Endogenous and artificial miRNAs explore a rich variety of conformations: a potential relationship between secondary structure and biological functionality.

Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.

Targeted deletion of c-Met in thymic epithelial cells leads to an autoimmune phenotype.

Hepatocyte growth factor (HGF) and its receptor c-Met signaling have been implicated in regulating various types of cells including epithelial cells. We have previously reported that c-Met is expressed by thymic epithelial cells (TECs), and that in vivo administration of hybrid cytokines containing IL-7 and the beta- or alpha-chain of HGF significantly increase the number of TECs. In order to study the role of c-Met signaling in TECs, we generated conditional knockout (cKO) mice in which c-Met was specifically deleted in TECs using a Foxn1-Cre transgene. We show here that c-Met deficiency in TECs results in age-progressive reduction in TEC number and reduced number of regulatory T cells. Consequently, c-Met TEC cKO mice displayed an autoimmune phenotype. Thus, c-Met signaling in TECs is important for the maintenance of TECs and immune self-tolerance.

Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH.

We have recently shown that hepatocyte-specific c-met deficiency accelerates the progression of nonalcoholic steatohepatitis in experimental murine models resulting in augmented production of reactive oxygen species and accelerated development of fibrosis. The aim of this study focuses on the elucidation of the underlying cellular mechanisms driven by Nrf2 overactivation in hepatocytes lacking c-met receptor characterized by a severe unbalance between pro-oxidant and antioxidant functions. Control mice (c-metfx/fx), single c-met knockouts (c-metΔhepa), and double c-met/Keap1 knockouts (met/Keap1Δhepa) were then fed a chow or a methionine-choline-deficient (MCD) diet, respectively, for 4 weeks to reproduce the features of nonalcoholic steatohepatitis. Upon MCD feeding, met/Keap1Δhepa mice displayed increased liver mass albeit decreased triglyceride accumulation. The marked increase of oxidative stress observed in c-metΔhepa was restored in the double mutants as assessed by 4-HNE immunostaining and by the expression of genes responsible for the generation of free radicals. Moreover, double knockout mice presented a reduced amount of liver-infiltrating cells and the exacerbation of fibrosis progression observed in c-metΔhepa livers was significantly inhibited in met/Keap1Δhepa. Therefore, genetic activation of the antioxidant transcription factor Nrf2 improves liver damage and repair in hepatocyte-specific c-met-deficient mice mainly through restoring a balance in the cellular redox homeostasis.

Hepatocyte growth factor in physiology and infectious diseases.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine composed of an α-chain and a ß-chain, and these chains contain four kringle domains and a serine protease-like structure, respectively. The receptor for HGF was identified as the c-met proto-oncogene product of transmembrane receptor tyrosine kinase. HGF-induced signaling through the receptor Met provokes dynamic biological responses that support morphogenesis, regeneration, and the survival of various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. Characterization of tissue-specific Met knockout mice has further indicated that the HGF-Met system modulates immune cell functions and also plays an inhibitory role in the progression of chronic inflammation and fibrosis. However, the biological actions that are driven by the HGF-Met pathway all play a role in the acquisition of the malignant characteristics in tumor cells, such as invasion, metastasis, and drug resistance in the tumor microenvironment. Even though oncogenic Met signaling remains the major research focus, the HGF-Met axis has also been implicated in infectious diseases. Many pathogens try to utilize host HGF-Met system to establish comfortable environment for infection. Their strategies are not only simply change the expression level of HGF or Met, but also actively hijack HGF-Met system and deregulating Met signaling using their pathogenic factors. Consequently, the monitoring of HGF and Met expression, along with real-time detection of Met activation, can be a beneficial biomarker of these infectious diseases. Preclinical studies designed to address the therapeutic significance of HGF have been performed on injury/disease models, including acute tissue injury, chronic fibrosis, and cardiovascular and neurodegenerative diseases. Likewise, manipulating the HGF-Met system with complete control will lead to a tailor made treatment for those infectious diseases.

MicroRNA-31 inhibits lung adenocarcinoma stem-like cells via down-regulation of MET-PI3K-Akt signaling pathway.

Accumulated evidences suggested that microRNAs (miRs) play an important role in non-small cell lung cancer (NSCLC). However, how miRs perform their functions in lung adenocarcinoma cancer stem cells (CSCs) remains unknown. Notably, most studies pay more attention to the effects of miRNAs on the metastasis traits whereas the growth activities of CSCs are rather undervalued. In our report, using A549CD133+cells, we examined the inhibitory effects and the underlying mechanisms of microRNA-31 (miR-31) on the growth of lung adenocarcinoma CSC-like cells. Initially, we determined the level of miR-31 in A549 and A549CD133+ cells. Over-expression of miR-31 was found in A549CD133+ cells by microarray and real-time quantitative PCR (RTqPCR) assays. Experiments in multiple NSCLC cell lines in vitro and A549CD133+ cells xenograft models in vivo confirmed that down regulation of miR-31 resulted in increase of A549CD133+ cells growth, whereas overexpression of miR-31 led to the inhibition of adenocarcinoma cell proliferation. Also, MET proto-oncogene has been determined to be a direct target of miR-31 by dual luciferase report, RT-qPCR and western blot analysis. Down regulation of MET inhibited viability of A549CD133+ cells. The levels of PI3Kinase, Akt and p-Akt as well as downstream proteins were consequently decreased. These results suggest that miR-31 might inhibit the growth of lung adenocarcinoma cancer stem-like cells via down regulation of the MET-PI3K-Akt signaling pathway.

MET is required for the recruitment of anti-tumoural neutrophils.

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

Knockdown of c-MET induced apoptosis in ABCB1-overexpressed multidrug-resistance cancer cell lines.

Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.

Adenovirus expressing dual c-Met-specific shRNA exhibits potent antitumor effect through autophagic cell death accompanied by senescence-like phenotypes in glioblastoma cells.

c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G2/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death.

Celastrol exerts synergistic effects with PHA-665752 and inhibits tumor growth of c-Met-deficient hepatocellular carcinoma in vivo.

PHA665752 (PHA), a selective small molecule c-Met Inhibitor, potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes of a variety of tumor cells including hepatocellular carcinoma cells. However, these effects were impaired in c-Met-deficient cancer cells. In the present study, we investigated the potential anti-human c-Met-deficient hepatocellular carcinoma effects of Celastrol, a novel triterpene, and its combination with PHA. Human hepatocellular carcinoma cells BEL-7402 (c-Met-positive) and Huh7 (c-Met-deficient) were treated with different dose of PHA with or without equal dose of Celastrol, and cell growth, cell cycle and apoptosis were evaluated, respectively, by MTT assay, flow cytometry and Caspase3/7 activity. Nude mice bearing Huh7 xenografts were used to assess the in vivo anti-tumor activity. Our results showed that Celastrol at high concentration (>1.0 µM) induced G2/M arrest and apoptosis with the activation of Caspase3/7 in Huh7 cells whereas at low concentration (<1.0 µM) had no obvious effects. Low concentration Celastrol presented significant combined effects with PHA on Huh7 cells and Huh7 xenografts in terms of growth inhibition, migration inhibition and apoptosis induction. These results suggest that Celastrol and its combination with PHA present the therapeutic potential on c-Met-deficient hepatocellular carcinoma, and deserve further preclinical and clinical studies.

Activation of hepatocyte growth factor receptor, c-met, in renal tubules is required for renoprotection after acute kidney injury.

Hepatocyte growth factor is a pleiotrophic protein that promotes injury repair and regeneration in multiple organs. Here, we show that after acute kidney injury (AKI), the HGF receptor, c-met, was induced predominantly in renal tubular epithelium. To investigate the role of tubule-specific induction of c-met in AKI, we generated conditional knockout mice, in which the c-met gene was specifically disrupted in renal tubules. These Ksp-met-/- mice were phenotypically normal and had no appreciable defect in kidney morphology and function. However, in AKI induced by cisplatin or ischemia/reperfusion injury, the loss of tubular c-met substantially aggravated renal injury. Compared with controls, Ksp-met-/- mice displayed higher serum creatinine, more severe morphologic lesions, and increased apoptosis, which was accompanied by an increased expression of Bax and Fas ligand and decreased phosphorylation/activation of Akt. In addition, ablation of c-met in renal tubules promoted chemokine expression and renal inflammation after AKI. Consistently, ectopic expression of hepatocyte growth factor in vivo protected the kidneys against AKI in control mice, but not in Ksp-met-/- counterparts. Thus, our results suggest that tubule-specific c-met signaling is crucial in conferring renal protection after AKI, primarily by its anti-apoptotic and anti-inflammatory mechanisms.

Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

Lack of hepatic c-Met and gp130 expression is associated with an impaired antibacterial response and higher lethality after bile duct ligation.

The prognosis of liver failure is often determined by infectious and cholestatic complications. As HGF/c-Met and interleukin (IL)-6/gp130 control hepatic cytoprotective pathways, we here investigated their cooperative role during the onset of cholestatic liver injury. Conditional hepatocyte-specific ((Δhepa)) c-Met, gp130 and c-Met/gp130 knockout mice (Cre-loxP system) were subjected to bile duct ligation (BDL) and lipopolysaccharide (LPS) stimulation. gp130(Δhepa) and c-Met/gp130(Δhepa) mice displayed increased lethality associated with severe bacteraemia early after BDL, whereas c-Met(Δhepa) and wild-type mice showed normal survival. Analysis of the innate immune response and the regulation of hepatic antibacterial pathways showed that the LPS-triggered hepatocellular response via the Toll-like receptor-4 pathway was regulated differentially by HGF/c-Met and IL-6/gp130. Activation of p38MAPK, c-Jun N-terminal kinase and signalling transducer and activator of transcription-3 was impaired in gp130(Δ) and c-Met(Δhepa) livers. In addition, the acute-phase response (APR) was reduced in c-Met(Δhepa) livers, whereas gp130(Δhepa) displayed a completely abolished APR. In contrast, TNF-α-dependent NF-κB activation was enhanced in gp130(Δhepa) and c-Met(Δhepa) mice and it was associated with a higher rate of apoptosis and inflammation. Moreover, expression of the neutrophil produced and secreted cathelin-related antimicrobial peptide and of genes related to the inflammasome complex correlated with the strength of the bacterial infection and with TNF-α expression. In conclusion, Gp130 and c-Met are involved in the hepatic antibacterial and innate immune response, control the APR and thus prevent sepsis and liver injury during cholestatic conditions.

Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer.

At diagnosis, the majority of pancreatic cancer patients present with advanced disease when curative resection is no longer feasible and current therapeutic treatments are largely ineffective. An improved understanding of molecular targets for effective intervention of pancreatic cancer is thus urgent. The Met receptor tyrosine kinase is one candidate implicated in pancreatic cancer. Notably, Met is over expressed in up to 80% of invasive pancreatic cancers but not in normal ductal cells correlating with poor overall patient survival and increased recurrence rates following surgical resection. However the functional role of Met signaling in pancreatic cancer remains poorly understood. Here we used RNA interference to directly examine the pathobiological importance of increased Met signaling for pancreatic cancer. We show that Met knockdown in pancreatic tumor cells results in decreased cell survival, cell invasion, and migration on collagen I in vitro. Using an orthotopic model for pancreatic cancer, we provide in vivo evidence that Met knockdown reduced tumor burden correlating with decreased cell survival and tumor angiogenesis, with minimal effect on cell growth. Notably, we report that Met signaling regulates the secretion of the pro-angiogenic chemokine interleukin-8/CXCL8. Our data showing that the interleukin-8 receptors CXCR1 and CXCR2 are not expressed on pancreatic tumor cells, suggests a paracrine mechanism by which Met signaling regulates interleukin-8 secretion to remodel the tumor microenvironment, a novel finding that could have important clinical implications for improving the effectiveness of treatments for pancreatic cancer.

The HGF receptor/Met tyrosine kinase is a key regulator of dendritic cell migration in skin immunity.

The Met tyrosine kinase has a pivotal role in embryonic development and tissue regeneration, and deregulated Met signaling contributes to tumorigenesis. After binding of its cognate ligand hepatocyte growth factor, Met signaling confers mitogenic, morphogenic, and motogenic activity to various cells. Met expression in the hematopoietic compartment is limited to progenitor cells and their Ag-presenting progeny, including dendritic cells (DCs). In this study, we demonstrate that Met signaling in skin-resident DCs is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. By using a conditional Met-deficient mouse model (Met(flox/flox)), we show that Met acts on the initial step of DC release from skin tissue. Met-deficient DCs fail to reach skin-draining lymph nodes upon activation while exhibiting an activated phenotype. Contact hypersensitivity reactions in response to various contact allergens is strongly impaired in Met-deficient mice. Inhibition of Met signaling by single-dose epicutaneous administration of the Met kinase-specific inhibitor SU11274 also suppressed contact hypersensitivity in wild-type mice. Additionally, we found that Met signaling regulates matrix metalloproteinase MMP2 and MMP9 activity, which is important for DC migration through extracellular matrix. These data unveil Met signaling in DCs as a critical determinant for the maintenance of normal immune function and suggest Met as a potential target for treatment of autoimmune skin diseases.

Loss of c-Met accelerates development of liver fibrosis in response to CCl(4) exposure through deregulation of multiple molecular pathways.

HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. HGF treatment accelerates resolution of fibrosis in experimental animal models. Here, we utilized Met(fl/fl);Alb-Cre(+/-) conditional knockout mice and a carbon tetrachloride(CCl(4))-induced liver fibrosis model to formally address the role of c-Met signaling in hepatocytes in the context of chronic tissue injury. Histological changes during injury (4weeks) and healing phase (4weeks) were monitored by immunohistochemistry; expression levels of selected key fibrotic molecules were evaluated by western blotting, and time-dependent global transcriptomic changes were examined using a microarray platform. Loss of hepatocyte c-Met signaling altered hepatic microenvironment and aggravated hepatic fibrogenesis. Greater liver damage was associated with decreased hepatocyte proliferation, excessive stellate cell activation and rapid dystrophic calcification of necrotic areas. Global transcriptome analysis revealed a broad impact of c-Met on critical signaling pathways associated with fibrosis. Loss of hepatocyte c-Met caused a strong deregulation of chemotactic and inflammatory signaling (MCP-1, RANTES, Cxcl10) in addition to modulation of genes involved in reorganization of the cytoskeletal network (Actb, Tuba1a, Tuba8), intercellular communications and adhesion (Adam8, Icam1, Itgb2), control of cell proliferation (Ccng2, Csnk2a, Cdc6, cdk10), DNA damage and stress response (Rad9, Rad52, Ercc4, Gsta1 and 2, Jun). Our study demonstrates that deletion of c-Met receptor in hepatocytes results in pronounced changes in hepatic metabolism and microenvironment, and establishes an essential role for c-Met in maintaining the structural integrity and adaptive plasticity of the liver under adverse conditions.

EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells.

Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met(flx/flx) and Met(-/-) oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-ß)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met(flx/flx) cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met(flx/flx) and Met(-/-) oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in normal, untransformed oval cells, c-Met and EGFR represent critical molecular players to control proliferation and survival that function independent of one another.

Hepatocyte growth factor/c-met signaling is required for stem-cell-mediated liver regeneration in mice.

UNLABELLED: Hepatocyte growth factor (HGF)/c-Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c-Met in stem-cell-mediated liver regeneration by utilizing mice harboring c-met floxed alleles and Alb-Cre or Mx1-Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC). Deletion of c-met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence-activated cell-sorted epithelial cell adhesion molecule (EpCam)-positive cells. Loss of c-Met receptor decreased the sphere-forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval- (A6 and EpCam) and hepatocyte-specific (i.e. hepatocyte nuclear factor 4-alpha) antibodies. Furthermore, lack of c-Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal-cell-derived factor 1. Using a combination of double immunofluorescence of cell-type-specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC-treated livers. The Mx1-Cre-driven c-met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c-Met(fl/fl); Alb-Cre(+/-) mice. However, in both models, genetic loss of c-met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice. CONCLUSION: These results establish a direct contribution of c-Met in the regulation of HSC response and support a unique role for HGF/c-Met as an essential growth-factor-signaling pathway for regeneration of diseased liver.

Activation of c-MET induces a stem-like phenotype in human prostate cancer.

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway.Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 ('stem-like signature'). We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f.In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.

c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase.

The progression and negative outcome of a variety of human carcinomas are intimately associated with aberrant activity of the c-Met oncogene. The underlying cause of this dysregulation, however, remains a subject of discussion, as the majority of cancer patients do not present with activating mutations in c-Met receptor itself. In this study, we show that the oncogenic protease matriptase is ubiquitously co-expressed with the c-Met in human squamous cell carcinomas and amplifies migratory and proliferative responses of primary epithelial cells to the cognate ligand for c-Met, pro-hepatocyte growth factor/scatter factor (proHGF/SF), through c-Met and Gab1 signaling. Furthermore, the selective genetic ablation of c-Met from matriptase-expressing keratinocytes completely negates the oncogenic potential of matriptase. In addition, matriptase-dependent carcinoma formation could be blocked by the pharmacological inhibition of the Akt-mammalian target of Rapamycin (mTor) pathway. Our data identify matriptase as an initiator of c-Met-Akt-mTor-dependent signaling axis in tumors and reveal mTor activation as an essential component of matriptase/c-Met-induced carcinogenesis. The study provides a specific example of how epithelial transformation can be promoted by epigenetic acquisition of the capacity to convert a widely available paracrine growth factor precursor to its signaling competent state.

Evidence of cell-nonautonomous changes in dendrite and dendritic spine morphology in the met-signaling-deficient mouse forebrain.

Human genetic findings and murine neuroanatomical expression mapping have intersected to implicate Met receptor tyrosine kinase signaling in the development of forebrain circuits controlling social and emotional behaviors that are atypical in autism-spectrum disorders (ASD). To clarify roles for Met signaling during forebrain circuit development in vivo, we generated mutant mice (Emx1(Cre)/Met(fx/fx)) with an Emx1-Cre-driven deletion of signaling-competent Met in dorsal pallially derived forebrain neurons. Morphometric analyses of Lucifer yellow-injected pyramidal neurons in postnatal day 40 anterior cingulate cortex (ACC) revealed no statistically significant changes in total dendritic length but a selective reduction in apical arbor length distal to the soma in Emx1(Cre)/Met(fx/fx) neurons relative to wild type, consistent with a decrease in the total tissue volume sampled by individual arbors in the cortex. The effects on dendritic structure appear to be circuit-selective, insofar as basal arbor length was increased in Emx1(Cre)/Met(fx/fx) layer 2/3 neurons. Spine number was not altered on the Emx1(Cre)/Met(fx/fx) pyramidal cell populations studied, but spine head volume was significantly increased (∼20%). Cell-nonautonomous, circuit-level influences of Met signaling on dendritic development were confirmed by studies of medium spiny neurons (MSN), which do not express Met but receive Met-expressing corticostriatal afferents during development. Emx1(Cre)/Met(fx/fx) MSN exhibited robust increases in total arbor length (∼20%). As in the neocortex, average spine head volume was also increased (∼12%). These data demonstrate that a developmental loss of presynaptic Met receptor signaling can affect postsynaptic morphogenesis and suggest a mechanism whereby attenuated Met signaling could disrupt both local and long-range connectivity within circuits relevant to ASD.