Regulation of growth hormone and epidermal growth factor receptors by progestins in breast cancer cells

A 24 hr incubation of T-47D human breast cancer cells with R5020, a synthetic progestin, resulted in a 200-250% increase in the specific binding of human growth hormone (hGH) and epidermal growth factor (EGF) by these cells. This effect was specific for progestins in that similar responses were observed with progesterone, medroxyprogesterone acetate and ORG 2058 but no significant increases in hGH or EGF binding were observed in cells incubated with testosterone, estradiol or hydrocortisone. Increased binding was due to an increase in the concentration of receptors (hGH, control = 6,490 +/- 500, progestin treated = 13,180 +/- 3,270 sites/cell; EGF, control = 33,380 +/- 7,410, progestin treated = 67,460 +/- 20,330 sites/cell) while the affinity constants for the hormone-receptor interactions were unchanged by progestin treatment. The specific binding of insulin, calcitonin, transferrin and concanavalin A was unaffected by these treatments. It is concluded that expression of hGH and EGF receptors in this breast cancer cell line is regulated by progestins.     

Receptors for epidermal growth factor in breast cancer

Epidermal growth factor receptor (EGFr) and cytosolic (cER) and nuclear (nER) estradiol receptors were quantified in 220 primary breast cancers. The EGFr was significantly more frequent (X2 = 5.9; P less than 0.025) and its concentration was significantly higher (P less than 0.001) among ER- tumors than in ER+ tumors. There was a significantly greater proportion (X2 = 6.4; P less than 0.05) of node involvement in EGFr+/ER+ tumors than in EFGr/ER+. Increases in the proportion of EGFr+ in ER- tumors are parallel to Scarff-Bloom scores (X2 = 6.1; P less than 0.05) and there is a significant trend towards increased EGFr concentrations with histologic dedifferentiation. In ER+ tumors the median concentrations of EGFr in the different age groups show linear correlation and follow a parallel profile with the medians of nER. These findings support the hypothesis that considers EGFr as a bad prognosis factor and suggest that EGFr expression and concentration in ER+ tumors might be considered an estrogenic action mediated through the binding of ER to their nuclear acceptors.     

Adrenocorticotropic hormone and growth factor receptors in breast cancer

Tissues from 100 cases of breast cancer were analysed immunohistochemically for the presence of adrenocorticotropic hormone (ACTH) or ACTH-like peptides and expression of c-erbB-2 oncoprotein, epidermal growth factor receptor (EGF-R) as well as oestrogen receptor (ER). Immunopositivity for ACTH was found in 15% cases of infiltrating duct carcinoma of the breast, whereas 38% and 36% breast tumours were positive for c-erbB-2 and EGF-R respectively. While 27% cases were positive for ER. The immunoexpressions of all parameters were higher in breast cancer cases with upper age group (45 years or above) than the patients below 45 years of age. A significant correlation was observed between the tumour grade and the expression of c-erbB-2 oncoprotein. Further, a positive association between the immunoexpression of c-erbB-2 and EGF-R was noticed. Interestingly, a statistically significant relationship was found between the immunopositivity of ACTH and ER. The study reflects a probable association of ACTH or ACTH-like peptides in pathological process of breast cancer.     

Processing of calcitonin and epidermal growth factor after binding to receptors in human breast cancer cells (T 47D).

125I-labelled calcitonin and 125I-labelled epidermal growth factor (EGF) bound to T 47D breast cancer cells at 37 degrees C in a manner that became increasingly resistant to removal by acid pH. Bound 125I-labelled EGF became resistant to acid removal more rapidly than did bound 125I-labelled calcitonin. The shift from acid accessibility to acid inaccessibility was energy-dependent since it was not seen at 4 degrees C and was inhibited in the presence of cell metabolic inhibitors. Radioactivity removed by acid represented intact hormone as assessed by trichloroacetic acid precipitation, whereas radioactivity released spontaneously by the cells was trichloroacetic acid-soluble. Inclusion of 10 mM-NH4Cl in the incubation medium resulted in an accumulation of cell-associated radioactivity without affecting the shift to acid inaccessibility. The accumulated radioactivity was relatively more trichloroacetic acid-precipitable in comparison with that associated with control cells. These data are consistent with internalization of receptor-bound EGF and a similar though slower mechanism of processing for receptor-bound calcitonin. The predominant route of hormone release from cells seems to occur via intracellular degradation rather than dissociation from cell-surface receptors.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

G protein-coupled receptors desensitize and down-regulate epidermal growth factor receptors in renal mesangial cells

Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine(2A), lysophosphatidic acid, angiotensin AT(1), bradykinin B(2)) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-kappaB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative beta-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis.     

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.     

Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells.

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EGF present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in "dense" and "sparse" cultures. Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.     

Inhibition of epidermal growth factor receptor by ferulic acid and 4-vinylguaiacol in human breast cancer cells

Objectives

To examine the potential of ferulic acid and 4-vinylguaiacol for inhibiting epidermal growth factor receptor (EGFR) in human breast cancer cells in vitro.

Results

Ferulic acid and 4-vinylguaiacol limit the EGF (epidermal growth factor)-induced breast cancer proliferation and new DNA synthesis. Western blot analysis revealed both ferulic acid and 4-vinylguaiacol exhibit sustained inhibition of EGFR activation through down-regulation of Tyr 1068 autophosphorylation. Molecular docking analysis shows ferulic acid forming hydrogen bond interaction with Lys 745 and Met 793 whereas, 4-vinylguaiacol forms two hydrogen bonds with Phe 856 and exhibits stronger hydrophobic interactions with multiple amino acid residues at the EGFR kinase domain.

Conclusions

Ferulic acid and 4-vinylguaiacol could serve as a potential structure for the development of new small molecule therapeutics against EGFR.

     

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.     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Somatostatin receptors 1 and 5 heterodimerize with epidermal growth factor receptor: Agonist-dependent modulation of the downstream MAPK signalling pathway in breast cancer cells

The role of somatostatin (SST) and epidermal growth factor (EGF) in breast cancer is undisputed; however, the molecular mechanisms underlying their antiproliferative or proliferative effects are not well understood. We initially confirmed that breast tumour tissues express all five somatostatin receptors (SSTR1-5) and four epidermal growth factor receptors (ErbB1-4). Subsequently, to gain insight into the function of SSTRs and ErbBs in oestrogen receptor (ER)-positive (MCF-7) or ERα-negative (MDA-MB-231) breast cancer cells, we defined SSTR1, SSTR5 and ErbB1 mRNA and protein expression in these two tumour cell lines. Consistent with previous studies showing SSTR1/SSTR5 heterodimerization and having seen cell-specific and ligand-selective alterations in receptor expression, we next elucidated whether SSTR1 and SSTR5 functionally interact with ErbB1 using pbFRET analysis. We subsequently determined the effects of SST and EGF either alone, or in combination, on selected downstream signalling molecules such as erk1/2, p38 and JNK. Here, we showed that both SST and EGF influenced erk1/2 phosphorylation and that SST modulated the effects of EGF in a cell-specific manner. We also demonstrated agonist-, time and cell-dependent regulation of p38 phosphorylation. We further investigated modulation of Grb2, SOS, Shc, SH-PTP1 and SH-PTP2. ErbB1 adaptor proteins known to play a role in MAPK activation, Shc, Grb2 and SOS, changed in an agonist- and cell-specific manner whereas, SH-PTP1 and SH-PTP2, adaptor proteins reported to interact with SSTRs, translocated from the cytosol to membrane in a cell-specific manner following SST and/or EGF treatment. Although several previous studies have shown crosstalk between RTKs and GPCRs, there are no reports describing SSTR (GPCR) modulation of ErbBs (RTK) in breast cancer. To the best of our knowledge, this is the first report describing crosstalk/interactions between SSTRs and ErbBs.     

Toxicity and effects of epidermal growth factor on glucose metabolism of MDA-468 human breast cancer cells

Epidermal growth factor (EGF) has an in vitro inhibitory effect on tumor cells which exhibit a high number of EGF receptors (EGFR). Studies were performed in order to delineate the effects of EGF on glucose metabolism of MDA-468 human breast cancer cells, which have a large number of EGFR. Glucose consumption and lactate production were found to be substantially increased in MDA-468 cells following EGF exposure, while no such effects were detected in MCF-7 breast cancer cells, which have a very low number of EGFR. When glucose levels in the growth medium were increased, the toxicity of EGF was diminished. The energetic status of MDA-468 cells perfused with growth medium containing EGF was monitored by 31P magnetic resonance spectroscopy, and no signs of compromised metabolic state or viability were noted for up to 36 h. The rate of glucose transport and phosphorylation was quantitated by 13C magnetic resonance spectroscopy, utilizing [6-13C]2-deoxyglucose, and a 97% increase was found in MDA-468 cells following EGF administration. The profound effects of EGF on glucose metabolism in cells with very high numbers of EGFR and the lack of toxicity in the perfused system may indicate that the growth-inhibitory effect is confined to the in vitro cultured cells.     

Adriamycin causes up regulation of epidermal growth factor receptors in actively growing cells

We have demonstrated a drug-dependent increase in the capacity of HeLa and 3T3 cells, grown in the presence of lethal and sublethal concentrations of adriamycin, to bind epidermal growth factor (EGF). Scatchard analysis ascribes this effect to an increase in the number of binding sites, with little change in affinity. The time course of binding of ¹²⁵I-EGF is unchanged by adriamycin treatment, in both 3T3 and HeLa cells, at both 0 and 37 °C. This increase appears gradually over 3 or 4 days' exposure to the drug and is reversible over a similar period. Although in HeLa cells the increase reaches a maximum of about 4-fold, regardless of cell density, the maximum observed in 3T3 cells, over 100-fold, is seen only at low cell densities. This could be related to the density-dependent growth regulation seen in 3T3 cells, but not in HeLa cells. We suggest that the ability of the anticancer agent adriamycin to alter the cellular response to a growth-regulatory substance may be related to the mechanism of its cytotoxic action.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Neuronal differentiation in response to epidermal growth factor of transfected murine P19 embryonal carcinoma cells expressing human epidermal growth factor receptors

The human epidermal growth factor receptor (hEGF-R) was introduced into murine P19 embryonal carcinoma (EC) cells, which do not express endogenous EGF-R. Undifferentiated stable P19 EC transfectants containing multiple copies of the hEGF-R complementary DNA were isolated. These cells express functional EGF-R, exhibiting characteristic biphasic EGF binding and intrinsic tyrosine protein kinase activity. Whereas normally EGF induces the expression of multiple nuclear protooncogenes, only junB expression is induced by EGF in the HER-transfected cells. This indicates that undifferentiated P19 EC cells contain at least part of a signal transduction machinery capable of coupling to the ectopically expressed hEGF-R. Interestingly, neuronal differentiation is induced in these cells in response to EGF under culture conditions resembling those during early preimplantation embryogenesis. These results indicate that neuronal differentiation of pluripotent P19 EC cells can be induced via activation of a tyrosine protein kinase signaling pathway.     

Membrane distribution of epidermal growth factor receptors in cells expressing different gangliosides.

Gangliosides have been found to reside in glycosphingolipid-enriched microdomains (GEM) of the plasma membrane and to be involved in the regulation of epidermal growth factor receptor (EGFr or ErbB1) activity. To gain further insight into the mechanisms involved in EGFr modulation by gangliosides, we investigated the distribution of EGFr family members in the plasma membrane of CHO-K1 cells, which were genetically modified to express different ganglioside molecules or depleted of glycolipids. Our data demonstrate that at least four different sets of endogenously expressed gangliosides, including GD3, did not have a significant effect on EGFr distribution in the plasma membrane. In addition, using confocal microscopy analysis we clearly demonstrated that the EGFr co-localizes only to a minor extent with GD3. We also explored the endogenous expression, in wild-type CHO-K1 cells, of the orphan receptor ErbB2 (which is the preferred heteroassociation partner of all other ErbB proteins) and the effect of GD3 expression on its membrane distribution. Our results showed that CHO-K1 cells endogenously express ErbB2 and that expression of the GD3 affected, to some extent, the membrane distribution of endogenous ErbB2. Finally, our findings support the notion that most EGFr are excluded from GEM, while an important fraction of ErbB2 is found to be associated with these microdomains in membranes from CHO-K1 cells.     

Characterization of insulin-like growth factor I (IGF-I) receptors of human breast cancer cells

Studies of binding of IGF-I to a plasma-membrane-enriched subcellular fraction prepared from MCF-7 human breast cancer cells reveal the presence of 0.2 pmols specific binding sites for this mitogen per mg membrane protein, with an equilibrium affinity constant of 1.45 nM-1. Competition studies with insulin, IGF-II, and an anti-IGF-I receptor antibody are consistent with the presence of specific IGF-I receptors, and SDS-PAGE showed binding to a 130 kDa subunit identical to that of receptors from human placenta. In addition, we show that IGF-I is more potent than estradiol and comparable to EGF in stimulating in vitro proliferation of MCF-7 cells, and that IGF-I-stimulated proliferation of these cells is inhibited by a blocking monoclonal antibody against the IGF-I receptor. These results demonstrate that IGF-I is an important mitogen for MCF-7 cells and that the mitogenic effect is mediated by specific IGF-I receptors.