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

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

Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARγ has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARγ has been validated through two supporting biological assays

Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth

Abstract Background Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. Methods We crossed AREG-null (AREG−/−) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG−/− PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. Results Intriguingly, PyMT-induced lesions progress more rapidly in AREG−/− mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG−/− mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG−/− PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. Conclusions Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer