The Burkholderia cenocepacia sensor kinase hybrid AtsR is a global regulator modulating quorum-sensing signalling

Burkholderia cenocepacia is commonly found in the environment and also as an important opportunistic pathogen infecting patients with cystic fibrosis. Successful infection by this bacterium requires coordinated expression of virulence factors, which is achieved through different quorum sensing (QS) regulatory systems. Biofilm formation and Type 6 secretion system (T6SS) expression in B. cenocepacia K56-2 are positively regulated by QS and negatively regulated by the sensor kinase hybrid AtsR. This study reveals that in addition to affecting biofilm and T6SS activity, the deletion of atsR in B. cenocepacia leads to overproduction of other QS-regulated virulence determinants including proteases and swarming motility. Expression of the QS genes, cepIR and cciIR, was upregulated in the ?atsR mutant and resulted in early and increased N-acylhomoserine lactone (AHL) production, suggesting that AtsR plays a role in controlling the timing and fine-tuning of virulence gene expression by modulating QS signalling. Furthermore, a ?atsR?cepI?cciI mutant could partially upregulate the same virulence determinants indicating that AtsR also modulates the expression of virulence genes by a second mechanism, independently of any AHL production. Together, our results strongly suggest that AtsR is a global virulence regulator in B. cenocepacia.

Potentiation of Inflammatory CXCL8 Signalling Sustains Cell Survival in PTEN-deficient Prostate Carcinoma

Background: Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised.

Objective: To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa.

Design, setting and participants: Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten(+/-)) mice harbouring inactivation of one PTEN allele.

Interventions: Small interfering RNA (siRNA)- or small hairpin RNA (shRNA)-directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions.

Outcome measurements and statistical analysis: Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays.

Results and limitations: Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten(+/-) murine prostate tissue relative to normal epithelium in wild-type PTEN (Pten(WT)) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study.

Conclusions: PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.

Constitutive and Treatment-Induced CXCL8-Signalling Selectively Modulates the Efficacy of Anti-Metabolite Therapeutics in Metastatic Prostate Cancer

Background: The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent.

Methods: The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression.

Results: Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P,0.001), and increased 5-FU-induced apoptosis in PC3 cells (P,0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU.

Conclusions: CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis. © 2012 Wilson et al.

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.

Analysis of alteration of p75NTR processing and signalling by PS2 mutation and gamma-secretase inhibition

The presenilins (PSs) were identified as causative genes in cases of early-onset familial Alzheimer's disease (AD) and current evidence indicates that PSs are part of the gamma-secretase complex responsible for proteolytic processing of type I membrane proteins. p75NTR, a common neurotrophin receptor, was shown to be subject to gamma-secretase processing. However, it is not clear if the p75NTR downstream signal is altered in response to gamma-secretase cleavage, and further there is a possibility that AD-related PS mutations may affect this cleavage, resulting in pathogenic alterations in signal transduction. In this study, we confirmed that p75NTR downstream signalling is altered by PS2 mutation or gamma-secretase inhibition in SHSY-5Y cells. The activity of the small GTPase RhoA is strongly affected by these treatments. This study demonstrates that gamma-secretase and PS2 play an important role in regulating neurotrophin signal transduction and either mutation of PS2 or inhibition of gamma-secretase disturbs this function.