Hepatocyte Growth Factor Activator Research Articles

Adipose‐derived stem cell‐containing hyaluronic acid/alginate hydrogel improves vocal fold wound healing

The purpose of this study was to investigate the regenerative efficacy of an injectable hyaluronic acid/mildly cross-linked alginate hydrogel (HA/ALG hydrogel) containing human adipose-derived mesenchymal stem cells (hAdMSCs) for vocal fold (VF) wound healing. Animal research. HA/ALG hydrogel containing hAdMSCs was injected into the VFs of rabbits immediately after direct injury. Endoscopic evaluations were performed at 1 and 3 months after injury, and functional evaluations of mucosal vibration and viscoelastic properties were carried out posteuthanization at 3 months after injury. Histopathologic and immunohistochemical evaluations of extracellular matrix (ECM) components and of hepatocyte growth factor (HGF) activity were conducted in injured VFs. The engraftment of implanted hAdMSCs was investigated by detecting fluorescent-labeled cells. The administration of hAdMSCs and hAdMSCs in HA/ALG hydrogel produced better macroscopic morphologies than the injection of phosphate-buffered saline (PBS). Histologic evaluations revealed that treatment with hAdMSCs in HA/ALG produced more favorable ECM changes than hAdMSC. In particular, the treatment of hAdMSCs in HA/ALG hydrogel ameliorated excessive deposition of collagen type I and increased HGF activity in regenerating VFs. hAdMSCs in HA/ALG-treated VFs also exhibited functional improvements in viscoelastic properties. hAdMSCs in HA/ALG remained viable in recipient VFs at 1 month after transplantation, and some were observed to be fostered to differentiate into fibroblasts. The findings of the present study suggest that HA/ALG hydrogel is a promising biomaterial for prolonging the retention time of stem cells in VFs and for promotion of VF wound healing.

Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

Matriptase is a member of the family of type II transmembrane serine proteases that is essential for development and maintenance of several epithelial tissues. Matriptase is synthesized as a single-chain zymogen precursor that is processed into a two-chain disulfide-linked form dependent on its own catalytic activity leading to the hypothesis that matriptase functions at the pinnacle of several protease induced signal cascades. Matriptase is usually found in either its zymogen form or in a complex with its cognate inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), whereas the active non-inhibited form has been difficult to detect. In this study, we have developed an assay to detect enzymatically active non-inhibitor-complexed matriptase by using a biotinylated peptide substrate-based chloromethyl ketone (CMK) inhibitor. Covalently CMK peptide-bound matriptase is detected by streptavidin pull-down and subsequent analysis by Western blotting. This study presents a novel assay for detection of enzymatically active matriptase in living human and murine cells. The assay can be applied to a variety of cell systems and species.

HAI-2 suppresses the invasive growth and metastasis of prostate cancer through regulation of matriptase

Dysregulation of cell surface proteolysis has been strongly implicated in tumorigenicity and metastasis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer (PCa) cell migration, invasion, tumorigenicity and metastasis using a human PCa progression model (103E, N1, and N2 cells) and xenograft models. N1 and N2 cells were established through serial intraprostatic propagation of 103E human PCa cells and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was revealed to gradually increase throughout the serial isolations (103E<N1<N2). In this series of cells, the expression of HAI-2 but not HAI-1 was significantly decreased throughout the progression and occurred in parallel with increased activation of matriptase. The expression level and activity of matriptase increased whereas the HAI-2 protein level decreased over the course of orthotopic tumor growth in mice, which was consistent with the immunohistochemical profiles of matriptase and HAI-2 in archival PCa specimens. Knockdown of matriptase reduced the PCa cell invasion induced by HAI-2 knockdown. HAI-2 overexpression or matriptase silencing in N2 cells downregulated matriptase activity and significantly decreased tumorigenicity and metastatic capability in orthotopically xenografted mice. These results suggest that during the progression of human PCa, matriptase activity is primarily controlled by HAI-2 expression. The imbalance between HAI-2 and matriptase expression led to matriptase activation, thereby increasing cell migration, invasion, tumorigenicity and metastasis.

Abstract B3: HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor

Abstract The Hepatocyte Growth Factor (HGF) also known as Scatter Factor, activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27KDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the motility in vitro and the remodeling of actin filaments induced by HGF. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired both the ability of ovarian cancer cells to form experimental haematogenous metastases and to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic Paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the pro-invasive and pro-metastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy. Grant sponsors: This work has been supported by grants to M.F.D.: 2012 IG grant and 2010 Special Program Molecular Clinical Oncology 5xMille of the Italian Association of Cancer Research (AIRC), Project n° 9970, and Grant of the CARIPLO Foundation. Citation Format: Simona Pavan, Daniele Musiani, Erica Torchiaro, Giorgia Migliardi, Jessica Erriquez, Martina Olivero, Maria Flavia Di Renzo. HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B3.

Imbalanced Matriptase Pericellular Proteolysis Contributes to the Pathogenesis of Malignant B-Cell Lymphomas

Membrane-associated serine protease matriptase is widely expressed by epithelial/carcinoma cells in which its proteolytic activity is tightly controlled by the Kunitz-type protease inhibitor, hepatocyte growth factor activator inhibitor (HAI-1). We demonstrate that, although matriptase is not expressed in lymphoid hyperplasia, roughly half of the non-Hodgkin B-cell lymphomas analyzed express significant amounts of matriptase. Furthermore, a significant proportion of these tumors express matriptase in the absence of HAI-1. Aggressive Burkitt lymphoma was more likely than indolent follicular lymphoma to express matriptase alone (86% versus 36%). In the absence of significant HAI-1 expression, the lymphoma cells activate and shed active matriptase when the cells are stimulated with mildly acidic buffer or the hypoxia-mimicking agent, CoCl2. The shed active matriptase can initiate pericellular proteolytic cascades by activating urokinase-type plasminogen activator on the cell surface of monocytes, and it can activate prohepatocyte growth factor. In addition, matriptase knockdown suppressed proliferation and colony-forming ability of neoplastic B cells in culture and growth as tumor xenografts in mice. Furthermore, exogenous expression of HAI-1 significantly suppressed proliferation of neoplastic B cells. These studies suggest that dysregulated pericellular proteolysis as a result of unregulated matriptase expression with limited HAI-1 may contribute to the pathological characteristics of several human B-cell lymphomas through modulation of the tumor microenvironment and enhanced tumor growth.

HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor

The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.

Matriptase Expression and Zymogen Activation in Human Pilosebaceous Unit

Studies of human genetic disorders and mouse models reveal the important roles of matriptase in hair growth. Here, we investigate matriptase expression and zymogen activation in hair follicles. We show: 1) layer-dependent distribution patterns, with much higher matriptase expression in cells of the outer root sheath and matrix cells of the hair bulb than in cells of the inner root sheath; 2) cycle-dependent expression patterns, with matriptase expressed in the anagen and catagen phases of the hair lifecycle, but not in the telogen phase; 3) reduced expression of the matriptase inhibitor, HAI-1, in the catagen phase, suggesting increased proteolytic activity in this phase; and 4) definitive matriptase zymogen activation patterns, with the highest matriptase activation observed in matrix cells and outer root sheath cells in the isthmus/bulge region. In sebaceous glands, matriptase is highly expressed in basal and ductal cells, with much lower expression in the differentiated, lipid-filled cells of the interior. We also show that matriptase potently activates hepatocyte growth factor (HGF) in vitro, and that the HGF receptor, c-Met, is co-expressed in those cells that express activated matriptase. Our observations suggest that the matriptase-HGF-c-MET pathway has the potential to be engaged, primarily in proliferative cells rather than terminally differentiated epithelial cells of the human pilosebaceous unit.

Down-regulation of HAI-1 is associated with poor-differentiation status of colorectal cancer

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor which is widely expressed in epithelial cells. The purpose of this study is to investigate the relationship between HAI-1 expression and differentiation status of colorectal epithelia. The expression of HAI-1 in clinical samples of both cancerous and normal colorectal tissues was evaluated by immunohistochemical staining. An in vitro epithelial differentiation model of Caco-2 cell was established, and the characteristics of differentiation of Caco-2 cells were observed by transmission electron microscopy. The expression of HAI-1 in Caco-2 cells at different differentiation stages was examined by western blot. Immunohistochemical staining of 52 human colorectal cancer tissues showed a definite correlation between HAI-1 expression and differentiation status: IHC score (mean±SE) of HAI-1 was higher for well- or moderately-differentiated colorectal cancer tissues than for poorly-differentiated colorectal cancer tissues, with significant differences in HAI-1 positive rate (P<0.01 and P<0.05 for well-differentiated vs. poorly-differentiated and moderately-differentiated vs. poorly-differentiated, respectively). Immunohistochemical staining of normal colorectal tissues showed positive HAI-1 expression in well-differentiated epithelial cells whereas the under-differentiated crypt cells showed very weak HAI-1 staining signals. The result of western blot also showed the gradual increasing of HAI-1 expression during the process of Caco-2 differentiation in vitro. HAI-1 expression correlates with the differentiation status of colorectal epithelia and could serve as a differentiation marker.

Prevention of Reg I-induced β-cell apoptosis by IL-6/dexamethasone through activation of HGF gene regulation

Reg (regenerating gene) product, Reg protein, is induced in pancreatic β-cells and acts as autocrine/paracrine growth factor for regeneration via the cell surface Reg receptor. However, high concentrations of Reg I protein induced β-cell apoptosis. In the present study, we found that hepatocyte growth factor (HGF) attenuated the β-cell apoptosis induced by the high concentrations of Reg I protein and that the combined stimulation of interleukin-6 (IL-6) and dexamethasone (Dx) induced the accumulation of HGF mRNA as well as Reg I mRNA in β-cells. The accumulation of the HGF mRNA was caused by the activation of the HGF promoter. Deletion analysis revealed that the region of −96 to −92 of the HGF gene was responsible for the promoter activation by IL-6+Dx. The promoters contain a consensus transcription factor binding sequence for signal transducer and activator of transcription (STAT). Site-directed mutations of STAT-binding motif in the region markedly attenuated the HGF promoter activity. Chromatin immunoprecipitation assay showed that STAT3 is located at the active HGF promoter in response to IL-6+Dx stimulation. These results strongly suggest that the combined stimulation of IL-6 and glucocorticoids induces the activation of both Reg and HGF genes and that the anti-apoptotic effects of HGF against the Reg I-induced apoptosis may help β-cell regeneration by Reg I protein.

Human placental multipotent mesenchymal stromal cells modulate trophoblast migration via Rap1 activation

IntroductionLittle is known about the interaction between human placental multipotent mesenchymal stromal cell (hPMSC) and trophoblast. We hypothesize that hPMSCs produce hepatocyte growth factor (HGF) which may interact with trophoblasts and regulate their migration during placentation. MethodshPMSCs were isolated from term placentas and conditioned medium was collected after 2 days of culture in hypoxic (<1% O2) or control (20% O2) conditions. Selective agonist and inhibitor or siRNA for protein kinase A (PKA) or Rap1 were combined with Rap1-GTP pull down assays, flow cytometry, integrin β1 activation assays and adhesion and migration studies to investigate HGF signaling effects in trophoblasts. The hPMSC abundance and HGF level in preeclamptic placentas were compared with gestational age-matched controls. ResultsHGF was expressed by hPMSCs and was decreased in hypoxia. HGF induced cAMP production and Rap1 activation in trophoblasts, which in turn activated integrin β1. The HGF and PKA activator 6-Bnz-cAMP induced Rap1 activation with increased trophoblast adhesion and migration. The alterations were inhibited by PKA inhibitor H89 or Rap1 siRNA. HGF and cAMP expression were reduced in preeclamptic placentas. hPMSC number was decreased in preeclamptic placenta compared to controls (0.68 ± 0.1% vs. 1.32 ± 0.5%; P = 0.026). hPMSC conditioned medium enhanced trophoblast migration which was inhibited by c-Met blocking antibody, but migration was reduced by conditioned medium from hPMSC cultured in hypoxia. ConclusionshPMSCs secrete HGF and increase trophoblast cAMP production. The cAMP effector PKA modulates adhesion and migration of trophoblast via signaling to Rap1 and integrin β1.

Impaired liver regeneration in Ldlr−/− mice is associated with an altered hepatic profile of cytokines, growth factors, and lipids

It is widely recognized that in the early stages of liver regeneration after partial hepatectomy, the hepatocytes accumulate a significant amount of lipids. The functional meaning of this transient steatosis and its effect on hepatocellular proliferation are not well defined. In addition, the basic mechanisms of this lipid accumulation are not well understood although some studies suggest the participation of the Low Density Lipoprotein Receptor (Ldlr). To address these questions, we studied the process of liver regeneration in Ldlr null mice and wild type mice following partial hepatectomy. Ldlr deficiency was associated with a significant decrease in serum albumin concentration, during early stages of liver regeneration, and a delayed hepatic regeneration. Remnant livers of Ldlr(-)(/)(-) showed a time-shifted expression of interleukin-6 (IL6) and a defective activation of tumor necrosis factor-α (TNFα) and hepatocyte growth factor (HGF) expression in early phases of liver regeneration. Unexpectedly, Ldlr(-)(/)(-) showed no significant differences in the content of lipid droplets after partial hepatectomy compared to wild type mice. However, lipidomic analysis of the regenerating liver from Ldlr(-)(/)(-) revealed a lipid profile compatible with liver quiescence: high content of cholesterol esters and ceramide, and low levels of phosphatidylcholine. Ldlr deficiency is associated with significant changes in the hepatic lipidome that affect cytokine-growth factor signaling and impair liver regeneration. These results suggest that the analysis of the hepatic lipidome may help predict the success of liver regeneration in the clinical environment, specifically in the context of pre-existing liver steatosis.

Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that β-AT, and not α-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans.

Identification of head and neck squamous cell carcinoma biomarker candidates through proteomic analysis of cancer cell secretome

Protein biomarker discovery for early detection of head and neck squamous cell carcinoma (HNSCC) is a crucial unmet need to improve patient outcomes. Mass spectrometry-based proteomics has emerged as a promising tool for identification of biomarkers in different cancer types. Proteins secreted from cancer cells can serve as potential biomarkers for early diagnosis. In the current study, we have used isobaric tag for relative and absolute quantitation (iTRAQ) labeling methodology coupled with high resolution mass spectrometry to identify and quantitate secreted proteins from a panel of head and neck carcinoma cell lines. In all, we identified 2,472 proteins, of which 225 proteins were secreted at higher or lower abundance in HNSCC-derived cell lines. Of these, 148 were present in higher abundance and 77 were present in lower abundance in the cancer-cell derived secretome. We detected a higher abundance of some previously known markers for HNSCC including insulin like growth factor binding protein 3, IGFBP3 (11-fold) and opioid growth factor receptor, OGFR (10-fold) demonstrating the validity of our approach. We also identified several novel secreted proteins in HNSCC including olfactomedin-4, OLFM4 (12-fold) and hepatocyte growth factor activator, HGFA (5-fold). IHC-based validation was conducted in HNSCC using tissue microarrays which revealed overexpression of IGFBP3 and OLFM4 in 70% and 75% of the tested cases, respectively. Our study illustrates quantitative proteomics of secretome as a robust approach for identification of potential HNSCC biomarkers. This article is part of a Special Issue entitled: An Updated Secretome.

AXL and MET crosstalk to promote gonadotropin releasing hormone (GnRH) neuronal cell migration and survival

The membrane tyrosine kinase receptors, AXL and MET, are implicated in GnRH neuron migration and/or survival. We hypothesized that the receptors with their ligands, GAS6 and HGF, respectively may cross-talk in GnRH neuronal function. In NLT GnRH neuronal cells, MET co-immunoprecipitated with AXL, although HGF or GAS6 did not transphosphorylate AXL or MET, respectively. Co-expression of a kinase dead AXL blocked HGF activation of MET and indirectly AKT and p38MAPK. Silencing of AXL decreased HGF’s ability to phosphorylate MET and activate AXL’s downstream effectors, p38MAPK and AKT. HGF/MET signaling modulated neuron migration dependent and independent of AXL co-expression and p38MAPK. Conversely, AXL’s control of GnRH neuronal survival was dependent on HGF/MET signaling. Together, these data support that the importance of membrane tyrosine kinase receptor crosstalk to regulate neuronal cell-specific developmental functions.

CD82 Blocks cMet Activation and Overcomes Hepatocyte Growth Factor Effects on Oligodendrocyte Precursor Differentiation

Mechanisms that regulate oligodendrocyte (OL) precursor migration and differentiation are important in normal development and in demyelinating/remyelinating conditions. We previously found that the tetraspanin CD82 is far more highly expressed in O4(+) OL precursors of the adult rat brain than those of the neonatal brain. CD82 has been physically linked to cMet, the hepatocyte growth factor (HGF) receptor, in tumor cells, and this interaction decreases downstream signaling. We show here that CD82 inhibits the HGF activation of cMet in neonatal and adult rat OL precursors. CD82 expression is sufficient to allow precursor differentiation into mature OLs even in the presence of HGF. In contrast, CD82 downregulation in adult O4(+)/CD82(+) cells inhibits their differentiation, decreases their accumulation of myelin proteins, and causes a reversion to less mature stages. CD82 expression in neonatal O4(+)/CD82(-) cells also blocks Rac1 activation, suggesting a possible regulatory effect on cytoskeletal organization and mobility. Thus, CD82 is a negative regulator of HGF/cMet during OL development and overcomes HGF inhibitory regulation of OL precursor maturation.

Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Suppressor of Intestinal Tumorigenesis

Hepatocyte growth factor activator inhibitor type 1 (HAI-1/SPINT1) is a membrane-bound serine protease inhibitor expressed on the surface of epithelial cells. Although HAI-1/SPINT1 is abundantly expressed in the intestinal epithelium, its role in intestinal tumorigenesis is not known. In this study, we investigated the role of Hai-1/Spint1 in intestinal tumorigenesis using mouse models. The membranous Hai-1/Spint1 immunoreactivity was decreased in murine Apc(Min/+) tumors and also in carcinogen (azoxymethane treatment followed by dextran sodium sulfate administration)-induced colon tumors compared with the adjacent non-neoplastic epithelium. The decreased immunoreactivity appeared to be due to sheddase activity of membrane-type 1 matrix metalloprotease. Then, we examined the effect of intestine-specific deletion of Spint1 gene on Apc(Min/+) mice. The loss of Hai-1/Spint1 significantly accelerated tumor formation in Apc(Min/+) mice and shortened their survival periods. Activation of HGF was enhanced in Hai-1/Spint1-deficient Apc(Min/+) intestine. Gene expression profiling revealed upregulation of the Wnt/β-catenin signaling circuit, claudin-2 expression, and angiogenesis not only in tumor tissue but also in the background mucosa without macroscopic tumors in Hai-1/Spint1-deficient Apc(Min/+) intestine. Intestinal deletion of Spint1 also enhanced the susceptibility to carcinogen-induced colon tumorigenicity of wild-type Apc mice. Our findings suggest that HAI-1/SPINT1 has a crucial role in suppressing intestinal tumorigenesis, which implies a novel link between epithelial cell surface serine protease inhibitors and protection from carcinogenic stimuli.

Abstract 926: The angiotensin IV analog Norleual acts as a hepatocyte growth factor/c-Met inhibitor in pancreatic cancer cells.

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

Abstract 2026: Co-targeting c-Met and COX-2 leads to enhanced inhibition of lung tumorigenesis in a murine model with heightened HGF signaling in the airways.

Abstract The hepatocyte growth factor (HGF)/c-Met signaling pathway is often dysregulated in lung cancer. We previously showed that HGF activation of c-Met in non-small cell lung cancer (NSCLC) cells led to a significant induction of cyclooxygenase-2 (COX-2) followed by secretion of PGE2. PGE2 caused secretion of TGFα by NSCLC cells, followed by both phosphorylation of EGFR and later, delayed phosphorylation of c-Met that does not require HGF. Secretion of PGE2 also appears to boost c-Met pathway signaling downstream via cross-activation by EGFR, in a reinforcing loop that is independent of HGF. We hypothesized that targeting of both c-Met and COX-2 might lead to enhanced anti-tumor effects by blocking both upstream and downstream pro-tumor events. Maximum effects on cell proliferation, invasion and EMT markers were observed in vitro in NSCLC cell lines with combination celecoxib and crizotinib treatment. We next tested whether targeting c-Met with the tyrosine kinase inhibitor crizotinib combined with celecoxib to target COX-2 would result in enhanced anti-tumor effects in an animal model with heightened HGF/c-Met pathway signaling in the airways. Mice transgenic for airway expression of human HGF were treated with the tobacco carcinogen, 4-(methylnitroso-amino)-1-(3-pyridyl)-1-butanone, from weeks 1-4. Crizotinib (40mg/kg), celecoxib (50mg/kg), combined crizotinib and celecoxib or placebo control was administered by oral gavage daily from week 3 until week 15, at which time lung tumors were evaluated. A highly significant decrease in the number of lung tumors per animal was observed with crizotinib treatment (mean 3.6, range 2-6), celecoxib treatment (mean 4.3, range 3-6) and combination treatment (mean 1.5, range 0-3) compared to placebo control (mean 9.9, range 6-13; P&amp;lt;0.001, Poisson regression). The number of tumors in the combination treatment group was also significantly lower than either single agent treatment (P&amp;lt;0.001). There was also a significant decrease in mean tumor size with crizotinib (mean 0.20mm2), celecoxib (mean 0.22mm2) and combination (mean 0.15mm2) compared to placebo (mean 0.38mm2, P&amp;lt;0.001, mixed effects modeling). Three biomarkers (COX-2, PGE2, and P-MAPK) were optimally down-modulated by combination treatment, compared to single treatment. Additionally, E-cadherin expression was up-regulated while the E-cadherin repressor, snail, was down-regulated with dual treatment. In a pulmonary environment with heightened HGF/c-Met pathway activity, dual inhibition of the c-Met and COX-2 pathways may enhance anti-tumor effects by targeting reinforcing up- and downstream signaling. Such a combination has potential clinical benefit for lung cancer patients with dysregulated c-Met/COX-2 pathways. Supported by R01 CA79882 and P50 CA090440. Citation Format: Laura P. Stabile, Mary E. Rothstein, Christopher T. Gubish, Nathan Lee, Diana E. Cunningham, Jill M. Siegfried. Co-targeting c-Met and COX-2 leads to enhanced inhibition of lung tumorigenesis in a murine model with heightened HGF signaling in the airways. [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 2026. doi:10.1158/1538-7445.AM2013-2026

Crystal Structures of Matriptase in Complex with Its Inhibitor Hepatocyte Growth Factor Activator Inhibitor-1

Matriptase, a type II trans-membrane serine protease of the S1 trypsin-like family, is expressed on the surface of nearly all normal human epithelium and found in biological fluid-like human milk. Matriptase overexpression has been implicated in tumor progression in certain epithelium-derived cancer cells. Matriptase is tightly regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). It has been demonstrated that the Kunitz domain I (KD1) but not Kunitz domain II (KD2) of HAI-1 is responsible for the inhibitory activity of HAI-1 against matriptase. To investigate the molecular basis of inhibition of matriptase by HAI-1, we solved several crystal structures of matriptase serine protease domain in complex with the fragments of HAI-1. Based on these structures, we found that the binding of KD1 was different from previously predicted binding mode. The P3 arginine residue occupies the S3 specificity pocket of matriptase, but not the S4 pocket as in the cases of hepatocyte growth factor activator·HAI-1 KD1 and matriptase·sunflower trypsin inhibitor-1 complexes. The long 60-loop of matriptase makes direct contact with HAI-1 but remains flexible even in the complexes, and its apex does not bind with KD1 tightly. The interactions between this unique 60-loop and KD1 may provide an opportunity to increase the specificity and inhibitory activity of KD1 for matriptase. Furthermore, comparison between KD1 and a homology model of HAI-1 KD2 rationalizes the structural basis of why KD1 but not KD2 is responsible for the inhibitory activity of HAI-1 against matriptase.

Role and Regulation of PDGFRα Signaling in Liver Development and Regeneration

Aberrant platelet-derived growth factor receptor-α (PDGFRα) signaling is evident in a subset of hepatocellular cancers (HCCs). However, its role and regulation in hepatic physiology remains elusive. In the current study, we examined PDGFRα signaling in liver development and regeneration. We identified notable PDGFRα activation in hepatic morphogenesis that, when interrupted by PDGFRα-blocking antibody, led to decreased hepatoblast proliferation and survival in embryonic liver cultures. We also identified temporal PDGFRα overexpression, which is regulated by epidermal growth factor (EGF) and tumor necrosis factor α, and its activation at 3 to 24 hours after partial hepatectomy. Through generation of hepatocyte-specific PDGFRA knockout (KO) mice that lack an overt phenotype, we show that absent PDGFRα compromises extracelluar signal-regulated kinases and AKT activation 3 hours after partial hepatectomy, which, however, is alleviated by temporal compensatory increases in the EGF receptor (EGFR) and the hepatocyte growth factor receptor (Met) expression and activation along with rebound activation of extracellular signal-regulated kinases and AKT at 24 hours. These untimely increases in EGFR and Met allow for normal hepatocyte proliferation at 48 hours in KO, which, however, are aberrantly prolonged up to 72 hours. Intriguingly, such compensation also was visible in primary KO hepatocyte cultures but not in HCC cells after siRNA-mediated PDGFRα knockdown. Thus, temporal activation of PDGFRα in liver development is important in hepatic morphogenesis. In liver regeneration, despite increased signaling, PDGFRα is dispensable owing to EGFR and Met compensation, which is unique to normal hepatocytes but not HCC cells.