Ligand Heparin-binding Epidermal Growth Factor Research Articles

Estrogen Receptor and Receptor Tyrosine Kinase Signaling: Use of Combinatorial Hormone and Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2–Targeted Therapies for Breast Cancer

Estrogen receptor (ER ) is expressed in nearly 70% of breast cancers and is a key driver of growth in ER-positive breast tumors. ER is primarily a nuclear protein that, when activated by estrogens, acts as a ligand-dependent transcription factor and promotes the expression of several genes that enhance cell survival, proliferation, and tumor progression (Fig 1). Examples of gene products upregulated by ER’s genomic actions include Bcl-2, cyclin D1, and the insulin-like growth factor 1 (IGF-1) receptor. Activated ER can also be localized to the cellular membrane and exert rapid, nongenomic effects. Here ER can colocalize with numerous different signaling molecules that lead to activation of growth and survival-promoting pathways, including the mitogen-activated protein kinase (MAPK) and Akt pathways. The use of hormone therapies, including aromatase inhibitors and selective estrogen receptor modulators, that target and downregulate ER signaling in the breast is a mainstay of treatment for patients with ER-positive breast cancers. Although these agents are extremely effective, a significant proportion of patients exhibit de novo or acquired resistance to hormone therapy. A proposed mechanism of resistance to hormone therapy is signaling crosstalk between ER and growth factor receptor tyrosine kinases (RTKs). In an accompanying article, Guarneri et al report the results of a randomized, double-blind, placebo-controlled, multicenter phase IIb neoadjuvant study of the aromatase inhibitor letrozole combined with the dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/ HER2) inhibitor lapatinib in postmenopausal hormone receptor– positive, HER2-negative early-stage breast cancer. Their results show that clinical response rates were similar for patients who received a combination of letrozole and lapatinib compared with patients receiving letrozole and placebo (70% v 63%, respectively). However, on further subset analysis, they found that patients with a PIK3CA mutation (37%) had a significantly higher likelihood of clinical response to the letrozole-lapatinib combination therapy versus those patients without PIK3CA mutations (overall response rate of 93% for patients with mutant PIK3CA v 63% for patients with wild-type PIK3CA). PIK3CA is the gene that encodes the PI3K catalytic subunit p110 , and the PI3K pathway is the most commonly mutated pathway in breast cancers, with PIK3CA recurrent hotspot mutations found at high frequency in ER-positive breast tumors. PI3K has been implicated as a key mediator of hormone resistance in preclinical models, and evidence suggests that hyperactivation of PI3K may enhance estrogen-independent and -dependent ER transcriptional activity. Although inhibition of the PI3K pathway has been postulated to sensitize cells to hormone therapy, several retrospective clinical studies have shown that activating mutations in PIK3CA paradoxically predict for a better long-term prognosis in patients with ER-positive breast cancers treated with hormonal therapy although other studies have shown contradictory results. It is known that activated RTKs and their downstream signaling partners can directly interact with ER and cause phosphorylation of key residues in the AF-1 domain of ER. This phosphorylation results in activation of ER in a ligand-independent fashion to promote cell growth and survival. In addition, membrane-activated ER can activate the EGFR pathway by increasing the production of the EGFR ligand heparin-binding epidermal growth factor. Furthermore, in vitro studies have shown that breast cancer cell lines with acquired resistance to hormone therapy have increased expression and activation of EGFR and HER2 along with their downstream signaling pathways. The use of ErbB-targeted tyrosine kinase inhibitors or the anti-HER2 monoclonal antibody trastuzumab significantly inhibits the growth of these cells after development of resistance to hormone therapies. In addition, sensitivity to hormone therapies can be restored when resistant cells are treated with lapatinib, a dual EGFR/ HER2 inhibitor. Together, these data suggest that enhanced RTK signaling and activationofPI3Ksignalingmaybekeycontributorstoresistancetohormone therapies in breast cancer. This provides the rationale for the dual JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 32 NUMBER 10 APRIL 1 2014

Epidermal Growth Factor (EGF) Ligand Release by Substrate-specific A Disintegrin and Metalloproteases (ADAMs) Involves Different Protein Kinase C (PKC) Isoenzymes Depending on the Stimulus

The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism

Cell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF. To determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration. Human coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, betaARK(CT) were also used. ATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gbetagamma inhibitor betaARK(CT) inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect. ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.

Tumor necrosis factor‐α‐stimulated membrane type 1‐matrix metalloproteinase production is modulated by epidermal growth factor receptor signaling in human gingival fibroblasts

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts. Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction. Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha). The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.

Prolactin and ErbB4/HER4 Signaling Interact via Janus Kinase 2 to Induce Mammary Epithelial Cell Gene Expression Differentiation

Differentiation of mammary epithelium in vivo requires signaling through prolactin and ErbB4/HER4-dependent mechanisms. Although stimulation of either the prolactin receptor or ErbB4/HER4 results in activation of the transcription factor signal transducer and activator of transcription 5A (STAT5A) and induction of lactogenic differentiation, how these pathways intersect is unknown. We show herein that prolactin signaling in breast cells cooperates with and is substantially enhanced by the receptor tyrosine kinase ErbB4/HER4. Prolactin and the ErbB4/HER4 ligand heparin-binding epidermal growth factor each induced STAT5A tyrosine phosphorylation and nuclear translocation; each pathway required the intracellular tyrosine kinase Janus kinase 2 (JAK2). We found that full prolactin-mediated STAT5A activation and binding to the endogenous beta-casein promoter required ErbB4/HER4 but did not require ErbB1/epidermal growth factor receptor. For example, prolactin-induced STAT5A activity was markedly diminished in cells overexpressing kinase inactive HER4, in cells transfected with small interfering RNAs to specifically knock down endogenous ErbB4/HER4 expression and in cells treated with a small molecule inhibitor that targets ErbB4 kinase. Interestingly, prolactin caused ErbB4/HER4 tyrosine phosphorylation in a JAK2 kinase-dependent manner. Finally, prolactin receptor, ErbB4/HER4, and JAK2 were coimmunoprecipitated from prolactin-treated but not untreated cells. These results suggest that prolactin signaling engages the ErbB4 pathway via JAK2 and that ErbB4 provides an important component of STAT5A-dependent lactogenic differentiation; this pathway integration may help explain the similar deficit in mammary development observed in gene-targeted mice deficient in prolactin receptor, JAK2, ErbB4, or STAT5A.

Insulin-induced epidermal growth factor activation in vascular smooth muscle cells is ADAM-dependent

With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. This study examines the role of insulin actions on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor-linked tyrosine kinases and is key to many of their responses. To determine the pathway of EGFR transactivation by insulin in human vascular smooth muscle cells (VSMC). VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the A disintegrin and metalloproteinase domain (ADAM) inhibitors tumor necrosis factor-alpha protease inhibitor (TAPI)-0 and TAPI-1, heparin-binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration-dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. This is the first description of cross-talk via ADAM between insulin and EGFR in VSMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target.

Heparin binding EGF is necessary for vasospastic response to endothelin

Endothelin-1 (ET-1), a powerful vasoconstrictor, is involved in vasospastic diseases such as coronary artery disease and subarachnoidal hemorrhage, as well as in renal and cardiovascular fibrotic remodeling. Transactivation of the epidermal growth factor receptor (EGFR) mediates ET-1 signaling in vascular smooth muscle cells (VSMCs) and isolated arteries. Moreover, EGFR is required for a full constrictive response to ET-1. However, the relevant mechanisms mediating EGFR transactivation in response to ET-1 have not been identified. The present study used isolated arteries and VSMCs to investigate the role of the EGFR ligand heparin binding-epidermal growth factor (HB-EGF) in ET-1-induced transactivation of EGFR, intracellular calcium mobilization, and VSMCs contraction. While baseline blood pressures were similar in HB-EGF-deficient and in wild-type littermate mice, the vasoconstrictor actions of ET-1 were attenuated in HB-EGF-/- animals. In isolated mouse carotid artery segments mounted in an arteriograph, ET-1 caused only a weak increase in isovolumetric tone in HB-EGF-deficient vessels, and this effect was mimicked by inhibition of EGFR tyrosine kinase or phosphoinositide 3-kinase (PI3K) in wild-type arteries with or without endothelium, indicating a specific role in VSMCs. EGFR or PI3K inhibitors had no effect on KCl-induced contraction, which was normal in HB-EGF-deficient mice. To confirm that the abnormal responses in HB-EGF-deficient mice were due to impaired EGFR signaling, we studied VSMCs from waved-2 (wa2) mice; these animals have a mutation causing a partial loss of function of EGFR tyrosine kinase activity. The ET-1-induced calcium peak was reduced by 30% in VSMCs from wa2 mice and from HB-EGF-/- mice. This effect was reproduced by preincubation of wild-type VSMCs with EGFR inhibitor AG1478 and PI3K inhibitors LY294002 and wortmannin. ProHB-EGF is bound to the cell membrane and released after cleavage by metalloproteinases; its action may contribute to effects of GPCR agonists on cell growth. Pretreatment of mouse VSMCs with batimastat, a metalloproteinase inhibitor, significantly attenuated ET-1-induced [Ca(2+)](i) response in wild-type cells. Human proHB-EGF has been shown to be the endogenous receptor for Corynebacterium diphteriae toxin (DT). Mutated DT toxin (CRM197) is devoid of toxicity but it neutralizes HB-EGF binding to EGFR. Pretreatment of human VSMCs from internal mammary arteries with CRM197 significantly blunted ET-1-stimulated calcium transients. In conclusion, these findings suggest that the mechanism of ET-1-induced vasoconstriction involves HB-EGF-mediated transactivation of the EGFR. This functional cascade requires modulation of agonist-induced calcium transient by EGFR and PI3K with extremely fast kinetics, suggesting a novel paradigm for GPCR-mediated calcium signaling, which may offer future therapeutic targets.

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway

Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.

A new mechanism of infertility associated with myoma: decreased production of heparin-binding epidermal growth factor in the endometrium

Background: The receptive uterus produces the epidermal growth factor receptor ligand heparin binding-epidermal growth factor (HB-EGF), a membrane-associated protein found in the luminal epithelium at the site adjacent to the blastocyte.Objective: To determine the role of HB-FGF in infertility associated with a myoma.Methods: Twenty primary infertiles having myoma and 20 controls were enrolled in the study to examine HB-EGF in the endometrium of the luteal phase of the menstrual cycle. Endometrial biopsy specimens collected during the luteal phase of the cycle underwent H & E staining for dating. Heparin-binding epidermal growth factor analysis cryostate sections were fixed, rehydrated, and incubated with goat polyclonal antibody against human HB-EGF 10 μg/mL. At the same time the negative control slide was incubated with nonimmune goat IgG, the slides were incubated with rabbit antigoat IgG, and the peroxidase reaction was scored using the following scheme: 0 = nonreaction; 1 = mild; 2 = moderate; and 3 = heavy.Results: There is a statistically significant reduction in the production of HB-EGF in the luteal phase of infertile myomatous than in the control group (P <0.005).Conclusion: A new cause of infertility associated with myoma (ie, decreased production of HB-EGF) is important in human implantation.