A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

It is well established that epidermal growth factor (EGF) is a potent mitogen in cells expressing EGF receptor (EGFR). However, a body of evidence indicated that the effects of mitogenic EGF signaling exhibit a non-monotonic, or biphasic dose response curve; EGF at low concentrations elicits a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, EGF inhibits cell growth. However, the molecular mechanism underlying this paradoxical effect of EGF on cell proliferation remains largely unknown. Here, we investigated the molecular mechanisms underlying the biphasic EGF signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which express endogenous EGFR. We found that EGF at low concentrations induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at high concentrations allowed Src-Y527 phosphorylation that inactivates Src. EGF at 10 ng/ml also induced phosphorylation of the MAPK/ERK and activated cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at a higher concentration (500 ng/ml). Our results thus demonstrated that Src functions as a switch of EGF signaling depending on concentrations of EGF.     

Effects of Endogenous Epidermal Growth Factor Receptor Signaling on DNA Synthesis and ERK Activation in a Cytokine-Dependent Hematopoietic Cell Line

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5.12. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic cells.     

EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis

Objectives

Cancer stem cells (CSCs) compose a subpopulation of cells within a tumour that can self‐renew and proliferate. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (b‐FGF) promote cancer stem cell proliferation in many solid tumours. This study assesses whether EGF, bFGF and IGF signalling pathways are essential for colon CSC proliferation and self‐renewal.

Material and methods

Colon CSCs were cultured in serum‐free medium (SFM) with one of the following growth factors: EGF, bFGF or IGF. Characteristics of CSC gene expression were evaluated by real time PCR. Tumourigenicity of CSCs was determined using a xenograft model in vivo. Effects of EGF receptor inhibitors, Gefitinib and PD153035, on CSC proliferation, apoptosis and signalling were evaluated using fluorescence‐activated cell sorting and western blotting.

Results

Colon cancer cell HCT116 transformed to CSCs in SFM. Compared to other growth factors, EGF was essential to support proliferation of CSCs that expressed higher levels of progenitor genes (Musashi‐1, LGR5) and lower levels of differential genes (CK20). CSCs promoted more rapid tumour growth than regular cancer cells in xenografts. EGFR inhibitors suppressed proliferation and induced apoptosis of CSCs by inhibiting autophosphorylation of EGFR and downstream signalling proteins, such as Akt kinase, extracellular signal‐regulated kinase 1/2 (ERK 1/2).

Conclusions

This study indicates that EGF signalling was essential for formation and maintenance of colon CSCs. Inhibition of the EGF signalling pathway may provide a useful strategy for treatment of colon cancer.     

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.     

Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function

We have used cross-linking reagents on cell lines expressing both p185neu and EGFR. The lysates of the cells were precipitated with anti-p185neu or anti-EGFR antibodies. These precipitates included a high molecular weight complex that was identified as an EGFR-p185neu heterodimer. Heterodimerization was found to be induced by exposure to EGR. The EGFR of these cells displayed three affinity states for EGF: low (Kd, approximately 10(-9) M), high (Kd, 10(-9) to 10(-10) M), and very high (Kd, 10(-11) M), as determined by Scatchard analyses. Relatively small levels of EGF had a dramatic biological effect on cells expressing very high affinity EGFR. The very high affinity EGFR disappeared after the cells were treated with anti-p185neu monoclonal antibodies that selectively down-regulated p185neu. EGF and TPA had differential effects on down-modulation of the EGFR in cells that express either one or both species of receptor proteins.     

Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.     

Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 ('Iressa') is independent of EGFR expression level

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.     

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.     

Evidence of an EGF/TGE-alpha--independent pathway for estrogen-regulated cell proliferation

To elucidate the relationship between epidermal growth factor (EGF)/transforming growth factor (TGF-alpha) and estradiol-17 beta (E) in cell proliferation, we examined their effects on the breast cancer cell line, CAMA-1. While E was able to consistently induce cell proliferation under a variety of experimental conditions, EGF/TGF-alpha was without effect. Despite the presence of the receptor (EGFR) gene, mature EGFR protein and mRNA were not detected by radioreceptor assay, 35S Met-labelling, and the Intron Differential RNA/PCR method under conditions in which cells remain responsive to E. Furthermore, TGF-alpha is not an autocrine factor in CAMA-1 cells. We demonstrated unequivocally that EGF/TGF-alpha interaction with EGFR is not an obligatory event in mediating estrogen-stimulated cell proliferation.     

Interferon-γ effect on growth regulation of cells with different levels of EGF receptor expression

Interferon gamma (IFNγ) is known to inhibit the proliferation of some transformed cell lines. Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNγ (Burova et al., 2007) and provided direct evidence for the dependence of IFNγ-induced EGFR transactivation on the EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNγ on human epithelial cell lines—A431 and HeLa that express high levels of EGFR, as well as HEK293 that expresses low levels of EGFR. To characterize the IFNγ-induced changes in these cells, we studied cell growth, the cell cycle, and induction of apoptosis. The response to IFNγ differed in the compared cell lines; cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as was shown by the cell count and MTT. The cell-cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNγ. On the contrary, in HEK293 cells, the IFNγ treatment did not alter distribution by cell cycle phases. Our results indicate that IFNγ produces an antiproliferative effect that depends on the increased expression of EGFR in A431 and HeLa cells. Furthermore, it was demonstrated that IFNγ induced the caspase 3 activation in A431 cells, which suggests the involvement of active caspase 3 in the IFNγ-induced apoptosis.     

Contact-dependent growth inhibition and apoptosis of epidermal growth factor (EGF) receptor-expressing cells by the membrane-anchored form of heparin-binding EGF-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) transduces mitogenic signals through the EGF receptor (EGFR). There are two forms of HB-EGF, the membrane-anchored form (pro-HB-EGF) and the soluble form (sHB-EGF). We studied the biological activity of pro-HB-EGF by using a model in which pro-HB-EGF-expressing effector cells was co-cultured with EGFR-expressing target cells. The DER cell, an EGFR-expressing derivative of the interleukin-3-dependent hematopoietic 32D cell line, grows well in the presence of EGF or sHB-EGF without IL-3. When DER cells were co-cultured on a monolayer of Vero-H cells overexpressing pro-HB-EGF, growth inhibition and subsequent apoptosis were induced in the DER cells even in the presence of excess amounts of EGF or sHB-EGF. Such growth inhibition of DER cells was abrogated when specific antagonists for pro-HB-EGF were added in the culture medium or when direct contact of DER cells with Vero-H cells was prevented, indicating that pro-HB-EGF is involved in this inhibitory effect. Pro-HB-EGF-induced apoptosis of DER cells was also observed even in the presence of IL-3. This rules out the possibility of simple competition between soluble EGFR ligands and pro-HB-EGF. Moreover, 32D cells expressing EGFR mutant composed of the extracellular and the transmembrane domain of EGFR and the cytoplasmic domain of erythropoietin receptor did not undergo apoptosis by co-culture with Vero-H cells, indicating that the inhibitory signal induced by pro-HB-EGF-expressing Vero-H cells is mediated to DER cells via EGFR and that the cytoplasmic domain of EGFR is essential for pro-HB-EGF-induced apoptosis. From these results, we concluded that pro-HB-EGF has unique biological activity through cell-cell contact that is distinct from the activity of sHB-EGF.     

Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells

Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.     

Expression of functional human EGF receptor on murine bone marrow cells.

The human epidermal growth factor-receptor (EGF-R) was introduced into primary mouse bone marrow cells (BMC), utilizing retrovirus mediated gene transfer. Cultivation of infected BMC in the presence of interleukin-3 (IL-3) led to the outgrowth of IL-3 dependent myeloid cells, which efficiently expressed functional EGF-R, exhibiting its two characteristic affinity states. EGF acts on these cells synergistically with IL-3 in stimulating DNA synthesis and cell proliferation even under IL-3 saturation conditions. However, EGF was not sufficient to replace the requirement for IL-3. In contrast, EGF was able to maintain proliferation of a factor-dependent hemopoietic cell line (FDC-P1) infected with the EGF-R retrovirus in the absence of IL-3, but these cells did not respond to EGF in the presence of IL-3. No influence of EGF on IL-3 induced mast cell differentiation of BMC expressing the EGF-R could be observed by histological criteria. These data show that the expression of EGF-R alone is not sufficient to induce or maintain cell proliferation in IL-3 dependent bone marrow derived cells, although it can do so in established hemopoietic cell lines.     

ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma

The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, 'Iressa') blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dose-dependent growth arrest at G0-G1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.     

Bile acids activate EGF receptor via a TGF-alpha-dependent mechanism in human cholangiocyte cell lines

Bile acids transactivate the EGF receptor (EGFR) in cholangiocytes. However, the mechanisms by which bile acids transactivate the EGFR remain unknown. Our aims were to examine the effects of bile acids on EGFR activation in human cholangiocyte cell lines KMBC and H-69. Bile acids stimulated cell growth and induced EGFR phosphorylation in a ligand-dependent manner. Although cells constitutively expressed several EGFR ligands, only transforming growth factor-alpha (TGF-alpha) antisera effectively blocked bile acid-induced EGFR phosphorylation. Consistent with the concept that matrix metalloproteinase (MMP) activity is requisite for TGF-alpha membrane release and ligand function, bile acid transactivation of EGFR and cell growth was blocked by an MMP inhibitor. In conclusion, bile acids activate EGFR via a TGF-alpha-dependent mechanism, and this EGFR activation promotes cellular growth.     

Monoclonal antibody 425 inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived EGF/TGF-alpha

Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha. Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors. Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha. Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media. All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha. EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels. EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media. In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha. Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media. Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

DNA microarray analysis of immediate response to EGF treatment in rat schwannoma cells

Epidermal growth factor (EGF) activates many intracellular effector molecules, which subsequently influence the expression levels of many genes involved in cell growth, apoptosis and signal transduction,etc. In this study, the early response of gene expressions due to EGF treatment was monitored using oligonucleotide DNA microarrays in rat schwannoma cell lines. An immunoblotting experiment showed the successful activation of EGF receptors and an effector protein, STATS, due to EGF treatment. The microarray study showed that 35 genes were significantly induced and 2 were repressed within 60 min after the treatment. The list of induced genes included early growth response 1, suppressor of cytokine signaling 3, c-fos, interferon regulatory factor 1 and early growth response 2,etc. According to the microarray data, six of these were induced by more than 10-fold, and showed at least two different induction patterns, indicating complicated regulatory mechanisms in the EGF signal transduction.