Hepatocyte growth factor/c-Met signaling axis in human diseases: Mechanistic insights and therapeutic potential.

The primary purpose of this study was to demonstrate that the hepatocyte growth factor (HGF) antagonist Norleual is capable of attenuating the cellular responses of pancreatic cancer cells to HGF. The angiotensin IV analog Norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] exhibits structural homology with the hinge (linker) region of HGF and acts to block HGF dimerization, a process required for its activation. Norleual competitively inhibited the binding of a H3-Hinge peptide sequence to the HGF, and binds directly to HGF with a Ki = 3.6x10-12M. Predictably, Norleual is able to block the cellular responses due to HGF activation in multiple cell types at concentrations in the picomolar range including HGF/c-Met sensitive pancreatic cancer cells. Norleual's ability to inhibit the growth and survival of pancreatic cancer cells was evaluated using viability testing and cell sorting tags to identify living, dead and apoptotic cells. HGF/c-Met activation increases the invasive potential of the pancreatic cancer cells, which provides a mechanistic basis for the ability of c-Met to support pancreatic tumor metastasis. In this regard Norleual as an HGF antagonist was able to block the migration and invasion of pancreatic cancer cells through collagen gels in a transwell chamber. Over-activation of the growth factor system HGF/c-Met is a critical contributor to pancreatic cancer's ability to disseminate rapidly and its refractoriness to standard chemotherapy. Norleual, which is a representative of a “first-in-class” group of molecules that blocks the activation step of HGF, represent a novel therapeutic approach to the treatment of pancreatic cancer. Citation Format: Leen H. Kawas, Phillip Harris, Kevin Church, John W. Wright, Joseph W. Harding. The angiotensin IV analog Norleual acts as a hepatocyte growth factor/c-Met inhibitor in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 926. doi:10.1158/1538-7445.AM2013-926

The primary purpose of this study was to demonstrate that the hepatocyte growth factor (HGF) antagonist norleual is capable of attenuating the cellular responses of cancer cells to HGF. The angiotensin IV analog norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] exhibits structural homology with the hinge (linker) region of HGF and acts to block HGF dimerization, a process required for its activation. Norleual competitively inhibited the binding of a H3-Hinge peptide sequence to the HGF, and binds directly to HGF with a Ki = 3.6x10-12M. Predictably, norleulal is able to block the cellular responses due to HGF activation in multiple cell types at concentrations in the picomolar range including HGF/c-Met sensitive pancreatic cancer cells. Norleual's ability to inhibit the growth and survival of several cell lines including pancreatic cancer cells was evaluated using viability testing and cell sorting tags to identify living, dead and apoptotic cells. HGF/c-Met activation increases the invasive potential of the pancreatic cancer cells, which provides a mechanistic basis for the ability of c-Met to support pancreatic tumor metastasis. In this regard norleual as an HGF antagonist was able to block the migration and invasion of cancer cells through collagen gels in a transwell chamber. Over-activation of the growth factor system HGF/c-Met is a critical contributor to cancer's ability to disseminate rapidly and its refractoriness to standard chemotherapy. Norleual, which is a representative of a “first-in-class” group of molecules that blocks the activation step of HGF, represent a novel therapeutic approach to the treatment of HGF/Met sensitive cancers including pancreatic cancer. Citation Format: Leen H. Kawas, Kevin J. Church, Malte Lange, Michelle Mcmicheal, Brent Yamamoto, Joseph W. Harding. Norleual a hepatocyte growth factor/c-Met inhibitor blocks malignant phenotypes in cancerous cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 729. doi:10.1158/1538-7445.AM2014-729

Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which affects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/ HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal fibroblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-dl cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/ NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis.

The study reported in the article by Smith et al that accompanies this editorial presents data on a novel orally bioavailable tyrosine kinase inhibitor (TKI), cabozantinib, with activity against multiple targets including mesenchymal-epithelial transition factor (MET), also known as hepatocyte growth factor receptor, and the vascular endothelial growth factor receptor 2 (VEGFR2). The authors describe 171 men with castration-resistant prostate cancer (CRPC), a subgroup of patients from a larger phase II randomized discontinuation trial that included multiple tumor types treated with cabozantinib. The trial included a lead-in stage of open-label therapy with cabozantinib and a planned blinded random assignment to cabozantinib or placebo for patients who achieved stable disease at 12 weeks. As reported by the authors, the trial was suspended after 122 patients with CRPC were enrolled onto the open-label portion of the study because of improvements in bone scans and a decrease in pain. At the time the study was halted, a group of 31 patients had been randomly assigned. In this group, there was a marked improvement in the primary end point of progression-free survival (PFS) in the patients receiving cabozantinib compared with placebo (median, 23.9 v 5.9 weeks, respectively; hazard ratio, 0.12; P .001). Although impressive as a small phase II study, the completion of ongoing phase III studies will be critical to confirm the survival benefit. COMET-1, with a primary end point of overall survival, is a placebo-controlled trial of 960 men with CRPC and additional prior therapy randomly assigned to cabozantinib or prednisone (NCT01605227). COMET-2 is a placebo-controlled phase III trial evaluating cabozantinib versus mitoxantrone and prednisone in men with previously treated symptomatic CRPC (NCT01522443), with an emphasis on the effect of cabozantinib on pain and bone disease. Clearly, the results reported by Smith et al support the completion of these larger more definitive studies; perhaps just as important, however, is the need to understand these data as a direction toward more precise multitargeted therapy in prostate cancer. As one potential target in prostate cancer, the understanding of the role of MET, or MET in conjunction with VEGFR, is the subject of ongoing studies. Increased expression of MET protein in tumor cells compared with normal tissue has been associated with tumor grade. An evaluation of the MET ligand hepatocyte growth factor (HGF) in patients from a prior Cancer and Leukemia Group B 9480 study of suramin revealed a correlation between high plasma levels of HGF and decreased patient survival. Studies have also demonstrated that androgen receptor (AR) signaling represses the expression of MET, which supports future studies of targeting the combination of the AR and MET pathways. With the understanding that cabozantinib also inhibits the VEGF pathway, it is of interest that prior laboratory studies have supported the importance of angiogenesis in prostate cancer progression and provide a rationale for combined MET and VEGF pathway inhibition. Additional laboratory studies have demonstrated HGF-mediated angiogenesis through VEGF regulation and an association between VEGF regulation and the activation of c-MET signaling in prostate cancer. Clinical results of antiangiogenic agents alone in prostate cancer, however, have not been optimal, which supports the hypothesis that targeting multiple pathways in addition to VEGF need to be considered to fully influence the complex nature of the tumor microenvironment. In this regard, efforts to understand the effect of agents such as cabozantinib on the MET pathway with additional multitargeted effects such as VEGF inhibition are likely to be important. In fact, there are multiple agents targeting the MET pathway under investigation that have varying degrees of specificity for additional targets. In addition to MET and VEGFR2, cabozantinib has been shown in preclinical models to inhibit RET, KIT, AXL and FLT3 pathways. Cabozantinib has also been demonstrated to have distinct biologic effects on angiogenesis, invasiveness, and metastasis in animal models, as would be expected from these molecular targets. Other MET pathway–focused therapeutic approaches under investigation include antibodies against HGF such as rilotumumab (which is being evaluated in lung, gastric, and gynecologic malignancies) and antibodies against MET such as onartuzumab (which is being evaluated in a phase III trial in combination with erlotinib v erlotinib alone in patients with MET-positive non–small-cell lung carcinoma [NSCLC; NCT01456325]). Receptor TKIs targeting MET are under investigation and include nonselective TKIs such as cabozantinib (as studied in the clinical trial by Smith et al), crizotinib (which targets MET as well as ALK and is approved for the treatment of EML4-ALK positive NSCLC), and foretinib (which inhibits c-MET, VEGFR2, PDGFR, and KIT). More target-selective MET TKIs are also under investigation including agents such as tivantinib (ARQ197; Arqule, Woburn, MA), EMD 1214063 (EMD Serono, Darmstadt, Germany), EMD 1204831 (EMD Serono), AMG 337 (Amgen, Thousand Oaks, CA), and INCB028060 (Incyte, Wilmington, DE). JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 4 FEBRUARY 1 2013

Lymphangiogenesis is key to the lymphatic spread of cancer cells. The current study examined the potential effect of hepatocyte growth factor (HGF), a factor known to have strong biological effects on endothelial cells, on the lymphangiogenic function of endothelial cells and the formation of lymphatic vessels using both in vitro and in vivo models. Human endothelial cells that have lymphatic characteristics, human prostate and breast cancer cells PC-3 and MDA MB 231, were used. Expression of lymphatic markers, podoplanin, Prox-1, vascular endothelial growth factor receptor 3 (VEGF-R3) and LYVE-1 was determined using reverse transcription polymerase reaction and quantitative PCR. In nude mice prostate and breast xenograft tumour models, either HGF or an HGF-producing fibroblast cell line MRC-5 was given with or without the HGF antagonist, NK4. The lymphangiogenic marker and lymphatic vessels in tumour tissues were also assessed using quantitative PCR and immunohistochemistry, respectively. In the mice tumour models, infusion of rhHGF significantly increased the levels of podoplanin and LYVE-1 in the tumour (p=0.05 for podoplanin and p<0.05 for LYVE-1 vs. without HGF in the prostate tumour model, p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF for the breast tumour model; p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF in the breast tumour model). The increased level of LYVE-1 transcript was supported by an increase in the number of LYVE-1-positive lymphatic vessels in tumours, using immunohistochemical analysis. Co-injection of MRC5 cells also increased the levels of LYVE-1 and number of LYVE-1-positive vessels in tumour tissues. The effects of HGF and MRC5 were significantly reduced by the HGF antagonist, NK4. In the in vitro models, rhHGF significantly increased the level of both podoplanin and LYVE-1, as shown by quantitative PCR analysis. Hepatocyte growth factor has potential lymphangiogenic activities, and this may have important implications in the nodal spread of cancer cells.

Advances in mesenchymal to epithelial transition factor signaling pathway and inhibitors

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), is active in colorectal cancer (CRC). However, response rates range from only 10% to 20%. Here, we investigate hepatocyte growth factor (HGF)-dependent mesenchymal-epithelial transition factor (MET) activation as a mediator of cetuximab resistance through signal diversification in CRC cell lines. DiFi, GEO, and LIM1215 cells were treated with varying concentrations and combinations of EGF, HGF, cetuximab, and PHA-665752 (a highly specific MET kinase inhibitor). Biological end points included proliferation, cell cycle arrest, and apoptosis. Proliferation was measured using WST-1 assays and synergy investigated via isobolograms. Expression and signaling were examined using immunoblotting. EGFR and MET are coexpressed in these CRC cell lines, and dual receptor activation synergistically increased proliferation. Cetuximab inhibited cell growth by 60%-80% with an associated dephosphorylation of EGFR, MAPK, and/or AKT. Addition of HGF to cetuximab-treated cells phosphorylated MET, but not EGFR or ErbB3, restimulated the MAPK and AKT pathways, restored cell proliferation, and rescued cells from G1 arrest and apoptosis. Importantly, this effect could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating MET expression with RNAi. HGF-induced MET activation is a novel mechanism of cetuximab resistance in CRC. Inhibition of the HGF-MET pathway may improve response to EGFR inhibitors in CRC, and combination therapy should be further investigated.

Chapter 15 - Use of MET kinase inhibitors to overcome cetuximab resistance in colorectal cancer

Mutations in the genes encoding for Met, Ret and Kit receptor tyrosine kinases invariably result in increased kinase activity and in the acquisition of transforming potential. However, the requirement of receptor ligands for the transformation process is still unclear. We have investigated the role of hepatocyte growth factor (HGF), the high-affinity ligand for Met, in mutant Met-mediated cell transformation. We provide evidence that the transforming potential displayed by mutant forms of Met found in human cancer is not only sensitive but entirely dependent on the presence of HGF, by showing that mutant Met transforms NIH3T3 fibroblasts, which produce endogenous HGF, but is not able to transform epithelial cells, unless exogenous HGF is supplied. Accordingly, mutant Met-induced transformation of NIH3T3 cells can be inhibited by HGF antagonists and increased by HGF stimulation. We also show that an engineered Met receptor which contains an oncogenic mutation but is impaired in its ability to bind HGF completely loses its transforming activity, which can be rescued by causing receptor dimerization using a monoclonal antibody. These results indicate that point mutations resulting in Met kinase activation are necessary but not sufficient to cause cell transformation, the latter being dependent on ligand-induced receptor dimerization. They also suggest that mutant Met-driven tumour growth depends on the availability and tissue distribution of active HGF, and provide proof-of-concept for the treatment of mutant-Met related pathologies by HGF-antagonizing drugs.

Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET

To explore the effects of hepatocyte growth factor (HGF) on retinal pigment epithelium (RPE) cell behaviors. The human adult retinal pigment epithelial cell line-19 (ARPE-19) were treated by HGF or mesenchymal-epithelial transition factor (MET) inhibitor SU11274 in vitro. Cell viability was detected by a Cell Counting Kit-8 assay. Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay, respectively. The expression levels of MET, phosphorylated MET, protein kinase B (AKT), and phosphorylated AKT proteins were determined by Western blot assay. The MET and phosphorylated MET proteins were also determined by immunofluorescence assay. HGF increased ARPE-19 cells' viability, proliferation and migration, and induced an increase of phosphorylated MET and phosphorylated AKT proteins. SU11274 significantly reduced cell viability, proliferation, and migration and decreased the expression of MET and AKT proteins. SU11274 suppressed HGF-induced increase of viability, proliferation, and migration in ARPE-19 cells. Additionally, SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins. HGF enhances cellular viability, proliferation, and migration in RPE cells through the MET/AKT signaling pathway, whereas this enhancement is suppressed by the MET inhibitor SU11274. HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.

Hepatocyte growth factor (HGF) plays a definitive role in invasive, angiogenic, and metastatic activities of tumor cells by binding to the c-Met receptor. NK4, a competitive antagonist for HGF and the c-Met receptor, prevents tumor cell growth and metastasis via its bifunctional properties to act as an HGF antagonist and angiogenesis inhibitor. In the present study, we investigated the inhibitory effectiveness of NK4 on hematogenous pulmonary metastasis of the CT26 murine colon cancer cell line, focusing on tumor cell adhesion to endothelial cells. In an in vitro adhesion assay, HGF facilitated adhesion of CT26 cells to a murine endothelial cell line (F-2) in a dose-dependent manner. Furthermore, the enhancing effect of HGF on CT26-F-2 cell interaction was blocked by NK4 as well as by anti-HGF antibody. Similarly, HGF-induced phosphorylation of focal adhesion kinase (FAK), downstream of integrin signaling, was reduced by NK4 and by anti-HGF antibody. However, distinct integrin expression on the surface of CT26 cells was not altered by HGF. In an in vivo experimental pulmonary metastasis assay, stable NK4 expression potently decreased the number of pulmonary metastatic foci. The NK4-induced suppression of pulmonary metastasis was partially reversed when HGF was intraperitoneally administered in an adhesive phase. These results suggest that NK4 could act on tumor cells to inhibit CT26 adhesion to endothelial cells by reducing FAK phosphorylation, which is regulated by inside-out HGF/c-Met signaling, and thereby suppress hematogenous pulmonary metastasis.

Hepatocyte growth factor (HGF), acting through the c‑Met receptor, plays an important role in solid tumors. Various malignant cells utilize the biological actions of the HGF/c‑Met pathway for their dissociative, invasive and metastatic behaviors. HGF also binds to the receptor expressed on endothelial cells that stimulates angiogenesis, a process critical to continued growth of solid tumors. It is known that HGF induces invitro expression of vascular endothelial growth factor (VEGF), a key agonist of tumor angiogenesis. In the present study, we showed using invitro co-culture system with fibroblasts that VEGF expression of CT26 cells was amplified through tumor-stromal interaction, i.e., the HGF paracrine loop. This action was inhibited by interruption of the HGF paracrine loop by gene transfer of NK4, an HGF antagonist. In invivo experiments, CT26 tumor growth and angiogenesis were markedly enhanced by fibroblast co-inoculation, while the effect of fibroblasts was not observed in NK4‑expressing CT26 cells. These findings suggest that NK4 exerted potent anti‑angiogenic action via indirectly inhibiting VEGF expression of tumor cells in addition to direct effects on endothelial cells. Thus, the HGF/c‑Met pathway may be a considerable candidate for molecular targeting strategy against tumor angiogenesis.

Crystal structure of the NK1 fragment of human hepatocyte growth factor at 2.0 A resolution.