Is Cadmium Genotoxicity Due to the Induction of Redox Stress and Inflammation? A Systematic Review.

The transition metal cadmium (Cd) is toxic to humans and can induce cellular redox stress and inflammation. Cd is a recognized carcinogen, but the molecular mechanisms associated with its genotoxicity and carcinogenicity are not defined. Therefore, a systematic review was undertaken to examine the scientific literature that has covered the molecular mechanism of Cd genotoxicity and its relationship to cellular redox stress and inflammation. An electronic database search of PubMed, Scopus, and the Web of Science Core Collection was conducted to retrieve the studies that had investigated if Cd genotoxicity was directly linked to the induction of redox stress and inflammation. Studies included exposure to Cd via in vitro and in vivo routes of administration. Of 214 publications retrieved, 10 met the inclusion criteria for this review. Preclinical studies indicate that Cd exposure causes the induction of reactive oxygen species (ROS) and, via concomitant activity of the transcription factor NF-κß, induces the production of pro-inflammatory cytokines and a cytokine profile consistent with the induction of an allergic response. There is limited information regarding the impact of Cd on cellular signal transduction pathways, and the relationship of this to genotoxicity is still inconclusive. Nevertheless, pre-incubation with the antioxidants, N-acetylcysteine or sulforaphane, or the necroptosis inhibitor, necrostatin-1, reduces Cd toxicity; indicative that these agents may be a beneficial treatment adjunct in cases of Cd poisoning. Collectively, this review highlights that Cd-induced toxicity and associated tissue pathology, and ultimately the carcinogenic potential of Cd, may be driven by redox stress and inflammatory mechanisms.

Investigating genomic, proteomic, and post-transcriptional regulation profiles in colorectal cancer: a comparative study between primary tumors and associated metastases.

INTRODUCTION: Approximately 50% of patients with primary colorectal carcinoma develop liver metastases. This study investigates the possible molecular discrepancies between primary colorectal cancer (pCRC) and their respective metastases. METHODS: A total of 22 pairs of pCRC and metastases were tested. Mutation profiling of 26 cancer-associated genes was undertaken in 22/22primary-metastasis tumour pairs using next-generation sequencing, whilst the expression of a panel of six microRNAs (miRNAs) was investigated using qPCRin 21/22 pairs and 22 protein biomarkers was tested using Reverse Phase Protein Array (RPPA)in 20/22 patients' tumour pairs. RESULTS: Among the primary and metastatic tumours the mutation rates for the individual genes are as follows:TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. The primary-metastasis tumour mutation status was identical in 54/60 (90%) loci. However, there was discordance in heterogeneity status in 40/58 genetic loci (z-score = 6.246, difference = 0.3793, P < 0.0001). Furthermore, there was loss of concordance in miRNA expression status between primary and metastatic tumours, and 57.14-80.95% of the primary-metastases tumour pairs showed altered primary-metastasis relative expression in all the miRNAs tested. Moreover, 16 of 20 (80%) tumour pairs showed alteration in at least 3 of 6 (50%) of the protein biomarker pathways analysed. CONCLUSION: The molecular alterations of primary colorectal tumours differ significantly from those of their matched metastases. These differences have profound implications for patients' prognoses and response to therapy.

Interaction between HER2 and ATM predicts poor survival in bladder cancer patients.

Human Epidermal Growth Factor Receptor 2 (HER2) overexpression is considered one of the interesting prognostic biomarkers in bladder cancer. However, the mechanism of bladder cancer development in relation to HER2 status remains to be elucidated. In this study, we investigated HER2-Ataxia telangiectasia mutated (ATM) kinase interaction and their impact on patient survival and cancer aggressiveness. Using the Cancer Genome Atlas (TCGA) cohorts, we demonstrated that ATM expression (protein/mRNA) is increased in HER2 deficient compared with proficient HER2 patients. This finding was then validated using the Gene Expression Omnibus database (GEO). Correlation analysis (using low expression vs high expression as a discriminator) revealed a significant association of ATM low and HER2 high status with several clinicopathological variables such as high tumour grade, late disease stage and tumour shape. Kaplan-Meier survival analysis indicated that ATM low and HER2 high is a powerful prognosticator of both overall survival (OS) and disease-free survival (DFS). Furthermore, using bioinformatics and protein/protein interaction analyses, we identified 66 putative overlapping proteins with direct link between HER2 and ATM most of which are functionally involved in transcription regulation, apoptotic process and cell proliferation. Interestingly, the results showed that these proteins are strongly linked with PI3K-Akt pathway, p53 pathway and microRNAs in cancer. Altogether, our data pinpoint an important biological role of the interconnection between HER2 and ATM. The latter appear to be an independent prognostic biomarker and may serve as targets to develop novel combination therapies to improve the outcome of patients with bladder cancer.

A Pro-Inflammatory Signature Constitutively Activated in Monogenic Autoinflammatory Diseases.

Autoinflammatory diseases (AIDs) are disorders characterised by recurrent inflammatory episodes in charge of different organs with no apparent involvement of autoantibodies or antigen-specific T lymphocytes. Few common clinical features have been identified among all monogenic AIDs (mAIDs), while the search for a common molecular pattern is still ongoing. The aim of this study was to increase knowledge on the inflammatory pathways in the development of mAIDs in order to identify possible predictive or diagnostic biomarkers for each disease and to develop future preventive and therapeutic strategies. Using protein array-based systems, we evaluated two signalling pathways known to be involved in inflammation and a wide range of inflammatory mediators (pro-inflammatory cytokines and chemokines) in a cohort of 23 patients affected by different mAIDs, as FMF, TRAPS, MKD, Blau syndrome (BS), and NLRP12D. Overall, we observed upregulation of multiple signalling pathway intermediates at protein levels in mAIDs patients' PBMCs, compared with healthy controls, with significant differences also between patients. FMF, TRAPS, and BS presented also peculiar activations of inflammatory pathways that can distinguish them. MAPK pathway activation, however, seems to be a common feature. The serum level of cytokines and chemokines produced clear differences between patients with distinct diseases, which can help distinguish each autoinflammatory disease. The FMF cytokine production profile appears broader than that of TRAPS, which, in turn, has higher cytokine levels than BS. Our findings suggest an ongoing subclinical inflammation related to the abnormal and constitutive signalling pathways and define an elevated inflammatory cytokine signature. Moreover, the upregulation of Th17-related cytokines emphasises the important role for Th17 and/or Th17-like cells also in monogenic AIDs.

Mutations in the binding site of TNFR1 PLAD reduce homologous interactions but can enhance antagonism of wild-type TNFR1 activity.

The tumour necrosis factor receptor superfamily (TNFRSF) members contain cysteine-rich domains (CRD) in their extracellular regions, and the membrane-distal CRD1 forms homologous interactions in the absence of ligand. The CRD1 is therefore termed a pre-ligand assembly domain (PLAD). The role of PLAD-PLAD interactions in the induction of signalling as a consequence of TNF-TNFR binding led to the development of soluble PLAD domains as antagonists of TNFR activation. In the present study, we generated recombinant wild-type (WT) PLAD of TNFR1 and mutant forms with single alanine substitutions of amino acid residues thought to be critical for the formation of homologous dimers and/or trimers of PLAD (K19A, T31A, D49A and D52A). These mutated PLADs were compared with WT PLAD as antagonists of TNF-induced apoptosis or the activation of inflammatory signalling pathways. Unlike WT PLAD, the mutated PLADs showed little or no homologous interactions, confirming the importance of particular amino acids as contact residues in the PLAD binding region. However, as with WT PLAD, the mutated PLADs functioned as antagonists of TNF-induced TNFR1 activity leading to induction of cell death or NF-κB signalling. Indeed, some of the mutant PLADs, and K19A PLAD in particular, showed enhanced antagonistic activity compared with WT PLAD. This is consistent with the reduced formation of homologous multimers by these PLAD mutants effectively increasing the concentration of PLAD available to bind and antagonize WT TNFR1 when compared to WT PLAD acting as an antagonist. This may have implications for the development of antagonistic PLADs as therapeutic agents.

Corrigendum: Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas Aeruginosa With Bronchial Epithelial Cells.

[This corrects the article DOI: 10.3389/fmicb.2018.03018.].

Tobacco smoke and nicotine suppress expression of activating signaling molecules in human dendritic cells.

Cigarette smoke has significant toxic effects on the immune system, and increases the risk of developing autoimmune diseases; one immunosuppressive effect of cigarette smoke is that it inhibits the T cell-stimulating, immunogenic properties of myeloid dendritic cells (DCs). As the functions of DCs are regulated by intra-cellular signaling pathways, we investigated the effects of cigarette smoke extract (CSE) and nicotine on multiple signaling molecules and other regulatory proteins in human DCs to elucidate the molecular basis of the inhibition of DC maturation and function by CSE and nicotine. Maturation of monocyte-derived DCs was induced with the TLR3-agonist poly I:C or with the TLR4-agonist lipopolysaccharide, in the absence or presence of CSE or nicotine. Reverse-phase protein microarray was used to quantify multiple signaling molecules and other proteins in cell lysates. Particularly in poly I:C-matured DCs, cigarette smoke constituents and nicotine suppressed the expression of signaling molecules associated with DC maturation and T cell stimulation, cell survival and cell migration. In conclusion, constituents of tobacco smoke suppress the immunogenic potential of DCs at the signaling pathway level.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

Characterization of Behavioral, Signaling and Cytokine Alterations in a Rat Neurodevelopmental Model for Schizophrenia, and Their Reversal by the 5-HT6 Receptor Antagonist SB-399885.

Post-weaning social isolation of rats produces neuroanatomical, neurochemical and behavioral alterations resembling some core features of schizophrenia. This study examined the ability of the 5-HT6 receptor antagonist SB-399885 to reverse isolation-induced cognitive deficits, then investigated alterations in hippocampal cell proliferation and hippocampal and frontal cortical expression of selected intracellular signaling molecules and cytokines. Male Lister hooded rats (weaned on post-natal days 21-24 and housed individually or in groups of 3-4) received six i.p. injections of vehicle (1% Tween 80, 1 mL/kg) or SB-399885 (5 or 10 mg/kg) over a 2-week period starting 40 days post-weaning, on the days that locomotor activity, novel object discrimination (NOD), pre-pulse inhibition of acoustic startle and acquisition, retention and extinction of a conditioned freezing response (CFR) were assessed. Tissue was collected 24 h after the final injection for immunohistochemistry, reverse-phase protein microarray and western blotting. Isolation rearing impaired NOD and cue-mediated CFR, decreased cell proliferation within the dentate gyrus, and elevated hippocampal TNFα levels and Cdc42 expression. SB-399885 reversed the NOD deficit and partially normalized CFR and cell proliferation. These effects were accompanied by altered expression of several members of the c-Jun N-terminal Kinase (JNK) and p38 MAPK signaling pathways (including TAK1, MKK4 and STAT3). Although JNK and p38 themselves were unaltered at this time point hippocampal TAK1 expression and phosphorylation correlated with visual recognition memory in the NOD task. Continued use of this neurodevelopmental model could further elucidate the neurobiology of schizophrenia and aid assessment of novel therapies for drug-resistant cognitive symptoms.

Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells.

Pseudomonas aeruginosa causes infections in patients with compromised epithelial barrier function. Multiple virulence factors produced by P. aeruginosa are controlled by quorum sensing (QS) via 2-alkyl-4(1H)-quinolone (AQ) signal molecules. Here, we investigated the impact of AQs on P. aeruginosa PAO1 infection of differentiated human bronchial epithelial cells (HBECs). The pqsA-E operon is responsible for the biosynthesis of AQs including the 2-alkyl-3-hydroxy-4-quinolones, 4-hydroxy-2-alkylquinolines, and 4-hydroxy-2-alkylquinoline N-oxides as exemplified by pseudomonas quinolone signal (PQS), 2-heptyl-4-hydroxyquinoline (HHQ), and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), respectively. PQS and HHQ both act as QS signal molecules while HQNO is a cytochrome inhibitor. PqsE contributes both to AQ biosynthesis and promotes virulence in a PQS-independent manner. Our results show that PQS, HHQ, and HQNO were produced during PAO1 infection of HBECs, but no differences in growth or cytotoxicity were apparent when PAO1 and an AQ-negative ΔpqsA mutant were compared. Both strains promoted synthesis of inflammatory cytokines TNF-α, interleukin (IL)-6, and IL-17C by HBECs, and the provision of exogenous PQS negatively impacted on this response without affecting bacterial growth. Expression of pqsE and the PQS-independent PqsE-regulated genes mexG and lecA was detected during HBEC infection. Levels were reduced in the ΔpqsA mutant, that is, in the absence of PQS, and increased by exogenous PQS. These results support an AQ-independent role for PqsE during initial infection of HBEC by P. aeruginosa and for PQS as an enhancer of PqsE and PqsE-controlled virulence determinants and as an immunomodulator.

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro-inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti-inflammatories based on their ability to down-regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through-put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS-associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro-inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10-fold concentrations on cell lines expressing both wild-type (WT) TNFR1 and TRAPS-associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse-phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y-TNFR1 transfectants towards the level seen in the WT-TNFR1 transfectants. Seven high-ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y-TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro-inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose-dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF-κB and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing.

Autoantibodies of IgM and IgG classes show differences in recognition of multiple autoantigens in chronic obstructive pulmonary disease.

Autoimmunity occurs in chronic obstructive pulmonary disease (COPD). We describe an antigen microarray for detecting serum autoantibodies (AAbs) to determine how IgM, as well as IgG, AAbs distinguish patients with COPD from controls with a history of smoking without COPD. All COPD patients' sera contained elevated levels of AAbs to some of 30 autoantigens. There were significant differences in the autoantigenic specificities of IgM AAbs compared to IgG AAbs in the COPD sera: for example, AAbs to histone and scl-70 were mainly IgG, whereas AAbs to CENP-B and La/ssB were mainly IgM; by contrast, IgM and IgG AAbs to collagen-V were equally prevalent. Thus, a combination of IgM and IgG AAbs specific for multiple autoantigens are detected in all cases of COPD at a level at which all non-COPD controls are negative for AAbs. This highlights the importance of different classes of AAbs to a range of autoantigens in COPD.

High prevalence of subclass-specific binding and neutralizing antibodies against Clostridium difficile toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic C. difficile infection.

OBJECTIVES: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. METHODS: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. RESULTS: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. CONCLUSION: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

The role of indoleamine 2,3-dioxygenase-aryl hydrocarbon receptor pathway in the TLR4-induced tolerogenic phenotype in human DCs.

A controlled inflammatory response is required for protection against infection, but persistent inflammation causes tissue damage. Dendritic cells (DCs) have a unique capacity to promote both inflammatory and anti-inflammatory processes. One key mechanism involved in DC-mediated immunosuppression is the expression of tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO has been implicated in diverse processes in health and disease but its role in endotoxin tolerance in human DCs is still controversial. Here we investigated the role of IDO in shaping DCs phenotype and function under endotoxin tolerance conditions. Our data show that TLR4 ligation in LPS-primed DCs, induced higher levels of both IDO isoforms together with the transcription factor aryl-hydrocarbon receptor (AhR), compared to unprimed controls. Additionally, LPS conditioning induced an anti-inflammatory phenotype in DCs - with an increase in IL-10 and higher expression of programmed death ligand (PD-L)1 and PD-L2 - which were partially dependent on IDO. Furthermore, we demonstrated that the AhR-IDO pathway was responsible for the preferential activation of non-canonical NF-κB pathway in LPS-conditioned DCs. These data provide new insight into the mechanisms of the TLR4-induced tolerogenic phenotype in human DCs, which can help the better understanding of processes involved in induction and resolution of chronic inflammation and tolerance.

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction.

OBJECTIVE: The three main types of killer cells - CD8+ T cells, NK cells and NKT cells - have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. METHODS: Peripheral CD8+ T cells (CD8+CD3+CD56-), NK cells (CD56+CD3-) and NKT-like cells (CD56+CD3+) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. RESULTS: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. CONCLUSIONS: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.

SMAD4 loss enables EGF, TGFß1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor ß1 (TGFß1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFß1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFß1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/ß-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFß1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFß1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/ß-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFß1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. METHODS: Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. RESULTS: MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER+ subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER- subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER+ and ER- cell lines. CONCLUSIONS: MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathway.

Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein.

Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51 N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51 N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51 N(+) was an independent predictor of longer BCSS (P < 0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients' outcome suggest that the trafficking of DDR proteins between the nucleus and cytoplasm might play a role in the development and progression of BC.

Human Blood Autoantibodies in the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpolated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC; stage I (n = 19), stage II (n = 40), stage III (n = 34) and stage IV (n = 6) was similar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice.

The prognostic significance of STAT3 in invasive breast cancer: analysis of protein and mRNA expressions in large cohorts.

Signal transducer and activator of transcription (STAT) transcription factors family are involved in diverse cellular biological functions. Reports regarding the prognostic impact of STAT3 expression in breast cancer (BC) are variable whether being a factor of poor or good prognosis. Immunohistochemical expression of phospho-STAT3 (pSTAT3) was studied in large series of invasive BC (n = 1270). pSTAT3 and STAT3 were quantified using reverse phase protein array (RPPA) on proteins extracted from macro-dissected FFPE tissues (n = 49 cases). STAT3 gene expression in the METABRIC cohort was also investigated. STAT3 gene expression prognostic impact was externally validated using the online BC gene expression data (n = 26 datasets, 4.177 patients). pSTAT3 was expressed in the nuclei and cytoplasm of invasive BC cells. Nuclear pSTAT3 overexpression was positively associated with smaller tumour size, lower grade, good NPI, negative lymphovascular invasion (LVI), ER+, PgR+, p53-, HER2-, and low Ki67LI and an improved breast cancer-specific survival (BCSS), independently of other factors. On RPPA, the mean pSTAT3 and STAT3 expressions were higher in ER+, PgR+, and smaller size tumours. Higher STAT3 transcripts in the METABRIC cohort were observed in cases with favourable prognostic criteria and as well as improved BCSS within the whole cohort, ER+ cohort with and without hormonal therapy, and ER- cohort including those who did not receive adjuvant chemotherapy. Pooled STAT3 gene expression data in the external validation cohort showed an association with improved patients' outcome (P < 0.001, HR = 0.84, 95 % CI 0.79-0.90). Results of this study suggest nuclear localisation of pSTAT3 as favourable prognostic marker in invasive BC, results re-enforced by analysis of STAT3 gene expression data. This good prognostic advantage was maintained in patients who received and who did not receive adjuvant therapy. Therefore, STAT3 could have context-dependent molecular roles of in BC, results which warrant further prospective verification in clinical trials.