A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR₁), and by PAR₁ inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR₁-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.     

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.     

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.Fundamental to cellular physiology is the ability to transmit extracellular signals across the cell membrane to trigger intracellular responses. Although the extracellular and intracellular portions of cell surface receptors are responsible for detecting ligands and initiating signal cascades, respectively, transmembrane (TM) domains are thought to play critical roles by specifically associating and propagating signals across the phospholipid bilayer. However, the mechanisms by which single-pass TM domains associate and conduct signals are poorly understood.The epidermal growth factor receptor (EGFR) is the prototypical type I TM receptor tyrosine kinase. EGFR and related members of the ErbB family—ErbB2, ErbB3, and ErbB4—contain a glycosylated extracellular ligand binding domain; a single-pass TM domain; and intracellular juxtamembrane, tyrosine kinase, and autophosphorylation domains. The extracellular domain of EGFR binds polypeptide growth factor ligands, such as epidermal growth factor (EGF), to stimulate an array of intracellular signaling cascades that regulate normal and oncogenic cellular growth and proliferation (3, 17, 36). In one model of growth factor-dependent EGFR activation, ligand binding promotes receptor dimerization and activation of intracellular protein tyrosine kinase activity (35); other models suggest that receptors are predimerized on the cell surface and ligand binding alters the equilibrium between inactive and active dimeric (or higher-order oligomeric) configurations (9, 29).Structural mechanisms of growth factor-mediated receptor dimerization and allosteric kinase domain activation have been proposed from recent crystal structures of isolated extracellular ligand binding domains (7) and intracellular tyrosine kinase domains (37). The orientation between the four extracellular domains is dramatically altered upon ligand binding, which frees interfaces that are masked in tethered, unliganded monomers to mediate dimer formation (7). Furthermore, an unusual asymmetric interface between two kinase domain monomers is linked to rearrangement of the kinase site to the active conformation (37). However, neither the position of the last extracellular domain, domain IV, nor association between the TM domains is well-defined experimentally in liganded receptors. The approximate location of domain IV has been suggested by models based on the orientation between domains III and IV in unliganded monomers (7, 12) and two-dimensional negative-stain electron microscopy (EM) averages (27); however, the position of domain IV in the liganded dimer has not been determined in previous crystal structures (13, 30). Thus, it is not known how the extracellular domain positions the TM domains for transmembrane signaling.Several lines of evidence suggest that the TM domain contributes directly to receptor dimerization and signaling. The neu oncogene encodes a Val → Glu substitution in the TM domain of ErbB2 that results in constitutive activation (34). Recombinant EGFR fragments consisting of the extracellular and TM domains have a 10⁵-fold higher affinity for dimerization than the isolated soluble extracellular domains (31). The TM domains of all four ErbB family members self-associate when expressed in bacterial inner membranes (26). A dimeric structure for isolated ErbB2 TM peptides in bicelles has been defined by nuclear magnetic resonance (NMR) imaging (4). However, ErbB2 does not bind ligand and does not physiologically homodimerize (17). Moreover, different ErbB family member TM domains utilize potentially distinct GxxxG sequence motifs to dimerize, as shown with fusion proteins in bacterial membranes (26). However, it is not clear how the TM domains contribute to dimerization and signaling in intact receptors on the cell surface.Here, we characterize the structural basis for EGFR transmembrane signaling. An improved crystal structure of the EGF-bound EGFR extracellular domain resolves domain IV in electron density maps and identifies a small domain IV dimerization interface, the mutation of which does not abolish signaling. The crystal structure and disulfide cross-linking demonstrate a flexible, dimeric linker between the extracellular and transmembrane domains. EGF-induced dimerization of the TM domains involves an interface far less extensive than that found in two receptors that dimerize in the absence of activation. Furthermore, mutagenesis shows that no unique interface is required for transmembrane signaling. Thus, we propose that signal transmission through the EGFR is communicated much more loosely than was previously thought.     

S-Nitrosylation of the epidermal growth factor receptor: A regulatory mechanism of receptor tyrosine kinase activity

Nitric oxide (NO) donors inhibit the epidermal growth factor (EGF)-dependent auto(trans)phosphorylation of the EGF receptor (EGFR) in several cell types in which NO exerts antiproliferative effects. We demonstrate in this report that NO inhibits, whereas NO synthase inhibition potentiates, the EGFR tyrosine kinase activity in NO-producing cells, indicating that physiological concentrations of NO were able to regulate the receptor activity. Depletion of intracellular glutathione enhanced the inhibitory effect of the NO donor 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) on EGFR tyrosine kinase activity, supporting the notion that such inhibition was a consequence of an S-nitrosylation reaction. Addition of DEA/NO to cell lysates resulted in the S-nitrosylation of a large number of proteins including the EGFR, as confirmed by the chemical detection of nitrosothiol groups in the immunoprecipitated receptor. We prepared a set of seven EGFR(C → S) substitution mutants and demonstrated in transfected cells that the tyrosine kinase activity of the EGFR(C166S) mutant was completely resistant to NO, whereas the EGFR(C305S) mutant was partially resistant. In the presence of EGF, DEA/NO significantly inhibited Akt phosphorylation in cells transfected with wild-type EGFR, but not in those transfected with C166S or C305S mutants. We conclude that the EGFR can be posttranslationally regulated by reversible S-nitrosylation of C166 and C305 in living cells.     

Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host

The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.     

Kinase Activator-Receiver Preference in ErbB Heterodimers Is Determined by Intracellular Regions and Is Not Coupled to Extracellular Asymmetry

The EGF receptor (EGFR) family comprises four homologs in humans collectively known as the ErbB or HER proteins. ErbB proteins are receptor tyrosine kinases that become activated when ligands bind to their extracellular regions and promote formation of specific homo- and heterodimers with enhanced tyrosine kinase activity. An essential feature of ErbB activation is formation of an asymmetric kinase dimer in which the C-terminal lobe of one kinase serves as the activator or donor kinase by binding the N-terminal lobe of a receiver or acceptor kinase and stabilizing its active conformation. ErbB extracellular regions are also thought to form active asymmetric dimers in which only one subunit binds ligand. The observation that the unliganded ErbB2 kinase preferentially serves as the activator kinase when paired with EGFR/ErbB1 implied that extracellular asymmetry in ErbB proteins might be coupled to intracellular asymmetry with unliganded partners favoring the activator kinase position. Using cell-based stimulation assays and chimeric ErbB proteins, we show that extracellular asymmetry is not coupled to intracellular asymmetry and that ErbB intracellular regions are sufficient to determine relative kinase activator-receiver orientation. We further show a hierarchy of activator-receiver preferences among ErbB proteins, with EGFR/ErbB1 being the strongest receiver, followed by ErbB2 and then ErbB4, and that cis-phosphorylation of EGFR and ErbB2 appears to be negligible. This hierarchy shapes the nature of signaling responses to different ligands in cells expressing multiple ErbB proteins.     

Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual EGFR/HER2 Tyrosine Kinase Inhibitors

4-Anilinoquinazolines as an important class of protein kinase inhibitor are widely investigated for epidermal growth factor receptor (EGFR) tyrosine kinase or epidermal growth factor receptor 2 (HER2) inhibition. A series of novel 6-salicyl-4-anilinoquinazoline derivatives 9–27 were prepared and evaluated for their EGFR/HER2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on three variant cancer cell lines (A431, MCF-7, and A549). The bioassay results showed most of the designed compounds exhibited moderate to potent in vitro inhibitory activity in the enzymatic and cellular assays, of which compound 21 revealed the most potent dual EGFR/HER2 inhibitory activity, with IC₅₀ values of 0.12 µM and 0.096 µM, respectively, comparable to the control compounds Erlotinib and Lapatinib. Furthermore, the kinase selectivity profile of 21 was accessed and demonstrated its good selectivity over the majority of the close kinase targets. Docking simulation was performed to position compound 21 into the EGFR/HER2 active site to determine the probable binding pose. These new findings along with molecular docking observations could provide an important basis for further development of compound 21 as a potent EGFR/HER2 dual kinase inhibitor.     

The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface.The epidermal growth factor receptor (EGFR) is often considered the “prototypical” receptor tyrosine kinase (RTK) and has been intensively studied. It is one of a family of four RTKs in humans, the others being ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 (Fig. 1). EGFR and its relatives are known oncogenic drivers in cancers such as lung cancer (Mok 2011), breast cancer (Arteaga et al. 2011), and glioblastoma (Libermann et al. 1985; Lee et al. 2006a; Vivanco et al. 2012), and inhibitors of these receptors have been among the most successful examples of targeted cancer therapies to date (Arteaga 2003; Moasser 2007; Zhang et al. 2007), including antibody therapeutics (e.g., trastuzumab and cetuximab) and small-molecule tyrosine kinase inhibitors (e.g., erlotinib, gefitinib, lapatinib).Open in a separate windowFigure 1.Schematic representation of EGFR/ErbB family receptors and their ligands. (A) The domain composition of human EGFR is shown. The extracellular region contains four domains: Domain I (amino acids 1–165), domain II (amino acids 165–310), domain III (amino acids 310–480), and domain IV (amino acids 480–620). Domains I and III are closely related in sequence, as are domains II and IV. Shown are representations of the structures of domains I and IV. Domain IV contains two types of disulfide-bonded module (C1 and C2). In C1 domains, a single disulfide constrains an intervening bow-like loop. In C2 modules, two disulfides link four successive cysteines in the patterns C1–C3 and C2–C4 to give a knot-like structure. A short extracellular juxtamembrane (eJM) region separates the extracellular region from the ∼23-amino-acid transmembrane (TM) domain. Within the cell, a short intracellular juxtamembrane (iJM) region separates the tyrosine kinase domain (TKD) from the membrane. A representative EGFR TKD structure is shown. The TKD is followed by a carboxy-terminal largely unstructured tail (amino acids 953–1186) that contains at least five tyrosine autophosphorylation sites. (B) EGFR is one of four members of the EGFR/ErbB family in humans. The other members are ErbB2/HER2, for which no soluble activating ligand is shown; ErbB3/HER3, which has a significantly impaired kinase domain (Jura et al. 2009b; Shi et al. 2010); and ErbB4/HER4. The primary active moiety of the ligands for these receptors is the EGF-like domain, shown as a cartoon structure (top right). EGFR is activated by the EGFR agonists: EGF itself, TGF-α (transforming growth factor α), ARG (amphiregulin), and EGN (epigen). The bispecific ligands regulate both EGFR and ErbB4: HB-EGF (heparin-binding EGF-like growth factor), EPR (epiregulin), and BTC (betacellulin). Neuregulins (NRGs) 1 and 2 regulate ErbB3 and ErbB4, whereas NRG3 and NRG4 appear to be specific for ErbB4 (Wilson et al. 2009).Far from being prototypical, however, it is now clear that regulation of EGFR family members is unique among RTKs (Ferguson 2008; Lemmon 2009; Lemmon and Schlessinger 2010). Structural studies have revealed how the ∼620-amino-acid isolated extracellular region is induced to dimerize after growth factor binding (Burgess et al. 2003) and how the isolated intracellular tyrosine kinase domain (TKD) becomes allosterically activated after forming an asymmetric dimer (Zhang et al. 2006; Jura et al. 2009a; Red Brewer et al. 2009). These findings have typically been interpreted in the context of a model in which EGF family receptors are regulated through ligand-induced receptor homodimerization or heterodimerization, with growth factor binding converting the receptor from an inactive monomeric configuration to an active dimeric conformation (Yarden and Schlessinger 1987; Schlessinger 1988, 2014; Ullrich and Schlessinger 1990). Although this original model has stood the test of time and initiated a whole field of studies of ligand-induced RTK dimerization, recent work has provided highly sophisticated views of a unique mode of allosteric regulation used by EGFR.     

The human tyrosine kinase gene (FER) maps to chromosome 5 and is deleted in myeloid leukemias with a del(5q)

A novel member of the SRC tyrosine kinase gene family was recently isolated and characterized (Hao et al., 1989). This FES/FPS-related gene, named FER, lacks the transmembrane and extracellular domains which characterize tyrosine kinases with receptor function. Expression of FER in a wide range of cell types indicates a general role in intracellular signalling or differentiation processes. We have now mapped FER to chromosome 5q14----q23 using in situ hybridization techniques and suggest a more precise location within bands 5q21----q22. This region lies adjacent to a complex domain of growth factors and receptors, many involved in regulation of haematopoiesis. FER maps within a critical segment frequently deleted from chromosome 5 in patients with acute myeloid leukemia or myelodysplastic syndromes and was shown to be deleted in two such patients. It also maps close to the familial polyposis coli locus at 5q22.     

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.     

Association between polymorphism in the insulin receptor-related receptor gene and growth traits in the Pacific oyster Crassostrea gigas

Insulin receptor-related receptor (IRR) is an orphan receptor tyrosine kinase of the insulin receptor family, and involved in the growth and reproduction processes of the Pacific oyster Crassostrea gigas. Polymorphisms of the IRR gene were evaluated for associations with growth performance of 336 individuals in five families, and further confirmed in 206 individuals from three selectively bred strains for fast growth. Two of the six identified synonymous mutations (C.1996G > A and C.2110C > T) were significantly associated with growth performance in the families and strains. Five diplotypes were constructed based on the two growth-related SNPs, and diplotypes analysis revealed that D₃ (GGTT) might be the most advantageous diplotype for growth traits. The results suggest that two SNPs (C.1996G > A and C.2110C > T) in IRR gene are potentially associated with growth performance of C. gigas, and could serve as genetic markers for fast growth in oyster breeding.     

Mer receptor tyrosine kinase signaling: prevention of apoptosis and alteration of cytoskeletal architecture without stimulation or proliferation

Mer is a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR, Mer chimera activation induced adherence and cell flattening in the normally suspension-growing 32D cells. The Mer chimera signal also blocked IL-3-dependent proliferation leading to G(1)/S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4-8 days) ligand-dependent differentiation of 32D cells, the combined Mer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Mer chimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases.     

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.     

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor,improves wound healing through cGMP/cGMP-dependent protein kinase

Nitric oxide (NO) donors have been shown to improve wound healing, but the mechanism is not well defined. Here we show that the novel NO donor nitrosyl-cobinamide (NO-Cbi) improved in vitro wound healing in several cell types, including an established line of lung epithelial cells and primary human lung fibroblasts. On a molar basis, NO-Cbi was more effective than two other NO donors, with the effective NO-Cbi concentration ranging from 3 to 10 μM, depending on the cell type. Improved wound healing was secondary to increased cell migration and not cell proliferation. The wound healing effect of NO-Cbi was mediated by cGMP, mainly through cGMP-dependent protein kinase type I (PKGI), as determined using pharmacological inhibitors and activators, and siRNAs targeting PKG type I and II. Moreover, we found that Src and ERK were two downstream mediators of NO-Cbi's effect. We conclude that NO-Cbi is a potent inducer of cell migration and wound closure, acting via cGMP, PKG, Src, and extracellular signal regulated kinase (ERK).     

Role of targeted therapy in the treatment of squamous cell head and neck cancer

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck cancer (HNC). Since EGFR has a large extracellular ligand binding as well as an intracellular tyrosine kinase domain, anti-EGFR therapy may involve anti-ligand binding domain antibody- or tyrosine kinase inhibitor therapies. Phase II-III studies confirmed the efficacy of anti-EGFR antibody therapy in case of squamous cell HNC. In combination with irradiation, anti-EGFR antibody therapy improved survival of locally advanced HNC patients. In case of recurrent or metastatic HNC, anti-EGFR antibody therapy in combination with chemotherapy significantly increased remission rate without increasing toxicity. Although studies on EGFR kinase inhibitors in HNC are in early phase, preliminary data are encouraging.     

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.     

The HDAC inhibitor,MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells

Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.     

Platycodin D inhibits migration,invasion, and growth of MDA-MB-231 human breast cancer cells via suppression of EGFR-mediated Akt and MAPK pathways

Platycodin D (PD), an active triterpenoid saponin from Platycodon grandiflorum, has been known to inhibit the proliferation of a variety of cancer cells, but the effect of PD on the invasiveness of cancer cells is largely unknown. In this study, we first determined the molecular mechanism by which PD inhibits the migratory and invasive abilities of the highly metastatic MDA-MB-231 breast cancer cell line. We demonstrated that a non-cytotoxic concentration of PD markedly suppressed wound healing migration, invasion through the matrigel, and adhesion to an ECM-coated substrate in a dose-dependent manner. Moreover, PD inhibited cell invasion by reducing matrix metalloproteinase (MMP)-9 enzyme activity and mRNA expression. Western blot analysis indicated that PD potently suppressed the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) as well as blocked the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling pathway. Furthermore, PD treatment inhibited the DNA binding activity of NF-κB, which is known to mediate the expression of epidermal growth factor receptor (EGFR), as observed by electrophoretic mobility shift assay. Specific mechanisms of action exerted by PD involved the downregulation of EGFR and the inhibition of EGF-induced activation of the EGFR, MAPK, and PI3K/Akt pathways. The in vivo studies showed that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in BALB/c nude mice. These results suggest that PD might be a potential therapeutic candidate for the treatment of breast cancer metastasis.     

Suppression of α-catenin and adherens junctions enhances epithelial cell proliferation and motility via TACE-mediated TGF-α autocrine/paracrine signaling

Sustained cell migration is essential for wound healing and cancer metastasis. The epidermal growth factor receptor (EGFR) signaling cascade is known to drive cell migration and proliferation. While the signal transduction downstream of EGFR has been extensively investigated, our knowledge of the initiation and maintenance of EGFR signaling during cell migration remains limited. The metalloprotease TACE (tumor necrosis factor alpha converting enzyme) is responsible for producing active EGFR family ligands in the via ligand shedding. Sustained TACE activity may perpetuate EGFR signaling and reduce a cell’s reliance on exogenous growth factors. Using a cultured keratinocyte model system, we show that depletion of α-catenin perturbs adherens junctions, enhances cell proliferation and motility, and decreases dependence on exogenous growth factors. We show that the underlying mechanism for these observed phenotypical changes depends on enhanced autocrine/paracrine release of the EGFR ligand transforming growth factor alpha in a TACE-dependent manner. We demonstrate that proliferating keratinocyte epithelial cell clusters display waves of oscillatory extracellular signal–regulated kinase (ERK) activity, which can be eliminated by TACE knockout, suggesting that these waves of oscillatory ERK activity depend on autocrine/paracrine signals produced by TACE. These results provide new insights into the regulatory role of adherens junctions in initiating and maintaining autocrine/paracrine signaling with relevance to wound healing and cellular transformation.