All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor α, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17−/− knockout mice corroborated the essential role of adam17−/− in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.
EGF receptor; EGF receptor ligands; ADAMs; ectodomain shedding; growth factor signaling
Autocrine, paracrine and juxtacrine are recognized modes of action for mammalian EGFR ligands that include EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells four-fold over TGFα or HB-EGF exosomes and five-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between non-transformed and transformed cells is unknown. We show that exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, non-transformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes may contribute to diverse cancer phenomena such as field effect and priming the metastatic niche.
Exosomes; EGF Receptor; amphiregulin; TGF-α; HB-EGF
Activation of the epidermal growth factor receptor (EGFR) requires cell surface cleavage of EGFR ligands, uptake of soluble ligand by the receptor, and initiation of EGFR tyrosine kinase activity. We define these collective events as the EGFR axis. Transforming growth factor-α (TGF-α) and amphiregulin are two EGFR ligands that are delivered preferentially to the basolateral surface of polarized epithelial cells where the EGFR resides. TACE/ADAM-17 (tumor necrosis factor-α converting enzyme/a disintegrin and metalloprotease) has been implicated in ectodomain cleavage of TGF-α and amphiregulin.
Using a human polarizing colorectal cancer (CRC) cell line, HCA-7, and a tissue array of normal colonic mucosa and primary and metastatic CRC, we determined the intracellular localization of TACE and the effects of EGFR axis inhibition in CRC.
Herein, we show that TACE is localized to the basolateral plasma membrane of polarized HCA-7 cells. TACE is overexpressed in primary and metastatic CRC tumors compared with normal colonic mucosa; the intensity of its immunoreactivity is inversely correlated with that of TGF-α and amphiregulin. Pharmacologic blockade of HCA-7 cells with an EGFR monoclonal antibody, a selective EGFR tyrosine kinase inhibitor, and a selective TACE inhibitor results in concentration-dependent decreases in cell proliferation and active, phosphorylated mitogen-activated protein kinase. Combining suboptimal concentrations of these agents results in cooperative growth inhibition, increased apoptosis, and reduced mitogen-activated protein kinase pathway activation. Furthermore, an EGFR tyrosine kinase – resistant clone of HCA-7 cells is growth-inhibited by combined monoclonal antibody and TACE inhibition.
These results implicate TACE as a promising target of EGFR axis inhibition in CRC.
Mammalian cells respond to environmental stress by activating a variety of protein kinases critical for cellular signal transmission, such as the epidermal growth factor receptor (EGFR) tyrosine kinase and different members of the mitogen-activated protein kinase (MAPK) family. EGFR activation by stress stimuli was previously thought to occur independently of stimulation by extracellular ligands. Here, we provide evidence that osmotic and oxidative stresses induce a metalloprotease activity leading to cell surface cleavage of pro-heparin-binding EGF (pro-HB-EGF) and subsequent EGFR activation. This ligand-dependent EGFR signal resulted from stress-induced activation of the MAPK p38 in human carcinoma cells and was mediated by the metalloproteases ADAM9, -10, and -17. Furthermore, stress-induced EGFR activation induced downstream signaling through the MAPKs extracellular signal-regulated kinases 1 and 2 and JNK. Interestingly, apoptosis induced by treatment of tumor cells with doxorubicin was strongly enhanced by blocking HB-EGF function. Together, our data provide novel insights into the mammalian stress response, suggesting a broad mechanistic relevance of a p38-ADAM-HB-EGF-EGFR-dependent pathway and its potential significance for tumor cells in evasion of chemotherapeutic agent-induced apoptosis.
Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.
The ectodomain shedding of epidermal growth factor receptor (EGFR) ligands, such as amphiregulin (AREG), by ADAMs (A Disintegrin And Metalloproteases) can be stimulated by G protein-coupled receptor (GPCR) agonists. Interactions between the CXCR4 GPCR and the CXCL12 chemokine have been shown to mediate gene transcription and cellular proliferation in non-transformed and transformed prostate epithelial cells, as well as motility/invasiveness in transformed cells.
In this report, we investigated the ability of CXCL12 to stimulate amphiregulin ectodomain shedding in non-transformed and transformed prostate epithelial cells that respond proliferatively to sub-nanomolar levels of CXCL12 and amphiregulin.
Materials and Methods
Non-transformed N15C6 and transformed PC3 prostate epithelial cells were assessed for amphiregulin shedding, ADAM activation, Src phosphorylation and EGFR activation using ELISA, immunoblot, and immunoprecipitation techniques, and for proliferation using cell counting after stimulation with CXCL12 or vehicle.
The results of these studies identify CXCL12 as a novel inducer of amphiregulin ectodomain shedding and show that both basal and CXCL12-mediated amphiregulin shedding are ADAM10- and Src kinase-dependent in non-transformed N15C6 cells. In contrast, amphiregulin shedding is not amplified subsequent to stimulation with exogenous CXCL12, and is not reduced subsequent to metalloprotease- or Src kinase-inhibition, in highly aggressive PC3 prostate cancer cells. These data also show that CXCL12-mediated cellular proliferation requires EGFR transactivation in a Src-and ADAM-dependent manner in non-transformed prostate epithelial cells. However, these same mechanisms are dysfunctional in highly transformed prostate cancer cells, which secrete amphiregulin in an autocrine manner that cannot be repressed through metalloprotease- or Src kinase inhibition.
These findings show that non-transformed and transformed prostate epithelial cells may employ different mechanisms to activate EGFR ligands and thereby utilize the EGFR axis to promote cellular proliferation.
The authors have previously demonstrated that wounding of human corneal epithelial cells (HCECs) transactivates epidermal growth factor (EGF) receptor (EGFR) and its downstream signaling pathways and that this EGFR signaling is required for epithelial wound healing. In this study, the authors sought to identify the underlying mechanisms for EGFR transactivation in response to wounding in HCECs.
SV40-immortalized HCEC (THCE) monolayer was wounded and allowed to heal in the presence or absence of a selective inhibitor of the Src family kinases PP2 and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Wound closure was monitored by photographing of the injury immediately or 24 hours after wounding. Activation of EGFR in THCE cells and in primary HCECs was analyzed by immunoprecipitation of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylation of extracellular signal–regulated kinase (ERK), AKT (a major substrate of phosphatidylinositol 3′-kinase [PI3K]), Src at tyrosine Y416, and EGFR at Y845 was analyzed by Western blotting with antibodies specific to phosphorylated proteins. Effects of PP2 on THCE cell migration were determined by Boyden chamber migration assay.
Among several inhibitors tested, PP2 blocked wound-induced EGFR phosphorylation in THCE cells. PP2 at 12.5 μM effectively inhibited EGFR transactivation in response to wounding and to the phosphorylation of ERK and AKT in THCE cells and primary HCECs. Consistent with the inhibition of EGFR transactivation, PP2 also attenuated epithelial migration and wound closure with or without exogenously added HB-EGF. PP2 at a concentration as high as 50 μM exhibited no effects on HB-EGF induced ERK phosphorylation. On the other hand, AKT phosphorylation was much more sensitive to PP2 than ERK or EGFR phosphorylation because 3.13 μM PP2 effectively inhibited wound- or HB-EGF-induced AKT phosphorylation.
These results suggest that Src kinase mediates wound-induced EGFR transactivation and participates in a pathway to activate the PI3K-AKT pathway downstream of EGFR in HCECs.
To compare the characteristics of HER receptors and their ligands deregulation between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).
Fifty five NSCLC primary tumors (PT) and corresponding brain metastases (BM) specimens were examined for the immunohistochemical expression of EGFR, phosphorylated (p)–EGFR, Her2, Her3, and p-Her3, and their ligands EGF, TGF-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, and neuregulins-1 and -2. Analysis of EGFR copy number using fluorescent in situ hybridization and mutation by PCR-based sequencing was also performed.
Metastases showed significantly higher immunohistochemical expression of EGF (membrane, BM 66.0 vs. PT 48.5; P=0.027; and nucleus, BM 92.2 vs. 67.4; P=0.008), amphiregulin (nucleus, BM 53.7 vs. PT 33.7; P=0.019), p-EGFR (membrane, BM 161.5 vs. PT 76.0; P<0.0001; and cytoplasm, BM 101.5 vs. PT 55.9; P=0.014), and p-Her3 (membrane, BM 25.0 vs. PT 3.7; P=0.001) than primary tumors (PT) did. Primary tumors showed significantly higher expression of cytoplasmic TGF–α (PT 149.8 vs. BM 111.3; P=0.008) and neuregulin-1 (PT 158.5 vs. BM 122.8; P=0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time points.
Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.
Communication between different signaling pathways enables cells to coordinate the responses to diverse environmental signals. Activation of the transmembrane growth factor precursors plays a critical role in this communication and often involves metalloprotease-mediated proteolysis. Stimulation of G protein–coupled receptors (GPCR) transactivates the EGF receptors (EGFRs), which occurs via a metalloprotease-dependent cleavage of heparin-binding EGF (HB-EGF). However, the metalloprotease mediating the transactivation remains elusive. We show that the integral membrane metalloprotease Kuzbanian (KUZ; ADAM10), which controls Notch signaling in Drosophila, stimulates GPCR transactivation of EGFR. Upon stimulation of the bombesin receptors, KUZ increases the docking and activation of adaptors Src homology 2 domain–containing protein and Gab1 on the EGFR, and activation of Ras and Erk. In contrast, transfection of a protease domain–deleted KUZ, or blocking endogenous KUZ by morpholino antisense oligonucleotides, suppresses the transactivation. The effect of KUZ on shedding of HB-EGF and consequent transactivation of the EGFR depends on its metalloprotease activity. GPCR activation enhances the association of KUZ and its substrate HB-EGF with tetraspanin CD9. Thus, KUZ regulates the relay between the GPCR and EGFR signaling pathways.
signal crosstalk; bombesin; HB-EGF; shedding; tetraspanin
Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-α causes receptor recycling. TGF-α therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-α, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking.
We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-α and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.
amphiregulin; betacellulin; endocytic trafficking; epidermal growth factor; epidermal growth factor receptor; epiregulin; heparin-binding EGF-like growth factor; transforming growth factor-α
Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of EGFR ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase (AP) -tagged EGF receptor (EGFR) ligand plasmids transfected into CCL-151 lung fibroblasts, and ELISAs to detect shedding of native ligands. LPA induced shedding of transfected AP-tagged HB-EGF, amphiregulin and TGF-alpha;non-transfected fibroblasts shed amphiregulin and HB-EGF under baseline conditions, and increased shedding of HB-EGF in response to LPA.. Treatment of fibroblasts with LPA (10 μM) resulted in elevated phosphorylation of ERK1/2 (3.3 ± 0.04 fold induction at 5 minutes), enhanced expression of mRNA for c-fos (59 ± 7.9-fold at 30 minutes), HB-EGF (28 ± 4.7-fold at 4 hours) and amphiregulin (5.7 ± 1.8-fold at 4 hours), and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA stimulated- CCL-151 cells, and found enhanced EGFR and ERK1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. About one-third of th response (37%, P < 0.05) was attributable to EGFR activation. These data demonstrate that LPA mediates EGF-family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells.
epidermal growth factor receptor; LPA; A549 cells
Proliferation and differentiation of the pulmonary epithelium after injury is a critical process in the defense against the external environment. Defects in this response can result in airway remodeling, such as mucus cell metaplasia (MCM), commonly seen in patients with chronic lung disease. We have previously shown that amphiregulin (AREG), a ligand to the epidermal growth factor receptor (EGFR), is induced during the repair/differentiation process elicited by naphthalene-induced lung injury. Thus, we hypothesized that AREG signaling plays an important role in epithelial proliferation and differentiation of the repairing airway. Mice deficient in AREG and lung epithelial EGFR were used to define roles for AREG-dependent EGFR signaling in airway repair and remodeling. We show that AREG and epithelial EGFR expression is dispensable to pulmonary epithelial repair after naphthalene-induced lung injury, but regulates secretory cell differentiation to a mucus-producing phenotype. We show that the pulmonary epithelium is the source of AREG, suggesting that naphthalene-induced MCM is mediated through an autocrine signaling mechanism. However, induction of MCM resulting from allergen exposure was independent of AREG. Our data demonstrate that AREG-dependent EGFR signaling in airway epithelial cells contributes to MCM in naphthalene-induced lung injury. We conclude that AREG may represent a determinant of nonallergic chronic lung diseases complicated by MCM.
Clara cells; epidermal growth factor receptor; amphiregulin; mucus cell metaplasia
Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)–ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells.
Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP.
In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor–sensitive manner.
HB-EGF released on wounding acts as an autocrine–paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.
Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR blocking-antibody that has been approved for the treatment of patients with cancers of the head and neck (HNSCC) and metastatic colorectal cancer (mCRC). Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin (AR), heparin-binding EGF (HB-EGF) and β-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and re-sensitization to cetuximab. In addition, expression of a nuclear localization sequence tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab.
EGFR; nuclear; cetuximab; resistance; Src-family kinases; dasatinib
The EGF receptor ligand amphiregulin (AREG) has been implicated as an important autocrine growth factor in several epithelial malignancies and in psoriasis, a hyperproliferative skin disorder. To characterize the mechanisms by which AREG regulates autocrine epithelial cell growth, we transduced human keratinocytes (KCs) with lentiviral constructs expressing tetracycline (TET)-inducible small hairpin RNA (shRNA). TET-induced expression of AREG shRNA markedly reduced autocrine ERK phosphorylation, strongly inhibited autocrine KC growth with an efficiency similar to metalloproteinase and EGFR inhibitors and induced several markers of KC differentiation including keratins 1 and 10. Addition of various concentrations of exogenous EGFR ligands to KC cultures reversed the growth inhibition in response to AREG blocking antibodies but not to shRNA-mediated AREG knockdown. Lentivirus-mediated expression of the full-length AREG transmembrane precursor, but not of the AREG extracellular domain, markedly reversed the shRNA-mediated growth inhibition and morphological changes, and strongly reduced the induction of multiple markers of KC differentiation. Taken together, our data demonstrate that autocrine human KC growth is highly dependent on the AREG transmembrane precursor protein and strongly suggest a previously unreported function of the metalloproteinase-processed carboxy-terminal domain of AREG.
epidermal growth factor receptor; amphiregulin; keratinocyte; proliferation; differentiation
Resistance to drug treatments underlies the high lethality of pancreatic ductal adenocarcinoma (PDA). We and others have recently identified that proteasome inhibition is a promising therapeutic option in this highly refractory disease. The pleiotropic effects of proteasome inhibition include the activation of apoptotic signaling pathways and also anti-apoptotic signaling pathways such as EGFR, AKT and the MAP kinases that reduce the apoptotic potential of this class of drug. In the current study, we sought to determine the mechanism behind the activation of EGFR in response to proteasome inhibition in pancreatic cancer cells. We found that the second generation proteasome inhibitor NPI-0052 induced the mRNA transcription of several EGFR family ligands (EGF, HB-EGF and epiregulin), however only increases in HB-EGF were detected at the protein level. Using both pharmacological inhibitors and lentiviral mediated shRNA knock down of EGFR ligand expression, we discovered that ligand cleavage by MMP/ADAM’s and HB-EGF expression is required for activation of EGFR in response to proteasome inhibition. Furthermore we discover that induction of HB-EGF is dependent on reactive oxygen species and p38-MAPK signaling but not ERK and that the transcription factor SP-1 is involved in NPI-0052 induced HB-EGF transcription. Together these results indicate that stress signaling leading to induction of HB-EGF expression and increases in MMP/ADAM dependant HB-EGF cleavage are responsible for proteasome inhibitor induced activation of EGFR in pancreatic cancer cells.
Proteasome inhibition; NPI-0052; EGFR; HB-EGF; Pancreatic cancer
Background & Aims
Helicobacter pylori infection disrupts the balance between gastric epithelial cell proliferation and apoptosis, which is likely to lower the threshold for the development of gastric adenocarcinoma. H. pylori infection is associated with EGF receptor (EGFR) activation through metalloproteinase-dependent release of EGFR ligands in gastric epithelial cells. Since EGFR signaling regulates cell survival, we investigated whether activation of EGFR following H. pylori infection promotes gastric epithelial survival.
Mouse conditionally immortalized stomach epithelial cells (ImSt) and AGS cells, as well as wild-type and kinase-defective EGFR (EGFRwa2) mice, were infected with the H. pylori cag+ strain 7.13. Apoptosis, caspase activity, EGFR activation (phosphorylation) and EGFR downstream targets were analyzed.
Inhibiting EGFR kinase activity or decreasing EGFR expression significantly increased H. pylori-induced apoptosis in ImSt. Blocking H. pylori-induced EGFR activation with a heparin-binding (HB)-EGF neutralizing antibody or abrogating a disintegrin and matrix metalloproteinase-17 (ADAM-17) expression increased apoptosis of H. pylori-infected AGS and ImSt, respectively. Conversely, pretreatment of ImSt with HB-EGF completely blocked H. pylori-induced apoptosis. H. pylori infection stimulated gastric epithelial cell apoptosis in EGFRwa2, but not in wild-type mice. Furthermore, H. pylori-induced EGFR phosphorylation stimulated PI3K-depnedent activation of the anti-apoptotic factor Akt, increased expression of the anti-apoptotic factor Bcl-2, and decreased expression of the pro-apoptotic factor Bax.
EGFR activation by H. pylori infection has an anti-apoptotic effect in gastric epithelial cells that appears to involve Akt signaling and Bcl family members. These findings provide important insights into the mechanisms of H. pylori-associated tumorigenesis.
Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Rα2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13–induced EGFR phosphorylation and the IL-13–reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.
asthma; bronchial epithelium; epithelial repair; epidermal growth factor; interleukin-13
Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, lead to significant tumor regressions in 10% to 15% of non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, 30% to 40% of NSCLC patients, majority of whom are EGFR wild-type, develop stable disease following EGFR tyrosine kinase inhibitor therapy. EGFR-directed antibodies (cetuximab) are effective treatments for head and neck squamous cell carcinomas, which seldom contain EGFR mutations. The determinant(s) of efficacy of EGFR-targeted therapies in EGFR wild-type cancers is not well defined.
We examined the relationship of EGFR ligands, EGF, transforming growth factor-α, and amphiregulin and the efficacy of gefitinib and cetuximab in EGFR wild-type NSCLC (n = 10) and head and neck squamous cell carcinoma (n = 4) cell lines. We compared amphiregulin expression using immunohistochemistry in EGFR wild-type NSCLC patients (n = 24) that developed either stable or progressive disease following erlotinib or gefitinib treatment.
Cell lines which produced ≥20 pmol/L amphiregulin, as detected by an ELISA, were significantly more likely to be growth inhibited by both gefitinib and cetuximab than those that produced minimal or no amphiregulin. In these cell lines, both cetuximab and gefitinib led to cell cycle arrest at the G1-S boundary and was associated with preferential inhibition of extracellular signal-regulated kinase 1/2 but not Akt signaling. Amphiregulin expression was significantly higher in NSCLC patients that developed stable disease compared with those that developed disease progression following gefitinib or erlotinib treatment.
Amphiregulin expression may help select EGFR wild-type patients who are likely to develop stable disease from EGFR-targeted therapies.
In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.
The metalloprotease ADAM17 (a.k.a. TACE) plays a pivotal role in the cleavage and activation of membrane-anchored receptor ligands. More recently, it has been revealed that ADAM17 is a potent sheddase of the epidermal growth factor (EGF) family of ligands and regulates epidermal growth factor receptor (EGFR) activity in a variety of tumors. EGFR is a key component of autonomous growth signaling in several tumors, and correlates with the malignancy grade of astrocytoma. In this study, we tested the hypothesis that over-expression of ADAM17 in cortical astrocytes derived from normal brain would induce a progression towards a malignant phenotype. Over-expression of human ADAM17 (hADAM17) in the CTX-TNA2 cortical astrocyte cell line resulted in non-adherent growth, increased proliferation, invasiveness, production of angiogenic factors, and expression of genes associated with immature and/or neoplastic cells. hADAM17 up-regulated EGFR and AKT phosphorylation, and increased proliferation and cell invasion were significantly dependent upon EGFR activity. When implanted in the nude mouse brain, CTX-TNA2 cells induced low histological grade, benign intraventricular gliomas. In contrast, the same astrocytes with hADAM17 formed large malignant gliomas. Taken together, these findings suggest that unregulated ADAM17 activity induces functional changes in astrocytes that significantly advance the malignant phenotype.
Previously, it was shown that a novel 4-(N)-stearoyl gemcitabine nanoparticle formulation was more effective than gemcitabine hydrochloride in controlling the growth of model mouse or human tumors pre-established in mice. In the present study, the feasibility of targeting the stearoyl gemcitabine nanoparticles (GemC18-NPs) into tumor cells that over-express epidermal growth factor receptor (EGFR) to more effectively control tumor growth was evaluated. EGFR is over-expressed in a variety of tumor cells, and EGF is a known natural ligand of EGFR. Recombinant murine EGF was conjugated onto the GemC18-NPs. The ability of the EGF to target the GemC18-NPs to human breast adenocarcinoma cells that expressed different levels of EGFR was evaluated in vitro and in vivo. In culture, the extent to which the EGF-conjugated GemC18-NPs were taken up by tumor cells was correlated to the EGFR density on the tumor cells, whereas the uptake of untargeted GemC18-NPs exhibited no difference among those same cell lines. The relative cytotoxicity of the EGF-conjugated GemC18-NPs to tumor cells in culture was correlated to EGFR expression as well. In vivo, EGFR-over-expressing MDA-MB-468 tumors in mice treated with the EGF-conjugated GemC18-NPs grew significantly slower than in mice treated with untargeted GemC18-NPs, likely due to that the EGF-GemC18-NPs were more anti-proliferative, anti-angiogenic, and pro-apoptotic. Fluorescence intensity data from ex vivo imaging showed that the EGF on the nanoparticles helped increase the accumulation of the GemC18-NPs into MDA-MB-468 tumors pre-established in mice by more than 2-fold as compared to the un-targeted GemC18-NPs. In conclusion, active targeting of the GemC18-NPs into EGFR-over-expressed tumors can further enhance their anti-tumor activity.
particle uptake; cytotoxicity; biodistribution; ex vivo imaging
The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. We previously showed that the pivotal effector of this pathway, YAP, is amplified in tumors and promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. Here, we report that overexpression of TAZ, a paralog of YAP, in human mammary epithelial cells promotes EMT and, in particular, some invasive structures in 3D cultures. TAZ also leads to cell migration and anchorage-independent growth in soft agar. Furthermore, we identified amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, as a target of TAZ. We show that AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. In addition, ablation of TEAD binding completely abolishes the TAZ-induced phenotype. Last, analysis of breast cancer patient samples reveals a positive correlation between TAZ and AREG in vivo. In summary, TAZ-dependent secretion of AREG indicates that activation of the EGFR signaling is an important non-cell-autonomous effector of the Hippo pathway, and TAZ as well as its targets may play significant roles in breast tumorigenesis and metastasis.
amphiregulin (AREG); epithelial-to-mesenchymal transition (EMT); Hippo pathway; TAZ; tumor metastasis
The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.
Amphiregulin; EGFR; degradation; localization; breast cancer