Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.     

Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells

The development of acquired resistance to antihormonal agents in breast cancer is a major therapeutic problem. We have developed a tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line to investigate the mechanisms behind this condition. Both epidermal growth factor receptor (EGFR) and c-erbB2 mRNA and protein expression were increased in TAM-R compared with wild-type MCF-7 cells, whereas comparable levels of c-erbB3 mRNA and protein were expressed in both cell lines. Under basal conditions, phosphorylated EGFR/c-erbB2, EGFR/c-erbB3 but not c-erbB2/c-erbB3 receptor heterodimers were detected in TAM-R cells in association with increased levels of phosphorylated extracellular-signal regulated kinase 1/2 (ERK1/2). Both cell lines were capable of generating a range of EGFR-specific ligands and increased expression of transforming growth factor alpha was observed in TAM-R cells. Treatment of TAM-R cells with ZD1839 (Iressa) or trastuzumab (Herceptin) blocked c-erbB receptor heterodimer formation and phosphorylation, reduced ERK1/2 activity, and strongly inhibited cell growth. The MAPK kinase inhibitor PD098059 specifically reduced phosphorylated ERK1/2 levels and inhibited TAM-R growth. All three agents abolished ERK1/2 activity in wild-type cells but caused only small reductions in cell proliferation. These results demonstrate that TAM-R MCF-7 cell growth is mediated by the autocrine release and action of an EGFR-specific ligand inducing preferential EGFR/c-erbB2 dimerization and downstream activation of the ERK pathway.     

Ligand and p185c-neu density govern receptor interactions and tyrosine kinase activation.

The neu protooncogene (also known as c-erbB2, NGL,and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosinekinase activity that resembles the receptor for epidermal growth factor. Thep185 gene and protein were originally identified in the brain and are thought toplay a critical role in neurogenesis. Aberrant c-erbB2 protein overexpressionalso occurs in several human adenocarcinomas. A ligand for p185, neu-activatingfactor (NAF), specifically binds to neu receptor and increases the p185c-neutyrosine phosphorylation in vitro and in vivo in a dose-dependent manner. We nowshow that NAF specifically binds to purified p185 expressed in baculovirus.Direct binding analysis showed that NAF binds with high affinity (Kd = 1.3 nM).We have investigated changes in the structure and association state ofbaculovirus-produced neu holoreceptor that are induced by ligand binding. Inthis study, we used sucrose gradients to show that purified p185c-neu existsmainly in the monomeric form at low concentrations, whereas at higherconcentrations p185c-neu exists as dimers or multimers. At low concentrations,but in the presence of ligand, p185c-neu sediments as a dimeric or multimericform. Monomer-oligomer interconversion is absolutely ligand dependent at lowreceptor concentrations. The high molecular weight form of the receptor isenzymatically more active, as a consequence of ligand-driven activation of thereceptor kinase. Oncogenic p185neu receptors sediment predominantly as highmolecular weight forms and have constitutively active kinases.     

The epidermal growth factor receptor and the product of the neu protooncogene are members of a receptor tyrosine phosphorylation cascade.

The protein product of the neu protooncogene, p185, is a tyrosine kinase with a high degree of sequence homology to the epidermal growth factor (EGF) receptor. Although p185 does not bind EGF, EGF stimulates tyrosine phosphorylation of p185. To determine the mechanism of this interaction we have used a vaccinia virus/bacteriophage T7-based transient gene expression system to induce production of normal and kinase-deficient forms of p185 in the absence and presence of EGF receptors. Tyrosine phosphorylation of kinase-deficient p185 was observed, but only in the presence of the EGF receptor. These findings strongly support the hypothesis that p185 is a substrate for the EGF receptor tyrosine kinase in a tyrosine kinase cascade.     

The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.     

Agonistic antibodies stimulate the kinase encoded by the neu protooncogene in living cells but the oncogenic mutant is constitutively active.

The neu protooncogene (also called c-erbB2 and HER-2) undergoes oncogenic activation through a single mutation. The product of the protooncogene, p185neu, probably functions as a receptor for a peptide growth factor. To circumvent the absence of a well-characterized ligand, I generated ligand-mimicking monoclonal antibodies directed to the presumed receptor. These antibodies stimulated tyrosine phosphorylation of p185neu in living cells and also accelerated the rate of endocytosis and degradation of p185neu. A monovalent Fab fragment of such an antibody was ineffective, suggesting a role for receptor dimerization in signal transduction. Unlike the product of the protooncogene, the transforming mutant was not affected by the ligand-like antibodies. However, it undergoes constitutively high phosphorylation on tyrosine residues in living cells, and its turnover rate is remarkably rapid. Nevertheless, the pattern of phosphorylation of the mutant protein is similar to the one exhibited by an antibody-stimulated p185neu, suggesting that the mutation mimics activation by the antibody. These results suggest that the kinase of p185neu is under allosteric control that may involve ligand-induced dimerization of receptors. This mechanism is deregulated in the oncogenic mutant, which is functionally equivalent to ligand-stimulated receptor.     

Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells

Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle cell growth, an important feature of hypertension and atherosclerosis. Many of these effects are dependent on the production of reactive oxygen species (ROS). Ang II induces phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules. Here, we provide novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. Pretreatment of vascular smooth muscle cells with the antioxidants diphenylene iodonium, Tiron, N-acetylcysteine, and ebselen significantly inhibited ( approximately 80% to 90%) tyrosine phosphorylation of the EGF-R by Ang II but not by EGF. Of the 5 autophosphorylation sites on the EGF-R, Ang II mainly phosphorylated Tyr1068 and Tyr1173 in a redox-sensitive manner. The Src family kinase inhibitor PP1, overexpression of kinase-inactive c-Src, or chelation of intracellular Ca(2+) attenuated EGF-R transactivation. Although antioxidants had no effects on the Ca(2+) mobilization or phosphorylation of Ca(2+)-dependent tyrosine kinase Pyk2, they inhibited c-Src activation by Ang II, suggesting that c-Src is 1 signaling molecule that links ROS and EGF-R phosphorylation. Furthermore, Ang II-induced tyrosine phosphorylation of the autophosphorylation site and the SH2 domain of c-Src was redox sensitive. These findings emphasize the importance of ROS in specific Ang II-stimulated growth-related signaling pathways and suggest that redox-sensitive EGF-R transactivation may be a potential target for antioxidant therapy in vascular disease.     

Suppressor of cytokine signaling (SOCS)-5 is a potential negative regulator of epidermal growth factor signaling

The suppressors of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing intracellular inhibitors of cytokine signal transduction that act by several different mechanisms. Recent evidence suggests that the action of the SOCS proteins may extend beyond the cytokine receptors to signaling initiated by members of the tyrosine kinase receptor family. In this study, the ability of SOCS-5 to negatively regulate signaling cascades downstream of the epidermal growth factor receptor (EGF-R) has been examined by using an EGF-responsive cell line engineered to constitutively express the EGF-R and SOCS-5 or SOCS-5 mutants. SOCS-5 associated with the EGF-R complex in an EGF-independent manner, and the mitogenic response to EGF of all SOCS-5-expressing cell lines was dramatically inhibited when compared with control cell lines. Furthermore, this effect was abrogated after deletion of the SOCS-5 SOCS box. This result suggests that the inhibition of signaling occurs through enhanced proteasomal degradation of the EGF-R through SOCS box recruitment of E3 ubiquitin ligase activity.     

Epidermal growth factor system regulates mucin production in airways

Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor alpha (TGFalpha), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor alpha (TNFalpha). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNFalpha. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogen-free rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNFalpha induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blue-periodic acid-Schiff staining (reflecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNFalpha, EGF, or TGFalpha alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNFalpha-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.     

The v-sea oncogene of avian erythroblastosis retrovirus S13: another member of the protein-tyrosine kinase gene family.

The cloning and sequencing of the oncogene of the avian erythroblastosis virus S13 is described. The oncogene, termed v-sea, was found to be another member of the protein-tyrosine kinase gene family. The oncogene was fused in frame with the retrovirus S13 envelope gene, thus generating a fusion protein with a structure resembling that of a growth factor receptor. Sequence comparisons revealed that the v-sea gene was most closely related to the insulin receptor family of protein-tyrosine kinases, the greatest similarity being with the human MET oncogene.     

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.     

Analogs of GnRH-I and GnRH-II inhibit epidermal growth factor-induced signal transduction and resensitize resistant human breast cancer cells to 4OH-tamoxifen

About 50-64% of human breast cancers express receptors for GnRH-I. Direct antiproliferative effects of analogs of GnRH-I on human breast cancer cell lines have been shown. They are at least in part mediated by antagonizing growth promoting effects of estradiol, epidermal growth factor (EGF) or insulin-like growth factor. Recently, expression of a putative receptor for GnRH-II in human tissues was demonstrated. Antiproliferative effects of GnRH-II in human endometrial and ovarian cancer cells were shown not to be mediated through the GnRH-I receptor. Now we demonstrate direct anti-proliferative effects of the GnRH-I analog Triptorelin and the GnRH-II analog [d-Lys(6)]GnRH-II in MCF-7 and T47D human breast cancer cells expressing GnRH-I receptors and putative GnRH-II receptors. Pretreatment with Triptorelin or [d-Lys(6)]GnRH-II blocked EGF-induced autophosphoryla-tion of EGF receptor and activation of mitogen-activated protein kinase (extracellular-signal-regulated kinase 1/2 (ERK1/2)) in these cells. In sublines of MCF-7 and T47D cells, which were developed to be resistant to 4OH-tamoxifen, HER-2/p185 was overexpressed. Pretreatment of these cell lines with Triptorelin or [d-Lys(6)]GnRH-II completely abolished resistance to 4OH-tamoxifen, assessed by 4OH-tamoxifen-induced apoptosis. Analogs of GnRH-I and GnRH-II counteract EGF-dependent signal transduction in human breast cancer cells with expression of receptors for GnRH-I and GnRH-II. Through this mechanism, they probably reverse acquired resistance to 4OH-tamoxifen mediated through overexpression or activation of receptors of the c-erbB family.     

Expression of tyrosine kinases in human malignancies as potential targets for kinase-specific inhibitors

Tyrosine kinase (TK) inhibition has been identified as a promising strategy in the treatment of human malignancies and several synthetic inhibitors have been developed. While the selective blockage of specific TKs is highly effective in vitro, clinical results have been less impressive. It has been suggested that the simultaneous inhibition of multiple TKs might lead to more favorable therapeutic results in vivo. We have therefore performed a systematic analysis of intratumoral TK expression in order to identify potential targets for a simultaneous kinase inhibition. To this end, we have analyzed the protein expression of membrane-associated epidermal growth factor receptor (EGF-R), Her-2/neu, platelet-derived growth factor receptor (PDGF-R), insulin-like growth factor receptor (IGF-R), c-Kit and of cytoplasmatic c-Abl in 500 human tumors of epithelial, stromal and mesenchymal origin by immunohistochemistry, and found a distinct pattern of kinase expression: EGF-R, PDGF-R and c-Abl were expressed in the majority of malignant tumors, whereas c-Kit, Her-2/neu and IGF-R protein expression was considerably less frequent. Overall, the EGF-R protein expression was correlated with PDGF-R, c-Kit and c-Abl immunoreactivity (P = 0.003, P = 0.001 and P < 0.001, respectively). c-Abl was co-expressed with IGF-R and PDGF-R (P = 0.003 and P < 0.001, respectively). Kinase co-expression was also seen in tumor subgroups and was particularly significant in breast cancer where IGF-R protein was expressed together with PDGF-R and c-Abl (P = 0.003 and P = 0.004, respectively), and in colon cancer where PDGF-R was correlated with EGF-R (P < 0.001). With the exception of Her-2/neu expression and age, intra-tumoral TK expression was not associated with parameters such as grading or histological subtypes. Taken together, we have found a specific pattern of kinase co-expression and have identified several potential targets for a tumor-specific multimodal TK inhibition.     

Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues.

An aberrant p210BCR-ABL protein that possesses constitutive protein-tyrosine kinase activity is presumed to be involved in the development of the neoplastic phenotype in chronic myelogenous leukemia (CML). Using a highly specific antibody against phosphotyrosine, we have isolated the tyrosine-phosphorylated p210BCR-ABL and several other proteins containing phosphotyrosine from a variety of CML cell lines. p210BCR-ABL isolated by the monoclonal anti-phosphotyrosine antibody possessed protein-tyrosine kinase activity in vitro comparable to that of the p210BCR-ABL isolated by antibody to a specific peptide sequence in the ABL protein-tyrosine kinase. Other prominent proteins containing phosphorylated tyrosine residues were observed at 185, 150, 120, 105, 63, 56, 36, and 32 kDa, and less prominent proteins were observed at 195, 155, 94, 53, 40, and less than 29 kDa. Staphylococcal V8 peptide mapping indicated that proteins of similar molecular weights were highly homologous to each other across cell lines, despite the diverse hematopoietic lineages of these cells and the genetic heterogeneity of the patients from whom the CML cell lines were derived. Phosphopeptide mapping also revealed that these proteins were distinct from each other as well as from p210BCR-ABL. Because virtually identical phosphotyrosine-containing proteins were found in peripheral blood leukocytes taken directly from CML patients, these proteins are not an artifact of long-term tissue culture but appear to be an integral part of the CML phenotype.     

Immunohistochemical analysis of expression of a 65 kDa oncofetal protein (p65), epidermal growth factor receptor (EGFR), oncogene c-erb B2 and tumor suppressor gene p53 protein products in breast cancer patients

Paraffin-embedded tissue slides from 88 infiltrating ductal breast carcinoma were examined by immunohistochemistry technique with the use of monoclonal antibody against human p65 antigen and polyclonal antibody against p65-like protein present in fetal bovine serum. Immunohistochemical analysis of expression of growth factor receptors (EGFR), protein product of oncogene c-erb B2 as well as protein product of mutated anti-oncogene p53 was also done. It was established that there is no correlation between p65 and c-erbB2, EGFR or p53 expression. In low differentiated tumors (grade III) high p53 index and high EGFR and c-erbB2 expression was connected with low p65 expression. The lack of c-erbB2 and EGFR and low p53 expression was combined usually with high p65 oncoprotein levels.     

Suppression of EGF-R signaling reduces the incidence of prostate cancer metastasis in nude mice

The activation of epidermal growth factor receptor (EGF-R) plays a key role in the promotion of proliferation and invasion in prostatic carcinoma (PCa). Gefitinib (Iressa; ZD1839), an orally active EGF-R tyrosine kinase inhibitor, has shown an important anti-proliferative activity in tumors expressing EGF-R both in vitro and in vivo. Our aim was to elucidate the role of gefitinib in the modulation of the metastatic spread of PCa cells. The therapeutic role of gefitinib was investigated by evaluating the proliferative and invasive ability of the PCa cell line PC3 and of its high metastatic sub-line, PCb2, by in vitro assays and intracardiac injection in nude mice. The inhibitory effect of gefitinib was tested in vivo by injecting PCa cells subcutaneously or in the left ventricle of nude mice and by administering daily 150 mg/kg of gefitinib. While xenograft growth was equally reduced in all PCa lines (about 50%), the bone metastasis formation was inhibited especially for the high metastatic PCb2 sub-line (81%) in comparison to PC3 cells (47%). The comparative in vitro analysis among PCa cell lines showed that PCb2 cells were more sensitive to the inhibitory effect of gefitinib in their invasive ability compared to parental PC3 cells but not in their proliferation rate. Moreover, PCb2 cells demonstrated an increased invasive ability in vitro in response to bone stromal cell conditioned medium (BCM). The simultaneous presence of 0.1 ng/ml gefitinib was sufficient to reduce the number of invaded cells in the presence of both EGF and BCM. The molecular characterization of the highly aggressive PCa sub-lines demonstrated that this phenomenon was associated with an increment in uPA/uPAR axis but not in EGF-R expression. In conclusion, our data suggest that the use of gefitinib as a therapeutic agent may be indicated in the control of PCa spreading to bone.     

Genetic evidence that Shp-2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals

By using both genetic and biochemical approaches, we have investigated the physiological role of Shp-2, a cytoplasmic tyrosine phosphatase with two Src homology 2 domains, in signaling pathways downstream of epidermal growth factor receptor (EGF-R). In previous studies, a targeted deletion mutation in the SH2-N domain of Shp-2 was introduced into the murine Shp-2 locus, which resulted in embryonic lethality of homozygous mutant (Shp-2(-/-)) mice at midgestation. By aggregating Shp-2(-/-) embryonic stem cells with wild-type embryos, we created Shp-2(-/-)/wild-type chimeric animals. Most chimeras had open eyelids at birth and abnormal skin development, a phenotype characteristic of mice with mutations in EGF-R signaling components. In genetic crosses, a heterozygous Shp-2 mutation dominantly enhanced the phenotype of a weak mutant allele of EGF-R (wa-2), resulting in distinctive growth retardation, developmental defects in the skin, lung, and intestine, and perinatal mortality that are reminiscent of EGF-R knockout mice. Biochemical analysis revealed that signal propagation proximal to the EGF-R upon EGF stimulation was significantly attenuated in wa-2 fibroblast cells, which was exacerbated by the additional Shp-2 mutation. Thus, we provide biological evidence here that protein-tyrosine phosphatase Shp-2 acts to enhance information flow from the EGF-R in mouse growth and development.