Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression

Epidermal growth factor receptor (EGFR) is a causal factor in carcinoma, yet many carcinoma patients are resistant to EGFR inhibitors. Potential insight into this resistance stems from prior work that showed EGFR in normal epithelial cells docks to the extracellular domain of the plasma membrane proteoglycan syndecan-4 (Sdc4) engaged with α3β1 and α6β4 integrins. We now report that this receptor complex is modified by the recruitment of syndecan-2 (Sdc2), the Recepteur d’Origine Nantais (RON) tyrosine kinase, and the cellular signaling mediator Abelson murine leukemia viral oncogene homolog 1 (ABL1) in triple-negative breast carcinoma and head and neck squamous cell carcinoma, where it contributes to EGFR kinase–independent proliferation. Treatment with a peptide mimetic of the EGFR docking site in the extracellular domain of Sdc4 (called SSTN_EGFR) disrupts the entire complex and causes a rapid, global arrest of the cell cycle. Normal epithelial cells do not recruit these additional receptors to the adhesion mechanism and are not arrested by SSTN_EGFR. Although EGFR docking with Sdc4 in the tumor cells is required, cell cycle progression does not depend on EGFR kinase. Instead, progression depends on RON kinase, activated by its incorporation into the complex. RON activates ABL1, which suppresses p38 mitogen-activated protein kinase and prevents a p38-mediated signal that would otherwise arrest the cell cycle. These findings add to the growing list of receptor tyrosine kinases that support tumorigenesis when activated by their association with syndecans at sites of matrix adhesion and identify new potential targets for cancer therapy.     

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.     

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice

The transforming growth factor alpha (TGF) 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 TGF and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGF is evident in the transgenic mice overexpressing TGF 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 TGF transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGF 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 TGF, 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.     

A putative human breast stem cell population is enriched for steroid receptor-positive cells

Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERalpha and PR), we examined the biology of these ERalpha/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERalpha and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21(CIP1) and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERalpha-positive cells and expressed several fold higher levels of the ERalpha, p21(CIP1) and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.     

BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.     

Calmodulin binds HER2 and modulates HER2 signaling

Human epidermal growth factor receptor 2 (HER2), a member of the ErbB family of receptor tyrosine kinases, has defined roles in neoplastic transformation and tumor progression. Overexpression of HER2 is an adverse prognostic factor in several human neoplasms and, particularly in breast cancer, correlates strongly with a decrease in overall patient survival. HER2 stimulates breast tumorigenesis by forming protein-protein interactions with a diverse array of intracellular signaling molecules, and evidence suggests that manipulation of these associations holds therapeutic potential. To modulate specific HER2 interactions, the region(s) of HER2 to which each target binds must be accurately identified. Calmodulin (CaM), a ubiquitously expressed Ca²⁺ binding protein, interacts with multiple intracellular targets. Interestingly, CaM binds the juxtamembrane region of the epidermal growth factor receptor, a HER2 homolog. Here, we show that CaM interacts, in a Ca²⁺-regulated manner, with two distinct sites on the N-terminal portion of the HER2 intracellular domain. Deletion of residues 676-689 and 714-732 from HER2 prevented CaM-HER2 binding. Inhibition of CaM function or deletion of the CaM binding sites from HER2 significantly decreased both HER2 phosphorylation and HER2-stimulated cell growth. Collectively, these data suggest that inhibition of CaM-HER2 interaction may represent a rational therapeutic strategy for the treatment of patients with breast cancer. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

The process of TNF-induced cytotoxicity is complex but appears to be mediated through a TNF-specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF-receptor modulatory peptides implicating a role for EGF-R in the process of TNF-induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF on several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF-sensitive and -resistant tumor cell lines. In TNF-sensitive ME-180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF-receptor (EGF-R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF-R kinase activity in ME-180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activation of the receptor by TNF correlated with increased 32P incorporation into EGF-R protein when receptor was immunoprecipitated from 32P-equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF-R protein isolated from TNF-treated ME-180 cells demonstrates an increase in the phosphotyrosine content of EGF-R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF-R tyrosine protein kinase activity and the state of EGF-receptor tyrosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF-R when incubated with extensively washed immunoprecipitates of EGF-R. In TNF-resistant T24 bladder carcinoma cells, TNF failed to alter EGF-R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF-R kinase in target cells may correlate with its cytotoxic actions on TNF-sensitive tumor cells. Other biochemical activities associated with the induction or regulation of cellular growth were examined in TNF- or EGF-treated tumor cells. EGF stimulated a rapid 8-16-fold increase in the expression of the proto-oncogene c-myc when analyzed by dot-blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c-myc expression in ME-180 cells when analyzed by either technique.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK−/−). Mammary glands from RonTK−/− mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK−/− epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK−/− glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.     

Mitogenic response to epidermal growth factor: Relationship to number,affinity, and down-regulation of EGF receptors in three murine embyro cell lines

Swiss 3T3 and C3H-M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H-10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10-nM EGF all three cell lines “down-regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.     

Neuregulins: functions,forms, and signaling strategies

The neuregulins (NRGs) are cell-cell signaling proteins that are ligands for receptor tyrosine kinases of the ErbB family. The neuregulin family of genes has four members: NRG1, NRG2, NRG3, and NRG4. Relatively little is known about the biological functions of the NRG2, 3, and 4 proteins, and they are considered in this review only briefly. The NRG1 proteins play essential roles in the nervous system, heart, and breast. There is also evidence for involvement of NRG signaling in the development and function of several other organ systems, and in human disease, including the pathogenesis of schizophrenia and breast cancer. There are many NRG1 isoforms, raising the question "Why so many neuregulins?" Study of mice with targeted mutations ("knockout mice") has demonstrated that isoforms differing in their N-terminal region or in their epidermal growth factor (EGF)-like domain differ in their in vivo functions. These differences in function might arise because of differences in expression pattern or might reflect differences in intrinsic biological characteristics. While differences in expression pattern certainly contribute to the observed differences in in vivo functions, there are also marked differences in intrinsic characteristics that may tailor isoforms for specific signaling requirements, a theme that will be emphasized in this review.     

Estrogen mitogenic action. II. Negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications

Summary In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoaldextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of ≤10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER⁺) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFβ) substituted for the serum-borne inhibitor. TGFβ did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor “activated” intracellular signaling pathways.     

Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.