Suppresses Hepatocyte Growth Factor (HGF)-Induced Epithelial–Mesenchymal Transition by Down-regulation of Mdm2 and Vimentin

Substantial activation of the hepatocyte growth factor (HGF)/c-Met pathway leads to cancer cell scattering and invasion and has been observed in several types of cancers, including prostate and colorectal cancers. The phosphorylation cascade downstream of HGF, particularly PI3K/Akt signaling, regulates epithelial-to-mesenchymal transition (EMT). How this signaling governs EMT and whether specific kinases respond to particular EMT effectors remain unclear. This study found specific increases in Mdm2 and vimentin rather than the coregulation of an array of EMT marker proteins in response to HGF-induced EMT in DU145 prostate cancer cells. Importantly, it was further found that diosgenin abrogated HGF-induced DU145 cell scattering and invasion. Moreover, diosgenin effectively inhibited the HGF-induced increases in Mdm2 and vimentin by down-regulating phosphorylated Akt and mTOR. In summary, the results suggest that diosgenin may be a potential compound for use in prostate cancer therapy to target the major HGF-induced EMT pathway.

Abstract 2914: A role for glycolysis in the c-Met/HGF signaling axis

Abstract Mitochondrial respiration is the most efficient form of energy production in the cell; however, in the absence of oxygen, such as within a solid tumor mass, cells rely on glycolysis as the main source of energy. Under hypoxic conditions the end product of glycolysis, pyruvate, is reduced to lactate by lactate dehydrogenase (LDH), which is then exported out of the cell. This reduction is essential to regenerate NAD+ consumed by the earlier steps of glycolysis. Recent evidence suggests that hepatocyte growth factor (HGF), the only known ligand for the c-Met receptor, causes an increase in glycolysis. This led our laboratory to hypothesize that glycolysis may be required for HGF-induced phenotypic changes in cancer cells. HGF signaling through the c-Met receptor induces an epithelial-mesenchymal transition (EMT), including loss of cell-cell adhesions through down-regulation of E-cadherin expression, as well as increased motility and cell scattering characteristic of the initiating steps leading to cancer cell invasion and metastasis. Using DU145 prostate cancer cells, we have found that inhibitors of glycolysis (2-DG) and lactate production via LDH activity (oxamate), were able to inhibit HGF-induced cell scattering. Using a wound healing assay, we observed that oxamate was able to prevent HGF-induced cell migration, suggesting a role for glycolysis in cell motility. Additionally, lactic acid was able to rescue cell scattering and wound healing blocked by oxamate, but not by 2-DG. Lactic acid measurements showed that oxamate prevented an increase in glycolysis stimulated by HGF. Measurement of ATP levels also suggested that 2-DG was able to block cell motility due to a reduction in ATP, while oxamate appeared to work through a yet undetermined method. Western blot analysis indicated that oxamate caused a decrease in HGF-induced phospho-Met and downstream phospho-Akt levels. Using H1993 lung cancer cells with constitutively active c-Met, we were able to determine that oxamate deactivated c-Met, while not affecting total c-Met levels. The ability of oxamate to reduce phosphorylation levels appeared to be Met specific based on its inability to block phosphorylation of other activated receptors. Oxamate not only inhibits LDH, but also aspartate aminotransferase (AAT), an enzyme involved in the malate-aspartate shuttle. Our data suggest, however, that the ability of oxamate to inhibit HGF-induced signaling and cell scattering is not through its ability to inhibit AAT. Taken together, our data suggest the c-Met/HGF signaling axis is dependent on glycolysis and perhaps lactic acid levels. Our long-term goals are to elucidate the specific mechanism for this dependence as a means of defining novel targets for preventing c-Met-mediated cancer cell invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2011-2914

Abstract 3845: Endocytic protein APPL2 as a regulator of glioblastoma cell growth

Abstract Recent discoveries have suggested that some endocytic proteins are involved in multiple steps of cellular signaling, which starts from endocytosis of activated receptors and is further transmitted from the cytoplasm to the nucleus. Two homologous adaptor proteins APPL1 and APPL2 seem to constitute good examples of such proteins, involved in both endocytosis (by interacting with small GTPase Rab5) and cellular signaling. They are able to translocate to the nucleus and may participate in the regulation of chromatin modification, raising a possibility of their involvement in the control of gene transcription. Deregulation of such important cellular functions may be a cause of neoplastic growth and tumorigenesis. In fact, there are some lines of evidence suggesting a possible role for APPL proteins in cancer development and/or progression: 1) APPL proteins participate in endocytic trafficking of several transmembrane receptors and defective degradation (via deregulation of endocytosis) of the receptor tyrosine kinases, like EGFR, leads to sustained signaling, changes in cell proliferation or transformation; 2) APPL proteins can also increase activity the key kinase involved in the regulation of tumorigenesis – AKT, by interacting with several components of its signaling pathway, like PI3K, AKT itself, GSK3β and TSC2. Indeed both APPL proteins appear to be required for HGF-induced cell survival and migration via activation of AKT. Many major genomic abnormalities in human glioblastoma (GBM) samples, recently cataloged by the TCGA Research Network, result in aberrations of signaling molecules and pathways potentially regulated by APPL proteins, like receptor tyrosine kinases and AKT, highlighting their importance for GBM development. Taking in consideration all the facts mentioned above, we have chosen GBM as a model to study the potential role of APPL proteins in the regulation of tumor growth. During our study we have discovered the following phenomena: i) The level of APPL2 protein (but not APPL1) is significantly increased in 40% of human GBM versus normal brain samples. ii) Silencing of APPL2 in GBM cell lines decreases both anchorage-independent growth and cell viability under reduced growth factor availability. These effects result from both: inhibition of cell proliferation and increased apoptosis. iii) Expression of some cell cycle- and apoptosis-related genes changes upon APPL2 silencing. To our knowledge these results for the first time show the upregulation of APPL2 protein in human tumor samples suggesting that this protein may provide some advantage for tumor growth. The results of in vitro experiments explain the mechanism by demonstrating that APPL2 influences both cell proliferation and resistance to apoptosis. Our results also highlight the surprising differences in the functions of homologous APPL1 and APPL2 proteins that will be further discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3845. doi:10.1158/1538-7445.AM2011-3845

Green tea (−)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.

Filamin a mediates HGF/c‐MET signaling in tumor cell migration

Deregulated hepatocyte growth factor (HGF)/c-MET axis has been correlated with poor clinical outcome and drug resistance in many human cancers. Identification of novel regulatory mechanisms influencing HGF/c-MET signaling may therefore be necessary to develop more effective cancer therapies. In our study, we show that multiple human cancer tissues and cells express filamin A (FLNA), a large cytoskeletal actin-binding protein, and expression of c-MET is significantly reduced in human tumor cells deficient for FLNA. The FLNA-deficient tumor cells exhibited poor migrative and invasive ability in response to HGF. On the other hand, the anchorage-dependent and independent tumor cell proliferation was not altered by HGF. The FLNA-deficiency specifically attenuated the activation of the c-MET downstream signaling molecule AKT in response to HGF stimulation. Furthermore, FLNA enhanced c-MET promoter activity by its binding to SMAD2. The impact of FLNA deficiency on c-MET expression and HGF-mediated cell migration in human tumor cells was confirmed in primary mouse embryonic fibroblasts deficient for Flna. These data suggest that FLNA is one of the important regulators of c-MET signaling and HGF-induced tumor cell migration.

Numb is a negative regulator of HGF dependent cell scattering and Rac1 activation

Numb is an endocytic adaptor protein that regulates internalization and post-endocytic trafficking of cell surface proteins. In polarized epithelial cells Numb is localized to the basolateral membrane, and recent work has implicated Numb in regulation of cell adhesion and migration, suggesting a role for Numb in epithelial–mesenchymal transition (EMT). We depleted MDCK cells of Numb and examined the effects downstream of EMT-promoting stimuli. While knockdown of Numb did not affect apicobasal polarity, we show that depletion of Numb destabilizes E-cadherin-based cell–cell adhesion and promotes loss of epithelial cell morphology. In addition, Numb knockdown in MDCK cells potentiates HGF-induced lamellipodia formation and cell dispersal. Examination of Rac1-GTP levels in Numb knockdown cells revealed hyperactivation of Rac1 following extracellular calcium depletion and HGF stimulation, which corresponds with enhanced loss of cell adhesions and lamellipodia formation. Furthermore, inhibition of Rac1 in Numb depleted cells stabilized cell–cell contacts following depletion of extracellular calcium. Together, these data indicate that Numb acts to suppress Rac1-GTP accumulation, and its loss leads to increased sensitivity toward extracellular signals that disrupt cell–cell adhesion to induce epithelial–mesenchymal transition (EMT) and cell dispersal.

The Tetraspanin CD151 Is Required for Met-dependent Signaling and Tumor Cell Growth

CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cell-substrate adhesion and cell migration through physical and functional interactions with integrin receptors. In contrast, little is known about the potential role of CD151 in controlling cell proliferation and survival. We have previously shown that β4 integrin, a major CD151 partner, not only acts as an adhesive receptor for laminins but also as an intracellular signaling platform promoting cell proliferation and invasive growth upon interaction with Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF). Here we show that RNAi-mediated silencing of CD151 expression in cancer cells impairs HGF-driven proliferation, anchorage-independent growth, protection from anoikis, and tumor progression in xenograft models in vivo. Mechanistically, we found that CD151 is crucially implicated in the formation of signaling complexes between Met and β4 integrin, a known amplifier of HGF-induced tumor cell growth and survival. CD151 depletion hampered HGF-induced phosphorylation of β4 integrin and the ensuing Grb2-Gab1 association, a signaling pathway leading to MAPK stimulation and cell growth. Accordingly, CD151 knockdown reduced HGF-triggered activation of MAPK but not AKT signaling cascade. These results indicate that CD151 controls Met-dependent neoplastic growth by enhancing receptor signaling through β4 integrin-mediated pathways, independent of cell-substrate adhesion.

The role of TRPC6 in HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells

Hepatocyte growth factor (HGF) is a glycoprotein that induces prostate cancer cell proliferation, migration and invasion. The activation of transient receptor potential canonical 6 (TRPC6) channels is considered important in promoting prostate cancer cell proliferation. In this study, we assessed the role of endogenous TRPC6 channels in the HGF-induced cell proliferation of prostate cancer. Reverse transcription-PCR and Western blotting were used to investigate TRPC6 expression. Electrophysiological techniques (whole-cell patch clamp configuration) and Ca(2+) imaging analysis were used to investigate the channel activity in cells. The effects of TRPC6 channels on cell cycle progression, cell apoptosis and cell growth were also examined. TRPC6 and c-MET were expressed in DU145 and PC3 cells. In addition, functional TRPC6 channels were present in DU145 and PC3 cells, and TRPC6 knockdown suppressed TRPC-like currents evoked by oleoyl-2-acetyl-sn-glycerol (OAG). Inhibition of TRPC6 channels in DU145 and PC3 cells abolished OAG- and HGF-induced Ca(2+) entry. Furthermore, inhibition of TRPC6 channels arrested DU145 and PC3 cells at the G(2)/M phase and suppressed HGF-induced cell proliferation. Collectively, our results indicate that TRPC6 has an important role in HGF-induced DU145 and PC3 cell proliferation.

Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.

Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.

Breast tumor kinase and extracellular signal-regulated kinase 5 mediate Met receptor signaling to cell migration in breast cancer cells

IntroductionBreast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.MethodsIn vitro kinase assays were performed to directly measure Brk kinase activity in response to MET and RON ligands. Transfection of Brk-targeted RNAi was used to knock down endogenous Brk or ERK5 in multiple cell lines. Kinase activities (downstream of MET signaling) were assayed by Western blotting using total and phospho-specific antibodies. Boyden chamber assays were used to measure cell migration in response to manipulation of Brk and downstream MET effectors. Rescue experiments were performed by knock down of endogenous Brk using RNAi (targeting the untranslated region (3′-UTR)) and transient transfection (re-expression) of either wild-type or kinase-inactive Brk.ResultsBrk gene silencing revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk and ERK5 copurified in HGF-induced protein complexes, and Brk/ERK5 complexes formed independently of Brk kinase activity. ERK5 was required for breast cancer cell but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knockdown. Notably, rescue experiments indicated that the kinase activity of Brk was not required for HGF-induced cell migration. Further, expression of either wild-type or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration.ConclusionsThese results have identified Brk and ERK5 as important downstream effectors of Met signaling to cell migration. Targeting ERK5 kinase activity or inhibiting the formation of Brk/ERK5 complexes may provide an additional means of blocking cell migration associated with breast cancer progression to metastasis.

Effects of transferred NK4 gene on proliferation, migration, invasion and apoptosis of human prostate cancer DU145 cells

We investigated the ability of NK4, an antagonist of human hepatocyte growth factor (HGF), to inhibit the influence of HGF on proliferation, migration, invasion and apoptosis of human prostate cancer cells. Expression vector pBudCE4.1-EGFP-NK4 containing NK4 cDNA was used to transfect human prostate cancer DU145 cells, and the effects of the autocrine NK4 on tumor cell proliferation, migration, invasion and apoptosis were assessed in vitro. In vivo, we subcutaneously implanted DU145 cells, mock-transfected clone (DU145/empty vector) cells and NK4-transfected clone (DU145/NK4) cells into nude mice, and then evaluated tumor growth, cell proliferation and cell apoptosis in vivo. We found that DU145/NK4 cells expressed NK4 protein. In the in vitro study, autocrine NK4 attenuated the HGF-induced tumor cell proliferation, migration and invasion, and stimulated apoptosis. Furthermore, autocrine NK4 effectively inhibited the HGF-induced phosphorylation of c-Met, extracellular signal-regulated kinase-1 (ERK1). and protein kinase B 1/2 (Akt1/2). Histological examination revealed that autocrine NK4 inhibited proliferation and accelerated apoptosis of prostate cancer cells. These results show that genetic modification of DU145 cells with NK4 cDNA yields a significant effect on their proliferation, migration, invasion and apoptosis. Molecular targeting of HGF/c-Met by NK4 could be applied as a novel therapeutic approach to prostate cancer.

Comment on “Effect of transferred NK4 gene on proliferation, migration, invasion, and apoptosis of human prostate cancer DU145 cells” by Dan Yue et al. in Asian Journal of Andrology

Comment on “Effect of transferred NK4 gene on proliferation, migration, invasion, and apoptosis of human prostate cancer DU145 cells” by Dan Yue et al. in Asian Journal of Andrology

Abstract 1783: Activated c-MET; a prognostic factor and potential therapeutic target for malignant peripheral nerve sheath tumors (MPNST)

Abstract There is a crucial need for sensitive prognosticators and novel therapeutic approaches for better management of MPNST. These tumors harbor a dismal prognosis due to their chemoresistant nature and enhanced metastatic propensity. A large body of evidence suggests a role for the tyrosine kinase receptor c-Met in cancer and its utility as a target for cancer therapy is currently being investigated. Here we aimed to evaluate the role of the c-Met signaling pathway in human MPNST. Expression level of c-Met, its ligand HGF and phosphorylated c-Met was determined immunohistochemically in a tissue microarray of human MPNSTs (n = 96) and benign neurofibromas (n = 31). All three markers were found to be over-expressed in MPNST as compared to their pre-malignant precursors (neurofibroma; p<0.05). Furthermore, univariable and multivariable analyses identified a statistically significant correlation between activated-c-Met expression and poor patient outcome (HR=2.32, p=0.012 and HR=1.48, p<0.001, respectively). Human MPNST cell lines expressed c-Met, HGF, and activated c-Met to varying levels. An activating c-Met mutation (Exon 2: N375S) was identified in one cell line (T265); all cells evaluated (n=3) harbored c-Met and HGF amplification based on aCGH. c-Met activation resulted in significantly increased MPNST cell motility and invasion. Furthermore, c-Met activation enhanced the pro-angiogenic effects of MPNST. c-Met blockade (via siRNA or a small molecule inhibitor) abrogated the HGF-induced downstream signaling and MPNST cell migration/invasion. Dual blockade of c-Met and VEGFR significantly abrogated MPNST local and metastatic growth in vivo. In conclusion, Human MPNST tumors and cell lines express the c-Met receptor which, upon activation, results in increased MPNST migration, invasion, and angiogenesis. Inhibition of c-Met-mediated signaling could impair MPNST metastatic capacity, and therefore merits further consideration as a potential MPNST therapeutic target. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1783.

Cyr61 Mediates Hepatocyte Growth Factor–Dependent Tumor Cell Growth, Migration, and Akt Activation

Certain tumor cell responses to the growth factor-inducible early response gene product CCN1/Cyr61 overlap with those induced by the hepatocyte growth factor (HGF)/c-Met signaling pathway. In this study, we investigate if Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells. A semiquantitative immunohistochemical analysis of 112 human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlate with tumor grade (P < 0.001) and with c-Met protein expression (r(2) = 0.4791, P < 0.0001). Purified HGF rapidly upregulated Cyr61 mRNA (peak at 30 minutes) and protein expression (peak at 2 hours) in HGF(-)/c-Met(+) human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway inhibitors reduced Cyr61 expression in HGF(+)/c-Met(+) human glioma cell lines in vitro and in HGF(+)/c-Met(+) glioma xenografts. Targeting Cyr61 expression with small interfering RNA (siRNA) inhibited HGF-induced cell migration (P < 0.01) and cell growth (P < 0.001) in vitro. The effect of Cyr61 on HGF-induced Akt pathway activation was also examined. Cyr61 siRNA had no effect on the early phase of HGF-induced Akt phosphorylation (Ser(473)) 30 minutes after stimulation with HGF. Cyr61 siRNA inhibited a second phase of Akt phosphorylation measured 12 hours after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target glycogen synthase kinase 3alpha. We treated preestablished subcutaneous glioma xenografts with Cyr61 siRNA or control siRNA by direct intratumoral delivery. Cyr61 siRNA inhibited Cyr61 expression and glioma xenograft growth by up to 40% in a dose-dependent manner (P < 0.05). These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell growth, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies.