Therapeutic Strategies for Ovarian Cancer in Point of HGF/c-MET Targeting.

Ovarian cancer is the fifth leading cause of cancer deaths in women and is regarded as one of the most difficult cancers to treat. Currently, studies are being conducted to develop therapeutic agents for effective treatment of ovarian cancer. In this review, we explain the properties of the hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-MET) and how the signaling pathway of HGF/c-MET is activated in different cancers and involved in tumorigenesis and metastasis of ovarian cancer. We present the findings of clinical studies using small chemicals or antibodies targeting HGF/c-MET signaling in various cancer types, particularly in ovarian cancer. We also discuss that HGF/c-MET-targeted therapy, when combined with chemo drugs, could be an effective strategy for ovarian cancer therapeutics.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

Local administration of anti-hepatocyte growth factor-neutralizing antibody reverts naturally occurring periodontitis.

OBJECTIVES: Periodontitis is a chronic inflammatory process associated with the loss of tooth-supporting tissue. The imbalance of epithelial-mesenchymal signaling is considered to drive disease progression, and hepatocyte growth factor (HGF) is one of the main mediators of this interaction. The aim of this study was to validate the role of HGF in the pathogenesis of periodontitis and to evaluate the effects of anti-HGF neutralizing antibodies. METHODS: Gingival tissues from cynomolgus monkeys, which naturally develop severe periodontitis, were isolated to establish an in vitro periodontitis model. Periodontitis-affected monkeys were treated by gingival injection of anti-HGF neutralizing antibodies. The therapeutic effects were documented by clinical examination (probing depth and bleeding on probing), histological examination of tissue, and reevaluation of gingival fibroblasts in the in vitro model. RESULTS: Periodontitis-affected monkeys contain periodontitis-associated fibroblasts (PAFs) with a pro-inflammatory phenotype that induced pronounced collagen degradation in vitro. This degradation was effectively inhibited by anti-HGF-neutralizing antibodies. Locally administered anti-HGF antibody to monkey gingiva clinically improved the severity of periodontitis. This was also reflected in the tissue histology with lower inflammatory cell infiltrates in treated gingiva than in non-treated gingiva. Moreover, fibroblasts isolated from anti-HGF-treated gingiva demonstrated reduced collagen degradation capacity. CONCLUSIONS: Our study confirmed the central role of HGF in the pathogenesis of severe periodontitis in relevant in vitro and in vivo models. The positive effect of anti-HGF treatment provides a strong rationale for the use of anti-HGF-neutralizing antibodies for the treatment of human periodontitis.

MiR-706 alleviates white matter injury via downregulating PKCα/MST1/NF-κB pathway after subarachnoid hemorrhage in mice.

Increasing numbers of patients with spontaneous subarachnoid hemorrhage(SAH) who recover from surgery and intensive care management still live with cognitive impairment after discharge, indicating the importance of white matter injury at the acute stage of SAH. In the present study, standard endovascular perforation was employed to establish an SAH mouse model, and a microRNA (miRNA) chip was used to analyze the changes in gene expression in white matter tissue after SAH. The data indicate that 17 miRNAs were downregulated, including miR-706, miR-669a-5p, miR-669p-5p, miR-7116-5p and miR-195a-3p, while 13 miRNAs were upregulated, including miR-6907-5p, miR-5135, miR-6982-5p, miR-668-5p, miR-8119. Strikingly, miR-706 was significantly downregulated with the highest fold change. Further experiments confirmed that miR-706 could alleviate white matter injury and improve neurological behavior, at least partially by inhibiting the PKCα/MST1/NF-κB pathway and the release of inflammatory cytokines. These results might provide a deeper understanding of the pathophysiological processes in white matter after SAH, as well as potential therapeutic strategies for the translational research.

A receptor-antibody hybrid hampering MET-driven metastatic spread.

BACKGROUND: The receptor encoded by the MET oncogene and its ligand Hepatocyte Growth Factor (HGF) are at the core of the invasive-metastatic behavior. In a number of instances genetic alterations result in ligand-independent onset of malignancy (MET addiction). More frequently, ligand stimulation of wild-type MET contributes to progression toward metastasis (MET expedience). Thus, while MET inhibitors alone are effective in the first case, combination therapy with ligand inhibitors is required in the second condition. METHODS: In this paper, we generated hybrid molecules gathering HGF and MET inhibitory properties. This has been achieved by 'head-to-tail' or 'tail-to-head' fusion of a single chain Fab derived from the DN30 MET antibody with a recombinant 'ad-hoc' engineered MET extracellular domain (decoyMET), encompassing the HGF binding site but lacking the DN30 epitope. RESULTS: The hybrid molecules correctly bind MET and HGF, inhibit HGF-induced MET downstream signaling, and quench HGF-driven biological responses, such as growth, motility and invasion, in cancer cells of different origin. Two metastatic models were generated in mice knocked-in by the human HGF gene: (i) orthotopic transplantation of pancreatic cancer cells; (ii) subcutaneous injection of primary cells derived from a cancer of unknown primary. Treatment with hybrid molecules strongly affects time of onset, number, and size of metastatic lesions. CONCLUSION: These results provide a strategy to treat metastatic dissemination driven by the HGF/MET axis.

YYB-101, a Humanized Antihepatocyte Growth Factor Monoclonal Antibody, Inhibits Ovarian Cancer Cell Motility and Proliferation.

BACKGROUND/AIM: Hepatocyte growth factor (HGF) acts as a key regulator in promoting ovarian cancer metastasis. Previously, we observed that YYB-101, a humanized anti-HGF antibody, effectively inhibits ovarian cancer cell migration, invasion, and progression. Here, we evaluated the signaling mechanisms affected by YYB-101 that are important in ovarian cancer cell progression. MATERIALS AND METHODS: Using cell migration, invasion and proliferation assays, we evaluated the effects of YYB-101 on A2780/luc and SKOV3 cells. The effects of YYB-101 on signaling molecules were determined by immunocytochemistry and immunoblot analysis. RESULTS: YYB-101 inhibited HGF-induced ovarian cancer cell motility by down-regulating paxillin phosphorylation and actin-cytoskeleton rearrangement. Also, YYB-101 inhibited ovarian cancer cell proliferation by reducing c-MET phosphorylation and activating apoptosis in vitro and in vivo. These effects were significantly enhanced by combining YYB-101 treatment with paclitaxel, a standard chemotherapy drug. CONCLUSION: YYB-101 can be examined as a new therapeutic agent for the treatment of patients with ovarian cancer.

First-in-Human Phase I Study of MP0250, a First-in-Class DARPin Drug Candidate Targeting VEGF and HGF, in Patients With Advanced Solid Tumors.

PURPOSE: A first-in-human study was performed with MP0250, a DARPin drug candidate. MP0250 specifically inhibits both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) with the aim of disrupting the tumor microenvironment. PATIENTS AND METHODS: A multicenter, open-label, repeated-dose, phase I study was conducted to assess the safety, tolerability, and pharmacokinetics of MP0250 in 45 patients with advanced solid tumors. In the dose-escalation part, 24 patients received MP0250 as a 3-hour infusion once every 2 weeks at five different dose levels (0.5-12 mg/kg). Once the maximum tolerated dose (MTD) was established, 21 patients were treated with a 1-hour infusion (n = 13, 8 mg/kg, once every 2 weeks and n = 8, 12 mg/kg, once every 3 weeks) of MP0250 in the dose confirmation cohorts. RESULTS: In the dose-escalation cohort, patients treated with 12 mg/kg MP0250 once every 2 weeks experienced dose-limiting toxicities. Therefore, MTD was 8 mg/kg once every 2 weeks or 12 mg/kg once every 3 weeks. The most common adverse events (AEs) were hypertension (69%), proteinuria (51%), and diarrhea and nausea (both 36%); hypoalbuminemia was reported in 24% of patients. Most AEs were consistent with inhibition of the VEGF and HGF pathways. Exposure was dose-proportional and sustained throughout the dosing period for all patients (up to 15 months). The half-life was about 2 weeks. Signs of single-agent antitumor activity were observed: 1 unconfirmed partial response with a time to progression of 23 weeks and 24 patients with stable disease, with the longest duration of 72 weeks and a median duration of 18 weeks. CONCLUSION: MP0250 is a first-in-class DARPin drug candidate with suitable tolerability and appropriate pharmacokinetic properties for further development in combination with other anticancer therapies.

Roles of the HGF/Met signaling in head and neck squamous cell carcinoma: Focus on tumor immunity (Review).

c­mesenchymal­epithelial transition (Met) is a transmembrane tyrosine kinase receptor of hepatocyte growth factor (HGF). HGF/Met signaling stimulates numerous pathways, including the Ras/mitogenactivated protein kinase (MAPK), phosphatidylinositol 3­kinase/protein kinase B and Wnt/ß­catenin pathways, which serve important roles in cell proliferation, survival, motility, invasion and angiogenesis, and promotes the development and progression of tumors. Aberrant HGF/Met signaling is associated with a poor prognosis in several types of tumors, including head and neck squamous cell carcinoma (HNSCC). Although, the HGF/MET pathway and HGF and/or Met inhibitors have been extensively reviewed, their role in tumor immunity remains elusive. The present review article summarizes the findings on the HGF/Met signaling in HNSCC, including gene and protein alterations, biological functions and patient outcomes. Furthermore, the role of HGF/Met in tumor immunity is discussed and the controversial association between the expression of HGF/Met and the prognosis of patients with HNSCC from the perspective of tumor immunity is clarified. Ultimately, the present review proposes a clinical approach that may improve the efficacy of Met therapy for HNSCC, namely the intratumoral administration of Met inhibitors in order to reduce the inhibitory effect on immune cell recruitment. However, further studies are required to provide an improved understanding of the effects of the HGF/Met pathway on the tumor microenvironment, and the effects of HGF and Met inhibitors on immune cells in the tumor environment should be the focus of future studies.

Cyclic Peptide-Based Biologics Regulating HGF-MET.

Using a random non-standard peptide integrated discovery system, we obtained cyclic peptides that bind to hepatocyte growth factor (HGF) or mesenchymal-epithelial transition factor. (MET) HGF-inhibitory peptide-8 (HiP-8) selectively bound to two-chain active HGF, but not to single-chain precursor HGF. HGF showed a dynamic change in its molecular shape in atomic force microscopy, but HiP-8 inhibited dynamic change in the molecular shape into a static status. The inhibition of the molecular dynamics of HGF by HiP-8 was associated with the loss of the ability to bind MET. HiP-8 could selectively detect active HGF in cancer tissues, and active HGF probed by HiP-8 showed co-localization with activated MET. Using HiP-8, cancer tissues with active HGF could be detected by positron emission tomography. HiP-8 seems to be applicable for the diagnosis and treatment of cancers. In contrast, based on the receptor dimerization as an essential process for activation, the cross-linking of the cyclic peptides that bind to the extracellular region of MET successfully generated an artificial ligand to MET. The synthetic MET agonists activated MET and exhibited biological activities which were indistinguishable from the effects of HGF. MET agonists composed of cyclic peptides can be manufactured by chemical synthesis but not recombinant protein expression, and thus are expected to be new biologics that are applicable to therapeutics and regenerative medicine.

RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation.

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.

Tivantinib Decreases Hepatocyte Growth Factor-Induced BCRP Expression in Hepatocellular Carcinoma HepG2 Cells.

Tivantinib, a mesenchymal-epithelial transition factor (cMET) inhibitor, is a molecular targeting drug that kills hepatocellular carcinoma (HCC) cells. Tivantinib alone does not affect the overall survival of patients with HCC, and combination treatment with tivantinib and other therapies has not been evaluated. This study was conducted to clarify the effect of the tivantinib in regulating breast cancer therapy-resistant protein (BCRP), a key transporter of 5-fluorouracil (5-FU), and dihydropyridine dehydrogenase (DPYD), a major metabolic enzyme of 5-FU. To this end, cMET gene expression was determined by RT-PCR in HepG2 (human hepatoma) cells. The transcriptional start sites of BCRP were determined by 5'-rapid amplification of cDNA ends (5'-RACE). BCRP and DPYD mRNA levels were determined by real-time RT-PCR, and promoter activities were measured by dual-luciferase assays. Results show that hepatocyte growth factor (HGF) upregulated the mRNA level of BCRP, but not DPYD, in HepG2 cells. The upregulation of BCRP expression by HGF was down-regulated by tivantinib. We also identified two transcriptional start sites (E1α, E1ß) in BCRP by 5'-RACE. The transcriptional activity of the region -287 to E1α of BCRP was upregulated by HGF, which was decreased by tivantinib, whereas activity of the region -297 to E1ßo f BCRP was not affected by tivantinib. Therefore, tivantinib regulates BCRP expression upstream of exon 1α. Combination treatment of tivantinib and 5-FU should be further evaluated for HCC therapy.

Bromodomain and Extraterminal (BET) protein inhibition suppresses tumor progression and inhibits HGF-MET signaling through targeting cancer-associated fibroblasts in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality. The bromodomain and extra-terminal domain (BET) inhibitors suppresses the gene expressions of various oncogenes and shows a good efficacy in the preclinical CRC models. We investigate the mechanism of action of BET inhibitors in CRC. METHODS: The effect of BET inhibitor (JQ1) on the HGF-MET signaling was assessed by qPCR, western blot and immunohistochemical staining in CRC and cancer-associated fibroblasts (CAFs). The effect of JQ1 on the CAFs was investigated using the primary CAFs derived from CRC tissues and induced-CAFs derived from isolating foreskin fibroblasts. The effect of JQ1 on the gene expression profile of CAFs was explored by RNA-sequence, qPCR and bioinformatic analysis. RESULTS: JQ1 decreased the mRNA and protein levels of MET in CRC cells and downregulated the mRNA and protein levels of HGF in both CRC cells and CAFs. JQ1 attenuated the pro-migratory activity of CAFs through downregulation of HGF expression in CAFs. Meanwhile, JQ1 also reduced the ability of contracting collagen gels, decreased the cell proliferation, induced G1 arrest and repressed the pro-inflammatory gene expressions in CAFs. MYC expression was suppressed by JQ1 in CAFs. Knockdown of MYC induced G1 arrest in CAFs. CONCLUSION: Our results demonstrate the inhibitory effect of BET inhibition on the HGF-MET signaling and the pro-tumor activity of CAFs, revealing a new mechanism by which BET inhibition suppresses CRC progression.

YAP Mediates Hair Cell Regeneration in Balance Organs of Chickens, But LATS Kinases Suppress Its Activity in Mice.

Loss of sensory hair cells causes permanent hearing and balance deficits in humans and other mammals, but for nonmammals such deficits are temporary. Nonmammals recover hearing and balance sensitivity after supporting cells proliferate and differentiate into replacement hair cells. Evidence of mechanical differences between those sensory epithelia and their supporting cells prompted us to investigate whether the capacity to activate YAP, an effector in the mechanosensitive Hippo pathway, correlates with regenerative capacity in acceleration-sensing utricles of chickens and mice of both sexes. After hair cell ablation, YAP accumulated in supporting cell nuclei in chicken utricles and promoted regenerative proliferation, but YAP remained cytoplasmic and little proliferation occurred in mouse utricles. YAP localization in supporting cells was also more sensitive to shape change and inhibition of MST1/2 in chicken utricles than in mouse utricles. Genetic manipulations showed that in vivo expression of the YAP-S127A variant caused robust proliferation of neonatal mouse supporting cells, which produced progeny that expressed hair cell markers, but proliferative responses declined postnatally. Expression of YAP-5SA, which more effectively evades inhibitory phosphorylation, resulted in TEAD-dependent proliferation of striolar supporting cells, even in adult utricles. Conditional deletion of LATS1/2 kinases abolished the inhibitory phosphorylation of endogenous YAP and led to striolar proliferation in adult mouse utricles. The findings suggest that damage overcomes inhibitory Hippo signaling and facilitates regenerative proliferation in nonmammalian utricles, whereas constitutive LATS1/2 kinase activity suppresses YAP-TEAD signaling in mammalian utricles and contributes to maintaining the proliferative quiescence that appears to underlie the permanence of sensory deficits.SIGNIFICANCE STATEMENT Loud sounds, ototoxic drugs, infections, and aging kill sensory hair cells in the ear, causing irreversible hearing loss and balance deficits for millions. In nonmammals, damage evokes shape changes in supporting cells, which can divide and regenerate hair cells. Such shape changes are limited in mammalian ears, where supporting cells develop E-cadherin-rich apical junctions reinforced by robust F-actin bands, and the cells fail to divide. Here, we find that damage readily activates YAP in supporting cells within balance epithelia of chickens, but not mice. Deleting LATS kinases or expressing YAP variants that evade LATS-mediated inhibitory phosphorylation induces proliferation in supporting cells of adult mice. YAP signaling eventually may be harnessed to overcome proliferative quiescence that limits regeneration in mammalian ears.

Resveratrol Suppresses Prostate Cancer Epithelial Cell Scatter/Invasion by Targeting Inhibition of Hepatocyte Growth Factor (HGF) Secretion by Prostate Stromal Cells and Upregulation of E-cadherin by Prostate Cancer Epithelial Cells.

Cancer mortality is primarily attributed to metastasis and the resulting compromise of organs secondary to the initial tumor site. Metastasis is a multi-step process in which the tumor cells must first acquire a migratory phenotype and invade through the surrounding tissue for spread to distant organs in the body. The ability of malignant cells to migrate and breach surrounding tissue/matrix barriers is among the most daunting challenges to disease management for men in the United States diagnosed with prostate cancer (CaP), especially since, at diagnosis, a high proportion of patients already have occult or clinically-detectable metastasis. The interaction between hepatocyte growth factor (HGF) secreted by the stroma, with its receptor c-Met located in the epithelium, must occur for epithelial CaP cells to become migratory. We studied the effects of grape-derived phytochemical resveratrol on the transition of epithelial tumor cells from sedentary to a mobile, penetrant phenotype. A time lapse microscopy assay was used to monitor the acquisition of the migratory phenotype by resveratrol. The results show that resveratrol inhibits HGF-mediated interaction between the stroma and epithelium and suppresses epithelial CaP cell migration by attenuating the control of epithelial-to-mesenchymal transition (EMT).

Targeting the HGF/c-MET pathway in advanced pancreatic cancer: a key element of treatment that limits primary tumour growth and eliminates metastasis.

BACKGROUND: Stromal-tumour interactions facilitate pancreatic cancer (PC) progression. The hepatocyte growth factor (HGF)/c-MET pathway is upregulated in PC and mediates the interaction between cancer cells and stromal pancreatic stellate cells (PSCs). This study assessed the effect of HGF/c-MET inhibition plus gemcitabine (G) on the progression of advanced PC. METHODS: Orthotopic PC was produced by implantation of luciferase-tagged human cancer cells + human PSCs into mouse pancreas. Tumours were allowed to develop without treatment for 4 weeks. Mice were then treated for 6 weeks with one of the following: IgG, G, HGF inhibitor (Hi), c-MET inhibitor (Ci), Hi + Ci, Hi + G, Ci + G, or Hi + Ci + G. RESULTS: Bioluminescence imaging showed similar tumour sizes in all mice at the initiation of treatments. Triple therapy (Hi + Ci + G): (1) completely eliminated metastasis; (2) significantly reduced tumour size as assessed by bioluminescence and at necropsy; (3) significantly reduced proliferating cancer cell density and stem cell marker DCLK1 expression in tumours. In vitro 3D culture studies supported our in vivo findings. CONCLUSION: Even at an advanced disease stage, a two-pronged approach, targeting (a) HGF/c-MET with relevant inhibitors and (b) cancer cells with chemotherapy, completely eliminated metastasis and significantly decreased tumour growth, suggesting that this is a promising treatment approach for PC.

NURR1 inhibition reduces hypoxia-mediated cardiomyocyte necrosis via blocking Mst1-JNK-mPTP pathway.

Context: Although many studies have investigated the molecular mechanisms underlying hypoxia-related cardiomyocyte damage, the role of necrosis in cardiomyocyte death.Objective: The aim of our study is to explore the pathological role of nuclear receptor related 1 protein (NURR1) in regulating cardiomyocyte viability under hypoxia stress.Materials and methods: Cardiomyocyte was treated with hypoxia and siRNA against NURR1 was transfected into cardiomyocyte. Pathway agonist was used to activate the Mst1-JNK-mPTP pathway in cardiomyocyte.Results: In our study, the expression of NURR1 was rapidly increased in cardiomyocyte transfected with NURR1. Knockout of NURR1 could promote cardiomyocyte survival, reduce cell death and repress inflammation response. Mechanistically, NURR1 upregulation was associated with an activation of Mst1-JNK pathway and the latter promoted the mPTP opening in cardiomyocyte. Excessive mPTP opening was followed by cardiomyocyte necrosis and this effect could be reversed by NURR1 deletion. Besides, re-activation of Mst1-JNK pathway could abolish the protective effects of NURR1 deletion on cardiomyocytes, as evidenced by increased cell survival and decreased necrosis. Besides, re-activation of Mst1-JNK pathway also abolished NURR1 deletion-mediated mPTP opening.Conclusions: Hypoxia-mediated cardiomyocyte death is associated with NURR1 upregulation which contributes to the activation Mst1-JNK-mPTP pathways.

Inhibitory effect of melatonin on Mst1 ameliorates myocarditis through attenuating ER stress and mitochondrial dysfunction.

Viral myocarditis has been found to be one of the leading causes of sudden death in young adults. However, no effective drugs have been developed to intervene the progression of myocarditis. Accordingly, the present study is carried out to explore the protective role played by melatonin in the setting of viral myocarditis with a focus on Mst1-Hippo pathway, mitochondrial dysfunction and ER stress. Cardiac function was determined via echocardiographic examination. Mitochondrial function and ER stress were detected via ELISA, western blots, and immunofluorescence. Our data demonstrated that virus injection induced cardiac dysfunction as evidenced by reduced contractile function in myocardium. Besides, LDH release assay and western blotting analysis demonstrated that cardiomyocyte death was activated by virus injection. Interestingly, melatonin treatment improved cardiac function and repressed virus-mediated cardiomyocyte apoptosis. At the molecular levels, mitochondrial dysfunction was induced by virus infection, as indicated by mitochondrial membrane potential reduction, mPTP opening rate elevation and caspase-9-related apoptosis activation. Besides, ER stress parameters were also elevated in virus-treated cardiomyocytes. Interestingly, melatonin treatment maintained mitochondrial dysfunction and repressed ER stress. To the end, we found that Mst1 was upregulated by virus infection; this effect was attenuated through supplementation with melatonin. However, Mst1 overexpression reduced the beneficial impact exerted by melatonin on cardiomyocyte viability, mitochondrial function and ER homeostasis. Our study illustrated that melatonin treatment attenuated viral myocarditis via sustaining cardiomyocyte viability, repressing mitochondrial dysfunction and inhibiting ER stress in a manner dependent on Mst1 inhibition.

Bone Metastasis Phenotype and Growth Undergo Regulation by Micro-Environment Stimuli: Efficacy of Early Therapy with HGF or TGFß1-Type I Receptor Blockade.

Hepatocyte growth factor (HGF) and transforming growth factor ß1 (TGFß1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFß1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFß1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFß1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGFß1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFß-activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy; a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGFß1-RI blockade.

M2 macrophages mediate sorafenib resistance by secreting HGF in a feed-forward manner in hepatocellular carcinoma.

BACKGROUND: Sorafenib is the only approved first line systemic therapy for advanced hepatocellular carcinoma (HCC) in the last decade. Tumour resistance to sorafenib has been of major obstacles to improve HCC patient survival. METHODS: We polarised THP-1 cells to M1 and M2 macrophages, performed various in vitro assays and developed sorafenib-resistant xenograft models to investigate the role of tumour-associated macrophages (TAM)-secreted molecules in HCC resistance to the targeted therapy. RESULTS: We demonstrated M2, but not M1, macrophages not only promote proliferation, colony formation and migration of hepatoma cells but also significantly confer tumour resistance to sorafenib via sustaining tumour growth and metastasis by secreting hepatocyte growth factor (HGF). HGF activates HGF/c-Met, ERK1/2/MAPK and PI3K/AKT pathways in tumour cells. Tumour-associated M2 macrophages were accumulated in sorafenib-resistance tumours more than in sorafenib-sensitive tumours in vivo and produced abundant HGF. HGF chemoattracts more macrophages migrated from surrounding area, regulates the distribution of M2 macrophages and increases hepatoma resistance to sorafenib in a feed-forward manner. CONCLUSIONS: Our results provide new insights into the mechanisms of sorafenib resistance in HCC and rationale for developing new trials by combining sorafenib with a potent HGF inhibitor such as cabozantinib to improve the first line systemic therapeutic efficacy.