Changes In Breast Cancer Cells Research Articles

Dysregulation of zinc metabolism as a marker of phenotypic and functional change in breast cancer cells

Dysregulation of zinc metabolism as a marker of phenotypic and functional change in breast cancer cells

Abstract 5089: Evaluation of BMP4 and BMP7 induced gene expression changes in breast cancer cells reveals time-dependent transcription patterns and a common synexpression group of genes

Abstract Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily of growth factors. They are well-known for their key roles in the regulation of various developmental processes and bone formation, but in recent years, evidence has also accumulated of their crucial contribution in tumor biology. In particular, BMP4 and BMP7 have been implicated in breast cancer pathogenesis. However, little is known about BMP target genes in the context of tumor cells. Here, we used whole-genome microarrays to explore the effects of BMP4 and BMP7 treatment on global gene expression levels in seven breast cancer cell lines during a 6-point time series. Several complementary methods, including hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data, were used for data analysis. Both BMP ligands had a strong influence on gene expression levels, although the response to BMP4 treatment was more pronounced as compared to BMP7. As might have been expected, the cellular functions most strongly affected by the stimulation of BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, where the regulation of gene expression and signal transduction was followed by changes in transcripts involved in metabolism and cell proliferation. Hierarchical clustering analysis revealed distinct differences in the response of individual cell lines to BMPs, but also underlined a common synexpression group of genes for both ligands. Remarkably, the majority of the genes within these synexpression groups were shared by the two ligands, and thus these genes are likely to represent the core molecular responses common to BMP4 and BMP7 signaling pathways. Taken together, our data demonstrate that BMP signaling has a substantial effect on gene transcription in breast cancer cells and that the cellular functions affected follow a logical temporal pattern. Our results also reveal components of the cellular transcriptional response that are common to BMP4 and BMP7. Finally, our study provides a list of putative novel BMP target genes relevant in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5089. doi:1538-7445.AM2012-5089

EphrinA1–EphA2 interaction‐mediated apoptosis and FMS‐like tyrosine kinase 3 receptor ligand‐induced immunotherapy inhibit tumor growth in a breast cancer mouse model

The receptor tyrosine kinase EphA2 is overexpressed in several types of cancers and is currently being pursued as a target for breast cancer therapeutics. The EphA2 ligand EphrinA1 induces EphA2 phosphorylation and intracellular internalization and degradation, thus inhibiting tumor progression. The hematopoietic growth factor, FMS-like tyrosine kinase 3 receptor ligand (Flt3L), promotes expansion and mobilization of functional dendritic cells. We tested the EphrinA1-EphA2 interaction in MDA-MB-231 breast cancer cells focusing on the receptor-ligand-mediated apoptosis of breast cancer cells. To determine whether EphrinA1-EphA2 interaction-associated apoptosis and Flt3L-mediated immunotherapy would have an additive effect in inhibiting tumor growth, we used an immunocompetent mouse model of breast cancer to evaluate intratumoral (i.t.) inoculation strategies with human adenovirus (HAd) vectors expressing either EphrinA1 (HAd-EphrinA1-Fc), Flt3L (HAd-Flt3L) or a combination of EphrinA1-Fc + Flt3L (HAd-EphrinA1-Fc + HAd-Flt3L). In vitro analysis demonstrated that an EphrinA1-EphA2 interaction led to apoptosis-related changes in breast cancer cells. In vivo, three i.t. inoculations of HAd-EphrinA1-Fc showed potent inhibition of tumor growth. Furthermore, increased inhibition in tumor growth was observed with the combination of HAd-EphrinA1-Fc and HAd-Flt3L accompanied by the generation of an anti-tumor adaptive immune response. The results obtained in the present study, indicating the induction of apoptosis and inhibition of mammary tumor growth, show the potential therapeutic benefits of HAd-EphrinA1-Fc. In combination with HAd-Flt3L, this represents a promising strategy for effectively inducing mammary tumor regression by HAd vector-based therapy.

Molecular analysis of melatonin-induced changes in breast cancer cells: microarray study of anti-cancer effect of melatonin

Melatonin is a pineal secretory product that acts as a chemical messenger for the dark/light cycle and plays a central role in synchronization of circadian rhythms. Melatonin has been reported to exhibit a variety of therapeutic effects, including antiaging, anti-oxidative, anti-inflammation, and anticancer (such as induction of tumor suppressor genes and suppression of the metabolism of tumor cells, including breast cancer cells). In this study, we investigated the genome-wide transcriptional responses of MCF-7 human breast cancer cells exposed to melatonin by microarray gene expression profiling. We identified 1,946 and 983 genes that were 2-fold up- or down-regulated within 72 h of 1 nM and 100 nM melatonin treatment. Gene Ontology (GO) enrichment analysis was performed for identification of the biological functions and biological processes affected by the differential expression of genes in melatonin-stimulated cells. The differentially expressed genes that were dysregulated in many biological functions included cell proliferation, immune responses, translation, cell adhesion, apoptosis, and cell cycle. Our findings support the view that melatonin-stimulated changes in gene expression contribute to the anticancer effect of melatonin in breast cancer cells.

Light Scattering Detects Changes In Subcellular Structure And Organization With Connections To Cell Function

By incorporating new models into our light scattering analysis techniques that better account for the ellipsoidal shape of cellular organelles, we can determine not only the average size, but also the average shape of an ensemble of cell nuclei in culture. This advance permitted new insight into the nucleus structure, and by providing an accurate depiction of its contribution to the light scattering signal, has also enabled an enhanced ability to analyze density correlations and therefore subcellular organization in biological cells. We will present verification of our methods and results of two new studies facilitated by these recent developments. In the first study, we used light scattering to detect statistically significant structural changes in breast cancer cells within one hour after treatment with apoptosis-inducing drugs. Two conclusions emerge: First, the ability of this technique to discern early onset of apoptosis makes it a promising tool for monitoring cancer treatments; and second, monitoring the organization of subcellular organelles could be a powerful method for studying the mechanisms of apoptosis, and perhaps other functional changes in cells. In the second study, we evaluated the deformation of stem cell nuclei as a response to engineered nanotopographical cues and the mechanical properties of their substrate. As verified by image analysis and comparison to control samples, the changes in nuclear shape due to materials' properties and nanotopography are highly significant. Additionally, these shape changes relate to modifications in stem cell adhesion and mobility, and provide a connection between environmental cues, nuclear deformation, and cellular behavior. Both studies solidified light scattering as a promising tool to assess structure in biological samples, and indicate the potential to link these structural changes to corresponding alterations in cell function.

Potential promotion of metastatis of circulating breast cancer cells through interactions with mesenchymal stem cells (MSCs).

Abstract Abstract #1052 Introduction
 Mesenchymal Stem Cells (MSCs) are a subset of pluripotent non-haematopoeitic stem cells within the bone marrow stroma. Chemokines secreted by MSCs have been shown to influence the morphology and proliferative activity of cells within their vicinity. Dynamic reciprocal interactions occurring between circulating breast cancer cells and MSCs within the bone marrow microenvironment may facilitate further metastatic propagation and dissemination. Monocyte Chemotactic Protein-1 (MCP-1/CCL2) and Stromal Cell Derived factor 1-α (SDF-1a/CXCL12) are inflammatory chemokines with tumorigenic potential that may facilitate the cultivation of a pre-metastatic niche. Both of these chemokines have been associated with promoting and directing metastatic spread in breast cancer patients, and are know to be secreted by MSCs. The aim of this study was to elucidate the impact MSCs have on breast cancer cells and to potentially identify factors mediating these effects.
 Methods
 MSCs were isolated from bone marrow aspirates of healthy volunteers, and their pluripotency confirmed prior to use. Breast Cancer Cell lines (MDA-MB-231 & T47D) were cultured individually and on a monolayer of MSCs for a period of 72hrs. Conditioned medium was harvested from cells cultured individually and co-culture populations. The concentrations of CCL2, CXCL12 and VEGF were quantified using ELISA. Breast cancer cells were retrieved following co-culture using epithelial specific magnetic beads. RNA was extracted and changes in expression of target genes following co-culture with MSCs was investigated using RQ-PCR. Targets included CCL2, CXCL12, their cognate receptors CCR2 and CXCR4, as well as Her2, Ki-67, MMP11 - genes previously related to breast cancer tumorigenicity.
 Results
 While there was no significant change in secretion of SDF-1α or VEGF upon direct co-culture of breast cancer cells with MSCs, there was a significant increase in MCP-1 secreted by the mixed cell populations compared to those cultured alone (P<0.05). CCL2 gene expression was upregulated in breast cancer cells retrieved following co-culture (MDA-MB-231: 4 fold increase; T47D: 1.7 fold increase) and expression of its principal receptor, CCR2, was also increased in T47D cells. CXCL12 gene expression was upregulated in MDA-MB-231 cells (4.2 fold). There was no significant change in CXCL12 expression in the T47D cells, however expression of its principal receptor, CXCR4, was upregulated. MMP11, a marker of invasive potential, was upregulated in both epithelial cell lines while Ki-67 and Her2 expression remained unchanged.
 Conclusion
 Direct contact with MSCs causes a distinct change in breast cancer cells as demonstrated by genetic modifications across a range of genes associated with breast cancer tumorigenicity. These results highlight the potential role MSCs play in facilitating the creation of a pre-metastatic niche and supporting the progression of engrafted tumour cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1052.

P31 Monitoring phenotypical changes of breast cancer cells, sorted in accordance to their CD44 and CD24 marker expression

P31 Monitoring phenotypical changes of breast cancer cells, sorted in accordance to their CD44 and CD24 marker expression

Quantitative Profiling of Drug-Associated Proteomic Alterations by Combined 2-Nitrobenzenesulfenyl Chloride (NBS) Isotope Labeling and 2DE/MS Identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.

Camptothecin Induces Nuclear Export of Prohibitin Preferentially in Transformed Cells through a CRM-1-dependent Mechanism

Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.

Linkage of Rapid Estrogen Action to MAPK Activation by ERα-Shc Association and Shc Pathway Activation

E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc (Src-homology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ERalpha and Src are upstream regulators of Shc. A rapid physical association between ERalpha and Shc upon E2 stimulation further evidenced the role of ERalpha on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ERalpha. Using a glutathione-S-transferase-Shc pull-down assay, we demonstrated that this ERalpha-Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ERalpha membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERalpha can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells

Bioactive Synthetic Peptide of NDF/Heregulin

A folded synthetic peptide analog of NDF (NDF5), which has the 52 amino acid EGF-like domain of NDFα2, has been characterized. The folded peptide stimulates tyrosine phosphorylation of Her2, Her3 and Her4 in breast cancer cells and competes with low affinity with full-length NDFα2 for binding to the cells, while the linear one does not. NDF5 also induces morphologic changes in breast cancer cells. After several days treatment with NDF5 or NDFα2, Her2-transfected MCF7 cells (Her2/MCF7) became similar morphologically to non-transfected MCF7. The biological activity of NDF5 is between 1/10 and 1/100 that of NDFα2. This suggests that other motifs, such as the Ig and spacer domains may be important elements in conferring full activity.

Interleukin-6 enhances motility of breast carcinoma cells.

Interleukin-6 (IL-6) is the major systemic mediator of the early host response to infection and injury (the "acute phase response"). Furthermore, IL-6 is often detected in the peripheral circulation and in the local neoplastic tissue in cancer patients. IL-6 has distinctive effect on epithelial cells depending upon the cell type examined. IL-6 enhances proliferation of normal human keratinocytes without affecting cell morphology. In contrast, IL-6 inhibits the proliferation of ductal breast carcinoma cell lines T-47D, ZR-71-1 and MCF-7. In addition to, but independent of, the inhibition of cell proliferation, IL-6 induces a cellular phenotype in the typically epitheliod T-47D and ZR-75-1 cells, which is characterized by fibroblastoid morphology, increased cell-cell separation even within preformed colonies, decreased adherens type junction formation (desmosomes and focal adhesions), and enhanced motility. Time-lapse cinemicrography of T-47D and wild-type ZR-75-1 cells reveals increased local movement of IL-6-treated cells and also movement of these cells over considerable distances. The effects of IL-6 on breast cancer cell proliferation and motility are reversible by removal of IL-6 from the culture medium. Time-lapse cinemicrography reveals that in clone B ZR-75-1 cells, which are not sensitive to the DNA synthesis-inhibitory effect of IL-6 or to its cell-separating effect on preformed colonies, IL-6 can still block rapid readherence of post-mitotic cells to their neighbors and to the substratum leading to enhanced dispersal of cancer cells into the culture medium. In wild-type ZR-75-1 cells, 12-O-tetradecanoyl phorbol ester (TPA) exerts a cell-scattering effect on breast cancer cells without inhibiting cell proliferation. Combined treatment with IL-6 and TPA produces a cell-scattering effect that greatly exceeds in magnitude and speed the phenotypic change elicited by either reagent alone. Staurosporine blocks cell-scattering caused by TPA but not that caused by IL-6 suggesting that IL-6 and TPA elicit similar phenotypic changes in breast cancer cells via different pathways. Taken together, these findings identify a previously unrecognized property of IL-6, that of enhancing cell motility.