Increased expression of the RIα subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIα subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIα expression in patients with ovarian cancer. We have evaluated the expression of RIα in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIα mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIα mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIα mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIα mRNA expression and differentiation state. RIα mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIα mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIα mRNA expression is associated with advanced stage disease. RIα expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Persistent donor chimaerism is consistent with disease-free survival following BMT for chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT) leading to long-term disease-free survival. Leukemia relapse, however, remains a significant clinical problem. Relapse following BMT presumably results from the expansion of small numbers of recipient leukaemic cells which have survived the conditioning therapy. In order to define patients who are at a high risk of leukaemia relapse, a variety of techniques have been employed to detect persistence of host haemopoiesis (mixed chimaerism, MC) or residual leukaemia (minimal residual disease, MRD). However, the precise relationship between the detection of MC and MRD post-BMT is unknown. We have investigated chimaerism and MRD status in 22 patients who were in clinical and haematological remission post-allogeneic BMT for chronic phase CML. Chimaerism was assessed using short tandem repeat PCR (STR-PCR) while BCR-ABL mRNA detection using reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the presence of MRD. Seventeen patients received unmanipulated marrow (non-TCD) while in five patients a T cell-depleted transplant (TCD) was performed as additional GVHD prophylaxis. Chimaerism was evaluated in 18 patients (14 non-TCD, four TCD). Mixed chimaerism was an uncommon finding in recipients of unmanipulated BMT (21%) when compared to TCD BMT (100%). No evidence of MRD, as identified using the BCR-ABL mRNA RT-PCR assay, was detected in those patients who were donor chimaeras. Early and transient MC and MRD was detected in four patients (two non-TCD, two TCD) who have subsequently converted to a donor profile. One patient has stable low-level MC but remains MRD negative 4 years post-BMT. Late MC and MRD was observed in two patients who relapsed >6 years after TCD BMT for CML. We conclude that mixed chimaerism is a rare event in recipients of unmanipulated BMT and that donor chimaerism as detected by STR-PCR assay is consistent with disease-free survival and identifies patients with a low risk of leukaemic relapse post-BMT for CML.

CCL2 is transcriptionally controlled by the lysosomal protease cathepsin S in a CD74-dependent manner

Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA depletion of CatS resulted in reduced CCL2 expression. This regulation is transcriptionally mediated, as evident from RT-PCR analysis and CCL2 promoter studies. We revealed that CatS regulation of CCL2 is modulated through CD74 (also known as the invariant chain), a known substrate of CatS and a mediator of NFkB activity. Furthermore, CatS and CCL2 show a strong clinical correlation in brain, breast and colon tumours. In summary, these results highlight a novel mechanism by which CatS controls CCL2, which may present a useful pharmacodynamic marker for CatS inhibition.

Pro-neural transcription factors as cancer markers

BACKGROUND: The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours.

RESULTS: Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers.

CONCLUSION: By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.

Comprehensive profiling and localisation of the matrix metalloproteinases in urothelial carcinoma

The matrix metalloproteinases (MMPs) are endopeptidases which break down the extracellular matrix and regulate cytokine and growth factor activity. Several MMPs have been implicated in the promotion of invasion and metastasis in a broad range of tumours including urothelial carcinoma. In this study, RNA from 132 normal bladder and urothelial carcinoma specimens was profiled for each of the 24 human MMPs, the four endogenous tissue inhibitors of MMPs (TIMPs) and several key growth factors and their receptors using quantitative real time RT-PCR. Laser capture microdissection (LCM) of RNA from 22 tumour and 11 normal frozen sections was performed allowing accurate RNA extraction from either stromal or epithelial compartments. This study confirms the over expression in bladder tumour tissue of well-documented MMPs and highlights a range of MMPs which have not previously been implicated in the development of urothelial cancer. In summary, MMP-2, MT1-MMP and the previously unreported MMP-28 were very highly expressed in tumour samples while MMPs 1, 7, 9, 11, 15, 19 and 23 were highly expressed. There was a significant positive correlation between transcript expression and tumour grade for MMPs 1, 2, 8, 10, 11, 12, 13, 14, 15 and 28 (P < 0.001). At the same confidence interval, TIMP-1 and TIMP-3 also correlated with increasing tumour grade. LCM revealed that most highly expressed MMPs are located primarily within the stromal compartment except MMP-13 which localised to the epithelial compartment. This work forms the basis for further functional studies, which will help to confirm the MMPs as potential diagnostic and therapeutic targets in early bladder cancer.

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.

Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration

BACKGROUND: Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure.

METHODS: BALB/cJ and Cx3cr1 (GFP/+) mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.

RESULTS: Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls.

CONCLUSIONS: Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

Natural Killer Cell-like signature observed in locally advanced rectal cancer after neoadjuvant chemoradiotherapy in a tumour regression grade dependent manner

Background: Around 10-15% of patients with locally advanced rectal cancer (LARC) undergo a pathologically complete response (TRG4) to neoadjuvant chemoradiotherapy; the rest of patients exhibit a spectrum of tumour regression (TRG1-3). Understanding therapy-related genomic alterations may help us to identify underlying biology or novel targets associated with response that could increase the efficacy of therapy in patients that do not benefit from the current standard of care.
Methods: 48 FFPE rectal cancer biopsies and matched resections were analysed using the WG-DASL HumanHT-12_v4 Beadchip array on the illumina iScan. Bioinformatic analysis was conducted in Partek genomics suite and R studio. Limma and glmnet packages were used to identify genes differentially expressed between tumour regression grades. Validation of microarray results will be carried out using IHC, RNAscope and RT-PCR.
Results: Immune response genes were observed from supervised analysis of the biopsies which may have predictive value. Differential gene expression from the resections as well as pre and post therapy analysis revealed induction of genes in a tumour regression dependent manner. Pathway mapping and Gene Ontology analysis of these genes suggested antigen processing and natural killer mediated cytotoxicity respectively. The natural killer-like gene signature was switched off in non-responders and on in the responders. IHC has confirmed the presence of Natural killer cells through CD56+ staining.
Conclusion: Identification of NK cell genes and CD56+ cells in patients responding to neoadjuvant chemoradiotherapy warrants further investigation into their association with tumour regression grade in LARC. NK cells are known to lyse malignant cells and determining whether their presence is a cause or consequence of response is crucial. Interrogation of the cytokines upregulated in our NK-like signature will help guide future in vitro models.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca²⁺ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. © 2008 Elsevier Inc. All rights reserved.

Substance P production by human dental pulp fibroblasts

The inflammatory response to pulpal injury or infection has major clinical significance. Neurogenic inflammation describes the local release of neuropeptides, notably substance P (SP), from afferent neurones, and may play a role in the pathogenesis of pulpal disease. The fibroblast is the most numerous cell type in the dental pulp and recent work has suggested that it is involved in the inflammatory response. Objectives: The aims of the study were to determine whether pulp fibroblasts could produce SP, and to investigate the expression of the SP receptor, NK-1, by these cells. Methods: Primary pulp fibroblast cell populations were isolated by enzymatic digestion from non-carious teeth extracted for orthodontic reasons. Whole pulp tissue was obtained from freshly extracted sound (n=35) and carious (n=39) teeth. Expression of SP and NK-1 mRNA was determined by RT-PCR. The effects of interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) on SP and NK-1 expression were also determined. The presence of NK-1 on fibroblast cell membranes was established by western blotting. The effects of the cytokines on each parameter were analysed by ANOVA. Radioimmunoassay (RIA) was carried out to quantify SP expression by pulp fibroblasts and in whole pulp tissue. Results: SP was expressed by pulpal fibroblasts both at the mRNA level and the protein level. In addition, NK-1 was detected in fibroblast cultures at the mRNA level and appeared as a double band on western blots of membrane extracts. IL-1β and TGF-β1 significantly stimulated the expression of SP and NK-1. SP levels were significantly greater (p<0.05) in carious compared to sound teeth. Conclusion: Pulp fibroblasts are capable of synthesising and secreting SP, as well as expressing the SP receptor, NK-1. These findings suggest that pulp fibroblasts play a role in neurogenic inflammation in pulpal disease. (Supported by the European Society of Endodontology.)