Molecular subtyping of breast cancer: Opportunities for new therapeutic approaches

Evidence is accumulating that breast cancer is not one disease but many separate diseases. DNA microarray-based gene expression profiling has demonstrated subtypes with distinct phenotypic features and clinical responses. Prominent among the new subtypes is 'basal-like' breast cancer, one of the 'intrinsic' subtypes defined by negativity for the estrogen, progesterone, and HER2/neu receptors and positivity for cytokeratins-5/6. Focusing on basal-like breast cancer, we discuss how molecular technologies provide new chemotherapy targets, optimising treatment whilst sparing patients from un-necessary toxicity. Clinical trials are needed that incorporate long-term follow-up of patients with well-characterised tumour markers. Whilst the absence of an obvious dominant oncogene driving basallike breast cancer and the lack of specific therapeutic agents are serious stumbling blocks, this review will highlight several promising therapeutic candidates currently under evaluation. Thus, new molecular technologies should provide a fundamental foundation for better understanding breast and other cancers which may be exploited to save lives.

Cell-to-Cell Interactions and Signals Involved in the Reconstitution of Peripheral CD8(+) T-CM and T-EM Cell Pools

We here describe novel aspects of CD8(+) and CD4(+) T cell subset interactions that may be clinically relevant and provide new tools for regulating the reconstitution of the peripheral CD8(+) T cell pools in immune-deficient states. We show that the reconstitution capacity of transferred isolated naive CD8(+) T cells and their differentiation of effector functions is limited, but both dramatically increase upon the co-transfer of CD4(+) T cells. This helper effect is complex and determined by multiple factors. It was directly correlated to the number of helper cells, required the continuous presence of the CD4(+) T cells, dependent on host antigen-presenting cells (APCs) expressing CD40 and on the formation of CD4/CD8/APC cell clusters. By comparing the recovery of (CD44(+)CD62L(high)) T-CM and (CD44(+)CD62L(low)) T-EM CD8(+) T cells, we found that the accumulation of TCM and TEM subsets is differentially regulated. T-CM-cell accumulation depended mainly on type I interferons, interleukin (IL)-6, and IL-15, but was independent of CD4(+) T-cell help. In contrast, TEM-cell expansion was mainly determined by CD4(+) T-cell help and dependent on the expression of IL-2R beta by CD8 cells, on IL-2 produced by CD4(+) T-cells, on IL-15 and to a minor extent on IL-6.

Induction of endothelial PAS domain protein-1 by hypoxia: Characterization and comparison with hypoxia-inducible factor-1 alpha

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1 alpha and aryl hydrocarbon nuclear receptor translocator (ARNT), In response to hypoxia, HIF-1 alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1 alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1 alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1 alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1 alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1 alpha transactivation) of the VEGF promoter than the LDH-A promoter. (C) 1998 by The American Society of Hematology.

Apo J/clusterin expression and secretion: Evidence for 15-deoxy-Δ12,14-PGJ2-dependent mechanism

Cyclooxygenase-2 (Cox-2) and Apo J/clusterin are involved in inflammatory resolution and have each been reported to inhibit NF-?B signalling. Using a well-validated rat pheochromocytoma (PC12) cell culture model of Cox-2 over-expression the current study investigated inter-dependence between Cox-2 and clusterin with respect to induction of expression and impact on NF-?B signalling. Both gene expression and immunoblot analysis confirmed that intracellular and secreted levels of clusterin were elevated in Cox-2 over-expressing cells (PCXII). Clusterin expression was increased in control (PCMT) cells in a time- and dose-dependent manner by 15-deoxy-? 12,14-prostaglandin J 2 (15d-PGJ 2), but not PGE 2, and inhibited in PCXII cells by pharmacological Cox inhibition. In PCXII cells, inhibition of two transcription factors known to be activated by 15d-PGJ 2, heat shock factor 1 (HSF-1) and peroxisome proliferator activated receptor (PPAR)?, by transcription factor oligonucleotide decoy and antagonist (GW9662) treatment, respectively, reduced clusterin expression. While PCXII cells exhibited reduced TNF-a-induced cell surface ICAM-1 expression, IkB phosphorylation and degradation were similar to control cells. With respect to the impact of Cox-2-dependent clusterin upregulation on NF-?B signalling, basal levels of I?B were similar in control and PCXII cells, and no evidence for a physical association between clusterin and phospho-I?B was obtained. Moreover, while PCXII cells exhibited reduced NF-?B transcriptional activity, this was not restored by clusterin knock-down. These results indicate that Cox-2 induces clusterin in a 15d-PGJ 2-dependent manner, and via activation of HSF-1 and PPAR?. However, the results do not support a model whereby Cox-2/15d-PGJ 2-dependent inhibition of NF-?B signalling involves clusterin.

Early target cells of measles virus after aerosol infection of non-human primates

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n?=?3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus

The lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognized, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entry of MV into the body. However, the lack of detectable CD150 on these cells raises the issue of their importance in the pathogenesis of measles. This study utilized a combination of in vitro, ex vivo and in vivo model systems to characterize the susceptibility of epithelial cells to wt-MV of proven pathogenicity. Low numbers of MV-infected epithelial cells in close proximity to underlying infected lymphocytes or myeloid cells suggested infection via the basolateral side of the epithelium in the macaque model. In primary cultures of human bronchial epithelial cells, foci of MV-infected cells were only observed following infection via the basolateral cell surface. The extent of infection in primary cells was enhanced both in vitro and in ex vivo cornea rim tissue by disrupting the integrity of the cells prior to the application of virus. This demonstrated that, whilst epithelial cells may not be the primary target cells for wt-MV, areas of epithelium in which tight junctions are disrupted can become infected using high m.o.i. The low numbers of MV-infected epithelial cells observed in vivo in conjunction with the absence of infectious virus release from infected primary cell cultures suggest that epithelial cells have a peripheral role in MV transmission.

PTEN regulates colorectal epithelial apoptosis through Cdc42 signalling.

BACKGROUND:
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear.

METHODS:
To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies.

RESULTS:
The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis.

CONCLUSION:
Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.

Short Isoforms of the Cold Receptor TRPM8 Inhibit Channel Gating by Mimicking Heat Action Rather than Chemical Inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria

The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4,4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.

Characterisation of Genome-Wide PLZF/RARA Target Genes

The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear. We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.

Novel Protective Properties of IGFBP-3 Result in Enhanced Pericyte Ensheathment, Reduced Microglial Activation, Increased Microglial Apoptosis, and Neuronal Protection after Ischemic Retinal Injury

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GF(P)+ hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity. (Am J Pathol 2011, 178:1517-1524; DOI: 10.1016/j.ajpath.2010.12.031)

The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages

Burkholderia cenocepacia is a Gram-negative opportunistic pathogen of patients with cystic fibrosis and chronic granulomatous disease. The bacterium survives intracellularly in macrophages within a membrane-bound vacuole (BcCV) that precludes the fusion with lysosomes. The underlying cellular mechanisms and bacterial molecules mediating these phenotypes are unknown. Here, we show that intracellular B. cenocepacia expressing a type VI secretion system (T6SS) affects the activation of the Rac1 and Cdc42 RhoGTPase by reducing the cellular pool of GTP-bound Rac1 and Cdc42. The T6SS also increases the cellular pool of GTP-bound RhoA and decreases cofilin activity. These effects lead to abnormal actin polymerization causing collapse of lamellipodia and failure to retract the uropod. The T6SS also prevents the recruitment of soluble subunits of the NADPH oxidase complex including Rac1 to the BcCV membrane, but is not involved in the BcCV maturation arrest. Therefore, T6SS-mediated deregulation of Rho family GTPases is a common mechanism linking disruption of the actin cytoskeleton and delayed NADPH oxidase activation in macrophages infected with B. cenocepacia.

Type IVB pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CM is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the Delta F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells. (C) 2011 Elsevier Ltd. All rights reserved.

A two-tier model of polymyxin B resistance in Burkholderia cenocepacia

P>Burkholderia cenocepacia is an environmental bacterium causing serious human opportunistic infections and is extremely resistant to multiple antibiotics including antimicrobial peptides, such as polymyxin B (PmB). Extreme antibiotic resistance is attributed to outer membrane impermeability ('intrinsic' resistance). Previous work showed that production of full-length lipopolysaccharide (LPS) prevents surface binding of PmB. We hypothesized that two tiers of resistance mechanisms rendering different thresholds of PmB resistance exist in B. cenocepacia. To test this notion, candidate genes were mutated in two isogenic strains expressing full-length LPS or truncated LPS devoid of heptose ('heptoseless LPS') respectively. We uncovered various proteins required for PmB resistance only in the strain with heptoseless LPS. These proteins are not involved in preventing PmB binding to whole cells or permeabilization of the outer membrane. Our results support a two-tier model of PmB resistance in B. cenocepacia. One tier sets a very high threshold mediated by the LPS and the outer membrane permeability barrier. The second tier sets a lower threshold that may play a role in PmB resistance only when outer membrane permeability is compromised. This model may be of general applicability to understanding the high antimicrobial peptide resistance of environmental opportunistic pathogens.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.