Estrogen receptor-alpha 36 mediates mitogenic antiestrogen signaling in ER-negative breast cancer cells

It is prevailingly thought that the antiestrogens tamoxifen and ICI 182, 780 are competitive antagonists of the estrogen-binding site of the estrogen receptor-alpha (ER-α). However, a plethora of evidence demonstrated both antiestrogens exhibit agonist activities in different systems such as activation of the membrane-initiated signaling pathways. The mechanisms by which antiestrogens mediate estrogen-like activities have not been fully established. Previously, a variant of ER-α, EP-α36, has been cloned and showed to mediate membrane-initiated estrogen and antiestrogen signaling in cells only expressing ER-α36. Here, we investigated the molecular mechanisms underlying the antiestrogen signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of endogenous ER-α36. We found that the effects of both 4-hydoxytamoxifen (4-OHT) and ICI 182, 780 (ICI) exhibited a non-monotonic, or biphasic dose response curve; antiestrogens at low concentrations, elicited a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, antiestrogens inhibited cell growth. Antiestrogens at l nM induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at 5 μM induced Src-Y527 phosphorylation that inactivates Src. Antiestrogens at 1 nM also induced phosphorylation of the MAPK/ERK and activated the Cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at 5 μM. Knock-down of ER-α36 abrogated the biphasic antiestrogen signaling in these cells. Our results thus indicated that ER-α36 mediates biphasic antiestrogen signaling in the ER-negative breast cancer cells and Src functions as a switch of antiestrogen signaling dependent on concentrations of antiestrogens through the EGFR/STAT5 pathway.     

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.     

Hepassocin activates the EGFR/ERK cascade and induces proliferation of L02 cells through the Src-dependent pathway

Hepassocin (HPS) is a secreted protein with mitogenic activity on primary hepatocytes and protects hepatocytes from chemically-induced injury. Our previous studies showed that HPS stimulates proliferation of hepatocytes in an ERK pathway-dependent manner. However, the molecular mechanism of HPS-induced activation of the ERK pathway remains unclear. In this study, we found that HPS induced the phosphorylation of the epidermal growth factor receptor (EGFR) in the human L02 hepatocyte cell line, and this event was concomitant with the activation of the non-receptor tyrosine kinase Src. Specific inhibition of EGFR kinase activity by gefitinib or down-regulation of EGFR by specific EGFR siRNAs prevented HPS-induced activation of the ERK pathway and proliferation of L02 cells. Furthermore, inhibition of Src activity significantly blocked HPS-induced activation of the EGFR, which was suggestive of a ligand-independent transactivation mechanism of EGFR itself as well as ERK phosphorylation and proliferation of L02 cells. These results indicate that EGFR plays an important role in the mitogenic signaling induced by HPS in L02 cell lines and may further stimulate research on the role of HPS in hepatocytes within biological processes in human health and disease.     

Insulin potentiates EGFR activation and signaling in fibroblasts

Insulin is an essential hormone for cell growth and potentiates the mitogenic actions of multiple growth factors, including EGF. While potentiation has been shown to be mediated by the upregulation of the cyclin/CDK system, the upstream mechanisms of such synergy have not been elucidated. Our study has examined whether insulin could mediate synergy by enhancing early signaling events of the EGF receptor (EGFR). Tyrosine phosphorylation at the cell periphery of confluent Swiss 3T3 fibroblasts induced by EGF was potentiated by insulin within 2 min of stimulation. Insulin potentiation of EGF-mediated phosphorylation of the EGFR occurred 2 min after stimulation. EGFR transactivation by insulin was not observed. In addition, downstream mitogenic signaling events including ERK1/2 activation and Elk-1 phosphorylation were enhanced in response to insulin and EGF coadministration. This study shows mitogenic synergy between insulin and EGF can occur at the earliest signaling event, receptor phosphorylation, and independent of transactivation.     

Lysophosphatidylcholine stimulates EGF receptor activation and mesangial cell proliferation: regulatory role of Src and PKC

Lysophosphatidylcholine (LPC), a major component of oxidized-low density lipoproteins (ox-LDL), modulates various pathobiological processes involved in vascular and glomerular diseases. Although several studies have shown increased plasma concentrations of ox-LDL as well as LPC in patients with renal disease, the role of LPC in mesangial cell proliferation and associated signaling mechanisms are not clearly understood. In this study, we have shown that LPC induced the phosphorylation of epidermal growth factor receptor (EGFR), as well as the p42/44 MAP kinases. LPC activated Src-kinase and protein kinase C (PKC), and both Src kinase inhibitor PP-2 and PKC inhibitor inhibited the activation of EGFR by LPC. LPC (5-25 microM) stimulated human mesangial cell proliferation by 4-5 fold. Preincubation of mesangial cells with the Src inhibitor (PP-2), or PKC inhibitor (bisindolylmaleimide GF109203-X), or EGF receptor kinase inhibitor (AG1478), or MEK inhibitor (PD98059) significantly inhibited LPC-mediated mesangial cell proliferation. The data suggest that LPC, by activating Src and PKC signaling pathways, stimulates EGF receptor transactivation and down-stream MAP kinase signaling resulting in mesangial hypercellularity, which is a characteristic feature of diverse renal diseases.     

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.     

Acute ligand-independent Src activation mimics low EGF-induced EGFR surface signalling and redistribution into recycling endosomes

Src, a non-receptor tyrosine kinase, is a key signal transduction partner of epidermal growth factor (EGF) receptor (EGFR). In human breast cancer, EGFR and Src are frequently over-expressed and/or over-activated. Although reciprocal activation is documented, mechanisms underlying Src:EGFR interactions are incompletely understood. We here exploited ts/v-Src thermo-activation in MDCK monolayers to test whether acute Src activation impacts on signalling and trafficking of non-liganded EGFR. We found that thermo-activation caused rapid Src recruitment to the plasma membrane, concomitant association with EGFR, and its phosphorylation at Y845 and Y1173 predominantly at the cell surface. Like low EGF concentrations, activated Src (i) decreased EGF surface binding without affecting the total EGFR pool; (ii) triggered EGFR endocytosis via clathrin-coated vesicles; (iii) and led to its sequestration in perinuclear/recycling endosomes with avoidance of multivesicular bodies and lysosomal degradation. Combined Src activation and EGF were synergistic for EGFR-Y845 and -Y1173 phosphorylation at some endosomes. We conclude that acute effects of Src in MDCK cells may mimic those of low EGF on EGFR activation and redistribution. Src:EGFR interactions may be sufficient to trigger EGFR activation and might contribute to its local signalling, without requiring either soluble extracellular signal or receptor over-expression.     

Microfluidic Generated EGF-Gradients Induce Chemokinesis of Transplantable Retinal Progenitor Cells via the JAK/STAT and PI3Kinase Signaling Pathways

A growing number of studies are evaluating retinal progenitor cell (RPC) transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR) and evaluated whether exposure to epidermal growth factor (EGF) can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.     

Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Role of epidermal growth factor and ErbB2 receptors in 3T3-L1 adipogenesis

Objective: Epidermal growth factor (EGF) stimulates proliferation in 3T3‐L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance. Research Methods and Procedures: We compared the effects of low (0.1 nM) vs. high (10 nM) EGF on the activation of EGF receptors, proximal signaling molecules Src and Shc, and the downstream mitogen‐activated protein kinase (MAPK) pathways extracellular regulated kinase (ERK) and p38 in proliferating and differentiated 3T3‐L1 cells. Results: Both low and high EGF activated ERK and p38 in preadipocytes. Src inhibitors PP1 and PP2 blocked ERK and p38 activation by low but not high EGF, and only high EGF increased Shc phosphorylation. Selective inhibition of the EGF receptor (EGFR) with AG1478 blocked ERK and p38 activation at both concentrations; however, selective inhibition of the ErbB2 receptor (EB2R) with AG825 or small interfering RNA (siRNA) blocked low but not high EGF activation of ERK and p38. Coimmunoprecipitation of EGFR with EB2R and Src was observed with low EGF in preadipocytes but at both concentrations in adipocytes. EB2R inhibition during differentiation decreased p38 activity and peroxisome proliferator‐activated receptor γ (PPARγ) abundance. Discussion: Our results show that EGFR homodimers mediate action of EGF at high abundance, but at low abundance, EGF promotes differentiation through EGFR/EB2R heterodimer activation of Src and p38. These results may partially explain the observations that high EGF concentrations inhibit, whereas low concentrations support, preadipocyte differentiation.     

Low‐concentration heparin suppresses ionomycin‐activated CAMK‐II/EGF receptor‐ and ERK‐mediated signaling in mesangial cells

Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK‐II. Many studies have used heparin at concentrations of 100 µg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low‐concentration (1 µg/ml) heparin on activation of several kinases and subsequent induction of the c‐fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK‐II (by 30 s), and subsequently of the EGF receptor (EGFR), c‐Src, and Erk 1/2. Low‐dose heparin suppresses the ionomycin‐dependent phosphorylation of EGFR, c‐Src, and Erk 1/2, but not of CaMK‐II, whereas inhibition of activated CaMK‐II reduces phosphorylation of EGFR, c‐Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK‐II, including EGFR, leading in turn to a decrease in Erk‐ (but not c‐Src‐) dependent induction of c‐fos. J. Cell. Physiol. 224: 484–490, 2010. © 2010 Wiley‐Liss, Inc.     

Control of proliferation of bovine vascular endothelial cells.

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.     

Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cells

In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3-K) have been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK-induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK- and EGF-dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK- and EGF-dependent effects. Inhibition of PI3-K by LY294002 attenuated BK-induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3-K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK-induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK-induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK-stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3-K/Akt.     

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.     

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation

Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc–GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.     

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.     

Growth hormone-releasing hormone induced transactivation of epidermal growth factor receptor in human triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subset of breast cancers which is negative for expression of estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2). Chemotherapy is currently the only form of treatment for women with TNBC. Growth hormone-releasing hormone (GHRH) and epidermal growth factor (EGF) are autocrine/paracrine growth factors in breast cancer and a substantial proportion of TNBC expresses receptors for GHRH and EGF. The aim of this study was to evaluate the interrelationship between both these signaling pathways in MDA-MB-468 human TNBC cells. We evaluated by Western blot assays the effect of GHRH on transactivation of EGF receptor (EGFR) as well as the elements implicated. We assessed the effect of GHRH on migration capability of MDA-MB-468 cells as well as the involvement of EGFR in this process by means of wound-healing assays. Our findings demonstrate that in MDA-MB-468 cells the stimulatory activity of GHRH on tyrosine phosphorylation of EGFR is exerted by two different molecular mechanisms: i) through GHRH receptors, GHRH stimulates a ligand-independent activation of EGFR involving at least cAMP/PKA and Src family signaling pathways; ii) GHRH also stimulates a ligand-dependent activation of EGFR implicating an extracellular pathway with an important role for metalloproteinases. The cross-talk between EGFR and GHRHR may be impeded by combining drugs acting upon GHRH receptors and EGFR family members. This combination of GHRH receptors antagonists with inhibitors of EGFR signalling could enhance the efficacy of both types of agents as well as reduce their doses increasing therapeutic benefits in management of human breast cancer.     

Heparin suppresses lipid raft-mediated signaling and ligand-independent EGF receptor activation

Heparin is well known to suppress vascular smooth muscle cell (VSMC) proliferation, and attempts to exploit this therapeutically have led to recognition of multiple pathways for heparin's anti-mitogenic actions. At low concentrations (ca. 1 microg.ml(-1)), these suppressive effects may reflect physiological activities of endogenous heparan sulfates, and appear to be rapid responses to extracellular or cell surface-associated heparin. Because heparin has been shown to influence expression of caveolin proteins, and caveolae/lipid rafts are critical structures modulating cell signaling, we examined the effect of heparin on signaling involving cholesterol-rich membrane microdomains. The VSMC line PAC-1 activates the MAP kinase Erk in response to the cholesterol-sequestering agents methyl-beta-cyclodextrin and nystatin. This follows a temporal sequence that involves Ras-GTP activation of MEK, and is independent of PKC, Src, and PI3 kinase. However, ligand-independent phosphorylation of the EGF receptor (EGFR) by removal of cholesterol precedes Ras activation, and the EGFR kinase inhibitor AG1478 blocks Erk phosphorylation, supporting occurrence of the signaling sequence EGFR-Ras-MEK-Erk. Phosphorylation of EGFR occurs predominantly in caveolin-rich microdomains as identified by Western blotting of fractions from density gradient centrifugation of membranes prepared under detergent-free conditions. In these situations, heparin inhibits phosphorylation of EGFR on the Src-dependent site Tyr(845), but not the autophosphorylation of Tyr(1173), and decreases Ras activation and Erk phosphorylation. We conclude that heparin can suppress Erk signaling in VSMC with effects on site-specific phosphorylation of EGFR localized in caveolin-enriched lipid rafts.     

Mathematical model for the effects of epidermal growth factor receptor trafficking dynamics on fibroblast proliferation responses.

We apply a mathematical model for receptor-mediated cell uptake and processing of epidermal growth factor (EGF) to analyze and predict proliferation responses to fibroblastic cells transfected with various forms of the EGF receptor (EGFR) to EGF. The underlying conceptual hypothesis is that the mitogenic signal generated by EGF/EGFR binding on the cell surface, via stimulation of receptor tyrosine kinase activity, is attenuated when the receptors are downregulated and growth factor is depleted by endocytic internalization and subsequent intracellular degradation. Hence, the cell proliferation rate ought to depend on receptor/ligand binding and trafficking parameters as well as on intrinsic receptor signal transduction properties. The goal of our modeling efforts is to formulate this hypothesis in quantitative terms. The mathematical model consists of kinetic equations for binding, internalization, degradation, and recycling of EGF and EGFR, along with an expression relating DNA synthesis rate to EGF/EGFR complex levels. Parameter values have been previously determined from independent binding and trafficking kinetic experiments on B82 fibroblasts transfected with wild-type and mutant EGFR. We show that this model can successfully interpret literature data for EGF-dependent growth of NR6 fibroblasts transfected with wild-type EGFR. Moreover, it successfully predicts the literature observation that NR6 cells transfected with a delta 973 truncation mutant EGFR, which is kinase-active but internalization-deficient, require an order of magnitude lower EGF concentration than cells with wild-type EGFR for half-maximal proliferation rate. This result demonstrates that it may be feasible to genetically engineer mammalian cell lines with reduced growth factor requirements by a rational, nonempirical approach. We explore by further model computations the possibility of exploiting other varieties of EGFR mutants to alter growth properties of fibroblastic cells, based on relationships between changes in the primary structure of the EGF receptor and the rates of specific receptor/ligand binding and trafficking processes. Our studies show that the ability to predict cell proliferation as a function of serum growth factors such as EGF could lead to the designed development of cells with optimized growth responses. This approach may also aid in elucidation of mechanisms underlying loss of normal cell proliferation control in malignant transformation, by demonstrating that receptor trafficking dynamics may in some cases play as important a role as intrinsic signal transduction in determining the overall resulting mitogenic response.