BRCA1 transcriptionally regulates genes associated with the basal-like phenotype in breast cancer

Expression profiling of BRCA1-deficient tumours has identified a pattern of gene expression similar to basal-like breast tumours. In this study, we examine whether a BRCA1-dependent transcriptional mechanism may underpin the link between BRCA1 and basal-like phenotype. In methods section, the mRNA and protein were harvested from a number of BRCA1 mutant and wild-type breast cancer cell lines and from matched isogenic controls. Microarray-based expression profiling was used to identify potential BRCA1-regulated transcripts. These gene targets were then validated (by in silico analysis of tumour samples) by real-time PCR and Western blot analysis. Chromatin immunoprecipitation (ChIP) assays were used to confirm recruitment of BRCA1 to specific promoters. In results, we demonstrate that functional BRCA1 represses the expression of cytokeratins 5(KRT5) and 17(KRT17) and p-Cadherin (CDH3) in HCC1937 and T47D breast cancer cell lines at both mRNA and protein level. ChIP assays demonstrate that BRCA1 is recruited to the promoters of KRT5, KRT17 and CDH3, and re-ChIP assays confirm that BRCA1 is recruited independently to form c-Myc and Sp1 complexes on the CDH3 promoter. We show that siRNA-mediated inhibition of endogenous c-Myc (and not Sp1) results in a marked increase in CDH3 expression analogous to that observed following the inhibition of endogenous BRCA1. The data provided suggest a model whereby BRCA1 and c-Myc form a repressor complex on the promoters of specific basal genes and represent a potential mechanism to explain the observed overexpression of key basal markers in BRCA1-deficient tumours.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β Production

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-alpha and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-alpha after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-alpha but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-kappa B activation, whereas TNF-alpha expression is blocked at the gene transcription level. Interferon beta plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon beta. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon beta and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal-like breast cancers

In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs. Oncogene (2012) 31, 3667-3678; doi:10.1038/onc.2011.531; published online 28 November 2011

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda, Ascaroidea)

The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF A SALMFAMIDE-LIKE NEUROPEPTIDE IN THE NERVOUS-SYSTEM OF ADULT AND LARVAL STAGES OF THE HUMAN BLOOD FLUKE, SCHISTOSOMA-MANSONI

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, S1-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

Introduction: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.

Methods: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.

Results: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.

Conclusion: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.

Airway epithelial cell apoptosis and inflammation in COPD, smokers and non-smokers

We hypothesised that primary bronchial epithelial cells (PBECs) from subjects with COPD respond differently to Pseudomonas aeruginosa lipopolysaccharide (PA LPS) after cigarette smoke extract (CSE) exposure than PBECs obtained from smokers without airflow obstruction (SWAO) and non-smokers (NS).PBECs from 16 COPD subjects, 10 SWAOand 9 NS were cultured at air-liquid interface. Cultures were incubated with CSE prior to stimulation with PA LPS. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 expression by FACS. Activation of NF-?B was determined by western blotting and ELISA, and MAPK and caspase-3 activity by western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) methods.Constitutive release of IL-8 and IL-6 was greatest from the COPD cultures.However, CSE pre-treatment followed by PA LPS stimulation reduced IL-8 release from COPD PBECs, but increased it from cells of SWAOand NS. TLR-4 expression,MAPK and NF-?B activation in COPD cultures were reduced after CSE treatment, but not in the SWAOor NS groups, which was associated with increased apoptosis.CSE attenuates inflammatory responses to LPS in cells from people with COPD but not those from non-smoking individuals and those who smoke without airflow obstruction.

Mitochondrial proteins Bnip3 and Bnip3L are involved in anthrax lethal toxin-induced macrophage cell death

Anthrax lethal toxin (LeTx) induces rapid cell death of RAW246.7 macrophages. We recently found that a small population of these macrophages is spontaneously and temporally refractory to LeTx-induced cytotoxicity. Analysis of genome-wide transcripts of a resistant clone before and after regaining LeTx sensitivity revealed that a reduction of two closely related mitochondrial proteins, Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) and Bnip3-like (Bnip3L), correlates with LeTx resistance. Down-regulation of Bnip3 and Bnip3L was also found in "toxin-induced resistance" whereby sublethal doses of LeTx induce resistance to subsequent exposure to cytolytic toxin doses. The role of Bnip3 and Bnip3L in LeTx-induced cell death was confirmed by showing that overexpression of either Bnip3 or Bnip3L rendered the resistant cells susceptible to LeTx, whereas down-regulation of Bnip3 and Bnip3L in wild-type macrophages conferred resistance. The down-regulation of Bnip3 and Bnip3L mRNAs by LeTx occurred at both transcriptional and mRNA stability levels. Inhibition of the p38 pathway by lethal factor was responsible for the destabilization of Bnip3/Bnip3L mRNAs as confirmed by showing that p38 inhibitors stabilized Bnip3 and Bnip3L mRNAs and conferred resistance to LeTx cytotoxicity. Therefore, Bnip3/Bnip3L play a crucial role in LeTx-induced cytotoxicity, and down-regulation of Bnip3/Bnip3L is a mechanism of spontaneous or toxin-induced resistance of macrophages.

Plasma concentrations of glucagon-like peptide-2 in adult patients with treated and untreated coeliac disease

Coeliac disease is a common chronic inflammatory enteropathy characterized by villous atrophy and crypt hyperplasia in the small intestine. The mechanism of the intestinal damage in coeliac disease remains unclear. Glucagon-like peptide (GLP)-2 is an enterotrophic peptide that causes crypt hyperplasia and intestinal cell proliferation. We postulate that GLP-2 may be involved in the mucosal changes found in coeliac disease.

Recurrence of Urothelial Carcinoma of the Bladder: A Role for Insulin-Like Growth Factor-II Loss of Imprinting and Cytoplasmic E-Cadherin Immunolocalization

Purpose:This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II.
Experimental Design: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pTanoninvasive and 44 pT1laminapropria invasive) using the commercially available Novocastra antibody.
Results: IGF-IILOI was evident in 7 of17 (41%) UCB tumors and 4 of11 (36%) tumor-associated normal urothelial samples.Two of four pT1grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-IImaintenance ofimprinting tumorshad concomitant CDH1cytoplasmiclocalization. UCB cell lines displaying cytoplasmic CDH1immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1staining. Finally, cytoplasmic CDH1staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage.
Conclusions: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase

We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.-Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase.