Hepatocyte Growth Factor Stimulation Research Articles

Spatiotemporal regulation of the hepatocyte growth factor receptor MET activity by sorting nexins 1/2 in HCT116 colorectal cancer cells.

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease.

Abstract 6003: The TNF-α-induced protein 3-interacting protein1 (TNIP1) suppresses HGF mediated NF-kB pathway activation and growth in gastric cancer cells

Abstract Background: TNF-α-induced protein 3-interacting protein 1 (TNIP1), an integral component in the regulation of immune responses and cellular pathways, is gaining increased attention in the scientific community. Its ability to interact with A20, a crucial ubiquitin-editing enzyme, forms a significant part of the body's mechanism to control inflammation and prevent unregulated immune responses, which are pivotal in the pathogenesis of autoimmune diseases. TNIP1's role extends beyond autoimmunity; it is also implicated in the regulation of cancer cell proliferation and survival, making it a potential target in cancer therapy. However, TNIP1 has not yet been elucidated on the tumorigenesis, proliferation, and invasion of human gastric cancer, and needs to be researched and discovered. In this study, the HGF-mediated association of TNIP1, nuclear factor kappa-B (NF-κB), and matrix metalloproteinase 9 (MMP9) and cancer cell proliferation and invasion were investigated in two types of human gastric cancer cell lines. Methods: In this study, cell culture, cDNA microarray analysis, western blotting, Real-Time Polymerase chain reaction, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TNIP1 knock-down with short hairpin RNA (shRNA), apoptosis, reagents and antibodies, standard two chamber invasion assay. Results: In our study on gastric cancer cells, we found that hepatocyte growth factor (HGF) stimulation altered TNIP1 expression at both mRNA and protein levels, indicating a dynamic interaction between HGF and TNIP1. The use of LY294002, a PI3K/AKT pathway inhibitor, revealed TNIP1's involvement in the NF-kB pathway, showing changes in NFkB and TNIP1 expression post-HGF stimulation. TNIP1-shRNA expressing cells exhibited altered expression of p53, BCL-2, NFkB, and MMP9, markers crucial for tumor progression and metastasis. Notably, TNIP1 knockdown led to increased cell proliferation and enhanced invasion capabilities in gastric cancer cells. Furthermore, TNIP1 knockdown cells showed a significant decrease in apoptosis rates upon HGF treatment, as confirmed by propidium iodide staining and FACS analysis. Conclusions: In conclusion, our results demonstrate that TNIP1 is closely associated with the NF-kB pathway and plays a critical role in modulating tumor invasion and apoptosis in gastric cancer cells. These findings suggest that TNIP1 could be a potential therapeutic target for the treatment of gastric cancer, providing new insights into the mechanisms of tumor progression and the development of targeted therapies. Citation Format: Jiyoon Jung, Sungae Koh, Kyunghee Lee. The TNF-α-induced protein 3-interacting protein1 (TNIP1) suppresses HGF mediated NF-kB pathway activation and growth in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6003.

AML Inhibits Lipid Uptake By the Liver through an HGF/c-MET Dependent Mechanism Which Results in an Increased Supply of Free Fatty Acid to the Tumor

Acute myeloid leukemia (AML) requires a high metabolic turnover to allow for constant expansion. We and others have previously shown that primary human AML blasts rely on fatty acid (FA)-oxidation for survival and proliferation. (Shafat MS, et al. 2017, Jones CL, et al. 2018). The liver plays a major role in carbohydrate, protein, amino acid, and lipid metabolism (Hodson L, and Gunn PJ. 2019). Given that AML is a FA dependent tumor, we have studied the interactions between AML and the liver, and specifically how these affect lipid metabolism, the availability of pro-tumoral FFA and ultimately the mechanism by which this supports AML growth. To understand the role of lipid metabolism we used two syngeneic models of AML (HOXA9/Meis1 or MN1). When engrafted with AML cells, mice body weight during the latter stages of disease. After sacrifice, the size of epididymal and inguinal fat pads were significantly reduced in mice with AML compared to controls. Mice with AML also had elevated free fatty acid (FFA) and reduced glucose in the plasma. We and others have shown that AML induces lipolysis of white adipose tissue for uptake by blasts and resulting in tumor expansion (Shafat MS, et al. 2017, Ye H, et al. 2016). Since the liver is the master regulator of available FA in the plasma, we isolated livers from AML engrafted mice. Transcriptomics revealed down-regulation of genes involved in FA uptake and metabolism. RT-qPCR of RNA isolated from liver samples confirmed these results showing down-regulation of FA transport proteins, (FABP1, SLC27A2, SLC27A5, and CD36) and FA metabolism genes (CPT1, ACADM, and HMGCS2). Furthermore, primary hepatocytes isolated and cultured with AML conditioned media showed a down-regulation of FA uptake and metabolism genes. These data support the hypothesis that AML secretes a factor that alters lipid metabolism in the liver. To discover the factor that AML secretes, and which regulates FA uptake and metabolism in the liver, a proteome profile of the serum from AML engrafted mice was compared to conditioned media from AML cultured cells. Pathway analysis revealed hepatocyte growth factor (HGF), IL-1B and Granulocyte colony stimulating factor (G-CSF) as possible key factors involved in liver FA metabolism (Jing Y, et al. 2019, Kaibori M, et al. 2002). In vitro gene expressions analysis of primary hepatocytes revealed that addition of HGF to the medium, but not Il-1b or G-CSF, inhibited expression of FA uptake genes. In addition, using Seahorse mitochondrial stress kit we confirmed that primary hepatocytes decreased FA oxidation when cultured with AML conditioned media or HGF. Next, we confirmed that AML derived HGF was responsible for inhibition of hepatocyte FA genes by using a neutralizing antibody to HGF which blocked the transcriptional changes previously observed in hepatocytes cultured in AML conditioned media. This showed that AML derived HGF downregulates hepatocyte FA uptake and metabolism. Finally, to determine the role of AML in the uptake of FA by hepatocytes, cultures of primary murine hepatocytes were pretreated with AML conditioned media or HGF with and without neutralizing antibody. A BODIPY conjugated long chain FA was then added to the culture. Images analysis showed hepatocytes had reduced lipid uptake in the presence of AML condition media or HGF, but this was restored with the addition of HGF neutralizing antibody. Here we report that AML inhibition of lipid uptake in the liver, through an HGF dependent mechanism, increases the availability of plasma FA and forms a fundamental part of the AML disease. Ongoing work to develop our understanding of how AML increases access to FA to aid proliferation, may aid in the development of new therapeutics to target liver metabolism in combination with other AML therapies.

Circular RNA encoded MET variant promotes glioblastoma tumorigenesis.

Activated by its single ligand, hepatocyte growth factor (HGF), the receptor tyrosine kinase MET is pivotal in promoting glioblastoma (GBM) stem cell self-renewal, invasiveness and tumorigenicity. Nevertheless, HGF/MET-targeted therapy has shown limited clinical benefits in GBM patients, suggesting hidden mechanisms of MET signalling in GBM. Here, we show that circular MET RNA (circMET) encodes a 404-amino-acid MET variant (MET404) facilitated by the N6-methyladenosine (m6A) reader YTHDF2. Genetic ablation of circMET inhibits MET404 expression in mice and attenuates MET signalling. Conversely, MET404 knock-in (KI) plus P53 knock-out (KO) in mouse astrocytes initiates GBM tumorigenesis and shortens the overall survival. MET404 directly interacts with the MET β subunit and forms a constitutively activated MET receptor whose activity does not require HGF stimulation. High MET404 expression predicts poor prognosis in GBM patients, indicating its clinical relevance. Targeting MET404 through a neutralizing antibody or genetic ablation reduces GBM tumorigenicity in vitro and in vivo, and combinatorial benefits are obtained with the addition of a traditional MET inhibitor. Overall, we identify a MET variant that promotes GBM tumorigenicity, offering a potential therapeutic strategy for GBM patients, especially those with MET hyperactivation.

Genetic Ablation of the MET Oncogene Defines a Crucial Role of the HGF/MET Axis in Cell-Autonomous Functions Driving Tumor Dissemination.

Cancer cell dissemination is sustained by cell-autonomous and non-cell-autonomous functions. To disentangle the role of HGF (Hepatocyte Growth Factor) and MET ligand/receptor axis in this complex process, we genetically knocked out the MET gene in cancer cells in which MET is not the oncogenic driver. In this way, we evaluated the contribution of the HGF/MET axis to cancer cell dissemination independently of its direct activities in cells of the tumor microenvironment. The lack of MET expression in MET-/- cells has been proved by molecular characterization. From a functional point of view, HGF stimulation of MET-/- cancer cells was ineffective in eliciting intracellular signaling and in sustaining biological functions predictive of malignancy in vitro (i.e., anchorage-independent growth, invasion, and survival in the absence of matrix adhesion). Cancer cell dissemination was assessed in vivo, evaluating: (i) the ability of MET-/- lung carcinoma cells to colonize the lungs following intravenous injection and (ii) the spontaneous dissemination to distant organs of MET-/- pancreatic carcinoma cells upon orthotopic injection. In both experimental models, MET ablation affects the time of onset, the number, and the size of metastatic lesions. These results define a crucial contribution of the HGF/MET axis to cell-autonomous functions driving the metastatic process.

HGF/c-MET pathway contributes to cisplatin-mediated PD-L1 expression in hepatocellular carcinoma.

Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.

Hepatocyte Growth Factor and Insulin-like Growth Factor-1 based Cellular Therapies for Oxidative Stress Injury.

Imbalance between free radicals and antioxidants causes oxidative stress by the accumulation of reactive oxygen species (ROS) in the tissues and organs. Oxidative stress occurs in many damaged conditions, and the increase of ROS and reduction of antioxidants enhances inflammation, apoptosis, fibrosis and may worsen the pathology leading to organ failure. The potential therapies aim to increase antioxidants and decrease ROS. Mesenchymal stem cells (MSCs) isolated from the stroma of various tissues are multipotent cells and have beneficial effects on several diseases with their immunomodulation and regeneration capacities. MSCs trigger the proliferation of the cells with various secretory factors, reduce oxidative stress and decrease apoptosis, inflammation, fibrosis and thus, increase regeneration. However, survival, engraftment, and differentiation problems of transplanted MSCs restrict their protective and regenerative effects. Preconditioning of MSCs with several factors, such as cytokines, hypoxia, chemical agents, pharmacological drugs, physical factors and growth factors, enhances their repairing efficacy for injury and disease models. This review is mainly focused on insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF), and discusses the research on MSC priming/induction with IGF-1 and HGF stimulation either by supplementation or overexpression that can enhance the regenerative potential of MSCs on various oxidative stress conditions such as acute/chronic kidney diseases, lung injury, cancer, metabolic and cardiovascular diseases.

Long-term Production of Glycogen and Hepatic-Derived, Cell-Invasion-Promoting Chemokines by Ultrasound-Driven Hepatic-Differentiated Human Bone Marrow Mesenchymal Stem Cells

The current treatment for liver failure is restricted to surgical liver transplantation, which is technically complicated, limited by the shortage of available organs and presents major risks to the patient. Bone marrow mesenchymal stem cells (BMSCs) represent promising sources of hepatocyte-like cells for cell transplantation treatment. However, a safe and efficient induction method for their differentiation remains to be defined. Here we further optimized an effective technique by combining high-dose treatment with hepatocyte growth factor (HGF) and ultrasound stimulation. The optimized ultrasound parameter (1.0 W/cm2 intensity, 1 MHz frequency, 20% duty cycle, 100 Hz pulse repetition frequency, 60-s irradiation duration, triple times in three days) combined with different HGF doses (10, 20 and 50 ng/ml) was used to treat BMSCs. The results showed that the specific hepatic markers, including α-fetoprotein (αFP/AFP), cytokeratin 18 (CK18), albumin (ALB) and glycogen, were increased in a dose-dependent manner. Their concentration was then further increased when ultrasound irradiation was administered (P < 0.05), as indicated by PCR, Western blot and immunofluorescence staining as well as a glycogen synthesis test. Furthermore, analysis of the hepatocyte-derived chemokines showed elevated stromal cell-derived factor 1alpha (SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) after HGF treatment. Again, concentrations of those chemokines were further increased by ultrasound radiation (P < 0.05). The observed increased effect was sustained for 21 days. To summarize, we further defined the optimal combination of HGF and ultrasound treatment to increase the differentiation and chemotaxis of BMSCs in a safe, sustained and efficient manner. These findings provide a new perspective for stem cell orientation in the field of tissue engineering.

Comparative characterization of the HGF/Met and MSP/Ron systems in primary pancreatic adenocarcinoma

Pancreatic cancer is an aggressive disease with a poor prognosis for which current standard chemotherapeutic treatments offer little survival benefit. Receptor tyrosine kinases (RTK)s have garnered interest as therapeutic targets to augment or replace standard chemotherapeutic treatments because of their ability to promote cell growth, migration, and survival in various cancers. Met and Ron, which are homologous RTKs activated by the ligands hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), respectively, are over-activated and display synergistic malignant effects in several cancers. Despite the homology between Met and Ron, studies that have directly compared the functional outcomes of these systems in any context are limited. To address this, we sought to determine if the HGF/Met and MSP/Ron systems produce overlapping or divergent contributions towards a malignant phenotype by performing a characterization of MSP and HGF driven signaling, behavioral, and transcriptomic responses in a primary pancreatic adenocarcinoma (PAAD) cell line in vitro. The impact of dual Met and Ron expression signatures on the overall survival of PAAD patients was also assessed. We found HGF and MSP both encouraged PAAD cell migration, but only HGF increased proliferation. RNA sequencing revealed that the transcriptomic effects of MSP mimicked a narrow subset of the responses induced by HGF. Analysis of clinical data indicated that the strong prognostic value of Met expression in primary PAAD does not appear to be modulated by Ron expression. The relatively reduced magnitude of MSP-dependent effects on primary PAAD cells are consistent with the limited prognostic value of Ron expression in this cancer when compared to Met. Although HGF and MSP produced a differing breadth of responses in vitro, overlapping pro-cancer signaling, behavioral, and transcriptional effects still point to a potential role for the MSP/Ron system in pancreatic cancer.

Up-regulation of Grb2-associated binder 1 promotes hepatocyte growth factor-induced endothelial progenitor cell proliferation and migration.

ObjectivesGrb2-associated binder 1 (Gab1), a scaffolding adaptor protein, plays an important role in transmitting key signals that control cell growth, migration, and function from multiple tyrosine kinase receptors. This study was designed to investigate the influence of upregulation of Gab1 in endothelial progenitor cells (EPCs) stimulated with hepatocyte growth factor (HGF), and the underlying molecular mechanisms.Materials and MethodsEndothelial progenitor cells isolated from human umbilical cord blood were identified and divided into four groups. EPCs in the Control group were cultured normally; those in the Control+HGF group were treated with HGF stimulation; those in the AD-Gab1 group were transfected with adenovirus containing the Gab1 gene but not treated with HGF stimulation; and, those in the AD-Gab1+HGF group were treated with both HGF stimulation and transfection with adenovirus containing the Gab1 gene. Subsequently, Gab1 expression and proliferation and migration ability were compared for EPCs grown under different conditions. Furthermore, we measured phosphorylation levels of three key proteins Gab1, SHP2, and ERK1/2.ResultsThe AD-Gab1+HGF group had the highest expression of Gab1 and higher proliferation and migration than the other three groups.ConclusionsUpregulation of Gab1 promoted HGF-induced EPC proliferation and migration. Mechanistically, HGF stimulated Gab1 tyrosine phosphorylation in EPCs, thus leading to activation of extracellular regulated MAP kinase 1/2, which is involved in proliferation and migration signaling.

Regorafenib reverses HGF-induced sorafenib resistance by inhibiting epithelial-mesenchymal transition in hepatocellular carcinoma.

Sorafenib resistance is one of the major obstacles towards achieving a better outcome in patients with advanced hepatocellular carcinoma (HCC), in which aberrant activation of the hepatocyte growth factor (HGF)/mesenchymal‐epithelial transition pathway is frequently observed. Here, we report that HCC cells develop sorafenib resistance following HGF stimulation. Furthermore, HGF activates the downstream extracellular signal‐related kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) pathway and induces epithelial–mesenchymal transition (EMT) by up‐regulating Snail in HCC cells. Inhibition of ERK and STAT3 abolished the rescue effect of HGF by down‐regulating Snail and EMT. Moreover, phosphoinositide 3‐kinase/Akt was also activated in HGF‐treated HCC cells, although it had no effect on Snail expression. Notably, we also found that regorafenib reversed HGF‐induced sorafenib resistance by inhibiting ERK and STAT3, and subsequently down‐regulating Snail and EMT. Taken together, our results indicate that HGF induces sorafenib resistance by activating phosporylated (P)‐ERK/Snail/EMT and P‐STAT3/Snail/EMT pathways. Inhibition of P‐ERK and P‐STAT3 by regorafenib can block HGF‐induced EMT, thereby reversing HGF‐induced sorafenib resistance.

C-Src Recruitment is Involved in c-MET-Mediated Malignant Behaviour of NT2D1 Non-Seminoma Cells.

c-MET pathway over-activation is the signature of malignancy acquisition or chemotherapy resistance of many cancers. We recently demonstrated that type II Testicular Germ Cell Tumours (TGCTs) express c-MET receptor. In particular, we elucidated that the non-seminoma lesions express c-MET protein at higher level, compared with the seminoma ones. In line with this observation, NTERA-2 clone D1 (NT2D1) non-seminoma cells increase their proliferation, migration and invasion in response to Hepatocyte Growth Factor (HGF). One of the well-known adaptor-proteins belonging to c-MET signaling cascade is c-Src. Activation of c-Src is related to the increase of aggressiveness of many cancers. For this reason, we focused on the role of c-Src in c-MET-triggered and HGF-dependent NT2D1 cell activities. In the present paper, we have elucidated that this adaptor-protein is involved in HGF-dependent NT2D1 cell proliferation, migration and invasion, since Src inhibitor-1 administration abrogates these responses. Despite these biological evidences western blot analyses have not revealed the increase of c-Src activation because of HGF administration. However, notably, immunofluorescence analyses revealed that cytoplasmic and membrane-associated localization of c-Src shifted to the nuclear compartment after HGF stimulation. These results shed new light in the modality of HGF-dependent c-Src recruitment, and put the basis for novel investigations on the relationship between c-Src, and TGCT aggressiveness.

Piperidine carbamate peptidomimetic inhibitors of the serine proteases HGFA, matriptase and hepsin.

Matriptase and hepsin are type II transmembrane serine proteases (TTSPs). Along with related S1 trypsin like serine protease HGFA (hepatocyte growth factor activator), their unregulated proteolytic activity has been associated with cancer including tumor progression and metastasis. These three proteases have two substrates in common, hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), the ligands for MET and recepteur d'origine nantais (RON) receptor tyrosine kinases. Mechanism-based tetrapeptide and benzamidine inhibitors of these proteases have been shown to block HGF/MET and MSP/RON cancer cell signaling. Herein, we have rationally designed a new class of peptidomimetic hybrid small molecule piperidine carbamate dipeptide inhibitors comparable in potency to much larger tetrapeptides. We have identified multiple compounds which have potent activity against matriptase and hepsin and with excellent selectivity over the off-target serine proteases factor Xa and thrombin.

Differential responses of MET activations to MET kinase inhibitor and neutralizing antibody

BackgroundAberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive.MethodsWith the goal to investigate the molecular basis of MET amplification (METamp) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring METamp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo.ResultsWe find that cancer cells driven by METamp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit METamp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors.ConclusionsOur results demonstrate that METamp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring METamp, a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.

C-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

Type II testicular germ cell tumors (TGCTs) represent the most frequent malignancy in Caucasian males (20–40 years). Even if diagnosed with disseminated disease, >80% of patients are cured; however, a small percentage of cases progress and result in death. It is commonly accepted that these cancers arise from a disturbed testicular embryonic niche that leads to the block of gonocyte differentiation. The subsequent development of the invasive seminomas and non-seminomas is due to a combination of genetic, epigenetic and microenvironment-based alterations (genvironment). Hepatocyte growth factor (HGF) is present in the testicular microenvironment, together with its receptor c-MET, from early embryonic development to an adult stage. In addition, c-MET is a well-known proto-oncogene involved in the onset and progression of various human cancers. Herein, we have investigated the expression and availability of HGF and c-MET in TCam-2, NCCIT and NT2D1 cells, which are type II (T)GCT representative cell lines, and the effect of c-MET activation/repression on the regulation of cancerous biological processes. We found that NT2D1 cells increase their proliferation, polarized migration, and invasion in response to HGF administration. NCCIT cells respond to HGF stimulation only partially, whereas TCam-2 cells do not respond to HGF, at least according to the investigated parameters. Interestingly, the immunohistochemical study of c-MET distribution in TGCTs confirm its presence in both seminoma and non-seminoma lesions with different patterns. Notably, we found the highest c-MET immunoreactivity in the epithelial elements of the various components of TGCTs: teratoma, yolk sac tumor and choriocarcinoma.

Correlation between HGF/c-Met and Notch1 signaling pathways in human gastric cancer cells.

In recent decades, research concerning gastric carcinogenesis has rapidly progressed. It is evident that hepatocyte growth factor (HGF) is clinically related to gastric cancer progression and metastasis. In addition, previous studies have found that expression of Notch ligand Jagged1 is correlated with the poor prognosis of gastric cancer. However, the interaction between the HGF/c-Met and Notch1 signaling pathways remains unknown. In the present study, we found that gastric cancer patients with positive c-Met expression exhibited poorer overall survival than patients without c-Met expression (P=0.043) and that Jagged1 expression was significantly correlated with c-Met expression (r=0.301; P=0.004) in human gastric cancer specimens. In addition, Jagged1 activity increased after HGF stimulation, which in turn increased the downstream expression of cyclooxygenase 2 (COX-2) in a time-dependent manner. After knockdown of Notch1 intracellular domain (N1IC), HGF was found to increase the proliferation and migration ability in human gastric cancer cells. However, overexpression of N1IC still had no effect after HGF stimulation. Our study found a feedback loop between HGF/c-Met and Jagged1/Notch1 signaling. Furthermore, both HGF/c-Met and Notch1 signaling triggered COX-2 activity. These results suggest that gastric cancer progression is not associated with a unique signaling pathway and that a feedback loop may exist between the HGF/c-Met and Notch1 signaling pathways, which may result in therapeutic resistance. Therefore, multi-modality therapies should be considered for treating gastric cancer.

Norleual, a hepatocyte growth factor and macrophage stimulating protein dual antagonist, increases pancreatic cancer sensitivity to gemcitabine.

Pancreatic cancer is a leading cause of cancer deaths in the USA and is characterized by an exceptionally poor long-term survival rate compared with other major cancers. The hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) growth factor systems are frequently over-activated in pancreatic cancer and significantly contribute to cancer progression, metastasis, and chemotherapeutic resistance. Small molecules homologous to the 'hinge' region of HGF, which participates in its dimerization and activation, had been developed and shown to bind HGF with high affinity, antagonize HGF's actions, and possess anticancer activity. Encouraged by sequence homology between HGF's hinge region and a similar sequence in MSP, our laboratory previously investigated and determined that these same antagonists could also block MSP-dependent cellular responses. Thus, the purpose of this study was to establish that the dual HGF/MSP antagonist Norleual could inhibit the prosurvival activity imparted by both HGF and MSP to pancreatic cancer cells in vitro, and to determine whether this effect translated into an improved chemotherapeutic impact for gemcitabine when delivered in combination in a human pancreatic cancer xenograft model. Our results demonstrate that Norleual does indeed suppress HGF's and MSP's prosurvival effects as well as sensitizing pancreatic cancer cells to gemcitabine in vitro. Most importantly, treatment with Norleual in combination with gemcitabine markedly inhibited in-vivo tumor growth beyond the suppression observed with gemcitabine alone. These results suggest that dual functional HGF/MSP antagonists like Norleual warrant further development and may offer an improved therapeutic outcome for pancreatic cancer patients.

Differential Response of MET inhibition by Glesatinib (MGCD265) and Sitravatinib (MGCD516) in Non‐small Cell Lung Cancer and Malignant Mesothelioma

Aberrant activation of the receptor tyrosine kinase (RTK) MET is frequently involved in malignant transformation and inhibiting its activity in dependent cancers is associated with improved clinical outcomes. Glesatinib (MGCD265) and sitravatinib (MGCD516) are next generation MET RTK inhibitors (RTKIs) and while glesatinib also targets the AXL RTK, sitravatinib inhibits a closely related spectrum of RTKs. The efficacy of these compounds in MET‐dependent non‐small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines that differed with respect to MET gene expression was evaluated. Both drugs inhibited cell growth and migratory activity with similar potency in NSCLC H1993 cells with clustered MET amplification and A459 cells with ‘wild type’ MET. Also, MPM cell lines H2373 and H2461 with elevated MET expression showed comparable levels of sensitivity to either drug. Kinomics analysis of the affected pathways suggested that both drugs acted similarly, yet distinct signaling was affected when different cell lines were tested. In particular, cellular pathways associated with transformation were inhibited whereas those promoting cell death were activated upon drug treatment. Furthermore, phosphorylation of RON, which interacts with MET, and the downstream signaling molecules AKT and p44/42MAPK were also inhibited in response to hepatocyte growth factor (HGF) stimulation. Importantly, in a pre‐clinical in vivo xenotransplant mouse model, both drugs caused significant tumor growth inhibition. Our data suggest that discerning the expression and activation status of MET and its associated signaling pathways may help guide treatment decisions concerning targeted therapies for NSCLC and MPM.Support or Funding InformationThe National Cancer Institute of the National Institutes of Health under award number P30CA033572 and Mirati Therapeutics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Attenuation of MET-mediated migration and invasion in hepatocellular carcinoma cells by SOCS1.

AIMTo investigate the role of suppressor of cytokine signaling 1 (SOCS1) in regulating MET-mediated invasive potential of hepatocellular carcinoma (HCC) cells.METHODSStable derivatives of mouse (Hepa1-6) and human (hep3B, HepG2) HCC cell lines expressing SOCS1 or control vector were evaluated for their ability to migrate towards hepatocyte growth factor (HGF) in the transwell migration assay, invade extracellular matrix in response to HGF stimulation in a 3-D invasion assay by confocal microscopy, and to undergo anchorage-independent proliferation in semisolid agar. Following intravenous and intrasplenic inoculation into NOD.scid.gamma mice, the ability of Hepa cells to form othotopic tumors was evaluated. Following HGF stimulation of Hepa and Hep3B cells, expression of proteins implicated in epithelial-to-mesenchymal transition was evaluated by western blot and qRT-PCR.RESULTSSOCS1 expression in mouse and human HCC cells inhibited HGF-induced migration through matrigel. In the 3-D invasion assay, HGF stimulation induced invasion of HCC cells across type-I collagen matrix, and SOCS1 expression significantly reduced the depth of invasion. SOCS1 expression also reduced the number and size of colonies formed by anchorage-independent growth in semisolid agar. Following intravenous inoculation, control Hepa cell formed large tumor nodules that obliterated the liver whereas the SOCS1-expressing Hepa cells formed significantly smaller nodules. Tumors formed by SOCS1-expressing cells showed reduced phosphorylation of STAT3 and ERK that was accompanied by reduced levels of MET protein expression. HGF stimulated Hepa cells expressing SOCS1 showed increased expression of E-cadherin and decreased expression of EGR1, SNAI1 and ZEB1. Comparable results were obtained with Hep3B cells. SOCS1 expressing HCC cells also showed reduced levels of EGR1 and SNAI1 transcripts.CONCLUSIONOur findings indicate that loss of SOCS1-dependent control over epithelial-to-mesenchymal transition may contribute to MET-mediated migration, invasion and metastatic growth of HCC.

60P - Understanding and targeting Met signalling in bladder cancer

Background: Bladder cancer affects 430,000 patients and leads to 165,000 deaths annually worldwide. With no major advances in the management of this disease in the last 2 decades, there is an urgent need to identify therapeutic targets with validated biomarkers. Overexpression of Met, a Receptor Tyrosine Kinase, was shown to correlate with poor prognosis in bladder cancer making it an attractive target. Cabozantinib, a Met inhibitor, showed clinical activity in patients with refractory bladder cancer in a clinical trial. However, little is known about how Met exactly signals in bladder cancer and there are no validated biomarkers. This study aims at unravelling Met signalling in bladder cancer. Methods: Western blots and confocal/low light microscopy were used to assess Met signalling and its role in wound healing in Transitional Cell Carcinoma (TCC) cells. Met expression was assessed by immunohistochemistry in tissue samples (n = 64). Results: Met is overexpressed in TCC cells and in 78% of invasive bladder cancer tissues. This was associated with a shorter median survival as compared to Low Met levels (12.97 Vs 18.05 months). Stimulation of TCC cells with Met ligand, Hepatocyte Growth Factor (HGF), triggered Met activation and downstream signalling as well as wound healing, all of which were reduced with Met pharmacological inhibitors including Cabozantinib. The PI3K downstream effector AKT was highly activated upon Met activation. Moreover, class I PI3K inhibition with GDC094 significantly inhibited HGF-dependent wound healing. Interestingly, HGF triggered rapid Met endocytosis in TCC cells. Furthermore, pharmacological inhibition of endocytosis reduced Met downstream signalling. Conclusions: We report that Met is a major target in invasive bladder cancer. Our results further suggest that PI3K may be considered as a co-target of Met to improve patients’ outcome. It may also be developed as a biomarker to help select patients who may respond to Met targeted therapy. Finally, we report for the first time that, upon HGF stimulation, Met gets rapidly endocytosed in TCC cells. Furthermore, inhibiting endocytosis reduced Met dependent signalling. All together, our results open the way for novel strategies to target invasive bladder cancer. Legal entity responsible for the study: Queen Mary University of London Funding: Barts Cancer Institute, Queen Mary University of London Disclosure: T. Powles: Honoraria: AstraZeneca, Bristol-Myers Squibb, Roche and Merck. Research Funding: AstraZeneca, Roche and Merck. L. Menard: Employee: AstraZeneca. All other authors have declared no conflicts of interest.