The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.

The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription.

Background:It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC.Methods:A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry.Results:KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables.Conclusions:This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Epithelial mesenchymal transition (EMT), as defined by loss of epithelial characteristics and gain of a mesenchymal phenotype, has been reported in vivo although the occurrence of events remains unclear. This study aims at exploration of EMT portraits of breast cancer (BC) with relevance to different molecular pathways, especially potential EMT triggers and BC molecular subtypes. Immunohistochemical (IHC) expression of markers/triggers of EMT was studied on a well-defined cohort of invasive non-lobular BC (n = 1,035), prepared as tissue microarrays. IHC panel of biomarkers included cadherins (cad; E-cad and N-cad), TGFβ1, PIK3CA, pAkt, and others. Reverse phase protein array (RPPA) was performed for quantitative analysis of proteins extracted from formalin fixed paraffin embedded tissues of a subset of cases from this cohort. Four combinatorial phenotypic groups representing cadherin switch were defined, including E-cad(+)/N-cad(-), E-cad(-)/N-cad(-), E-cad(+)/N-cad(+), and E-cad(-)/N-cad(+). Statistically significant association was noticed between these phenotypes and histological tumour grade, tumour type and size and NPI staging classes. The E-cad/N-cad switch occurred more frequently in the triple negative molecular class, both basal and non-basal, and in the HER2(+) subtype than in luminal BC. Significant outcome differences were observed between cadherin switch combinatorial groups regarding BCSS and DMFS (p < 0.001). Results of RPPA confirm those observed using IHC regarding differential expressions of EMT markers/triggers. EMT/cadherin switch programs in BC appear to occur in synergy with TGFβ1 and PIK3/Akt pathways activation. These data explain, at translational proteomic level, the molecular heterogeneity and in turn the varied clinical behaviour of BC molecular subtypes. RPPA is a promising high-throughput technique in monitoring subtle quantitative changes in protein expression in archival material.

No abstract

Although the role of BRCA1 and the homologous recombination (HR) pathway in breast cancer (BC) has been extensively studied, the alternative repair pathway for DNA double-strand breaks (DSBs), non-homologous end-joining (NHEJ) remains to be defined. Ku proteins bind to DNA DSB ends and play a key role in NHEJ. In this study we aimed to assess the expression and biological significance of the KU70/KU80 heterodimer in the different molecular classes of BC. The expression of KU70/KU80 was assessed immunohistochemically in a well-characterised and annotated series of 1302 unselected invasive BC cases with a long-term follow-up together with 25 cases with known BRCA1 mutations. The results were correlated with clinicopathological parameters, other DNA repair proteins and patient outcome. The expression of KU70/KU80 protein was further evaluated in various BC cell lines using western blotting and reverse-phase protein microarray (RPPA). Nuclear KU70/KU80 expression was correlated with features of poor prognosis including higher histological grade, lymphovascular invasion, negative oestrogen receptor expression, basal-like phenotype, P53 and CHK1 positivity. KU70/KU80 was expressed in all BRCA1-associated tumours and showed an inverse correlation with nuclear BRCA1 protein and aberrant cytoplasmic RAD51 expression. RPPA confirmed these results and showed higher expression of KU70/KU80 in BRCA1-deficient cell line compared to BRCA1-proficient cell line. KU70/KU80 expression showed an association with disease-free interval; however, it was not an independent predictor of outcome. As a conclusion, KU70/KU80 may play a role in DNA DSBs repair in HR-deficient tumours. Further study of other NHEJ markers in sporadic BC is warranted.

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.

Mutations in TNFRSF1A encoding TNF receptor 1 (TNFR1) cause the autosomal dominant TNF receptor-associated periodic syndrome (TRAPS): a systemic autoinflammatory disorder. Misfolding, intracellular aggregation, and ligand-independent signaling by mutant TNFR1 are central to disease pathophysiology. Our aim was to understand the extent of signaling pathway perturbation in TRAPS. A prototypic mutant TNFR1 (C33Y), and wild-type TNFR1 (WT), were expressed at near physiological levels in an SK-Hep-1 cell model. TNFR1-associated signaling pathway intermediates were examined in this model, and in PBMCs from C33Y TRAPS patients and healthy controls. In C33Y-TNFR1-expressing SK-Hep-1 cells and TRAPS patients’ PBMCs, a subtle, constitutive upregulation of a wide spectrum of signaling intermediates and their phosphorylated forms was observed; these were associated with a proinflammatory/antiapoptotic phenotype. In TRAPS patients’ PBMCs, this upregulation of proinflammatory signaling pathways was observed irrespective of concurrent treatment with glucocorticoids, anakinra or etanercept, and the absence of overt clinical symptoms at the time that the blood samples were taken. This study reveals the pleiotropic effect of a TRAPS-associated mutant form of TNFR1 on inflammatory signaling pathways (a proinflammatory signalome), which is consistent with the variable and limited efficacy of cytokine-blocking therapies in TRAPS. It highlights new potential target pathways for therapeutic intervention.