BRCA1 Suppresses Osteopontin-mediated Breast Cancer

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor alpha, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorage independent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.

BRCA1 regulates the interferon gamma-mediated apoptotic response

BRCA1 is a tumor suppressor gene implicated in transcriptional regulation. We have generated cell lines with inducible expression of BRCA1 as a tool to identify downstream targets that may be important mediators of BRCA1 function. Oligonucleotide array-based expression profiling identified 11 previously described interferon regulated genes that were up-regulated following inducible expression of BRCA1. Northern blot analysis revealed that a subset of the identified targets including IRF-7, MxA, and ISG-54 were synergistically up-regulated by BRCA1 in the presence of interferon gamma (IFN-gamma) but not interferons alpha or beta. Importantly, IFN-gamma-mediated induction of IRF-7 and MxA was attenuated in the BRCA1 mutant cell line HCC1937, an effect that was rescued following reconstitution of exogenous wild type BRCA1 in these cells. Furthermore, reconstituted BRCA1 sensitized HCC1937 cells to IFN-gamma-induced apoptotic cell death. This study identifies BRCA1 as a component of the IFN-gamma-regulated signaling pathway and suggests that BRCA1 may play a role in the regulation of IFN-gamma-mediated apoptosis.

ICln, a novel integrin alpha(11b)beta3-associated protein, functionally regulates platelet activation.

A critical role for the conserved -integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin IIb3. To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with IIb3 by surface plasmon resonance. The affinity of this interaction was 82.2 ± 24.4 nM in a cell free assay. ICln co-immunoprecipitates with IIb3 in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 µM to 5 mM), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10–100 µM) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.

DUB-3, a Cytokine inducible Deubiquitinating Enzyme That Blocks Profileration

Previous studies have identified the DUB family of cytokine-regulated murine deubiquitinating enzymes, which play a role in the control of cell proliferation and survival. Through data base analyses and cloning, we have identified a human cDNA (DUB-3) that shows significant homology to the known murine DUB family members. Northern blotting has shown expression of this gene in a number of tissues including brain, liver, and muscle, with two transcripts being apparent (1.6 and 1.7 kb). In addition, expression was observed in cell lines including those derived from a number of hematopoietic tumors such as the Burkitt's lymphoma cell line RAJI. We have also demonstrated that DUB-3, which was shown to be an active deubiquitinating enzyme, is induced in response to interleukin-4 and interleukin-6 stimulation. Finally, we have demonstrated that constitutive expression of DUB-3 blocks proliferation and can initiate apoptosis in both IL-3-dependent Ba/F3 cells and NIH3T3 fibroblasts. These findings suggest that human DUB-3, like the murine DUB family members, is transiently induced in response to cytokines and can, when constitutively expressed, block growth factor-dependent proliferation.

Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids

TRPM8 represents an ion channel activated by cold temperatures and cooling agents, such as menthol, that underlies the cold-induced excitation of sensory neurons. Interestingly, the only human tissue outside the peripheral nervous system, in which the expression of TRPM8 transcripts has been detected at high levels, is the prostate, a tissue not exposed to any essential temperature variations. Here we show that the TRPM8 cloned from human prostate and heterologously expressed in HEK-293 cells is regulated by the Ca(2+)-independent phospholipase A(2) (iPLA(2)) signaling pathway with its end products, lysophospholipids (LPLs), acting as its endogenous ligands. LPLs induce prominent prolongation of TRPM8 channel openings that are hardly detectable with other stimuli (e.g. cold, menthol, and depolarization) and that account for more than 90% of the total channel open time. Down-regulation of iPLA(2) resulted in a strong inhibition of TRPM8-mediated functional responses and abolished channel activation. The action of LPLs on TRPM8 channels involved either changes in the local lipid bilayer tension or interaction with the critical determinant(s) in the transmembrane channel core. Based on this, we propose a novel concept of TRPM8 regulation with the involvement of iPLA(2) stimulation. This mechanism employs chemical rather than physical (temperature change) signaling and thus may be the main regulator of TRPM8 activation in organs not exposed to any essential temperature variations, as in the prostate gland.