High Levels Of Hepatocyte Growth Factor Research Articles

Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression.

Prognostic Significance of Targetable Angiogenic and Growth Factors in Patients Undergoing Resection for Gastric and Gastroesophageal Junction Cancers

Circulating factors in patients with gastric/gastroesophageal junction (GEJ) cancers may promote tumor progression and metastasis and may be targeted for therapy. Serum levels of ligands-vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF)-from four targetable pathways were measured before surgery, and levels were correlated to clinicopathologic characteristics and overall survival (OS). In 147 patients who underwent potentially curative resection for gastric/GEJ adenocarcinoma, VEGF-A levels were higher in patients with R1 versus R0 resection (p = 0.037). High EGF levels were associated with poorly differentiated tumors (p = 0.02). Elevated FGF2 levels were found in Lauren diffuse-type tumors (p = 0.017) and tumors with seven or more metastatic nodes (N3) (p < 0.042). Patients with advanced-staged tumors had higher HGF levels (p = 0.012). At a median follow-up of 35 months, 46 patients (31 %) had died. Increased VEGF and HGF levels were correlated with decreased OS (p = 0.009 and 0.005). An adjusted total value (ATV) of all factors was better than any single factor in stratifying patients into good and poor prognosis groups (5-year OS 84.1 vs. 53.9 %, p = 0.005). By multivariate analysis, serum VEGF-A and ATV were significant independent prognostic factors (along with T and N category) for OS (p = 0.028 and 0.013, respectively). In patients undergoing resection for gastric and GEJ cancer, high levels of angiogenic and growth factors are associated with unfavorable tumor characteristics and poorer overall survival. Thus levels of these factors can help delineate tumor biology and stratify prognosis.

Effective inhibition of c-MET-mediated signaling, growth and migration of ovarian cancer cells is influenced by the ovarian tissue microenvironment

The signaling mediated by c-MET and its ligand, hepatocyte growth factor (HGF), has been implicated in malignant progression of cancer involving stimulation of proliferation, invasion, and metastasis. We studied the c-MET/HGF axis as a mediator of tumor-stromal interaction in ovarian cancer and the value of targeting c-MET for the treatment of ovarian cancer. To assess c-MET signaling, we established in vitro models of the microenvironment using primary and immortalized human fibroblasts from normal ovary and tumor samples and epithelial ovarian cancer cell lines. We found that fibroblast from normal ovaries secreted high levels of HGF (1,500 to 3,800 pg/mL) as compared to tumor-derived fibroblasts (undetectable level) and could elicit cellular biological responses on c-MET expressing ovarian cancer cells including increase of cell proliferation and migration (2- to 140-fold increase). HGF secreted by fibroblasts was also found sequestered within extracellular matrices (ECMs) and when degraded this ECM-derived HGF stimulated cancer cell migration (1.5- to 24-fold). In cells containing constitutive c-MET phosphorylation, recombinant HGF and fibroblast-derived HGF negligibly affect c-MET phosphorylation on Tyr1234 and Tyr1003. However, both sources of HGF increased the phosphorylation of c-MET on Tyr1349, the multi-substrate docking site, by more than 6-fold and led to activation of downstream signaling transducers. DCC-2701 (Deciphera Pharmaceuticals, LLC), a novel c-MET/TIE-2/VEGFR inhibitor was able to effectively reduce tumor burden in vivo and block c-MET pTyr1349-mediated signaling, cell growth, and migration as compared to a HGF antagonist in vitro. Importantly, DCC-2701’s anti-proliferative activity was dependent on c-MET activation induced by stromal human fibroblasts and to a lesser extent exogenous HGF. Our data suggest for the first time that DCC-2701 may be superior to HGF antagonists that are in clinical trials and that pTyr1349 levels might be a good indicator of c-MET activation and likely response to targeted therapy as a result of signals from the microenvironment.

Phase II Study Of The c-MET Inhibitor ARQ 197 (Tivantinib) In Patients With Relapsed Or Relapsed/Refractory Multiple Myeloma (RRMM)

Backgroundc-MET receptor tyrosine kinase (RTK) activity has been implicated in establishing the oncogenic phenotype across several human cancers with high levels of the activating c-MET ligand, hepatocyte growth factor (HGF). Malignant plasma cells secrete HGF-activator (HGFA), which converts HGF to its active form, and high HGF levels are correlated with a poor prognosis in multiple myeloma. Syndecan 1 (CD138) on malignant plasma cells binds HGF and potentiates interleukin-6-induced growth and migration. HGF stimulation of myeloma cells also activates autophosphorylation of c-MET and other critical downstream signaling pathways promoting oncogenesis. Finally, pre-clinical studies have shown that suppression of c-MET signaling with a number of small molecules, including ARQ 197, induced myeloma cell apoptosis. Tivantinib-mediated cytotoxic response was observed at concentrations of less than 5 µM, which are achievable in the clinic. These findings supported the hypothesis that suppression of the HGF/c-MET signaling axis could be a rational strategy against RRMM. MethodsIn this phase II study, the efficacy and safety of ARQ 197, a non-competitive and highly selective inhibitor of the c-MET RTK, was studied in patients with RRMM. Primary objectives were to determine the overall response rate (ORR) to single-agent tivantinib in patients who had received one to four prior lines of therapy, and to define the toxicities in this population. A Simon's Minimax 2-stage design was used for the study. ARQ 197 was administered at a starting oral dose of 360 mg twice daily with meals for each day of every 4-week treatment cycle. This dose was selected from prior phase I investigations in solid tumors, and at this dose level, steady-state plasma levels of ARQ 197 were 7 µM. Treatment could continue providing that patients did not experience undue toxicities, or disease progression. Tivantinib is provided through the Cancer Therapy Evaluation Program (CTEP), and this study was supported by CTEP, as well as the MD Anderson Cancer Center SPORE in Multiple Myeloma. ResultsA total of 16 patients were enrolled and treated to date, including 9 men and 7 women, who had received a median of 1 prior line of therapy (range 1-3), including stem cell transplant in ten. The mean patient age was 66 (range 49-76), with ethnicity including 13 Caucasian Americans, 2 African Americans, and 1 Asian American. Patients have received a median of 3 cycles of therapy to date (1-11) with one patient continuing on study, and all were evaluable for toxicity, while 11 were evaluable for response based on having completed two treatment cycles. The most common adverse events (AEs) of any grade seen in at least 25% of patients and felt to be at least possibly drug related included fatigue or decreased neutrophils (94% each), pain (81%), myalgias (56%), diarrhea (38%), memory impairment, respiratory disorders, and rash (31% each), and hypertension (25%), and these were predominantly grade 1 or 2. Grade 3 or 4 AEs included neutropenia (31% and 25%, respectively), syncope, infection, pain (13% of each, all grade 3), and anal fissure, cough, fatigue, hypertension, and pulmonary embolism (6% each, all grade 3). Stable disease (SD) has been seen as the best response in 4/11 (36%) evaluable patients, which was maintained for up to 11 cycles, while the remaining patients showed evidence of disease progression. ConclusionSingle-agent tivantinib has been well tolerated in patients with RRMM, and the ability to achieve stable disease in patients with previously progressing myeloma does support the possibility that targeting c-MET has some promise. Future studies with rationally designed combination regimens incorporating a c-MET inhibitor and other novel agents may better define the role of this class of drugs in our armamentarium against myeloma. Disclosures:Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees. Thomas:Millenium: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Immunomedics: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Shah:Celgene: Consultancy, Research Funding; Array: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Paracrine activation of MET promotes peritoneal carcinomatosis in scirrhous gastric cancer

Scirrhous gastric cancer is associated with abundant stroma and frequently develops into peritoneal carcinomatosis with malignant ascites. Although malignant ascites is among the most deadly diseases worldwide, its molecular pathogenesis is poorly understood. We investigated the role of hepatocyte growth factor (HGF) in the production of peritoneal carcinomatosis with malignant ascites. We examined three scirrhous and three non-scirrhous human gastric cancer cell lines for the production of peritoneal carcinomatosis in vivo and responses to HGF in vitro. Furthermore, clinical scirrhous gastric cancer specimens were examined for HGF production. Among the six cell lines examined, only two scirrhous cell lines (NUGC4 and GCIY) produced peritoneal carcinomatosis with massive ascites after intraperitoneal injection in nude mice. Their proliferation was stimulated by exogenous HGF in vitro. On the other hand, a non-scirrhous cell line, MKN45, with MET amplification generated peritoneal tumors but not ascites. MET tyrosine kinase inhibitors, crizotinib and TAS-115, inhibited HGF-stimulated proliferation of NUGC4 and GCIY as well as constitutive proliferation of MKN45. Furthermore, crizotinib and TAS-115 prolonged the survival of mice bearing established tumors by NUGC4 or MKN45. In clinical specimens, HGF was markedly produced by stromal fibroblasts. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high levels of HGF. Our results strongly suggest that paracrine HGF-induced activation of MET-mediated signaling pathways plays an important role in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, MET signaling pathway may be a potential therapeutic target for peritoneal carcinomatosis of gastric cancer, even without MET amplification.

Signaling Control by Epidermal Growth Factor Receptor and MET: Rationale for Cotargeting Strategies in Lung Cancer

In a report accompanying our article, Spigel et al present their findings on combining erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), with onartuzumab (MetMAb; Genentech, South San Francisco, CA), a humanized monovalent monoclonal antibody that blocks ligand activation of MET receptor tyrosine kinase (RTK). MET-positive patients (defined by immunohistochemistry) treated with erlotinib and onartuzumab demonstrated improved progression-free and overall survival, whereas MET-negative patients had worse outcomes. This article provides an overview of signaling control by EGFR and MET and the biologic rationale for cotargeting strategies. EGFR and MET are membrane-bound RTKs consisting of extracellular domains that bind to growth factors and intracellular domains that contain a kinase domain (Fig 1). MET is activated by its cognate ligand, hepatocyte growth factor (HGF), which is produced by stromal cells, also in an autocrine fashion. EGFR is activated by several ligands, including EGF, transforming growth factor alpha (TGF ), and heparin-binding EGF. On receptor binding of the ligands, the kinase domain becomes activated, leading to transautophosphorylation of tyrosine residues on the cytoplasmic tail. Phosphorylation of these tyrosine residues creates intramolecular docking sites for proteins containing Src homology 2 domains that serve as readers for tyrosine phosphorylation. This includes adaptor proteins such as growth factor receptor–bound protein 2 (GRB2), GAB1 (GRB2-associated binding protein 1), and Src homology 2–containing transforming protein 1 (SHC1). These proteins serve as scaffolds to support signal transduction to the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), phospholipase C (PLC), and signal transducer and activator of transcription (STAT) pathways. EGFR and MET use a highly overlapping repertoire of signaling adaptors and downstream effector pathways, highlighting their combined ability to codrive oncogenic signaling. Three general approaches are being employed in clinic to target EGFR and MET. The first uses small-molecule TKIs, such as erlotinib for EGFR and crizotinib for MET, that bind the ATP-binding pocket in the kinase domain and inhibit the kinase function. The second approach uses antibody therapy directed against the extracellular domain of the MET receptor (onartuzumab) or the HGF ligand (ficlatuzumab). The third approach targets downstream proteins involved in signaling, such as inhibitors of PI3K, or molecules regulating receptor trafficking and degradation, such as heat shock proteins. The EGFR pathway can be activated in lung cancer through genomic events, such as activating EGFR mutations or gene amplification, or through tumor environmental events, such as autocrine production of EGFR-activating ligands. Likewise, MET activation occurs through HGF produced in the tumor microenvironment and through MET gene amplification. MET amplification by fluorescent in situ hybridization occurs in approximately 5% to 15% of lung adenocarcinomas. Increased MET is associated with advanced stage of disease, poor outcome, brain metastasis, and high levels of EGFR. Coactivation of EGFR and MET occurs in subsets of lung cancer cell lines and tumor tissues and is associated with aggressive disease. In cells lacking EGFR mutation or high levels of MET, activation of either receptor can produce extensive signaling crosstalk to the opposing RTK and downstream substrates. EGFR activation can induce accumulation of activated MET protein through a c-SRC– dependent mechanism, and dual MET/EGFR inhibition has shown increased activity in xenograft models. Phosphoproteomic analysis of lung tumors and cell lines can identify tumors with concomitant EGFR and MET activation. EGFR, either through mutation or ligand-mediated activation, can also regulate MET levels through hypoxia-inducible factor-1 . Importantly, MET signaling is one actionable mechanism driving resistance to EGFR TKIs. MET activation can drive intrinsic as well as acquired resistance to EGFR through genomic and tumor microenvironment–mediated mechanisms. Primary resistance to anti-EGFR antibody therapy has been associated with MET activation in primary explant models of lung cancer. Approximately 5% of EGFR mutation–positive tumors with acquired resistance to EGFR TKIs are found to have MET gene amplification. Mechanistically, amplified MET co-opts use of ERBB3 to maintain the PI3K pathway activation. Combining MET and EGFR TKIs or EGFR/HSP90 inhibition has been effective in mouse models of lung cancer with T790M EGFR and MET amplification. High levels of HGF are associated with intrinsic or acquired resistance to EGFR TKIs in patients with lung cancer with activating EGFR mutations and in those with other solid tumors. HGF production by stromal fibroblasts leads to fibroblast-induced JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 31 NUMBER 32 NOVEMBER 1

Multipotent Human Mesenchymal Stromal Cells Mediate Expansion of Myeloid-Derived Suppressor Cells via Hepatocyte Growth Factor/c-Met and STAT3

SummaryMesenchymal stromal cells (MSCs) are multilineage progenitors with immunomodulatory properties, including expansion of immunomodulatory leukocytes such as regulatory T lymphocytes (Tregs) and tolerogenic dendritic cells. We report that human MSCs can expand CD14−CD11b+CD33+ human myeloid-derived suppressor cells (MDSCs). MSC-expanded MDSCs suppress allogeneic lymphocyte proliferation, express arginase-1 and inducible nitric oxide synthase, and increase the number of Tregs. This expansion occurs through the secretion of hepatocyte growth factor (HGF), with effects replicated by adding HGF singly and abrogated by HGF knockdown in MSCs. In wild-type mice, the liver, which secretes high levels of HGF, contains high numbers of Gr-1+CD11b+ MDSCs, and injection of HGF into mice significantly increases the number of MDSCs. Expansion of MDSCs by MSC-secreted HGF involves c-Met (its receptor) and downstream phosphorylation of STAT3, a key factor in MDSC expansion. Our data further support the strong immunomodulatory nature of MSCs and demonstrate the role of HGF, a mitogenic molecule, in the expansion of MDSCs.

Abstract 1627: Fibroblast HGF elicits c-MET-mediated signaling and migration in ovarian cancer cells.

Abstract Background: Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynecologic malignancies diagnosed in the U.S. due to its rapid progression without symptom. Stromal components in the ovary contribute to rapid progression of cancer and interruption of their interaction with cancer cells are being explored as important therapeutic target. Hepatocyte growth factor (HGF) is expressed primarily in mesenchymal cells and acts on epithelial cells. HGF is also a unique cytokine that induces pleiotropic biological responses, involving proliferation, invasion, and dissemination of cancer cells. We studied c-MET/HGF axis as a mediator of tumor-stromal interactions in ovarian cancer using human ovarian fibroblast and their derived extracellular matrices (ECM). Methods: Six EOC cells were each cultured alone or in the presence of three human ovarian fibroblast cultures and their derived ECM in Transwell plates and the number of migrated cells was quantitated. ELISA was used to measure the concentration of HGF secreted by cancer cells and fibroblasts or released from fibroblast-derived ECM. EOC cells were treated with conditioned media derived from fibroblast that secretes high level of HGF and c-MET mediated signalling was assessed by immunoblotting. Results: Five out of the six EOC cells tested highly expressed c-MET and none produced detectable levels of HGF. IHFNO-303, a human ovarian fibroblast line, secreted the highest level of HGF (∼3,800 pg/mL) and markedly enhanced migration (2 to 140-fold increase) of the EOC cells that express c-MET. Recombinant HGF and a HGF neutralizing antibody were used to validate HGF as a major migratory factor secreted by fibroblast. HGF sequestered within the fibroblast-derived ECM was also able to stimulate cell migration by 1.5 to 24-fold, when released via EOC-associated degradation of the ECM. In cells containing constitutive c-MET phosphorylation, recombinant HGF and fibroblast-derived HGF negligibly affect c-MET phosphorylation on Tyr1234 and Tyr1003. However, both sources of HGF increased the phosphorylation of c-MET on Tyr1349, the multisubstrate docking site, by more than 6-fold and led to activation of downstream signalling transducers, e.g., AKT and ERK, in all c-MET expressing EOC cells. Therefore, signalling mediated by c-MET and HGF likely occurs in a paracrine manner in ovarian cancer. Conclusion: Our study demonstrates the functional contribution of c-MET and HGF signalling in malignant progression driven by important tumor-stromal interactions. We demonstrate for the first time that phosphorylation of c-MET on Tyr1349 requires extraneous HGF and phosphorylated c-MET level on Try1349 might be a good indicator of c-MET activation in the microenvironment and likely responsive to c-MET targeted therapy. Citation Format: Youngjoo Kwon, Yan Zhou, Andrew K. Godwin. Fibroblast HGF elicits c-MET-mediated signaling and migration in ovarian 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 1627. doi:10.1158/1538-7445.AM2013-1627

Cabozantinib in Prostate Cancer: The Beginning of a Precision Paradigm?

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

Phase II Study of the c-Met Inhibitor ARQ 197 (Tivantinib) in Patients with Relapsed Multiple Myeloma.

Abstract 2976 Background:c-Met receptor tyrosine kinase (RTK) activity has been implicated in establishing the oncogenic phenotype across several human cancers with high levels of the activating c-Met ligand, hepatocyte growth factor (HGF). Malignant plasma cells secrete HGF-activator (HGFA), which converts HGF to its active form, and high HGF levels are correlated with a poor prognosis in multiple myeloma (MM). Syndecan 1 (CD138) on malignant plasma cells binds HGF and potentiates interleukin-6-induced growth and migration. HGF stimulation of myeloma cells also activates autophosphorylation of c-Met and other critical downstream signaling pathways promoting oncogenesis. Finally, pre-clinical studies have shown that suppression of c-Met signaling with a number of small molecules, including ARQ 197, induced myeloma cell apoptosis. Tevantinib-mediated cytotoxic response was observed at concentrations of less than 5 μM, which are achievable in the clinic. These findings supported the hypothesis that suppression of the HGF/c-Met signaling axis could be a rational strategy against relapsed multiple myeloma. Methods:In this phase II study, the efficacy and safety of ARQ 197, a non-competitive and highly selective inhibitor of the c-Met RTK, is being studied in patients with relapsed multiple myeloma. Primary objectives were to determine the overall response rate (ORR) to single-agent tivantinib in patients with relapsed multiple myeloma who had received one to four prior lines of therapy, and to define the toxicities in this population. ARQ 197 was administered at a starting oral dose of 360 mg twice daily with meals for each day of every 4-week treatment cycle. This dose was selected from prior phase I investigations in solid tumors, and at this dose level, steady-state plasma level sof ARQ-197 were 7 μM. Treatment could continue providing that patients did not experience undue toxicities, or disease progression. Tivantinib is provided through the Cancer Therapy Evaluation Program (CTEP), and this study is supported by CTEP, as well as the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results:A total of 10 patients have been enrolled and treated to date, all of whom were evaluable for toxicity, with 8 evaluable for response based on having completed two treatment cycles. Patients had received from 1–3 prior lines of therapy for their disease, and 7/10 (70%) had presented with International Staging System stage I disease at diagnosis. All patients on study had an ECOG performance status of 1 or better, and received a median of 3.5 cycles of tivantinib (range 1–7). The most common adverse events (AEs) of any grade seen in at least 30% of patients included diarrhea (30%), dizziness (30%), dry eyes (30%), shortness of breath (30%), memory change (30%), myalgias (40%), fatigue (60%), and neutropenia (60%). Serious AEs (SAEs) occurred in 2 patients, including one patient with grade 3 syncope, and another with grade 4 neutropenia and a grade 3 anal fissure. Stable disease (SD) has been seen as the best response in 5/7 (71%) evaluable patients, which was maintained for up to 7 cycles, while the remaining patients showed evidence of disease progression. Conclusion:Enrollment is continuing to this first study of any c-Met inhibitor in patients with relapsed multiple myeloma to better define the role of single-agent tivantinib in this setting. To date, tivantinib has been tolerated well, and some evidence of activity has been seen, with stable disease in 63% of patients, all of whom were progressing at the time of enrollment. Updated toxicity and efficacy data will be presented at the time of the Annual Meeting. Correlative studies are also underway with the goal of identifying potential predictive biomarkers. Disclosures:Off Label Use: Tivantinib is being evaluated for patients with relapsed myeloma, but is not yet approved in this setting.

Targeting the Hepatocyte Growth Factor–cMET Axis in Cancer Therapy

The hepatocyte growth factor (HGF) and its receptor, the transmembrane tyrosine kinase cMET, promote cell proliferation, survival, motility, and invasion as well as morphogenic changes that stimulate tissue repair and regeneration in normal cells but can be co-opted during tumor growth. MET overexpression, with or without gene amplification, has been reported in a variety of human cancers, including breast, lung, and GI malignancies. Furthermore, high levels of HGF and/or cMET correlate with poor prognosis in several tumor types, including breast, ovarian, cervical, gastric, head and neck, and non-small-cell lung cancers. Gene amplification and protein overexpression of cMET drive resistance to epidermal growth factor receptor family inhibitors, both in preclinical models and in patients. It is increasingly apparent that the HGF-cMET axis signaling network is complex, and rational combinatorial therapy is needed for optimal clinical efficacy. Better understanding of HGF-cMET axis signaling and the mechanism of action of HGF-cMET inhibitors, along with the identification of biomarkers of response and resistance, will lead to more effective targeting of this pathway for cancer therapy.

Mesenchymal Stem Cells Overexpressing Hepatocyte Growth Factor (HGF) Inhibit Collagen Deposit and Improve Bladder Function in Rat Model of Bladder Outlet Obstruction

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly male. This study was performed to examine the capability of human mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF) to inhibit collagen deposition in rat model of bladder outlet obstruction (BOO). HGF is known for its antifibrotic effect and the most promising agent for treating bladder fibrosis. BM3.B10 stable immortalized human MSC line (B10) was transduced to encode human HGF with a retroviral vector was prepared (B10.HGF). Two weeks after the onset of BOO, B10, and B10.HGF cells were injected into the rat's bladder wall. After 4 weeks, bladder tissues were harvested and Masson's trichrome staining was performed. Transgene expression in HGF-expressing B10 cells was demonstrated by reverse transcriptase polymerase chain reaction and immunohistochemical staining, and the high levels of HGF secreted by B10.HGF cells was confirmed by ELISA. The mean bladder weight in BOO rats was 5.8 times of the normal controls, while in animals grafted with B10.HGF cells, the weight was down to four times of the control [90.2 ± 1.6 (control), 89.9 ± 2.8 (sham), 527.9 ± 150.9 (BOO), 447.7 ± 41.0 (BOO + B10), and 362.7 ± 113.2 (BOO + B10.HGF)]. The mean percentage of collagen area increased in BOO rats, while in the animals transplanted with B10.HGF cells, the collagen area decreased to the normal control level [12.2 ± 1.3, (control), 12.8 ± 1.1 (sham), 26.6 ± 2.7 (BOO), 19.9 ± 6.0 (BOO + B10), and 13.3 ± 2.1 (BOO + B10.HGF)]. The expression of collagen and TGF-b protein increased after BOO, while the expression of HGF and c-met protein increased in the group with B10.HGF transplantation after BOO. Intercontraction interval decreased after BOO, but it recovered after B10.HGF transplantation. Maximal voiding pressure (MVP) increased after BOO, and it recovered to levels of the normal control after transplantation of B10.HGF cells. Residual urine volume (RU) increased after BOO, but the RU increase was not reversed by transplantation of B10.HGF cells. Human MSCs overexpressing HGF inhibited collagen deposition and improved cystometric parameters in bladder outlet obstruction of rats. The present study indicates that transplantation of MSCs modified to overexpress HGF could serve as a novel therapeutic strategy against bladder fibrosis in patients with bladder outlet obstruction.

The levels and clinical significances of hepatocyte growth factor in the hepatic tissue and serum of hepatocellular carcinoma

Objective To investigate hepatocyte growth factor (HGF) levels in the tissue and serum of patients with chronic hepatitis, cirrhosis or hepatocellular carcinoma (HCC), and analyze the clinical significances of HGF for HCC. Method Surgical specimens from 97 patients were collected during Dec.2003 to Aug.2008 in the Third Central Hospital.The patients were prospectively enrolled and categorized into four groups:normal subjects (n=11), chronic hepatitis B or C (n=6), cirrhosis (n=20) and HCC (n= 60) including well-differentiated (n= 21), moderately differentiated (n= 23), poorly differentiated (n= 16) specimens.N0 (n=24), N1 (n=21), N2 (n=54) and N3 (n=43) were tissues respectively removed from liver at 0, 1, 2 or 3 cm beyond the margin of tumor.HGF mRNA expression in liver tissues was determined by real-time quantitative reverse transcription- (RT)-PCR.Serum HGF levels in the other cases of normal subjects (n=20), chronic hepatitis B or C (n=20), cirrhosis (n=20) and HCC (n=57) were measured by ELISA.The Kaplan-Meier method with log-rank test was employed for survival analysis.Univariate and multivariate analyses were performed to identify prognostic factors in each group. Results The HGF mRNA in normal subjects, chronic hepatitis, cirrhosis, N3, N2, N1, N0 and HCC were 0.99(0.78-1.66), 2.15(1.06-3.40), 1.78(1.18-2.73), 4.59(2.67-8.63), 3.86(2.25-6.45)，3.12(1.59-5.74), 2.92(0.88-5.99) and 0.48(0.19-1.06) respectively.The serum concentration of HGF in the normal subjects, chronic hepatitis, cirrhosis and HCC patients were (0.31±0.05), (0.65±0.07), (1.27±0.30) and （2.06±0.66） μg/L respectively.The highest level of HGF mRNA was found in N3, while the HGF mRNA expression in HCC was ［（2.14±0.52）μg/L］ lower than that not only in the non-tumor tissues, but also in the normal control (U=196.50, P=0.03).The serum concentration of HGF was significantly higher in patients with chronic hepatitis, cirrhosis or HCC than in normal subjects.The serum HGF level of HCC was bounced after hepatectomy (t=2.70, P＜0.01).On the logistic regression analysis，the tumor numbers and Child-pugh were related with the levels of the tissue HGF mRNA and serum HGF of HCC, OR were 0.15 (95%CI：0.03-0.72, P<0.05) and 0.13 (95%CI：0.27-0.89, P<0.05), respectively.Univariate analysis using the Cox proportional hazards model in the complication groups revealed that the levels of the tissue HGF mRNA and serum HGF were significant risk factors of death for HCC, OR were 0.02 (95%CI:0.00-0.52, P<0.05) and 10.01 (95%CI:1.16-86.23, P< 0.05), respectively.On the Log-rank analysis, no statistically difference in the cumulative survival was found between the two groups categorized by median (0.49) of tissue HGF mRNA 2-ΔΔCT (χ2=0.13，P=0.72). While the HCC patients were dichotomized by their the median(0.69 μg/L) of serum HGF concentration, the death risk for the patients with higher levels of HGF was increased 2.84 fold than those with lower levels (95%CI:1.03-7.92，P<0.05). Conclusion HGF mRNA expression is decreased in tumor tissues, while its level in tumor adjacent live and serum is significantly elevated and is in association with shortened postoperative survival of HCC patients.（Chin J Lab Med,2012,35:598-603） Key words: Liver neoplasms; Carcinoma，hepatocellular; Hepatocyte growth factor; Reverse transcription-quantitative polymerase chain; Enzyme-linked immunosorbent assay

Abstract 1896: Hepatocyte growth factor induces resistance to anti-epidermal growth factor receptor antibody in lung cancer

Abstract Introduction: Epidermal growth factor receptor (EGFR) is an attractive drug target in lung cancer, with several anti-EGFR antibodies and small-molecule inhibitors showing efficacy in lung cancer patients. Patients, however, may develop resistance to EGFR inhibitors. We demonstrated previously that hepatocyte growth factor (HGF) induced resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) in lung cancers harboring EGFR mutations. We therefore determined whether HGF could induce resistance to the anti-EGFR antibody (EGFR Ab) cetuximab in lung cancer cells, regardless of EGFR gene status. Methods: Cetuximab sensitivity and signal transduction in lung cancer cells were examined in the presence or absence of HGF, HGF-producing fibroblasts, and cells tranfected with the HGF gene in vitro and in vivo. Results: HGF induced resistance to cetuximab in H292 (EGFR wild) and Ma-1(EGFR mutant) cells. Western blotting showed that HGF-induced resistance was mediated by the Met/Gab1/Akt signaling pathway. Resistance of H292 and Ma-1 cells to cetuximab was also induced by co-culture with lung fibroblasts producing high levels of HGF and by cells stably transfected with the HGF gene. This resistance was abrogated by treatment with anti-HGF neutralizing antibody. Conclusions:HGF-mediated resistance is a novel mechanism of resistance to EGFR Ab in lung cancers, with fibroblast-derived HGF inducing cetuximab resistance in H292 tumors in vivo. The involvement of HGF-Met-mediated signaling should be assessed in acquired resistance to EGFR Ab in lung cancer, regardless of EGFR gene status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1896. doi:1538-7445.AM2012-1896

Hepatocyte Growth Factor Induces Resistance to Anti-Epidermal Growth Factor Receptor Antibody in Lung Cancer

Epidermal growth factor receptor (EGFR) is an attractive drug target in lung cancer, with several anti-EGFR antibodies and small-molecule inhibitors showing efficacy in lung cancer patients. Patients, however, may develop resistance to EGFR inhibitors. We demonstrated previously that hepatocyte growth factor (HGF) induced resistance to EGFR tyrosine kinase inhibitors in lung cancers harboring EGFR mutations. We therefore determined whether HGF could induce resistance to the anti-EGFR antibody (EGFR Ab) cetuximab in lung cancer cells, regardless of EGFR gene status. Cetuximab sensitivity and signal transduction in lung cancer cells were examined in the presence or absence of HGF, HGF-producing fibroblasts, and cells tranfected with the HGF gene in vitro and in vivo. HGF induced resistance to cetuximab in H292 (EGFR wild) and Ma-1(EGFR mutant) cells. Western blotting showed that HGF-induced resistance was mediated by the Met/Gab1/Akt signaling pathway. Resistance of H292 and Ma-1 cells to cetuximab was also induced by coculture with lung fibroblasts producing high levels of HGF and by cells stably transfected with the HGF gene. This resistance was abrogated by treatment with anti-HGF neutralizing antibody. HGF-mediated resistance is a novel mechanism of resistance to EGFR Ab in lung cancers, with fibroblast-derived HGF inducing cetuximab resistance in H292 tumors in vivo. The involvement of HGF-Met-mediated signaling should be assessed in acquired resistance to EGFR Ab in lung cancer, regardless of EGFR gene status.

Gene Transfer of c-met Confers Protection Against d-Galactosamine/Lipopolysaccharide-Induced Acute Liver Failure

Acute liver failure (ALF) is characterized by impaired regeneration of hepatocytes, partially resulting from the defective signaling between elevated levels of hepatocyte growth factor (HGF) and the downregulation of its receptor, c-met. In this study, we assess the therapeutic efficacy of in vivo c-met gene transfer in ALF. We established a D: -galactosamine/lipopolysaccharide (D: -GalN/LPS)-induced ALF model in rats. The levels of HGF and c-met in D: -GalN/LPS rats were compared with vehicle-injected rats and those undergoing liver regeneration following partial hepatectomy (PH). To deliver c-met in vivo, pcDNA-c-met was constructed and transfected into the rat liver via hydrodynamic injection. Control rats were transfected with the pcDNA3.1 plasmid. The expressions of c-met in different tissues were examined by immunohistochemistry. Hepatocyte proliferation and apoptosis were determined by PCNA staining and TUNEL assay, respectively. The survival of rats in the control and c-met-expressing rats was compared by Kaplan-Meier analysis. Compared with control rats, D: -GalN/LPS or PH -treated rats had significantly higher levels of HGF (P < 0.01). D: -GalN/LPS also led to significantly lower c-met expression in the liver (P < 0.01) than PH. The in vivo c-met gene transfer led to its specific expression on hepatocytes, which was accompanied by the enhancement of hepatocyte proliferation, a reduction in apoptosis, as well as significant improvement in overall survival (P < 0.01). This study provides the initial evidence that c-met may serve as a novel target for gene therapy, and may be of clinical benefit in the treatment of patients with ALF.

Hepatocyte growth factor in patients with coronary artery disease and its relation to periodontal condition

Hepatocyte growth factor (HGF) is an angiogenic, cardioprotective factor important for tissue and vascular repair. High levels of HGF are associated with chronic inflammatory diseases, such as coronary artery disease (CAD) and periodontitis, and are suggested as a marker of the ongoing atherosclerotic event in patients with CAD. Periodontal disease is more prevalent among patients with CAD than among healthy people. Recent studies indicate a reduced biological activity of HGF in different chronic inflammatory conditions. Biologically active HGF has high affinity to heparan sulfate proteoglycan (HSPG) on cell-membrane and extracellular matrix. The aim of the study was to investigate the serum concentration and the biological activity of HGF with ELISA and surface plasmon resonance (SPR), respectively, before and at various time points after percutaneous coronary intervention (PCI) in patients with CAD, and to examine the relationship with periodontal condition. The periodontal status of the CAD patients was examined, and the presence of P. gingivalis in periodontal pockets was analyzed with PCR. The HGF concentration was significantly higher, at all time-points, in patients with CAD compared to the age-matched controls (P< 0.001), but was independent of periodontal status. The HGF concentration and the affinity to HSPG adversely fluctuated over time, and the biological activity increased one month after intervention in patients without periodontitis. We conclude that elevated concentration of HGF but with reduced biological activity might indicate a chronic inflammatory profile in patients with CAD and periodontitis.

Circulating levels of hepatocyte growth factor and left ventricular remodelling after acute myocardial infarction (from the REVE-2 study)

Aim As experimental studies suggest that hepatocyte growth factor (HGF) is cardioprotective after myocardial infarction (MI), this study sought to investigate relationships between circulating levels of HGF and left ventricular (LV) remodelling in patients after acute MI. This prospective multicentre study included 246 patients with a first anterior Q-wave MI. Serial echocardiographic studies were performed at hospital discharge and 3 and 12 months after MI; quantitative analysis was performed at a core echocardiography laboratory. Blood samples to measure HGF, brain natriuretic peptide (BNP), and C-reactive protein were obtained at discharge and at the 1, 3, and 12 month follow-up visits. Plasma HGF levels were high at baseline, decreased at 1 month, and remained stable thereafter. In the post-MI period (at 3 and 12 months), HGF levels were positively associated with LV volumes, wall motion systolic index, E/Ea, and BNP; and negatively with LV ejection fraction. High HGF levels were associated with higher C-reactive protein levels. Multivariate analysis showed that both BNP (P < 0.0001) and C-reactive protein (P < 0.0001) were independently associated with HGF levels at 3 and 12 months. Patients who died or were rehospitalized for heart failure during follow-up had higher HGF levels at 1 month (P = 0.0006), 3 months (P = 0.018), and 1 year (P = 0.006) after MI. Circulating HGF levels correlate with all markers of LV remodelling after MI and are associated with rehospitalization for heart failure.

Clinical Correlations and Prognostic Relevance of Tissue Angiogenic Factors in Patients with Gastric Cancer

AimsTo evaluate the relationship between vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) levels in gastric cancer tissue and clinicopathological features and to determine whether these factors were correlated with survival. Materials and methodsWe analysed tissue samples from 58 patients with gastric cancer and used 24 normal gastric mucosae as controls. Tissue levels of VEGF and HGF were measured in tissue extracts by enzyme-linked immunosorbent assay. ResultsHGF and VEGF levels were significantly higher in gastric cancer tissue than in matched normal gastric mucosa. VEGF levels were significantly increased in cancer tissue from cases involving lymphatic invasion. HGF levels were significantly increased according to the disease stage. Patients with high levels of VEGF or HGF showed significantly worse survival rates than patients with low levels. Using multivariate analysis, a high level of VEGF or HGF was an independent factor predicting poor survival. ConclusionsIntratumoral levels of HGF and VEGF are an important prognostic determinant in gastric cancer. The current findings suggest that high concentrations of HGF and VEGF may induce aggressive tumour growth and metastasis.

Abstract A67: The aromatase gene (CYP19A1) variants and circulating hepatocyte growth factor in postmenopausal women

Abstract Background: In postmenopausal women, estrogen biosynthesis takes place predominantly in adipose tissue through the catalyzed conversion from androgens by the aromatase enzyme. Variation in the CYP19A1 gene, which encodes the aromatase enzyme, is involved in the regulation of estrogen levels. Recent evidence also suggests that estrogen regulates several cancer-related adipocytokines including hepatocyte growth factor (HGF). HGF acts as an angiogenic growth factor in both an autocrine and paracrine fashion in various cell types to promote cell migration, proliferation, and invasion. It is plausible that the CYP19A1 gene which alters estrogen production also influences HGF concentrations. We accordingly conducted an evaluation of the CYP19A1 gene in relation to circulating HGF levels among postmenopausal women in the Women's Health Initiative Observational Study (WHI-OS). Methods: We included in the present analysis a total of 45 CYP19A1 functional and common variants that were in Hardy-Weinberg equilibrium and had a minor allele frequency of ≥5%. As the distribution of HGF levels was highly skewed (p value for normality &amp;lt;0.001), we transformed HGF concentrations for all women into a log-, ranked-, or normal score (van der Waerden (VW) normal score method)-scale value. The associations between the CYP19A1 gene variants and the transformed HGF concentrations were assessed through a linear regression as implemented in PLINK with an additive scoring for 0, 1, or 2 copies of the minor allele of each variant. The analyzed models were adjusted for age (continuous) and body mass index (continuous). We also used linear regression to estimate haplotype-specific association with the rest of the haplotypes as the referents. To control for comparisons for multiple SNPs (or haplotypes), we performed 10,000 permutations to generate a gene-specific (familywise) empirical p value for each SNP (or haplotype) and to determine how frequently the identified association would occur by chance. We performed these permutations using the max(T) permutation option in PLINK. Results: We observed an association between the rs7172156, rs1008805, rs6493494, rs749292, and rs11636639 variants and circulating HGF levels in ranked and normal score scales (corrected p values ≤0.04). Specifically, each additional copy of the minor allele of rs7172156 (T) and rs1008805 (C) was associated with lower mean HGF levels. By contrast, the minor alleles of rs6493494 (T), rs749292 (T), and rs11636639 (C) were each associated with higher HGF levels. These 5 SNPs, which were in linkage disequilibrium (pairwise D′ ≥97%, r2 ≥56%), collectively contributed ≥9% of total variance in ranked- and normal score-transformed HGF. Additionally, these 5 variants constituted block #6 of the CYP19A1 gene with 2 common haplotypes. One common haplotype (42%) containing the minor alleles of rs7172156 (T) and rs1008805 (C) was associated with lower HGF levels, whereas the other haplotype (40%) with the minor alleles of rs6493494 (T), rs749292 (T), and rs11636639 (C) was associated with higher HGF levels. The association of these 5 SNPs with log-scale HGF was not significant (corrected p values ≥0.17). Conclusion: Our study suggests a potential association between the CYP19A1 gene variants and circulating HGF levels in ranked or normal score scales. However, our findings are exploratory and require confirmation in larger observational studies. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A67.