Hepatocyte growth factor (HGF) is the most potent mitogen for mature hepatocytes and seems to act as a hepatotropic factor that has not been purified over the past 30 years. HGF was first purified from rat platelets in 1986. HGF is a hetrodimer molecule composed of 69-kDa alpha-subunit and 34-beta-subunit. In 1989, cDNAs of both human and rat HGF were cloned and primary structure of HGF was determined. HGF is derived from preproprecursor of of 728 amino acids, which is proteolytically processed to form mature HGF. The alpha-chain contains four kringle domains and it has 38% homology with plasmin. HGF mRNA and HGF activity increase markedly in the liver of rats after various liver injuries such as hepatitis, ischemia, physical crush, and partial hepatectomy. Production of HGF in the liver occurs in Kupffer cells and sinusoidal endothelial cells, but not in parenchymal hepatocytes. HGF mRNA is also markedly increased even in the intact lung, kidney, and spleen after injuries of the liver. Therefore, HGF may act as a trigger for liver regeneration through two mechanisms: a paracrine mechanism and an endocrine mechanism. Moreover, HGF mRNA increases markedly in the kidney after various renal injuries, thus it suggests that HGF may act not only as a hepatotropic factor but also as a renotropic factor. HGF receptor with a Kd of 20 to 30 pM is widely distributed in various epithelial cells including hepatocytes. HGF receptor was recently identified as the product of c-met protooncogene, which encodes a 190-kDa transmembrane protein possessing tyrosine kinase domain. HGF has recently been shown to be a pleiotropic factor. HGF stimulates growth of various epithelial cells, including renal tubular cells (Mitogen). It is worth noting that HGF strongly enhances motility of epithelial cells (Motogen) and induces epithelial tubule formation (Morphogen), while it strongly inhibits growth of several tumor cells. All these findings indicate that HGF may have important roles in organogenesis, morphogenesis, carcinogenesis, as well as in organ regeneration.

Osteopontin (OPN) is a secreted protein that is overexpressed in a number of human cancers, and has been associated with increased metastatic burden and poor prognosis in breast cancer patients. The OPN protein contains several conserved structural elements including heparin- and calcium-binding domains, a thrombin-cleavage site, a CD44 binding site, and two integrin-binding sites. Experimental studies have shown that the ability of OPN to interact with a diverse range of factors, including cell surface receptors (integrins, CD44), secreted proteases (matrix metalloproteinases, urokinase plasminogen activator), and growth factor/receptor pathways (TGFalpha/EGFR, HGF/Met) is central to its role in malignancy. These complex signaling interactions can result in changes in gene expression, which ultimately lead to alterations in cell properties involved in malignancy such as adhesion, migration, invasion, enhanced tumor cell survival, tumor angiogenesis, and metastasis. Therefore, OPN is not merely associated with cancer, but rather it plays a multi-faceted functional role via complex molecular cross-talk with other factors. This review will focus on the role of OPN in breast cancer, in particular on the malignancy-promoting aspects of OPN that may reveal opportunities for new approaches to the clinical management of breast cancer.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver cells, epithelial cells, and endothelial cells and may play an important role in liver regeneration following hepatic injury. HGF is homologous to plasminogen and is first synthesized and secreted as an inactive single-chain precursor and then activated to a heterodimeric form by endoproteolytic processing. Recently, a novel serine protease responsible for this processing (HGF activator) has been purified from fetal bovine serum (Shimomura, T., Ochiai, M., Kondo, J., and Morimoto, Y. (1992) Cytotechnology 8, 219-229). In this study, we purified HGF activator from human serum and determined its partial amino acid sequence. Based on the amino acid sequence, we have molecularly cloned the cDNA for human HGF activator. The nucleotide sequence of the cDNA revealed that HGF activator is derived from the COOH-terminal half region of a precursor protein of 655 amino acids and that the precursor consists of multiple putative domains homologous to those observed in blood coagulation factor XII. These domains may be involved in the conversion of the precursor to the active form of HGF activator.

Wnt-1, a secreted glycoprotein, participates in development of the nervous system and contributes to mammary oncogenesis when overexpressed. We show that GSK3 activity is decreased in mouse mammary cells transformed by Wnt-1. These cells also exhibit a substantial Wnt-1 dependent increase in the uncomplexed population of beta-catenin. Wnt-1 signaling does not change the steady state level of either GSK3 alpha or GSK3 beta but instead leads to an increased association between GSK3 beta and beta-catenin. HGF/SF treatment of mouse mammary cells also leads to a transient decrease in GSK3 activity and a parallel, selective increase in the uncomplexed pool of beta-catenin. Both Wnt-1 and HGF/SF lead to nuclear accumulation of beta-catenin and activation of a LEF/Tcf responsive reporter gene. This study defines a pivotal signal transduction pathway, activated by both Wnt-1 and HGF/SF, leading to decreased GSK3 beta activity and consequently an increase in the free pool and nuclear accumulation of beta-catenin and changes in gene expression.

Previously, we demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is expressed by human bone stromal cells and is a powerful mitogen to prostatic epithelial cells in culture. Based on these observations, we hypothesized that, if prostate cancer cells in the prostate or bone environment respond to HGF/SF as a mitogen, then they must express the HGF/SF receptor, which is coded by the c-met proto-oncogene. We used immunohistochemical techniques to: 1) assess the presence and localization of c-met protein in benign and malignant human prostate tissues and 2) correlate the presence of c-met protein with tumor stage, grade and androgen sensitivity. c-met protein immunostaining was consistently observed in the basal epithelial layer of normal prostate glands but was absent in luminal epithelial cells of the peripheral and transition zones. c-met protein immunostaining was detected in 10 of 11 foci (91%) of high grade prostatic intraepithelial neoplasia (PIN). Overall, c-met protein staining was noted in 36 of 43 (84%) primary prostate cancer samples versus 2 of 11 (18%) benign prostate hyperplasia samples (p < 0.0001) and in 4 of 4 (100%) lymph node metastases, 23 of 23 (100%) bone marrow metastases and 1 of 3 (33%) other metastatic sites. There was a clear relationship between c-met protein staining and higher grade adenocarcinomas (p < 0.001). c-met protein is frequently detected in PIN and higher grade prostate cancers; future studies should evaluate the biological significance of these findings.

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids.

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

There is much evidence that rheumatoid arthritis is closely linked to angiogenesis. Important angiogenic mediators have been demonstrated in synovium and tenosynovium of rheumatoid joints. VEGF (Vascular Endothelial Growth Factor), expressed in response to soluble mediators such as cytokines and growth factors and its receptors are the best characterized system in the angiogenesis regulation of rheumatoid joints. Moreover, other angiogenic mediators such as platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, IL-8, IL-13, IL-15, IL-18, angiogenin, platelet activating factor (PAF), angiopoietin, soluble adhesion molecules, endothelial mediator (endoglin) play an important role in angiogenesis in rheumatoid arthritis. On the other hand, endostatin, thrombospondin-1 and -2 are angiogenic inhibitors in rheumatoid arthritis. The persistence of inflammation in rheumatoid joints is a consequence of an imbalance between these inducers and inhibitors of angiogenesis.

There is increasing evidence that epithelial-vascular interactions are essential for tissue patterning. Here we identified components of the molecular cross talk between respiratory epithelial cells and pulmonary capillaries necessary for the formation of the gas exchange surface of the lung. Selective inactivation of the Vegf-A gene in respiratory epithelium results in an almost complete absence of pulmonary capillaries, demonstrating the dependence of pulmonary capillary development on epithelium-derived Vegf-A. Deficient capillary formation in Vegf-A deficient lungs is associated with a defect in primary septae formation, a morphogenetic process critical for distal lung morphogenesis, coupled with suppression of epithelial cell proliferation and decreased hepatocyte growth factor (Hgf) expression. Lung endothelial cells express Hgf, and selective deletion of the Hgf receptor gene in respiratory epithelium phenocopies the malformation of septae, confirming the requirement for epithelial Hgf signaling in normal septae formation and suggesting that Hgf serves as an endothelium-derived factor that signals to the epithelium. Our findings support a mechanism for primary septae formation dependent on reciprocal interactions between respiratory epithelium and the underlying vasculature, establishing the dependence of pulmonary capillary development on epithelium-derived Vegf-A, and identify Hgf as a putative endothelium-derived factor that mediates the reciprocal signaling from the vasculature to the respiratory epithelium.

Liver sinusoidal endothelial cells (LSECs) have long been noted to contribute to liver regeneration after liver injury. In normal liver, the major cellular source of HGF is the hepatic stellate cell, but after liver injury, HGF expression has been thought to increase markedly in proliferating LSECs. However, emerging data suggest that even after injury, LSEC expression of HGF does not increase greatly. In contrast, bone marrow progenitor cells of LSECs (BM SPCs), which are rich in HGF, are recruited to the liver after injury. This Review examines liver regeneration from the perspective that BM SPCs that have been recruited to the liver, rather than mature LSECs, drive liver regeneration.

Bone-marrow-derived human MSCs (mesenchymal stem cells) support repair when administered to animals with TBI (traumatic brain injury) in large part through secreted trophic factors. We directly tested the ability of the culture medium (or secretome) collected from human MSCs under normoxic or hypoxic conditions to protect neurons in a rat model of TBI. Concentrated conditioned medium from cultured human MSCs or control medium was infused through the tail vein of rats subjected to TBI. We have demonstrated that MSCs cultured in hypoxia were superior to those cultured in normoxia in inducing expression of both HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor) in the cultured medium. We showed further that rats treated with the secretome from both normoxic- and hypoxic-preconditioned MSCs performed significantly better than the controls in both motor and cognitive functional test. Subsequent post-mortem evaluation of brain damage at the 4-day time point confirmed that both normoxic- and hypoxic-preconditioned MSC secretome-treated rats had significantly greater numbers of newly forming neurons, but significantly less than the controls in brain damaged volume and apoptosis. The TBI rats treated with hypoxic-preconditioned MSC secretome performed significantly better in both motor and cognitive function tests and neurogenesis, and had significantly less brain damage than the TBI rats treated with the normoxic-preconditioned MSC secretome. Collectively, these findings suggest that MSCs secrete bioactive factors, including HGF and VEGF, that stimulate neurogenesis and improve outcomes of TBI in a rat model. Hypoxic preconditioning enhances the secretion of these bioactive factors from the MSCs and the therapeutic potential of the cultured MSC secretome in experimental TBI.

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca(2+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

KAI1/CD82, a tetraspanin protein, was first identified as a metastasis suppressor in prostate cancer. How loss of CD82 expression promotes cancer metastasis is unknown. Restoration of CD82 expression to physiological levels in the metastatic prostate cell line PC3 inhibits integrin-mediated cell migration and invasion, but does not affect integrin expression. Integrin-dependent activation of the receptor kinase c-Met is dramatically reduced in CD82-expressing cells, as is c-Met activation by its ligand HGF/SF. CD82 expression also reduced integrin-induced activation and phosphorylation of the cytoplasmic tyrosine kinase Src, and its downstream substrates p130Cas and FAK Y861. Inhibition of c-Met expression or Src kinase function reduced matrigel invasion of PC3 cells to the same extent as CD82 expression. These data indicate that CD82 functions to suppress integrin-induced invasion by regulating signaling to c-Met and Src kinases, and suggests that CD82 loss may promote metastasis by removing a negative regulator of c-Met and Src signaling.

Squamous cell cancers comprise the most common type of human epithelial cancers. One subtype, esophageal squamous cell carcinoma (ESCC), is an aggressive cancer with poor prognosis due to late diagnosis and metastasis. Factors derived from the extracellular matrix (ECM) create an environment conducive to tumor growth and invasion. Specialized cancer-associated fibroblasts (CAFs) in the ECM influence tumorigenesis. We have shown previously that the nature and activation state of fibroblasts are critical in modulating the invasive ability of ESCC in an in vivo-like organotypic 3D cell culture, a form of human tissue engineering. Dramatic differences in invasion of transformed esophageal epithelial cells depended on the type of fibroblast in the matrix. We hypothesize that CAFs create an environment primed for growth and invasion through the secretion of factors. We find that fibroblast secretion of hepatocyte growth factor (HGF) fosters the ability of transformed esophageal epithelial cells to invade into the ECM, although other unidentified factors may cooperate with HGF. Genetic modifications of both HGF in fibroblasts and its receptor Met in epithelial cells, along with pharmacologic inhibition of HGF and Met, underscore the importance of this pathway in ESCC invasion and progression. Furthermore, Met activation is increased upon combinatorial overexpression of epidermal growth factor receptor (EGFR) and p53(R175H), two common genetic mutations in ESCC. These results highlight the potential benefit of the therapeutic targeting of HGF/Met signaling in ESCC and potentially other squamous cancers where this pathway is deregulated.

Lung cancer is a heterogeneous group of disorders that is now being subdivided into molecular subtypes with dedicated targeted therapies. The MET receptor tyrosine kinase has been identified as aberrantly overexpressed, potentially having activating mutations, and amplified in certain subsets of lung cancers. The ligand hepatocyte growth factor (HGF) can also be overexpressed in lung cancer or expressed in stroma, and both the MET receptor and the HGF ligand can be targets for therapeutics, especially in lung cancer. Activation of MET leads to a plethora of biochemical and biologic changes both in normal and cancerous cells. Preclinically, it has been shown that silencing or inactivating MET leads to decreased viability of cancer cells. There are a number of compounds against MET/HGF in clinical trials that have been shown to be active in lung cancers. This review will summarize the biology of MET as well as its therapeutic inhibition in lung cancer.

Activation of NFkappaB is a fundamental cellular event central to all inflammatory diseases. Hepatocyte growth factor (HGF) ameliorates both acute and chronic inflammation in a multitude of organ systems through modulating NFkappaB activity; nevertheless, the exact molecular mechanism remains uncertain. Here we report that HGF through inactivation of GSK3beta suppresses NFkappaB p65 phosphorylation specifically at position Ser-468. The Ser-468 of RelA/p65 situates in a GSK3beta consensus motif and could be directly phosphorylated by GSK3beta both in vivo and in vitro, signifying Ser-468 of RelA/p65 as a putative substrate for GSK3beta. In addition, the C terminus of RelA/p65 harbors a highly conserved domain homologue of the consensus docking sequence for GSK3beta. Moreover, this domain was required for efficient phosphorylation of Ser-468 and was indispensable for the physical interaction between RelA/p65 and GSK3beta. HGF substantially intercepted this interaction by inactivating GSK3beta. Functionally, phosphorylation of Ser-468 of RelA/p65 was required for the induced expression of a particular subset of proinflammatory NFkappaB-dependent genes. Diminished phosphorylation at Ser-468 by HGF resulted in a gene-specific inhibition of these genes' expression. The action of HGF on proinflammatory NFkappaB activation was consistently mimicked by a selective GSK3beta inhibitor or GSK3beta knockdown by RNA interference but largely abrogated in cells expressing the mutant uninhibitable GSK3beta. Collectively, our findings suggest that HGF has a potent suppressive effect on NFkappaB activation, which is mediated by GSK3beta, an important signaling transducer controlling RelA/p65 phosphorylation specificity and directing the transcription of selective proinflammatory cytokines implicated in inflammatory kidney disease.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs), which can differentiate into several lineages, have immunomodulatory properties similar to those of bone marrow-derived MSCs. However, the specific mechanism by which the immunomodulatory effect of MSCs occurs is not clear. In this study, we isolated canine AD-MSCs (cAD-MSCs) and induced their development into adipocyte, osteocyte, and neuron-like cells. We then investigated their phenotype and cytokine expression to determine whether they were able to exert an immunomodulatory effect and what the underlying mechanisms of this effect were. cAD-MSCs expressed CD44, CD90, and MHC class I and were also partially positive for the expression of CD34; however, they did not express CD14 and CD45. In addition, they expressed the mRNA of transforming growth factor beta (TGF-beta), IL-6, IL-8, CCL2, CCL5, vascular endothelial growth factor, hepatocyte growth factor (HGF), tissue inhibitor metalloproteinase-1/2, and cyclooxygenase-2 but not that of IL-10. Further, leukocyte proliferation induced by mitogens was suppressed when they were cocultured with irradiated cAD-MSCs, as well as with culture supernatants of cAD-MSCs alone. Moreover, TNF-alpha production significantly decreased, whereas TGF-beta, IL-6, and interferon-gamma production significantly increased in cAD-MSCs that were cocultured with leukocytes. Finally, immonomodulatory factors of MSCs, such as TGF-beta, HGF, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in cAD-MSCs that were cocultured with leukocytes; however, the production of PGE2 and IDO showed different kinetics, and leukocyte proliferation was effectively restored by PGE2 and IDO inhibitors. Taken together, these results indicate that the immunomodulatory effects of cAD-MSCs are associated with soluble factors (TGF-beta, HGF, PGE2, and IDO). Therefore, it is suggested that cAD-MSCs have a potential therapeutic use in the treatment of immune-mediated disease.

OBJECTIVE

Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance.

METHODS

We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab.

RESULTS

Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET-dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR-MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells.

CONCLUSIONS

These results suggest that overexpression of TGF-α through induction of EGFR-MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer.

BACKGROUND

MicroRNAs (miRNAs) have been suggested to play a vital role in tumor initiation and progression by negatively regulating oncogenes and tumor suppressors. Quite recently, studies have identified some miRNAs operating to promote or suppress tumor invasion or metastasis via regulating metastasis-related genes, providing potential therapeutic targets on anti-metastasis strategy. Metastasis-associated in colon cancer-1 (MACC1) has been newly identified to express highly in colorectal cancer (CRC) and promote tumor metastasis through transactivating metastasis-inducing HGF/MET signaling pathway. In this study, we investigated whether miRNA 143 is involved in the regulation of MACC1 and thus plays a functional role in CRC.

RESULTS

Using both in silico prediction and western blot assay, we found the previously reported tumor suppressive miR-143 targeted MACC1 in CRC. The direct interaction between them was confirmed by 3' UTR luciferase reporter gene. In concordance with the inhibitory effects induced by siRNA mediated knockdown of MACC1, restoration of miR-143 by mimics in SW620 cells significantly attenuated cell growth, migration and invasion. It is notable that combined treatment of miR-143 mimics and MACC1 siRNA induced synergistic inhibitory effects compared to either miR-143 mimics or MACC1 siRNA treatment alone. Conversely, reduction of miR-143 by inhibitors in SW480 cells apparently stimulated these phenotypes. Furthermore, we observed that miR-143 level was inversely correlated with MACC1 mRNA expression in CRC tissues.

CONCLUSIONS

Our findings newly described miR-143/MACC1 link and provided a potential mechanism for MACC1 dysregulation and contribution to CRC cell invasion. It may help to estimate the therapeutic utility of miR-143 in CRC.