Exogenous Hepatocyte Growth Factor Research Articles

Activation of Hepatocyte Growth Factor/MET Signaling as a Mechanism of Acquired Resistance to a Novel YAP1/TEAD Small Molecule Inhibitor.

Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of cases are considered YAP1/TEAD dependent. Identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs, which may be necessary for YAP1 protein interactions, has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TEAD complex formation and transcriptional activity. We report the discovery and characterization of a novel YAP1/TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TEAD activity. In vivo, MRK-A showed a favorable tolerability profile in mice and demonstrated pharmacokinetics suitable for twice daily oral dosing in preclinical efficacy studies. Importantly, monotherapeutic targeting of YAP1/TEAD in preclinical models generated regressions in a mesothelioma CDX model; however, rapid resistance to therapy was observed. RNA-sequencing of resistant tumors revealed mRNA expression changes correlated with the resistance state and a marked increase of hepatocyte growth factor (HGF) expression. In vitro, exogenous HGF was able to fully rescue cytostasis induced by MRK-A in mesothelioma cell lines. In addition, co-administration of small molecule inhibitors of the MET receptor tyrosine kinase suppressed the resistance generating effect of HGF on MRK-A induced growth inhibition. In this work, we report the structure and characterization of MRK-A, demonstrating potent and specific inhibition of YAP1/TAZ-TEAD-mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling, including factors resulting in acquired drug resistance.

Remote limb ischemic postconditioning inhibits microglia pyroptosis by modulating HGF after acute ischemia stroke.

The repetitive inflation-deflation of a blood pressure cuff on a limb is known as remote limb ischemic postconditioning (RIPostC). It prevents brain damage induced by acute ischemia stroke (AIS). Pyroptosis, executed by the pore-forming protein gasdermin D (GSDMD), is a type of regulated cell death triggered by proinflammatory signals. It contributes to the pathogenesis of ischemic brain injury. However, the effects of RIPostC on pyroptosis following AIS remain largely unknown. In our study, linear correlation analysis confirmed that serum GSDMD levels in AIS patients upon admission were positively correlated with NIHSS scores. RIPostC treatment significantly reduced GSDMD level compared with patients without RIPostC at 3 days post-treatment. Besides, middle cerebral artery occlusion (MCAO) surgery was performed on C57BL/6 male mice and RIPostC was induced immediately after MCAO. We found that RIPostC suppressed the activation of NLRP3 inflammasome to reduce the maturation of GSDMD, leading to decreased pyroptosis in microglia after AIS. Hepatocyte growth factor (HGF) was identified using the high throughput screening. Importantly, HGF siRNA, exogenous HGF, and ISG15 siRNA were used to reveal that HGF/ISG15 is a possible mechanism of RIPostC regulation in vivo and in vitro.

Function of hepatocyte spheroids in bioactive microcapsules is enhanced by endogenous and exogenous hepatocyte growth factor

The ability to maintain functional hepatocytes has important implications for bioartificial liver development, cell-based therapies, drug screening, and tissue engineering. Several approaches can be used to restore hepatocyte function in vitro, including coating a culture substrate with extracellular matrix (ECM), encapsulating cells within biomimetic gels (Collagen- or Matrigel-based), or co-cultivation with other cells. This paper describes the use of bioactive heparin-based core-shell microcapsules to form and cultivate hepatocyte spheroids. These microcapsules are comprised of an aqueous core that facilitates hepatocyte aggregation into spheroids and a heparin hydrogel shell that binds and releases growth factors. We demonstrate that bioactive microcapsules retain and release endogenous signals thus enhancing the function of encapsulated hepatocytes. We also demonstrate that hepatic function may be further enhanced by loading exogenous hepatocyte growth factor (HGF) into microcapsules and inhibiting transforming growth factor (TGF)-β1 signaling. Overall, bioactive microcapsules described here represent a promising new strategy for the encapsulation and maintenance of primary hepatocytes and will be beneficial for liver tissue engineering, regenerative medicine, and drug testing applications.

Abstract 552: Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC

Abstract Despite inducing strong and durable remissions, inhibitors of mutant ALK (ALKi) are not curative for advanced ALK+ lung cancers, as residual tumors eventually develop resistance and relapse. Apart from tumor cells’ intrinsic ability to escape from therapy, there is a growing body of evidence suggesting the contribution of extrinsic factors, produced by cancer-associated fibroblasts (CAFs). Despite the multitude of studies that demonstrated the ability of multiple CAF-produced factors to reduce the sensitivity of tumor cells to ALKi, the contribution of these effects toward responses in vivo remains unresolved. To study the impact of stroma on the ability of tumor cells to survive and develop resistance to ALKi, we derived multiple isolates of fibroblasts from clinical samples. Consistent with previous reports, we found that co-culture with CAFs or CAF-conditioned media protects tumor cells against ALKi. We observed that the degree of protection varies between different CAF isolates. This variability in the extent of protection could be attributed to variability in the levels of secreted hepatocyte growth factor (HGF) a known paracrine mediator of environmental resistance. Moreover, exogenous HGF phenocopied the effect of CAFs while blocking HGF-cMET signaling with neutralizing antibodies or pharmacologically abrogated the protective effect. To test the relevance of these findings in vivo, we took advantage of the inability of murine HGF to activate human cMET by comparing the response of ALK+ xenograft tumors to front-line ALKi alectinib between NSG mice and NSG-derivative strain with humanized HGF. In contrast to the in vitro data, HGF status had only a minimal impact on the remission-relapse dynamics of xenograft tumors. This lack of differences reflected a strong HGF-independent sheltering effect of the stromal niche. Our histological analyses of samples at different points of remission-relapse response revealed that while alectinib potently suppressed tumor cell proliferation, proximity to stroma reduced this cytostatic effect, without impacting cell proliferation in the absence of therapy. To gain insights into the mechanistic underpinning of this HGF-independent effect, we used spatial transcriptomics, comparing stroma-proximal and stroma-distant tumor regions. These analyses, as well as functional validation studies, indicate that stroma-sheltering effects are mediated by multiple mechanisms, acting in an additive fashion. In summary, our studies indicate that therapy resistance of tumor cells reflects the combined action of both intrinsic and microenvironmental factors. Our findings indicate that focusing on a single resistance mechanism at a time is unlikely to induce strong, durable responses. Instead, tackling the issue of therapy resistance necessitates the consideration of multiple resistance mechanisms as well as the moving target nature of tumors under therapy. Citation Format: Bina Desai, Tatiana Miti, Daria Miroshnychenko, Viktoriya Marusyk, Chandler Gatenbee, Menkara Henry, Uwe Rix, Alexander Anderson, Eric Haura, David Basanta, Andriy Marusyk. Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 552.

Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition

The hepatocyte growth factor (HGF)/MET signaling pathway has been proposed to be involved in the resistance to radiotherapy of glioblastoma via proinvasive and DNA damage response pathways.Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models. We find that the murine glioma cell lines GL-261, SMA-497, SMA-540 and SMA-560 express HGF and its receptor MET and respond to exogenous HGF with MET phosphorylation. Glioma cell viability or proliferation are unaffected by genetic or pharmacological MET inhibition using tepotinib or CRISPR/Cas9-engineered Met gene knockout and MET inhibition fails to sensitize glioma cells to irradiation in vitro. In contrast, the combination of tepotinib with radiotherapy prolongs survival of orthotopic SMA-560 or GL-261 glioma-bearing mice compared with radiotherapy or tepotinib treatment alone. Synergy is lost when such experiments are conducted in immunodeficient Rag1−/− mice, and, importantly, also when Met gene expression is disrupted in the tumor cells. Combination therapy suppresses a set of pro-inflammatory mediators including matrix metalloproteases that are upregulated by radiotherapy alone and that have been linked to poor outcome in glioblastoma. Several of these mediators are positively regulated by transforming growth factor (TGF)-β, and pSMAD2 levels as a surrogate marker of TGF-β pathway activity are suppressed by combination treatment. We conclude that synergistic suppression of experimental syngeneic glioma growth by irradiation and MET inhibition requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined strategy in newly diagnosed glioblastoma is warranted.

Abstract P6-12-01: ALCAM (Activated Leukocyte Cell Adhesion Molecule) is associated with HGF/MET signalling pathway in different subtypes of breast cancer

Abstract Introduction. ALCAM (Activated Leukocyte Cell Adhesion Molecule), also known as CD166, is a cell adhesion molecule which belongs to the immunoglobulin superfamily and is widely expressed in various human tissues. It has been demonstrated that ALCAM plays an important role in the progression of malignant diseases and tumour metastasis in multiple cancer types including breast cancer. However, the molecular mechanism of ALCAM and cancer progression is currently unclear. The present study performed protein array analyses using ALCAM genetically manipulated cell models to select potential protein partners of ALCAM. The study focused on MET (Hepatocyte Growth Factor (HGF) Receptor), a prominent ALCAM interacting protein kinase and a protooncogene contributing to cancer progression and spread. Method. Human breast cancer cell lines MCF-7 and MDA-MB-231 were selected to create ALCAM knockdown cell models. A range of other breast cancer cell line with differing hormone receptor status were also used. Protein samples of transfected cells were used to perform Kinexus protein kinase microarray analysis. The protein interaction between ALCAM and other prospective protein kinases including MET was verified by the method of immunoprecipitation. Additionally, the ALCAM knockdown model was also assessed for the impact of ALCAM and HGF/MET on the biological functions by Electric Cell-substrate Impedance Sensing (ECIS). Results. MCF-7 and MDA MB-231 cells both were strongly expressed ALCAM. Cells models with ALCAM knocking down were successfully created by way of anti-ALCAM shRNA. We have shown on the protein kinase microarray analysis that the hepatocyte growth factor (HGF) receptor, MET was one of the kinases significantly affected following ALCAM knocking down. It was also found that the alteration of protein interaction between the MET protein kinase and ALCAM protein showed an opposite pattern between ER positive and ER negative ALCAM knocking down cells. This protein interaction was observed by immunoprecipitation in MDA-MB-361 and MDA-MB-231 cell lines, but not in MCF-7 cells. The biological analyses using the ECIS technologies showed that MET kinase inhibitors and exogenous recombinant HGF affected the ALCAM-mediated cell adhesion in different breast cancer subtypes. Conclusion. ALCAM is associated with HGF/MET signalling and the interaction between ALCAM and MET is different amongst subtypes of breast cancer which have different hormone receptor status. Citation Format: Yiming Yang, Andrew J. Sanders, Wen G. Jiang. ALCAM (Activated Leukocyte Cell Adhesion Molecule) is associated with HGF/MET signalling pathway in different subtypes of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-01.

Loss of MEN1 leads to renal fibrosis and decreases HGF-Adamts5 pathway activity via an epigenetic mechanism.

BackgroundRenal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear.MethodsKidney histology was examined on paraffin sections stained with hematoxylin‐eosin staining. Masson’s trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real‐time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA‐sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP‐seq) was carried out for identification of menin‐ and H3K4me3‐enriched regions within the whole genome in the mouse kidney tissue. ChIP‐qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh‐HGF on renal fibrosis.ResultsThe expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α‐SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial‐to‐mesenchymal transition (EMT) induced by TGF‐β treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO‐induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency.ConclusionsThese findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

HGF Secreted by Mesenchymal Stromal Cells Promotes Primordial Follicle Activation by Increasing the Activity of the PI3K-AKT Signaling Pathway

Primordial follicle activation is fundamental for folliculogenesis and for the maintenance of fertility. An effective therapeutic strategy for patients with premature ovarian insufficiency (POI) is to promote the activation of residual primordial follicles. The secretome of human umbilical cord mesenchymal stromal cells (hUC-MSC-sec) contains several components that might promote the activation of primordial follicles. In the present study, we revealed that treatment with the hUC-MSC-sec significantly increased the proportion of activated primordial follicles in mouse ovaries both in vitro and in vivo. The activating effects of hUC-MSC-sec on primordial follicles were attributed to the activation of the PI3K-AKT signaling pathway by hepatocyte growth factor (HGF). While the effect of the hUC-MSC-sec was attenuated by the neutralizing antibodies against HGF, application of exogenous HGF alone also promoted the activation of primordial follicles. Furthermore, we demonstrated that HGF promoted the expression of KITL in granulosa cells by binding with the HGF receptor c-Met, thereby increasing the activity of the PI3K-AKT signaling pathway to activate primordial follicles. Taken together, our findings demonstrate that hUC-MSC-sec promotes primordial follicle activation through the functional component HGF to increase the PI3K-AKT signaling activity, highlighting the application of the hUC-MSC-sec or HGF for the treatment of POI patients.Graphical abstract

Hepatocyte growth factor and fish oil facilitated reversal of D-galactosamine–induced toxicity in primary hepatocyte cultures of albino mice

D-galactosamine (Ga1N), a well-known hepatotoxic agent, induces liver injury resembling human viral hepatitis usually followed by the regeneration processes. Hepatocyte growth factor (HGF) is a cytoprotective factor involved in regeneration of the injured liver. However, the effects of exogenous HGF remain poorly understood because of its rapid clearance by the liver. This study was undertaken to find out whether HGF and fish oil facilitated the reversal of GalN–induced toxicity in primary hepatocyte cultures of albino mice. Primary hepatocytes cultures were established from mice liver tissue. The study involved the effect of GalN on hepatocytes and also determination of the protective role of fish oil on hepatocyte cultures. Cell proliferation tests and liver function tests were done to determine the degree of GalN effect on cultured hepatocytes. Biochemical parameters of cultured cells were also performed to check the recovery effect of fish oil on GalN–induced hepatotoxicity. The combination of Ga1N and HGF triggered cell proliferation in primary hepatocyte cultures specifying activation of regeneration through HGF. However, hepatocyte function tests revealed that although the regeneration process was initiated, its function was slightly altered by Ga1N. Therefore, to control its effect at a functional level, we tested fish oil doses and indicated its influence. This work can be a useful tool for studying hepatotoxic-induced cell regeneration in vitro; moreover, the data indicates that HGF and fish oil has hepatoprotective activity against Ga1N and may aid as a suitable adjuvant in clinical conditions associated with liver damage.

HGF can reduce accumulation of inflammation and regulate glucose homeostasis in T2D mice.

Hepatocyte growth factor (HGF) has been studied as a protective factor for the survival of islet β cells and regulatory glucose transport and metabolism in many studies. The addition of exogenous HGF to cells or mice is the most common way to study HGF, but the persistence and stability of the administered HGF are unclear. In this experiment, wild-type C57BL6 (WT) mice and HGF-overexpressing transgenic (HGF-Tg) mice were divided into a normal diet (ND) group and an HFD group. The HGF protein level in the liver, kidney, spleen, pancreas, and VAT of HGF-Tg-ND mice was upregulated compared to that of WT-ND mice, and it was also upregulated in HGF-Tg-HFD mice compared to that in WT-HFD mice. In the ND group, though the HGF-Tg-ND mice showed higher fasted blood glucose levels and larger integrated density (IOD) of glucagon-positive cells than WT-ND mice, we found that HGF-Tg-ND mice can still maintain normal glucose tolerance based on an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin tolerance test (IPITT). In the HFD group, the HGF-Tg-HFD mice showed insulin sensitivity in IPGTT and IPITT and had larger areas and higher IOD values of islet β cells and smaller areas and IOD values of islet α cells than the WT-HFD mice. HGF-Tg-HFD mice had lower level of serum insulin than WT-HFD mice. The HGF-Tg-HFD mice showed inhibited accumulation of CD4+ T cells, CD8+ T cells, Ly6G+ neutrophils, and F4/80+ macrophages in the blood and tissues and protected liver and kidney functions. Oil Red O-stained liver sections revealed that WT-HFD mice had larger areas and higher IOD values of Oil Red O-positive cells than HGF-Tg-HFD mice, and WT-HFD mice had higher score of NASH. PAS-stained kidney sections found WT-HFD has higher mesangial area/glomerular area × 100% than HGF-Tg-HFD mice. Comparative analyses demonstrated that HGF reduces the proportions of inflammatory cells in the blood and tissues, and protects liver and kidney tissues by regulating glucose homeostasis of type 2 diabetic mice.

Metformin reduces HGF-induced resistance to alectinib via the inhibition of Gab1

Alectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor that has sufficient clinical efficacy and satisfactory safety in ALK-positive non-small cell lung cancer (NSCLC) patients with or without brain metastasis. Alectinib has now become an important drug in the first-line treatment of advanced ALK-positive NSCLC; however, resistance is almost inevitable. The increased expression of hepatocyte growth factor (HGF) and its physiological receptor tyrosine kinase MET have been shown to be linked to acquired resistance to various tyrosine kinase inhibitors (TKIs), and this phenomenon has been observed in some ALK-positive NSCLC tumour tissues. In this study, we found that HGF levels in the culture supernatant of an ALK-positive cell line tended to increase with time and could be further increased by alectinib in a time-dependent manner. Exogenous or endogenous HGF did not cause resistance to the ALK/MET double-targeted small molecule inhibitor crizotinib, but it was an important cause of alectinib resistance. Furthermore, Gab1 was a key effector in the HGF/MET signal transduction pathway that mediated alectinib resistance. The antidiabetic drug metformin combined with alectinib overcame alectinib resistance triggered by HGF/MET through disrupting the complex between MET and Gab1, thereby inhibiting Gab1 phosphorylation and the activation of downstream signal transduction pathways. These results suggest that metformin combined with alectinib may be useful for overcoming alectinib resistance induced by the activation of the HGF/MET signalling pathway and improving the efficacy of alectinib.

Effect of c-Met inhibitor AMG-102 on radiosensitivity in laryngeal squamous carcinoma cells

Objective: To investigate the effect of c-Met inhibitor AMG-102 on proliferation and radiosensitivity in laryngeal squamous carcinoma cells. Methods: The effects of AMG-102 on proliferation and radiosensitivity of laryngeal squamous carcinoma cell lines Hep-2 and KBV200 were detected by 3-(4, 5-dimethy-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and colony formation assay, respectively. The apoptosis of Hep-2 and KBV200 cells was detected by flow cytometry. The expression levels of c-Met, phospho-Met (p-Met), cleaved caspase-3 and Akt/p-Akt, Erk/p-Erk were detected by Western blot. Specific small interfering RNA targeting c-Met or plasmid of c-Met were transfected into Hep-2 and KBV200 cells to investigate the cell sensitivity to AMG-102. Results: Compared with KBV200 cells, Hep-2 cells were more sensitive to AMG-102 with IC(50) of 14 and 9 μmol/L, respectively. The relative expression levels of c-Met and p-Met proteins in Hep-2 cells were 194.48±0.57 and 177.76±1.53, respectively, which were significantly higher than those in KBV200 cells (171.24±1.00 and 115.37±0.56, respectively, P<0.001 for both). Exogenous hepatocyte growth factor (HGF) was added to increase the expression level of p-Met protein in KBV200 cells. The results showed that AMG-102 significantly reduced the expression of p-Met in KBV200 cells treated with HGF (P<0.001). Compared with the dimethyl sulfoxide (DMSO) group, AMG-102 treatment combined with radiotherapy significantly increased the radiosensitivity of Hep-2 cells (SER=1.28, P<0.001). However, AMG-102 had little effect on the radiosensitivity of KBV200 cells (SER=1.18, P=0.002). Compared with the 4 Gy radiotherapy alone group and the 5 μmol/L of AMG-102 alone treatment group, the apoptosis rate of Hep-2 cells in the combined treatment group was significantly increased. Meanwhile, the expression level of cleaved caspase-3 protein was also markedly increased. However, there were no significant changes in the apoptotic rate and cleaved caspase-3 expression in each treatment group of KBV200 cells. Compared with DMSO treatment group, the expression levels of p-Met, p-Akt and p-Erk were significantly decreased in the 4 Gy radiotherapy group, 5 μmol/L of AMG-102 treatment group and combined treatment group of Hep-2 cells. And the levels of p-Met, p-Akt and p-Erk in the combined treatment group were significantly lower than those in the 4 Gy radiotherapy alone group and 5 μmol/L of AMG-102 treatment alone group. By contrast, in KBV200 cells, the expression of p-Met, p-Akt and p-Erk in each group was not changed. The relative expression of p-Met in Hep-2 cells before and after radiotherapy at 30 min, 1 h, 4 h, 8 h, 24 h were 99.89±0.61, 138.62±1.00, 163.07±5.00, 87.80±1.85, 90.67±0.65 and 94.09±1.41, respectively. The level of p-Met was slightly increased after radiotherapy at 30 min and 1 h (P<0.001 for all), whereas it was significantly decreased from 4 h to 24 h after radiotherapy (P<0.05 for all). By contrast, the expression of p-Met in KBV200 cells did not change with time after radiotherapy (P>0.05). The sensitivity of Hep-2 cells to AMG-102 was decreased after silencing of c-Met, while the sensitivity of KBV200 cells to AMG-102 was not significantly changed (P>0.05). Moreover, the radiosensitivity of Hep-2 cells in c-Met knockdown group had a slightly increasing trend (SER=1.07, P=0.068). After the treatment with 10 μmol/L of AMG-102, the proliferation rate of c-Met ectopically expressed KBV200 cells was 60.05%±3.23%, It was significantly lower than that of the blank control 90.08%±1.04% and siRNA negative control (90.12%±1.01%, P<0.001). The results suggested that the overexpression of c-Met in KBV200 cells increased the radiosensitivity to AMG-102, whereas depletion of c-Met resulted in resistance to AMG-102 in Hep-2 cells. Furthermore, the radiosensitivity of KBV200 cells that overexpressed c-Met showed a decreased trend (SER=0.7, P=0.005). Conclusions: c-Met inhibitor AMG-102 has a significant inhibitory effect on the proliferation of c-Met overexpressing laryngeal squamous carcinoma cells, leading to increased radiosensitivity. It suggests that molecular targeted therapy against c-Met receptor is more effective in c-Met overexpressed subtype of laryngeal squamous cell carcinoma.

Amelioration of Endoplasmic Reticulum Stress by Mesenchymal Stem Cells via Hepatocyte Growth Factor/c-Met Signaling in Obesity-Associated Kidney Injury.

Recent advances in the understanding of lipid metabolism suggest a critical role of endoplasmic reticulum (ER) stress in obesity‐induced kidney injury. Hepatocyte growth factor (HGF) is a pleiotropic cytokine frequently featured in stem cell therapy with distinct renotropic benefits. This study aims to define the potential link between human induced pluripotent stem cell‐derived mesenchymal stem cells (iPS‐MSCs)/bone marrow‐derived MSCs (BM‐MSCs) and ER stress in lipotoxic kidney injury induced by palmitic acid (PA) in renal tubular cells and by high‐fat diet (HFD) in mice. iPS‐MSCs or BM‐MSCs alleviated ER stress (by preventing induction of Bip, chop, and unfolded protein response), inflammation (Il6, Cxcl1, and Cxcl2), and apoptosis (Bax/Bcl2 and terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling‐positive cells) in renal cortex of animals exposed to HFD thus mitigating histologic damage and albuminuria, via activating HGF/c‐Met paracrine signaling that resulted in enhanced HGF secretion in the glomerular compartment and c‐Met expression in the tubules. Coculture experiments identified glomerular endothelial cells (GECs) to be the exclusive source of glomerular HGF when incubated with either iPS‐MSCs or BM‐MSCs in the presence of PA. Furthermore, both GEC‐derived HGF and exogenous recombinant HGF attenuated PA‐induced ER stress in cultured tubular cells, and this effect was abrogated by a neutralizing anti‐HGF antibody. Taken together, this study is the first to demonstrate that MSCs ameliorate lipotoxic kidney injury via a novel microenvironment‐dependent paracrine HGF/c‐Met signaling mechanism to suppress ER stress and its downstream pro‐inflammatory and pro‐apoptotic consequences. stem cells translational medicine 2019;8:898&910

Application of Hepatocyte Growth Factor for Acute Spinal Cord Injury: The Road from Basic Studies to Human Treatment.

Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes, and has also gained attention as a strong neurotrophic factor in the central nervous system. We found that during the acute phase of spinal cord injury (SCI) in rats, c-Met, the specific receptor for HGF, increases sharply, while the endogenous HGF up-regulation is relatively weak. Introducing exogenous HGF into the spinal cord by injecting an HGF-expressing viral vector significantly increased the neuron and oligodendrocyte survival, angiogenesis, and axonal regeneration, to reduce the area of damage and to promote functional recovery in rats after SCI. Other recent studies in rodents have shown that exogenously administered HGF during the acute phase of SCI reduces astrocyte activation to decrease glial scar formation, and exerts anti-inflammatory effects to reduce leukocyte infiltration. We also reported that the intrathecal infusion of recombinant human HGF (intrathecal rhHGF) improves neurological hand function after cervical contusive SCI in the common marmoset, a non-human primate. Based on these collective results, we conducted a phase I/II clinical trial of intrathecal rhHGF for patients with acute cervical SCI who showed a modified Frankel grade of A/B1/B2 72 h after injury onset, from June 2014 to May 2018.

Failures in preclinical and clinical trials of c-Met inhibitors: evaluation of pathway activity as a promising selection criterion.

C-Met is a frequently overexpressed or amplified receptor tyrosine kinase involved in metastatic-related functions, including migration, invasion, cell survival, and angiogenesis. Because of its role in cancer progression and metastasis, many inhibitors have been developed to target this pathway. Unfortunately, most c-Met inhibitor clinical trials have failed to show significant improvement in survival of cancer patients. In these trials tumor type, protein overexpression, or gene amplification are the primary selection criteria for patient inclusion. Our data show that none of these criteria are associated with c-Met pathway activation. Hence, it is conceivable that the majority of c-Met inhibitor clinical trial failures are the consequence of a lack of appropriate patient selection. Further complicating matters, c-Met inhibitors are routinely tested in preclinical studies in the presence of high levels of exogenous Hepatocyte Growth Factor (HGF), its activating ligand. In our studies, several tumor cell lines showed sensitivity to a c-Met inhibitor at high HGF concentrations (50 ng/mL). However, when the tumor lines were tested at HGF levels typically detected in human serum (0.4 to 0.8 ng/mL), inhibitor activity was lost. Thus testing c-Met inhibitors at non-physiological concentrations of HGF may lead to incorrect predictions of drug efficacy in vivo.

Ginkgolic acid suppresses the invasion of HepG2 cells via downregulation of HGF/c‑Met signaling.

Liver cancer is one of the most devastating types of cancer worldwide. Despite years of improvements in treatment, the prognosis of patients with this type of malignancy remains poor due to frequent recurrence and metastasis after surgical resection. Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgobiloba L. that possesses a wide range of bioactive properties. However, to the best of our knowledge, whether GA can inhibit the invasion of liver cancer cells and the underlying mechanisms remains unknown. The aim of the present study was to investigate the effects of GA on the migration and invasion abilities of liver cancer cells and the underlying molecular mechanism. The results revealed that GA suppressed the migration and invasion abilities of HepG2 cells. In addition, GA treatment inhibited the expression of invasion‑related molecules (MMP‑2 and MMP‑9) and prevented the epithelial‑mesenchymal transition (EMT) of HepG2 cells. Further experiments revealed that GA‑reduced hepatocyte growth factor (HGF) production and suppressed c‑Met phosphorylation may be the underlying mechanisms. Exogenous recombinant HGF supplementation improved the cell invasion ability impaired by GA. Moreover, the invivo experiment revealed that GA inhibited the tumor growth of liver cancer and prevented EMT. Collectively, these data indicated that GA effectively suppressed the invasion and EMT of HepG2 cells via downregulation of HGF/c‑Met signaling, thus GA may serve as a novel chemotherapeutic agent for the treatment of HCC.

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells.

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.

Abstract 330: Systemic Blocking Exosome Formation/Release Improves Ischemic Hindlimb Repair in Diabetic db/db Mice

Background: Recent studies demonstrated that plasma exosomes from diabetic patients and animals lose their ability to maintain physiological properties of cardiomyocytes. Exosome inhibitor GW 4869 (GW) tended to improve heart function in diabetic mice with myocardial infarction. We tested the hypotheses that systemic blocking exosome synthesis/formation improves ischemic hindlimb repair in diabetes. Methods and Results: Unilateral hindlimb ischemia (HLI) surgery was conducted by ligation of left femoral artery in 12-week male db/db and db/+ mice which were administrated with either GW (2 mg/kg body weight, i.p) or vehicle for 4 weeks starting from one week before HLI. Blood perfusion in ischemic hindlimbs was examined by Laser Doppler at pre-HLI, and 0, 3, 7, 14 and 21 days post-HLI. Plasma exosomes were isolated by standard ultracentrifugation method and counted by NanoSight. GW increased blood concentration of CD31 + /Sca-1 + cell 3-day post-HLI and decreased necrosis and loss of toe/toenail; improved blood flow; enhanced capillary/arterial density determined by CD31 and SMA-α staining and decreased fibrosis in the muscles of ischemic hindlimbs in db/db mice 21-day post-HLI. Plasma exosomes from db/db mice significantly impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration. Mechanistically, hepatocyte growth factor (HGF) expression was decreased in bone marrow-derived endothelial progenitor cells (EPCs) from db/db mice and HCMVECs treated with diabetic plasma exosomes. Administration of GW increased HGF level in diabetic EPCs. Furthermore, diabetic plasma exosomes decreased H3K4me3 (active maker of transcription) whereas increased H3K27me3 (suppressive marker of transcription) at HGF promoter. Finally, treatment of HCMEVs with exogenous HGF rescued diabetic exosomes impaired tube formation of HCMVEC. Conclusions: Systemic inhibition of exosome synthesis/formation by GW increased ischemic limb repair in diabetic db/db mice, at least partially, via histone methylation mediated suppression of HGF. Our findings suggest that therapeutic targeting of systemic dysfunctional exosomes could represent a new avenue for therapeutics of ischemic tissue injury in patients with diabetes.

Abstract 3923: Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model

Abstract C-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair. In tumors, including breast, prostate, glioblastoma, and thyroid cancer, Met protein levels are often elevated and associated with disease progression and metastasis. The c-Met pathway is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). HGF is produced by mesenchymal cells and binds to the c-Met protein, leading to phosphorylation and upregulation of a variety of downstream signaling pathways that result in increased tumor cell migration, proliferation, survival, and induction of angiogenesis. C-Met is associated with disease progression and metastasis. Consequently, there has been significant interest in the development of c-Met inhibitors as anti-cancer therapeutics. The efficacy of such agents usually initially are determined in vitro utilizing HGF concentrations of 25-50 ng/mL. However, HGF serum levels in humans typically range from 0.4-0.8 ng/mL. In our studies, we examined the prostate carcinoma cell line DU145, and BMS-777607. Migration and phosphorylation of c-Met increased in a dose-dependent manner with HGF treatment. Additionally, c-Met inhibition with BMS-777607 reduced migration and invasion when in the presence of 25-50 ng/mL HGF. However, at physiologically relevant HGF concentrations the drug had no effect on migration or invasion of this cell line. In vivo models of c-Met inhibition have been limited mainly to mice genetically engineered to express HGF, on the basis that mouse HGF does not stimulate human c-Met. However, in our studies, mouse HGF did stimulate c-Met in the DU145 tumor cell line. Subsequent in vivo evaluation revealed treatment with BMS-777607 had no reduction on DU145 tumor cell-induced angiogenesis in cells pretreated with the drug, nor in mice receiving oral administration of the drug. An experimental metastasis model also resulted in no significant reduction in metastasis formation, as assessed by a tail vein injection model. To characterize HGF concentration in vivo, an ELISA was used to determine an average concentration of 7.7 ng/g of DU145 tumor tissue. Further evaluations of phospho-Met are underway utilizing immunofluorescence of FFPE samples. Preliminary results indicate low levels of phospho-Met in DU145 tumor sections that can be induced by treating tumor-bearing mice with exogenous HGF. Our results suggest that in vitro evaluation of c-Met inhibitors utilizing non-physiological concentrations of HGF may produce an abnormally high phospho-Met that can subsequently be reduced by treatment with c-Met inhibition. In vitro studies performed at high HGF concentrations may not accurately predict the in vivo drug efficacy. Further characterization of HGF concentrations within the tumor is necessary to properly conduct in vitro evaluation of c-Met inhibitors. Citation Format: Veronica S. Hughes, Mumtaz Rojiani, Dietmar Siemann. Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3923.

Abstract B157: Effect of HGF concentration on c-Met phosphorylation and inhibition in paracrine-activated tumor cells

Abstract c-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair. In tumors, including breast, prostate, glioblastoma, and thyroid cancer, Met protein levels are often elevated and associated with disease progression and metastasis. The c-Met pathway is activated by the endogenous ligand hepatocyte growth factor (HGF). HGF is produced by mesenchymal cells and binds to the c-Met protein, leading to phosphorylation and upregulation of a variety of downstream signaling pathways that result in increased tumor cell migration, proliferation, survival, and induction of angiogenesis. Consequently, there has been significant interest in the development of c-Met inhibitors as anticancer therapeutics. The efficacy of such agents usually initially are determined in vitro utilizing HGF concentrations of 25-50 ng/mL. However, HGF serum levels in humans typically range from 0.4-0.8 ng/mL. To examine the impact of exogenous HGF levels on c-Met inhibitor activity, human prostate (PC-3, DU145) and lung (A549) cancer cell lines were utilized in this study. These cell lines have been previously characterized as paracrine-activated: cell lines that express the c-Met receptor, but not HGF. In the presence of 25-50 ng/mL HGF, these cell lines demonstrated a reduction in migration, invasion, and phosphorylation of Met after treatment with the small-molecule c-Met inhibitor BMS-777607. However, such antitumor efficacy was absent when these paracrine-activated cell lines were treated with BMS-777607 in the presence of physiologically normal concentrations of HGF (0.4-0.8 ng/mL). A human HGF-expressing mouse model has been developed for xenograft studies of human c-Met inhibition, on the basis that mouse HGF does not activate human c-Met. However, we found that mouse HGF was able to induce phosphorylation of Met in the human DU145 cell line. In vivo, DU145 xenografts in NSG mice treated with BMS-777607 showed no reduction in c-Met phosphorylation unless the mice were administered exogenous HGF during tumor growth. Taken together, these findings indicate that the utilization of nonphysiologic concentrations of HGF in tissue culture evaluations of c-Met targeting agents may result in an overestimation of agent activity not likely to be observed in the tumor microenvironment. Citation Format: Veronica S. Hughes, Dietmar W. Siemann. Effect of HGF concentration on c-Met phosphorylation and inhibition in paracrine-activated tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B157.