Corinne M. Silva

Tyrosine kinase signalling in breast cancer: Epidermal growth factor receptor and c-Src interactions in breast cancer

Both the non-receptor tyrosine kinase, c-Src, and members of the epidermal growth factor (EGF) receptor family are overexpressed in high percentages of human breast cancers. Because these molecules are plasma membrane-associated and involved in mitogenesis, it has been speculated that they function in concert with one another to promote breast cancer development and progression. Evidence to date supports a model wherein c-Src potentiates the survival, proliferation and tumorigenesis of EGF receptor family members, in part by associating with them. Phosphorylation of the EGF receptor by c-SRC is also critical for mitogenic signaling initiated by the EGF receptor itself, as well as by several G-protein coupled receptors (GPCRs), a cytokine receptor, and the estrogen receptor. Thus, c-Src appears to have pleiotropic effects on cancer cells by modulating the action of multiple growth-promoting receptors.

Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II.

ABSTRACT When co-overexpressed, the epidermal growth factor receptor (EGFR) and c-Src cooperate to cause synergistic increases in EGF-induced DNA synthesis, soft agar colony growth, and tumor formation in nude mice. This synergy is dependent upon c-Src-mediated phosphorylation of a unique tyrosine on the EGFR, namely, tyrosine 845 (Y845). Phenylalanine substitution of Y845 (Y845F) was found to inhibit EGF-induced DNA synthesis without affecting the catalytic activity of the receptor or its ability to phosphorylate Shc or activate mitogen-activated protein kinase. These results suggest that synergism may occur through alternate signaling pathways mediated by phosphorylated Y845 (pY845). One such pathway involves the transcription factor Stat5b. Here we describe another pathway that involves cytochrome c oxidase subunit II (CoxII). CoxII was identified as a specific binding partner of a pY845-containing peptide in a phage display screen. EGF-dependent binding of CoxII to the wild type but not to the mutant Y845F-EGFR was confirmed by coimmunoprecipitation experiments. This association also required the kinase activity of c-Src. Confocal microscopy, as well as biochemical fractionation, indicated that the EGFR translocates to the mitochondria after EGF stimulation, where it colocalizes with CoxII. Such translocation required the catalytic activity of the receptor but not phosphorylation of Y845. However, ectopic expression of the Y845F-EGFR prevented the EGF from protecting MDA-MB-231 breast cancer cells from adriamycin-induced apoptosis, whereas two mutants of Stat5b, a dominant-interfering mutant (DNstat5b) and a tyrosine mutation at 699 (Y699F-Stat5b) did not. Taken together, these data suggest that, through the ability of EGFR to translocate to the mitochondria, the binding of proteins such as CoxII to pY845 on the EGFR may positively regulate survival pathways that contribute to oncogenesis.

Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b.

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Signal transducer and activator of transcription 5b: a new target of breast tumor kinase/protein tyrosine kinase 6

IntroductionSignal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor signaling. In recent years, STAT5b has emerged as a key regulator of tumorigenesis. STAT5b phosphorylation and activation is mediated by several kinases known to be overexpressed in breast cancer, such as epidermal growth factor receptor, HER2, and c-Src. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase expressed in more than 60% of breast cancers. Only a few substrates of the Brk tyrosine kinase have been identified, the most recent being STAT3. In the present article we investigate the potential role of Brk in the phosphorylation and activation STAT5b.MethodsTo determine whether Brk can phosphorylate STAT5b, transient transfection and in vitro kinase assays were performed. Luciferase reporter assays were used to measure Brk-induced STAT5b transcriptional activity. siRNA technology was utilized to investigate the biological significance of Brk-induced activation of STAT5b in breast cancer cell models.ResultsPhosphospecific antibodies, mutational analysis, and in vitro kinase assays demonstrated that Brk specifically mediated STAT5b phosphorylation at the activating tyrosine, Y699. Transient transfection of Brk into the Brk-negative BT-549 breast cancer cell line enhanced STAT5b transcriptional activity, as measured by a STAT5-specific luciferase reporter. Furthermore, overexpression of kinase active c-Src enhanced Brk-induced STAT5b transcriptional activity. In Brk-positive breast cancer cell lines BT-20 and SKBr3, knockdown of Brk protein or of STAT5b protein using siRNA methodology resulted in a decrease in DNA synthesis. Knockdown of Brk and STAT5b together did not further decrease DNA synthesis compared with each alone, suggesting that Brk and STAT5b converge on the same pathway, ultimately leading to cellular proliferation.ConclusionOur studies demonstrate that Brk phosphorylates STAT5b on Y699, leading to increased STAT5b transcriptional activity. Furthermore, analysis of DNA synthesis suggests that STAT5b and Brk are converging upon the same proproliferative signaling pathway in breast cancer cells. We propose that Brk, like other tyrosine kinases, signals downstream to STAT5b to mediate proliferation of breast cancer cells. These results further establish STAT5b as well as Brk as potential targets for breast cancer therapy.

Signal transducer and activator of transcription 5b, c-Src, and epidermal growth factor receptor signaling play integral roles in estrogen-stimulated proliferation of estrogen receptor-positive breast cancer cells.

17beta-Estradiol (E2) acts through the estrogen receptor alpha (ERalpha) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERalpha and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.

A novel role for signal transducer and activator of transcription 5b (STAT5b) in β1-integrin-mediated human breast cancer cell migration

IntroductionSignal transducer and activator of transcription (STAT) 5b is a transcription factor involved in pro-proliferative and pro-survival signaling in a number of solid tumors, including breast cancer. The contribution of STAT5b to breast cancer cell motility has not been explored. This work aims to elucidate the role of STAT5b in breast cancer cell migration.MethodsSTAT5b was knocked down by using siRNA in two aggressive, highly migratory breast cancer cell lines (BT-549 and MDA-MB-231), and transwell migration assays were performed to determine the importance of STAT5b for their migration. Knockdown-rescue experiments were used to validate the specificity of STAT5b knockdown and to determine which regions/functions of STAT5b are necessary for its role in migration. Live-cell imaging of wound healing and spreading was carried out to examine cell morphology and motility after STAT5b knockdown.ResultsKnockdown of STAT5b, but not STAT5a, inhibited migration of BT-549 and MDA-MB-231 breast cancer cells to serum by 60% to 80%, and inhibited migration equally over a range of serum concentrations (0.1% to 10% serum). Migratory inhibition upon STAT5b knockdown could be rescued by reintroduction of wild-type STAT5b, as well as Y699F- and dominant-negative STAT5b mutants, but not an SH2 domain defective R618K-STAT5b mutant. β1- integrin-mediated migration of breast cancer cells to fibronectin was inhibited with STAT5b knockdown, and loss of STAT5b correlated with loss of directional migration and formation of multiple, highly contractile protrusions upon attachment to fibronectin.ConclusionsThe data presented here demonstrate that STAT5b is integral to breast cancer cell migration and identify a novel, SH2-dependent function of STAT5b in regulating β1-integrin-mediated migration of highly aggressive breast cancer cells.

Signal transduction defects in growth hormone insensitivity

Clayton PE, Freeth JS, Whatmore AJ, Ayling RM, Norman MR, Silva CM. Signal transduction defects in growth hormone insensitivity. Acta Pædiatr 1999; Suppl 428: 174–8. Stockholm. ISSN 0803–5326

STAT6 expression in glioblastoma promotes invasive growth

BackgroundGlioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.MethodsSTAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring 3H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt [1] public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.ResultsSTAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time.STAT6-deficient GBM cells showed a reduction in 3H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in 3H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt [1] gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.ConclusionsTaken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

A Mechanistic Study of the Effect of Doxorubicin/Adriamycin on the Estrogen Response in a Breast Cancer Model

Objective: Estrogen treatment limits the cytotoxic effects of chemotherapy in estrogen receptor-positive (ER+) breast cancer cell lines, suggesting that estrogen pathway signaling may confer chemotherapeutic resistance. This study investigates the molecular responses of ER+ breast cancer cell lines to the chemotherapeutic agent, doxorubicin, in the presence or absence of estrogen. Methods: ER+ MCF-7 and T47-D cells were cultured in hormone-starved or estrogen-containing media with or without doxorubicin at concentrations mimicking the low concentrations seen in plasma and tumor microenvironments in humans following typical bolus administration. Protein levels, phosphorylations, and interactions of estrogen-signaling molecules were assessed following these treatments, as well the effects of ER signaling inhibitors on cell proliferation. Results: Surprisingly, estrogen and doxorubicin co-treatment markedly induced pro-growth alterations compared to doxorubicin alone and modestly enhanced estrogen alone-induced changes. Several inhibitors suppressed cell proliferation in the presence of doxorubicin and estrogen. Conclusions: These findings demonstrate that molecular changes caused by doxorubicin in ER+ breast cancer cells can be reversed by estrogen, providing molecular evidence for the poorer responses of ER+ tumors to doxorubicin in the presence of physiologic estrogen levels. Our results also suggest that the addition of drugs targeting the ER, EGFR, the SFKs, MEK, PI3K, and/or the MMP proteins to a conventional chemotherapy regimen may improve chemosensitivity.

Cytokines in endocrine function.

Publisher Summary This chapter discusses the effects of cytokines on endocrine function. These hormones have pronounced effects on cellular metabolism and proliferation depending on the cell type. It explains how growth hormone can induce different effects through either pulsitile or continuous delivery or by the target cells themselves having different signal transduction pathways. The integral role of growth hormone (GH) in metabolism and the overall growth of the organism are well established, clinically. Growth hormone acts both directly and indirectly through the regulation of hepatic insulin-like growth factor-1 (IGF-1) to stimulate longitudinal bone growth at the epiphyseal growth plate. The regulation of growth by GH involves effects on tissue differentiation, cell proliferation, and protein synthesis. Elucidation of the signal transduction pathways is activated by the GH receptor. Many of the pathways activated are consider in detail and the role of cell-type-specific activation of these pathways is characterized. This continued understanding of GH signaling has profound implications in the treatment of the clinical effects of GH excess, deficit, and impairment of GH action at the cellular level.