Adverse prognostic and predictive significance of low DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression in early-stage breast cancers

Background: DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a serine threonine kinase belonging to the PIKK family (phosphoinositide 3-kinase-like-family of protein kinase), is a critical component of the non-homologous end joining (NHEJ) pathway required for the repair of DNA double strand breaks. DNA-PKcs may be involved in breast cancer pathogenesis. Methods: We evaluated clinicopathological significance of DNA-PKcs protein expression in 1161 tumours and DNA-PKcs mRNA expression in 1950 tumours. We correlated DNA-PKcs to other markers of aggressive phenotypes, DNA repair, apoptosis and cell cycle regulation. Results: Low DNA-PKcs protein expression was associated with higher tumour grade, higher mitotic index, tumour de-differentiation and tumour type (ps<0.05). Absence of BRCA1, low XRCC1/SMUG1/APE1/Polβ were also more likely in low DNA-PKcs expressing tumours (ps<0.05). Low DNA-PKcs protein expression was significantly associated with worse breast cancer specific survival (BCCS) in univariate and multivariate analysis (ps<0.01). At the mRNA level, low DNA-PKcs was associated with PAM50.Her2 and PAM50.LumA molecular phenotypes (ps<0.01) and poor BCSS. In patients with ER positive tumours who received endocrine therapy, low DNA-PKcs (protein and mRNA) was associated with poor survival. In ER negative patients, low DNA-PKcs mRNA remains significantly associated with adverse outcome. Conclusions: Our study suggests that low DNA-PKcs expression may have prognostic and predictive significance in breast cancers

ERα suppresses slug expression directly by transcriptional repression

Two of the most common signalling pathways in breast cancer are the ER (oestrogen receptor) ligand activation pathway and the E-cadherin snai1 slug EMT (epithelial–mesenchymal transition) pathway. Although these pathways have been thought to interact indirectly, the present study is the first to observe direct interactions between these pathways that involves the regulation of slug expression. Specifically we report that ligand-activated ERα suppressed slug expression directly by repression of transcription and that knockdown of ERα with RNA interference increased slug expression. More specifically, slug expression was down-regulated in ERα-negative MDA-MB-468 cells transfected with ERα after treatment with E2 (17β-oestradiol). The down-regulation of slug in the ERα-positive MCF-7 cell line was mediated by direct repression of slug transcription by the formation of a co-repressor complex involving ligand-activated ERα protein, HDAC1 (histone deacetylase 1) and N-CoR (nuclear receptor co-repressor). This finding was confirmed by sequential ChIP (chromatin immunoprecipitation) studies. In the MCF-7 cell line, slug expression normally was low. In addition, knockdown of ERα with RNA interference in this cell line increased slug expression. This effect could be partially reversed by treatment of the cells with E2. The efficacy of the effect of ERα on slug repression was dependent on the overall level of ERα. These observations confirmed that slug was an E2-responsive gene

Insulin-like growth factor - Oestradiol crosstalk and mammary gland tumourigenesis

Development and differentiation of the mammary gland are dependent on the appropriate temporal expression of both systemically acting hormones and locally produced growth factors. A large body of evidence suggests that molecular crosstalk between these hormonal and growth factor axes is crucial for appropriate cell and tissue function. Two of the most important trophic factors involved in this process are the oestrogen (E) and insulin-like growth factor (IGF) molecular axes. The reciprocal crosstalk that exists between these pathways occurs at transcriptional/post-transcriptional and translational/post-translational levels regulate the expression and activity of genes involved in this process. In a clinical context an important consequence of such crosstalk in the mammary gland is the role which it may play in the aetiology, maintenance and development of breast tumours. Although oestradiol (E2) acting through oestrogen receptors α and β (ERα/β) is important for normal mammary gland function it can also provide a mitogenic drive to ER+ breast tumours. Therefore over several years anti-oestrogen therapeutic regimens in the form of selective oestrogen receptor modulators (SERMs - e.g. tamoxifen), aromatase inhibitors (AI e.g. anastrozole) or selective oestrogen receptor down regulators (SERDs - e.g. fulvestrant) have been used in an adjuvant setting to control tumour growth. Although initial response is usually encouraging, large cohorts of patients eventually develop resistance to these treatments leading to tumour recurrence and poor prognosis. There are potentially many routes by which breast cancer (BC) cells could escape anti-oestrogen based therapeutic strategies and one of the most studied is the possible growth factor mediated activation of ER(s). Because of this, growth factor modulation of ER activity has been an intensively studied route of molecular crosstalk in the mammary gland. The insulin-like growth factors (IGF-1 and -2) are amongst the most potent mitogens for mammary epithelial cells and there is accumulating evidence that they interact with the E2 axis to regulate mitogenesis, apoptosis, adhesion, migration and differentiation of mammary epithelial cells. Such interactions are bi-directional and E2 has been shown to regulate the expression and activity of IGF axis genes with the general effect of sensitising breast epithelial cells to the actions of IGFs and insulin. In this short review we discuss the evidence for the involvement of crosstalk between the insulin-like growth factor (IGF) and oestrogen axes in the mammary gland and comment on the relevance of such studies in the aetiology and treatment of BC

Social and Clinical Factors Associated with End-Stage Renal Disease (ESRD): A Retrospective Cohort of Older Adult Veterans with Severe Chronic Kidney Disease (CKD)

As the aging population continues to grow so does the prevalence of chronic kidney disease (CKD) and the need for adequate CKD management in those 65 or over who are highly heterogenous in their disease progression. Hospitalization rates for acute kidney injury (AKI) are increasing, especially amongst older adults (≥65) who are at elevated risk given their high comorbidity burden and susceptibility to nephrotoxins. Previous epidemiologic analyses of AKI have focused on hospitalized populations which may bias results towards sicker populations; particularly when results are extrapolated to ambulatory CKD populations. Studies on social determinants of health (SDOH) and disease progression in CKD populations are limited, both in number and range of key areas, especially among older adults who become more vulnerable to stressors as they age. To fill these gaps in the literature, we obtained information on select social and clinical factors and examined their relationship with progression to end-stage renal disease (ESRD) in Veterans ≥65 years of age with incident CKD stage 4 using national VA administrative data. We hypothesize several points: first, that AKI is associated with ESRD, and age is an effect modifier of this relationship; second, an AKI and a rapidly declining kidney function are jointly associated with ESRD; third, a high social vulnerability at the census tract level based on the CDC’s Social Vulnerability Index (SVI) is associated with ESRD. This dissertation identified several clinically relevant joint effects associated with ESRD. However, more Veterans died than progressed to ESRD. Traditional methods of CKD management through means of achieving appropriate target controls can help mitigate the risk of death in this population who have a high comorbidity burden. Outsourcing to primary care or specialty care to alleviate the burden on nephrology should be considered in this group

Geographic distribution and risk of upper urothelial carcinomas in Croatia, 2001–2011

Background: Strong associations exist between Balkan endemic nephropathy (BEN) and upper urothelial carcinomas (UUCs). However, the common etiology between the two remains unclear and there are no studies to date that visualize UUC risks in Croatia. In Croatia, 14 villages in the southwestern part of Brod-Posavina County are considered endemic for BEN. The aim of this ecological study is to map cancer risks and describe the case distribution of UUCs in Croatia at the county level during 2001-2011. ----- Methods: A total of 608 incident cases from the Croatian National Cancer Registry were identified. Indirect standardization was employed to compute standardized incidence ratios (SIRs). ----- Results: Counties with SIRs greater than 1 were concentrated around the agricultural region of Slavonia and the coastal region of Dalmatia. However, only Brod-Posavina County and Vukovar-Srijem County had a statistically significant risk of UUC development, where there were 390 and 210% more UUC cases observed than expected, respectively. Only unique to Brod-Posavina County, females were at higher risk (SIR 4.96; 95% CI 3.59-6.34) of developing UUCs than males (SIR 3.03; 95% CI 2.04-4.01) when compared to their Croatian counterparts. Although Brod-Posavina County only made up 3.7% of the total Croatian population (as of 2011), it had the highest frequency of incident UUC cases after the capital City of Zagreb. No elevated cancer risks were noted in the City of Zagreb, even after stratifying by sex. ----- Conclusion: Our findings suggest that Brod-Posavina County had the highest cancer risk for UUCs, especially among females, when compared to Croatia as a whole during 2001-2011. Given that a majority of BEN patients develop associated UUCs, concurrent screening programs for UUCs and BEN should be considered not only in endemic areas of BEN but also the surrounding rural areas and amongst at-risk groups such as those undergoing hemodialysis, who frequently develop UUCs, to help clarify BEN-UUC associations by identifying common risk factors while standardizing disease estimates across endemic regions for BEN

GSK-3β controls NF-kappaB activity via IKKγ/NEMO

The NF-κB signaling pathway is central for the innate immune response and its deregulation is found in multiple disorders such as autoimmune, chronic inflammatory and metabolic diseases. IKKγ/NEMO is essential for NF-κB activation and NEMO dysfunction in humans has been linked to so-called progeria syndromes, which are characterized by advanced ageing due to age-dependent inflammatory diseases. It has been suggested that glycogen synthase kinase-3β (GSK-3β) participates in NF-κB regulation but the exact mechanism remained incompletely understood. In this study, we identified NEMO as a GSK-3β substrate that is phosphorylated at serine 8, 17, 31 and 43 located within its N-terminal domain. The kinase forms a complex with wild-type NEMO while point mutations of NEMO at the specific serines abrogated GSK-3β binding and subsequent phosphorylation of NEMO resulting in its destabilization. However, K63-linked polyubiquitination was augmented in mutated NEMO explaining an increased binding to IKKα and IKKβ. Even IκBα was found degraded. Still, TNFα-stimulated NF-κB activation was impaired pointing towards an un-controlled signalling process. Our data suggest that GSK-3β is critically important for ordered NF-κB signalling through modulation of NEMO phosphorylation

Adverse prognostic and predictive significance of low DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression in early-stage breast cancers

Background: DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a serine threonine kinase belonging to the PIKK family (phosphoinositide 3-kinase-like-family of protein kinase), is a critical component of the non-homologous end joining (NHEJ) pathway required for the repair of DNA double strand breaks. DNA-PKcs may be involved in breast cancer pathogenesis. Methods: We evaluated clinicopathological significance of DNA-PKcs protein expression in 1161 tumours and DNA-PKcs mRNA expression in 1950 tumours. We correlated DNA-PKcs to other markers of aggressive phenotypes, DNA repair, apoptosis and cell cycle regulation. Results: Low DNA-PKcs protein expression was associated with higher tumour grade, higher mitotic index, tumour de-differentiation and tumour type (ps<0.05). Absence of BRCA1, low XRCC1, low SMUG1, low APE1 and low Polβ were also more likely in low DNA-PKcs expressing tumours (ps<0.05). Low DNA-PKcs protein expression was significantly associated with worse breast cancer specific survival (BCCS) in univariate and multivariate analysis (ps<0.01). At the mRNA level, low DNA-PKcs was associated with PAM50.Her2 and PAM50.LumA molecular phenotypes (ps<0.01) and poor BCSS. In patients with ER positive tumours who received endocrine therapy, low DNA-PKcs (protein and mRNA) was associated with poor survival. In ER negative patients, low DNA-PKcs mRNA remains significantly associated with adverse outcome. Conclusions: Our study suggests that low DNA-PKcs expression may have prognostic and predictive significance in breast cancers

Left atrial enlargement and clinical considerations in patients with or without a residual interatrial shunt after closure of the left atrial appendage with the WATCHMAN™-device

Abstract Background Interventional closure of the left atrial appendage (LAA) in patients with non-valvular atrial fibrillation, high thromboembolic and bleeding risk or bleeding history is an alternative therapeutic strategy to oral anticoagulation. It is not known if the exclusion of the LAA from the blood circulation affects the left atrial volume (LAV) and consequently its prognostic value or the circulatory performance of the heart in humans. Methods We aimed to prospectively assess potential changes in baseline LAV, left ventricular ejection fraction (LVEF), NT-proBNP-level and the covered distance in the 6-min walk-test 6 weeks and 6 months after LAA closure with the WATCHMAN™ device. We used serial 3-dimensional transthoracic and transesophageal echocardiography to assess LAV, residual interatrial shunt and device performance in 58 consecutive patients with successful LAA closure. Results Accurate 3D–echocardiographic data for LAV measurements were evaluable for 51 (91%) patients. Maximum LAV (LAVmax) at baseline was 102.8 ± 30.8 ml and increased significantly to 107.7 ± 32.8 ml after 6 weeks (p < 0.01) and 113.5 ± 34.2 ml after 6 months (p < 0.01). Minimal LAV (LAVmin) increased from 76.9 ± 29.5 ml at baseline to 81.8 ± 30.2 ml after 45 days (p < 0.01) and 82.1 ± 33.3 ml after 6 months (p < 0.01). Similarly, their indexes to BSA (LAVImax and LAVImin) increased significantly, as well. Patients without a residual left-to-right interatrial shunt showed a significantly higher increase in LAVmax or LAVmin. Baseline LVEF, NT-proBNP-level or the distance covered at the 6-min walk test did not significantly change 6 weeks or 6 months after LAA closure. Conclusions LAVmax and LAVmin increase significantly after interventional LAA closure. LA enlargement does not correlate with clinical progression of heart failure. Persistent left-to-right interatrial shunt counteracts the LA enlargement. A reduced LA compliance after exclusion of the LAA from the blood circulation with consecutive increase in LA pressure may be a potential cause of LA enlargement and warrants further investigation. Trial registration German Clinical Trials Register ID: DRKS00010768 ; Registration Date 07.07.2016

IKKγ/NEMO Localization into Multivesicular Bodies

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3β leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3β and V-ATPase in NF-κB signaling activation