51 research outputs found
Receptor and secreted targets of Wnt-1/beta-catenin signalling in mouse mammary epithelial cells.
BackgroundDeregulation of the Wnt/ beta-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in beta-catenin stabilization and accumulation, and the aberrant modulation of beta-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.MethodsGene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of beta-catenin was used to identify target genes of this pathway.ResultsThe differential expression in response to DeltaNbeta-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/beta-catenin tumour cells and between the tumour and its microenvironment. One of these genes, Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon DeltaNbeta-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.ConclusionsHIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer
Clarification of the C-terminal proteolytic processing site of human Amphiregulin
AbstractAmphiregulin, like other ErbB ligands, is synthesized as a pro-protein which requires cleavage at the cell surface to release the active signaling domain. Prior studies using a variety of approaches have not yielded a consensus about the precise cleavage site. Here we report the purification and protein sequencing of the cell-associated human Amphiregulin stalk which remains following cleavage of the signaling domain. These data indicate that human Amphiregulin is cleaved at Lysine 187, a site homologous to the cleavage site reported in the mouse protein and distinct from the Lysine 184 site previously reported for the human protein
Retroviral vectors for establishing tetracycline-regulated gene expression in an otherwise recalcitrant cell line
BACKGROUND: Tetracycline-regulated systems have been used to control the expression of heterologous genes in such diverse organisms as yeast, plants, flies and mice. Adaptation of this prokaryotic regulatory system avoids many of the problems inherent in other inducible systems. There have, however, been many reports of difficulties in establishing functioning stable cell lines due to the cytotoxic effects of expressing high levels of the tetracycline transactivator, tTA, from a strong viral promoter. RESULTS: Here we report the successful incorporation of tetracycline-mediated gene expression in a mouse mammary epithelial cell line, HC11, in which conventional approaches failed. We generated retroviruses in which tTA expression was controlled by one of three promoters: a synthetic tetracycline responsive promoter (TRE), the elongation factor 1-alpha promoter (EF1α) or the phosphoglycerate kinase-1 promoter (PGK), and compared the resulting cell lines to one generated using a cytomegalovirus immediate early gene promoter (CMV). In contrast to cells produced using the CMV and PGK promoters, those produced using the EF1α and TRE promoters expressed high levels of β-galactosidase in a tetracycline-dependent manner. CONCLUSIONS: These novel retroviral vectors performed better than the commercially available system and may have a more general utility in similarly recalcitrant cell lines
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3D culture models of normal and malignant breast epithelial cells
This report describes a robust and generalized method for the clustering of various human breast cell lines in 3D and describes the preparation of cellular extracts from these cultures for molecular analysis
Assessment of the TRPM8 inhibitor AMTB in breast cancer cells and its identification as an inhibitor of voltage gated sodium channels
To assess levels of the calcium permeable transient receptor potential cation channel, subfamily melastatin, member 8 (TRPM8) in breast cancer molecular subtypes and to assess the consequences of TRPM8 pharmacological inhibition with AMTB (an inhibitor of TRPM8) on breast cancer cell lines.Cell viability and migration of breast cancer cells was determined using MTS assays and wound healing assays, respectively. RNA-Seq analysis of breast tumours and qPCR in breast cancer cell lines were used to assess mRNA levels of ion channels. Membrane potential assays were employed to assess the effects of AMTB against specific voltage gated sodium channels (Na).TRPM8 levels were significantly higher in breast cancers of the basal molecular subtype. AMTB decreased viable cell number in MDA-MB-231 and SK-BR-3 breast cancer cell lines (30 and 100 μM), and also reduced the migration of MDA-MB-231 cells (30 μM). However, these effects were independent of TRPM8, as no TRPM8 mRNA was detected in MDA-MB-231 cells. AMTB was identified as an inhibitor of Naisoforms. Na1.1-1.9 were expressed in a number of breast cancer cell lines, with Na1.5 mRNA highest in MDA-MB-231 cells compared to the other breast cancer cell lines assessed.TRPM8 levels may be elevated in basal breast cancers, however, TRPM8 expression appears to be lost in many breast cancer cell lines. Some of the effects of AMTB attributed to TRPM8 may be due to effects on Nachannels
Calcium channel TRPV6 as a potential therapeutic target in estrogen receptor negative breast cancer
Calcium signaling is a critical regulator of cell proliferation. Elevated expression of calcium channels and pumps is a characteristic of some cancers, including breast cancer. We show that the plasma membrane calcium channel TRPV6, which is highly selective for Ca(2+), is overexpressed in some breast cancer cell lines. Silencing of TRPV6 expression in a breast cancer cell line with increased endogenous TRPV6 expression lead to a reduction in basal calcium influx and cellular proliferation associated with a reduction in DNA synthesis. TRPV6 gene amplification was identified as one mechanism of TRPV6 overexpression in a sub-set of breast cancer cell lines and breast tumor samples. Analysis of two independent microarray expression datasets from breast tumor samples showed that increased TRPV6 expression is a feature of estrogen receptor negative breast tumors encompassing the basal-like molecular subtype, as well as HER2-positive tumors. Breast cancer patients with high TRPV6 levels had decreased survival compared to patients with low or intermediate TRPV6 expression. Our findings suggest that inhibitors of TRPV6 may offer a novel therapeutic strategy for the treatment of estrogen receptor-negative breast cancers
The morphologies of breast cancer cell lines in three-dimensionalassays correlate with their profiles of gene expression
3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments
Keystone symposium: The role of microenvironment in tumor induction and progression, Banff, Canada, 5–10 February 2005
The first Keystone symposium on the role of microenvironment in tumor induction and progression attracted 274 delegates from 13 countries to Banff in the heart of the Canadian Rockies. The meeting was organized by Mina Bissell, Ronald DePinho and Luis Parada, and was held concurrently with the Keystone symposium on cancer and development, chaired by Matthew Scott and Roeland Nusse. The 30 oral presentations and over 130 posters provided an excellent forum for discussing emerging data in this rapidly advancing field
Remodeling of Purinergic Receptor-Mediated Ca2+ Signaling as a Consequence of EGF-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells
Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis
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