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.     

A differential requirement for the COOH-terminal region of the epidermal growth factor (EGF) receptor in amphiregulin and EGF mitogenic signaling

The epidermal growth factor receptor (EGFR) mediates the actions of a family of bioactive peptides that include epidermal growth factor (EGF) and amphiregulin (AR). Here we have studied AR and EGF mitogenic signaling in EGFR-devoid NR6 fibroblasts that ectopically express either wild type EGFR (WT) or a truncated EGFR that lacks the three major sites of autophosphorylation (c'1000). COOH-terminal truncation of the EGFR significantly impairs the ability of AR to (i) stimulate DNA synthesis, (ii) elicit Elk-1 transactivation, and (iii) generate sustained enzymatic activation of mitogen-activated protein kinase. EGFR truncation had no significant effect on AR binding to receptor but did result in defective GRB2 adaptor function. In contrast, EGFR truncation did not impair EGF mitogenic signaling, and in c'1000 cells EGF was able to stimulate the association of ErbB2 with GRB2 and SHC. Elk-1 transactivation was monitored when either ErbB2 or a truncated dominant-negative ErbB2 mutant (ErbB2-(1-813)) was overexpressed in cells. Overexpression of full-length ErbB2 resulted in a strong constitutive transactivation of Elk-1 in c'1000 but only slightly stimulated Elk-1 in WT or parental NR6 cells. Conversely, overexpression of ErbB2-(1-813) inhibited EGF-stimulated Elk-1 transactivation in c'1000 but not in WT cells. Thus, the cytoplasmic tail of the EGFR plays a critical role in AR mitogenic signaling but is dispensable for EGF, since EGF-activated truncated EGFRs can signal through ErbB2.     

Effect of epidermal growth factor receptor internalization on regulation of the phospholipase C-gamma1 signaling pathway

The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGFalpha-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-gamma1 (PLC-gamma1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGFalpha stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP2) hydrolysis over those stimulated by TGFalpha. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP2 hydrolysis, PLC-gamma1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-gamma1 phosphorylation and PIP2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-gamma1 to its substrate in endocytic trafficking organelles.     

Stimulation of cell proliferation by endosomal epidermal growth factor receptor as revealed through two distinct phases of signaling

Strong evidence indicates that endosome-localized epidermal growth factor receptor (EGFR) plays an important role in cell signaling. However, elimination of endosomal signaling does not attenuate EGF-induced physiological outcomes, arguing against physiological relevance. Recently we established a system to specifically activate endosome-associated EGFR in the absence of any plasma membrane activation of EGFR and showed that endosomal EGFR signaling is sufficient to support cell survival. However, this pure endosomal signaling of EGFR does not stimulate cell proliferation, because EGFR only remained activated for less than 2 h following its stimulation at endosomes, while DNA synthesis generally requires growth factor exposure for 8 h or more. Here we report that the prolonged requirement for EGF to stimulate epithelial cell proliferation can be substituted for with two short pulses of EGF. By combining the two short pulses of EGF stimulation with our previously established method to generate endosomal EGFR signaling, we are able to generate two pulses of endosomal EGFR signaling. In this way, we demonstrated that two pulses of endosomal EGFR signaling are sufficient to stimulate cell proliferation. The first pulse of EGFR signaling induces exit from quiescence into G(1) phase and appears to render cells responsive to subsequent mitogenic stimulus. This second pulse, required several hours later, drives cells through the restriction point of late G(1) and into S phase. We further showed that the two pulses of endosomal EGFR signaling engaged cell cycle machinery the same way as the two pulses of standard EGFR signaling. Moreover, two pulses of endosomal EGFR signaling stimulated downstream signaling cascades in a similar way to the two pulses of standard EGFR activation. The data therefore demonstrate that signals transduced from internalized EGFR, with or without a contribution from the plasma membrane, fully satisfy the physiological requirements for S-phase entry.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin-1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand-stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of 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.     

Specific epidermal growth factor receptor autophosphorylation sites promote mouse colon epithelial cell chemotaxis and restitution

Upon ligand binding, epidermal growth factor (EGF) receptor (R) autophosphorylates on COOH-terminal tyrosines, generating docking sites for signaling partners that stimulate proliferation, restitution, and chemotaxis. Specificity for individual EGFR tyrosines in cellular responses has been hypothesized but not well documented. Here we tested the requirement for particular tyrosines, and associated downstream pathways, in mouse colon epithelial cell chemotactic migration. We compared these requirements to those for the phenotypically distinct restitution (wound healing) migration. Wild-type, Y992/1173F, Y1045F, Y1068F, and Y1086F EGFR constructs were expressed in EGFR(-/-) cells; EGF-induced chemotaxis or restitution were determined by Boyden chamber or modified scratch wound assay, respectively. Pharmacological inhibitors of p38, phospholipase C (PLC), Src, MEK, JNK/SAPK, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC) were used to block EGF-stimulated signaling. Pathway activation was determined by immunoblot analysis. Unlike wild-type EGFR, Y992/1173F and Y1086F EGFR did not stimulate colon epithelial cell chemotaxis toward EGF; Y1045F and Y1068F EGFR partially stimulated chemotaxis. Only wild-type EGFR promoted colonocyte restitution. Inhibition of p38, PLC, and Src, or Grb2 knockdown, blocked chemotaxis; JNK, PI 3-kinase, and PKC inhibitors or c-Cbl knockdown blocked restitution but not chemotaxis. All four EGFR mutants stimulated downstream signaling in response to EGF, but Y992/1173F EGFR was partially defective in PLCγ activation whereas both Y1068F and Y1086F EGFR failed to activate Src. We conclude that specific EGFR tyrosines play key roles in determining cellular responses to ligand. Chemotaxis and restitution, which have different migration phenotypes and physiological consequences, have overlapping but not identical EGFR signaling requirements.     

Epidermal Growth Factor (EGF)-enhanced Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression Promotes Macrophage and Glioblastoma Cell Interaction and Tumor Cell Invasion

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCϵ- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.     

Co-inhibition of epidermal growth factor receptor and type 1 insulin-like growth factor receptor synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis

Inhibition of epidermal growth factor receptor (EGFR) signaling sensitizes human malignant glioma cells to death ligand-induced apoptosis. However, tumor cells may compensate the loss of EGFR signaling by activation of the type 1 insulin-like growth factor receptor (IGF-1R). We here report that antagonism of the IGF-1R with the small-molecule inhibitor AG1024 in combination with inhibitors of the EGFR synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis. This cell death is p53-independent, but requires caspase 8 activity. The levels of the receptor, CD95, are not altered by the inhibitors alone or in combination. Analysis of the downstream signaling pathways reveals synergistic inhibition of ribosomal protein S6 phosphorylation by inhibitor co-treatment, suggesting an involvement of the mammalian target of rapamycin pathway. These findings suggest that adding inhibitors of IGF-1R may be a strategy to overcome escape from the anti-apoptotic effects of EGFR inhibition in malignant gliomas.     

Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.     

Gene 33 is an endogenous inhibitor of epidermal growth factor (EGF) receptor signaling and mediates dexamethasone-induced suppression of EGF function

We report a mechanism by which the adapter protein Gene 33 (also called RALT and MIG6) regulates epidermal growth factor receptor (EGFR) signaling. We find that Gene 33 inhibits EGFR autophosphorylation and specifically blunts epidermal growth factor (EGF)-induced activation and/or phosphorylation of Ras, ERK, JNK, Akt/PKB, and retinoblastoma protein. The Ack homology domain of Gene 33, which contains the previously identified EGFR binding domain, is both necessary and sufficient for this inhibition of EGFR autophosphorylation. The endogenous Gene 33 polypeptide is induced by EGF, platelet-derived growth factor, serum, and dexamethasone (Dex) in Rat 2 rat fibroblasts. Dex induces Gene 33 expression and inhibits EGFR phosphorylation and EGF signaling. RNA interference-mediated silencing of Gene 33 significantly reverses this effect. Overexpression of Gene 33 completely blocks EGF-induced protein and DNA synthesis in Rat 2 cells, whereas gene 33 RNA interference substantially enhances EGF-induced protein and DNA synthesis in Rat 2 cells. Our results indicate that Gene 33 is a physiological feedback inhibitor of the EGFR, functioning to inhibit EGFR phosphorylation and all events induced by EGFR activation. Our results also indicate a role for Gene 33 in the suppression, by Dex, of EGF signaling pathways. We propose that Gene 33 may function in the cross-talk between EGF signaling and other mitogenic and/or stress signaling pathways.     

Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.     

Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm²), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By ¹²⁵I-EGF binding analysis and [³⁵S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8–10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2–3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley-Liss, Inc.     

Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.     

Semapimod Sensitizes Glioblastoma Tumors to Ionizing Radiation by Targeting Microglia

Glioblastoma is the most malignant and lethal form of astrocytoma, with patients having a median survival time of approximately 15 months with current therapeutic modalities. It is therefore important to identify novel therapeutics. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of glioblastoma tumors. In this paper we show that microglia, in addition to stimulating glioblastoma cell invasion, also promote glioblastoma cell proliferation and resistance to ionizing radiation in vitro. We found that semapimod, a drug that selectively interferes with the function of macrophages and microglia, potently inhibits microglia-stimulated GL261 invasion, without affecting serum-stimulated glioblastoma cell invasion. Semapimod also inhibits microglia-stimulated resistance of glioblastoma cells to radiation, but has no significant effect on microglia-stimulated glioblastoma cell proliferation. We also found that intracranially administered semapimod strongly increases the survival of GL261 tumor-bearing animals in combination with radiation, but has no significant benefit in the absence of radiation. In conclusion, our observations indicate that semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia and/or infiltrating macrophages.