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

Oh what a tangled web it weaves: BRCA1 and DNA decatenation

SummaryBRCA1 has significant roles in DNA repair and cell cycle checkpoint control, and is important in the maintenance of genomic stability. Defects in these pathways likely underpin the cancer susceptibility of BRCA1 mutation carriers. Now, a new function for BRCA1 in DNA decatenation—removing the tangles introduced into chromosomes as a consequence of DNA replication—is suggested in a new paper by Lou et al. (2005) in Nature Structural and Molecular Biology. Ineffective DNA decatenation may lead to chromosome breakage and inappropriate repair, adding to the roll call of defects in BRCA1 mutant cells

Evolutionary rate of a gene affected by chromosomal position

AbstractGenes evolve at different rates depending on the strength of selective pressure to maintain their function. Chromosomal position can also have an influence [1,2]. The pseudoautosomal region (PAR) of mammalian sex chromosomes is a small region of sequence identity that is the site of an obligatory pairing and recombination event between the X and Y chromosomes during male meiosis [3–6]. During female meiosis, X chromosomes can pair and recombine along their entire length. Recombination in the PAR is therefore approximately 10 times greater in male meiosis compared with female meiosis [4–6]. The gene Fxy (also known as MID1[7]) spans the pseudoautosomal boundary (PAB) in the laboratory mouse (Mus musculus domesticus, C57BL/6) such that the 5′ three exons of the gene are located on the X chromosome but the seven exons encoding the carboxy-terminal two-thirds of the protein are located within the PAR and are therefore present on both the X and Y chromosomes [8]. In humans [7,9], the rat, and the wild mouse species Mus spretus, the gene is entirely X-unique. Here, we report that the rate of sequence divergence of the 3′ end of the Fxy gene is much higher (estimated at 170-fold higher for synonymous sites) when pseudoautosomal (present on both the X and Y chromosomes) than when X-unique. Thus, chromosomal position can directly affect the rate of evolution of a gene. This finding also provides support for the suggestion that regions of the genome with a high recombination frequency, such as the PAR, may have an intrinsically elevated rate of sequence divergence

MAP kinase phosphatases

Mitogen-activated protein MAP kinases are key signal-transducing enzymes that are activated by a wide range of extracellular stimuli. They are responsible for the induction of a number of cellular responses, such as changes in gene expression, proliferation, differentiation, cell cycle arrest and apoptosis. Although regulation of MAP kinases by a phosphorylation cascade has long been recognized as significant, their inactivation through the action of specific phosphatases has been less studied. An emerging family of structurally distinct dual-specificity serine, threonine and tyrosine phosphatases that act on MAP kinases consists of ten members in mammals, and members have been found in animals, plants and yeast. Three subgroups have been identified that differ in exon structure, sequence and substrate specificity

Signalling Pathways Implicated in Early Mammary Gland Morphogenesis and Breast Cancer

Specification of mammary epithelial cell fate occurs during embryogenesis as cells aggregate to form the mammary anlage. Within the embryonic mammary bud, a population of epithelial cells exists that will subsequently proliferate to form a ductal tree filling the stromal compartment, and which can produce milk upon terminal differentiation after birth. Subsequently, these structures can be remodelled and returned to a basal state after weaning before regenerating in future pregnancies. The plasticity of the mammary epithelial cell, and its responsiveness to hormone receptors, facilitates this amazing biological feat, but aberrant signalling may also result in unintended consequences in the form of frequent malignancies. Reflecting this intimate connection, a considerable number of signalling pathways have been implicated in both mammary gland morphogenesis and carcinogenesis

A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers.

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers

Brca2 and Trp53 deficiency cooperate in the progression of mouse prostate tumourigenesis.

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches

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

Neuregulin3 alters cell fate in the epidermis and mammary gland

<p>Abstract</p> <p>Background</p> <p>The Neuregulin family of ligands and their receptors, the Erbb tyrosine kinases, have important roles in epidermal and mammary gland development as well as during carcinogenesis. Previously, we demonstrated that Neuregulin3 (Nrg3) is a specification signal for mammary placode formation in mice. Nrg3 is a growth factor, which binds and activates Erbb4, a receptor tyrosine kinase that regulates cell proliferation and differentiation. To understand the role of Neuregulin3 in epidermal morphogenesis, we have developed a transgenic mouse model that expresses Nrg3 throughout the basal layer (progenitor/stem cell compartment) of mouse epidermis and the outer root sheath of developing hair follicles.</p> <p>Results</p> <p>Transgenic females formed supernumerary nipples and mammary glands along and adjacent to the mammary line providing strong evidence that Nrg3 has a role in the initiation of mammary placodes along the body axis. In addition, alterations in morphogenesis and differentiation of other epidermal appendages were observed, including the hair follicles. The transgenic epidermis is hyperplastic with excessive sebaceous differentiation and shows striking similarities to mouse models in which c-Myc is activated in the basal layer including decreased expression levels of the adhesion receptors, α6-integrin and β1-integrin.</p> <p>Conclusion</p> <p>These results indicate that the epidermis is sensitive to Nrg3 signaling, and that this growth factor can regulate cell fate of pluripotent epidermal cell populations including that of the mammary gland. Nrg3 appears to act, in part, by inducing c-Myc, altering the proliferation and adhesion properties of the basal epidermis, and may promote exit from the stem cell compartment. The results we describe provide significant insight into how growth factors, such as Nrg3, regulate epidermal homeostasis by influencing the balance between stem cell renewal, lineage selection and differentiation.</p