HGF Production Research Articles

FORRAJE VERDE HIDROPÓNICO COMO ALTERNATIVA FORRAJERA EN LA ALIMENTACIÓN ANIMAL

<p><strong>Background. </strong>Animal feeding and nutrition constitute the main limitation in livestock farming in arid and semi-arid zones, because it depends to a large extent on imported concentrated feed. Hydroponic green fodder (FVH) represents an efficient and economical alternative to obtain food with high nutritional value for domestic animals. However, little has been used due to ignorance of the method to produce it and its benefits. <strong>Objective. </strong>To analyze the scientific information available on the agronomic, productive and nutritional attributes of HGF, and its potential as a forage resource in animal feed. <strong>Methodology. </strong>A bibliographic review was carried out based in the following databases: Scopus, NCBI, Springer, Science direct, Google Scholar, Redalyc, ResearchGate and other digital repositories, using keywords in Spanish and English, such as hydroponic green fodder, hydroponic technology, hydroponic food and alternative fodder. <strong>Results.</strong> The HGF production cycle is basically carried out in six stages: <em>i</em>) seed selection, <em>ii</em>) seed washing and disinfection, <em>iii</em>) seed hydration, <em>iv</em>) sowing and germination, <em>v</em>) growth, and <em>vi</em>) harvest. Biomass production increases progressively in relation to a higher seed sowing density. The optimum harvest period for forage is from 12 to 14 days after sowing. However, the volume and yield of production must be assessed in terms of the nutritional quality of harvested fodder. In prolonged harvest periods, the crude protein content increases, while the dry matter content decreases. In cattle and goats, partial diet supplementation with HGF improves milk production and composition, as well as the weight gain rate and feed ratio, due to increased feed intake and nutrients digestibility. In monogastrics, the total replacement of conventional feed with HGF affects feed intake and growth rate. <strong>Implications. </strong>More research is required to determine the adequate portion of HGF in the supplementation of animal diets, without affecting their productive performance, especially in the case of monogastrics. <strong>Conclusion. </strong>The HGF technology represents a viable and economical alternative to counteract the scarcity of fresh and nutritious food, especially in dry seasons of arid and semi-arid areas; as well as in urban or suburban areas where there is limited land area for conventional fodder production.</p>

Modulation of Fibroblast Phenotype by Colorectal Cancer Cell-Secreted Factors Is Mostly Independent of Oncogenic KRAS.

KRAS mutations have been shown to extend their oncogenic effects beyond the cancer cell, influencing the tumor microenvironment. Herein, we studied the impact of mutant KRAS on the modulation of the pro-tumorigenic properties of cancer-associated fibroblasts (CAFs), including α-SMA expression, TGFβ1 and HGF production, extracellular matrix components and metalloproteinases expression as well as collagen contraction and migration capacities. To do so, CCD-18Co normal-like colon fibroblasts were challenged with conditioned media from control and KRAS silenced colorectal cancer (CRC) cells. Our results showed that the mutant KRAS CRC cell-secreted factors were capable of turning normal-like fibroblasts into CAF-like by modulating the α-SMA expression, TGFβ1 and HGF production and migration capacity. Oncogenic KRAS played a secondary role as its silencing did not completely impair the capacity of CRC cells to modulate most of the fibroblast properties analyzed. In summary, our work suggests that mutant KRAS does not play a major role in controlling the CRC cell-secreted factors that modulate the behavior of fibroblasts. The fact that CRC cells retain the capacity to modulate the pro-tumorigenic features of fibroblasts independently of KRAS silencing is likely to negatively impact their response to KRAS inhibitors, thus standing as a putative mechanism of resistance to KRAS inhibition with potential therapeutical relevance.

Analysis of hepatocyte growth factor immunostaining in the placenta of HIV-infected normotensive versus preeclamptic pregnant women

ObjectiveHepatocyte Growth Factor (HGF) plays a role in the migration and morphogenesis of different cell types and tissues. Preeclampsia (PE) is associated with deficient trophoblast invasion and placental insufficiency; hence HGF production is expected to be compromised. This study therefore aimed to immunolocalize and morphometrically analyse placental HGF in normotensive versus PE pregnancies stratified by HIV status and gestational age. Study designNormotensive (N; n = 40) and preeclamptic (PE; n = 80) women were stratified by HIV status (HIV- and HIV+), and gestational age i.e. early onset of PE (EOPE; <34 weeks) and late onset of PE (LOPE; ≥34 weeks). Placental tissues were stained using conventional immunohistochemistry, performed using mouse anti-human HGF antibody. Morphometric image analysis was performed using Zeiss Axio-Vision software. ResultsHGF was immuno-localized within the syncytiotrophoblast, cytotrophoblast, endothelial and fibroblast-like cell populations of both conducting and exchange villi. Based on pregnancy type, HGF immunoexpression within the conducting villi was significantly different between Nvs EOPE (p = 0.0372) and EOPE vs LOPE (p = 0.0006). Within the exchange villi, no significant difference of HGF immunostaining was noted between N vs EOPE and N vs LOPE. A down-regulation of HGF immuno-expression was observed in LOPE compared to other groups within both villi types, albeit non-significant. Based on HIV status, no significant difference in HGF immuno-expression was demonstrated between HIV- vs HIV + within the exchange and conducting villi. However, the expression of HGF in HIV- group was elevated in both villi types. Across the groups, a significant difference was found between N+ vs EOPE- (p = 0.0207), EOPE+ vs LOPE- (p = 0.0036) and EOPE- vs LOPE- (p = 0.0016) of the conducting villi while no significant difference was found within the exchange villi. ConclusionThis novel study demonstrates that HGF was two-fold higher in conducting compared to exchange villi irrespective of the pregnancy type. HIV infection does not influence HGF expression within the conducting and exchange villi. The HGF/c-MET receptor complex may modulate the ligand expression within the placenta.

MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

BackgroundFibroblasts are crucial mediators of tumor-stroma cross-talk through synthesis and remodeling of the extracellular matrix and production of multiple soluble factors. Nonetheless, little is still known about specific determinants of fibroblast pro-tumorigenic activity in lung cancer. Here, we aimed at understanding the role of miRNAs, which are often altered in stromal cells, in reprogramming fibroblasts towards a tumor-supporting phenotype.MethodsWe employed a co-culture-based high-throughput screening to identify specific miRNAs modulating the pro-tumorigenic potential of lung fibroblasts. Multiplex assays and ELISA were instrumental to study the effect of miRNAs on the secretome of both primary and immortalized lung fibroblasts from lung cancer patients and to evaluate plasmatic levels of HGF in heavy smokers. Direct mRNA targeting by miRNAs was investigated through dual-luciferase reporter assay and western blot. Finally, the pro-tumorigenic activity of fibroblasts and their conditioned media was tested by employing in vitro migration experiments and mouse xenografts.ResultsWe identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers’ plasma correlates with levels of tobacco exposure. Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts reduced their ability to promote tumor growth and that HGF plays a central role in the pro-tumorigenic activity of fibroblasts.ConclusionsOverall, these results uncover a central role for miR-16 in regulating HGF production by lung fibroblasts, thus affecting their pro-tumorigenic potential. Correlation between smoking exposure and miR-16 levels could provide novel clues regarding the formation of a tumor-proficient milieu during the early phases of lung cancer development.

Efficacy of combined use of plasmid constructs containing HGF and angiopoietin-1 genes to restore blood flow in ischemic tissues

New methods to stimulate blood supply of the ischemic organs and tissues are being intensively developed worldwide. These approaches are based on revascularization and remodeling of the newly formed blood vessels. This strategy was called therapeutic angiogenesis. Using in vitro, ex vivo and in vivo models we investigated the specific biological activity and angiogenic potential of Vascopoietin, which contained the plasmids for HGF and angiopoietin-1 expression. Vascopoietin stimulated vascular cell migration, proliferation and the formation of capillary-like structures in vitro and ex vivo. Using in vivo model of posterior limb ischemia in mice we demonstrated that Vascopoietin administration mediated stable HGF and angiopoietin-1 production resulting in new blood vessel formation and their stabilization in the ischemic muscles. In addition, Vascopoietin injection led to the restoration of the blood flow, decrease in the size of necrosis in ischemic limb and the reduction in the amputation frequency. The current data suggest Vascopoietin a promising drug for therapeutic angiogenesis.

The human kidney capsule contains a functionally distinct mesenchymal stromal cell population.

We recently demonstrated that the adult human kidney cortex contains a perivascular stromal cell (kPSC) that shows organotypic properties and is important for repair and stabilisation of kidney function. Not only the kidney cortex but also the kidney capsule contains stromal cells that are important for the three dimensional organisation of the kidney during nephrogenesis. They provide the barrier function of the capsule which is critical for homeostatic processes such as pressure natriuresis. We postulated that stromal cells derived from the kidney capsule may therefore also have specific properties and functions. To this end, we isolated these capsule mesenchymal stromal cells (cMSC) from human cadaveric kidneys that were not suitable for transplantation. There were several similarities between cMSCs and kPSCs including support of vascular plexus formation, phenotypic marker expression and resistance against myofibroblast transformation. However, compared to kPSCs, cMSCs showed distinct mRNA and miRNA expression profiles, showed increased immunosuppressive capacity, and displayed strongly reduced HGF production, contributing to the inability to enhance kidney epithelial repair. Therefore cMSCs are a distinct, novel human kidney-derived MSC-population and these data underpin the large functional diversity of phenotypic similar stromal cells in relation to their anatomic site, even within one organ.

Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip.

Physiologically, four major types of hepatic cells - the liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and hepatocytes - reside inside liver sinusoids and interact with flowing peripheral cells under blood flow. It is hard to mimic an in vivo liver sinusoid due to its complex multiple cell-cell interactions, spatiotemporal construction, and mechanical microenvironment. Here we developed an in vitro liver sinusoid chip by integrating the four types of primary murine hepatic cells into two adjacent fluid channels separated by a porous permeable membrane, replicating liver's key structures and configurations. Each type of cells was identified with its respective markers, and the assembled chip presented the liver-specific unique morphology of fenestration. The flow field in the liver chip was quantitatively analyzed by computational fluid dynamics simulations and particle tracking visualization tests. Intriguingly, co-culture and shear flow enhance albumin secretion independently or cooperatively, while shear flow alone enhances HGF production and CYP450 metabolism. Under lipopolysaccharide (LPS) stimulations, the hepatic cell co-culture facilitated neutrophil recruitment in the liver chip. Thus, this 3D-configured in vitro liver chip integrates the two key factors of shear flow and the four types of primary hepatic cells to replicate key structures, hepatic functions, and primary immune responses and provides a new in vitro model to investigate the short-duration hepatic cellular interactions under a microenvironment mimicking the physiology of a liver.

Peritumoral stromal neutrophils are essential for c-Met-elicited metastasis in human hepatocellular carcinoma

ABSTRACTInflammation is a component of tumor progression mechanisms. Neutrophils are a common inflammatory infiltrate in many tumors, but their regulation and functions in neoplasia are not understood. Here, we showed, in detailed studies of c-Met molecule in 225 untreated patients with hepatocellular carcinoma (HCC), that high infiltration of neutrophils in HCC tissues determined malignant cell c-Met-associated clinical outcome of patients. High infiltration of neutrophils in HCCs determined malignant cell c-Met-associated clinical outcome of patients. Neutrophils were enriched predominantly in invading tumor edge of HCCs; the accumulated neutrophils were the major source of c-Met ligand HGF in HCCs. Exposure to HCC environments resulted in neutrophil activation and the following HGF production. Inhibiting the activities of Erk1/2, p38, and NF-κB, but not the phosphorylation of AKT or JNK, successfully attenuated the neutrophil HGF production induced by HCC environments. Further investigation revealed that GM-CSF was an important determinant in malignant cell-elicited neutrophil HGF production in vitro and in vivo. Moreover, we demonstrated that tumor neutrophils, via HGF/c-Met interaction, actively enhanced the metastasis of malignant cells in vitro and in vivo. These data provide direct evidence supporting the critical role of neutrophils in human tumor progression and reveal a fine-tuned collaborative action between cancer cells and immune cells in tumor milieu, which reroutes the immune activation into a tumor-promoting direction.

Abstract 1219: Sym015, a novel antibody mixture targeting non-overlapping epitopes of MET, effectively inhibits growth of MET dependent tumors and overcomes resistance to a single monoclonal antibody

Abstract The tyrosine kinase receptor MET is involved in progression of a variety of human cancers and constitutes a promising therapeutic target. Particularly, subsets of tumors originating from lung or gastric tissues appear to be truly MET dependent. MET dependency is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, kinase activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents in lung and colorectal cancers. Sym015, a novel antibody mixture comprising two monoclonal antibodies targeting non-overlapping epitopes on the SEMA domain of MET, was shown to effectively inhibit cell growth in vitro through effective MET degradation. In the present study, we screened a large panel of highly annotated human cancer cell lines for sensitivity to Sym015 in order to identify potential markers of response. Sym015 effectively inhibited growth of cell lines with MET-amplification, MET-exon 14 deletion, and autocrine HGF production, including MET-amplified cell lines with acquired resistance to EGFR targeting agents. To validate the in vitro findings, a range of cell line- and patient-derived xenograft models with MET amplification or Exon 14 deletion were tested for sensitivity to Sym015 and an analogue of the clinical stage anti-MET monoclonal antibody emibetuzumab (LY2875358). Sym015 effectively inhibited growth of tumors with autocrine HGF production, MET-amplification, and/or Exon 14 deletion, and had superior activity compared to the emibetuzumab analogue in many of the models. Importantly, tumors with a partial response to the emibetuzumab analogue were strongly inhibited by subsequent treatment with Sym015 in two MET-amplified models, one of which also harbors a MET-exon 14 deletion. In summary, our findings demonstrate a potent antitumor effect of Sym015 in MET-dependent models. The data thus strongly support initiation of clinical trials for patients with MET-amplification and Exon 14 deletions. Citation Format: Thomas T. Poulsen, Michael M. Grandal, Helle J. Jacobsen, Dorte S. Hansen, Trine Lindsted, Mikkel W. Pedersen, Ivan D. Horak, Michael Kragh, Johan Lantto. Sym015, a novel antibody mixture targeting non-overlapping epitopes of MET, effectively inhibits growth of MET dependent tumors and overcomes resistance to a single monoclonal antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1219.

Abstract 1218: A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer models

Abstract The receptor tyrosine kinase MET (Hepatocyte Growth Factor Receptor, HGFR) has been associated with development and progression of a range of human tumors due to its regulation of cell proliferation, migration, invasion, and angiogenesis. A subset of human tumors, particularly of lung or gastric origin, appear to have a primary dependency on MET, which is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents. Sym015 is a mixture of two humanized monoclonal antibodies against non-overlapping epitopes on MET. The specific pair of antibodies was identified in a high-throughput cell based screen searching for antibody mixtures with superior growth inhibitory activity against four MET-dependent cell lines. Both antibodies bind the SEMA domain of MET with high affinity. Synergistic activity was confirmed by dose-response curves in several MET-dependent cell lines and cell line and patient-derived xenograft models. Mechanistically, Sym015 blocks HGF binding to MET, induces MET internalization and degradation effectively diminishing MET oncogenic signaling. Sym015 also induces higher levels of antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro compared to individual mAbs. In conclusion, Sym015, a novel monoclonal antibody mixture against MET, shows strong inhibitory activity against MET driven cell lines and xenografts due to a combined effect of MET degradation and secondary effector functions. These data support initiation of clinical trials. Citation Format: Michael M. Grandal, Thomas T. Poulsen, Klaus Koefoed, Karsten W. Eriksen, Anna Dahlman, Paolo Conrotto, Thomas Bouquin, Helle Jacobsen, Ivan D. Horak, Michael Kragh, Johan Lantto, Mikkel W. Pedersen. A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1218.

PDGFRα plays a crucial role in connective tissue remodeling.

Platelet derived growth factor (PDGF) plays a pivotal role in the remodeling of connective tissues. Emerging data indicate the distinctive role of PDGF receptor-α (PDGFRα) in this process. In the present study, the Pdgfra gene was systemically inactivated in adult mouse (α-KO mouse), and the role of PDGFRα was examined in the subcutaneously implanted sponge matrices. PDGFRα expressed in the fibroblasts of Pdgfra-preserving control mice (Flox mice), was significantly reduced in the sponges in α-KO mice. Neovascularized areas were largely suppressed in the α-KO mice than in the Flox mice, whereas the other parameters related to the blood vessels and endothelial cells were similar. The deposition of collagen and fibronectin and the expression of collagen 1a1 and 3a1 genes were significantly reduced in α-KO mice. There was a significantly decrease in the number and dividing fibroblasts in the α-KO mice, and those of macrophages were similar between the two genotypes. Hepatocyte growth factor (Hgf) gene expression was suppressed in Pdgfra-inactivated fibroblasts and connective tissue. The findings implicate the role of PDGFRα-dependent ECM and HGF production in fibroblasts that promotes the remodeling of connective tissue and suggest that PDGFRα may be a relevant target to regulate connective tissue remodeling.

Identification of core active disaccharides in heparin for HGF-inducing activity.

Objectives:To ascertain the positions of sulfated groups for HGF-inducing activity using differently sulfated heparin disaccharides and to investigate whether the heparin disaccharide elevates HGF levels in plasma in vivo and exerts protective effects on acute liver injury.Materials and Methods:The heparin disaccharides ΔUA-GlcNS, ΔUA (2S)-GlcN, ΔUA-GlcNAc (6S), ΔUA-GlcNS (6S), ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S), ΔUA-GlcNAc and ΔUA (2S)-GlcNS (6S) were added to MRC-9 fibroblasts and HGF concentrations in culture media were determined by enzyme-linked immunosorbent assay. Furthermore, ΔUA-GlcNS (100 μg/head) was injected into C57BL/6 mice and plasma levels of HGF measured at 12 h. After acute hepatitis was induced by CCl4 (15 mg/kg) in mice, liver specimens were stained with hematoxylin and eosin (H and E). Levels of aspartate aminotransferase and alanine aminotransferase were measured at 24 h.Results:Among the disaccharides investigated, ΔUA-GlcNS, ΔUA-GlcNAc (6S) and ΔUA-GlcNS (6S) stimulated HGF production in MRC-9 fibroblasts. However, none of the 2-O-sulfated disaccharides [ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S) and ΔUA (2S)-GlcNS (6S)] showed any activity despite the presence of N-sulfated and/or 6-O-sulfated disaccharides. Thus, 2-O-sulfation of hexuronic acid has an inhibitory effect. Moreover, ΔUA-GlcNS administration increased plasma levels of HGF in normal mice and prevented CCl4-induced liver injury in mice.Conclusions:N-sulfation and/or 6-O-sulfation of glucosamine with nonsulfated hexuronic acid provides a structural basis for the HGF-inducing activity of disaccharides. ΔUA-GlcNS increases plasma levels of HGF and protects against CCl4-induced acute liver injury.

Abstract 3935: A perfused 3D co-culture model of vemurafenib-resistant melanoma

Abstract Background: Vemurafenib(VF) is highly active against melanomas with a BRAFV600E mutation. Although remarkable clinical responses occur, progression ensues, in part due to innate resistance driven by the tumor micro-environment (TME). 2D co-culture screens have demonstrated that the cMet-HGF axis is an important mediator of TME-associated resistance. However, many aspects of the TME including cell-extracellular matrix (ECM), ligand-receptor interactions and signaling are different in 3D versus 2D cultures, thus potentially limiting the clinical utility of a 2D co-culture screen. We report our initial results of a 3D perfused co-culture model of VF-resistant melanoma as a clinically relevant screen for identifying agents and combinations to revert VF resistance. Methods: Media conditions, 3D hydrogel scaffolds and cell ratios were optimized prior to establishing 3D co-cultures. Stromal components and tumor cells were assessed for adaptation to 3D perfused conditions using the 3DKUBE™ segregated co-culture plasticware. Multi-syringe pumps achieved flow rates of 10ul/min. Analysis consisted of cell viability, dsDNA quantification, confocal imaging and immunoassays (secreted HGF and c-Met). Results: Fibroblast HGF production correlated with increasing cell numbers in 3D perfusion, but not in 2D and there was significantly more HGF produced by fibroblasts in 3D perfusion (2504 v 1639 pg/ml, p&amp;lt;.05). Melanoma spheres maintained cell viability better in Matrigel ™ versus Matrigel:Collagen mix and retained 3D structure in perfused co-culture for 7 days. VF showed increased activity in melanoma spheroids in 3D ECM-free static cultures versus 2D monolayer (IC50 0.016uM v 15.79µM). There was a concentration-dependent response but reduced activity of VF against perfused 3D melanoma spheroids embedded in ECM, whereas there was no effect of VF against perfused melanoma spheroids in co-culture. Conclusion: V600E melanoma cells are extremely sensitive to BRAF targeting in 3D ECM free spheroids as compared with 2D monolayers. Clinical resistance is mimicked by a more complex 3D TME comprising ECM, perfusion and segregated co-culture of HGF-secreting fibroblasts in 3D. Mechanistic studies are ongoing to identify specific factors critical for VF resistance, including HGF/cMet axis interrogation. Future Direction: The ultimate goal is to establish clinically relevant 3D perfused co-culture models to identify novel agents or combinations which will overcome TME-mediated resistance to VF and other targeted agents. Future efforts are focused on incorporation of well characterized primary malignant epithelial cells with defined stromal components in the 3D perfused co-culture model in order to better identify combinations of multitargeted agents in both the preclinical(lead compound selection) and the co-clinical process (patient selection). Citation Format: Chaitra Cheluvaraju, Stephen Shuford, Christina Mattingly, Teresa DesRochers, Matthew Gevaert, David E. Orr, Hal E. Crosswell. A perfused 3D co-culture model of vemurafenib-resistant melanoma. [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 3935. doi:10.1158/1538-7445.AM2014-3935

Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients

BackgroundHepatocyte growth factor (HGF) is a pleiotropic cytokine which can lead to cancer cell proliferation, migration and metastasis. In multiple myeloma (MM) patients it is an abundant component of the bone marrow. HGF levels are elevated in 50% of patients and associated with poor prognosis. Here we aim to investigate its source in myeloma.MethodsHGF mRNA levels in bone marrow core biopsies from healthy individuals and myeloma patients were quantified by real-time PCR. HGF gene expression profiling in CD138+ cells isolated from bone marrow aspirates of healthy individuals and MM patients was performed by microarray analysis. HGF protein concentrations present in peripheral blood of MM patients were measured by enzyme-linked immunosorbent assay (ELISA). Cytogenetic status of CD138+ cells was determined by fluorescence in situ hybridization (FISH) and DNA sequencing of the HGF gene promoter. HGF secretion in co-cultures of human myeloma cell lines and bone marrow stromal cells was measured by ELISA.ResultsHGF gene expression profiling in both bone marrow core biopsies and CD138+ cells showed elevated HGF mRNA levels in myeloma patients. HGF mRNA levels in biopsies and in myeloma cells correlated. Quantification of HGF protein levels in serum also correlated with HGF mRNA levels in CD138+ cells from corresponding patients. Cytogenetic analysis showed myeloma cell clones with HGF copy numbers between 1 and 3 copies. There was no correlation between HGF copy number and HGF mRNA levels. Co-cultivation of the human myeloma cell lines ANBL-6 and JJN3 with bone marrow stromal cells or the HS-5 cell line resulted in a significant increase in secreted HGF.ConclusionsWe here show that in myeloma patients HGF is primarily produced by malignant plasma cells, and that HGF production by these cells might be supported by the bone marrow microenvironment. Considering the fact that elevated HGF serum and plasma levels predict poor prognosis, these findings are of particular importance for patients harbouring a myeloma clone which produces large amounts of HGF.

Astragalosides IV inhibits high glucose-induced cell apoptosis through HGF activation in cultured human tubular epithelial cells

Astragaloside IV (ASI) in Radix Astragali is believed to be the active component. The study aims to investigate whether ASI inhibits tubular epithelial cells apoptosis induced by high glucose and its mechanisms. Tubular epithelial cells in this paper were isolated from human kidney. The cells apoptosis was detected by TUNEL and caspase 3 assay. The protein levels of HGF and TGF-β1 were measured by ELISA. The phospho-p38 production, ERK and JNK were determined by Western blot. ASI could inhibit cells apoptosis induced by high glucose (25 mmol/L) in dose-dependent and time-dependent manners. ASI also inhibited high glucose-induced expression of TGF-β1 and activation of p38 MAPK pathway at the protein level. Furthermore, ASI increased HGF production in human tubular epithelial cells. The ASI inhibition of tubular epithelial cells apoptosis and reduction of TGF-β1 expression induced by high glucose may represent a new treatment for diabetic kidney injury. The mechanism underlying this inhibitory effect may be related to the inhibition of p38 MAPK signaling pathway activation and HGF overproduction.

Coordinated induction of cyclooxygenase-2/prostaglandin E2 and hepatocyte growth factor by apoptotic cells prevents lung fibrosis

Apoptotic cell instillation after bleomycin induces persistent HGF production and protects from pulmonary fibrosis, but the underlying mechanism remains unclear. We investigated immediate and prolonged effects of in vivo instillation of apoptotic cells into bleomycin-stimulated mouse lungs (2 days old) on COX-2 expression in lung tissue and alveolar macrophages and PGE2 production in BALF. Furthermore, functional interaction between these molecules and HGF, following apoptotic cell instillation in a bleomycin-induced lung fibrosis model, was assessed. Apoptotic cell instillation results in enhanced immediate and prolonged expression of COX-2 and PGE2 when compared with those from bleomycin-only-treated mice. Coadministration of the COX-2-selective inhibitor NS398 or the selective PGE2R EP2 inhibitor AH6809 inhibited the increase in HGF production. Inhibition of HGF signaling using PHA-665752 inhibited increases in COX-2 and PGE2. Long-term inhibition of COX-2, PGE2, or HGF reversed the reduction of TGF-β, apoptotic and MPO activities, protein levels, and hydroxyproline contents. Up-regulation of COX-2/PGE2 and HGF through a positive-feedback loop may be an important mechanism whereby apoptotic cell instillation exerts the net results of anti-inflammatory, antiapoptotic, and antifibrotic action.

754 Tumorigenesis Promoted by Colibactin-Producing Escherichia coli Explained by the Dark Side of Cellular Senescence

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

757 Impact of Metabolic Syndrome on Incident or Recurrent Advanced Adenoma After Colonoscopy

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

755 Obesity Accelerates Helicobacter felis-Induced Gastric Carcinogenesis by Enhancing TH17 Responses and Immature Myeloid Cell Trafficking

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

Abstract 3042: LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2.

Abstract Under normal conditions MET receptor activation, internalization, and degradation is a highly controlled process regulated by the binding of its ligand, HGF. In many cancers however, MET becomes highly dysregulated, and MET overexpression, with or without gene amplification, is associated with a poorer prognosis. Aberrant activation of MET can also occur through mutation, or increased autocrine or paracrine HGF production. MET activation leads to increased oncogenic activities including cell migration, proliferation, invasion, survival, tumor neovascularization and decreased apoptosis. Several inhibitors of MET, including type I and type II kinase inhibitors, are currently being investigated in clinical trials. LY2801653 is a type II MET kinase inhibitor being investigated in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). In addition to MET, LY2801653 also inhibits ROS1 kinase, and the serine/threonine kinases MKNK1/2, with cell-based IC50 of 23nM and 7nM respectively. ROS1 is an orphan receptor that can be constitutively activated as a fusion protein either by genomic microdeletion or translocation. ROS1 fusion proteins are found in glioblastoma, NSCLC and cholangiocarcinoma. MKNK1/2 are kinases downstream of ERK and p38 and are capable of regulating translation through the direct phosphorylation of eIF4E. Increased MKNK1/2-dependent phosphorylation of the translation factor eIF4E is observed in head and neck squamous cell carcinoma and is correlated with a poor prognosis. This study evaluated the relative inhibitory effects of LY2801653, Crizotinib, XL184, XL880, and PF4217903 in a variety of cell lines against MET, ROS1, MKNK1/2, ERK, and other downstream molecules. In HCC78, a ROS1-fusion protein bearing cell line, and in NIH3T3 cells stably transfected with the FIG-ROS(s) fusion, LY2801653, XL184 and XL880 were more potent inhibitors of ROS1 compared with Crizotinib. Moreover, LY2801653 and Crizotinib were both efficacious in NIH3T3 FIG-ROS(s) xenografts, although LY2801653 was more potent in reducing p-ROS1 in these tumors than Crizotinib. In NCI-H2122 and Hs746T cells (mutated MET), A2780 cells (MET-negative), KP-4 cells (MET autocrine), H441 cells (HGF-independent; MET positive), HCT116 (mutated KRAS), and the cholangiocarcinoma cell lines EGI-1 and TFK-1, LY2801653 demonstrated more potent inhibitory activity against MKNK1/2 versus treatment with XL184 and XL880, while Crizotinib had no observed inhibitory activity against MKNK1/2. LY2801653 also showed inhibitory activity against MKNK1/2 in MET-amplified MKN45 cells. These findings suggests there may be clinical merits to testing LY2801653 in tumor types associated with increased MKNK1/2 activity or ROS1 fusion proteins alone, or in conjunction with aberrant MET activity, such as colorectal carcinoma, head and neck squamous cell carcinoma, and cholangiocarcinoma. Citation Format: Sau-Chi B. Yan, Megan N. Thobe, Bruce W. Konicek, Victoria L. Peek, Suzane L. Um, Richard A. Walgren, Huaxing Pei, Timothy P. Burkholder, Jeremy R. Graff. LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2. [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 3042. doi:10.1158/1538-7445.AM2013-3042