Association of the Ras to mitogen-activated protein kinase signal transduction pathway with microfilaments. Evidence for a p185(neu)-containing cell surface signal transduction particle linking the mitogenic pathway to a membrane-microfilament association site.
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Microvilli of the aggressive 13762 ascites mammary adenocarcinoma contain a large, microfilament-associated signal transduction particle whose scaffolding is a stable glycoprotein complex (Li, Y., Hua, F., Carraway, K. L., and Carraway, C. A. C. (1999) J. Biol. Chem. 274, 25651-25658) associated with the growth factor receptor p185(neu). The receptor is constitutively tyrosine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interaction site. Immunoprecipitation of cell lysates with anti-phosphotyrosine and immunoblotting showed phosphorylated forms of the mitogenic pathway proteins Shc and MAPK in addition to p185(neu), suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185(neu)-containing microvillar fractions revealed the presence in each of stably associated Shc, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk. All of these pathway components co-immunoprecipitated with p185(neu) from cleared lysates of microvilli solubilized under microfilament-depolymerizing conditions. The recruitment of constitutively phosphorylated p185(neu) and the activated mitogenic pathway proteins to this membrane-microfilament interaction site provides a physical model for integrating the assembly of the mitogenic pathway with the transmission of growth factor signal to the cytoskeleton. This linkage is probably a requisite step in the global cytoskeleton remodeling accompanying mitogenesis. (+info)
Expression of C-erbB-2/HER-2 in patients with metastatic breast cancer undergoing high-dose chemotherapy and autologous blood stem cell support.
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C-erbB-2/HER-2 (designated HER-2) is overexpressed in both primary and metastatic breast cancer and predicts poor prognosis. We investigated the expression of HER-2 in patients with metastatic breast cancer undergoing high-dose chemotherapy (HDCT) with autologous blood stem cell (ABSC) support and correlated the presence (positive) or absence (negative) of HER-2 overexpression in these patients with response to treatment, progression-free survival (PFS) and overall survival (OS). The level of HER-2 expression was analyzed in 57 patients with metastatic breast cancer undergoing HDCT with ABSC support. Plasma from peripheral blood was taken at three different time points during the course of treatment and was analyzed using an enzyme immunoassay (EIA) to detect circulating levels of the extracellular portion of HER-2. HER-2 levels were elevated (>0.2 U/mg protein) in 27/57 (47.4%) patients at one or more time points during treatment. The level of HER-2 varied during the course of treatment. Following induction chemotherapy (ICT), five patients who were negative initially, showed overexpression of HER-2. Three patients overexpressed HER-2 only after HDCT/ABSC. Response to treatment was similar in patients independent of plasma HER-2 levels. Overexpression of HER-2 was associated with a significantly shorter PFS (P = 0.004, log rank) and OS (P = 0.003, log rank) after HDCT/ABSC. HER-2 overexpression, patient age, estrogen receptor status, progesterone receptor status, and previous hormone treatment were assessed by univariate and multivariate analysis. Univariate analysis determined that only HER-2 overexpression correlated significantly with decreases in progression free survival (P = 0.005, Cox regression). Decreased overall survival correlated significantly with HER-2 overexpression (P = 0.004) and decreased expression of both estrogen receptor (P = 0.032) and progesterone receptor (P = 0.039). In multivariate analysis of these variables, only HER-2 expression levels proved to be of independent statistical significance in predicting outcome for both PFS (P = 0.007) and OS (P = 0.002). These results suggest that overexpression of HER-2, measured by EIA in plasma may predict a shorter PFS and OS in patients with metastatic breast cancer treated with HDCT and ABSC support. (+info)
Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors.
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Tumors that overexpress HER-2/neu receptor or exhibit enhanced EGFR signaling have been reported to possess constitutively activated Src family kinases, especially pp60c-Src. High levels of pp60c-Src activity have also been reported for cell lines that overexpress the EGFR or the chimeric EGFR-HER-2 receptor. It has therefore been suggested that Src kinases may contribute significantly to the oncogenic phenotype of these cells and to the degree of malignancy of tumors that overexpress EGFR family receptors. In this study we show that the induced expression of c-SRC antisense RNA or the application of a selective Src kinase inhibitor induces growth arrest, programmed cell death and reverses the transformed properties of cells that overexpress EGFR or HER-2 receptors. We show that inhibition of Src kinase expression or activity results in the reduction of Stat3 tyrosine phosphorylation, decline of Bcl-XL expression, and induction of cell death. Using a construct in which the promoter of Bcl-X, which possesses putative Stat3 sites, is tethered to the luciferase reporter gene, we show that inhibition of Src activity or expression induces a decline in Bcl-X expression. We also show that the expression of activated Src induces activation of the Bcl-X promoter. This activation is inhibited by the expression of kinase dead Src or of Stat3beta, the dominant-negative form of Stat3. Taken together, these results support the hypothesis that Src positively regulates the transformed phenotype of cells overexpressing EGFR family kinases. Furthermore, these results also suggest that Src positively regulates Bcl-XL expression via Stat3 activation and thus acts not only as a potent mitogenic signaling element, but also as an anti-apoptotic signaling protein. The combination of both activities probably confers upon activated Src its oncogenic activity. Since Src kinase is activated in many tumors, pp60c-Src kinase inhibitors may prove useful as anti-cancer agents for many types of cancer. (+info)
erbB-2 overexpression but no activation of beta-Catenin gene in extramammary Paget's disease.
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Our previous study in extramammary Paget's disease showed neither p53 mutations nor allelic loss at selected loci implicated in other cancers, suggesting a pathogenesis of this skin cancer different from other common epithelial malignancies. To examine further the genetic defects in extramammary Paget's disease, we carried out molecular genetic analyses in 31 tumor samples obtained from 27 cases of extramammary Paget's disease without underlying malignancies. Immunohistochemistry using CB-11 monoclonal antibody revealed either membrane or cytoplasmic erbB-2 oncoprotein overexpression in none of the 13 primary in situ tumors, but in one recurrent in situ tumor, 10 of 13 invasive primary tumors and two of four lymph node metastases. Sensitive dual color fluorescence in situ hybridization analysis using probes for erbB-2 gene locus and chromosome 17 pericentromere, however, revealed different erbB-2 gene status in the erbB-2 overexpressing tumors. One recurrent in situ tumor and one lymph node metastasis showed definite gene amplification characterized by multiple scattered signals or a few large clustered erbB-2 signals, whereas four tumors with predominantly cytoplasmic erbB-2 overexpression were thought to have low-grade gene amplification. The remaining six tumors overexpressing erbB-2 showed no increase of erbB-2 copy numbers. No evidence of abnormal activation of the beta-catenin gene, a critical mediator of Wnt signaling pathway, in any tumor by immunohistochemical staining and by direct sequencing and reverse transcription-polymerase chain reaction analysis was found. Frequent overexpression of erbB-2 by either gene amplification or possible transcriptional activation in invasive primary tumors and metastases suggests an important part for this oncogene in the progression of extramammary Paget's disease. (+info)
Serum c-erB-2 levels in monitoring of operable breast cancer patients.
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BACKGROUND: Various methods and criteria are used to determine protein overexpression of c-erbB-2 and the clinical utility of c-erbB-2 is under investigation. We have reported previously that the levels of cytosol c-erbB-2 in breast cancer were significantly different between the clinical stages. METHODS: The levels of c-erbB-2 protein were determined in sera from 210 breast cancer patients using a sandwich enzyme immunoassay between November 1996 and March 1998. The cut-off level was set at 5.4 ng/ml for healthy female blood donors. RESULTS: First, serum c-erbB-2 levels were analyzed in 73 preoperative breast cancer patients with stage I-IIIB disease. The range and median values were 2.3-32.3 and 4.8 ng/ml, respectively. The positive rate was 38%. Overexpression of serum c-erbB-2 was significantly associated with tumor size, clinical stage, histological grade, lymphatic invasion, nodal status and overexpression of cytosol c-erbB-2, but not with hormonal receptor status and other clinico-pathological factors. Second, c-erbB-2, CEA and CA15-3 in sera were examined in 157 postoperative breast cancer patients. In the 137 disease-free patients, specificities of c-erbB-2, CEA and CA15-3 were 72, 93 and 99%, respectively, but in the 20 first recurrent patients, these sensitivities were 80, 25 and 25%, respectively. CONCLUSIONS: These results suggest that serum c-erbB-2 protein is a useful marker for predicting aggressive behavior and first recurrence of breast cancer. (+info)
A role for phospholipase C-gamma-mediated signaling in tumor cell invasion.
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The invasive and metastatic transformation of cancers often results in death. However, the mechanisms that promote this transformation remain unclear. Two closely related receptors, the epidermal growth factor receptor (EGFR) and ErbB2, are overexpressed in a significant percentage of breast and prostate carcinomas, among others, with this up-regulated signaling correlating with tumor progression. Previous studies in our laboratory have demonstrated that an EGFR-phospholipase C (PLC)gamma-mediated motility-associated signaling pathway is rate-limiting for tumor cell invasion in vitro and in vivo in one model of prostate carcinoma. Therefore, we investigated whether this PLCgamma signaling pathway also was rate-limiting for invasion in other tumor cell lines and types and whether this EGFR activity is subsumed by the closely related ErbB2. We determined the effects of PLCgamma signal abrogation by pharmacological (U73122) and molecular (expression of the dominant-negative PLCz) means on the in vitro invasiveness of tumor cells. Inhibition of PLCgamma signaling concomitantly decreased invasiveness of de novo-occurring transgenic adenocarcinoma mouse prostate (TRAMP) lines and the human breast cancer cell lines MDA-468 and MDA-231; these lines present up-regulated EGFR signaling. Because the prostate and breast cancer lines usually present autocrine stimulatory loops involving EGFR, we also examined transgenic adenocarcinoma mouse prostate C1 and MDA-468 treated with the EGFR-specific kinase inhibitor PD153035 to determine whether invasiveness is dependent on EGFR signaling. PD153035 reduced invasiveness to levels similar to those seen with U73122, suggesting that the autocrine EGFR stimulatory loop is functioning to promote invasiveness. To determine whether this signaling pathway also promotes invasiveness of ErbB2-overexpressing tumors, we examined the human breast carcinoma line MDA-361; again, U73122 inhibition of PLCgamma decreased invasiveness. In all situations, the inhibition of PLCgamma signaling did not decrease mitogenic signaling. Thus, the motility-associated PLCgamma signaling pathway is a generalizable rate-limiting step for tumor cell progression. (+info)
Stimulation of beta1-integrin function by epidermal growth factor and heregulin-beta has distinct requirements for erbB2 but a similar dependence on phosphoinositide 3-OH kinase.
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Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the beta1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest beta1-dependent increases in cell adhesion and motility, heregulin-beta (HRGbeta) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGbeta-mediated adhesion and potently blocked HRGbeta- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate beta1-integrin function. (+info)
Fluorescence in situ hybridisation detection of erbB2 amplification in breast cancer fine needle aspirates.
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AIM: To develop a method for the detection of amplification of the erbB2 oncogene in breast cancer fine needle aspirates using fluorescence in situ hybridisation (FISH) and to compare amplification with immunohistochemical detection of the erbB2 protein. METHODS: A digoxigenin labelled probe to the erbB2 gene was hybridised to 15 aspirates prepared from operative breast cancer specimens. A chromosome 17 centromere probe was also hybridised to the aspirates either separately or in combination with the erbB2 probe. The aspirates were scored for erbB2 amplification and chromosome 17 centromere number. Subsequently, paraffin wax embedded sections of the tumours were stained with the antibody CB11 and scored for the presence of membrane staining. RESULTS: Three of the 15 tumour aspirates showed high level amplification of erbB2 detected by FISH. These three tumours also showed chromosome 17 polysomy and diffuse membrane staining by immunohistochemistry. CONCLUSIONS: FISH can be used to detect erbB2 amplification in fine needle aspirates and results correlate with conventional immunohistochemical staining. Difficulties were encountered in the visualisation of the signals in non-amplified cases without the use of specialised digital imaging. (+info)