Hepatocyte Growth Factor/scatter Factor Research Articles

Xeroderma Pigmentosum Type C Primary Skin Fibroblasts Overexpress HGF and Promote Squamous Cell Carcinoma Invasion in the Absence of Genotoxic Stress.

Xeroderma pigmentosum (XP) is a very rare recessive disease caused by the incapacity to resolve ultraviolet-induced DNA lesions through Nucleotide Excision Repair (NER). Most XP patients suffer from aggressive skin carcinoma and melanoma at a very early age (<8). Our previous results showed that primary XP fibroblasts isolated from healthy (non-photo-exposed) skin negatively impact the extracellular matrix and fail to activate the innate immune system. Here, we show for the first time that XP-C fibroblasts also play a major role in cancer cell invasion ex vivo and in vivo through the overexpression of Hepatocyte Growth Factor/Scatter Factor (HGF/SF) in the absence of genotoxic attacks. The use of inhibitors of the activation of the HGF/SF pathway counteracted the effects of XP fibroblasts on the growth of cancer cells, suggesting new perspectives in the care of XP patients.

Aberrant MET Receptor Tyrosine Kinase Signaling in Glioblastoma: Targeted Therapy and Future Directions.

Brain tumors represent a heterogeneous group of neoplasms characterized by a high degree of aggressiveness and a poor prognosis. Despite recent therapeutic advances, the treatment of brain tumors, including glioblastoma (GBM), an aggressive primary brain tumor associated with poor prognosis and resistance to therapy, remains a significant challenge. Receptor tyrosine kinases (RTKs) are critical during development and in adulthood. Dysregulation of RTKs through activating mutations and gene amplification contributes to many human cancers and provides attractive therapeutic targets for treatment. Under physiological conditions, the Met RTK, the hepatocyte growth factor/scatter factor (HGF/SF) receptor, promotes fundamental signaling cascades that modulate epithelial-to-mesenchymal transition (EMT) involved in tissue repair and embryogenesis. In cancer, increased Met activity promotes tumor growth and metastasis by providing signals for proliferation, survival, and migration/invasion. Recent clinical genomic studies have unveiled multiple mechanisms by which MET is genetically altered in GBM, including focal amplification, chromosomal rearrangements generating gene fusions, and a splicing variant mutation (exon 14 skipping, METex14del). Notably, MET overexpression contributes to chemotherapy resistance in GBM by promoting the survival of cancer stem-like cells. This is linked to distinctive Met-induced pathways, such as the upregulation of DNA repair mechanisms, which can protect tumor cells from the cytotoxic effects of chemotherapy. The development of MET-targeted therapies represents a major step forward in the treatment of brain tumours. Preclinical studies have shown that MET-targeted therapies (monoclonal antibodies or small molecule inhibitors) can suppress growth and invasion, enhancing the efficacy of conventional therapies. Early-phase clinical trials have demonstrated promising results with MET-targeted therapies in improving overall survival for patients with recurrent GBM. However, challenges remain, including the need for patient stratification, the optimization of treatment regimens, and the identification of mechanisms of resistance. This review aims to highlight the current understanding of mechanisms underlying MET dysregulation in GBM. In addition, it will focus on the ongoing preclinical and clinical assessment of therapies targeting MET dysregulation in GBM.

Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent MET receptor agonist.

Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in postnatal life of epithelial organs such as the liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search of HGF/SF mimics for therapy. In this work, we describe the rational design, production, and characterization of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF in tandem orientation. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent of MET activation.

A MET-PTPRK kinase-phosphatase rheostat controls ZNRF3 and Wnt signaling.

Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that targets Wnt receptors for ubiquitination and lysosomal degradation. Previously, we showed that dephosphorylation of an endocytic tyrosine motif (4Y motif) in ZNRF3 by protein tyrosine phosphatase receptor-type kappa (PTPRK) promotes ZNRF3 internalization and Wnt receptor degradation (Chang et al 2020). However, a responsible protein tyrosine kinase(s) (PTK) phosphorylating the 4Y motif remained elusive. Here we identify the proto-oncogene MET (mesenchymal-epithelial transition factor) as a 4Y kinase. MET binds to ZNRF3 and induces 4Y phosphorylation, stimulated by the MET ligand HGF (hepatocyte growth factor, scatter factor). HGF-MET signaling reduces ZNRF3-dependent Wnt receptor degradation thereby enhancing Wnt/β-catenin signaling. Conversely, depletion or pharmacological inhibition of MET promotes the internalization of ZNRF3 and Wnt receptor degradation. We conclude that HGF-MET signaling phosphorylates- and PTPRK dephosphorylates ZNRF3 to regulate ZNRF3 internalization, functioning as a rheostat for Wnt signaling that may offer novel opportunities for therapeutic intervention.

Molecular Mechanisms of Canine Osteosarcoma Metastasis.

Osteosarcoma (OSA) represents the most common bone tumor in dogs. The malignancy is highly aggressive, and most of the dogs die due to metastasis, especially to the lungs. The metastatic process is complex and consists of several main steps. Assessment of the molecular mechanisms of metastasis requires in vitro and especially in vivo studies for a full evaluation of the process. The molecular and biological resemblance of canine OSA to its human counterpart enables the utilization of dogs as a spontaneous model of this disease in humans. The aim of the present review article is to summarize the knowledge of genes and proteins, including p63, signal transducer and activator of transcription 3 (STAT3), Snail2, ezrin, phosphorylated ezrin-radixin-moesin (p-ERM), hepatocyte growth factor-scatter factor (HGF-SF), epidermal growth factor receptor (EGFR), miR-9, and miR-34a, that are proven, by in vitro and/or in vivo studies, to be potentially involved in the metastatic cascade of canine OSA. The determination of molecular targets of metastatic disease may enhance the development of new therapeutic strategies.

Abstract PO-089: Morphokinetic single-cell analysis and machine learning as a tool to characterize breast cancer cell motility and response to therapy

Abstract Triple-negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2). TNBC patients have a high propensity for distant metastasis and limited treatment options. Cancer cell motility and invasion are fundamental steps in metastasis. MET, a tyrosine kinase receptor, and its ligand, Hepatocyte Growth Factor/Scatter Factor (HGF/SF), induce specific signal transduction in tumor cells leading to cell motility and metastasis. MET's induced tumorigenesis and metastatic processes make it an ideal target for anti-cancer therapy. We characterized the motility patterns of m-Cherry labeled TNBC cells (BT-549, MDA-MB-231) and ER-positive cells (MCF7) subjected to time-lapse fluorescence wound healing assay. We also studied the effect of MET inhibition, chemotherapy, and their combined impact on breast cancer motility. Time-lapse images were subjected to commercial packages for segmentation and tracking - allowing us to extract morphokinetic (MK) information at a single-cell resolution. To better understand cell motility patterns, we developed the Tool for Analysis of Single-Cell (TASC) infrastructure, which utilizes unsupervised machine learning methods to demonstrate a high-dimensional feature set at a single-cell resolution. We compared our experimental MK results to simulations of three modified classical physical models: Lévy flight (LF), fractal Brownian motion, and random walk (RW) with or without a back-propagating wave (BPW). TASC analysis demonstrated a fundamental difference between ER-positive and TNBC cells: both cell types contained two subpopulations harboring similar MK characteristics. TNBC cells presented an additional subpopulation characterized by 1) dominantly increasing cumulative kinetics values (mean square displacement - MSD), 2) highest temporal wave values (velocity starting-time), and 3) a decrease in non-cumulative kinetics values. The combined treatment of TNBC cells with MET inhibition and chemotherapy eliminates this high-motility group. Cell motility models and motion simulations were compared using Wasserstein's analysis. Untreated TNBC cells showed high similarities to RW with BPW simulations, while MET-activated TNBC cells transformed into LF without BPW simulations, indicating more aggressive behavior. TASC analysis revealed a previously undefined high motility subpopulation of TNBC that was eradicated by combining MET inhibition and chemotherapy. The primary benefit of TASC is the ability to cluster and characterize similar behaviors across different motility models while still detecting subtle changes within each one. MK analysis combined with TASC can lead to discovering novel targets for precise therapy. Moreover, this infrastructure may determine patient tumor susceptibility to chemotherapy and biological treatments and operate as an analytical tool for personalized, targeted therapy. Citation Format: Ilan Tsarfaty, Or Megides, Ohad Doron, Hagar Alaloof, Ori Moskowitz, Judith Horev. Morphokinetic single-cell analysis and machine learning as a tool to characterize breast cancer cell motility and response to therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-089.

Abstract PO-067: AI and CT-radiomics as a tool for breast cancer prognosis and radiomics-gene isolation: Activated MET induced mammary carcinoma as a model

Abstract Triple-negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2). TNBC represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel driver and modifier genes of TNBC, representing targets to treat the disease. MET tyrosine kinase is the receptor for hepatocyte growth factor/scatter factor (HGF/SF). MET overexpression and constitutively activated mutation promotes tumor growth, cell motility and metastases, making MET an ideal target for anti-metastatic precision therapy. In mice carrying MET constitutive activation mutation (METmut), the gene initiates tumorigenicity associated with inherited driver modifier genes (IDMGs). The Collaborative Cross (CC) animal model consists of recombinant inbred lines generated by reciprocal crosses between numerous founder lines. It is a reference to apply genetic traits mapping with high-resolution phenotypes such as radiomic features. In this study, we established a new machine learning prognostic model of overall survival (OS) of METmut-induced mammary carcinoma. QTL (Quantitative Trait Locus) mapping analysis was used to identify candidate genes that may contribute to intra-tumor heterogenicity and OS. Two CT images cohorts were included: METmut-FVB/N and METmut-CC mice that developed mammary carcinoma (n=13 and n=15, respectively). Each segmented tumor was divided into 3D cubes, and radiomic features were extracted from each cube using a novel in-house tool. We performed Principle Component Analysis (PCA) and cluster analysis (k-means) on the radiomic features to produce cube cluster groups with similar radiomic characteristics, such as tumor region intensity and morphology. We then assessed each cluster group's proportion per tumor – the cluster proportion - as a radiomic phenotype for QTL. QTL analysis identified loci in chromosomes #6 and #15 that code for new radiomics morphological candidate genes that may alter MET signaling and reduce OS. To analyze tumor heterogenicity, median and standard deviation of cubes PCA values were calculated per tumor and used as covariates in Cox regression against OS. Four out of the eight covariates were significantly correlated with OS (P &amp;lt; 0.01, Xi 21.59 on 8 df). Using PCA and cluster analysis in radiomic feature analysis can create a unique prognostic signature for breast cancer patients. CT-radiomics can be used to isolate tumor genes, substituting tumor biopsies in patients with breast cancer metastases to brain and providing novel targets for precision therapy. Citation Format: Ilan Tsarfaty, Eran Dotan, Michal Bloom, Judith Horev, Fuad Iraqi. AI and CT-radiomics as a tool for breast cancer prognosis and radiomics-gene isolation: Activated MET induced mammary carcinoma as a model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-067.

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.

Preclinical evaluation of WXSH0011, a selective small molecule dual inhibitor of FGFR/c-Met.

e15641 Background: FGFRs (fibroblast growth factor receptors) are a family of receptor tyrosine kinases. c-Met is a receptor tyrosine kinase with a high-affinity ligand, hepatocyte growth factor/scatter factor (HGF/SF). Aberrations in the FGFR pathway are associated with a variety of human cancers. c-Met and HGF have been implicated in carcinogenesis and metastatic tumor progression because of their ability to enhance angiogenesis, cancer cell proliferation, migration, and invasion. WXSH0011 is an orally bioavailable novel potent and selective FGFR/c-Met dual kinase inhibitor, which displayed significant antitumor activity in preclinical models of solid cancer with FGFRs/c-Met overexpression. Preclinical studies demonstrated that WXSH0011 has potential to be effective in solid cancer, such as HCC and Gastric. Methods: Kinase inhibiting activity of WXSH0011 was determined with FGFRs/ c-Met kinase assays. Cellular anti-proliferative activity was evaluated with FGFR overexpressed NCl-H1581, SNU-16, RT112/84, Hep3B, c-Met overexpressed MHCC97H, EBC-1 cell line and FGFR, c-Met both overexpressed PDC cell line (LI-03-0141 (HCC) and ST-02-0063 (Gastric) PDC). The antitumor activity of WXSH0011 was evaluated in vivo cell derived-xenograft (CDX) models of Gastric Cancer SNU-16 and HCC Hep3B. Results: WXSH0011 displayed potent kinase inhibiting activity for FGFR1/2/3/4 and c-Met/ with IC50 0.19 nM/0.21 nM/1.15 nM/1.49 nM /15.9 nM, respectively. WXSH0011 inhibited cell proliferation in FGFR overexpressed NCl-H1581, SNU-16, RT112/84, Hep3B with IC50 10 nM, 3 nM, 2 nM, 63 nM, c-Met overexpressed MHCC97H and EBC-1 cell line with IC50 0.832 µM, 0.52 µM, FGFR, c-Met dual overexpressed LI-03-0141 (HCC) and ST-02-0063 (Gastric) PDC with IC50 0.19 µM, 0.08 µM. Kinome panel screening revealed that WXSH0011 demonstrated good overall kinase selectivity. WXSH0011 showed antitumor efficacy in gastric cancer SNU-16 (TGI = 100% @5 mpk, BID), as well as in the liver cancer Hep3B model (TGI = 86.92% @20 mpk, QD). Conclusions: We have identified a novel selective and potent dual FGFR/c-Met inhibitor WXSH0011. Preclinical studies shows antitumor efficacy of WXSH0011 in gastric and liver cancer models. It is currently at IND submission stage.

Inhibition of the MET Kinase Activity and Cell Growth in MET-Addicted Cancer Cells by Bi-Paratopic Linking

MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.

The effect of low-intensity ultrasound and met signaling on cellular motility and morphology

During the past decade, the potential of low-intensity ultrasound (LIUS) was demonstrated in medical research including microvascular remodeling, wound repair, blood flow restoration, angiogenesis and activation of mechanosensitive signaling pathways. However, the current clinical application of LIUS hasn’t been extended beyond physiotherapy. In cancer therapy, LIUS related research is focused on ultrasound-mediated techniques for chemotherapeutic drug activation, drug delivery enhancement and gene transfection improvement. While clinical trials show highly promising results, the ambiguity regarding the mechanism of ultrasound-cell interaction is a major limitation in the road to clinical applications. In 2011, the bilayer sonophore (BLS) model suggested that cell intramembrane cavitation is the underlying mechanism for ultrasound-induced bio-effects. According to the BLS model, ultrasound affects cell shape and functionality by inducing intramembrane gas bubbles that deform the cell membrane. This mechanical effect might involve alterations in the bio-chemical signaling pathways. Many biological processes such as cell motility, proliferation and angiogenesis are triggered by MET tyrosine kinase growth factor receptor and its ligand HGF/SF (Hepatocyte growth factor/Scatter Factor). The activation of MET receptor increases cancer cell motility via membrane alterations, leading to epithelial to mesenchymal transformation (EMT), invasion and metastasis. The aim of this work is to examine the crosstalk between MET-activation and LIUS under the assumption that both induce cell membrane alterations whereby affecting cell morphology and motility. In this paper, we present preliminary observations regarding the effect of MET-activation, LIUS exposure and their combined effect on living cells. Measurements of morphokinetic parameters using single cell time-lapse analysis are presented as well. The analysis demonstrates that LIUS inhibits the motility and modulates the morphology of MET-activated cells. These findings demonstrate the balance between the inner cellular processes (MET signaling pathways) and the external forces (LIUS exposure). Understanding the mechanism of LIUS on motile cells might improve the performance of existing LIUS-based cancer treatment modalities and help develop new ones.

Abstract B24: Met and VDAC1 as target for breast cancer therapy

Abstract During the past decade, considerable efforts have been devoted to the identification of agents that selectively kill neoplastic breast cancer cells based on their signaling and metabolic alterations. We hypothesis that combination tyrosine kinase and metabolic inhibitors are potential novel therapies for breast cancer. Aberrant signaling through Met-tyrosine kinase receptor and its ligand hepatocyte growth factor/scatter factor (HGF/SF) has been shown to play a critical role in development of tumors and metastasis in a variety of cancers, including breast cancer. We have previously shown that HGF/SF-induced Met-activation leads to diverse metabolic alteration in the tumor. Activation of Met signaling increases glycolytic flux into lactate and increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. The voltage-dependent anion channel 1 (VDAC1), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, thereby controlling crosstalk between mitochondria and the rest of the cell. VDAC1 is also a key player in mitochondria-mediated apoptosis. Thus, in addition to regulating the metabolic and energetic functions of mitochondria, VDAC1 appears to act as a convergence point for a variety of cell survival and cell death signals, mediated by its association with various ligands and proteins. Shoshan-Barmatz’s lab developed the retro-Tf-D-LP4 peptide, a cell-penetrating VDAC1-based peptide that acts by inducing apoptosis and impairment of cell energy and metabolism. To study the possible therapeutic effect of Met inhibition and alteration of VDAC1 combined therapy on breast tumor cells, we evaluated cell proliferation and motility in BC cells. Using our newly developed single-cell 35 parameters morphokinetic analysis infrastructure, we demonstrate that after 24 hours, the peptide decreased all cell lines velocity (5µM; MDMDA-MB-231_velocity=8.2%, p=0.024. 15µM; MDMDA-MB-231_velocity=62.4%, p&amp;lt;0.001). The peptide in combination with Met inhibitor further decreased cell velocity and acceleration (MDMDA-MB-231_Velocity=85.9%, p&amp;lt;0.001; MDMDA-MB-231_Acceleration=76%, p&amp;lt;0.001). The combined treatment increased cell coordination but abrogated the back-propagating wave. The morphologic effect of the treatment was dependent on the cell line. Cluster analysis of morphokinetic parameters demonstrated that untreated or low doses of treatment MDA-MB-231 and HB2 are clustered together; MCF7 and T47D have slower motility parameters. Upon high-dose combined treatment MDA-MB-231 cells change are clustered with slow-velocity T47D and MCF7. Combined Met inhibition and VDAC1 alteration dramatically reduced cell motility and increased coordinated motility. In vitro experiments show a slight inhibitory effect of VDAC1 on MDA-MB-231 cell proliferation. However, in vivo experiments demonstrate an inhibitory effect on tumor growth. These results indicate that the combined therapy can reduce cell motility and metastasis in triple-negative breast cancer tumors. Citation Format: Guy Namir, Varda Shoshan-Barmatz, Ilan Tsarfaty. Met and VDAC1 as target for breast cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B24.

Isoquercitrin, ingredients in Tetrastigma hemsleyanum Diels et Gilg, inhibits hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

ABSTRACTAberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the screening assay of extracts from the root tuber of Tetrastigma hemsleyanum Diels et Gilg, isoquercitrin inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering. Further analysis revealed that isoquercitrin specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met. We also found that isoquercitrin decreased HGF-induced migration and invasion by parental or HGF/SF-transfected bladder carcinoma cell line NBT-II cells. Furthermore, isoquercitrin inhibited HGF/SF-induced epithelial mesenchymal transition in vitro and the invasion/metastasis of HGF/SF-transfected NBT-II cells in vivo. Our data suggest the possible use of isoquercitrin in human cancers associated with dysregulated HGF/SF-Met signaling.

Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain

The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the “compact”, InternalinB-bound conformation, but not when MET is in the “open” conformation. These findings provide further support for the importance of the “compact” conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.

Novel pleiotropic effects of bioactive phospholipids in human lung cancer metastasis.

We previously proposed that one of the unwanted side effects of chemotherapy and radiotherapy is the increase in several peptide- and non-peptide based chemoattractants in damaged tissues, leading to induction of a prometastatic microenvironment for remaining cancer cells. Herein, we turned out our attention to a potential role of bioactive phospholipids (BphsLs), such as sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and lysophosphatidic acid (LPA) in lung cancer (LC) metastasis. We report that LC cells express several functional BphL receptors (for S1P, LPC, and LPA) as well as several enzymes involved in their metabolism and that BphsLs are potent chemokinetic and adhesion factors for these cells. We also demonstrate for the first time the novel role of C1P as a prometastatic factor in LC cells. In addition to their chemokinetic activities, BphsLs also sensitize or prime the chemotactic responsiveness of LC cells to known prometastatic factors such as hepatocyte growth factor/scatter factor (HGF/SF). Thus, for the first time we demonstrate a prometastatic effect that is based on the priming of a cell's responsiveness to chemotactic factors by chemokinetic factors. To our surprise, none of the bioactive lipids induced proliferation of LC cells or ameliorated toxic effects of vincristine treatment. Interestingly, BphsLs increase adhesion of LC cells to bone marrow-derived stromal cells and stimulate these cells to release ExNs, which additionally increase LC cell motility. In conclusion, our results show that BphsLs are important modulators of prometastatic environment. Therefore, their inhibitors could be considered as potential anti-metastatic drug candidates to be included as a part of post radio- and/or chemo- therapy treatment.

MicroRNA-182 Suppresses HGF/SF-Induced Increases in Retinal Pigment Epithelial Cell Proliferation and Migration through Targeting c-Met.

As increases in hepatocyte growth factor/scatter factor (HGF/SF) induce retinal pigment epithelial (RPE) migration and proliferation into the vitreous cavity and contribute to proliferative vitreoretinopathy (PVR) development, we determined if changes in miR-182 expression affect such behavioral changes. We found that miR-182 expression was less in PVR clinical samples than in primary RPE cells whereas c-Met was upregulated. Ectopic miR-182 inhibited RPE cell proliferation, cell cycle, and migration. Bioinformatic analysis identified c-Met as a miR-182 target, which was confirmed with the luciferase reporter assay. Transfection of miR-182 into RPE cells induced c-Met downregulation, which led to reduced cell proliferation and migration through declines in p-Akt formation. MiR-182 downregulation along with c-Met upregulation in PVR tissues suggest that these two opposing effects play important roles in PVR development. As ectopic miR-182 expression suppressed RPE cell proliferation and migration, strategies to selectively upregulate miR-182 expression in a clinical setting may provide a novel option to treat this disease.

Role of phytochemicals in the inhibition of epithelial–mesenchymal transition in cancer metastasis

Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells become mesenchymal cells, gaining fibroblast-like properties and displaying reduced intercellular adhesion and increased motility. EMT plays an important role in cancer metastasis. Suppressing or reversing EMT is therefore an important way of controlling various cancers. Phytochemicals are important sources of anticancer lead molecules. Natural products have been shown to safely suppress or reverse EMT via the inhibition of associated signalling pathways in various cancer cells. In the present review, we discuss the relevant factors and the role of EMT in cancer metastasis. We then present examples of phytochemicals with a role in the inhibition and reversal of EMT in various cancers, focusing on reports from 2012 to 2016.

Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing.

Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

Commentary: Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure.

Commentary: Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure.

Abstract 4981: Angiotensin-(1-7) targets c-Met signaling by up-regulating the phosphatase PTP1b to reduce triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with no known targeted therapies, since the tumors cells lack expression of the estrogen receptor and do not overexpress the HER2/Neu oncoprotein. Our laboratory showed that angiotensin-(1-7) [Ang-(1-7)], an endogenous seven amino acid peptide hormone, specifically activates the G protein-coupled receptor mas to inhibit the growth of lung, breast and prostate cancer cells and tumors. Using the 4T1 syngeneic mouse TNBC model, we examined the molecular signaling involved in the anti-tumor effects of Ang-(1-7). Ang-(1-7) significantly reduced tumor volume when compared to the control group (a 46% reduction by day 18, n = 6-7, p&amp;lt;0.05), with a concomitant decrease in tumor weight (2.0±0.3 g in untreated mice vs. 1.3±0.1 g in treated mice, p&amp;lt;0.05). A significant reduction in the proliferation marker Ki67 was observed in tumors from Ang-(1-7)-treated mice compared to tumors from untreated mice (18.1±3.7 Ki67(+) cells vs 103.9±17.2, p&amp;lt;0.005). Hepatocyte growth factor/scatter factor (HGF/SF) is a potent inducer of cell proliferation and cancer cell invasion by activating the receptor tyrosine kinase (RTK) c-Met and HGF/SF/c-Met signaling is elevated in TNBC. Ang-(1-7) attenuates c-Met RTK activation by HGF [phospho-c-Met(Y1234/1235)] in human MDA-MB-231 and mouse 4T1 TNBC cells by 30.3% (p&amp;lt;0.001) and 32.1% (p&amp;lt;0.005), respectively. The phosphatase PTP1b, a negative regulator of c-Met RTK activation, was significantly up-regulated by 75% in response to Ang-(1-7) treatment in the 4T1 cells (from 0.8±0.1 to 1.4±0.1, p&amp;lt;0.05), suggesting that the heptapeptide hormone increases PTP1b to negatively regulate c-Met signaling. A significant decrease in immunoreactive phospho-c-Met(Y1234/1235) was also observed in 4T1 tumors treated with Ang-(1-7) as compared to untreated tumors (from 47.0 to 19.7% of field, n = 5, p&amp;lt;0.05). The Ang-(1-7)-mediated suppression of HGF/SF-mediated c-Met activation was abrogated by D-alanine7-Ang-(1-7) [D-Ala], a mas receptor antagonist. In agreement with the Ang-(1-7)-mediated reduction of the tumor volume of 4T1 tumors, HGF-induced activation of the MAPKs ERK1 and ERK2 was reduced by the heptapeptide hormone in vitro (by 59.7 and 74.8%, respectively, n = 5). Further, Ang-(1-7) treatment significantly attenuated HGF/SF-stimulated cell invasion through Matrigel (by 53±2, n = 8, p&amp;lt;0.05 compared to HGF/SF stimulation); this effect was abrogated by the mas receptor antagonist D-Ala. Collectively, our data suggest that Ang-(1-7) in part suppresses the progression of triple negative breast tumors by activating the mas receptor to upregulate the phosphatase PTP1b and attenuate HGF/SF-induced c-Met activation, resulting in decreased ERK1/2 signaling, reduced tumor growth and diminished cell invasion, providing support for the use of Ang-(1-7) as a novel therapeutic agent for patients with TNBC. Citation Format: Guorui Deng, Linda Metheny-Barlow, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) targets c-Met signaling by up-regulating the phosphatase PTP1b to reduce triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4981. doi:10.1158/1538-7445.AM2015-4981