EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.     

Rab21 attenuates EGF-mediated MAPK signaling through enhancing EGFR internalization and degradation

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is regulated by endocytosis where many Rab proteins play an important role in the determination of the receptor recycle or degradation. In an effort to better understand how EGF signaling is regulated, we examined the role of Rab21 in regulation of the degradation and signal transduction of the EGFR. Using a transient expression protocol in HEK293T and HeLa cells, we found that Rab21 enhanced the degradation of EGFR through accelerating its internalization in both EGF-independent and EGF-dependent manners. We further demonstrated that Rab21 interacted with EGFR by immunoprecipitation experiments. Interestingly, we observed that overexpression of Rab21 attenuated EGF-mediated mitogen-activated protein kinase (MAPK) signaling by inducing EGFR degradation. Taken together, these data suggest that Rab21 plays a negative role in the EGF-mediated MAPK signaling pathway.     

Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations.     

Epidermal Growth Factor Receptor Translocation to the Mitochondria: REGULATION AND EFFECT*

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.     

Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21(WAF-1), and p27(KIP-1) levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G(1) arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells

Amphiregulin (AR) autocrine loops have been associated with several types of cancer. We demonstrate that SUM149 breast cancer cells have a self-sustaining AR autocrine loop. SUM149 cells are epidermal growth factor (EGF)-independent for growth, and they overexpress AR mRNA, AR membrane precursor protein, and secreted AR relative to the EGF-dependent human mammary epithelial cell line MCF10A. MCF10A cells made to overexpress AR (MCF10A AR) are also EGF-independent for growth. Treatment with the pan-ErbB inhibitor CI1033 and the anti-EGF receptor (EGFR) antibody C225 demonstrated that ligand-mediated activation of EGFR is required for SUM149 cell proliferation. AR-neutralizing antibody significantly reduced both SUM149 EGFR activity and cell proliferation, confirming that an AR autocrine loop is required for mitogenesis in SUM149 cells. EGFR tyrosine phosphorylation was dramatically decreased in both SUM149 and MCF10A AR cells after inhibition of AR cleavage with the broad spectrum metalloprotease inhibitor GM6001, indicating that an AR autocrine loop is strictly dependent on AR cleavage in culture. However, a juxtacrine assay where fixed SUM149 cells and MCF10A AR cells were overlaid on top of EGF-deprived MCF10A cells showed that the AR membrane precursor can activate EGFR. SUM149 cells, MCF10A AR cells, and MCF10A cells growing in exogenous AR were all considerably more invasive and motile than MCF10A cells grown in EGF. Moreover, AR up-regulates a number of genes involved in cell motility and invasion in MCF10A cells, suggesting that an AR autocrine loop contributes to the aggressive breast cancer phenotype.     

Autophosphorylation of EGFR at Y954 Facilitated Homodimerization and Enhanced Downstream Signals

Asymmetric dimer formation of epidermal growth factor receptor (EGFR) is crucial for EGF-induced receptor activation. Even though autophosphorylation is important for activation, its role remains elusive in the context of regulating dimers. In this study, employing overlapping time series analysis to raster image correlation spectroscopy (RICS), we observed time-dependent transient dynamics of EGFR dimerization and found EGFR kinase activity to be essential for dimerization. As a result of which, we hypothesized that phosphorylation could influence dimerization. Evaluating this point, we observed that one of the tyrosine residues (Y954) located in the C-terminal lobe of the activator kinase domain was important to potentiate dimerization. Functional imaging to monitor Ca²⁺ and ERK signals revealed a significant role of Y954 in influencing downstream signaling cascade. Crucial for stabilization of EGFR asymmetric dimer is a “latch” formed between kinase domains of the binding partners. Because Y954 is positioned adjacent to the latch binding region on the kinase domain, we propose that phosphorylation strengthened the latch interaction. On the contrary, we identified that threonine phosphorylation (T669) in the latch domain negatively regulated EGFR dimerization and the downstream signals. Overall, we have delineated the previously anonymous role of phosphorylation at the latch interface of kinase domains in regulating EGFR dimerization.     

Oncogenic mutations counteract intrinsic disorder in the EGFR kinase and promote receptor dimerization

The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.     

Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization

Gangliosides are shed by tumor cells and can bind to normal cells in the tumor microenvironment and affect their function. Exposure of fibroblasts to exogenous gangliosides increases epidermal growth factor (EGF)-induced fibroblast proliferation and enhances EGF receptor (EGFR)-mediated activation of the mitogen-activated protein kinase signaling pathway (Li, R., Liu, Y., and Ladisch, S. (2001) J. Biol. Chem. 276, 42782-42792). Here we report that the EGFR itself is the target of this ganglioside effect: Preincubation of normal human dermal fibroblasts with G(D1a) ganglioside enhanced both EGF-induced EGFR autophosphorylation and receptor-tyrosine kinase activity. The enhancement was rapid (within 30 min), not due to alteration of time kinetics of the EGFR response to EGF, and reproduced in purified G(D1a)-enriched cell membranes isolated from ganglioside-preincubated fibroblasts. Evaluating the initial steps underlying activation, EGF binding, and EGFR dimerization, we found that G(D1a) enrichment of the cell membrane increased EGFR dimerization and the effective number of high affinity EGFR without increasing total receptor protein. Unexpectedly, G(D1a) enrichment also triggered increased EGFR dimerization in the absence of growth factor. This resulted in enhanced activation of the EGFR signal transduction cascade when EGF was added. We conclude that membrane ganglioside enrichment of normal fibroblasts (such as by tumor cell ganglioside shedding) facilitates receptor-receptor interactions (possibly by altering membrane topology), causing ligand-independent EGFR dimerization and, in turn, enhanced EGF signaling.     

Cyclooxygenase-2-derived prostaglandin E2 promotes human cholangiocarcinoma cell growth and invasion through EP1 receptor-mediated activation of the epidermal growth factor receptor and Akt

Cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis has recently been implicated in human cholangiocarcinogenesis. This study was designed to examine the mechanisms by which COX-2-derived prostaglandin E2 (PGE2) regulates cholangiocarcinoma cell growth and invasion. Immunohistochemical analysis revealed elevated expression of COX-2 and the epidermal growth factor (EGF) receptor (EGFR) in human cholangiocarcinoma tissues. Overexpression of COX-2 in a human cholangiocarcinoma cell line (CCLP1) increased tumor cell growth and invasion in vitro and in severe combined immunodeficient mice. Overexpression of COX-2 or treatment with PGE2 or the EP1 receptor agonist ONO-DI-004 induced phosphorylation of EGFR and enhanced tumor cell proliferation and invasion, which were inhibited by the EP1 receptor small interfering RNA or antagonist ONO-8711. Treatment of CCLP1 cells with PGE2 or ONO-DI-004 enhanced binding of EGFR to the EP1 receptor and c-Src. Furthermore, PGE2 or ONO-DI-004 treatment also increased Akt phosphorylation, which was blocked by the EGFR tyrosine kinase inhibitors AG 1478 and PD 153035. These findings reveal that the EP1 receptor transactivated EGFR, thus activating Akt. On the other hand, activation of EGFR by its cognate ligand (EGF) increased COX-2 expression and PGE2 production, whereas blocking PGE2 synthesis or the EP1 receptor inhibited EGF-induced EGFR phosphorylation. This study reveals a novel cross-talk between the EP1 receptor and EGFR signaling that synergistically promotes cancer cell growth and invasion.     

Plakophilin-2 Promotes Activation of Epidermal Growth Factor Receptor

While growth factor-driven dimerization of receptor tyrosine kinases (RTKs) is a simple and intuitive mechanism of activating RTKs, K.-I. Arimoto et al. (Mol. Cell. Biol. 34:3843–3854, 2014, doi:10.1128/MCB.00758-14) describe a novel means of promoting the activity of RTKs. Namely, plakophilin-2 (PKP2) associates with the epidermal growth factor receptor (EGFR) and enhances its ligand-dependent and ligand-independent activity. This discovery suggests that antagonizing PKP2 may be a new therapeutic opportunity to combat tumors in which activation of EGFR contributes to pathogenesis.     

Cool-1 functions as an essential regulatory node for EGF receptor- and Src-mediated cell growth

Cool-1 (cloned-out of library 1) has a key role in regulating epidermal growth factor receptor (EGFR) degradation. Here, we show that Cool-1 performs this function by functioning as both an upstream activator and downstream target for Cdc42. EGF-dependent phosphorylation of Cool-1 enables it to act as a nucleotide exchange factor for Cdc42 and to form a complex with the E3 ligase Cbl, thus regulating Cbl-catalysed EGFR degradation. The EGF-dependent phosphorylation is normally transient; however, Cool-1 phosphorylation is sustained in cells expressing v-Src and is essential for cellular transformation, as well as for v-Src-induced tumour formation in mice. These findings demonstrate that the regulated phosphorylation of Cool-1 is necessary to maintain the balance between normal signalling by EGFR and Src versus aberrant growth and transformation.     

SOCS2 induces neurite outgrowth by regulation of epidermal growth factor receptor activation

Suppressor of cytokine signaling (SOCS) 2 is a negative regulator of growth hormone (GH) signaling that regulates body growth postnatally and neuronal differentiation during development. SOCS2 binds to the GH receptor and inhibits GH signaling, including attenuation of STAT5 activation. Here we describe a new function and mechanism of action for SOCS2. Overexpression of SOCS2 in central nervous system neurons promoted neurite outgrowth, and in PC12 cells, neurite outgrowth was induced under nondifferentiating conditions, leading to inhibition of the neurite-inhibitory GTPase Rho and activation of the neurite-promoting GTPase Rac1. Addition of the epidermal growth factor receptor (EGFR) inhibitors PP3 or AG490 or the Src kinase inhibitor PP2 blocked the SOCS2-induced neurite outgrowth. The overexpressed SOCS2 bound to the EGFR, which was constitutively phosphorylated at Tyr845, the Src binding site. Overexpression of the phosphatase SHP-2 reduced the constitutive EGFR phosphorylation and subsequent neurite outgrowth. SOCS2 expression also resulted in a modest 30% decrease in phosphorylation of STAT5b at Tyr699, which is the primary site on STAT5 phosphorylated by GH; however, total tyrosine phosphorylation of STAT5 was decreased by 75-80% under basal and epidermal growth factor-stimulated conditions. Our findings suggest that SOCS2 regulates EGFR phosphorylation, leading to regulation of neurite outgrowth through a novel pathway that is distinct from GH.     

Activation of EGFR/ERBB2 via pathways involving ERK1/2, P38 MAPK, AKT and FOXO enhances recovery of diabetic hearts from ischemia-reperfusion injury

This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.     

The roles of phospholipase D in EGFR signaling

Epidermal growth factor receptor (EGFR) is a representative model of receptor tyrosine kinases (RTKs), and offers a means of understanding their common principles and fundamental mechanisms. Furthermore, EGFR plays an essential role in cell proliferation and migration, and the disruption of EGFR signaling has been implicated in the development and growth of cancer. Phospholipase D (PLD) is a key mediator of EGFR function, and can be directly regulated by upstream binding partners in an EGF-dependent manner. PLD regulates downstream molecules by generating phosphatidic acid (PA), but it also dynamically interacts with a variety of intracellular molecules and these interactions spatiotemporally regulate EGFR function and serve as a hub that orchestrates signaling flow. This review summarizes the interrelationship between PLD and its binding molecules in the context of EGFR signaling, and addresses the roles of PLD in the mediation and coordination of this signaling.