transforming-growth-factor-alpha and Breast-Neoplasms

Breast cancer is the second leading cause of death in women worldwide. The extremely fast rate of metastasis and ability to develop resistance mechanism to all the conventional drugs make them very difficult to treat which are the causes of high morbidity and mortality of breast cancer patients. Scientists throughout the world have been focusing on the early detection of breast tumor so that treatment can be started at the very early stage. Moreover, conventional treatment processes such as chemotherapy, radiotherapy, and local surgery suffer from various limitations including toxicity, genetic mutation of normal cells, and spreading of cancer cells to healthy tissues. Therefore, new treatment regimens with minimum toxicity to normal cells need to be urgently developed.. Iron oxide nanoparticles have been widely used for targeting hyperthermia and imaging of breast cancer cells. They can be conjugated with drugs, proteins, enzymes, antibodies or nucleotides to deliver them to target organs, tissues or tumors using external magnetic field.. Iron oxide nanoparticles have been successfully used as theranostic agents for breast cancer both in vitro and in vivo. Furthermore, their functionalization with drugs or functional biomolecules enhance their drug delivery efficiency and reduces the systemic toxicity of drugs.. This review mainly focuses on the versatile applications of superparamagnetic iron oxide nanoparticles on the diagnosis, treatment, and detecting progress of breast cancer treatment. Their wide application is because of their excellent superparamagnetic, biocompatible and biodegradable properties.

Topics: Breast Neoplasms; Cell Line, Tumor; Drug Delivery Systems; Female; Ferric Compounds; Fever; Gonadotropin-Releasing Hormone; Humans; Hyaluronan Receptors; Integrins; Nanoparticles; Phototherapy; Theranostic Nanomedicine; Transferrin; Transforming Growth Factor alpha; Trastuzumab

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Communication; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Female; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Humans; Mice; Neuregulins; Phosphatidylinositol 3-Kinases; rac GTP-Binding Proteins; Receptors, G-Protein-Coupled; Signal Transduction; Transforming Growth Factor alpha

Topics: Animals; Breast Neoplasms; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Receptors, Estrogen; Signal Transduction; Transcription Factors; Transcription, Genetic; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A

Recent progress in the study of c-Myc has convincingly demonstrated that it possesses a dual role in regulating both proliferation and apoptosis; however, the manner in which c-Myc influences these cellular response pathways remains incompletely characterized. Deregulation of c-Myc expression, via many mechanisms, is a common feature of multiple cancers and is an especially prominent feature of many breast cancers. Of significant interest to those who study mammary gland development and neoplasia is the unresolved nature and contribution of apoptosis to breast tumorigenesis. Recently, the use of transgenic mice and gene-knockout mice has allowed investigators to evaluate the pathological mechanisms by which different genes influence tumor development and progression. In this review, we address two distinct c-myc-containing bitransgenic murine mammary tumor models and discuss the contribution and possible future directions for resolution of cancer-relevant molecular pathways influenced by c-Myc.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Division; Female; Genes, myc; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Milk Proteins; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Transforming growth factor alpha (TGFalpha) is a principal molecule in the normal and neoplastic development of the mammary gland. Binding of TGFalpha to the epidermal growth factor receptor (EGFR), activates the EGFRs' endogenous tyrosine kinase activity and stimulates growth of the epithelium in the virgin and pregnant mouse mammary gland. TGFalpha expression can be detected in breast cancer cells in vivo and in vitro and overexpression can elicit partial transformation or immortalized human and rodent mammary epithelial cells. Despite evidence implicating TGFalpha in the development of mammary neoplasia, the actual mechanism of TGFalpha-induced transformation is unclear. Transgenic mouse models targeting heterologus TGFalpha to the mammary gland have established TGFalpha overexpression can induce hyperproliferation, hyperplasia and occasional carcinoma. These transgenic studies demonstrated a facilitating, proliferative role for TGFalpha in the development of neoplasia and implicated several oncogenes that can cooperate with TGFalpha to transform the mammary epithelium. From studies of EGFR signaling pathways, inhibitory and modulating agents such as anti-EGFR antibodies and specific kinases inhibitors have been used to block the action of this pathway and prevent the development of TGFalpha-induced neoplasia and tumor formation. Studies in Stat5a knockout mice have established that the JAK2/Stat5a pathway can facilitate the survival of the mammary epithelium and can impact the progression of TGFalpha-mandated mammary tumorigenesis. Together these experiments indicate that TGFalpha and the EGFR signaling pathway are potentially amenable to therapies for treatment of human breast disease.

Topics: Animals; Breast Neoplasms; Cell Division; Disease Models, Animal; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Pregnancy; Signal Transduction; Transforming Growth Factor alpha

The growth factor transforming growth factor alpha (TGFalpha) and the nuclear transcription factor c-myc often are overexpressed by human breast cancer cells. To produce models of breast disease with these etiologies, mice were generated that carried TGF-alpha- or c-myc-encoding transgenes. Transgene targeting employed the whey acidic protein (WAP) gene promoter, which is expressed in pregnant and lactating mammary epithelial cells. Non-virgin WAP-TGFalpha transgenic mice displayed accelerated mammary development during pregnancy, delayed post-parturient mammary involution, a progressive increase in the number of hyperplastic alveolar nodules (HANs), and development of mammary carcinoma with a mean latency of 9 months. Non-virgin WAP-c-myc transgenic mice displayed accelerated mammary gland development during pregnancy and development of mammary carcinomas with a latency of 8 months. Bitransgenic mice carrying both WAP-TGFalpha and WAP-c-myc displayed a dramatic acceleration of tumor development. These models identify the overexpression of TGFalpha or c-myc as etiological factors in the development of mammary neoplasia and demonstrate the increased severity of disease when both molecular alterations are present in the same cell.

Topics: Animals; Breast Neoplasms; Female; Genes, myc; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Pregnancy; Transforming Growth Factor alpha

Topics: Animals; Breast Neoplasms; Drug Resistance, Neoplasm; Estrogen Antagonists; Estrogens; Female; Humans; Osteoporosis, Postmenopausal; Piperidines; Raloxifene Hydrochloride; Tamoxifen; Transforming Growth Factor alpha; Uterine Neoplasms

Based on the scientific literature, there are several molecular markers which might be used for the prognosis of breast cancer. Possible molecular prognostic markers are: BRCA-1, BRCA-2, p53, erbB oncogenes, loss of heterozygosity (LOH), chromosomal aberrations, microsatellite instability, transforming growth factor alpha (TGFalpha), and the multiple drug resistance (MDR) gene. In this chapter, we discuss the possible role of these prognostic markers in breast cancer.

Topics: BRCA2 Protein; Breast Neoplasms; Chromosome Aberrations; Drug Resistance, Multiple; Female; Genes, BRCA1; Genes, erbB-2; Genes, p53; Humans; Loss of Heterozygosity; Microsatellite Repeats; Neoplasm Proteins; Prognosis; Transcription Factors; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGFalpha)4 and/or the EGF receptor (EGFR) are frequently overexpressed by human and rodent breast tumors, as well as tumor-derived cell lines. Additionally, various observations suggest a role for TGFalpha and the EGFR signaling system in normal mouse mammary gland development. Recently, several laboratories have established TGFalpha transgenic mice with which to study the role of this growth factor in normal and neoplastic mammary biology. Examination of these mice revealed that overexpression of TGFalpha has profound consequences for this tissue. Most strikingly, transgenic mice expressing TGFalpha under the control of tissue-specific and nonspecific promoters stochastically developed focal mammary tumors with an incidence and latency that was markedly affected by pregnancy. Most TGFalpha-induced tumors were well-differentiated adenomas/adenocarcinomas, although some were undifferentiated and locally invasive. Distant metastases were only occasionally observed. Administration of the genotoxic carcinogen, 7,12-dimethylbenzanthracene (DMBA), dramatically accelerated mammary tumorigenesis induced by the TGFalpha transgene, raising the possibility that TGFalpha acts as a promoter in this tissue. Mice harboring dual transgenes encoding TGFalpha and either wild-type ERBB2 or c-myc displayed markedly accelerated tumorigenesis compared to mice carrying any of the single transgenes alone, indicative of potent cooperativity. Moreover, tumorigenesis in the bitransgenic mice was less dependent on pregnancy, and tumors were generally more malignant in appearance. Finally, TGFalpha also affected mammary gland dynamics. TGFalpha transgenic mice consistently displayed precocious alveolar development, were variably impaired with respect to lactation, and showed markedly reduced postlactional involution. As a result, the glands of multiparous females accumulated hyperplastic lesions that generally resembled milk-producing alveoli. Limited data support the hypothesis that these lesions were precursors to TGFalpha-induced tumors. In summary, these various findings underscore the potential importance of TGFalpha for cellular differentiation and transformation in the mammary gland. They also establish TGFalpha transgenic mice as a powerful model with which to study the role of EGFR signaling molecules in this dynamic tissue.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Breast Neoplasms; Cell Transformation, Neoplastic; ErbB Receptors; Female; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Pregnancy; Transforming Growth Factor alpha

Normal mammary gland development is the result of complex interactions between a number of hormones and growth factors. Normal and malignant human mammary epithelial cells are able to synthesize and to respond to various different, locally acting growth factors and growth inhibitors. Among these, the EGF-related peptides play an important role in regulating the proliferation and differentiation of human mammary epithelial cells. EGF4 and TGF4 are able to stimulate the lobulo-alveolar development of the mammary gland in vivo as well they are involved in the pathogenesis of human breast cancer. Experimental evidence suggests that estrogen-induced proliferation of breast carcinoma cells is mediated in part by EGF-related growth factors. It has also been demonstrated that activation of certain cellular protooncogenes such as c-Ha-ras in human mammary epithelial cells results in cellular transformation and in an increased production of several EGF-related growth factors such as TGFalpha and amphiregulin. Coexpression of both EGF-related peptides and their own receptors frequently occurs in human breast carcinomas and in human breast cancer cell lines, suggesting that an autocrine pathway of uncontrolled cell growth sustains neoplastic transformation.

Topics: Animals; Breast; Breast Neoplasms; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Mammary Glands, Animal; Transforming Growth Factor alpha

There has been increased interest in the last few years in seeking a better understanding of the local regulation of polypeptide growth factors by systemic hormones, such as sex steroids and by polypeptide hormones. Growth factors and systemic hormones play pivotal roles in hormone-regulated cancers such as breast cancer. In this review, we discuss the regulation of members of the epidermal growth factor (EGF) family by sex steroids and by regulators of the polypeptide hormone signal transduction enzyme termed protein kinase C (PKC). Regulation of the EGF family of genes will be discussed as a model system to evaluate interactions between these two important types of regulatory pathways in breast cancer.

Topics: Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Gonadal Steroid Hormones; Humans; Male; Phorbol Esters; Prostatic Neoplasms; Protein Kinases; Receptors, Estrogen; Signal Transduction; Steroids; Transforming Growth Factor alpha

Although valuable initial information can be gathered about transformation from in vitro studies, human cancer occurs in the context of a complex interaction with its environment and must ultimately be studied in living animals. Transgenic animal models have been used to study breast transformation for a number of years and have yielded valuable information on the subject. In this paper, we will summarize results from our laboratories, and others, regarding the use of transgenic mice to study breast tumorigenesis. We will also suggest future directions for the use of transgenic models to understand, and hopefully, one day to cure the disease.

Topics: Animals; Breast Neoplasms; Disease Models, Animal; Female; Forecasting; Humans; Mice; Mice, Transgenic; Oncogene Proteins, Viral; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha

Synthesis of oestrogens within breast tissues makes an important contribution to the high concentrations of oestradiol which are found in breast tumours. The activities of the enzymes involved in oestrogen synthesis, i.e. the aromatase, oestradiol dehydrogenase (E2DH) and oestrone sulphatase (E1-STS), can be stimulated by several growth factors and cytokines. As it is possible that some of these factors may be derived from cells of the immune system (macrophages and lymphocytes), the effects of basic fibroblast growth factor (bFGF) and interleukin-2 (IL-2), which are produced by these cells, on E2DH activity was examined in MCF-7 cells. Treatment of these cells with bFGF resulted in a dose-dependent increase in E2DH reductive activity whereas IL-2 was inactive at the concentration tested. To obtain further evidence that factors produced by macrophages and lymphocytes can modulate the activities of enzymes involved in oestrogen synthesis, conditioned medium was collected from these cells and found to stimulate both E1-STS and E2DH activities. In addition to understanding the control of oestrogen synthesis in breast tumours an inhibitor to block the synthesis of oestrone via the oestrone sulphatase pathway was developed. Oestrone-3-O-sulphamate (EMATE) is a potent, irreversible, inhibitor of E1-STS. A single dose of EMATE (10 mg/kg) inhibited tissue E1-STS activity in rats by more than 95% for up to 7 days, indicating that this compound may have considerable therapeutic potential for the treatment of breast cancer. Evidence is also reviewed that another steroid sulphatase, dehydroepiandrosterone sulphate sulphatase, may have a crucial role in regulating cytokine production and that this may indirectly control tumour oestrogen synthesis.

Topics: Breast Neoplasms; Cytokines; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Estradiol Dehydrogenases; Estrogens; Estrone; Fibroblast Growth Factor 2; Growth Substances; Humans; In Vitro Techniques; Insulin-Like Growth Factor I; Interleukin-2; Lymphocytes; Macrophages; Sulfatases; Transforming Growth Factor alpha; Tumor Cells, Cultured

Data obtained using long-established human breast cancer cell lines have suggested that autocrine growth factors secreted by the cells are important for their growth in vitro. Such data alone cannot definitively establish a role for autocrine growth factors in human breast cancer cell proliferation in vivo, but they create a paradigm that can be tested by analysis of data obtained with primary breast cancer cells and tissues. This review is aimed at examining experimental data obtained using fresh human breast cancer cells and tissues to determine whether the results obtained are consistent with predictions made by autocrine models of human breast cancer cell proliferation. Conceptually, for autocrine loops to be of primary importance in human breast cancer cell proliferation, breast cancer cells in vivo must 1) synthesize biologically active growth factors that are available to growth factor receptors, 2) synthesize the cognate growth factor receptors, 3) require the specific factors for proliferation, and 4) express the autocrine loop as a pathologic, rather than a physiologic, process. Since the proportion of tumors that express growth factors of a given family is consistently higher than the proportion of tumors that express the cognate receptors, it is likely that growth factor synthesis has an important, nonautocrine role in breast cancer progression as well. Data obtained with primary human breast cancer specimens indicate that growth factors synthesized by breast cancer cells have an important role in tumor development and progression but that these factors act in a true autocrine fashion only in a subset of tumors.

Topics: Breast Neoplasms; Cell Division; Disease Progression; Epidermal Growth Factor; Humans; Transforming Growth Factor alpha

Emerging evidence suggests that breast cancer may originate during early life. In particular, offspring of mothers who during pregnancy exhibited behaviors that are associated with increased incidence of breast cancer, may be at risk. These behaviors include intake of high fat diet or alcohol, or stressful life style. We have found that neonatal exposure to handling that leads to improved ability to cope with stress, reduces 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. Further, our results indicate that maternal exposure to high fat diet increases the incidence of DMBA-induced mammary tumors in female offspring. High fat diet also increases serum 17 beta-estradiol (E2) levels in pregnant animals. These results support the hypothesis that in utero concentrations of estrogens play a critical role in the vulnerability to develop breast cancer. The mechanism of estrogen action might be related to its effect on the induction of epithelial hyperplasia and altered breast differentiation. These events then increase the rate of genetic/epigenetic changes that increase the possibility of neoplastic transformation. Increased pregnancy estrogens may also lead to behavioral alterations in the offspring. This could explain the proposed association between certain behavioral patterns and increased tumorigenity. Our results in transgenic mice overexpressing transforming growth factor alpha (TGF alpha) are in accordance with this interpretation. The male TGF alpha mice exhibit elevated serum E2 levels, impaired ability to cope with stress, increased voluntary alcohol intake and high incidence of spontaneous hepatocellular tumors. These findings indicate that animal models offer a unique opportunity to investigate the role of timing of risk behaviors on breast cancer. They are also useful in the attempts to understand the mechanism of early estrogen action on mammary tumorigenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antidepressive Agents; Breast Neoplasms; Cocarcinogenesis; Diet; Dietary Fats; Estradiol; Estrogens; Ethanol; Female; Handling, Psychological; Humans; Incidence; Liver Neoplasms, Experimental; Male; Mammary Neoplasms, Experimental; Maternal-Fetal Exchange; Mice; Mice, Transgenic; Personality; Pregnancy; Pregnancy Complications; Prenatal Exposure Delayed Effects; Retrospective Studies; Risk Factors; Stress, Psychological; Transforming Growth Factor alpha

A number of different epidermal growth factor (EGF)-related peptides such as EGF, transforming growth factor alpha (TGF alpha), amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1), are coexpressed to varying degrees in both normal and malignant mammary epithelial cells. However, in general the frequency and level of expression of TGF alpha, AR, and CR-1 are higher in malignant breast epithelial cells than in normal mammary epithelium. In addition, several of these peptides such as TGF alpha and AR can function as autocrine and/or juxtacrine growth factors in mammary epithelial cells, and their expression is stringently regulated by mammotrophic hormones such as estrogens, activated proto-oncogenes that have been implicated in the pathogenesis of breast cancer, and other growth factors. The redundancy of expression that is observed for a number of these structurally related peptides in both normal and malignant mammary epithelial cells suggests that some of these peptides may be involved in regulating other aspects of cellular behavior such as differentiation in addition to proliferation.

Topics: Amino Acid Sequence; Amphiregulin; Biomarkers, Tumor; Breast; Breast Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Neuregulins; Protein Structure, Secondary; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha

Topics: Antibodies, Monoclonal; Breast Neoplasms; Female; Growth Substances; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Prognosis; Receptors, Cell Surface; Receptors, Somatomedin; Transforming Growth Factor alpha; Transforming Growth Factor beta

Breast cancer patients who acquire tamoxifen resistance may respond to second-line hormonal therapy or progress to true endocrine resistance. The biological basis for these processes are poorly understood. Following successful therapy with tamoxifen there is little evidence at relapse for change in either the host endocrine environment or drug metabolic profile to account for the development of acquired resistance. Many tamoxifen resistant tumours still retain a structurally and functionally normal oestrogen receptor (ER) and yet will grow independent of oestrogen. The oestrogen-regulated molecular events which normally govern the growth of hormone-sensitive breast cancer involve a complex autocrine and paracrine interaction between several peptide growth factors (including TGF alpha, IGF-1 and TGF beta), their receptors and signal transduction pathways. Evidence now exists that constitutive activity of many of these mediators of the mitogenic signal can bypass the cell's dependence on oestrogen and provide a mechanism for hormone-independent growth. Research into these molecular mechanisms may result in a better understanding of how to overcome the clinical problem of tamoxifen resistance.

Topics: Breast Neoplasms; Drug Resistance; ErbB Receptors; Female; Humans; Insulin-Like Growth Factor I; Oncogenes; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta

Topics: Animals; Breast Neoplasms; Female; Gene Expression; Genes, ras; Humans; Mammary Neoplasms, Experimental; Rats; Transforming Growth Factor alpha

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Division; Cell Line; Estrogen Antagonists; Estrogens; Female; Growth Substances; Humans; Transforming Growth Factor alpha; Tumor Cells, Cultured

In this review I summarize the experimental data in favor of the notion that control of epidermal growth factor (EGF) receptor (R) and/or c-erbB-2 protooncogene expression by specific autocrine growth factors and certain classical endocrine hormones serves as a transducer of extracellular signals that ultimately lead to growth responses in breast carcinoma cells. I summarize some new results on the role of epidermal growth factor (EGF), transforming growth factor (TGF) alpha, and TGF beta in the control of EGF-R protooncogene expression in human breast carcinoma cells. Furthermore, the data embracing the hypothesis that the growth actions of hormone receptors that are homologous to the v-erbA oncogene (estrogens, progesterone, thyroid hormones, retinoic acid, and vitamin D) are mediated, in part, by modulating EGF-R and/or c-erbB-2 protooncogene transcription are reviewed. Finally, I develop the theme that cooperation of certain c-erb-A-related, c-erbB-2 and/or EGF-R gene products contribute to the uncontrolled growth of human mammary carcinoma cells. From the evidence reviewed, one can infer that elucidation of the molecular control of EGF-R/c-erbB-2 gene expression by c-erbA-related gene products may lead to both a better understanding of breast carcinogenesis and a new therapeutic approach directed at controlling the transcriptional responses of EGF-R/c-erbB-2 genes.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Division; Cholecalciferol; Epidermal Growth Factor; ErbB Receptors; Estrogens; Gene Expression Regulation, Neoplastic; Humans; Hydrocortisone; Progesterone; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tretinoin; Triiodothyronine

A wealth of recently derived information has strongly implicated the protooncogene erbB-2 (also termed HER-2 or neu) and its protein product as critically involved in human breast cancer as well as other important epithelial malignancies. Because of its substantial homology with the EGF receptor, erbB-2 has long been assumed to encode a growth factor receptor, although until recently definitive identification of ligand(s) has remained elusive. Both in a mutated form and when overexpressed in a non-mutated form, erbB-2 is capable of inducing malignant transformation of many target cells including immortalized breast epithelium. We have recently identified and purified a 30 kDa size growth factor secreted by some human breast cancer cells. The factor is related to transforming growth factor-alpha (TGF-alpha) in its ability to bind to the epidermal growth factor (EGF) receptor (though with about 10 fold lower apparent affinity), its ability to phosphorylate EGF receptor and its ability to induce cloning of normal rat kidney (NRK) fibroblasts. However, it is distinct from TGF-alpha as determined by peptide mapping and its ability to induce activation of erbB-2. TGF-alpha and EGF are incapable of directly inducing phosphorylation of erbB-2. However, in a variety of spontaneously occurring tumor cells as well as cell lines transfected with erbB-2 prepared in our laboratory, 30 kDa glycoprotein (gp30) is capable of inducing direct phosphorylation of erbB-2. The ability to induce phosphorylation of erbB-2 is not inhibited by an anti-EGF receptor blocking antibody. In cells that overexpress erbB-2, the gp30 low concentrations is stimulatory of both standard mitogenesis assays and in clonogenic assays. At higher concentrations, the ligand is growth inhibitory in both of these assays. Because of the ability of gp30 to compete for binding with antibodies directed against erbB-2 which inhibit growth, the gp30 ligand is capable of reversing antibody-induced inhibition of growth. In addition, the gp30 ligand can overcome inhibitory effects seen in cells which overexpress erbB-2 which are induced by extracellular domain fragments of the erbB-2 receptor, once again suggesting a specific pathway of action for the gp30 ligand mediated for interaction with erbB-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Breast Neoplasms; Cell Division; CHO Cells; Cricetinae; ErbB Receptors; Female; Growth Substances; Humans; Phosphorylation; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF alpha) is one growth factor that has been circumstantially implicated in regulating the autocrine growth of breast cancer cells. Expression of TGF alpha can be modulated by activated cellular protooncogenes such as ras and by estrogens. For example, the epidermal growth factor (EGF)-responsive normal NOG-8 mouse and human MCF-10A mammary epithelial cell lines can be transformed with either a point-mutated c-Ha-ras protooncogene or with a normal or point-mutated c-neu (erbB-2) protooncogene. In ras transformed NOG-8 and MCF-10A cells but not in neu transformed cells there is a loss in or an attenuated response to the mitogenic effects of EGF. This response may be due in part to an enhanced production of endogenous TGF alpha that is coordinately and temporally linked to the expression of the activated ras gene and to the acquisition of transformation-associated properties in these cells. TGF alpha mRNA and TGF alpha protein can also be detected in approximately 50-70% of primary human breast tumors. In addition, approximately 2- to 3-fold higher levels of biologically active and immunoreactive TGF alpha can also be detected in the pleural effusions from breast cancer patients as compared with the TGF alpha levels in the serous effusions of noncancer patients. Over-expression of a full-length TGF alpha cDNA in NOG-8 and MCF-10A cells is capable of transforming these cells. Finally, expression of TGF alpha mRNA and production of biologically active TGF alpha protein is also found in normal rodent and human mammary epithelial cells.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Cells, Cultured; Gene Expression; Humans; Pleural Effusion; Proto-Oncogenes; Transforming Growth Factor alpha

While steroid hormones act as endocrine effectors of growth and development of normal breast and of carcinogenesis and progression of malignant breast, recent evidence suggests that local hormonal effectors also exist. These are the growth regulatory growth factors. This article summarizes current status of our understanding of structure and function of growth factors secreted by the normal and malignant mammary epithelium. While growth inhibitory factors and their receptors generally suppress development of the transformed phenotype and promote differentiation, growth stimulatory factors and their receptors may be necessary for both normal proliferation and early stages of malignant progression of breast cancer. Overexpression of two receptors, c-erbB-2 and EGF receptor, have also been associated with poor prognosis in the clinical disease.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Transformation, Neoplastic; Epidermal Growth Factor; Growth Substances; Humans; Oncogenes; Transforming Growth Factor alpha

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Estrogen Antagonists; Estrogens; Female; Genes, ras; Humans; Neoplasms, Hormone-Dependent; Oncogenes; Phenotype; Platelet-Derived Growth Factor; Receptors, Estrogen; Somatomedins; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Cells, Cultured

Topics: Animals; Breast Neoplasms; Epithelial Cells; Epithelium; Humans; In Vitro Techniques; Mammary Neoplasms, Experimental; Molecular Weight; Oncogenes; Rats; Transforming Growth Factor alpha; Transforming Growth Factors

Bone metastasis (BM) is the advanced complication of breast cancer, while bone marrow-derived mesenchymal stem cells (BMSCs) in the microenvironment unclearly contribute to cancer metastasis. This study investigated potential roles of transforming growth factor- (TGF-)

Topics: Aged; Bone Marrow Cells; Bone Neoplasms; Breast Neoplasms; Cytokines; Female; Humans; Mesenchymal Stem Cells; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Transforming Growth Factor alpha

WWOX is a tumor-suppressive steroid dehydrogenase, which relationship with hormone receptors was shown both in animal models and breast cancer patients. Herein, through nAnT-iCAGE high-throughput gene expression profiling, we studied the interplay of estrogen receptors and the WWOX in breast cancer cell lines (MCF7, T47D, MDA-MB-231, BT20) under estrogen stimulation and either introduction of the WWOX gene by retroviral transfection (MDA-MB-231, T47D) or silenced with shRNA (MCF7, BT20). Additionally, we evaluated the consequent biological characteristics by proliferation, apoptosis, invasion, and adhesion assays. TGFα-EGFR signaling was found to be significantly affected in all examined breast cancer cell lines in response to estrogen and strongly associated with the level of WWOX expression, especially in ER-positive MCF7 cells. Under the influence of 17β-estradiol presence, biological characteristics of the cell lines were also delineated. The study revealed modulation of adhesion, invasion, and apoptosis. The obtained results point at a complex role of the WWOX gene in the carcinogenesis of the breast tissue, which seems to be closely related to the presence of estrogen α and/or β receptors.

Topics: Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; ErbB Receptors; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Receptors, Estrogen; Transforming Growth Factor alpha; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase

The human rhomboid-5 homolog-1 (RHBDF1) is a multi-transmembrane protein present mainly on the endoplasmic reticulum. RHBDF1 has been implicated in the activation of epidermal growth factor receptor (EGFR)-derived cell growth signals and other activities critical to cellular responses to stressful conditions, but details of this activation mechanism are unclear. Here, we report a RHBDF1 mRNA transcript alternative splicing variant X6 (RHBDF1 X6 or RHX6) that antagonizes RHBDF1 activities. We found that while the RHBDF1 gene is marginally expressed in breast tumor-adjacent normal tissues, it is markedly elevated in the tumor tissues. In sharp contrast, the RHX6 mRNA represents the primary RHBDF1 variant in normal breast epithelial cells and tumor-adjacent normal tissues but is diminished in breast cancer cells and tumors. We demonstrate that, functionally, RHX6 acts as an inhibitor of RHBDF1 activities. We show that artificially overexpressing RHX6 in breast cancer cells leads to retarded proliferation, migration, and decreased production of epithelial-mesenchymal transition-related adhesion molecules. Mechanically, RHX6 is able to inhibit the maturation of TACE, a protease that processes pro-TGFα, a pro-ligand of EGFR, and to prevent intracellular transportation of pro-TGFα to the cell surface. Additionally, we show that the production of RHX6 is under the control of the alternative splicing regulator RNA binding motif protein-4 (RBM4). Our findings suggest that differential splicing of the RHBDF1 gene transcript may have a regulatory role in the development of epithelial cell cancers.

Topics: Alternative Splicing; Breast Neoplasms; Cell Line, Tumor; ErbB Receptors; Female; Humans; Membrane Proteins; RNA-Binding Proteins; RNA, Messenger; Transforming Growth Factor alpha

Genes associated with growth factors were previously analyzed in a radiation- and estrogen-induced experimental breast cancer model. Such in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line, MCF-10F, to low doses of high linear energy transfer (LET) α particle radiation (150 keV/μm) and subsequent growth in the presence or absence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles. In the present report, the profiling of differentially expressed genes associated with growth factors was analyzed in their relationship with clinical parameters. Thus, the results indicated that Fibroblast growth factor2 gene expression levels were higher in cells transformed by radiation or in the presence of ionizing radiation; whereas the fibroblast growth factor-binding protein 1gene expression was higher in the tumor cell line derived from this model. Such expressions were coincident with higher values in normal than malignant tissues and with estrogen receptor (ER) negative samples for both gene types. The results also showed that transforming growth factor alpha gene expression was higher in the tumor cell line than the tumorigenic A5 and the transformed A3 cell line, whereas the transforming growth factor beta receptor 3 gene expression was higher in A3 and A5 than in Tumor2 cell lines and the untreated controls and the E cell lines. Such gene expression was accompanied by results indicating negative and positive receptors for transforming growth factor alpha and the transforming growth factor beta receptor 3, respectively. Such expressions were low in malignant tissues when compared with benign ones. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expressions were found to be present in all BRCA patients that are BRCA-Basal, BRCA-LumA, and BRCA-LumB, except in BRCA-Her2 patients. The results also indicated that the insulin growth factor receptor gene expression was higher in the tumor cell line Tumor2 than in Alpha3 cells transformed by ionizing radiation only; then, the insulin growth factor receptor was higher in the A5 than E cell line. The insulin growth factor receptor gene expression was higher in b

Topics: Breast Neoplasms; Cell Line, Tumor; Estrogens; Female; Fibroblast Growth Factors; Humans; Insulin; Insulin, Regular, Human; Radiation, Ionizing; Receptors, Transforming Growth Factor beta; Transforming Growth Factor alpha

Emerging evidence indicates that microRNAs (miRNAs) play a critical role in breast cancer development. We recently reported that a higher expression of miR-374b in tumor tissues was associated with a better disease-free survival of triple-negative breast cancer (TNBC). However, the functional significance and molecular mechanisms underlying the role of miR-374b in breast cancer are largely unknown. In this current study, we evaluated the biological functions and potential mechanisms of miR-374b in both TNBC and non-TNBC. We found that miR-374b was significantly downregulated in breast cancer tissues, compared to adjacent tissues. MiR-374b levels were also lower in breast cancer cell lines, as compared to breast epithelial cells. In vitro and in vivo studies demonstrated that miR-374b modulates the malignant behavior of breast cancer cells, such as cell proliferation in 2D and 3D, cell invasion ability, colony-forming ability and tumor growth in mice. By using bioinformatics tools, we predicted that miR-374b plays a role in breast cancer cells through negatively regulating cyclin D1 (CCND1) and transforming growth factor alpha (TGFA). We further confirmed that CCND1 and TGFA contribute to the malignant behavior of breast cancer cells in vitro and in vivo. Our rescue experiments showed that overexpressing CCND1 or TGFA reverses the phenotypes caused by miR-374b overexpression. Taken together, our studies suggest that miR-374b modulates malignant behavior of breast cancer cells by negatively regulating CCND1 and TGFA genes. The newly identified miR-374b-mediated CCND1 and TGFA gene silencing may facilitate a better understanding of the molecular mechanisms of breast cancer progression.

Topics: Animals; Breast Neoplasms; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; MCF-7 Cells; Mice; MicroRNAs; Neoplasm Invasiveness; Transforming Growth Factor alpha

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; ErbB Receptors; Female; Humans; Multidrug Resistance-Associated Proteins; Protein Binding; Recombinant Proteins; Transforming Growth Factor alpha

Stemness and epithelial-mesenchymal transition (EMT) are two fundamental characteristics of metastasis that are controlled by diverse regulatory factors, including transcription factors. Compared with other subtypes of breast cancer, basal-type or triple-negative breast cancer (TNBC) has high frequencies of tumor relapse. However, the role of alpha-globin transcription factor CP2 (TFCP2) has not been reported as an oncogenic driver in those breast cancers. Here, we show that TFCP2 is a potent factor essential for EMT, stemness, and metastasis in breast cancer. TFCP2 directly bound promoters of EGF and TGFα to regulate their expression and stimulate autocrine signaling via EGFR. These findings indicate that TFCP2 is a new antimetastatic target and reveal a novel regulatory mechanism in which a positive feedback loop comprising EGF/TGFα and AKT can control malignant breast cancer progression. SIGNIFICANCE: TFCP2 is a new antimetastatic target that controls TNBC progression via a positive feedback loop between EGF/TGFα and the AKT signaling axis.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Disease Progression; DNA-Binding Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Feedback, Physiological; Female; Gene Knockdown Techniques; Heterografts; Humans; MCF-7 Cells; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Transcription Factors; Transforming Growth Factor alpha; Up-Regulation

Epidermal growth factor receptor (EGFR) signalling is critical in epithelial cancer development. Human rhomboid family-1 (RHBDF1) facilitates the secretion of TGFα, an EGFR ligand, in breast cancer; however, the underlying mechanism remains unclear. We evaluated the role for RHBDF1 in clathrin-coated vesicle (CCV)-dependent pro-TGFα membrane trafficking in breast cancer cells upon stimulation by G-protein coupled receptor (GPCR) agonists.. RHBDF1 was silenced in various breast cancer cells using shRNA. TGFα levels, subcellular localization, and secretion were evaluated using ELISA, immunofluorescent staining, and coimmunoprecipitation. Phosphorylation and expression of relevant proteins were measured by western blotting. RHBDF1-dependent cell viability and invasion were measured.. RHBDF1 mediates GPCR agonist-induced EGFR phosphorylation by promoting TGFα secretion in various types of breast cancer cells. RHBDF1 not only mediates ADAM17-dependent shedding of TGFα, but is essential in membrane trafficking of pro-TGFα. RHBDF1 silencing results in blocking of clathrin uncoating from CCV, a crucial step for the plasma membrane release of pro-TGFα. Interaction of RHBDF1 with auxilin-2, a CCV protein, determines the recruitment of HSC70 to CCV to facilitate clathrin uncoating. RHBDF1 function is required for the proliferation and mobility of breast cancer cells upon stimulation by Sphingosine 1 Phosphate (S1P), a GPCR agonist. We demonstrate a significant correlation between RHBDF1 overexpression and EGFR activation in breast cancer tissues.. RHBDF1 is an indispensable component of the protein trafficking machinery involved in GPCR-mediated EGFR transactivation, and is an attractive therapeutic target for cancer. FUND: National Natural Science Foundation of China (81,672,740 to ZSZ, 81,272,356 and 81,330,029 to LYL).

Topics: ADAM17 Protein; Auxilins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Clathrin-Coated Vesicles; Disease Progression; ErbB Receptors; Female; HSC70 Heat-Shock Proteins; Humans; Ligands; Membrane Proteins; Models, Biological; Protein Binding; Protein Transport; RNA, Small Interfering; Transforming Growth Factor alpha

Adropin is a peptide hormone that has been implicated in insulin resistance and as a potential regulator of growth. The aim of this study is to determine the effect of calorie restriction on circulating levels of adropin in the MMTV-TGFα breast cancer mouse model and investigate the effects of adropin peptide on the viability of MCF-7 and MDA-231 breast cancer cells in culture. Ten-week-old mice were assigned to either ad libitum-fed (AL), chronic calorie-restricted, or intermittent calorie-restricted groups. Concentrations of serum adropin were measured using an enzyme-linked immunosorbent assay. Results showed an inverse correlation between serum adropin levels and mouse age that was attenuated by calorie restriction. In the AL group the level of adropin was significantly lower at week 50 compared to levels at week 10. However, among the calorie-restricted groups, serum levels of adropin remained high at week 50. The cell-line-specific effects were observed after treatment of cancer cell lines with a series of adropin concentrations (5, 10, 25, 50 ng/mL). Flow cytometry analysis showed that MCF-7 cells entered the early phase of apoptosis after treatment with 50 ng/mL for 24 h. Adropin may be involved in the protective effects that calorie restriction has on breast cancer risk.

Topics: Age Factors; Animals; Apoptosis; Blood Proteins; Body Weight; Breast Neoplasms; Caloric Restriction; Cell Line, Tumor; Cell Survival; Female; Humans; Intercellular Signaling Peptides and Proteins; Male; MCF-7 Cells; Mice, Inbred C57BL; Mice, Transgenic; Proteins; Transforming Growth Factor alpha

Breast cancer remains the second most common disease worldwide. Radiotherapy, alone or in combination with chemotherapy, is widely used after surgery as a treatment for cancer with proven therapeutic efficacy manifested by reduced incidence of loco-regional and distant recurrences. However, clinical evidence indicates that relapses occurring after radiotherapy are associated with increased metastatic potential and poor prognosis in the breast. Among the anticarcinogenic and antiproliferative agents, curcumin is a well-known major dietary natural yellow pigment derived from the rhizome of the herb Curcuma longa (Zingiberaceae). The aim of the present study was to analyze the differential expression of metastatic genes in radiation- and estrogen-induced breast cancer cell model and the effect of curcumin on such metastatic genes in breast carcinogenesis. Expression levels of TGF-α and TGFβ1 genes were upregulated in MCF-10F and downregulated in Tumor2 cell lines treated with curcumin. Expression levels of other genes such as caspase 9 and collagen 4 A2 were upregulated in both MCF-10F and Tumor2-treated cell lines. Integrin α5 and cathepsin B and D decreased its expression in Tumor2, whereas E-Cadherin, c-myc and CD44 expressions were only increased in MCF-10F. It can be concluded that metastatic genes can be affected by curcumin in cancer progression and such substance can be used in breast cancer patients with advanced disease without side-effects commonly observed with therapeutic drugs.

Topics: Antioxidants; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Curcuma; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Prognosis; Transforming Growth Factor alpha; Transforming Growth Factor beta1

Recent research has attempted to direct superantigens towards tumors by means of tumor-targeted superantigen (TTS) strategy. In this study, we explored the antitumor property of TTS by fusing the third loop of transforming growth factor α (TGFαL3) to staphylococcal enterotoxin type B (SEB) and investigated the possibility of the therapeutic application of TGFαL3-SEB as a novel antitumor candidate in mice bearing breast cancer. Treatment was performed through intratumoral and intravenous injection of TGFαL3-SEB. Tumor size/volume, long-term survival, and cytokine secretion were assessed. In addition, the toxicity of each treatment on liver and kidneys was examined. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral TGFaL3-SEB (p < 0.05). Surprisingly, 5 out of the 14 mice were cleared from the tumor thoroughly in 10-25 days after intratumoral administration of TGFaL3-SEB. Quantification of cytokines clearly showed that the mice receiving intratumoral SEB significantly secreted higher interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the other groups (p < 0.05). The antitumor effect was followed by inhibition of cell proliferation (Ki-67) and micro vascularization (CD31). The highest and lowest levels of tumor necrosis were observed in the intratumoral administration of TGFαL3-SEB (85 %) and PBS (14 %), respectively. Intratumoral injection of TGFαL3-SEB increased the lifespan of the mice so 37.5 % of them could survive for more than 6 months (p < 0.05). Overall, our findings indicated that intratumoral administration of TGFαL3-SEB effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis without systemic toxicity.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Enterotoxins; Female; Humans; Immunotherapy; Interferon-gamma; Mice; Neovascularization, Pathologic; Oncogene Proteins, Fusion; Superantigens; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha; Xenograft Model Antitumor Assays

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Epithelium; Humans; Mice, SCID; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta

Elucidating the mechanisms that underlie metastasis is of paramount importance to understanding tumor progression and to the development of novel therapeutics. Epithelial to Mesenchymal Transition (EMT) plays a vital role in tumor cell dissemination and is regulated by a core cassette of transcription factors. Despite recent advances, the molecular pathways that regulate the EMT program have not yet been fully delineated. We show that Siah ubiquitin ligases regulate Zeb1 protein, a key EMT transcription factor. The induction of EMT in breast cancer cells leads to the down-regulation of Siah, while the loss of Siah induces a mesenchymal phenotype, concurrent with an up-regulation of Zeb1. Overexpression of Siah in vitro mediates Zeb1 degradation, which can be blocked with a Siah peptide inhibitor. Thus, this work demonstrates that Siah is a novel regulator of EMT. This work is the first to identify a mechanism of post-translational regulation of the key Epithelial to Mesenchymal Transition transcription factor Zeb1.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; MCF-7 Cells; Mice; Microscopy, Fluorescence; Models, Genetic; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Transcription Factors; Transforming Growth Factor alpha; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Zinc Finger E-box-Binding Homeobox 1

Fibronectin (FN), an extracellular matrix ligand, plays a pivotal role in cell adhesion, migration, and oncogenic transformation. Aberrant FN expression is associated with poor prognoses in various types of cancer, including breast cancer. In the current study, we investigated the relationship between FN induction and HER2 expression in breast cancer cells. Our results showed that the level of FN expression increased in response to HER family ligands, EGF and TGF-α in a time- and dose-dependent manner. On the other hand, EGF-induced FN expression decreased in response to trastuzumab, which is a HER2-targeted monoclonal antibody. However, EGF-induced FN expression was not affected by trastuzumab in JIMT-1 breast cancer cells, which are trastuzumab insensitive cells. Next, we introduced the HER2 gene into MDA-MB231 cells to verify the relationship between FN and HER2. The level of FN expression significantly increased in HER2-overexpressed MDA-MB231 cells. In contrast, the induction of FN by HER2 was significantly decreased in response to trastuzumab treatment. In addition, the induction of FN by HER2 was down-regulated by the MEK 1/2 specific inhibitor, U0126. Using conditioned culture media of vec- and HER2-overexpressed MDA-MB231 cells, we observed the cell morphology, adhesion, and invasion of MDA-MB231 cells. Interestingly, in conditioned culture media of HER2-overexpressed MDA-MB231 cells, the cell morphology was altered, and adhesion and invasion of MDA-MB231 cells significantly increased. In addition, our results showed that recombinant human FN augmented cell adhesion and invasion of MDA-MB231 cells while these inductions decreased in response to an FN inhibitor. Therefore, we demonstrated that the induction of FN by HER2 triggers cell adhesion and invasion capacities.

Topics: Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Shape; Epidermal Growth Factor; Female; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Receptor, ErbB-2; Transcriptional Activation; Transforming Growth Factor alpha

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Dinoprostone; Female; Humans; Linoleic Acid; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Transforming Growth Factor alpha

Conventional molecular profiling methods using immunochemical assays have limits in terms of multiplexity and the quantification of biomarkers in investigation of cancer cells. In this paper, we demonstrate a quantum dot (QD)-based microfluidic multiple biomarker quantification (QD-MMBQ) method that enables labeling of more than eight proteins immunochemically on cell blocks within 1 h, in a quantitative manner. An internal reference, β-actin, was used as a loading control to compensate for differences in not only the cell number but also in staining quality among specimens. Furthermore, the microfluidic blocking method exhibited less nonspecific binding of QDs than the conventional static blocking method.

Topics: Betacellulin; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Estrogen Receptor alpha; Female; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Ki-67 Antigen; Microfluidics; Quantum Dots; Receptor, ErbB-2; Receptor, ErbB-3; Receptors, Progesterone; TOR Serine-Threonine Kinases; Transforming Growth Factor alpha

Expression of the CD44 gene is upregulated in breast cancer cells and is correlated with patient survival. Aberrant CD44 expression promotes tumor progression and metastasis. In the present study, we investigated the role of zerumbone (ZER) on regulatory mechanisms of CD44 expression in breast cancer cells. Our results showed that CD44 expression was significantly increased by epidermal growth factor receptor (EGFR) ligands in SKBR3 breast cancer cells. In contrast, EGF-induced CD44 expression was decreased by a MEK1/2 inhibitor, UO126, or STAT3 inhibitor, STAT3 VI, respectively. Notably, ZER downregulated the basal level of CD44 expression in CD44+ breast cancer cells. In addition, the induction of CD44 expression by EGFR ligands, EGF or TGF-α, was markedly decreased by ZER treatment. Finally, we investigated the inhibitory mechanism of ZER on EGF-induced CD44 expression. Our results showed that EGF-induced phosphorylation of STAT3 was completely suppressed by ZER. Collectively, ZER suppressed EGF-induced CD44 expression through inhibition of the STAT3 pathway. Therefore, we suggested that ZER may act as a promising therapeutic drug for the treatment of breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Sesquiterpenes; Signal Transduction; STAT3 Transcription Factor; Transcriptional Activation; Transforming Growth Factor alpha

MT4-MMP (MMP-17) is a glycosylphosphatidyl inositol-anchored matrix metalloprotease expressed on the surface of cancer cells that promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of cancer cell proliferation through CDK4 activation and retinoblastoma protein inactivation. We also determine a functional link between MT4-MMP and the growth factor receptor EGFR. Mechanistic experiments revealed direct association of MT4-MMP and its positive effects on EGFR phosphorylation in response to TGFα and EGF in cancer cells. Notably, the effects of MT4-MMP on proliferation and EGFR activation did not rely on metalloprotease activity. Clinically, MT4-MMP and EGFR expressions were correlated in human triple-negative breast cancer specimens. Altogether, our results identify MT4-MMP as a positive modifier of EGFR outside-in signaling that acts to cooperatively drive cancer cell proliferation.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Chlorocebus aethiops; COS Cells; Cyclin-Dependent Kinase 4; Epidermal Growth Factor; ErbB Receptors; Female; HeLa Cells; Humans; Matrix Metalloproteinases, Membrane-Associated; Mice; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor alpha; Triple Negative Breast Neoplasms

We demonstrate here increased expression of ADAM17 protein in cancer-associated fibroblasts (CAFs) extracted from human breast carcinomas compared with donor-matched normal fibroblasts, and TGF-α secretion positively correlates with ADAM17 expression in these cells. In SK-BR-3 cells co-cultured with CAFs, CAF-secreted TGF-α promotes cell proliferation by activation of EGFR, Akt, and ERK, but it does not promote cell migration. Furthermore, anti-TGF-α neutralizing antibodies antagonize the CAF-dependent increase in proliferation and activation of EGFR, Akt and ERK. Thus, pharmacologic inhibition of ADAM17 and TGF-α may have therapeutic potential for the treatment of breast cancer when fibroblast-directed therapy is considered.

Topics: ADAM Proteins; ADAM17 Protein; Breast; Breast Neoplasms; Cell Movement; Cell Proliferation; Chemotaxis; Disease Progression; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Transforming Growth Factor alpha

The epidermal growth factor receptor (EGFR) is frequently expressed in triple-negative breast cancer (TNBC) and is a marker of poor prognosis in this patient population. Because activating mutations in this kinase are very rare events in breast cancer, we screened breast tumor gene expression profiles to examine the distribution of EGFR ligand expression. Of the six known EGFR ligands, transforming growth factor alpha (TGFα) was expressed more highly in triple-negative breast tumors than in tumors of other subtypes. TGFα is synthesized as a transmembrane precursor requiring tumor necrosis factor alpha converting enzyme (TACE)/ADAM17-dependent proteolytic release to activate its receptor. In our study, we show that an inhibitor of this proteolytic release blocks invasion, migration and colony formation by several TNBC cell lines. Each of the effects of the drug was reversed upon expression of a soluble TGFα mutant that does not require TACE activity, implicating this growth factor as a key metalloproteinase substrate for these phenotypes. Together, these data demonstrate that TACE-dependent TGFα shedding is a key process driving EGFR activation and subsequent proliferation and invasion in TNBC cell lines.

Topics: ADAM Proteins; ADAM17 Protein; Breast Neoplasms; Cell Line, Tumor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neoplasm Invasiveness; Neoplasms; Phosphorylation; Signal Transduction; Transforming Growth Factor alpha

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.

Topics: Amphiregulin; Animals; Autocrine Communication; Breast Neoplasms; Cell Line, Tumor; Disease Progression; EGF Family of Proteins; ErbB Receptors; Female; Gene Knockdown Techniques; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Macrophages; Mice; Mice, Nude; Neoplasms, Basal Cell; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha

Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors.. Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs.. In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents.. It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.

Topics: ADAM Proteins; ADAM17 Protein; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; ErbB Receptors; Female; Humans; Molecular Targeted Therapy; Phosphorylation; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha

A hallmark of human cancer is heterogeneity, reflecting the complex series of changes resulting in the activation of oncogenes coupled with inactivation of tumor suppressor genes. Breast cancer is no exception and indeed, many studies have revealed considerable complexity and heterogeneity in the population of primary breast tumors and substantial changes in a recurrent breast tumor that has acquired metastatic properties and drug resistance. We have made use of a Myc-inducible transgenic mouse model of breast cancer in which elimination of Myc activity following tumor development initially leads to a regression of a subset of tumors generally followed by de novo Myc-independent growth. We have observed that tumors that grow independent of Myc expression have gene profiles that are distinct from the primary tumors with characteristics indicative of an epithelial-mesenchymal transition (EMT) phenotype. Phenotypic analyses of Myc-independent tumors confirm the acquisition of an EMT phenotype suggested to be associated with invasive and migratory properties in human cancer cells. Further genomic analyses reveal mouse mammary tumors growing independent of myc have a higher probability of exhibiting a gene signature similar to that observed for human 'tumor-initiating' cells. Collectively, the data reveal genetic alterations that underlie tumor progression and an escape from Myc-dependent growth in a transgenic mouse model that can provide insights to what occurs in human cancers as they acquire drug resistance and metastatic properties.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Neoplastic Stem Cells; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha; Transforming Growth Factor beta

To investigate the relation between neutrophil elastase (NE) and proliferation of breast cancer cells and whether the NE inhibitor sivelestat could both contribute and be applied to therapy for anti-epithelial growth factor receptor 2 (HER2)-positive breast cancers.. The proliferation or inhibition of breast cancer cell line SKBR-3 by each agent was evaluated by methylthiazole tetrazolium (MTT) assay. Signal transduction and expression of signaling molecules were evaluated by Western blot analysis.. The auto tumor progression mechanism initiated by NE through tumor growth factor-α (TGF-α) was present in breast cancer cells, and this mechanism was intensively suppressed by sivelestat. The effect of trastuzumab was suppressed, and trastuzumab-induced HER2 down-regulation was impaired by TGF-α. TGF-α not only promoted cell proliferation as a ligand but also enhanced resistance to trastuzumab by impairing HER2 down-regulation. Furthermore, combined use of trastuzumab and sivelestat suppressed cell proliferation more intensively than either drug alone and did not provoke impairment by TGF-α of HER2-induced down-regulation.. Combinatorial use of sivelestat and trastuzumab might be a novel therapeutic strategy for HER2-positive breast cancer.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Glycine; Humans; Leukocyte Elastase; Receptor, ErbB-2; Serine Proteinase Inhibitors; Signal Transduction; Sulfonamides; Transforming Growth Factor alpha; Trastuzumab

Here we describe gross and microscopic sweat gland tumors found in a transgenic mouse model of breast cancer, which had transforming growth factor α under the control of mouse mammary tumor virus promoter (MMTV-TGFα). Initially, 20% of the mice in the colony were affected. Cystic lesions formed on the phalanges, palmar surfaces of the metacarpals, and plantar surfaces of the metatarsals. The lesions were multifocal and nonulcerated with straw-colored fluid, ranging in size from 1 to 30 mm at the largest dimension. The colony was monitored for 6 mo; during that time, the prevalence of lesions increased to 52% of the mice. Histologically, in most cases the cyst walls were lined by 1 or 2 layers of normal-appearing epithelial cells that resembled basal cells, indicating adenoma. However, 2 cysts from 2 different mice had papillary proliferative projections and extensive disorganized glandular structures that protruded into the cyst cavities, indicating adenocarcinoma. In these 2 cases, the neoplastic cells revealed architectural and cytologic atypia with rare mitoses. Similar findings have previously been observed in sweat gland tumors; however, multiple sweat-gland tumors have not been reported in mice.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cysts; Extremities; Female; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; Prevalence; Promoter Regions, Genetic; Sweat Gland Neoplasms; Transforming Growth Factor alpha

Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15- 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis.. Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability.. Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.. Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

Topics: Animals; Brain; Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; ErbB Receptors; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Mice; Mice, Nude; Neoplasm Metastasis; Transforming Growth Factor alpha; Transplantation, Heterologous

Lapatinib and capecitabine combination therapy is effective in trastuzumab-resistant human epidermal growth factor receptor 2 (HER2)-positive breast cancer. We investigated the biomarkers from serum of patients receiving lapatinib and capecitabine Patients received lapatinib 1,250 mg once daily and capecitabine 2,000 mg/m(2)/day, day 1-14, every 3 weeks. Serum samples were obtained before treatment initiation. Levels of transforming growth factor-α (TGF-α), epidermal growth factor (EGF), extracellular domains of EGFR and HER2 were measured by enzyme-linked immunosorbent assay. The effect of TGF-α on in vitro sensitivity of SK-BR-3 cells to lapatinib was investigated. Sixty-four patients were included. Response rate was significantly higher in patients with low serum TGF-α (≤ 3.75 pg/ml) compared to high TGF-α (>3.75 pg/ml) [61.1% (11/18) vs. 17.4% (8/46), respectively; P = 0.001]. Low serum TGF-α was independently associated with better response in multivariate analysis [adjusted odds ratio, 8.96; 95% confidence interval (CI) 2.4-34.2]. Time-to-progression tended to be shorter in patients with high serum TGF-α compared to low TGF-α [median 3.8 months (95% CI 2.3-5.4) vs. 6.5 (95% CI 6.1-6.8), respectively; P = 0.067]. We confirmed that TGF-α diminished the sensitivity of SK-BR3-cells to lapatinib in vitro. The in vitro antiproliferative effect of cetuximab in combination with lapatinib was higher than that of lapatinib alone in SK-BR3-cells exposed to TGF-α. These data suggest that TGF-α plays a role in resistance to lapatinib and capecitabine therapy among HER2-positive breast cancer.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Capecitabine; Cell Line, Tumor; Cell Proliferation; Chi-Square Distribution; Deoxycytidine; Disease-Free Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme-Linked Immunosorbent Assay; Female; Fluorouracil; Humans; Kaplan-Meier Estimate; Lapatinib; Logistic Models; Middle Aged; Quinazolines; Receptor, ErbB-2; Republic of Korea; Risk Assessment; Risk Factors; Time Factors; Transforming Growth Factor alpha; Treatment Outcome; Up-Regulation

Invasion of MDA-MB-231 breast cancer cells into three-dimensional (3-D) type I-collagen matrices depends on TGF-α. We characterized the steps of invasion mediated by TGF-α. Cell migration, as observed by videomicroscopy, was effectively stimulated by collagen, suggesting that TGF-α may specifically participate in the invasion of a 3-D collagen matrix. We assessed the role of small GTPases of the Rho family in the invasion. Cdc42 was found to be necessary for invasion but dispensable for cell migration. These results suggest that TGF-α mediates invasion into 3-D collagen matrices by initiating the formation of protrusions into collagen, likely through activation of Cdc42.

Topics: Breast Neoplasms; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Collagen Type I; Female; Humans; Signal Transduction; Transforming Growth Factor alpha

Activation of epidermal growth factor receptor (EGFR)-induced signaling pathways has been correlated with tumor progression, invasion and metastasis in a variety of cancers including breast carcinoma, but the underlying mechanism is not well understood. Matrix metalloproteinases (MMPs) have been implicated in cancer invasion and metastasis for their extracellular matrix (ECM)-proteolytic activity. However, the correlation of EGFR pathway with MMP expression in breast cancer has not been established. The aim of this study was to elucidate the interaction between EGFR ligands and their signaling pathway and MMP expression which might be closely related with breast cancer pathogenesis. We investigated the effect of EGF ligands on the MMP1 expression in SK-BR3 cell lines using RT-PCR, Western blot, ELISA and EMSA. Treatments with EGFR ligands, EGF and TGF-α enhanced MMP1 expression at the level of both transcription and translation in SK-BR3 breast cancer cells. EGF and TGF-α treatment resulted in phosphorylation of EGFR, and consequent activation of ERK1/2 pathway. Tyrosine kinase inhibitors of HER family, erlotinib, lapatinib and canertinib suppressed EGF-ligands mediated MMP1 overexpression. The specific MEK inhibitor, U0126, significantly blocks EGF and TGF-α-mediated ERK1/2 activation and subsequent MMP1 induction in SK-BR3 cells. Inhibition of the Akt pathway with LY294002 paradoxically augmented MMP1 expression by reciprocal activation of ERK1/2 pathway. These data suggest that invasive potential of SK-BR3 cell would be affected by these drugs by suppression of EGFR ligands-induced MMP1 expression.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ligands; Matrix Metalloproteinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Neuregulin-1; Nitriles; Proto-Oncogene Proteins c-akt; Transforming Growth Factor alpha

The present study aimed to investigate heating-induced alterations of breast cancer cell invasion abilities and the potential mechanisms associated with TGF-β1 expression. MCF-7 cells were heated at 43, 45, 47 and 37 °C for 30 min. In vitro cell invasion ability was evaluated by matrigel invasion assay. The activity of matrix metalloproteinase (MMP)-2/9 was investigated by gelatin zymographic assays. Expression of vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) was investigated by immunocytochemistry and RT-PCR. Apoptosis was analysed by flow-cytometry. The invasive potential of MCF-7 cells was reduced by heating, and MMP-2/9 secretion and enzymatic activity were suppressed. Furthermore, VEGF and TGF-β1 mRNA and proteins were suppressed by hyperthermia. These results suggest that down-regulation of the expression of TGF-β1, EGF and MMPs by hyperthermia probably accounts for the inhibition of the invasive abilities of MCF-7 cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Hyperthermia, Induced; Immunohistochemistry; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.

Topics: Adenocarcinoma; Angiogenic Proteins; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; ErbB Receptors; Female; Humans; Ligands; Mesenchymal Stem Cells; Metabolic Networks and Pathways; Neovascularization, Pathologic; Osteoblasts; Protein Kinase Inhibitors; Transforming Growth Factor alpha

CD44, the transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. The expression of CD44 and its variants is associated with poor prognosis in breast cancer. Here, we investigated the effect of silibinin (a polyphenolic flavonolignan of the herbal plant of Silybum marianum, milk thistle) on the epidermal growth factor (EGF) ligand-induced CD44 expression in human breast cancer cells. The levels of CD44 mRNA and protein expression were greatly increased by EGF and by TGF-α in SKBR3 and BT474 breast cancer cells. In contrast, EGFR ligand-induced CD44 expression was reduced by EGFR inhibitors, AG1478 and lapatinib, respectively. Interestingly, we observed that EGFR ligand-induced CD44 and matrix metalloproteinase-9 (MMP-9) expression was reduced by silibinin treatment in a dose-dependent manner. In addition, silibinin suppressed the EGF-induced phosphorylation of EGFR and extracellular signal-regulated kinase1/2 (ERK1/2), a downstream signaling molecule of EGFR. Therefore, we suggest that silibinin prevents the EGFR signaling pathway and may be used as an effective drug for the inhibition of metastasis of human breast cancer.

Topics: Antioxidants; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hyaluronan Receptors; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Real-Time Polymerase Chain Reaction; RNA, Messenger; Silybin; Silymarin; Transforming Growth Factor alpha

The levels of expression of the four receptors and eleven ligands composing the epidermal growth factor family were measured using immunohistochemical staining in one hundred cases of breast cancer. All of the family were expressed to some degree in some cases; however, individual cases showed a very wide range of expression of the family from essentially none to all the factors at high levels. The highest aggregate level of expression of a receptor was HER2 followed by HER1, then HER3, then HER4. The ligands (including two splice variants of the NRG1 and NRG2 genes) broadly fell into three groups, those with the highest aggregate expression were Epigen, Epiregulin, Neuregulin 1alpha, Neuregulin 2alpha, Neuregulin 2beta, Neuregulin 4 and TGFalpha, moderate expression was seen with EGF, Neuregulin 1beta and Neuregulin 3, and relatively low levels of expression were seen of HB-EGF, Betacellulin and Amphiregulin. Statistical analysis using Spearman's Rank Correlation showed a positive correlation of expression between each of the factors. Analysing the data using the Cox Proportional Hazards model showed that, in this dataset, the most powerful predictors of relapse free interval and overall survival were the combined measurement of only Epigen and Neuregulin 4.

Topics: Amphiregulin; Betacellulin; Breast Neoplasms; Disease-Free Survival; EGF Family of Proteins; Epidermal Growth Factor; Epigen; ErbB Receptors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, erbB; Genes, erbB-1; Genes, erbB-2; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Multigene Family; Neoplasm Proteins; Neuregulins; Prognosis; Proportional Hazards Models; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Receptors, Growth Factor; Transforming Growth Factor alpha

Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-alpha (MR(1))], its binding site the epidermal growth factor receptor [EGFR (MR(2))] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR(4))], and the androgen receptor [AR (MR(5))]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-alpha and EGFR mRNA [TGF-alpha/EGFR (MR(12)) and EGFR/TGF-alpha (MR(21))]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR(24)) and bcl-2/EGFR (MR(42))]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4(45)) and AR/bcl-2 (MR(54))]. Oligo pairs differ in their 5'-3' linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD(50) dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.

Topics: Androgen Receptor Antagonists; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Combined Modality Therapy; ErbB Receptors; Female; Gene Targeting; Humans; Male; Oligonucleotides, Antisense; Paclitaxel; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Sirolimus; Transforming Growth Factor alpha

Most breast cancers that occur in women express estrogen receptor alpha (ERalpha). However, a large subset of these cancers either does not initially respond to anti-estrogen therapy or develops resistance to such treatment modalities. One postulated mechanism of this failure is signaling cross talk between hormones and local growth factors. To examine these complex interactions in vivo, we assessed the effects of estrogen on transforming growth factor alpha (TGFalpha)- and prolactin (PRL)-induced mammary tumorigenesis in transgenic mice. Both PRL and estrogen reduced the latency of TGFalpha-induced oncogenesis, resulting in tumors that were variably ERalpha-positive, but were progesterone receptor-negative. However, despite elevated ERalpha levels in NRL-PRL/TGFalpha glands, tumor latency was not reduced with increasing estrogen levels, nor increased after ovariectomy. Furthermore, PRL and TGFalpha in combination blocked the mitogenic effects of estrogen, dramatically reduced progesterone receptor levels, and diminished ERalpha down-regulation in response to circulating estrogen levels, in contrast to the other genotypes. Notably, however, ductal morphology remained responsive to estrogen, indicating that TGFalpha and PRL in combination can inhibit some, but not all, estrogenic signals. Both in vitro and in vivo, PRL and TGFalpha cooperatively enhanced Akt phosphorylation, which is associated with endocrine resistance in human disease. These findings provide insight into the interactions of PRL with growth factors during mammary oncogenesis and suggest combinatorial approaches that may result in improved therapeutic efficacy.

Topics: Animals; Breast Neoplasms; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation; Growth Substances; Humans; Mammary Glands, Animal; Mice; Mice, Inbred Strains; Mice, Transgenic; Ovariectomy; Prolactin; Receptor Cross-Talk; Transforming Growth Factor alpha; Tumor Cells, Cultured

In HER2-overexpressing mammary epithelial cells, transforming growth factor beta (TGF-beta) activated phosphatidylinositol-3 kinase (PI3K)/Akt and enhanced survival and migration. Treatment with TGF-beta or expression of an activated TGF-beta type I receptor (Alk5 with the mutation T204D [Alk5(T204D)]) induced phosphorylation of TACE/ADAM17 and its translocation to the cell surface, resulting in increased secretion of TGF-alpha, amphiregulin, and heregulin. In turn, these ligands enhanced the association of p85 with ErbB3 and activated PI3K/Akt. RNA interference of TACE or ErbB3 prevented TGF-beta-induced activation of Akt and cell invasiveness. Treatment with TGF-beta or expression of Alk5(T204D) in HER2-overexpressing cells reduced their sensitivity to the HER2 antibody trastuzumab. Inhibition of Alk5, PI3K, TACE, or ErbB3 restored sensitivity to trastuzumab. A gene signature induced by Alk5(T204D) expression correlated with poor clinical outcomes in patients with invasive breast cancer. These results suggest that by acting on ErbB ligand shedding, an excess of TGF-beta may result in (i) conditioning of the tumor microenvironment with growth factors that can engage adjacent stromal and endothelial cells; (ii) potentiation of signaling downstream ErbB receptors, thus contributing to tumor progression and resistance to anti-HER2 therapies; and (iii) poor clinical outcomes in women with breast cancer.

Topics: ADAM Proteins; ADAM17 Protein; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cells, Cultured; Enzyme Activation; Female; Gene Expression Profiling; Humans; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; RNA Interference; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Trastuzumab; Treatment Outcome

To examine the effects of triiodothyronine (T3), 17beta-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments.. Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3- mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities.. We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma; Cell Culture Techniques; Down-Regulation; Drug Interactions; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Tamoxifen; Transforming Growth Factor alpha; Triiodothyronine; Tumor Cells, Cultured

The aim of this study is to provide an expression profile of ErbB/HER ligands in breast cancer. We analysed the relationships with their receptors, the bio-pathological features and prognosis.. Epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), amphiregulin (AREG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EREG) and neuregulins1-4 (NRG1-4) were quantified in 363 tumours by real-time reverse transcription-polymerase chain reaction using TaqMan probes.. Ligands were detected in 80%-96% of the cases, except NRG3 (42%) and EREG (45.5%). At least one ligand was expressed in 304 cases (cut-off: upper quartile). Almost all combinations of receptor and ligand co-expressions were observed, but TGFalpha is preferentially expressed in tumours co-expressing EGFR/HER3, NRG3 in those co-expressing EGFR/HER4, AREG and EREG in those co-expressing HER2/HER4. EGF and AREG were associated with estradiol receptors, small tumour size, low histoprognostic grading, high HER4 levels. TGFalpha, HB-EGF and NRG2 were negatively related to these parameters. In Cox univariate analyses, EGF was a prognostic factor.. Our study demonstrates that (i) ErbB/HER ligands, including BTC and EREG, are expressed in most breast cancers; and (ii) TGFalpha, HB-EGF and NRG2 high expressions are related to the biological aggressiveness of the tumours.

Topics: Amphiregulin; Betacellulin; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Disease-Free Survival; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Profiling; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuregulin-1; Neuregulins; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2), are efficacious against the UACC 897 breast, PC-3 and LNCaP prostate, and T98G glioblastoma tumor lines in both in vitro and in vivo studies. Oligos against the anti-apoptosis protein bcl-2 (MR4) are also efficient against PC-3 and LNCaP tumors in similar in vitro experiments. To enhance activity, and also to introduce a derivative type of multifunctional oligo into this field, "bispecifics" were constructed containing two truncated complementary DNA sequences (from either MR1 or MR2) designed to bind targeted mRNA about their respective AUG initiation codons, and/or a similar sequence adjacent to the AUG site of mRNA encoding bcl-2. Tandem pairs of bispecifics were constructed: The first had complementary sequences for TGF-alpha and EGFR mRNA, but differed in 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences); a second pair had binding sites associated with EGFR and bcl-2, also differing in orientation (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). In studies targeting PC-3 and LNCaP cells, bispecifics demonstrated significant in vitro activity, and the second pair was significantly better than the original monospecifics. These studies are now extended to the MCF-7 breast cancer model in order to determine whether these particular bispecifics have similar anti-breast cancer activity and if they are significantly better than monospecific oligos from which they were derived. We conclude that bispecific oligos significantly inhibit MCF-7 growth, however, in contrast to results obtained with PC-3 and LNCaP, the monospecific oligos directed against EGFR and bcl-2 have significantly greater activity than the bispecifics targeting a combination of TGF-alpha, EGFR, or bcl-2. These data suggest that the relative activities of oligos, whether mono- or bispecific, change with tumor type. Bispecific oligos which target different proteins, possibly those which regulate estrogen utilization, may be more effective against MCF-7 cells and warrant additional investigation, particularly if co-administered with traditional chemotherapeutics.

Topics: Breast Neoplasms; Cell Line, Tumor; ErbB Receptors; Female; Humans; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor alpha

Male breast cancer is rare and has been the focus of limited research. Although the etiology is unclear, conditions increasing circulating prolactin (PRL), as well as estrogen, increase the risk of tumorigenesis. We modeled exposure to elevated PRL in transgenic mice, using the mammary-selective, estrogen-insensitive promoter neu-related lipocalin (NRL), to drive PRL expression. Male NRL-PRL mice did not develop mammary tumors. However, in cooperation with the well-characterized oncogene transforming growth factor-alpha (TGF-alpha), PRL induced mammary tumors in 100% of male bitransgenic mice. Similar to disease in human males, these tumors expressed variable levels of estrogen receptor-alpha (ER-alpha) and androgen receptors. However, carcinogenesis was not responsive to testicular steroids because castration did not alter latency to tumor development or tumor ER-alpha expression. Interestingly, both NRL-TGF-alpha/PRL and NRL-PRL males demonstrated increased ductal development, which occurred during puberty, similar to female mice. This outgrowth was diminished in NRL-PRL males treated with ICI 182,780, suggesting that PRL enhances ER-mediated growth. Treatment of MCF-7-derived cells with PRL increased phosphorylation of ER-alpha at residues implicated in unliganded ER-alpha activity. Together, these studies suggest that PRL expands the pool of cells susceptible to tumorigenesis, which is then facilitated by PRL and TGF-alpha cross talk. Activation of ER-alpha is one mechanism by which PRL may contribute to breast cancer and points to other therapeutic strategies for male patients.

Topics: Animals; Breast Neoplasms; Breast Neoplasms, Male; Cell Line, Tumor; Coculture Techniques; Estrogen Receptor alpha; Estrogens; Humans; Male; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Prolactin; Sex Factors; Steroids; Transforming Growth Factor alpha

Diets high in n-6 fatty acids are associated with an increased risk of bone metastasis from prostate carces (PCa). The molecular mechanism underlying this phenomenon is largely unknown. Arachidonic acid (AA) and its precursor linoleic acid can be metabolized to produce pro-inflammatory cytokines that act as autocrine and paracrine regulators of cancer behavior. We and other authors have previously reported that factors released by PCa cells excite an aberrant response in bone marrow stromal cells (BMSCs). We planned to study how AA may modulate in vitro the interaction between PCa cells and human BMSCs. First, we observed that AA is a potent mitogenic factor for PCa cells through the production of both 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) metabolites. While 5-LOX controls cell survival through the regulation of the Bcl-2/Bax ratio, COX-2 activity stimulates the release of transforming growth factor-alpha (TGF-alpha) and pro-inflammatory cytokines. The blockade of COX-2 activity through a specific inhibitor is sufficient to repress AA-induced gene transcription. The over-expression of transforming growth factor -alpha (TGF-alpha), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) by AA-primed PCa cells resulted particularly effective in modifying cell behavior of cultured human BMSCs. In fact, we observed an increment in the cell number of BMSCs, due prevalently to the action of TGF-alpha, the number of osteoblasts, and the production of receptor activator for nuclear factor kappa B ligand (RANKL), events mainly controlled by inflammatory cytokines. These findings provide a possible molecular mechanism by which dietary n-6 fatty acids accumulating in bone marrow may influence the formation of PCa-derived metastatic lesions and indicate new molecular targets for the therapy of metastatic PCa.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blotting, Western; Bone Marrow; Breast Neoplasms; Cell Proliferation; Colony-Forming Units Assay; Cyclooxygenase 2; DNA Primers; Epidermal Growth Factor; Female; Humans; Interleukin-1beta; Male; Prostatic Neoplasms; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stromal Cells; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The ability to proliferate independently of signals from other cell types is a fundamental characteristic of tumor cells. Using a 3D culture model of human breast cancer progression, we have delineated a protease-dependent autocrine loop that provides an oncogenic stimulus in the absence of proto-oncogene mutation. Targeting this protease, TNF-alpha-converting enzyme (TACE; also referred to as a disintegrin and metalloproteinase 17 [ADAM17]), with small molecular inhibitors or siRNAs reverted the malignant phenotype in a breast cancer cell line by preventing mobilization of 2 crucial growth factors, TGF-alpha and amphiregulin. We show that TACE-dependent ligand shedding was prevalent in a series of additional breast cancer cell lines and, in all cases examined, was amenable to inhibition. Using existing patient outcome data, we demonstrated a strong correlation between TACE and TGFA expression in human breast cancers that was predictive of poor prognosis. Tumors resulting from inappropriate activation of the EGFR were common in multiple tissues and were, for the most part, refractory to current targeted therapies. The data presented here delineate the molecular mechanism by which constitutive EGFR activity may be achieved in tumor progression without mutation of the EGFR itself or downstream pathway components and suggest that this important oncogenic pathway might usefully be targeted upstream of the receptor.

Topics: ADAM Proteins; ADAM17 Protein; Amphiregulin; Base Sequence; Breast Neoplasms; Cell Line, Tumor; EGF Family of Proteins; ErbB Receptors; Female; Gene Expression; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Prognosis; Proto-Oncogene Mas; RNA, Small Interfering; Substrate Specificity; Transforming Growth Factor alpha

We have investigated mechanisms of acquired resistance to the HER2 antibody trastuzumab in BT-474 human breast cancer cells.. BT-474 xenografts established in athymic nude mice were eliminated by trastuzumab. Continuous cell lines (HR for Herceptin resistant) were generated from tumors that recurred in the presence of continuous antibody therapy.. The isolated cells behaved resistant to trastuzumab in culture as well as when reinjected into nude mice. They retained HER2 gene amplification and trastuzumab binding and were exquisitely sensitive to peripheral blood mononuclear cells ex vivo in the presence of the antibody. The HR cells exhibited higher levels of phosphorylated epidermal growth factor receptor (EGFR) and EGFR/HER2 heterodimers. Phosphorylation of HER2 in HR cells was inhibited by the EGFR tyrosine kinase inhibitors erlotinib and gefitinib. Gefitinib also inhibited the basal association of p85 with phosphorylated HER3 in HR cells. Both inhibitors as well as the dual EGFR/HER2 inhibitor, lapatinib, induced apoptosis of the HR cells in culture. Growth of established HR5 xenografts was inhibited by erlotinib in vivo. In addition, the HR cells overexpressed EGFR, transforming growth factor alpha, heparin-binding EGF, and heregulin RNAs compared with the parental trastuzumab-sensitive cells.. These results are consistent with the inability of trastuzumab to block the heterodimerization of HER2 and suggest that amplification of ligand-induced activation of ErbB receptors is a plausible mechanism of acquired resistance to trastuzumab that should be investigated in primary mammary cancers.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Female; Ligands; Mice; Mice, Inbred BALB C; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor alpha; Trastuzumab

The development of cancer prevention strategies depends on the elucidation of molecular pathways underlying oncogenesis. In a previous proteomic study of matched normal breast ducts and Ductal Carcinoma in Situ (DCIS), we identified overexpression of Rab11a in DCIS. Rab11a is not well studied in cancer, but is known to regulate the recycling of internalized cell surface proteins and receptors from the early endosome through the trans-Golgi network. Using immunohistochemistry, we confirmed our observation, noting increased Rab11a expression in 19 of 22 (86%) DCIS cases compared to matched normal breast epithelium. To study the function of Rab11a, immortal, nontumorigenic MCF10A breast cells were stimulated with ligands to the EGF receptor (EGFR) after transfection with empty vector (control), Rab11a, or a S25N dominant-negative (DN) Rab11a. Using an iodinated ligand:receptor recycling assay, transfection of Rab11a accelerated, while DN-Rab11a postponed EGFR recycling in vitro. The signaling and in vitro phenotypic consequences of Rab11a expression and function were studied. Transfection of DN-Rab11a increased Erk1/2 activation downstream of EGF, but exerted no effect on the Akt pathway. Expression of DN-Rab11a inhibited MCF10A proliferation by 50-60%, and also inhibited anchorage-dependent colonization. Notably, DN-Rab11a transfection increased motility toward EGFR ligands. The data provide a first demonstration that Rab11a modulates EGFR recycling, and promotes the proliferation but inhibits the motility of an immortal breast line, consistent with the DCIS phenotype.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Phosphorylation; rab GTP-Binding Proteins; Transforming Growth Factor alpha

Hypoxia-inducible factor-1 (HIF-1) is the key transcription factor regulating the cellular response to hypoxia, including angiogenesis. Growth factors play an important role in tumour growth and angiogenesis and some have been shown to be induced by HIF-1 in vitro. This study investigated if angiogenesis or growth factors or their receptors are associated with HIF-1alpha in invasive breast cancer.. High levels of HIF-1alpha, detected by immunohistochemistry in 45 breast cancers, were positively associated with increased microvessel density (as a measure of angiogenesis) (P = 0.023). Furthermore, high levels of HIF-1alpha were associated with epithelial expression (> or = 10%) of epidermal growth factor receptor (EGFR) (P = 0.011), platelet-derived growth factor (PDGF)-BB (P < 0.001), and basic fibroblast growth factor (bFGF) (P = 0.045). A positive, yet insignificant, trend for HIF-1alpha to be associated with epithelial expression of transforming growth factor (TGF)-alpha (P = 0.081) and vascular endothelial growth factor (VEGF) (P = 0.109) was noticed as well as an inverse association with stromal expression of TGF-beta-R1 (P = 0.070).. In invasive breast cancer, HIF-1alpha is associated with angiogenesis, and expression of growth factors bFGF and PDGF-BB, and the receptor EGFR. Thus, agents targeting HIF-1 may combine different pathways of inhibiting breast cancer growth, including angiogenesis and growth factors.

Topics: Autocrine Communication; Becaplermin; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Epithelial Cells; ErbB Receptors; Eukaryotic Initiation Factor-3; Female; Fibroblast Growth Factor 2; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Lymph Nodes; Neoplasm Invasiveness; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Proteins; Proto-Oncogene Proteins c-sis; Stromal Cells; Transcription Factors; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A

The HER2 gene encodes a tyrosine kinase receptor overexpressed in 25-30% of human breast cancers. Clinical trials have shown the efficacy of the anti-HER2 monoclonal antibody Trastuzumab in metastatic breast cancer patients. Nevertheless, 70% of patients are unresponsive from start of treatment and nearly all become unresponsive during treatment. Possible mechanisms for these failures could depend on impairment of the machinery responsible for receptor downregulation. To test this hypothesis, we analysed the genomic sequences encoding regions known to be critical for HER2 downregulation, of both HER2 and of the ubiquitin ligase Cbl. We investigated 63 breast cancers, and found no mutations in these regions. We thus considered alternative mechanisms -- such as TGFalpha production -- possibly interfering with HER2 downregulation. In selected cases, by comparing breast cancer neoplastic tissue before and after Trastuzumab treatment, we found induction of TGFalpha expression. Moreover, by in vitro expression of exogenous TGFalpha in breast cancer cells, we observed a dramatic reduction in Trastuzumab-induced HER2 endocytosis, downregulation and cell growth inhibition. Our results suggest that unresponsiveness to Trastuzumab may not be due to intrinsic defects in the machinery responsible for HER2 downregulation, but can be associated with a TGFalpha-related mechanism of escape to HER2 downregulation.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Down-Regulation; Female; Genes, erbB-2; Humans; Middle Aged; Mutation; Protein Structure, Tertiary; Transforming Growth Factor alpha; Trastuzumab; Tumor Cells, Cultured

There is considerable evidence that the epidermal growth factor receptor (EGFR) and IGF-I receptor (IGF-IR) cross-talk in breast cancer cells. In the present study, we have examined whether EGFR/IGF-IR cross-talk exists in EGFR-positive tamoxifen-resistant variants of MCF-7 (Tam-R) and T47D (T47D-R) breast cancer cell lines. Although Tam-R cells expressed reduced IGF-IR protein levels compared with their wild-type MCF-7 counterparts, phosphorylated IGF-IR protein levels were equivalent in the two cell lines under basal growth conditions, possibly as a consequence of increased IGF-II expression in Tam-R cells. IGF-II activated both IGF-IR and EGFR in Tam-R cells, whereas only activation of IGF-IR was observed in wild-type cells. In contrast, epidermal growth factor rapidly induced EGFR, but not IGF-IR, phosphorylation in Tam-R cells. IGF-II promoted direct association of c-SRC with IGF-IR, phosphorylated c-SRC, and increased EGFR phosphorylation at tyrosine 845, a c-SRC-dependent phosphorylation site. Pretreatment with either AG1024 (IGF-IR-specific inhibitor) or an IGF-II neutralizing antibody inhibited basal IGF-IR, c-SRC, and EGFR phosphorylation, and AG1024 significantly reduced Tam-R basal cell growth. The c-SRC inhibitor SU6656 also inhibited growth, reduced basal and IGF-II-induced c-SRC and EGFR phosphorylation, and blocked EGFR activation by TGFalpha. Similarly, in T47D-R cells, AG1024 and SU6656 inhibited basal and IGF-II-induced phosphorylation of c-SRC and EGFR, and SU6656 reduced TGFalpha-induced EGFR activity. These results suggest the existence of a unidirectional IGF-IR/EGFR cross-talk mechanism whereby IGF-II, acting through the IGF-IR, regulates basal and ligand-activated EGFR signaling and cell proliferation in a c-SRC-dependent manner in Tam-R cells. This cross-talk between IGF-IR and EGFR is not unique to Tam-R cells because this mechanism is also active in a tamoxifen-resistant T47D-R cell line.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Female; Humans; Insulin-Like Growth Factor II; Phosphorylation; Proto-Oncogene Proteins pp60(c-src); Receptor Cross-Talk; Receptor, IGF Type 1; Signal Transduction; Tamoxifen; Transforming Growth Factor alpha

The polycyclic aromatic hydrocarbon 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) is related to the industrial byproduct dioxin and is a weak agonist and partial antagonist at the aryl hydrocarbon receptor (AhR). Tamoxifen is used for the treatment and prevention of breast cancer and interferes with the interaction of estrogen with estrogen receptor alpha (ER). The combination of MCDF and tamoxifen lowered the effective dose of both drugs required to inhibit 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats and protected against the estrogenic effects of tamoxifen on the uterus in rats (A. McDougal et al., Cancer Res 2001;61:3902-7), pointing to the potential use of MCDF in breast cancer treatment. Potential AhR-ER cross-talk is evidenced by the antiestrogenic activity of MCDF and the degradative effect of MCDF on ER protein levels. Our studies confirmed that MCDF degraded the ER. MCDF displayed antiestrogenic activity at higher concentrations in MCF-7 human breast cancer cells, but MCDF alone (10(-6) M) stimulated the growth of MCF-7 cells. MCDF also activated an estrogen response element (ERE)-luciferase reporter and increased mRNA levels of the estrogen-responsive gene transforming growth factor (TGF)-alpha. The estrogenic effects of MCDF are ER dependent because they were blocked by the pure antiestrogen ICI 182,780. MCDF induced ER-coactivator interaction in glutathione S-transferase pull-down assays and the formation of an ER.ERE complex in gel mobility shift assays, further indicating that the estrogenic actions of MCDF are mediated by the ER. In addition, knockdown of the AhR with small interfering RNA did not affect MCDF-induced ERE-luciferase activity. Overall, these data support the conclusion that MCDF is a partial agonist at the ER. This study provides the first evidence for the direct interaction of the ER with MCDF and challenges the view that MCDF is simply an AhR-specific ligand.

Topics: Basic Helix-Loop-Helix Transcription Factors; Benzofurans; Breast Neoplasms; Cell Division; Cell Line, Tumor; Estradiol; Estrogen Receptor alpha; Humans; Models, Molecular; Receptor Cross-Talk; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor alpha

Resveratrol, a hydroxystilbene found in grapes and wine, has previously been shown to be a non-flavonoid phytoestrogen, and to act as an estrogen receptor (ER) superagonist in MCF-7 cells transiently transfected with estrogen-responsive reporter constructs. Several additional hydroxystilbenes, including diethylstilbestrol (DES) and piceatannol, were tested, and all showed ER agonism or partial agonism, but superagonism was specific to resveratrol. Moreover, superagonism was observed in cells carrying a stably integrated reporter gene, indicating that this phenomenon is not a result of transient transfection. To examine the role of the transcriptional activation function (AF) domains of ERalpha in resveratrol agonism, we compared the effects of resveratrol and estradiol (E2) on expression of exogenous reporter genes and an endogenous estrogen-regulated gene (TGFalpha) in MDA-MB-231 cells stably transfected with wild-type (wt) ERalpha or mutants with deleted or mutated AF domains. In reporter gene assays, cells expressing wtERalpha showed a superagonistic response to resveratrol. Deletion of AF-1 or mutation of AF-2 attenuated the effect of resveratrol disproportionately compared to that of E2, while deletion of AF-2 abrogated the response to both ligands. In TGFalpha expression assays, resveratrol acted as a full agonist in cells expressing wtERalpha. Deletion of AF-1 attenuated stimulation by E2 more severely than that by resveratrol, as did deletion of AF-2. In contrast, mutation of AF-2 left both ligands with a limited ability to induced TGFalpha expression. In summary, the effect of modifying or deleting AF domains depends strongly on the ligand and the target gene.

Topics: Breast Neoplasms; Cell Line, Tumor; Estrogens; Humans; Resveratrol; RNA, Messenger; Stilbenes; Transforming Growth Factor alpha

The mechanism of action of ZR2002, a chimeric amino quinazoline designed to possess mixed EGFR tyrosine kinase (TK) inhibitory and DNA targeting properties, was compared to those of ZR01, a reversible inhibitor of the same class and PD168393, a known irreversible inhibitor of EGFR. ZR2002 exhibited 4-fold stronger EGFR TK inhibitory activity than its structural homologue ZR01 but was approximately 3-fold less active than the 6-acrylamidoquinazoline PD168393. It preferentially blocked EGF and TGFalpha-induced cell growth over PDGF and serum. It also inhibited signal transduction in heregulin-stimulated breast tumour cells, indicating that it does not only block EGFR but also its closely related erbB2 gene product. In contrast to its structural homologues, ZR2002 was capable of inducing significant levels of DNA strand breaks in MDA-MB-468 cells after a short 2 hr drug exposure at a concentration as low as 10 microM. Reversibility studies using whole cell autophosphorylation and growth assays in human breast cell lines showed that in contrast to its reversible inhibitor counterpart ZR01, ZR2002 induced irreversible inhibition of EGF-stimulated autophosphorylation in MDA-MB-468 cells and irreversible inhibition of cell growth. Moreover despite possessing a weaker binding affinity than PD168393, it induced a significantly more sustained antiproliferative effect than the latter after a pulse 2 hr exposure. More importantly, in contrast to ZR01 and PD168393, ZR2002 was capable of inducing significant levels of cell death by apoptosis in MDA-MB-468 cells. The results in toto suggest that the superior antiproliferative potency of ZR2002 may be due to its ability to induce a protracted blockade of receptor tyrosine kinase-mediated signaling while damaging cellular DNA, a combination of events that may trigger cell-killing by apoptosis.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; DNA Damage; DNA, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Molecular Structure; Oncogene Proteins v-erbB; Phosphorylation; Platelet-Derived Growth Factor; Quinazolines; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

Topics: Blotting, Western; Breast Neoplasms; Butadienes; COUP Transcription Factor I; COUP Transcription Factor II; COUP Transcription Factors; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Oncostatin M; Peptides; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

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.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Division; Dimerization; Drug Resistance, Neoplasm; ErbB Receptors; Flavonoids; Gefitinib; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Quinazolines; Receptor, ErbB-2; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Trastuzumab; Tumor Cells, Cultured

Resveratrol (Res) is a phytoestrogen found in grapes and present in red wine. Res has been shown to function as an estrogen receptor (ER) agonist, but it remains unclear whether it may also exert antagonist activity. Our aim was to study the effects of Res at both the molecular (TGFalpha gene activation) and the cellular (cell growth) levels in breast cancer cells stably transfected with wild-type (wt) ER(D351) and mutant (mut) ER (D351Y). TGFalpha mRNA induction was used as a specific marker of estradiol (E(2)) responsiveness. Res caused a concentration-dependent (10(-8)-10(-4) M) stimulation of TGFalpha mRNA, indicating that it acts as an estrogen agonist in these cell lines. The pure antiestrogen ICI 182,780 (ICI) blocked Res-induced activation of TGFalpha, consistent with action through an ER-mediated pathway. Further studies that combined treatments with E(2) and Res showed that Res does not act as an antagonist in the presence of various (10(-11)-10(-8) M) concentrations of E(2). To determine whether Res can be classified as a type I or type II estrogen (Jordan et al., Cancer Res 2001;61:6619-23,), we examined Res with the D351G ER in the TGFalpha assay and found that Res belongs to the type I estrogens. Both Res and E(2) had concentration-dependent growth inhibitory effects in cells expressing wtER and D351Y ER. Although the pure antiestrogen ICI blocked the growth inhibitory effects of E(2), it did not block the inhibitory effects of Res, suggesting that the antiproliferative effects of Res also involve ER-independent pathways. Interestingly, Res differentially affected the levels of ER protein in these 2 cell lines: Res down-regulated wtER levels while significantly up-regulating the amount of mutD351Y ER. Co-treatment with ICI resulted in strongly reduced ER levels in both cell lines. Gene array studies revealed Res-induced up-regulation of more than 80 genes, among them a profound activation of p21(CIP1)/WAF1, a gene associated with growth arrest. The p21(CIP1)/WAF1 protein levels measured by Western blotting confirmed Res-induced significant up-regulation of this protein in both cell lines. In summary, Res acts as an ER agonist at low doses but also activates ER-independent pathways, some of which inhibit cell growth.

Topics: Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Receptors, Estrogen; Resveratrol; RNA, Messenger; Stilbenes; Transcriptional Activation; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

The factors and mechanisms that transduce the intracellular signals sent upon activation of the receptor for the epidermal growth factor (EGFR) and related receptors are reasonably well understood and, in fact, are the targets of anti-tumor drugs. In contrast, less is known about the mechanisms implicated in sending the signals that activate these receptors. Here we show that when its proteolytic shedding is prevented, the transmembrane form of the transforming growth factor-alpha (proTGF-alpha) interacts with, but does not activate, the EGFR. Thus, shedding seems to control not only the availability of the soluble form of the growth factor (TGF-alpha) but also the activity of the transmembrane form. The activity of the protease responsible for the shedding of proTGF-alpha, tumor necrosis factor-alpha converting enzyme (TACE), is required for the activation of the EGFR in vivo and for the development of tumors in nude mice, indicating a crucial role of TACE in tumorigenesis. In agreement with this view, TACE is dramatically overexpressed in the majority of mammary tumors analyzed. Collectively, this evidence points to TACE as a promising target of anti-tumor therapy.

Topics: ADAM Proteins; ADAM17 Protein; Amino Acid Sequence; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Breast Neoplasms; Carcinogenicity Tests; CHO Cells; Cricetinae; Culture Media, Conditioned; Endopeptidases; ErbB Receptors; Female; HeLa Cells; Humans; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Mice; Mice, Nude; Neoplasms; Transforming Growth Factor alpha; Transplantation, Heterologous

Osteopontin (OPN) is a secreted, integrin-binding glycophosphoprotein that has been implicated in breast cancer. We previously showed that OPN-induced cell migration of mammary epithelial cells (MEC) depends on binding to cell surface integrins and involves activation of the hepatocyte growth factor (HGF) receptor, Met. Here, we show that OPN-induced migration of MEC also requires activation of the epidermal growth factor (EGF) pathway. Synergism was seen between EGF and OPN in inducing cell migration. Furthermore, incubation of cells with exogenous OPN increased ligand (TGFalpha> EGF) and EGF receptor (EGFR) mRNA expression, as well as EGFR kinase activity. Treatment of cells with anti-TGFalpha or anti-EGFR antibody, or with tyrphostin-25 (EGFR inhibitor), significantly impaired the cell migration response to OPN. Other more broad-spectrum tyrosine kinase inhibitors and the growth factor/ receptor interaction inhibitor, suramin, also inhibited OPN-induced migration. Using specific signal transduction pathway inhibitors, we have screened for involvement of MEK (MAP kinase kinase), phosphatidylinositol 3-kinase, phospholipase C (PLC), and protein kinase C (PKC). Results implicated all of these pathways in OPN-induced cell migration, the most pronounced effect being seen with PLC and PKC inhibitors. These results suggest that induction of MEC migration by OPN involves a cascade of events including at least two growth factor/receptor pathways and multiple downstream signal transduction pathways. A number of potential targets are thus provided for strategies aimed at blocking the malignancy-promoting effects of OPN.

Topics: Adenocarcinoma; Breast; Breast Neoplasms; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neoplasm Proteins; Oligonucleotides, Antisense; Osteopontin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Proto-Oncogene Proteins c-met; Recombinant Fusion Proteins; Recombinant Proteins; RNA, Messenger; Sialoglycoproteins; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured; Type C Phospholipases

Topics: Breast Neoplasms; Crystallography, X-Ray; Epidermal Growth Factor; Humans; Protein Binding; Receptor, ErbB-2; Transforming Growth Factor alpha

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.

Topics: Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Movement; ErbB Receptors; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Maleimides; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Myosin-Light-Chain Kinase; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Protein Serine-Threonine Kinases; rho-Associated Kinases; Transforming Growth Factor alpha; Tyrosine

Long-term tamoxifen treatment of breast cancer can result in tamoxifen-stimulated breast cancer, in which estrogen inhibits tumor growth after tamoxifen withdrawal. We investigated the molecular mechanism(s) of estradiol-induced tumor regression by using an in vivo model of tamoxifen-stimulated human breast cancer.. Growth of parental estradiol-stimulated MCF-7E2 and long-term tamoxifen-stimulated MCF-7TAMLT xenografts in athymic mice was measured during treatment with vehicle, estradiol, estradiol plus tamoxifen, tamoxifen alone, estradiol plus fulvestrant, or fulvestrant alone. Apoptosis was detected by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Protein expression was assessed by western blot analysis. mRNA expression was assessed by real-time reverse transcription-polymerase chain reaction. All statistical tests were two-sided.. MCF-7E2 tumor growth was stimulated by estradiol (cross-sectional area at week 13 = 1.06 cm2, 95% confidence interval [CI] = 0.82 to 1.30 cm2; P<.001) compared with control (0.06 cm2, 95%CI = -0.02 to 0.14 cm2), but tumor growth was inhibited by tamoxifen or fulvestrant. MCF-7TAMLT tumor growth was stimulated by tamoxifen) cross-sectional area at week 10 = 0.60 cm2, 95% CI = 0.50 to 0.70 cm2; P<.001) compared with control (0.02 cm2, 95% CI = 0.00 to 0.04 cm2). For MCF-7TAMLT tumors that were initially 0.35 cm2, estradiol-induced regression to 0.18 cm2 (95% CI = 0.15 to 0.21 cm2; P<.001), and tamoxifen or estradiol plus fulvestrant enhanced tumor growth to 1.00 cm2 (95% CI = 0.88 to 1.22 cm2). Estradiol increased the number of apoptotic cells in tumors by 23% (95% CI = 20% to 26%; P<.001) compared with all other treatments, decreased estrogen receptor alpha(ERalpha) protein expression, increased the expression of Fas mRNA and protein, decreased the expression of HER2/neu mRNA and protein and nuclear factor kappaB (NF-kappaB) protein but did not affect Fas ligand protein expression compared with control. Paradoxically, fulvestrant reversed this effect and stimulated MCF-7TAMLT tumor growth apparently through ERalpha-mediated regulation of Fas, HER2/neu, and NF-kappaB.. Physiologic levels of estradiol induced regression of tamoxifen-stimulated breast cancer tumors, apparently by inducing the death receptor Fas and suppressing the antiapoptotic/prosurvival factors NF-kappaB and HER2/neu.

Topics: Animals; Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Estradiol; Estrogen Receptor Modulators; Fas Ligand Protein; Female; Fulvestrant; Humans; In Situ Nick-End Labeling; Membrane Glycoproteins; Mice; Mice, Nude; Polymerase Chain Reaction; Receptor, ErbB-2; Tamoxifen; Transforming Growth Factor alpha; Transplantation, Heterologous

To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed approximately 3-5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced approximately 1.5-2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor alpha and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions.

Topics: Breast Neoplasms; Cell Division; Cell Line, Tumor; Chemokine CXCL12; Chemokines, CXC; Drug Resistance, Neoplasm; Estradiol; Female; Gene Expression; Gene Expression Profiling; Humans; Ligands; Oligonucleotide Array Sequence Analysis; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

We previously reported stable transfection of estrogen receptor alpha (ERalpha) into the ER-negative MDA-MB-231 cells (S30) as a tool to examine the mechanism of action of estrogen and antiestrogens [J. Natl. Cancer Inst. 84 (1992) 580]. To examine the mechanism of ERbeta action directly, we have similarly created ERbeta stable transfectants in MDA-MB-231 cells. MDA-MB-231 cells were stably transfected with ERbeta cDNA and clones were screened by estrogen response element (ERE)-luciferase assay and ERbeta mRNA expression was quantified by real-time RT-PCR. Three stable MDA-MB-231/ERbeta clones were compared with S30 cells with respect to their growth properties, ability to activate ERE- and activating protein-1 (AP-1) luciferase reporter constructs, and the ability to activate the endogenous ER-regulated transforming growth factor alpha (TGFalpha) gene. ERbeta6 and ERbeta27 clones express 300-400-fold and the ERbeta41 clone express 1600-fold higher ERbeta mRNA levels compared with untransfected MDA-MB-231 cells. Unlike S30 cells, 17beta-estradiol (E2) does not inhibit ERbeta41 cell growth. ERE-luciferase activity is induced six-fold by E2 whereas neither 4-hydroxytamoxifen (4-OHT) nor ICI 182, 780 activated an AP-1-luciferase reporter. TGFalpha mRNA is induced in response to E2, but not in response to 4-OHT. MDA-MB-231/ERbeta clones exhibit distinct characteristics from S30 cells including growth properties and the ability to induce TGFalpha gene expression. Furthermore, ERbeta, at least in the context of the MDA-MB-231 cellular milieu, does not enhance AP-1 activity in the presence of antiestrogens. In summary, the availability of both ERalpha and ERbeta stable breast cancer cell lines now allows us to compare and contrast the long-term consequences of individual signal transduction pathways.

Topics: Breast Neoplasms; Cell Division; Cell Line, Tumor; DNA, Complementary; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Receptors, Estrogen; Response Elements; RNA, Messenger; Tamoxifen; Transcription Factor AP-1; Transcription, Genetic; Transfection; Transforming Growth Factor alpha

To study the suppressive effects of curcumin on breast carcinoma cells and the mechanism.. Estrogen receptor (ER) positive human breast cancer line MCF-7 and ER negative human breast cancer line MDA-MB-231 were cultured 17-beta estradiol and curcumin were added into the culture. Northern blot hybridization and Western blots were performed to detect the expression of mRNA and protein. The human ERE promoter activity was analyzed by transient transfection with CAT-reporter constructs. Invasion experiments were conducted with a Matrigel invasion chamber.. Curcumin inhibit the proliferation in both estrogen receptor (ER) positive MCF-7 cells and ER negative MDA-MB-231 cells. Curcumin's antiproliferative effects are estrogen dependent in ER positive MCF-7 cells. Curcumin inhibits the expression of ER downstream genes including pS2 and TGF-alpha (transforming growth factor-alpha) in ER-positive MCF-7 cells, and this inhibition is also dependent on the presence of estrogen. In addition, curcumin exerts strong anti-invasive effects in vitro which was not estrogen dependent in the ER-negative MDA-MB-231 breast cancer cells. These anti-invasive effects appeared to be mediated through the down-regulation of MMP-2 (matrix metalloproteinase) and the up-regulation of TIMP-1 (tissue inhibitor of metalloproteinase). Curcumin inhibited the transcript levels of two major angiogenesis factors VEGF (vascular endothelial growth factor) and b-FGF (basic fibroblast growth factor) in ER-negative MDA-MB-231 cells.. Curcumin exerts multiple suppressive effects on breast carcinoma cells;it's mechanism of chemoprevention is pleiotropic.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line, Tumor; Curcumin; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Humans; Proteins; Receptors, Estrogen; Transforming Growth Factor alpha; Trefoil Factor-1; Tumor Suppressor Proteins

Long-term estrogen deprivation causes human breast cancer cells to develop hypersensitivity to the mitogenic effect of estradiol (E(2)). Our prior studies demonstrated an association between enhanced MAPK activation and hypersensitivity in long-term estrogen-deprived (LTED) MCF-7 cells. Herein, we report that MAPK is constitutively activated in LTED cells and not dependent on serum factors. Additionally, activated MAPK levels fall upon reversion of the hypersensitivity. Importantly, we now provide direct evidence that enhanced MAPK causes hypersensitivity to E(2). We activated MAPK in wild-type MCF-7 cells using TGFalpha, and demonstrated a 2-3 log enhancement of sensitivity to E(2). PD98059 abrogated the TGFalpha-induced effect, indicating that MAPK activation is responsible for E(2) hypersensitivity. To determine the level at which MAPK activation enhanced E(2) sensitivity, we examined the dose-response effects of E(2) on several transcriptional readouts, including ERE-reporter activity and the levels of progesterone receptor and pS2. Wild-type and LTED cells exhibited nearly identical responses to E(2), suggesting that mechanisms downstream of estrogen receptor-mediated transcription are involved in inducing hypersensitivity. In support of this possibility, LTED and TGFalpha-treated wild-type cells were hypersensitive to the effects of E(2) on the key cell cycle regulator, E2F1.

Topics: Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Division; DNA-Binding Proteins; Dose-Response Relationship, Drug; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Activation; Enzyme Inhibitors; Estradiol; Flavonoids; Gene Expression; Humans; Mitogen-Activated Protein Kinases; Proteins; Receptors, Estrogen; Receptors, Progesterone; Response Elements; Transcription Factors; Transcription, Genetic; Transforming Growth Factor alpha; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Amino acid Asp-351 in the ligand binding domain of estrogen receptor alpha (ERalpha) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERalpha complexes. 4-Hydroxytamoxifen is a full agonist at a transforming growth factor alpha target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERalpha. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERalpha allosterically silences activation function-1 activity in the 4-hydroxytamoxifen-ERalpha complex with the complete loss of estrogen-like activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERalpha expression vectors. Profound differences in the agonist and antagonist actions of Ralcenter dotERalpha complexes were noted only in stable transfectants. The agonist activity of the Ralcenter dotERalpha complex was enhanced with D351E and D351Y ERalpha, but raloxifene lost its agonist activity with D351F ERalpha. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERalpha. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERalpha complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERalpha complex.

Topics: Breast Neoplasms; Estrogen Antagonists; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Nuclear Receptor Coactivator 2; Proteins; Raloxifene Hydrochloride; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Structure-Activity Relationship; Tamoxifen; Transcription Factors; Transfection; Transforming Growth Factor alpha; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

In our study, we present experimental evidence suggesting that curcumin exerts multiple different suppressive effects on human breast carcinoma cells in vitro. Our experiments demonstrate that curcumin's antiproliferative effects are estrogen dependent in ER (estrogen receptor)-positive MCF-7 cells, being more pronounced in estrogen-containing media and in the presence of exogenous 17-beta estradiol. Curcumin inhibits the expression of ER downstream genes including pS2 and TGF-beta (transforming growth factor) in ER-positive MCF-7 cells, and this inhibition is also dependent on the presence of estrogen. Curcumin also decreases ERE (estrogen responsive element)-CAT activities induced by 17-beta estradiol. In addition, we demonstrate that curcumin exerts strong anti-invasive effects in vitro that are not estrogen dependent in the ER-negative MDA-MB-231 breast cancer cells. These anti-invasive effects appear to be mediated through the downregulation of MMP-2 (matrix metalloproteinase) and the upregulation of TIMP-1 (tissue inhibitor of metalloproteinase), 2 common effector molecules that have been implicated in regulating tumor cell invasion. Our study also demonstrates that curcumin inhibits the transcript levels of 2 major angiogenesis factors VEGF (vascular endothelial growth factor) and b-FGF (basic fibroblast growth factor) mainly in ER-negative MDA-MB-231 cells.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Division; Curcumin; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dose-Response Relationship, Drug; Estrogen Antagonists; Female; Humans; Neoplasm Invasiveness; Neovascularization, Pathologic; Proteins; Receptors, Estrogen; Response Elements; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor alpha; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Constitutive bcl-2 overexpression increases the tumorigenic and metastatic potential of doxorubicin-resistant, estrogen-independent, MCF-7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl-2-overexpressing MCF-7 ADR clones and control neomycin-transfected MCF-7 ADR neo cells. The 2 bcl-2-overexpressing MCF-7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF-7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF-7 ADR neo and bcl-2-overexpressing MCF-7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor-alpha (TGF-alpha). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose-dependent manner (IC(50) of approximately 0.1 microm). This effect was accompanied by a dose-dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF-alpha, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl-2-overexpressing MCF-7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose-dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl-2 expression in bcl-2-overexpressing MCF-7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone-independent, multidrug-resistant, human breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Bridged-Ring Compounds; Cell Division; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; ErbB Receptors; Female; Fibroblast Growth Factor 2; Gefitinib; Humans; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Taxoids; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Activation of mitogen-activated protein kinase (Erk/MAPK) is a critical signal transduction event for estrogen (E(2))-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E(2) induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E(2)-induced Erk activation is dependent on new protein synthesis, suggesting that E(2)-induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E(2) in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60-70% of E(2)-induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E(2)-treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10-20% of E(2)-induced Erk activation, suggesting that E(2)-induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-delta-mediated signaling by a dominant negative mutant or the relatively specific PKC-delta inhibitor rottlerin blocked most of the E(2)-induced Erk activation but had no effect on TGF alpha-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E(2)- and TGF alpha-induced Erk activation. This evaluation of downstream signaling revealed that E(2)-induced Erk activation is mediated by a HRG/HER-2/PKC-delta/Ras pathway that could be crucial for E(2)-dependent growth-promoting effects in early stages of tumor progression.

Topics: Blotting, Western; Breast Neoplasms; Cell Division; Cycloheximide; Disease Progression; DNA, Complementary; Enzyme Activation; ErbB Receptors; Estradiol; Genes, Dominant; Humans; Isoenzymes; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Monomeric GTP-Binding Proteins; Neuregulin-1; Precipitin Tests; Protein Binding; Protein Kinase C; Protein Kinase C-delta; Protein Synthesis Inhibitors; Receptor, ErbB-2; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Neoplasms, Hormone-Dependent; Protein Kinase C; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Up-Regulation

Human prolactin (hPRL) has been shown to be one of the important survival/growth factors that promotes the proliferation of breast cancer cells in an autocrine/paracrine manner. In our recent studies, we demonstrated that a hPRL antagonist with a single amino acid substitution mutation (hPRL-G129R) was able to inhibit breast cancer cell proliferation via induction of apoptosis (1). In this study three independent yet related experiments were carried out regarding the effects of hPRL-G129R in breast cancer cells. We investigated the possible mechanism(s) of hPRL-G129R induced apoptosis in breast cancer cells. It is well documented that transforming growth factors (TGF) in conjunction with hormones such as estrogen and PRL play a major role in modulating the proliferation and apoptosis of mammary cells. We first investigated the relationships between hPRL/hPRL-G129R and TGFs. We show that hPRL is able to down-regulate TGF beta 1 (apoptotic factor) secretion and up-regulate TGF alpha (survival factor) secretion in a dose-dependent manner in T-47D cells. More importantly the hPRL antagonist up-regulates TGF beta 1 and down-regulates TGF alpha secretion. When hPRL-G129R was applied together with hPRL, it blocked the effects of hPRL. Secondly, we tested the possible involvement of caspases in hPRL-G129R induced apoptosis. We have shown that caspase-3 is activated by hPRL-G129R at a concentration of 250 ng/ml in T-47D breast cancer cells. Thirdly, we explored the additive effects of an anti-neoplastic drug, cisplatin, with the hPRL-G129R in T47D breast cancer cells. We show that cisplatin and hPRL-G129R when applied together resulted in about 40% growth inhibition in T-47D cells.

Topics: Amino Acid Substitution; Antineoplastic Combined Chemotherapy Protocols; Arginine; Breast Neoplasms; Caspase 3; Caspases; Cell Division; Cisplatin; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Female; Glycine; Humans; Point Mutation; Prolactin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

4-Hydroxytamoxifen (4-OHT), a selective estrogen receptor modulator, is an agonist at a transforming growth factor-alpha (TGF-alpha) target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with wild-type human ERalpha. In contrast, raloxifene (Ral) is a complete antiestrogen silencing activation function (AF) 1 and AF2 in this system. A natural mutation D351YERalpha enhances 4-OHT agonist activity and changes Ral-like compounds from antagonists to partial agonists. We reasoned that: either the conformation of the Ral-D351YERalpha is altered, thereby reactivating AF2 in the ligand binding domain, or the change at amino acid 351 allosterically reactivates AF1 in the Ral-D351YERalpha complex. Unlike the estradiol-ERalpha complex, agonist activity of 4-OHT and raloxifene through ERalpha and D351YERalpha were not attributed to coactivator (such as SRC-1, AIB1) binding to the ligand binding domain. We conclude that the classic AF2 is not responsible for the agonist activities of 4-OHT-ERalpha, 4-OHT-D351YERalpha, and Ral-D351YERalpha. To address the role of AF1, stable transfectants of ERalpha or D351YERalpha with an AF1 deletion (D351deltaAF1, D351YdeltaAF1) were generated in MDA-MB-231 cells. Additionally, D538A/E542A/D545A triple mutations within helix 12 (D351-3m, D351Y3m) or the COOH-terminal 537 deletion (D351delta537) were tested. The agonist activities of 4-OHT and raloxifene were lost in these stable transfectants, but antiestrogenic action was retained. The reactivation of an estrogen-like property of the Ral-ERalpha complex through AF1 with the D351Y mutation illustrates a novel allosteric mechanism for the selective estrogen receptor modulator ERalpha complex.

Topics: Allosteric Regulation; Breast Neoplasms; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; Protein Conformation; Raloxifene Hydrochloride; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Tamoxifen; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Epidermal growth factor receptor (EGFR) is a transmembrane receptor whose overexpression in breast cancer predicts for poor prognosis and is inversely correlated with expression of estrogen receptor (ER). This study was designed to investigate whether estrogen plays an active role in suppression of EGFR expression in estrogen-responsive breast cancer cell lines expressing low levels of EGFR. Upon withdrawal of estrogen, EGFR mRNA and protein increased 3-6 fold in MCF-7, T47D, and BT474 ER+ breast cancer cells. This was reversible upon addition of estradiol back to the culture media, but only after prolonged treatment. Nuclear run-on assays and studies with the transcription inhibitor actinomycin D demonstrated that regulation is at the transcriptional level. These results indicate that in the presence of estrogen, ER+ breast cancer cells possess active mechanisms to suppress EGFR expression. Up-regulation of EGFR in response to estrogen depletion and growth inhibition could represent an attempt to rescue cell growth by utilizing an alternative pathway. Indeed, we found that estrogen-depleted breast cancer cells are more sensitive to the mitogenic effects of EGF and TGF-alpha, and simultaneous blockade of both estrogen and EGFR signaling pathways induced cell death. J. Cell. Biochem. Suppl. 36: 232-246, 2001.

Topics: Blotting, Western; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor Modulators; Estrogens; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Neoplasms, Hormone-Dependent; Phosphorylation; Precipitin Tests; Receptors, Estrogen; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

We have shown that ER-negative and invasive human breast cancer cell lines MDA-MB-468 and MDA-MB-231 have constitutively higher mitogen activated protein kinase (ERK1&2/MAPK) when compared to the ER-positive and non-invasive MCF-7 human breast cancer cells. In MCF-7 cells, TGFalpha stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFalpha stimulation induced sustained MAPK activation, which correlated with enhanced cell motility and in vitro invasion. Serum stimulation activates ERK/MAPK activity persistently in both ER-positive and ER-negative breast cancer cells, leading to enhanced and sustained cell migration. Inhibition of MAPK activation by anti-sense MEK expression in MDA-MB-468 cells significantly inhibits cell migration and in vitro invasion. In contrast, MCF-7 cells expressing constitutively activated MEK show a significant increase in MAPK activity and cell migration, but this failed to enhance in vitro invasion. The kinetic profiles of MAPK activation and inhibition show a relationship between the duration and magnitude of MAPK activation and cell migration in both ER-positive and ER-negative human breast cancer cells. These studies show that cell motility is modulated by the magnitude and the duration of MAPK activation; but increased activation of MAPK may not be sufficient to allow in vitro invasion in non-invasive MCF-7 breast cancer cells.

Topics: Breast Neoplasms; Enzyme Activation; Humans; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Estrogen; Transforming Growth Factor alpha; Tumor Cells, Cultured

Estrogens are involved in a multiplicity of programmed events in target tissues e.g.: uterus, breast, and pituitary gland, and hormone-responsive tumors occur at these target sites. We have addressed the possibility that all of the estrogens do not produce the same conformation of estrogen receptor alpha (ER). A novel assay in vitro was used to activate the transforming growth factor alpha (TGF-alpha) gene in situ in MDA-MB-231 cells stably transfected with cDNA for D351 ER or D351G ER. Three estrogen types were used: estradiol, diethylstilbestrol, and a triphenylethylene (TPE) derivative of tamoxifen without the antiestrogenic side chain. Computer molecular modeling was used to interpret data. A flat estrogen such as estradiol or diethylstilbestrol can induce TGF-alpha through a correctly positioned activating function 2 (AF2) and bind SRC-1. The TPE did not activate AF2 but activated the TGF-alpha gene through AF2b. This was demonstrated because D351 but not D351G ER activated the TGF-alpha gene with the TPE. We propose two classes of estrogens with different ER complexes that may incorporate different coactivators to function. Phytoestrogens and environmental xenoestrogens will fall into different classes based on structure and may exhibit selective actions and carcinogenic potential based on different ER conformations.

Topics: Binding Sites; Breast Neoplasms; Estradiol Congeners; Estrogens; Humans; Models, Molecular; Protein Conformation; Receptors, Estrogen; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

CITED1, a CBP/p300-binding nuclear protein that does not bind directly to DNA, is a transcriptional coregulator. Here, we show evidence that CITED1 functions as a selective coactivator for estrogen-dependent transcription. When transfected, CITED1 enhanced transcriptional activation by the ligand-binding/AF2 domain of both estrogen receptor-alpha (ERalpha) and ERbeta in an estrogen-dependent manner, but it affected transcriptional activities of other nuclear receptors only marginally. CITED1 bound directly to ERalpha in an estrogen-dependent manner through its transactivating domain, and this binding activity was separable from its p300-binding activity. CITED1 was strongly expressed in nulliparous mouse mammary epithelial cells and, when expressed in ER-positive MCF-7 breast cancer cells by transduction, exogenous CITED1 enhanced sensitivity of MCF-7 cells to estrogen, stabilizing the estrogen-dependent interaction between p300 and ERalpha. The estrogen-induced expression of the transforming growth factor-alpha (TGF-alpha) mRNA transcript was enhanced in the CITED1-expressing MCF-7 cells, whereas estrogen-induced expression of the mRNA transcripts for progesterone receptor or pS2 was not affected. Chromatin immunoprecipitation assay revealed that endogenous CITED1 is recruited to the chromosomal TGF-alpha promoter in MCF-7 cells in an estrogen-dependent manner but not to the pS2 promoter. These results suggest that CITED1 may play roles in regulation of estrogen sensitivity in a gene-specific manner.

Topics: Animals; Base Sequence; Breast Neoplasms; DNA Primers; E1A-Associated p300 Protein; Estrogens; Mice; Nuclear Proteins; Promoter Regions, Genetic; RNA, Messenger; Trans-Activators; Transcriptional Activation; Transforming Growth Factor alpha; Tumor Cells, Cultured

The transforming growth factor alpha (TGFalpha) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGFalpha and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGFalpha is evident in the transgenic mice overexpressing TGFalpha in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGFalpha transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGFalpha double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGFalpha, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.

Topics: Animals; Aromatase; Breast Neoplasms; ErbB Receptors; Female; Gene Expression; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Neoplasms, Hormone-Dependent; Transforming Growth Factor alpha

Aberrrant signaling by the epidermal growth factor receptor [EGFR (HER1, erbB1)] and/or HER2/neu tyrosine kinases is present in a cohort of breast carcinomas. Because HER2 is constitutively phosphorylated in some breast tumors, we speculated that, in these cancers, transmodulation of HER2 may occur via EGFR signaling. To test this possibility, we examined the effect of EGFR-specific kinase inhibitors against the HER2-overexpressing human breast tumor lines BT-474, SKBR-3, MDA-361, and MDA-453. ZD1839 (Iressa) is an ATP-mimetic that inhibits the purified EGFR and HER2 kinases in vitro with an IC(50) of 0.033 and >3.7 microM, respectively. The specificity of ZD1839 against EGFR was confirmed in Rat1 fibroblasts transfected with EGFR or HER2 chimeric receptors activated by synthetic ligands without the interference of endogenous receptors. Treatment of all breast cancer cell lines (except MDA-453) with 1 microM ZD1839 almost completely eliminated HER2 phosphorylation. In contrast, the incorporation of [gamma-(32)P]ATP in vitro onto HER2 receptors isolated from BT-474 cells was unaffected by 1 microM ZD1839. EGFR is expressed by BT-474, SKBR-3, and MDA-361 but not by MDA-453 cells, suggesting that ZD1839-mediated inhibition of the EGFR kinase explained the inhibition of HER2 phosphorylation in vivo. In SKBR-3 cells, ZD1839 exhibited a greater growth-inhibitory effect than Herceptin, a monoclonal antibody against the HER2 ectodomain. In both SKBR-3 and BT-474 cells, treatment with ZD1839 plus Herceptin induced a greater apoptotic effect than either inhibitor alone. Finally, ZD1839 completely prevented growth of BT-474 xenografts established in nude mice and enhanced the antitumor effect of Herceptin. These data imply that EGFR tyrosine kinase inhibitors will be effective against HER2-overexpressing breast tumor cells that also express EGFR and support their use in combination with HER2 antibodies, such as Herceptin, against mammary carcinomas with high levels of the HER2 proto-oncogene.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Catalytic Domain; Cell Cycle; Cell Division; Cell Survival; Drug Synergism; Enzyme Inhibitors; ErbB Receptors; Female; Gefitinib; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Proto-Oncogene Mas; Quinazolines; Receptor, ErbB-2; Signal Transduction; Substrate Specificity; Transforming Growth Factor alpha; Trastuzumab; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Animals; Breast Neoplasms; Disease Models, Animal; Genes, myc; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; Signal Transduction; Transforming Growth Factor alpha

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.

Topics: Base Sequence; Breast Neoplasms; Cathepsin D; Cell Division; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Genes, Reporter; Growth Substances; Humans; Insulin-Like Growth Factor I; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Receptors, Estrogen; Sequence Deletion; Transcriptional Activation; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

We investigated tamoxifen's effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation.. Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-alpha, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques.. Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P <. 003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-alpha, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens.. These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.

Topics: Adrenomedullin; Antineoplastic Agents, Hormonal; Breast Neoplasms; Endometrial Neoplasms; Endometrium; Female; Gene Expression Regulation, Neoplastic; Genes, Regulator; Humans; Hyperplasia; Immunohistochemistry; Ki-67 Antigen; Lactoferrin; Peptides; Receptors, Estrogen; Receptors, Progesterone; Receptors, Tumor Necrosis Factor; Tamoxifen; Transforming Growth Factor alpha

The ErbB receptor tyrosine kinase family consists of the epidermal growth factor (EGF) receptor (ErbB1) and three related receptors (ErbB2, ErbB3, ErbB4). Their intrinsic tyrosine kinases can be activated by receptor-dimerization induced by numerous ligands or overexpression. ErbB receptors are frequently overexpressed in breast cancer, and their overexpression is associated with protection from apoptosis. To directly assess their role in apoptosis sensitivity of breast cancer cells, we established MCF-7 breast carcinoma cell lines overexpressing each ErbB receptor alone or in all possible pairs. Overexpression of ErbB1, ErbB2 and ErbB4 receptors was enough to activate them as judged by their phosphorylation, whereas co-expression of other ErbB receptors was necessary for the phosphorylation of the ErbB3. Surprisingly, overexpression of the ErbB receptors even when combined with treatment with their ligands (EGF, transforming growth factor alpha, betacellulin, neuregulins) failed to protect the MCF-7 cells from cell death induced by either tumor necrosis factor (TNF) or serum starvation. During starvation TGF-alpha, however, increased the cell size of the ErbB1 overexpressing cell line, and neuregulin1-beta1 increased that of all cell lines. In conclusion, our data does not support the role of ErbB receptors in the regulation of cell death induced by TNF or serum starvation, and the observed association in breast cancer may be due to other concomitant changes.

Topics: Breast Neoplasms; Cell Count; Cell Death; Cell Size; Culture Media, Serum-Free; ErbB Receptors; Female; Humans; Ligands; Neuregulin-1; Phosphorylation; Receptor, ErbB-2; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tyrosine

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

Topics: Animals; Base Sequence; Breast Neoplasms; DNA Footprinting; Drosophila; Estradiol; GC Rich Sequence; Humans; Molecular Sequence Data; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Sequence Deletion; Transcriptional Activation; Transforming Growth Factor alpha; Tumor Cells, Cultured

A pilot study of a novel translational research method to simultaneously assay multiple molecular markers and DNA in fine-needle aspirates (FNA) of mammographically detected breast lesions is described. Specimen mammography-guided 20-gauge FNAs obtained from 86 lesions and 22 areas of normal tissue were analyzed by multiparameter flow cytometry for DNA content, her2/neu, transforming growth factor alpha (TGF alpha), and the epithelial marker cytokeratin (CK) simultaneously. Epithelial cell her2/neu positivity was detected in 12 of 44 (27%) of invasive ductal carcinomas and 3 of 9 (33%) ductal carcinoma in situ (DCIS), 10 of 30 (33%) benign lesions, and 4 of 22 (18%) normal tissue aspirates. All lesions and normal tissue showed a similar positive rate for TGFalpha ranging from 61 to 76%. The CK(+)TGF alpha(-)her2/neu(+) immunophenotype was more frequently positive in aneuploid tumors (22%) than all other lesions (7%) (P < 0.05). Specimen mammography-guided FNAs provide fresh cells for flow cytometric multiple marker analysis and immunophenotyping of clinically occult breast lesions and normal tissue.

Topics: Adult; Aged; Aged, 80 and over; Aneuploidy; Biomarkers, Tumor; Biopsy, Needle; Breast; Breast Neoplasms; Carcinoma; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Diploidy; DNA; Female; Flow Cytometry; Humans; Immunophenotyping; Keratins; Mammography; Middle Aged; Pilot Projects; Receptor, ErbB-2; Transforming Growth Factor alpha

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.

Topics: Anisomycin; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; DNA-Binding Proteins; Doxorubicin; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Paclitaxel; Phenotype; Precipitin Tests; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Epidermal growth factor receptor (EGF-R) and its ligand, transforming growth factor-alpha (TGF-alpha), play an important role through the autocrine growth-regulation system in several human cancers, including breast cancer. However, the clinical significance of co-expression of EGF-R and TGF-alpha has not been elucidated. One hundred seventy-three female patients diagnosed as invasive ductal carcinoma who had undergone a mastectomy (159 patients) or breast-conserving surgery (14 patients) were followed up for 81 to 119 months (median 94 months) post-operatively. Immunoreactivity for EGF-R, TGF-alpha, p53 and c-erbB-2 with paraffin-embedded carcinoma tissue was investigated using labeled streptavidin-biotin methods. Positive rates of carcinoma cells were 27%, 33%, 32% and 26% for EGF-R, TGF-alpha, p53 and c-erbB-2, respectively. Expression of EGF-R only was observed in 16% (28/173), of TGF-alpha only in 22% (38/173), of both EGF-R and TGF-alpha in 11% (19/173) and of neither in 51% (88/173). By univariate analysis, significant differences in overall survival and disease-free survival were noted according to the co-expression of EGF-R and TGF-alpha (p< 0.0001, p<0.0001), co-expression of EGF-R and c-erbB-2 (p = 0.0029, p = 0.0028), nodal status (p = 0.0028, p = 0.0001), tumor size (p = 0.0001, p<0.0001) and c-erbB-2 expression (p = 0.0034, p = 0.018), respectively. The status of p53 expression (p = 0.01), estrogen receptor (p = 0.042) and progesterone receptor (p = 0.046) showed significant differences in overall survival. According to Cox's multivariate analysis, co-expression of EGF-R and TGF-alpha had the most significant effect on disease-free survival (p<0.0001) and overall survival (p<0.0001), followed by nodal status. Co-expression of EGF-R and TGF-alpha by immunohistochemical detection is an independent prognostic indicator, and it may be helpful for determining the group of breast-cancer patients with an aggressive phenotype.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Disease-Free Survival; ErbB Receptors; Female; Humans; Middle Aged; Multivariate Analysis; Predictive Value of Tests; Proportional Hazards Models; Receptor, ErbB-2; Survival Analysis; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

The immunologic response to prosthetic biomaterial particles is characterized by macrophage-rich inflammatory infiltrate, formation of multinucleated giant cells, and aseptic loosening at the site of arthroplasty. We investigated the in vivo expression and tissue distribution of transforming growth factor alpha, macrophage colony-stimulating factor, and the receptor for colony-stimulating factor-1 at the site of bone erosion in patients with clinically failed orthopaedic implants (n = 30). The expression was further compared with that detected in the inflamed synovial membranes from patients with rheumatoid arthritis or osteoarthritis (n = 15) and one patient with osteoclastoma (giant cell tumour of bone). Immunostaining of the tissue demonstrated positivity for transforming growth factor alpha within the inflammatory macrophage and multinucleated giant cell infiltrate in the diseased synovial membrane and the bone-implant interface. A comparative analysis between the synovium and retrieval interface membranes (pseudosynovium) revealed a high level of expression of transforming growth factor alpha, with intense membrane staining on multinucleated giant cells in all failed arthroplasties with pseudosynovium. In addition, the frequency, antigenic phenotype, and pattern of transforming growth factor alpha expression on multinucleated giant cells in the interface were markedly similar to those observed for multinucleated giant cells in osteoclastoma. Multinucleated giant cells within the interface lacked the expression of macrophage colony-stimulating factor and colony-stimulating factor-1 receptor, whereas those at the bone surfaces exhibited strong immunoreactivity. The predominant expression of transforming growth factor alpha by multinucleated giant cells in the bone-implant interface and its similarity to osteoclastoma highlight the importance of assessing transforming growth factor alpha as a possible contributor to the development of bone-resorbing giant cells at the site of failed orthopaedic implants.

Topics: Arthritis, Rheumatoid; Arthroplasty, Replacement, Hip; Arthroplasty, Replacement, Knee; Bone Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Fluorescent Antibody Technique, Indirect; Giant Cell Tumor of Bone; Giant Cells; Hip Prosthesis; Humans; Knee Prosthesis; Macrophage Colony-Stimulating Factor; Osteoarthritis; Osteolysis; Receptors, Colony-Stimulating Factor; Synovial Membrane; Transforming Growth Factor alpha

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.

Topics: Amphiregulin; Biomarkers, Tumor; Breast Neoplasms; Cell Division; Cell Survival; EGF Family of Proteins; Epidermal Growth Factor; Female; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Polymerase Chain Reaction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Breast Neoplasms; Humans; Immunohistochemistry; Transforming Growth Factor alpha

Resveratrol is a natural phytoalexin compound found in grapes and other food products. In this study, the effect of resveratrol on the growth of human breast cancer cells was examined. Results show that resveratrol inhibits the growth of estrogen receptor(ER)-positive MCF-7 cells in a dose-dependent fashion. Detailed studies with MCF-7 cells demonstrate that resveratrol antagonized the growth-promoting effect of 17-beta-estradiol (E2) in a dose-dependent fashion at both the cellular (cell growth) and the molecular (gene activation) levels. At 5 x 10(-6) M, resveratrol abolished the growth-stimulatory effect mediated by concentrations of E2 up to 10(-9) M. The antiestrogenic effect of resveratrol could be observed at a concentration of 10(-6) M and above. The antiestrogenic effect of resveratrol was also demonstrated at the molecular level. Resveratrol in a dose-dependent fashion antagonized the stimulation by E2 of progesterone receptor gene expression in MCF-7 cells. Moreover, expression of transforming growth factor-alpha and insulin-like growth factor I receptor mRNA was inhibited while the expression of transforming growth factor beta2 mRNA was significantly elevated in MCF-7 cells cultivated in the presence of resveratrol (10(-5) M). In summary, our results show that resveratrol, a partial ER agonist itself, acts as an ER antagonist in the presence of estrogen leading to inhibition of human breast cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Division; Culture Media; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Gene Expression Regulation, Neoplastic; Humans; Receptor, IGF Type 1; Resveratrol; RNA, Messenger; Rosales; Stilbenes; Transcriptional Activation; Transforming Growth Factor alpha; Tumor Cells, Cultured

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth an

Topics: Antibodies; Breast Neoplasms; Butadienes; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Carcinoma, Squamous Cell; Cell Death; Cell Division; DNA, Antisense; Dose-Response Relationship, Radiation; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Protein Kinases; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine

Transforming growth factor-alpha (TGF-alpha) contributes to the progression of mammary carcinogenesis in part through synergistic augmentation of estradiol (E2) action. To investigate this further, we sought to determine (1) whether the duration of TGF-alpha treatment might influence the nature of the TGF-alpha/E2 interaction, and (2) whether TGF-alpha would behave in a similar manner when combined with phytoestrogens. To this end, we transfected T47-D breast cancer cells with an estrogen-responsive reporter and then treated the cells (for 4-48 h) with varying concentrations of TGF-alpha, E2, the antiestrogen 4-hydroxy-tamoxifen (HOT), and/or one of three phytoestrogens. Our findings revealed that TGF-alpha has short-term synergistic and long-term inhibitory effects on E2- and phytoestrogen-regulated gene expression. Furthermore, this secondary inhibition of E2 action by TGF-alpha was similar in magnitude to that imposed by HOT. These findings demonstrate a novel role for TGF-alpha and invite reevaluation of current models regarding TGF-alphas interactions with E2 in breast cancer cells. Our results also raise the possibility that phytoestrogens, which interact with TGF-alpha in a manner conceptually identical to that of E2, may subserve a regulatory function in breast cancer cells.

Topics: Adenocarcinoma; Breast Neoplasms; Drug Synergism; Estrogen Antagonists; Estrogens; Estrogens, Non-Steroidal; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Luciferases; Phytoestrogens; Plant Preparations; Receptors, Estrogen; Tamoxifen; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

MDA-468 human breast cancer cells overexpress the EGFR and exhibit a functional TGFalpha-EGFR autocrine pathway. Loss of EGFR expression following stable transfection with an antisense EGFR cDNA containing plasmid down-regulates type I cAMP-dependent protein kinase (PKAI) expression with acquisition of cell growth resistance to the PKAI inhibitor 8-Cl-cAMP. These results suggest that PKAI expression and function are controlled by a TGFalpha-EGFR autocrine pathway in human breast cancer cells overexpressing the EGFR.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Breast Neoplasms; Cyclic AMP-Dependent Protein Kinases; DNA, Antisense; Down-Regulation; ErbB Receptors; Female; Humans; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Autocrine Communication; Biomarkers, Tumor; Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Neoplasms, Hormone-Dependent; Paracrine Communication; Prognosis; Transforming Growth Factor alpha

The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17beta-estradiol and raloxifene [A. M. Brzozowski et al., Nature (Lond.), 389: 753-758, 1997] now provides a molecular basis for the biological activity of complexes as either agonists or antagonists. It is well established that the critical structural feature of antiestrogens is a correctly positioned alkylaminoethoxy side chain. The X-ray crystallography clearly shows that the alkylaminoethoxy side chain of raloxifene causes a specific and inappropriate molecular perturbation of the LBD and that the nitrogen in the side chain must hydrogen bond with aspartate 351 in the LBD of ER. We previously identified and characterized a naturally occurring mutation in the ER from a tamoxifen-stimulated transplantable human breast tumor line. The mutation is at AA351 of LBD, where the aspartate is changed to tyrosine (Asp351Tyr). In this report, we compared and contrasted the pharmacology of raloxifene to block or induce E2-stimulated increase in TGF-alpha mRNA in stable transfectants of ER-negative human breast cancer cells with the cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mutant-AA 351 ER. Our results show that the mutation at AA 351 that replaces aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen. By contrast, a mutation at AA 400 does not, and the antiestrogenic properties of raloxifene are retained. These data and the fact that the nitrogen in the side chain must specifically interact with aspartate 351 makes this the key to the antiestrogenic activity of raloxifene.

Topics: Aspartic Acid; Blotting, Northern; Breast Neoplasms; Estradiol; Estrogen Antagonists; Female; Humans; Mutagenesis, Site-Directed; Piperidines; Point Mutation; Raloxifene Hydrochloride; Receptors, Estrogen; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Oestrogens and antioestrogens modulate the synthesis of transforming growth factor alpha (TGF-alpha) in breast cancer cells. The purpose of the present report was to examine regulation of TGF-alpha gene expression by oestradiol (E2) and antioestrogens in MDA-MB-231 breast cancer cells transfected with either the wild-type or mutant oestrogen receptor (ER). We recently reported the concentration-dependent E2 stimulation of TGF-alpha mRNA in MDA-MB-231 ER transfectants (Levenson et al, 1997). We now report that 4-hydroxytamoxifen (4-OHT) shows oestrogen-like effects on the induction of TGF-alpha gene expression in our transfectants. Accumulation of TGF-alpha mRNA in response to both E2 and 4-OHT but not in response to the pure antioestrogen ICI 182,780 suggests that E2-ER and 4-OHT-ER complexes can bind to an oestrogen response element (ERE), located in the promoter region of the TGF-alpha gene and can activate transcription of the gene. Surprisingly, no activation of luciferase expression was observed after transient transfection of the TGF-alpha ERE/luciferase reporter constructs. Possible activation of an alternative ER-mediated pathway responsible for the regulation of TGF-alpha gene expression in the ER transfectants is discussed.

Topics: Breast Neoplasms; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogens; Female; Fulvestrant; Humans; Receptors, Estrogen; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha

The presence of epidermal-growth-factor receptors (EGFR) and of its ligands (TGFalpha and amphiregulin) in breast-cancer tissues suggests that they play a paracrine/autocrine role in tumor growth or progression. This hypothesis was tested on 3 cell lines, S2T2, NS2T2A and NS2T2A1. These epithelial cells are derived from a normal human breast-epithelial-cell culture transformed by SV40-T Ag, are of the same clonal origin, have respectively increasing levels of EGFR, TGFalpha, amphiregulin and of thymidine-kinase activity associated with increasing tumorigenic potential in nude mice (tumor intake and tumor volume). The monoclonal antibody MAb 425, which blocks ligands interaction with EGFR, reduced by more than 90% anchorage-independent growth of the most tumorigenic cells, NS2T2A1. Another anti-EGFR MAb, 528, reduced to 25% of controls the mean tumor mass after NS2T2A1 grafting in mice. Anti-sense RNA expression of EGFR in these cells confirmed the importance of this receptor in tumor progression, since it reduced significantly the tumor volume and tumor weight of NS2T2A1 cells to 16% of those in mock-transfected control cells.

Topics: Amphiregulin; Animals; Antibodies, Monoclonal; Breast Neoplasms; Cell Division; Cell Line, Transformed; Disease Progression; EGF Family of Proteins; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Nude; Neoplasm Transplantation; RNA, Antisense; Transforming Growth Factor alpha; Tumor Cells, Cultured

The EGF domain of heregulin has all the receptor binding characteristics of full-length heregulin and has strong homology to the ligands for erbB-1. Despite this, it does not bind erbB-1 but instead binds erbB-3 and erbB-4. The sequence similarity between HRG and the erbB-1 ligands suggest that a few residues are responsible for receptor binding specificity. To determine the sequences involved in receptor binding, we performed homologue scanning mutagenesis on the EGF domain of HRGalpha using sequences of TGFalpha or EGF. We found three sets of mutations in the N-terminal subdomain that were responsible for receptor binding specificity. Mutations in the C-terminal subdomain affected the binding affinity, but did appear to confer any specificity.

Topics: Adenocarcinoma; Amino Acid Sequence; Binding, Competitive; Breast Neoplasms; Epitope Mapping; Glycoproteins; Humans; Ligands; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Growth Factors; Neuregulins; Receptor, ErbB-2; Receptors, Nerve Growth Factor; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Transforming Growth Factor alpha; Tumor Cells, Cultured

Antisense oligonucleotides (oligos) are artificial sequences of nucleotide bases which may be synthesized complementary to known regions within specific mRNAs. When these constructed oligos interact with protein encoding mRNA they may regulate expression of various growth factors and/or their receptors. Oligos directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), were employed: A) in vitro to affect the growth of hormone insensitive human derived PC-3 prostate cancer cells as well as the human derived UACC-893 breast cancer cell line; and B) in vivo to treat tumors established by these cell lines in athymic nude mice. The in vitro results for each oligo, and their combination, produced significant inhibition of both prostate and breast cell lines. In addition, the combination of oligos most efficiently diminished the immunohistochemical expression of both TGF-alpha and EGFR in PC-3 cells. Direct in vivo inoculation of oligos into established PC-3 or UACC-893 tumors in nude mice produced hemorrhagic necrosis within 2-3 days. Such therapy could represent a new tier of therapy for recurrent, hormone insensitive, tumors based upon the concept of growth factor deprivation.

Topics: Animals; Binding Sites; Breast Neoplasms; ErbB Receptors; Growth Substances; Humans; Male; Mammary Neoplasms, Animal; Mice; Mice, Nude; Oligonucleotides, Antisense; Prostatic Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Human breast cancer cells synthesize and release a variety of growth-modulating substances in response to estrogen stimulation, and it is generally accepted that the growth-promoting effects of estrogens are due at least in part to this autocrine/paracrine mechanism. Several of these growth-modulating substances, including transforming growth factor-alpha (TGF alpha) and its analogs, have been shown to require pericellular proteolysis for activation or release. Recently, we reported that MCF-7 human breast cancer cells are able to synthesize alpha 1-antitrypsin (alpha 1-AT), the major elastase inhibitor in human serum, and that there is a negative correlation between anchorage-independent growth of MCF-7 cells in soft agar and synthesis of alpha 1-AT. The studies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGF alpha from its membrane-bound precursor on MCF-7 cells is blocked by alpha 1-AT whether the cells were maintained in the presence or absence of estradiol and that there is a clear dose-response relationship between the alpha 1-AT concentration and both the release of TGF alpha and growth in soft agar. Consistent with this, TGF alpha release was increased in the presence of antibody to alpha 1-AT. In contrast, TGF alpha release and growth in soft agar were not blocked by peptide inhibitors specific for trypsin- or chymotrypsin-like enzymes. The alpha 1-AT concentration required for a half-maximal effect is lower for inhibition of TGF alpha release than it is for inhibition of colony formation (0.4 vs. 1.5 mumol/L). However, both values are in the range of concentrations one might expect at the cell surface in vivo. A new MCF-7 cell subline producing 10-fold higher levels of alpha 1-AT than its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of alpha 1-AT) with an alpha 1-AT complementary DNA. Growth in soft agar and release of TGF alpha were significantly decreased in cells transfected with the alpha 1-AT complementary DNA compared to those in cells transfected with vector alone, although, TGF alpha expression was the same. The above observations support a model for growth regulation in human breast ductal epithelial cells in which growth factor activation and release are dependent on the coordinate action of proteases and protease inhibitors. This model would predict that alpha 1-AT can act as a tumor sup

Topics: alpha 1-Antitrypsin; Breast Neoplasms; Cell Membrane; Dose-Response Relationship, Drug; Endopeptidases; Estradiol; Female; Humans; Solubility; Transforming Growth Factor alpha; Tumor Cells, Cultured

Epidemiological and experimental data suggest a role for polyunsaturated fatty acids in the etiology of breast cancer. In this report we have studied arachidonic acid and linoleic acid metabolism in the human breast carcinoma cell line BT-20 which overexpresses both EGF receptor and the homologous erbB-2 oncogene product. EGF and TGF alpha stimulated DNA synthesis in these cells which was attenuated by the addition of a lipoxygenase inhibitor, NDGA. The addition of a prostaglandin H synthase inhibitor did not alter DNA synthesis. Analytical studies reveal little arachidonic acid metabolism while linoleic acid was metabolized to 13-hydroxyoctadecadienoic acid (13-HODE). The formation of 13-HODE was inhibited by the addition of NDGA and was dependent on EGF or TGF alpha. These results suggest the metabolism of linoleic acid by a n-6 or 15-lipoxygenase regulated by EGF/TGF alpha, RT-PCR was used to isolate a clone, and sequenced the cDNA for this enzyme and it was found to be identical to the human 15-lipoxygenase previously characterized from human pulmonary tissue. EGF/TGF alpha did not alter the expression of this enzyme suggesting a potential post-translational regulation of activity. This study provides a link between metabolism of linoleic acid and growth factor regulation of cell proliferation in a human breast carcinoma cell line.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acid; Breast Neoplasms; Cyclooxygenase Inhibitors; DNA; Epidermal Growth Factor; Female; Humans; Indomethacin; Linoleic Acid; Linoleic Acids; Lipoxygenase Inhibitors; Masoprocol; Polymerase Chain Reaction; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Treatment of estrogen receptor (ER)-negative MDA-MB-468 human breast cancer cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced formation of a nuclear aryl hydrocarbon (Ah) receptor complex as determined by ligand-binding and gel electrophoretic mobility shift assays. TCDD also induced CYP1A1-dependent activity in MDA-MB-468 cells, which represents the first ER-negative Ah receptor-positive human breast cancer cell line that has been identified. Treatment of this cell line with TCDD and related compounds also caused a 50% inhibition of cell growth, which resembled the growth inhibitory effects previously reported for epidermal growth factor (EGF). However, EGF expression is minimal in this cell line and is not induced by TCDD; moreover, EGF and TCDD induced a different pattern of oncogene expression and apoptosis in MDA-MB-468 cells. In contrast, TCDD caused a rapid and sustained induction of transforming growth factor alpha (TGF alpha) gene expression and secreted protein (nearly 2-fold); moreover, the growth-inhibitory effects of TCDD could be blocked by antibodies to the EGF receptor. In a separate experiment, it was shown that TGF alpha also inhibited growth of MDA-MB-468 cells. The results of this study indicate that the mechanism of growth inhibition of MDA-MB-468 cells by TCDD is due to induction of TGF alpha, which is a potent antimitogen in this cell breast cancer line.

Topics: Aryl Hydrocarbon Receptor Nuclear Translocator; Breast Neoplasms; Cell Division; Cell Nucleus; Cytochrome P-450 CYP1A1; Diethylstilbestrol; DNA-Binding Proteins; DNA, Neoplasm; Estradiol; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Growth Inhibitors; Humans; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

Peptide growth factors (GFs), including epidermal GF (EGF) and transforming GF-alpha (TGF-alpha), are presumed to play an important role in the local regulation of breast cell proliferation. Breast fluid collected by nipple aspiration provides a potential means to assess the concentration of these factors in contact with the ductal epithelium. Although identification of immunoreactive EGF-like GFs in breast fluid has been reported previously, we performed this study to evaluate the sensitivity and reliability of newer RIA methods and to characterize the sources and amounts of both intra- and intersubject variability. We also evaluated the relationship of breast fluid EGF and TGF-alpha levels to each other and to plasma levels of estradiol and progesterone. Breast fluid and plasma samples were obtained two to four times at weekly intervals from 18 healthy, premenopausal women. EGF and TGF-alpha were measured by competitive binding RIA. Both GFs were detected with good precision in all breast fluid samples analyzed, using dilutions as low as 1:100 for EGF (1 microliter) and 1:25 for TGF-alpha (4 microliters). The correlations between the right and left breasts, sampled concurrently, were r = 0.78 (P = 0.003) for EGF and r = 0.89 (P = 0.0001) for TGF-alpha. For both GFs, the variation between women was substantially greater than the variation between breasts or over time in an individual woman, particularly for EGF, for which there were 100-fold differences between women in mean levels. When samples from multiple women were analyzed together, we found no apparent relationships between EGF and TGF-alpha levels or between either GF level and menstrual cycle phase or plasma hormone concentrations. However, in random effects analyses, EGF levels within an individual were significantly associated overall with both TGF-alpha (P = 0.02) and plasma estradiol levels (P = 0.01). These data, which are the first comprehensive results on the feasibility of measuring mitogenic GFs in breast fluid, support the conclusion that women secrete consistent and individually distinct levels of EGF and TGF-alpha and that, in at least some women, EGF secretion in vivo covaries with both TGF-alpha in breast fluid and circulating estradiol.

Topics: Adult; Breast Neoplasms; Epidermal Growth Factor; Estradiol; Exudates and Transudates; Female; Humans; Menstrual Cycle; Middle Aged; Nipples; Progesterone; Radioimmunoassay; Reproducibility of Results; Sensitivity and Specificity; Suction; Transforming Growth Factor alpha

Among the peptide growth factors active in breast glandular cell proliferation epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are thought to play a major role in tumour development. They operate through binding to and activation of a common membrane receptor, defined as EGF-R. Their production is modulated by hormones and local growth factors. After it was shown by previous investigation in this laboratory that EGF-R could be detected in 90% of the tumours, but was masked by endogenous ligand in 36% of them, the question was raised as to the level of the ligand's expression in tumour tissue biopsies. Therefore, we investigated the expression of EGF and TGF alpha mRNA in 146 breast cancer biopsies by slot blot analysis using specific 32P-labelled probes. The data were correlated with sex steroids and EGF receptor content. Our results showed that EGF and TGF alpha coexisted in all tumour samples, and that their level of mRNA expression was similar in half of the tumours. Northern blot and polymerase chain reaction (PCR) analysis validated these findings. A significant direct correlation was found between the level of TGF alpha/EGF mRNA expression and the ER/progesterone receptor (PGR) content. TGF alpha and EGF mRNA levels were significantly higher in ER+ (P = 0.0015 and P = 0.0001, respectively) and in PGR+ tumours (P < 0.005 and P = 0.0001) than in their negative counterparts. Moreover, TGF alpha mRNA expression negatively correlated with the number of EGF-R binding sites measured by the standard method (P = 0.02), and it was significantly related to the number of sites occupied by endogenous ligand. In conclusion, it was shown that TGF alpha and EGF mRNA were coexpressed in all the tumour biopsies tested and that their level was higher in the hormone receptor positive than in negative samples. The correlation between the presence of ER/PGR sites, high level of TGF alpha/EGF mRNA and EGF-R occupancy by endogenous ligand is in favour of ER mediated control of TGF alpha and EGF production.

Topics: Biopsy; Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Receptors, Estradiol; Receptors, Progesterone; Receptors, Steroid; RNA, Messenger; RNA, Neoplasm; Statistics, Nonparametric; Transforming Growth Factor alpha

The estrogen receptor (ER) functions as a ligand-activated transcription factor which mediates the actions of estrogens and antiestrogens in target tissues. Other investigators have shown that artificial point mutations in the transcriptional activation domain AF-2 of the ligand binding domain (LBD) of the ER can increase the estrogenic properties of antiestrogens, determined by transcriptional activation of estrogen-responsive reporter constructs cotransfected into cells. Although these data provide valuable information about ER function there is no evidence that these mutations occur naturally. We have taken a different approach and examined the naturally occurring codon 351 asp --> tyr mutation in the LBD of ER to stimulate the expression of an endogenous target gene. This approach avoids dependence on artificial reporter constructs and their idealized estrogen response elements (EREs). In this report we describe the regulation of transforming growth factor alpha (TGF alpha) mRNA by estradiol and the antiestrogens keoxifene and ICI 182,780 in our stable transfectants of ER-negative MDA-MB-231 breast cancer cells, which express either the wild-type (S30 cells) or codon 351 asp --> tyr mutant ER (BC-2 cells). The mutant receptor was identified in a tamoxifen-stimulated human breast tumor. Our results demonstrate, for the first time, that a naturally occurring mutation in the ER changes the pharmacology of the antiestrogen keoxifene by increasing estrogenic activity, and that keoxifene exhibits a gene-specific estrogen-like effect with mutant ER but not with wild-type ER. The pure antiestrogen ICI 182,780 maintained complete antagonistic activities in both ER transfectants, demonstrating that its action is unaffected by the mutation.

Topics: Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Mutation; Piperidines; Raloxifene Hydrochloride; Receptors, Estrogen; RNA, Messenger; RNA, Neoplasm; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

The response of two endogenous, estrogen-induced genes, LIV-1 and pS2, to growth factor stimulation of MCF-7 cells was examined. Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-1 (IGF-1) were each able to induce an increase in the two mRNAs in the absence of estradiol, and their effects were additive to that of an optimally inducing concentration (10(-8) M) of the hormone. Induction by EGF and TGF alpha, but not by IGF-1, were also additive to induction by a saturating concentration (2 microg/ml) of insulin. TGFbeta, an antimitogenic growth factor for MCF-7 cells, did not induce LIV-1 or pS2 mRNA but inhibited induction by estradiol. Increases in mRNA were shown to reflect increases in specific gene transcription. Induction by growth factors, but not by estradiol, was dependent upon protein synthesis. Induction by both growth factors and estradiol was inhibited by the pure antiestrogen, ICI 164384 (ICI), and by the mixed agonist/antagonist, tamoxifen. Despite differences in patterns of expression in vivo and in vitro, both LIV-1 and pS2 appeared to be responsive to growth factors via a mechanism distinct from that of estradiol but requiring the estrogen receptor.

Topics: Breast Neoplasms; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Insulin-Like Growth Factor I; Neoplasm Proteins; Peptides; Protein Biosynthesis; Proteins; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database.. The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity.. EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM.. Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; Cluster Analysis; Colonic Neoplasms; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Ovarian Neoplasms; Receptor, ErbB-2; RNA, Messenger; Structure-Activity Relationship; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

Localization of growth factors such as transforming growth factor alpha (TGF-alpha) and beta1 (TGF-beta1), insulin-like growth factor 1 (IGF-1), and epidermal growth factor receptor (EGFR) in breast cancer tissue is controversial. We immunohistochemically investigated expression patterns of these growth factors and EGFR along with estrogen receptor (ER) status in 36 breast carcinomas (21 invasive ductal, 11 invasive lobular, 4 noninvasive ductal) and compared the results with those found in 10 fibroadenomas. Twenty-four of 36 carcinomas and all of the 10 fibroadenomas showed positivity for ER. TGF-alpha was immunoreactive in all of the carcinomas and fibroadenomas. TGF-beta1 was negative in all of the invasive ductal carcinomas and positive in all of the fibroadenomas and in five lobular carcinomas. EGFR was regularly expressed preferentially in the myoepithelial cells of mammary ducts in the fibroadenomas and in nontumorous glands. Six of the 36 carcinomas were positive for EGFR. Those tumors were negative for ER (P < .001). There was IGF-1 expression in all of the cases of carcinoma and fibroadenoma. We conclude that TGF-alpha is expressed abundantly in invasive and intraductal breast carcinomas and in fibroadenomas. EGFR expression significantly correlates with negative ER status in breast carcinoma. In breast carcinoma, IGF-1 is broadly expressed by the tumor as well as by stromal cells and might act as a growth stimulator in endocrine, paracrine, and autocrine manners.

Topics: Adenocarcinoma; Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; ErbB Receptors; Female; Fibroadenoma; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Middle Aged; Transforming Growth Factor alpha; Transforming Growth Factor beta

8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amphiregulin; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Division; Colonic Neoplasms; Colorectal Neoplasms; EGF Family of Proteins; Endothelial Growth Factors; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Membrane Glycoproteins; Mice; Mice, Nude; Neoplasm Proteins; Neovascularization, Pathologic; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A stromal fibroblast-mediated paracrine regulation of epithelial tumor cell proliferation and differentiation plays an important role in the development and progression of breast tumors. We have studied the paracrine growth regulation of various phenotypically different breast cancer cell lines using conditioned serum-free media (C-SFM) from primary breast fibroblasts. Fibroblast cultures were established from malignant primary tumors and adjacent normal breast tissue, benign fibroadenomas, cosmetic reduction mammoplasties and breast skin tissues. All fibroblast-conditioned media were shown to stimulate the proliferation of breast cancer cell lines. However, the C-SFM-induced MCF-7 proliferative response was shown to be significantly higher than the proliferative response observed with any of the other cell lines tested. More importantly, the MCF-7 proliferative response obtained with malignant tumor tissue fibroblast C-SFM was shown to be significantly higher than the response to C-SFM from paired (and unpaired) normal adjacent breast tissue fibroblasts. The MCF-7 proliferative response to fibroblast C-SFM from normal tissue (adjacent to the tumor) was further shown to be comparable to the MCF-7 response using benign or reduction mammoplastic tissue fibroblast C-SFM. In addition, we show that IGFs are only partly responsible for the observed proliferative effect of the C-SFMs, while EGF, TGF alpha and basic-FGF are shown not to be involved. We conclude that stromal fibroblasts can differentially regulate breast cancer cell proliferation. Both the fibroblast's tissue source as well as the target tumor cell's phenotype will determine the extent of the proliferative response.

Topics: Breast; Breast Neoplasms; Cell Division; Epidermal Growth Factor; Female; Fibroblasts; Humans; Somatomedins; Transforming Growth Factor alpha; Tumor Cells, Cultured

The expression of growth factors, such as transforming growth factor alpha (TGF alpha), amphiregulin (AR) and CRIPTO, a type-1 tyrosine-kinase growth factor receptor (erbB-2), and a tumor-suppressor gene (p53), that have been implicated in the development and/or the progression of breast cancer, was evaluated by immunohistochemistry in 100 human primary infiltrating breast carcinomas (IBC). AR and CRIPTO immunoreactivity was also assessed in 55 human breast ductal carcinomas in situ (DCIS). Within the 100 IBC, 80, 50, 73, 17, and 34 tumors expressed moderate to high levels of TGF alpha, AR, CRIPTO, erbB-2, and p53 respectively. In addition, AR and CRIPTO immunoreactivity were found in 11 and in 26 out of 55 DCIS respectively. In contrast, only 4, 3, and 2 out of 10 normal mammary-gland samples were weakly positive for TGF alpha, AR, and CRIPTO expression, respectively, whereas none was positive for erbB-2 or p53. Within the 100 IBC, expression of erbB-2 significantly correlated with high histologic and nuclear grading, with high growth fraction, and with estrogen-receptor (ER)- and progesterone-receptor (PgR)-negative tumors. A statistically significant correlation was also observed between p53 expression and high histologic grading, high growth fraction, and PgR-negative tumors. In contrast, no significant correlations were found between TGF alpha, AR, and CRIPTO immunoreactivity and various clinicopathological parameters, with the exception of a positive correlation between TGF alpha and ER expression. These data demonstrate that TGF alpha, AR, and CRIPTO expression are significantly increased in malignant mammary epithelium relative to normal epithelium. In particular, the differential expression of CRIPTO may serve as a potential tumor marker for breast carcinogenesis.

Topics: Adult; Amphiregulin; Breast; Breast Neoplasms; Carcinoma in Situ; EGF Family of Proteins; Epidermal Growth Factor; Epithelium; Female; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Receptors, Estrogen; Receptors, Progesterone; Transforming Growth Factor alpha

We examined mRNA expressions of urokinase-type plasminogen activator (u-PA), its specific receptor (u-PR), and plasminogen activator inhibitors (PAI-1 and PAI-2) in 50 human breast cancers by the reverse transcriptase-polymerase chain reaction method. The expressions of the genes are discussed in relation to the clinicopathological findings. In the overall population in breast cancers, a low level of PAI-2 expression was significantly associated with lymph node involvement (P < 0.0001). The u-PA, u-PR, and PAM expressions tended to be at high levels in such metastatic cancers. Also, positive expression of u-PA, u-PR, and PAI-1 was significantly correlated with negative expression of PAI-2. These results indicate that PAI-2 may play a critical role in the regulation of extracellular matrix degradation during tumor cell invasion and metastasis, and the expression of PAI-2 may be useful as a marker to evaluate the prognosis of breast cancers.

Topics: Adult; Aged; alpha Catenin; Base Sequence; beta Catenin; Blotting, Southern; Breast Neoplasms; Cadherins; Cytoskeletal Proteins; Female; Fibroblast Growth Factor 2; Humans; Lymphatic Metastasis; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Plasminogen Activator Inhibitor 2; Polymerase Chain Reaction; Receptors, Estrogen; RNA, Messenger; Trans-Activators; Transforming Growth Factor alpha; Tumor Cells, Cultured

Expression of oestrogen receptor (ER), epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF alpha) proteins was assessed by immunocytochemistry on primary breast cancer specimens obtained before and following short-term (7-day) presurgical exposure to pure anti-oestrogen (7 alpha- [9- (4,4,5,5,5-pentafluoropentylsulphinyl) nonyl] estra-1,3,5, (10)-triene-3,17 beta-diol, ICI 182780) treatment and compared with no-treatment controls. Paired needle-core and mastectomy samples were obtained from 21 patients. Effects of ICI 182780 (10(-7)M) on MCF7 breast cancer cell ER, EGFR and TGF alpha expression were also examined over 14 days. ER protein was significantly suppressed by ICI 182780 in vivo (P = 0.009) and comparative analysis of short term ICI 182780 effects in vitro, using ER-positive MCF7 cells, gave largely equivalent results. EGFR and TGF alpha protein levels were unaltered by treatment. ICI 182780 suppresses ER without a concomitant rise in either EGFR or TGF alpha.

Topics: Breast Neoplasms; ErbB Receptors; Estradiol; Estrogen Antagonists; Female; Frozen Sections; Fulvestrant; Humans; Immunohistochemistry; Postmenopause; Receptors, Estrogen; Transforming Growth Factor alpha; Tumor Cells, Cultured

The erbB-2 oncoprotein is overexpressed in 30% of tumors from breast and ovarian cancer patients and it is related to poor overall and disease-free survival. In vitro studies on erbB-2-overexpressing cells have found a strong correlation between this oncogene overexpression and relative resistance to lymphokine-activated killer (LAK) cell lysis. gp30/heregulin/NDF (neu differentiation factor), indirect activators of erbB-2, are able to induce a more differentiated phenotype on erbB-2-overexpressing, erbB-3- and/or erbB-4-positive breast cancer cells. We tested the ability of these highly homologous growth factors to stimulate LAK cell lysis of breast cancer cells. Our experiments demonstrated a marked increase in LAK cell cytotoxicity towards an erbB-2-overexpressing, erbB-3-positive cell line by treatment of these cells with heregulin for 72 h. In contrast we did not observe any enhancement of lysis of MCF-7, a cell line that does not overexpress erbB-2 and is positive for the erbB-3 and erbB-4 receptors, after treatment with heregulin. The increased lysis was associated with upregulation of intercellular adhesion molecule 1 (ICAM-1), down-regulation of erbB-2 and increased binding between breast cancer cells and LAK cells. Pre incubation of target (SKBR3) cells with blocking anti-ICAM-1 antibody completely abrogated the enhanced cytotoxicity. A similar effect was observed by pretreatment of the effector (LAK) cells with antibodies directed against LFA-1, the receptor for ICAM-1. These results suggest the possible utilization of gp30/heregulin in the treatment of breast cancer patients by its ability to stimulate patient immune responses.

Topics: Breast Neoplasms; Female; Gene Expression; Glycoproteins; Humans; Intercellular Adhesion Molecule-1; Killer Cells, Lymphokine-Activated; Receptor, ErbB-2; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

To investigate the molecular mechanisms underlying the two- to three-fold induction of human transforming growth factor-alpha (hTGF-alpha) mRNA and two- to five-fold induction of hTGF-alpha protein observed following estrogen treatment of hormone-responsive human breast cancer cell lines, the hTGF-alpha promoter was assayed for ERE-like sequences able to mediate estrogen induction of a heterologous gene. Transient co-transfection of a chloramphenicol acetyl transferase (CAT) construct consisting of either 1100 bp or 330 bp of hTGF-alpha promoter sequence and an estrogen receptor expression vector into either COS-7 cells or hormonally responsive MCF-7 human breast cancer cells resulted in a two- to five-fold induction of CAT activity by estrogen. Although no consensus estrogen response element (ERE) exists in the hTGF-alpha promoter, a sequence consisting of two imperfect ERE palindromes separated by 20 bp is located at -200 to -252. This sequence was inserted into a mouse mammary tumor virus (MMTV) based CAT construct and assayed for its ability to confer estrogen regulation of CAT expression to a heterologous promoter. Transient co-transfection of this construct with an estrogen receptor expression vector into either COS-7 cells or MCF-7 cells resulted in an average 30-fold estrogen induction of CAT activity. Gel shift assays with human recombinant estrogen receptor (ER) and 32P-labelled fragments revealed that the ER could specifically bind to this sequence. These results indicate that this 53 bp sequence can function as an ERE, and is likely to be responsible for the observed induction of TGF-alpha message and protein in response to estrogen. These data also indicate that the level of estrogen inducibility mediated by this element may be positively or negatively modulated by interaction or competition with other transcription factors.

Topics: Animals; Base Sequence; Breast Neoplasms; Carcinoma; Chloramphenicol O-Acetyltransferase; Consensus Sequence; COS Cells; DNA; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Mammary Tumor Virus, Mouse; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Fusion Proteins; Repetitive Sequences, Nucleic Acid; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

MCF-7 (estrogen receptor positive--ER+) and MDA-MB-231 (estrogen receptor negative--ER-) are human breast cancer cell lines which express functional thyroid hormone receptors (c-erb A alpha1 and c-erb beta1) as indicated by stimulation of mitochondrial alpha-glycerophosphate dehydrogenase. In MCF-7, mimicking E2, T3 stimulated growth in a dose-dependent (10(10) M - 10(-8) M) manner, induced the expression of progesterone receptor and growth factor TGFalpha mRNAs and inhibited that of TGFbeta mRNA; T3 also increased progesterone binding and LDH5 isozyme activities. None of these effects were observed in (ER-) MDA-MB-231 cells. 10(-6) M tamoxifen (TAM) reverted growth stimulation, suppressed progesterone receptor and TGFalpha mRNA induction and restored TGFbeta mRNA to control levels in T3-treated MCF-7 cells. That T3 is acting in MCF-7 cells via its binding to ER is suggested by the immunoprecipitation of pre-bound 125I-T3 from MCF-7 nuclear extracts by an ER-specific monoclonal antibody and by the displacement of 3H-estradiol binding to ER by radioinert T3.

Topics: Breast Neoplasms; Carcinoma; Cell Division; Estradiol; Estrogen Antagonists; Gene Expression Regulation, Neoplastic; Glycerolphosphate Dehydrogenase; Humans; Isoenzymes; L-Lactate Dehydrogenase; Receptors, Estrogen; Receptors, Progesterone; Receptors, Thyroid Hormone; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Triiodothyronine; Tumor Cells, Cultured

The levels of the matrix metalloproteinase MMP1 mRNA in three breast tumour cell lines with varying numbers of epidermal growth factor (EGF) receptors, MDA-MB-231, T47D and MCF7, were investigated following treatment with EGF or TGF alpha in serum-free medium for up to 24 h. A higher level of MMP1 mRNA was found in both control and treated MDA-MB-231 cells compared with the other two cell lines. A 2-fold increase in MMP1 transcripts was observed in MDA-MB-231 cells following a 30 min treatment with EGF and 2 h with TGF alpha. An increase in MMP1 transcripts following serum deprivation in the absence of growth factor stimulation was also seen. This effect was not evident with the other cell lines. In MDA-MB-231 cells, low concentrations of MMP1 protein were detected in medium from treated cells and was only significantly increased after 24 h but it was inhibited by cycloheximide. The early effect of EGF on MMP1 expression was not inhibited by cycloheximide. Treatment with cycloheximide for longer periods produced increased transcripts of MMP1, TGF alpha and EGF-receptor, suggesting the activation of processes for tissue breakdown and subsequent repair may occur on prolonged inhibition of protein synthesis. These results confirm a relationship between EGF-receptor stimulation and MMP1 expression in some EGF-receptor positive tumour cells, which, in part, occurs at the transcriptional level, and have implications for the invasive progression of EGF-receptor positive tumours particularly in areas of nutritional deprivation.

Topics: Blotting, Northern; Breast Neoplasms; Collagenases; Culture Media, Serum-Free; Cycloheximide; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Matrix Metalloproteinase 1; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured

A series of breast cancer biopsies from 204 women were analysed immunohistochemically for the expression of transforming growth factor alpha (TGF-alpha). Expression of TGF-alpha was intense in 119 cases (58%), weak in 63 (31%), and totally absent in 22 (11%) of the cases. No correlation was observed between the expression of TGF-alpha and tumour size, metastasis at diagnosis, histological type and grade, ER and PR status, DNA index, S-phase fraction or the expression of TGF-beta1 or beta2. However, the expression of TGF-alpha was significantly related to axillary lymph node metastasis and to low survival probability during the follow-up. These data support the earlier observations on the in vitro studies, suggesting that TGF-alpha most probably exerts an in vivo growth stimulation of female breast cancer cells.

Topics: Adult; Aged; Aged, 80 and over; Biopsy; Breast Neoplasms; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Middle Aged; Predictive Value of Tests; Prognosis; Survival Analysis; Transforming Growth Factor alpha

Twenty-four normal or benign breast tissues were examined for the expression of transforming growth factor-alpha (TGF alpha), epidermal growth factor receptor (EGFR), and P-glycoprotein, the product of the mdr-1 gene. Specific staining for all three proteins was observed in the majority of the samples. P-glycoprotein staining was present in most (88%), and confined to the lumenal surface of the ductal epithelium. Membranous EGFR expression was observed in epithelial cells in 92% of the specimens and 42% displayed both myoepithelial and epithelial cell staining. TGF alpha staining was intense and uniformly distributed through the cytoplasm (96%). Coexpression of EGFR, TGF alpha, and P-glycoprotein in normal human breast tissues suggests a role for each of those proteins in normal breast physiology. An interaction may be present in normal breast tissue between the EGF receptor pathway and P-glycoprotein.

Topics: Adolescent; Adult; Aged; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Breast; Breast Diseases; Breast Neoplasms; ErbB Receptors; Fibroadenoma; Humans; Immunohistochemistry; In Situ Hybridization; Middle Aged; Transforming Growth Factor alpha

The effects of the polypeptide Decapeptyl (a gonadotropin-releasing hormone (GnRH) agonist analogue) and of transforming growth factor-alpha (TGF-alpha), on estrone sulfate-sulfatase activities in the homogenates of various breast cancer cell lines were studied in the presence of heparin. In hormone-dependent MCF-7 breast cancer cells, Decapeptyl can inhibit sulfatase activity, and this effect is significantly augmented in the presence of heparin. In the other hormone-dependent T-47D breast cancer cell line, the decrease of sulfatase activity was only significant when Decapeptyl was associated with heparin. No significant effect on sulfatase activity elicited by heparin, Decapeptyl or a mixture of both was found in the hormone-independent MDA-MB-231 breast cancer cells. TGF-alpha stimulates sulfatase activity in the MDA-MB-231 cells but has no effect in the MCF-7 cells; in contrast, TGF-alpha combined with heparin provokes a decrease of the sulfatase activity in both cell lines. It is concluded that the sulfatase activity in some types of breast cancer cell can be inhibited by heparin combined with the polypeptides Decapeptyl or TGF-alpha.

Topics: Arylsulfatases; Breast Neoplasms; Dose-Response Relationship, Drug; Drug Interactions; Heparin; Humans; Steryl-Sulfatase; Transforming Growth Factor alpha; Triptorelin Pamoate; Tumor Cells, Cultured

The aim of this study was to examine whether changes in growth factor or cytokine expression could be responsible for the growth inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the human breast cancer MCF-7 cell line. Treatment of MCF-7 cells with 10 nM TCDD for 7 days reduced the cell growth to 60% of control; this effect was partly abolished by cotreatment of the cells with 100 nM 17 beta-estradiol (E2). The inhibition of cell growth by TCDD was accompanied by an enhanced secretion of transforming growth factor-beta (TGF-beta) and the TGF-beta content in cell culture supernatants was 2-fold higher than in controls. Using reverse transcription polymerase chain reaction (RT-PCR), the effect of TCDD on the expression of TGF-beta isoforms, transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) was investigated. It was demonstrated that incubation with 1, 10 and 100 nM TCDD for 24 h increased mRNA levels of TGF-alpha, TNF-alpha and IL-1 beta. The strongest effect was found on IL-1 beta, the mRNA level of which was dose-dependently increased. TCDD had a minor effect on TGF-alpha and TNF-alpha mRNA. The mRNA levels were significantly increased after treatment with 10 and 100 nM TCDD. The mRNA expression of TGF-beta 1 and TGF-beta 2 was unchanged, whereas the TGF-beta 3 mRNA level was enhanced 2 to 3-fold after TCDD treatment. From the results, we suggest that TCDD-induced growth inhibition in MCF-7 cells is related to the growth inhibitory action of a set of growth factors and cytokines which have a contextual action on MCF-7 cell proliferation.

Topics: Analysis of Variance; Breast Neoplasms; Cytokines; Growth Substances; Humans; Interleukin-1; Lung Neoplasms; Polychlorinated Dibenzodioxins; Polymerase Chain Reaction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In situ methodologies allow qualitative and semi-quantitative analysis of spatial gene expression in whole organisms or tissues. We have applied quantitative autoradiography to in situ hybridizations of sections from human breast tumor xenografts to measure mRNA levels for ornithine decarboxylase, estrogen receptor, transforming growth factor alpha, and glyceraldehyde-3-phosphate dehydrogenase. Comparisons of control and tamoxifen-treated animals show significant decreases in MCF-7 tumor estrogen receptor mRNA levels in the drug-treated animals. Combining quantitative autoradiography with in situ hybridization allows measurement of absolute rather than relative mRNA levels for genes of interest, and to monitor effector-induced changes in these mRNAs in vivo.

Topics: Animals; Autoradiography; Breast Neoplasms; Carbon Radioisotopes; Cell Line; DNA Probes; Gene Expression; Humans; In Situ Hybridization; Mice; Mice, Nude; Molecular Sequence Data; Ornithine Decarboxylase; Receptors, Estrogen; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transplantation, Heterologous; Tritium; Tumor Cells, Cultured

The Seventeenth Annual Interdisciplinary Cancer Research Workshop was held at the University of New Orleans on March 4, 1994. It was again sponsored by the Cancer Association of Greater New Orleans, a United Way Agency. As all the previous workshops in this highly successful series, it was organized by Peter Politzer (University of New Orleans), with the assistance of Anita H. Buckel (University of New Orleans) and James R. Jeter (Tulane University School of Medicine, New Orleans). The three invited speakers were Robert J. Coffey (Vanderbilt University, Nashville, TN), Suzanne A. W. Fuqua (University of Texas Health Science Center, San Antonio, TX), and Frank M. Torti (Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC). A one-hour-discussion period in the afternoon presented ample opportunity for an exchange of ideas and research findings among the speakers and the workshop participants. James R. Jeter served as the moderator of this lively discussion session.

Topics: Animals; Breast Neoplasms; Cells, Cultured; Cytokines; Epithelium; Homeostasis; Humans; Iron; Mutation; Receptors, Estrogen; Transforming Growth Factor alpha

Topoisomerase II (Topo II) is an essential enzyme that catalyzes the breakage of double-strand DNA and is the target of several effective anticancer drugs, including the epipodophyllotoxins. The regulatory subunits of the cyclic AMP-dependent protein kinase are differentially expressed in normal and cancer cells. The RIalpha subunit is overexpressed in cells transformed by transforming growth factor-alpha (TGF-alpha) or Ha-ras oncogene. It has been shown that murine cells transformed by Ha-ras become hypersensitive to Topo II-targeting anticancer drugs. In this report we have tested whether any correlation exists between the expression of RIalpha protein and cellular sensitivity of Topo II-targeting drugs. Normal human breast MCF-10A cells and their derivatives overexpressing TGF-alpha, Ha-ras, or the different protein kinase subunits were treated with either Topo II inhibitors, such as etoposide, teniposide, or amsacrine, or with drugs which act independently of Topo II, such as bleomycin. Here we show that MCF-10A TGF-alpha and MCF-10A Ha-ras cells overexpress the RIalpha protein and become hypersensitive to epypodophyllotoxins and amsacrine but not to bleomycin. Direct introduction of the RIalpha gene into MCF-10A induces hypersensitivity to Topo II inhibitor drugs. In contrast, the overexpression of the other protein kinase subunits, RIIbeta or Calpha, does not modify the drug sensitivity of MCF-10A cells. No differences in the mRNA/protein content or in the activity of Topo II were found between hypersensitive cells and parental MCF-10A cells, suggesting that RIalpha may influence drug sensitivity via modulation of events downstream of the Topo II-DNA cleavable complex.

Topics: Amsacrine; Bleomycin; Breast Neoplasms; Cell Division; Cell Survival; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; DNA Topoisomerases, Type II; Etoposide; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Recombinant Proteins; Teniposide; Topoisomerase II Inhibitors; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Breast cancer cell lines have been shown to secrete transforming growth factor alpha (TGF alpha) and other polypeptide growth factors in response to estrogen (E2) stimulation. In this study, we investigated whether cellular-derived TGF alpha mediates the growth-stimulatory effects of E2 in ER-positive breast cancer cells. To test this hypothesis, we introduced an antisense TGF alpha mRNA expression vector under control of a human metallothionein promoter into E2-responsive T47D human breast cancer cells. In stably transfected cells, cadmium induced antisense mRNA and reduced expression of TGF alpha mRNA and protein in antisense clones (AS1). TGF alpha expression was increased in sense clones (S2), while wild-type T47D cells (W3) or pSV2 neomycin resistance-transfected cells showed no change in TGF alpha expression in response to cadmium. The basal proliferative capacity of antisense transfected cells was equivalent to that of the wild-type. E2 increased TGF alpha synthesis and cell proliferation in transfected and wild-type cells. In AS1 cells, the simultaneous addition of cadmium with E2 blocked most of the E2-induced increase in TGF alpha mRNA and protein and nearly abolished the stimulatory effects of E2 on DNA synthesis and cell number. In contrast, no reduction in cell proliferation was observed with cadmium in antisense-transfected cells with a low level of antisense expression or in the S2 or W3 cells. Our results are compatible with the hypothesis that autocrine production of TGF alpha may be an important contributor to E2-induced growth in T47D cells.

Topics: Breast Neoplasms; Cell Division; Cell Line; Culture Media, Conditioned; DNA, Neoplasm; Estradiol; Gene Expression; Genetic Vectors; Humans; Metallothionein; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

The relationship between the effects of EGF and mevalonate on proliferation of the breast cancer cell line Hs578T was investigated. When Hs578T cells were depleted of serum their proliferation was drastically retarded. This was partially counteracted by insulin or IGF-1 but not by EGF. However, if the activity of HMG-CoA reductase was inhibited, there was a significant increase in DNA synthesis of EGF-treated cells. This effect was not seen in cells stimulated by insulin or IGF-1, and was prevented by addition of mevalonate. The results suggest that mevalonate, or some of its products, inhibits steps in the EGF signal pathway.

Topics: Breast Neoplasms; Cell Division; Cell Line; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Insulin; Insulin-Like Growth Factor I; Kinetics; Lovastatin; Mevalonic Acid; Signal Transduction; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

In the last decade we have come to understand that the growth of cancer cells in general and of breast cancer in particular depends, in many cases, upon growth factors that will bind to and activate their receptors. One of these growth factor receptors is the erbB-2 protein which plays an important role in the prognosis of breast cancer and is overexpressed in nearly 30% of human breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. Our recent discovery of the gp30 has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2 receptor. These endpoints of growth, invasiveness, and differententiation te have clear implications for the emergence, maintenance and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. We have shown that gp30 induces a biphasic growth effect on cells with erbB-2 over-expression. We have recently determined the protein sequence of gp30 and cloned its full length cDNA sequence. We have also cloned two additional forms to the ligand, that are believed to be different isoforms. We are currently expressing the different forms in order to determine their biological effects. To elucidate the cellular mechanisms underlying cell growth inhibition by gp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells which overexpress erbB-2 and cells which express low levels of this protooncogene.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Breast Neoplasms; Cadherins; Carcinoma, Intraductal, Noninfiltrating; Cell Differentiation; Cell Division; Chemotaxis; Cloning, Molecular; Disease Progression; Endocytosis; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Receptor, ErbB-2; Recombinant Fusion Proteins; Transcription Factor AP-1; Transforming Growth Factor alpha; Tumor Cells, Cultured

Growth response of human breast cancer cells to epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) was tested both in culture and in vivo in nude mice. Human breast cancer cells were obtained from palpable tumors resulting from xenografted primary breast cancers in nude mice. In collagen gel culture, the breast cancer cells grew autonomously as expanding spherical masses of loosely adherent cells in the basal medium and the supplementation of growth factors had no additional stimulatory effect. To determine whether this in vitro response is reflected in vivo, the collagen gel embedded human breast cancer cells were transplanted into athymic nude mice and the growth response to EGF was studied in vivo. In contrast to the situation in vitro, exogenous EGF was growth promoting in vivo. Our results demonstrate the importance of the combined in vitro-in vivo approach in studying physiologically relevant growth regulation. In addition, the use of collagen gel embedded human breast cancer cells for transplantation studies may more closely model the clinical situation in view of the close histopathological resemblance of the recovered gels to the surgical breast specimens.

Topics: Animals; Breast Neoplasms; Cell Division; Collagen; Culture Media; Epidermal Growth Factor; Gels; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Stimulation, Chemical; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

Hormonal management of breast cancer is confounded by an almost inevitable progression of cell growth from a steroid-regulated to a steroid-autonomous state. We have experimentally induced this progression in the estrogen growth-responsive MCF-7 human breast cancer cell line by long-term culture in the absence of steroids. After an initial period (10-12 weeks) of slowed growth in response to steroid deprivation, rapid, steroid-independent growth rates were consistently established. In these cells, which contained 3-fold elevated, functional estrogen receptor levels (as determined by induction of PgR and transactivation of a transiently transfected estrogen-responsive gene construct), antiestrogens still effectively suppressed cell proliferation, although estrogens only minimally increased the proliferation rate. Depletion of steroids from the growth media also resulted in a marked (70-80%) transient decrease in transforming growth factor (TGF) alpha mRNA and TGF-alpha protein production at 2 weeks that was followed by a progressive, partial return to the initial parental TGF-alpha mRNA and protein levels. In contrast, the mRNAs for TGF-beta 1, -beta 2, and -beta 3 and bioactive TGF-beta proteins transiently increased (3-10-fold) at 2 to 10 weeks of steroid deprivation and then returned by 24 weeks to the lower levels of the parental MCF-7 cells. These results suggest that the cells acquired steroid-independent means to regulate the production of these peptides. The long-term steroid-deprived sublines showed a loss of regulation of proliferation by TGF-alpha or anti-TGF-alpha antibodies and a 10-fold decrease in sensitivity to the growth-suppressive effects of TGF-beta 1, despite little change in receptor levels for these factors. The diminished contributions of TGF-alpha and TGF-beta s to the regulation of cell proliferation in long-term steroid-deprived MCF-7 breast cancer cells suggest that the TGFs do not act as major growth regulators in these estrogen-autonomous sublines. However, the marked, transient alterations in the levels of these growth factors indicate that they may play a role in the events which accompany the progression from estrogen-responsive to estrogen-autonomous growth. In addition, continued exposure to estrogen may be needed for the long-term maintenance of cell responsiveness to these TGFs.

Topics: Breast Neoplasms; Cell Division; Culture Media; Dose-Response Relationship, Drug; Estradiol; Humans; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Thymidine; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The protein product of the bcl-2 gene is though to be involved in inhibition of apoptosis; it may therefore be important in the modulation of hormonal/anti-hormonal responsiveness exhibited by tumours. This study immunocytochemically investigates (i) relationships between bcl-2 protein expression in primary breast cancers and other markers of prognostic and therapeutic value and (ii) associations of the bcl-2 protein with breast cancer responsiveness to endocrine therapy. The bcl-2 protein was found within the tumour epithelial cell cytoplasm of 32/46 breast cancer specimens; inter-patient staining was heterogeneous. Immunostaining for steroid hormone receptors was strongly associated with that for the bcl-2 protein, and it is thus possible that this protein, like progesterone receptor, is under oestrogen regulation via oestrogen receptor. The protein was inversely related to 2 markers of endocrine insensitivity, epidermal growth factor receptor (EGFR) and c-erbB-2 oncoprotein, while no associations were observed with either transforming growth factor (TGF)-alpha or Ki-67 proliferative status. A highly significant relationship was observed between response to endocrine therapy and the presence of bcl-2 protein. Indeed, bcl-2 immunostaining proved to be a more accurate predictor of response than oestrogen receptor status. Patients with elevated bcl-2 immunostaining (particularly those who coexpressed high oestrogen receptor levels) appeared to derive the greatest benefit from endocrine therapy. Our results are paradoxical since it was expected that the bcl-2 protein would counteract the tumour inhibitory effects of endocrine therapies as it is thought to prevent programmed cell death.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Nucleus; Cytoplasm; Epithelium; ErbB Receptors; Female; Goserelin; Humans; Immunoenzyme Techniques; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Transforming Growth Factor alpha

Previous studies suggest that transforming growth factor-alpha (TGF alpha) may have the potential of a tumor marker. Since the levels of serum TGF alpha in cancer patients and healthy individuals have not been reported, we determined the serum TGF alpha levels of 83 breast cancer patients and 74 healthy individuals by using a TGF alpha radioimmunoassay kit. All of the cancer patients' sera were positive for TGF alpha; their TGF alpha concentrations ranged from 210 to 740 pg/ml, with a mean of 353 +/- 98 pg/ml. Sixty-seven percent (50 cases) of normal sera were positive for TGF alpha; the levels ranged from 120 to 207 pg/ml, with a mean of 144 +/- 17 pg/ml. The difference in serum TGF alpha levels between cancer patients of different disease stages and healthy individuals was found to be statistically significant by Student t-test and the Mann-Whitney test. No correlation was found between serum carcinoembryonic antigen and TGF alpha levels. The potential of serum immunoreactive TGF alpha as a marker for breast cancer warrants further investigation.

Topics: Adult; Aged; Breast Neoplasms; Carcinoembryonic Antigen; Female; Humans; Male; Middle Aged; Neoplasm Staging; Reference Values; Transforming Growth Factor alpha

Immunoreactive alpha-transforming growth factor (alpha-TGF) was shown by immunocytochemistry to be present in the rat mammary gland at various stages of development, the staining being most intense in mature myoepithelial cells. Alpha-TGF was also detected in the secretions of the mammary glands of pregnant and lactating rats. alpha-TGF in the extracts of rat mammary glands at each stage of development, and in several rat mammary cell lines and in culture medium in which they had been grown, was shown by Western blotting to consist primarily of a protein of molecular weight 50 kDa. The amount of this protein was greater in the mammary gland of the lactating rat than in resting or involuting glands. alpha-TGF was also found in some, but not all, human breast carcinomas, and in benign hyperplastic breast diseases.

Topics: Animals; Blotting, Western; Breast; Breast Diseases; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line; Culture Media, Conditioned; Humans; Immunohistochemistry; Lactation; Mammary Glands, Animal; Molecular Weight; Rats; Transforming Growth Factor alpha

Studies on radiation-induced changes in gene expression are likely to be very important in developing a better understanding of cellular responses to ionizing radiation. While there is some information on the activation of cellular signal transduction pathways after radiation, few late reacting target genes have been identified. This study focuses on the characterization of expression modulation of two critical growth regulatory genes, estrogen receptor and epidermal growth factor-receptor in malignant mammary epithelial cells in response to single and repeated ionizing radiation exposures.. MCF-7 cells were used for single radiation exposure (2-50 Gy) experiments and MCF-IR-3 cells, generated by exposure to cumulative doses of 60 Gy in 2 Gy fractions, respectively, were used to study the effects of repeated exposures. Steady-state messenger ribonucleic acid levels for estrogen receptor, epidermal growth factor-receptor, and transforming growth factor-alpha were determined by ribonucleic acid protection experiments. Estrogen receptor and epidermal growth factor-receptor protein expression was quantitated by competitive binding studies with 3H-estradiol and 125I-EGF.. MCF-IR-3 cells showed a permanent three-fold down-regulation of the estrogen receptor messenger ribonucleic acid and protein, while epidermal growth factor-receptor was upregulated about nine-fold. Epidermal growth factor-receptor was substantially up-regulated in MCF-7 cells, at both the mRNA and protein levels, within 24 h of a single 2 Gy exposures, while there was a two-fold concomitant increase in transforming growth factor-alpha messenger ribonucleic acid expression. A decrease in estrogen receptor messenger ribonucleic acid and protein was suggested only after higher doses of single radiation exposures.. Single and repeated radiation exposures modulate the expression of two critical growth promoting genes, estrogen receptor and epidermal growth factor-receptor, in MCF-7 cells. The inverse expression of estrogen receptor and epidermal growth factor-receptor established for estrogen receptor-positive malignant mammary epithelial cells is maintained in MCF-7 cells after single and repeated exposures suggesting that radiation acts through common regulatory circuits and may modulate the cellular phenotype.

Topics: Breast Neoplasms; Cell Cycle; Cell Survival; Dose-Response Relationship, Radiation; ErbB Receptors; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Phenotype; Receptors, Estrogen; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

The function of different growth factors in the development and progression of malignant tumors and the role of cytotoxic cytokines in the host response generated against neoplasms have been recently studied. Anti-TGF-alpha and anti-TNF-alpha monoclonal antibody families have been developed and characterized previously by our laboratory. Libraries of anti-TGF-alpha and anti-TNF-alpha monoclonal antibodies were selected for equal immunoreactivity both in native (frozen) and in formaldehyde fixed, paraffin embedded histological sections. No differences were found between native and fixed samples demonstrated in 10 cases in the present prospective study. Retrospective investigation was performed in 35 histopathological specimens of breast cancer patients detailed clinically and observed during 5 years after the surgical treatment. Correlation between TGF-alpha and/or TNF-alpha expression and clinical staging--TNM score, lymph node metastasis, tumor recurrence and survival time--was analyzed. According to our present study, the TGF-alpha positive patients had worse clinical prognosis than the TNF-alpha positive and double positive cases during long term observation.

Topics: Adult; Aged; Breast Neoplasms; Female; Humans; Immunohistochemistry; Middle Aged; Prognosis; Prospective Studies; Retrospective Studies; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Epidermal growth factor (EGF) receptors are expressed in high levels by some poor prognosis breast tumors. We have examined the cytotoxic effect of the tumor growth factor alpha (TGF alpha)-delta Cys-Pseudomonas exotoxin (PE40) recombinant fusion protein on normal and tumorigenic human breast epithelial cells in vitro and in vivo. The MDA-468, MDA-231, BT-20, and MCF-7ADR estrogen receptor-negative, EGF receptor-rich breast cancer lines were exquisitely sensitive in vitro to TGF alpha-delta Cys-PE40 with a 50% inhibitory concentration of < or = 0.02 nM. The estrogen receptor-positive, low EGF receptor MCF-7, ZR75-1, and T47D cells were less sensitive to the fusion toxin with a 50% inhibitory concentration of > 0.2 nM. The nontumorigenic cell lines 184, 184A1, and 184B5 were relatively resistant to TGF alpha-delta Cys-PE40 despite exhibiting high levels of EGF receptors. Continuous i.p. administration of TGF alpha-delta Cys-PE40 via an osmotic minipump at a dose of 0.4 microgram/g/day over 7 days inhibited MDA-468, MA-231, and BT-20 but not MCF-7 tumor growth in female athymic mice. Host tissue toxicity was not observed with this dose of TGF alpha-delta Cys-PE40. Mixed MDA-468/MCF-7 tumors were established in nude mice after coinoculation of both cell types in estrogen-supplemented animals. EGF receptor immunohistochemistry and immunoblot procedures indicated that TGF alpha-PE40 eliminated the MDA-468 cells while sparing the adjacent MCF-7 cells. By immunoblot, EGF receptors were consistently more abundant in tumor tissue than in adjacent nontumor tissue from the same mastectomy specimen (n = 7). These data support the notion that EGF receptors can be selectively targeted in human breast cancer cells for the delivery of antitumor agents. Further clinical studies with TGF alpha-delta Cys-PE40 and other chimeric toxins using the same cellular target will address this possibility.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Drug Administration Schedule; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Female; Humans; Mice; Mice, Nude; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

The expression of transforming growth factor-alpha (TGF-alpha) has been evaluated in 51 breast cancers of known responsiveness to endocrine therapy using immunohistochemistry. High levels of TGF-alpha were observed in 65% of tumors and showed no relationship with tumor estrogen receptor or epidermal growth factor receptor status or Ki67 immunostaining. TGF-alpha levels did, however, relate to the endocrine sensitivity of the disease, with unresponsive tumors frequently showing high levels of TGF-alpha immunoreactivity. This relationship was observed in estrogen receptor-positive disease and was independent of the epidermal growth factor receptor status of the tumor. No quantitative association between TGF-alpha and Ki67 immunostaining was observed in any of the subcategories of tumors. These data infer a role for TGF-alpha in the development of endocrine insensitivity in estrogen receptor-positive breast cancer by mechanisms which appear independent of tumor growth fraction, as determined by Ki67 immunostaining.

Topics: Biomarkers, Tumor; Breast Neoplasms; ErbB Receptors; Female; Goserelin; Humans; Immunohistochemistry; Ki-67 Antigen; Menopause; Neoplasm Proteins; Nuclear Proteins; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor alpha

The expression of three epidermal growth factor (EGF)-related peptides, transforming growth factor alpha (TGF-alpha), amphiregulin (AR) and cripto-1 (CR-1), was examined by immunocytochemistry (ICC) in 68 primary infiltrating ductal (IDCs) and infiltrating lobular breast carcinomas (ILCs), and in 23 adjacent non-involved human mammary tissue samples. Within the 68 IDC and ILC specimens, 54 (79%) expressed immunoreactive TGF-alpha, 52 (77%) expressed AR and 56 (82%) expressed CR-1. Cytoplasmic staining was observed with all of the antibodies, and this staining could be eliminated by preabsorption of the antibodies with the appropriate peptide immunogen. Cytoplasmic staining with all of the antibodies was confined to the carcinoma cells, since no specific immunoreactivity could be detected in the surrounding stromal or endothelial cells. In addition to cytoplasmic reactivity, the AR antibody also exhibited nuclear staining in a number of the carcinoma specimens. No significant correlations were found between the percentage of carcinoma cells that were positive for TGF-alpha, AR or CR-1 and oestrogen receptor status, axillary lymph node involvement, histological grade, tumour size, proliferative index, loss of heterozygosity on chromosome 17p or overall patient survival. However, a highly significant inverse correlation was observed between the average percentage of carcinoma cells that expressed AR in individual tumours and the presence of a point-mutated p53 gene. Likewise, a significantly higher percentage of tumour cells in the ILC group expressed AR as compared with the average percentage of tumour cells that expressed AR in the IDC group. Of the 23 adjacent, non-involved breast tissue samples, CR-1 could be detected by ICC in only three (13%), while TGF-alpha was found in six (26%) and AR in ten (43%) of the non-involved breast tissues. These data demonstrate that breast carcinomas express multiple EGF-related peptides and show that the differential expression of CR-1 in malignant breast epithelial cells may serve as a potential tumour marker for breast cancer.

Topics: Amphiregulin; Biomarkers, Tumor; Breast Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; Female; Genes, p53; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Point Mutation; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

A series of rat monoclonal antibodies (MAbs) has been generated against the extracellular domain of the receptor for EGF which block the binding of EGF and TGF alpha to the receptor and inhibit the growth in vitro of a range of carcinoma cell lines that over-express the receptor for EGF. Some of these antibodies were able also to induce the complete regression of xenografts of EGFR-over-expressing tumours when treatment was started, either at the time of tumour inoculation or later when the tumours were established. The most effective of these antibodies was ICR62, which was also able to activate host immune effector functions. We conclude that antibodies which block growth-factor-ligand interaction can have a profound influence on the proliferative capacity of tumour cells in vivo and may have useful clinical application.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Head and Neck Neoplasms; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Ovarian Neoplasms; Rats; Rats, Inbred Strains; Recombinant Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vulvar Neoplasms

The sensitivity of human breast and lung cancer cell lines to TGF-alpha-PE40, a novel chimeric recombinant cytotoxin composed of two independent domains, (i) TGF-alpha and (ii) a 40 kDa segment of the Pseudomonas exotoxin protein, PE-40, was investigated. Toxicity varied widely, correlated with epidermal growth factor receptor (EGFR) levels (P = 0.01) and was greatly reduced by EGF, indicating that binding of TGF-alpha-PE40 to EGFR is important in mediating toxicity. Cell lines expressing low EGFR levels were most highly protected by EGF, indicating that normal (low EGFR-expressing) tissue may be selectively protected by EGF in vivo. P-glycoprotein did not confer resistance to TGF-alpha-PE40, and toxicity was unaffected by multidrug resistance-modulating agents (cyclosporin A, tamoxifen, verapamil), indicating a role for TGF-alpha-PE40 in the clinical management of drug-resistant tumours.

Topics: Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Cell Line; Cyclosporine; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Female; Humans; Lung Neoplasms; Recombinant Fusion Proteins; Recombinant Proteins; Tamoxifen; Transforming Growth Factor alpha; Tumor Cells, Cultured; Verapamil

A number of studies suggest that an inverse correlation exists between the epidermal growth factor-receptor and the estrogen receptor expression in primary human breast carcinoma as well as in established human breast carcinoma cell lines. Recent studies suggest that the epidermal growth factor-receptor does not regulate the estrogen receptor gene expression. Whether the estrogen receptor regulates the epidermal growth factor-receptor gene expression is not known. We addressed this question by stably transfecting the estrogen receptor cDNA into the estrogen receptor-negative human breast carcinoma cell line MDA-MB-231. Constitutive expression of functional estrogen receptors in the transfectants resulted in increased mRNA levels of both epidermal growth factor-receptor and transforming growth factor alpha. Estradiol treatment of transfected cells, although enhancing transforming growth factor alpha mRNA levels, did not modulate epidermal growth factor-receptor mRNA levels. The estrogen receptor-transfected cells grown in estrogenic regular medium, however, exhibited lower constitutive levels of epidermal growth factor-receptor mRNA than in steroid-stripped medium, suggesting that estrogens coupled with some factors normally present in the regular medium may indeed downmodulate epidermal growth factor-receptor mRNA. Sodium butyrate treatment enhanced epidermal growth factor-receptor mRNA levels in nontransfected cells grown in regular estrogenic as well as in steroid stripped medium. Sodium butyrate enhancement of epidermal growth factor-receptor mRNA levels was completely abolished in estrogen receptor-transfected cells grown in regular estrogenic medium and blunted in steroid stripped medium. Using various epidermal growth factor-receptor gene promoter-CAT constructs in transient transfection assays, we further demonstrate that sodium butyrate enhanced transcription of the epidermal growth factor-receptor gene. The putative sodium butyrate responsive element(s) appears to localize within the proximal 384 bp of the epidermal growth factor-receptor gene promoter region. Although the interactions between estrogen receptor and epidermal growth factor-receptor are rather complex, taken together, our data suggest that estrogen receptor can indeed modulate the epidermal growth factor-receptor mRNA expression.

Topics: Breast Neoplasms; Butyrates; Butyric Acid; Carcinoma; Clone Cells; Down-Regulation; ErbB Receptors; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Receptors, Estrogen; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Interaction of transforming growth factor-alpha (TGF-alpha) with its receptor, epidermal growth factor receptor (EGFR), has been implicated as an autoregulatory autocrine mechanism of breast epithelial proliferation.. To examine the interrelationship and clinical relevance of TGF-alpha and EGFR in breast carcinoma, methanol-fixed cryostat sections from 73 patients were immunostained with monoclonal antibodies to epidermal growth factor (EGF), EGFR, and TGF-alpha.. Neither EGFR nor TGF-alpha staining was diagnostic or specific for the detection of malignant neoplastic cells. Both exhibited staining along the basal lamina of most benign ducts and lobules. TGF-alpha staining was observed in neoplastic cells in 41% and in non-neoplastic cells (peritumoral stroma and benign duct/lobular epithelium) in 36% of patients. Staining for EGF and TGF-alpha failed to correlate with node status or grade; however, TGF-alpha negative tumors were more frequently positive for estrogen receptor (ER) (70% versus 14%; P = 0.03). The presence of EGFR correlated with positive lymph node status (P = 0.004), poor differentiation (P = 0.001), and negative ER status (P = 0.0001). EGFR staining was more common in neoplasms which recurred, but this approached significance only in the group with node-negative disease (mean follow-up, 52 months; P = 0.06), and neoplastic cell TGF-alpha correlated with disease recurrence in patients with node-positive disease (no recurrence, -13% positive versus recurrence, -52% positive; P = 0.01). Concurrent TGF-alpha/EGFR staining, present in 18% of tumors, also was predictive of disease recurrence (no recurrence, 3% positive for both versus recurrence, 31% positive for both, P = 0.03).. TGF-alpha is heterogeneously expressed in neoplastic and host-derived components of breast tumors. Concurrent EGFR/TGF-alpha immunostaining may characterize a clinically aggressive subset of breast carcinomas, possibly reflecting autocrine interaction, and conferring growth advantage or metastatic phenotype.

Topics: Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Lymphatic Metastasis; Neoplasm Recurrence, Local; Receptors, Estrogen; Transforming Growth Factor alpha

Vitamin A or retinol is an important agent in the normal differentiation and growth of cells. Retinol is an effective inhibitor of the growth of many transformed cells in vitro and in vivo but its mechanism of action is unclear. Transforming growth factor alpha (TGF alpha) is a known mitogen. We examined the effect of retinol treatment on TGF alpha stimulation of two human mammary carcinoma cell lines, one which is growth inhibited by retinol and one which is not. Pretreatment of both cell lines for 48 hours with retinol resulted in inhibition of TGF alpha stimulation of growth. In the T47D cell line the mechanism was not related to an effect on the cellular content of TGF alpha, epidermal growth factor (EGF) receptor protein, EGF receptor mRNA, or on the binding of TGF alpha to the EGF receptor. However, TGF alpha-induced stimulation of the EGF receptor substrate, phospholipase C-gamma 1, was abrogated in the T47D cell line with retinol pretreatment. In the MDA-MB-468 cell line, pretreatment with retinol resulted in a decrease in tyrosine phosphorylation of the EGF receptor. These results suggest that pretreatment with retinol decreases cellular proliferation seen with TGF alpha treatment by altering phospholipase C-gamma 1 response and/or EGF receptor tyrosine kinase activity. Alteration of phospholipase C-gamma 1 activity does not appear to be responsible for the inhibition of cell growth seen in the absence of TGF alpha stimulation.

Topics: Breast Neoplasms; Cell Division; ErbB Receptors; Female; Gene Expression; Humans; In Vitro Techniques; Phosphotyrosine; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine; Vitamin A

Using the breast carcinoma cell line MDA-MB 468 as immunogen, we have produced six new rat monoclonal antibodies (mAbs) against the human EGF receptor (EGFR) and are investigating their use for diagnostic and therapeutic applications in cancer patients whose tumours overexpress these receptors. The mAbs (three IgG2b and one each of IgG2a, IgG1 and IgA) were selected on the basis that they bound to the extracellular domain of the EGFR and blocked growth factor-receptor interaction. Competitive assays showed that, with the exception of antibody ICR65, the mAbs bound to one of two distinct epitopes on the external domain of the EGFR. ICR65, however, cross-reacted with mAbs binding to both epitopes. All of the mAbs immunoprecipitated the 170 kDa glycoprotein from cells expressing the EGFR but not the 185 kDa product of the related c-erbB-2 proto-oncogene. Unlike EGF and TGF alpha none of the mAbs stimulated the growth of quiescent human foreskin fibroblasts but they inhibited the EGF and TGF alpha induced growth stimulation of these cells in vitro. When tested for their effect on tumour cells the mAbs were found to inhibit the growth in vitro of a number of human tumours that overexpressed the EGFR (e.g. HN5, HN6, HN15, A431, MDA-MB 468) but they were without effect on tumour cell lines expressing low or undetectable amounts of the receptor. Our initial results indicate that this new generation of antibodies which bind with high affinity to the EGFR, block growth factor-receptor interaction and inhibit the growth of human squamous carcinoma cell lines overexpressing the receptor have potential for clinical application.

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Cell Division; Cross Reactions; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Humans; Hybridomas; Immunoglobulin G; Mice; Proto-Oncogene Mas; Proto-Oncogene Proteins; Rats; Rats, Inbred Strains; Rats, Nude; Receptor, ErbB-2; Transforming Growth Factor alpha; Tumor Cells, Cultured; Vulvar Neoplasms

Growth factors play a major role in the control of human breast cancer cell proliferation but their acute effects on cell cycle progression have not been well studied in these cells. T-47D cells, growth-inhibited by serum deprivation, were induced to re-enter the cell cycle in a concentration- and time-dependent manner by addition of insulin, insulin-like growth factor (IGF)-I, epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) or basic fibroblast growth factor (bFGF). After a lag of approximately 10 h semi-synchronous entry into S phase was observed. The relative potencies of maximal concentrations of growth factors were in the order: insulin approximately IGF-I approximately bFGF > TGF alpha > EGF, identifying bFGF as among the most potent mitogens for these cells. Insulin or IGF-I alone resulted in growth rates comparable with those observed in fetal calf serum. These data demonstrate that single growth factors can induce a significant proportion of T-47D cells to traverse the cell cycle. The kinetics for entry into S phase were similar, indicating that the basis of differential sensitivity to the growth factors tested was the proportion of cells that responded and ultimately entered S phase.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours. The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer cells is modulated by such growth factors as EGF, TGF alpha, and IL-1. The present study deals with the immunocytochemically detectable cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGF alpha. In MCF-7 cells growing logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns as seen in MCF-7 cells were true for the stimulated MaTu cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies, Monoclonal; Blotting, Northern; Breast Neoplasms; Epidermal Growth Factor; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; RNA, Messenger; Thromboplastin; Transforming Growth Factor alpha; Tumor Cells, Cultured

Ligands for the epidermal growth factor receptor (EGFR) (e.g. EGF and transforming growth factor alpha, TGF alpha) may be included in membrane preparations from human breast tumors, either complexed or not to EGFR. Assessment of EGFR in human placental membranes (HPM) after preincubation with human EGF (hEGF) or human TGF alpha (hTGF alpha) indicated that the presence of these ligands in a membrane preparations did not affect the apparent number of binding sites, but only resulted in an increased apparent dissociation constant (Kd). To obviate the effect of endogenously bound EGFR ligands in a radioligand binding assay, an acid treatment procedure has been used recently. In the present study we show that acid treatment of mammary tumor membranes does not result in increased EGFR levels but does result in an EGFR enriched membrane preparation by elimination of contaminating cytosol proteins. This however also occurred by washing the membranes with assay buffer at neutral pH. From these experiments we conclude that endogenous ligands, if present in membrane fractions from human breast tumors and occupying EGFR, do not interfere in a multipoint radioligand binding assay.

Topics: Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Radioligand Assay; Transforming Growth Factor alpha

The effects of interferon-alpha (IFN) plus tamoxifen (TMX) in the treatment of advanced breast cancer were assessed. Changes of in vivo biologic determinants including hormone receptors, P24 protein, Ki-67 and growth factor expression were evaluated. Seven patients with advanced, heavily pretreated, breast cancer with accessible disease, underwent biopsy prior to and after sequential treatment with IFN and IFN plus TMX. Clinically 4/7 patients responded to treatment with one complete and three partial remissions. Apart from the favourable response rate the sequential in vivo changes in expression of tumour variables were of considerable interest. IFN treatment consistently increased the expression of the estrogen receptor (ER) and of the estrogen regulated protein P24 while decreasing the expression of the proliferation associated antigen Ki-67. Addition of TMX on the other hand resulted in a reduction of ER expression to pre-IFN levels and a rise in progesterone receptor (PR) expression. When the effect of either IFN or IFN plus TMX on the expression of two growth factors was assessed they were found to be somewhat variable. While PDGF expression tended to be suppressed, there was no clinical correlation with response to therapy. TGF beta expression was found in all patients prior to treatment and while all non-responders showed reduction of TGF beta following treatment, the alterations were variable amongst responders (including two patients with increased expression, one with no change, and one with decreased expression). It is concluded that both IFN and TMX exert multiple effects on the expression of tumour biologic variables and that while the study confirmed some of the predictions from in vitro models, the in vivo effect are more complex than has been appreciated from the models. From the clinical point of view, it might be expected that treatment which enhances the expression of ER in tumours should have a positive effect on the response to TMX.

Topics: Adult; Antibodies, Monoclonal; Biomarkers, Tumor; Biopsy, Needle; Breast Neoplasms; Drug Therapy, Combination; Female; Humans; Immunohistochemistry; Interferon alpha-2; Interferon-alpha; Ki-67 Antigen; Menopause; Middle Aged; Neoplasm Proteins; Nuclear Proteins; Platelet-Derived Growth Factor; Receptors, Estrogen; Receptors, Progesterone; Recombinant Proteins; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta

To ascertain if 17 beta-estradiol (E2)-induced proliferation could be attenuated by blocking the expression of endogenous transforming growth factor alpha (TGF alpha), estrogen receptor (ER)-positive, estrogen-responsive MCF-7 or ZR-75-1 cells and ER-negative, estrogen-nonresponsive MDA-MB-468 or HS-578T cells were infected with a recombinant amphotropic, replication-defective retroviral expression vector containing a 435 base pair (bp) Apa1-Eco R1 coding fragment of the human TGF alpha cDNA oriented in the 3' to 5' direction and under the transcriptional control of an internal heavy metal-inducible mouse metallothionein (MT-1) promoter and containing the neomycin (neo) resistance gene. E2-stimulated expression of endogenous TGF alpha mRNA was inhibited by 4-5-fold, and the production of TGF alpha protein was inhibited by 50-80% when M-1 mass-infected MCF-7 or MZ-1 mass-infected ZR-75-1 cells were treated with 0.75-1 microM CdCl2, whereas in comparably treated parental MCF-7 or ZR-75-1 cells there was no significant effect upon these parameters. E2-stimulated anchorage-dependent growth (ADG) and anchorage-independent growth (AIG) of the M-1 or MZ-1 cells was inhibited by 60-90% following CdCl2 treatment. In contrast, neither the ADG nor AIG of the parental noninfected MCF-7 or ZR-75-1 cells that were maintained in the absence or presence of E2 was affected by comparable concentrations of CdCl2. The ADG and AIG of TGF alpha antisense MD-1 mass-infected MDA-MB-468 cells that express high levels of endogenous TGF alpha mRNA were also inhibited by 1 microM CdCl2, whereas the ADG and AIG of MH-1 mass-infected HS-578T cells, a TGF alpha-negative cell line, were unaffected by CdCl2 treatment. These results suggest that TGF alpha may be one important autocrine intermediary in regulating estrogen-induced cell proliferation.

Topics: Breast Neoplasms; Cell Division; Estradiol; Humans; RNA, Antisense; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Tumor Cells, Cultured

Three approaches have been taken to elucidate further the biological importance of intratumoural aromatase activity. (i) MCF7 and T47D hormone-dependent breast cancer cell lines both showed detectable aromatase activity in vitro. The up-regulation of this by TGF alpha indicates the possible existence of an autocrine growth stimulatory loop involving aromatase. (ii) Both tamoxifen and cytotoxic chemotherapy caused the suppression of aromatase activity in breast carcinomas in vivo. Aromatase activity prior to treatment did not predict for clinical response to tamoxifen. (iii) Transfection of aromatase into MCF7 cells led to their growth being stimulated by low doses of androgens and this was inhibited by the aromatase inhibitor CGS 16949A.

Topics: Aromatase; Breast Neoplasms; Bucladesine; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fadrozole; Female; Humans; Testosterone; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor alpha (TGF alpha) and Transforming growth factor beta-1 (TGF-beta 1) are growth regulatory for breast cancer cell lines in vitro and several studies have suggested that levels of the receptor for TGF alpha, the epidermal growth factor (EGFR) in tumour biopsies predict relapse and survival. We have examined the prognostic significance of TGF alpha, TGF-beta 1 and EGFR mRNA expression in a series of patients with primary breast cancer with a median follow up period of 60 months. In 167 patients the expression of TGF-beta 1 was inversely correlated with node status (P = 0.065) but not ER status, tumour size or menopausal status. Patients with high levels of TGF-beta 1 had a longer disease free interval with a significantly longer probability of survival at 80 months although the overall relapse free survival was not increased. EGFR mRNA expression was measured in 106 patients and was inversely correlated with ER status (P = 0.018). EGFR levels did not predict for early relapse or survival. TGF alpha mRNA levels were measured in 104 patients, no correlation was seen tumour size, node status, Er status, or clinical outcome.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; ErbB Receptors; Female; Follow-Up Studies; Humans; Immunoblotting; Middle Aged; Prognosis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factors

TGF alpha and beta expression was examined using rabbit polyclonal antibodies and immunohistochemistry on a series of 195 breast carcinomas. TGF alpha immunoreactivity was observed in all but nine of the tumours, with over 50 per cent staining strongly. The polyclonal TGF alpha antibody (CIM1), when compared with a commercially available mouse monoclonal TGF alpha antibody used on the same sections, gave a good correlation (r = 0.52, P < 0.001). Both TGF alpha antibodies produced a granular cytoplasmic staining pattern, that with CIM1 being coarser, suggestive of binding to an aggregated protein or organelle. Eighty-one per cent of tumours stained with the TGF beta antibody, 35 per cent strongly. There was significant co-expression of TGF alpha and TGF beta (P < 0.001). However, they were not found to be useful prognostic indicators, lacking any significant correlation with histological classification, tumour size, nodal status, oestrogen receptor status, S-phase fraction, or overall survival over a 9-12 year period. The expression of these growth factors in most breast carcinomas suggests that they have important biological roles, but the exact nature of these roles remains unclear at the moment.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Immunoenzyme Techniques; Middle Aged; Prognosis; Transforming Growth Factor alpha; Transforming Growth Factor beta

Ligands binding to epidermal growth factor-receptors (EGF-Rs) as epidermal growth factor-like activity (EGF-like activity), along with EGF-R levels, were determined from human primary breast cancer samples. Receptor analysis revealed a single class of high affinity binding sites. Ng/mg protein EGF-like activity quantities were measured in cytosols, and were inversely correlated with EGF-R contents (p < 0.01), estimated by two-point assays.

Topics: Breast Neoplasms; Cytosol; Epidermal Growth Factor; ErbB Receptors; Humans; Transforming Growth Factor alpha

To investigate whether estrogen treatment of hormone-responsive human breast cancer cells was associated with activation of members of the jun family of immediate early response genes, the expression of these oncogenes in human breast cancer cells was examined. 17 beta-Estradiol had little effect on expression of c-jun, jun B, jun D, or c-fos mRNA by MCF-7 cells over 12 h, although it stimulated c-myc expression 4-fold within 30 min. In contrast, several peptide growth factors, including transforming growth factor-alpha (TGF-alpha), rapidly and transiently induced expression of c-jun, jun B, and c-fos mRNA 4- to 10-fold over control. A similar pattern of expression was seen in two other estrogen-responsive human breast cancer cell lines, ZR-75-1 and T47D. Inhibition of protein synthesis by cycloheximide did not abrogate induction of c-jun or jun B mRNA by TGF-alpha in MCF-7 cells, suggesting that new protein synthesis was not required. In addition, nuclear runoff transcription analysis demonstrated that increased expression of c-jun and jun B mRNA after TGF-alpha treatment of MCF-7 cells was regulated at least in part at the transcriptional level. Chronic exposure of MCF-7 cells to 17 beta-estradiol for 24-48 h was associated with decreased expression of jun B mRNA only, while similar treatment with TGF-alpha did not change mRNA expression of any jun family member. Thus, expression of jun family members is induced by peptide growth factors like TGF-alpha but not 17 beta-estradiol in human breast cancer cells. These results suggest that these nuclear protooncogenes play different roles in modulating gene expression by MCF-7 cells after exposure to TGF-alpha or 17 beta-estradiol.

Topics: Breast Neoplasms; Cycloheximide; DNA, Neoplasm; Estradiol; Gene Expression; Genes, fos; Genes, jun; Humans; In Vitro Techniques; RNA, Neoplasm; Time Factors; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

Previous work from our laboratory has demonstrated that gamma-interferon (IFN) inhibits growth of the human breast carcinoma cell line MDA 468, while enhancing expression of epidermal growth factor receptor (EGFR). Epidermal growth factor at high levels is known to inhibit growth of this cell line. Because MDA 468 cells produce low levels of transforming growth factor (TGF)-alpha (a ligand for epidermal growth factor receptor), we reasoned that IFN-induced cytotoxicity could be partially mediated by enhanced secretion of TGF-alpha. Therefore, we determined the ability of IFN to modulate the endogenous expression of TGF-alpha by MDA 468 cells. IFN-gamma, at 500 units/ml, increased the levels of TGF-alpha in serum-free conditioned media of MDA 468 cells 3-fold as measured by radioimmunoassay. TGF-alpha mRNA was similarly increased approximately 3-fold after 5 days of IFN treatment as determined by dot blot and Northern analysis. IFN increased expression of TGF-alpha in conditioned media in a dose-dependent fashion. Increased secretion of TGF-alpha into conditioned media was not observed at Days 1 and 3. Similarly, increases in TGF-alpha mRNA were not observed at those time points. These results demonstrate that IFN-gamma enhanced secretion of TGF-alpha by MDA 468 cells. Although exogenous TGF-alpha inhibited MDA 468 cell growth, the role that the enhanced endogenous production of TGF-alpha plays in the cessation of cell growth induced by IFN remains to be determined.

Topics: Breast Neoplasms; Cell Division; Cloning, Molecular; Female; Humans; Interferon-gamma; Kinetics; Radioimmunoassay; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

To investigate growth regulation and radiosensitivity in surviving clonogens after fractionated irradiation.. Four breast carcinoma cell lines isolated from the primary tumor (21NT, 21PT) and metastases (21MT-1, 21MT-2) of a single patient were exposed to cumulative radiation doses of 30 Gy yielding cell lines designated-IR with respect to their parent. The irradiated lines were then compared to their parent for serum-and growth factor-requirements under defined media conditions, ability to proliferate in soft agar, concentration of TGF-alpha in conditioned medium, and radiosensitivity.. The irradiated lines showed no change in proliferative doubling times under serum- and growth factor-supplemented media conditions. A single line, 21MT-1-IR, acquired a limited ability to proliferate in serum-and growth factor-deplete medium with a day 2-4 doubling time of 44.5 hr. Three lines, 21MT-1-IR, 21MT-2-IR, and 21NT-IR, formed colonies in soft agar in contrast to none of the unirradiated parent lines. There were significant 6-8 fold increases in conditioned media TGF-alpha concentrations for 21MT-2-IR and 21NT-IR cells. The 21MT-1-IR and 21NT-IR cells were significantly less radiosensitive than their respective parent lines. This decrease in radiosensitivity appeared to be at least partially mediated by a released factor as the radiosensitivity of 21MT-1 cells was significantly decreased by pre-incubation with conditioned medium from 21MT-1-IR cells.. Radiation-induced changes in growth phenotype vary with respect to clonal origin of the cell line and may influence the radiosensitivity of surviving clonogens after fractionated treatment.

Topics: Breast Neoplasms; Cell Division; Dose-Response Relationship, Radiation; Female; Humans; In Vitro Techniques; Phenotype; Radiation Tolerance; Transforming Growth Factor alpha; Tumor Cells, Cultured

The effects of estradiol (E2), dihydrotestosterone (DHT) and dehydro-3-epiandrosterone (DHEA) on proliferation and progesterone receptor induction were studied in a breast cancer cell line (T47D) expressing estrogen, androgen, and progesterone receptors. A significant enhancement of growth and progesterone receptor expression was observed after treatment with E2 and DHEA, which was antagonized by the antiestrogen tamoxifen and not altered by the antiandrogen flutamide, supporting the involvement of estrogen receptors. When cells were treated with either E2 or DHEA, transforming growth factor-alpha mRNA was induced. DHT treatment did not alter growth but was effective in stimulating androgen receptors and down-regulating progesterone receptors.

Topics: Androgens; Breast Neoplasms; Cell Division; Dehydroepiandrosterone; Dihydrotestosterone; Down-Regulation; Estradiol; Female; Flutamide; Humans; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Breast Neoplasms; Cell Division; Female; Humans; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The influence of tamoxifen treatment on transforming growth factor-alpha (TGF-alpha) levels in human breast cancer rarely has been studied in vivo.. Postmenopausal patients with estrogen receptor (ER)-positive and progesterone receptor (PR)-positive primary breast cancer underwent two fine-needle aspiration biopsies (FNA) of the tumors. Between the two FNAs, 10 patients received no treatment (control group), and the other 10 patients received tamoxifen (20 mg/day) for 10 (8-12) days (TAM group). TGF-alpha levels in FNA samples were assayed by enzyme immunoassay.. No significant difference was found in TGF-alpha levels between the first and second FNA samples in the control group. On the other hand, in the TAM group, TGF-alpha levels in the second FNA samples (2.5 +/- 0.5; mean +/- SEM ng/mg.DNA) were significantly (P < 0.01) lower than those in the first (4.5 +/- 0.8). Studies on the influence of tamoxifen treatment on TGF-alpha levels in ER-negative and PR-negative breast cancer showed that TGF-alpha levels were not affected by tamoxifen treatment. Positivity of epidermal growth factor receptor (EGFR) was 60% in ER-negative and PR-negative breast cancer and 30% in ER-positive and PR-positive breast cancer.. Tamoxifen downregulates TGF-alpha levels in ER-positive and PR-positive breast cancers through ER. The significance of TGF-alpha as an autocrine growth factor appears to be more important in ER-negative and PR-negative breast cancer with high EGFR positivity than in ER-positive and PR-positive breast cancer with low EGFR positivity.

Topics: Biopsy, Needle; Breast; Breast Neoplasms; Down-Regulation; ErbB Receptors; Female; Humans; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Transforming Growth Factor alpha

The antiestrogen tamoxifen has been successfully used to control estrogen receptor (ER) and progesterone receptor positive breast cancer. However, the development of antiestrogen resistance is frequently observed in patients following long term treatment. We have studied the development of antiestrogen resistance in vitro and established an antiestrogen resistant variant of MCF-7 cells (clone 5C) after long term culture in estrogen free medium. The growth of clone 5C cells was not altered by either estradiol-17 beta or the antiestrogens 4-hydroxytamoxifen and ICI 164,384. Estrogen-stimulated progesterone receptor and reporter gene expression were markedly reduced in 5C cells compared to wild type MCF-7 cells. Only minor alteration in the levels of ER and no alteration in the affinity of ER for ligand were found in 5C cells. No mutation of ER cDNA in 5C cells was detected by polymerase chain reaction and DNA sequencing. This study demonstrates that change(s) in ER-mediated gene expression rather than the amino acid sequence of the ER itself may be associated with the development of at least one form of antiestrogen resistance.

Topics: Breast Neoplasms; Cell Division; Clone Cells; DNA, Neoplasm; Drug Resistance; ErbB Receptors; Estradiol; Estrogen Antagonists; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Polymerase Chain Reaction; Polyunsaturated Alkamides; Progesterone; Receptors, Estrogen; Recombinant Fusion Proteins; Signal Transduction; Tamoxifen; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Exposure of MCF-7 breast carcinoma cells to estradiol results in an increase in transforming growth factor alpha (TGF-alpha) synthesis and secretion. Since TGF-alpha is a potent inducer of proliferation in MCF-7 cells, the increase in TGF-alpha production by estradiol is thought to play an important role in the estrogen stimulation of growth of these cells. Retinoic acid inhibits the proliferation of MCF-7 cells and antagonizes the estrogen stimulation of growth. Addition of retinoic acid resulted in a greater than 70% inhibition of estradiol-induced TGF-alpha synthesis and secretion in MCF-7 cells. The increase in TGF-alpha mRNA expression by estradiol was also inhibited by exposure of the cells to retinoic acid. Pretreatment of the cells with retinoic acid for 24 or 72 h caused more than 50 and 90% inhibition, respectively, of the estradiol-enhanced expression of TGF-alpha mRNA. Expression of pS2 mRNA in MCF-7 cells was stimulated approximately 8-fold by estradiol. Retinoic acid treatment suppressed by greater than 80% both the basal and estradiol-induced pS2 mRNA expression. Retinoic acid modulation of the estrogen receptor gene mRNA was not responsible for the retinoic acid inhibition of the stimulation of pS2 and TGF-alpha gene expression by estradiol, since estrogen receptor gene expression was increased rather than decreased in the presence of retinoic acid. The nuclear retinoic acid receptors alpha and gamma mRNA were expressed in MCF-7 cells and its retinoic acid-resistant derivative RROI. Addition of estradiol to MCF-7 cells resulted in a decreased expression of retinoic acid receptor gamma mRNA; this reduction is prevented by the presence of retinoic acid. These results indicate that retinoic acid can inhibit estradiol-induced TGF-alpha and pS2 mRNA expression in MCF-7 cells. The suppression of TGF-alpha expression may represent one possible mechanism by which retinoic acid antagonizes the stimulation of MCF-7 proliferation by estradiol.

Topics: Breast Neoplasms; Carrier Proteins; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Humans; Receptors, Retinoic Acid; RNA, Messenger; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

To determine at what stage there is increased expression of transforming growth factor alpha (TGF alpha) in preneoplastic diseases of the breast and to determine if this would assist in the histological diagnosis of different intraduct epithelial proliferations.. Specimens were retrieved from the archives of 17 cases of ductal hyperplasia, six cases of atypical ductal hyperplasia and 13 cases of ductal carcinoma in situ together with 12 'normal' breast biopsy specimens. Sections were stained immunohistochemically for TGF alpha. The staining was assessed semi-quantitatively taking into account both the staining intensity and the proportion of cells stained.. Minimal expression of TGF alpha was observed in normal breast tissue. Increased levels of expression were seen in ductal hyperplasia, atypical ductal hyperplasia, and ductal carcinoma in situ. Increased levels of expression of TGF alpha were also found in morphologically normal ducts immediately adjacent to areas of intraduct epithelial proliferation.. Increased expression of TGF alpha occurs in the early stages of intraduct epithelial proliferation and will not help the histopathologist distinguish atypical ductal hyperplasia from either ductal hyperplasia or ductal carcinoma in situ. The molecular changes within a cell may precede the morphological changes observed by light microscopy thereby reflecting the biological potential of the epithelium.

Topics: Adult; Aged; Breast; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Hyperplasia; Middle Aged; Precancerous Conditions; Transforming Growth Factor alpha

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Breast Neoplasms; Cell Adhesion; Cell Division; Cell Line; Chromatography, Affinity; Chromatography, Gel; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Female; Glycoproteins; Growth Substances; Humans; Kinetics; Molecular Weight; Peptide Mapping; Phosphorylation; Protein Biosynthesis; Protein-Tyrosine Kinases; Radioimmunoassay; Radioligand Assay; Receptors, Estrogen; Transforming Growth Factor alpha; Tunicamycin

The mechanism by which transforming growth factor-alpha (TGF-alpha) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 beta (E2)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF-alpha and E2 on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF-alpha was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF-alpha effect was relatively modest, ranging from 23% to 42%. E2 effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E2 did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E2 did seem to stimulate de novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF-alpha-stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF-alpha (MCF-7J) or E2 (MCF-7B). The role of increased PI synthesis in E2-stimulated MCF-7 cell growth remains to be established.

Topics: Breast Neoplasms; Calcimycin; Cell Division; Estradiol; Female; Humans; Phosphatidylinositols; Transforming Growth Factor alpha; Tumor Cells, Cultured

In this investigation, 83 human mammary carcinomas were examined for the expression of oestrogen receptor (ER), epidermal growth factor receptor (EGF-R), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), c-erbB-2, histological grade, mitotic index and nodal status, all of which are reportedly prognostically significant factors (Bloom and Richardson 1957; Baak et al. 1985; Wright et al. 1989). ER expression was biochemically recognized in 43.4% of mammary carcinomas, and EGF-R, EGF, TGF-alpha and c-erbB-2 were histochemically recognized in 25.3, 14.5, 27.7 and 18.0% of mammary carcinomas examined respectively, using conventional sections of buffered formalin-fixed, paraffin-embedded tissue and monoclonal or polyclonal antibodies. There were significant relationships between negative ER and positive EGF-R or TGF-alpha; positive EGF-R and TGF-alpha; positive EGF-R and c-erbB-2; and positive c-erbB-2 and TGF-alpha. The single changes which were the negative ER and the positive c-erbB-2 correlated with histological grade and mitotic index. Co-expression of EGF-R and TGF-alpha correlated with positive nodal status. Therefore, the present investigation indicates that the negative ER, single expression of c-erbB-2 and co-expression of EGF-R and TGF-alpha are important markers which contribute indirectly to prognosis, which reconfirms previous findings on the former two while adding the new finding that immunohistochemical demonstration of expression of EGF-R and TGF-alpha may provide useful information for selecting the appropriate treatment.

Topics: Biomarkers, Tumor; Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lymphatic Metastasis; Mitotic Index; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptors, Cell Surface; Receptors, Estrogen; Transforming Growth Factor alpha

MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-alpha (TGF-alpha) gene in MCF-10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF-10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-10A Ha-ras and MCF-10A TGF-alpha cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-10A TGF-alpha/c-erbB-2 (MCF-10A TE) and 10 MCF-10A Ha-ras/c-erbB-2 (MCF-10A HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-10A TE and MCF-10A HE clones expressed a 10-fold to 40-fold increase in p185 erbB-2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF-alpha compared with MCF-10A cells. Moreover, both MCF-10A TE and MCF-10A HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-10A Ha-ras, MCF-10A c-erbB-2, or MCF-10A TGF-alpha clones. However, neither MCF-10A TE nor MCF-10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erbB-2, and TGF-alpha genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Epithelium; ErbB Receptors; Female; Genes, ras; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha

Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.

Topics: Base Sequence; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Molecular Sequence Data; Ovarian Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

In order to evaluate the potential role of calcium as an intracellular messenger for IGF-I and TGF-alpha action on breast cancer cell proliferation, we determined whether these growth factors induce any change in [Ca2+]i using fura-2 loaded cells. The hormone independent BT-20 and MDA-MB-231 cells were refractory to the mitogenic actions of exogenously added IGF-I and TGF-alpha. TGF-alpha administration, however, stimulated [Ca2+]i transients in the BT-20 cells. IGF-I and TGF-alpha stimulated DNA synthesis in the MCF-7 and T47D cells. These growth factors did not, however, stimulate any changes in [Ca2+]i in these cells. These data support the idea that receptor-mediated phospholipid hydrolysis does not serve a major signalling function for driving human breast cancer cells into DNA synthesis.

Topics: Adenosine Triphosphate; Biological Transport; Breast Neoplasms; Calcium; DNA; Female; Humans; Insulin-Like Growth Factor I; Transforming Growth Factor alpha; Tumor Cells, Cultured

Expression of tissue factor, the initiator of the extrinsic coagulation protease cascade, is a feature of certain malignant tumours. To study the modulation of tissue factor expression we incubated the breast cancer cell line MCF-7 with several growth factors. Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and interleukin-1 (IL-1) rapidly increased tissue factor expression of MCF-7 cells peaking at 6-8 h after starting point of incubation, as determined by clotting test, enzyme linked immunosorbent assay and flow cytometry. The data presented support the hypothesis that modulation of constitutive tissue factor expression in tumour cells by TGF alpha and IL-1 could also occur in vivo possibly resulting from interactions of stromal and cancer cells. The meaning for tumour biology, however, remains unclear.

Topics: Blood Coagulation Tests; Breast Neoplasms; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Flow Cytometry; Growth Substances; Humans; Interleukin-1; Thromboplastin; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

A number of growth factors have been implicated in the control of the proliferation of breast cancer cells and some have been reported to mediate the proliferative effects of oestradiol. MCF-7 cells were treated with growth factors in the presence and absence of oestradiol. Oestradiol increased the response of cells to the proliferative effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and basic fibroblast growth factor (bFGF). Platelet derived growth factor (PDGF) and cathepsin D had no effect in the presence or absence of oestradiol while TGF-beta slightly reduced the stimulation by oestradiol. In the absence of oestradiol, there was little effect of combinations of growth factors although the effects of bFGF and IGF-I were additive. In the presence of oestradiol, the effects of bFGF and TGF-alpha were additive whereas bFGF acted as an IGF-I antagonist. Overall, bFGF had the greatest effect on cell proliferation although this was less marked than the previously described effect of the IGFs and insulin. The effects of oestradiol on the sensitivity of cells to the proliferative effects of bFGF did not appear to result from regulation of bFGF receptor expression.

Topics: Breast Neoplasms; Cathepsin D; Cell Count; Cell Division; Drug Interactions; Epidermal Growth Factor; Estrogens; Fibroblast Growth Factor 2; Growth Substances; Humans; Insulin-Like Growth Factor I; Platelet-Derived Growth Factor; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.

Topics: ADP Ribose Transferases; Bacterial Toxins; Base Sequence; Binding, Competitive; Breast Neoplasms; Cell Line; Cell Survival; Chromosome Deletion; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Female; Humans; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Biosynthesis; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Virulence Factors

Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.

Topics: Amphiregulin; Blotting, Northern; Breast; Breast Neoplasms; Cell Division; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelial Cells; Epithelium; ErbB Receptors; Fibroblast Growth Factors; Glycoproteins; Growth Substances; Heparin; Humans; Intercellular Signaling Peptides and Proteins; Precipitin Tests; Transforming Growth Factor alpha; Tumor Cells, Cultured

Estradiol 17 beta-hydroxysteroid dehydrogenase acts to convert estrone to the biologically active estrogen, estradiol, in breast tumors and MCF-7 breast cancer cells in vitro. In this study we have examined the ability of albumin to influence the effect of growth factors (insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha)) and cytokines (interleukin (IL)-1, IL-6) on estradiol 17 beta-hydroxysteroid dehydrogenase activity in MCF-7 cells. IGF-I (80 ng/ml) or albumin (30 micrograms/ml) stimulated estradiol 17 beta-hydroxysteroid dehydrogenase activity by 144% and 102% (p less than 0.01). The combination of IGF-I and albumin, however, produced a marked (704%) synergistic stimulation of estradiol 17 beta-hydroxysteroid dehydrogenase activity. EGF or TGF alpha failed to stimulate estradiol 17 beta-hydroxysteroid dehydrogenase activity and no synergism with albumin was detected. IL-1 (10 ng/ml), but not IL-6, also stimulated estradiol 17 beta-hydroxysteroid dehydrogenase activity and acted synergistically with albumin to stimulate enzyme activity. MCF-7 cells were shown to specifically bind 125I-albumin and binding is increased by pretreatment of cells with IGF-I (80 ng/ml) for 48 h. It is concluded that the synergism that results from treating MCF-7 cells with albumin and IGF-I may result from increased albumin uptake and subsequent biological effect.

Topics: Albumins; Breast Neoplasms; Epidermal Growth Factor; Estradiol; Estradiol Dehydrogenases; Growth Substances; Humans; Insulin-Like Growth Factor I; Interleukin-1; Interleukin-6; Mass Spectrometry; Transforming Growth Factor alpha; Tumor Cells, Cultured

We investigated the effects of a benzoate of an estradiol-chlorambucil conjugate (KM2210) and chlorambucil on growth, estrogen receptor, and secretion of transforming growth factor (TGF)-alpha in the hormone-dependent human breast cancer cell line MCF-7. In the presence of 10(-10)-10(-6) M KM2210, the estrogen-induced growth of MCF-7 was completely inhibited. Inhibited growth of MCF-7 treated with 10(-8) or 10(-6) M KM2210 for 4 days was not rescued by removal of the drug and the addition of estradiol. By treatment of MCF-7 with KM2210 for 4 days, estrogen receptor-binding sites were decreased at 10(-8) M and were not detected at 10(-6) M but were unaltered by 10(-8) M chlorambucil. Moreover, estrogen receptor immunoreactivity and the level of estrogen receptor mRNA were decreased through treatment with 10(-6) M KM2210 for 4 days. These suppressions occurred prior to the onset of inhibitory action on MCF-7 growth. Secretion of TGF-alpha from MCF-7 was decreased by 4 days of treatment with 10(-8) and 10(-6) M KM2210 but not with chlorambucil. The addition of exogenous TGF-alpha generally restored the growth of MCF-7 treated with 10(-8) M KM2210. We concluded that KM2210 has irreversible or at least long-standing inhibitory effect on estrogen-dependent growth of MCF-7. It is conceivable that the decrease of estrogen receptor renders the cell unable to respond to estrogen with increased TGF-alpha secretion and succeeding cell growth.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Chlorambucil; Drug Screening Assays, Antitumor; Estradiol; Humans; Receptors, Estrogen; Transforming Growth Factor alpha; Tumor Cells, Cultured

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.

Topics: beta-Galactosidase; Breast; Breast Neoplasms; Cathepsin D; Cell Line, Transformed; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; Diethylstilbestrol; DNA, Viral; Epithelium; Estradiol; Female; Gene Expression Regulation; Genetic Vectors; Humans; Papillomaviridae; Receptors, Estrogen; Recombinant Proteins; RNA, Messenger; Transfection; Transforming Growth Factor alpha

The major objectives of this study were twofold: to determine 1) if growth factors or growth factor receptors were expressed similarly or differently in a clinically well-characterized group of breast cancer patients and 2) if these phenotypic characteristics were associated with any of the commonly used prognostic parameters. Formalin-fixed paraffin-embedded tumor tissue from 51 node-positive breast cancer patients were analyzed for the expression of neu, epidermal growth factor-receptor (EGF-R), and transforming growth factor alpha (TGF alpha) using immunoperoxidase staining. Positive membranous staining for neu was observed in 15 (29%) tumors. Over-expression of neu was observed in high-grade, estrogen-receptor-negative tumors (P less than 0.05). Epidermal growth factor receptor was expressed in 22 (43%) of the tumors analyzed and found to a greater degree in estrogen-receptor-negative and high-grade tumors (P less than 0.025). A significant correlation between neu and EGF-R expression was also noted. Tumors expressing membranous staining of neu had a greater than 70% chance of expressing EGF-R (P less than 0.01). Expression of TGF alpha was found in 68% of tumors and TGF alpha was detected in grade 1 and 2 tumor to a greater degree than EGF-R. The authors conclude that assaying tumors for these antigens may give additional phenotypic characteristics that can give further insight into the biology of breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Breast Neoplasms; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Lymph Nodes; Middle Aged; Neoplasm Invasiveness; Proto-Oncogene Proteins; Receptor, ErbB-2; Staining and Labeling; Transforming Growth Factor alpha

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Base Sequence; Breast Neoplasms; Cats; Cells, Cultured; DNA, Neoplasm; Dose-Response Relationship, Drug; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Molecular Sequence Data; Plasmids; Progesterone; Radioimmunoassay; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

In an effort to explore the use of polypeptide growth factors as potential markers for cancer detection, we have identified the presence of transforming growth factor-alpha (TGF-alpha) in pooled urine of patients with metastatic breast cancer by a commercial radioimmunoassay (RIA) based on a rabbit antiserum raised to the C-terminal 17aa synthetic fragment of rat TGF-alpha. This TGF-alpha RIA detected both high molecular weight (HMW) and low molecular weight (LMW) forms of TGF-alpha in the conditioned media of a breast cancer cell line (MDA-MB-231) and in the urine of healthy women and those with breast cancer. The ratio of HMW to LMW species of TGF-alpha by RIA after Bio-Gel P-100 chromatography was approximately equal in pooled urine samples from both healthy women and those with breast cancer, and in the conditioned media from the cell line MDA-MB-231. Using established procedures for concentrating urinary proteins from 24-h urine samples by adsorption onto methyl-bonded microparticulate silica and selective elution by acetonitrile, TGF-alpha RIA results from women with disseminated breast carcinoma were compared with those of healthy pre- and post-menopausal control women. Analysis indicated a median TGF-alpha value of 981 ng/g urinary creatinine for urine samples from cancer patients (range 608 to 1737) and 642 ng/g creatinine (range 417 to 941) for control urine samples. Although the difference was statistically significant (p less than 0.05), urinary TGF-alpha detection with this assay method appears to have limited usefulness as a diagnostic marker for metastatic human adenocarcinoma of the breast.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Molecular Weight; Neoplasm Proteins; Radioimmunoassay; Transforming Growth Factor alpha; Tumor Cells, Cultured

The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E2), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E2- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor-alpha (TGF-alpha) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E2-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-beta 1, -beta 2, -beta 3 similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF-beta antibody. We conclude that IGFs and TGF-alpha are important mediators of E2-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF-beta appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.

Topics: Agar; Antibodies; Breast Neoplasms; Culture Media; Drug Interactions; Epidermal Growth Factor; Estradiol; Female; Growth Substances; Hormones; Humans; Insulin-Like Growth Factor I; Neoplasms, Hormone-Dependent; Progesterone; Prolactin; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

Breast tumors are a complex mix of epithelial, stromal, and vascular elements. We examined primary cultures of breast fibroblasts derived from benign and malignant lesions for expression of various growth factors. All fibroblast cultures, regardless of whether they were derived from benign or malignant lesions, expressed platelet-derived growth factor A chain, basic fibroblast growth factor, fibroblast growth factor 5, and transforming growth factor beta 1 mRNA. None expressed platelet-derived growth factor B chain or transforming growth factor alpha mRNA. However, examination of mRNA expression for the insulin-like growth factors revealed that 7 of 8 fibroblasts derived from benign lesions expressed insulin-like growth factor I (IGF-I) mRNA, while only 1 of 9 fibroblasts derived from malignancies expressed IGF-I mRNA. The opposite picture was seen for insulin-like growth factor II (IGF-II) mRNA expression, in which 1 of 9 benign-derived fibroblasts expressed IGF-II mRNA, while 5 of 9 malignant-derived fibroblasts expressed IGF-II. This correlated with previous in situ hybridization data, which showed IGF-I mRNA expression confined to the stroma of benign breast tissue. PDGF treatment of tumor fibroblasts resulted in a 3-fold increase in IGF-II mRNA. Thus there was an apparent dichotomy between IGF-I mRNA expression in the majority of fibroblasts derived from benign lesions and IGF-II mRNA expression in the majority of tumor-derived fibroblasts. Since the insulin-like growth factors are potent mitogens for breast tumor epithelial cells, this further supports the notion of a paracrine growth-promoting role for the insulin-like growth factors in breast lesions and suggests that IGF-II may be the more important growth promoter in malignant lesions.

Topics: Breast Diseases; Breast Neoplasms; Cell Division; Cells, Cultured; Female; Fibroblast Growth Factor 5; Fibroblast Growth Factors; Fibroblasts; Fluorescent Antibody Technique; Growth Substances; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Platelet-Derived Growth Factor; Ribonucleases; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

These experiments were designed to test polyamine (PA) involvement in the secretion and action of transforming growth factor alpha (TGF-alpha) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E2) and TGF-alpha stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E2-stimulated growth, administration of the PA synthesis inhibitor alpha-difluoromethylornithine (DFMO) did not influence either basal or E2-induced TGF-alpha secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-alpha. However, when the culture conditions were changed to serum-free medium, TGF-alpha and E2-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E2-stimulated TGF-alpha secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF-alpha and E2-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.

Topics: Breast Neoplasms; Cell Division; Eflornithine; Estrogens; Humans; In Vitro Techniques; Neoplasms, Hormone-Dependent; Polyamines; Putrescine; Serum Albumin, Bovine; Spermidine; Spermine; Transforming Growth Factor alpha; Tumor Cells, Cultured

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.

Topics: Base Sequence; Blotting, Southern; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Estradiol; Gene Expression; Humans; Molecular Sequence Data; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Tumor Cells, Cultured

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

Topics: Blotting, Northern; Breast Neoplasms; Cell Cycle; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Kinetics; Mifepristone; Pregnenediones; Progesterone Congeners; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Transforming Growth Factor alpha; Tumor Cells, Cultured

We and others have previously reported that transforming growth factor-alpha (TGF alpha) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5'-flanking region of the TGF alpha gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormone-stimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGF alpha 5'-flanking region (-373 to -59 relative to the TGF alpha translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGF alpha gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGF alpha-luciferase reporter construct, indicating it is the TGF alpha promoter. A 1.1-kilobase segment of the TGF alpha 5'-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5'-flanking region to -3500 results in marked suppression of reporter gene expression. These results indicate that the TGF alpha gene 5'-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene.

Topics: Base Sequence; Breast Neoplasms; Dexamethasone; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; In Vitro Techniques; Luciferases; Molecular Sequence Data; Sequence Homology, Nucleic Acid; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Transforming Growth Factor alpha; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

The progesterone receptor belongs to a class of ligand binding transcription factors that regulate transcription by interacting with specific DNA sequences on hormone regulated genes. In human mammary tumor T47D cells that contain both progesterone and epidermal growth factor (EGF) receptors, the progestin-induced transactivation at various hormone regulated promoters is enhanced by EGF. The effect of EGF is rapid and does not require new protein synthesis. EGF treatment does not alter the DNA binding activity of the progesterone receptor nor does it affect the total ligand-dependent phosphorylation of this receptor. These results suggest that EGF enhances the transactivation property of the progesterone receptor through mechanisms other than those involving a direct interaction of this receptor with its cognate binding sites.

Topics: Base Sequence; Blotting, Northern; Breast Neoplasms; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Molecular Sequence Data; Progestins; RNA, Neoplasm; Transforming Growth Factor alpha; Tumor Cells, Cultured

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Breast Neoplasms; Cell Division; ErbB Receptors; Estradiol; Female; Humans; Mammary Neoplasms, Experimental; Progesterone; Prolactin; Rats; Rats, Inbred Strains; Transforming Growth Factor alpha; Tumor Cells, Cultured

Two primary and two metastatic cell lines with distinct phenotypes and genotypes have been established from a patient diagnosed as having infiltrating and intraductal mammary carcinoma (21T series). All four lines can be cultured in the same medium, DFCI-1, which also supports long-term growth of normal epithelial cells. Therefore, we have been able to compare normal and tumor cells at the cellular and molecular levels. The mammary origin of the 21T series was confirmed by using antibodies against the human milk fat globule antigen-2 epitope. The two primary tumor lines (21NT and 21PT) are both immortal and aneuploid, although only 21NT is tumorigenic in the nude mouse assay. The two populations derived from the metastatic pleural effusion (21MT-1 and 21MT-2) each exhibit distinct characteristics in morphology and growth factor requirements. The erbB2 gene is amplified and overexpressed in all of these cell lines compared to normal epithelial cell controls. These four tumor cell lines from a single patient represent a mammary tumor progression series that has been established in long-term cell culture.

Topics: Adult; Antibodies, Monoclonal; Breast; Breast Neoplasms; Carcinoma; Cell Division; DNA Fingerprinting; ErbB Receptors; Gene Expression; Growth Substances; Humans; Karyotyping; Neoplasm Proteins; Pleural Effusion; Polymorphism, Restriction Fragment Length; Proto-Oncogene Proteins; Receptor, ErbB-2; RNA, Neoplasm; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Breast Neoplasms; DNA-Directed DNA Polymerase; DNA, Neoplasm; Female; Humans; Polymorphism, Restriction Fragment Length; Taq Polymerase; Transforming Growth Factor alpha

We have established an estrogen receptor- and progesterone receptor-negative, hormone-nonresponsive breast cancer cell line from a receptor-positive, hormone-responsive line grown under estrogen-free conditions. T47D breast cancer cells were cultured under estrogenized conditions (in phenol red-containing medium supplemented with whole fetal bovine serum) and cloned to produce line T47D:A18. The parental T47D line was also estrogen deprived (in phenol red-free medium supplemented with dextran-coated charcoal-treated fetal bovine serum) for more than 1 year and subsequently clone T47D:C4 was established. T47D:A18 was estrogen receptor and progesterone receptor positive as determined by both ligand binding assay analysis and enzyme immunoassay analysis. T47D:C4 cells were estrogen receptor and progesterone receptor negative and mRNA for these receptors was not detected. Incubation of hormone-responsive T47D:A18 cells with 17 beta-estradiol caused a 3-fold increase in cell growth over 8 days when compared to control. This stimulation of growth was completely inhibited by the anti-estrogens 4-hydroxytamoxifen (0.1 microM) and ICI 164,384 (1.0 microM). Receptor-negative T47D:C4 cells were refractory to the effects of both 17 beta-estradiol and the antiestrogens. T47D:A18 cells grown under both estrogen-containing and estrogen-free conditions expressed low levels of transforming growth factor (TGF)-alpha and epidermal growth factor receptor mRNA. In the presence of estrogen, high levels of TGF-beta 1 mRNA were detected in T47D:A18 cells. These levels decreased when T47D:A18 cells were grown in estrogen-free media. Conversely, TGF-beta 2 mRNA was not detected in T47D:A18 cells cultured under estrogenic conditions; however, message was detected after the cells were cultured under estrogen-free conditions. T47D:C4 cells expressed low levels of TGF-alpha, epidermal growth factor receptor, TGF-beta 1, and TGF-beta 2 mRNA. These studies characterize a novel hormone-nonresponsive cell line which has been established from a hormone-responsive cell line grown under estrogen-free and drug-free conditions. Further analysis of these lines should provide valuable information concerning the development of antiestrogen-resistant breast cancer.

Topics: Blotting, Northern; Breast Neoplasms; Cell Division; Clone Cells; Cytidine; DNA Fingerprinting; DNA, Neoplasm; ErbB Receptors; Estradiol; Gene Expression; Humans; In Vitro Techniques; Polyunsaturated Alkamides; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured