Inhibition Of Epidermal Growth Factor Receptor Phosphorylation Research Articles

Combined modality therapy of A431 human epidermoid cancer using anti-EGFr antibody C225 and radiation.

Monoclonal antibodies (mAb) to epidermal growth factor receptor (EGFr) inhibit tumor cell proliferation and enhance cytotoxicity of chemotherapeutic agents. The purpose of this study was to investigate the interaction of the anti-EGFr antibody C225 combined with radiotherapy (RT) on EGFr expressing A431 human epidermoid cancer cells. Cell proliferation, apoptosis, EGFr expression and phosphorylation, and clonogenic survival were assayed in vitro. A431 tumor growth inhibition and immunohistochemistry analysis of EGFr expression and apoptosis were assessed in vivo. C225 plus RT produced greater inhibition of A431 cell proliferation than C225 or RT alone which was corroborated by enhanced apoptosis. Similar clonogenic survival occurred following the addition of C225 to RT, although colonies were smaller in the presence of C225. C225 produced inhibition of EGF-induced phosphorylation of EGFr without concurrent down-regulation of surface receptor, which was not altered by RT. Combined treatment of mice bearing tumors demonstrated enhancement of complete regressions, reduction in time to tumor size doubling, and prolongation of survival. Significant apoptosis occurred in xenograft tumors treated with C225 with or without RT. These data demonstrate an interaction between C225 and RT. C225-mediated apoptosis and inhibition of EGFr phosphorylation may be critical in the interaction. Studies to define the precise influence of combined modality treatment on the EGFr signal transduction cascade need to be pursued. The combination of growth factor receptor antibodies and RT has potential application in clinical oncology.

The transmembrane protein tyrosine phosphatase RPTPsigma modulates signaling of the epidermal growth factor receptor in A431 cells.

Attenuation of epidermal growth factor receptor signaling by the ganglioside G(M3) has previously been found to involve activation of an unknown protein-tyrosine phosphatase (PTP). In transient expression experiments we tested different PTPs for activation towards EGF receptor by G(M3). The transmembrane PTP RPTPsigma but not RPTPalpha or the SH2-domain PTP SHP-1 exhibited elevated activity towards EGF receptor in G(M3)-treated cells. The possible relevance of RPTPsigma for regulation of EGF receptor signaling activity was further explored in stable A431 cells lines inducibly expressing RPTPsigma or RPTPsigma antisense RNA. RPTPsigma expression clearly reduced EGF receptor phosphorylation. Also, soft agar colony formation of respective cell lines was reduced upon RPTPsigma expression whereas RPTPsigma antisense RNA expression augmented both, EGF receptor phosphorylation and soft agar colony formation. In addition, RPTPsigma antisense RNA expression rendered A431 cells resistant to inhibition of EGF receptor phosphorylation by G(M3). We propose that RPTPsigma participates in EGF receptor dephosphorylation in A431 cells, becomes activated by G(M3) via an unknown mechanism and is thereby capable to mediate attenuation of EGF receptor phosphorylation by G(M3).

Inhibition of epidermal growth factor receptor gene expression and function decreases proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells.

Previous reports have shown that fresh tissues and cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress transforming growth factor alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies confirm that TGF-alpha and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting an autocrine growth pathway. Using three strategies, we examined the hypothesis that downmodulation of EGFR would reduce the proliferation of SCCHN cells. We targeted EGFR mRNA using antisense oligonucleotides and the mature EGFR protein at two sites, the ligand-binding domain and the kinase domain, and determined the effects of this targeting on SCCHN proliferation. Treatment of several SCCHN cell lines with a pair of antisense oligodeoxynucleotides directed against the translation start site and first intron-exon splice junction of the human EGFR gene resulted in decreased EGFR protein production and inhibited growth by 86% compared to a 13% reduction in cells treated with sense oligonucleotides (P=0.03). Growth inhibition was specific for carcinoma cells since the same EGFR antisense oligonucleotides had no effect on the proliferation of normal mucosa cells harvested from non-cancer patients. Two monoclonal antibodies which block ligand binding to EGFR (MAbs 425 and 528) inhibited the growth of several SCCHN cell lines by up to 97% which suggests that EGFR is participating in an autocrine pathway in SCCHN that is, at least in part, external. An EGFR-specific tyrosine kinase inhibitor (PD 153035) was found to inhibit EGFR phosphorylation in SCCHN cell lines and to reduce growth by 68% although it had no effect on the growth rate of normal mucosal epithelial cells. These experiments indicate that EGFR gene expression and function is critical for SCCHN cell growth but not for growth of normal mucosa cells and therefore may serve as a tumor-specific target for preventive and therapeutic strategies in head and neck cancer.

Inhibition of chemomigration of a human prostatic carcinoma cell (TSU‐pr1) line by inhibition of epidermal growth factor receptor function

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 μg/ml anti-hEGFR monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease. © 1996 Wiley-Liss, Inc.

Inhibition of chemomigration of a human prostatic carcinoma cell (TSU-pr1) line by inhibition of epidermal growth factor receptor function.

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 microgram/ml anti-hEGF monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

Identification and characterization of an anti-tyrosine kinase factor in cystic gliomas

In view of the frequent activation of the epidermal growth factor receptor (EGF-R) in gliomas and autocrine hypothesis, we searched for ‘EGF-like’ factor(s) in cystic fluids (CFs) associated with gliomas. Membranes of A431 cells, which overexpress EGF-R, were used to explore such activity in 20 CFs. In all cases CFs induced inhibition of EGF-R phosphorylation. Biochemical analysis revealed an anti-tyrosine kinase activity which was identified as a 18 kDa proteic factor. Effectiveness at high dilution and anti-proliferative effect on living cells in culture suggest that this factor may be involved in the negative regulation of glial oncogenesis.

Müllerian inhibiting substance blocks epidermal growth factor receptor phosphorylation in fetal rat lung membranes

Neonatal males develop respiratory distress syndrome more frequently than females for unknown reasons. The fetal testis secretes testosterone and müllerian inhibiting substance (MIS); MIS has been shown to inhibit fetal lung maturation in vitro and in vivo and to block phosphorylation of epidermal growth factor (EGF) receptors in A431 cells. We hypothesized that MIS would also inhibit membrane phosphorylation of EGF receptors in fetal lung, and that ultrastructural study of MIS-exposed lung might complement the biochemical data by assessing the effect of MIS on tissue morphology. Lung membranes were prepared from 19.5-day fetal rats and phosphorylation assays performed with 3 to 4 μg of membrane protein, with or without EGF (26 nmol/L), 0.025 mCi AT 32P (0.136 μmol/L), and either recombinant human MIS (rhMIS, 30 pmol) from media of Chinese hamster ovary (CHO) cells, rhMIS dialysis buffer, or wild-type CHO media. The 170,000 molecular weight EGF receptor, visualized by autoradiography of polyacrylamide gels, was phosphorylated in both female and male membranes. rhMIS, when added to EGF-stimulated membranes, caused significant inhibition of EGF receptor phosphorylation (females: 32.42% ± 11.5%; males: 32.3% ± 19.1%, P < 0.001; rhMIS-treated v EGF-stimulated state, P = NS, male v female, Cerenkov counting). Electron microscopy (EM) of rhMIS-exposed lung showed decreased lamellar bodies (LB) in both male alveolar spaces and female parenchyma, and, unexpectedly, increased numbers in female alveoli. Immunoabsorption experiments using coincubation of rhMIS with anti-rhMIS IgG polyclonal antibodies or equiprotein normal IgG demonstrated MIS antibody-specific reversal of rhMIS activity in membrane phosphorylation. These observations in fetal rat lung membranes extend the previous data showing inhibitory effects by MIS in fetal lung, confirm findings in natural tissue previously confined to tumor cells (A431), and suggest that rhMIS signal transduction could be mediated via inhibition of EGF receptor autophosphorylation.