Sauerstoffabhängige Regulation des Wilms-Tumor Gens WT1 in Neuroblastomzellen

Sauerstoffmangel (Hypoxie) fördert ein aggressives Wachstum von Tumoren. Dies gilt insbesondere für das Neuroblastom, den häufigsten extrakraniellen soliden Tumor des Kindesalters. Neuroblastome sind Malignome des peripheren sympathischen Nervensystems, deren Tumorzellen von der embryonalen Neuralleiste abstammen. In Neuroblastomen korreliert die Akkumulation von Hypoxie-induzierbarem-Faktor-2α (HIF-2α) mit einem hochmalignen Tumorphänotyp und einem ungünstigen klinischen Verlauf. Hypoxie-induzierbare-Faktoren (HIF) bilden eine Familie von heterodimeren Transkriptionsfaktoren, die bei der zellulären Adaptation an ein niedriges Sauerstoffniveau in normalem Gewebe und in Tumoren eine zentrale Rolle spielen. Neben der Anreicherung von HIF-2α ist auch eine hohe Expression von Wilms-Tumor-Gen 1 (WT1) mit einer schlechten Prognose von Neuroblastomen assoziiert. WT1 kodiert einen Transkriptionsfaktor, der ursprünglich als Suppressor für das Wachstum von Nierentumoren bei Kindern (Wilms-Tumor, Nephroblastom) charakterisiert wurde. Neuere Untersuchungen haben ergeben, dass WT1 in vielen malignen Tumoren verstärkt exprimiert wird und dort möglicherweise onkogene Eigenschaften besitzt. Vor diesem Hintergrund sollte in der vorliegenden Arbeit überprüft werden, ob die Expression von WT1 in Neuroblastomzellen sauerstoffabhängig reguliert wird. Weiterhin sollte gegebenenfalls der molekulare Mechanismus analysiert werden, über den ein Sauerstoffmangel die WT1-Expression stimuliert. Im Rahmen der durchgeführten Untersuchungen konnte gezeigt werden, dass die Expression von WT1 in Kelly-Neuroblastomzellen tatsächlich durch Hypoxie stimuliert wird. Der Knockdown von HIF-2α mittels RNA-Interferenz bzw. Deletion von HIF-2α mittels CRISPR/Cas9 bewirkte eine signifikante Abnahme der WT1 Expression in hypoxischen Kelly-Zellen. Die Inaktivierung von HIF-1α hatte hingegen keinen signifikanten Einfluss auf das WT1 Niveau. Weiterhin wurde in silico ein HIF-Bindungsmotiv innerhalb eines DNase-sensitiven Bereichs in Intron 3 des WT1 Gens identifiziert. Mittels Chromatin-Immunpräzipitation (ChIP) und Elektromobilitätsshiftassays konnte eine Bindung von HIF-2 an diese Sequenz experimentell nachgewiesen werden. In Reportergenassays, die an transient transfizierten Kelly-Zellen durchgeführt wurden, übertrug ein 256 bp langes DNA-Fragment unter Einschluss des HIF-Bindungsmotivs aus Intron 3 des WT1-Gens eine Hypoxieempfindlichkeit auf die ansonsten hypoxieresistenten SV40- und WT1-Promotoren. Funktionell konnte nachgewiesen werden, dass WT1 die Migration von SK-NA-S-Neuroblastomzellen stimuliert. R2-Datenanalysen von großen Kohorten von Neuroblastompatienten zeigten, dass sowohl HIF-2α als auch WT1 in Subgruppen von Neuroblastompatienten mit einer erhöhten Mortalität assoziiert sind. Mit diesen Ergebnissen wird erstmalig ein sauerstoffempfindlicher Enhancer in Intron 3 des WT1 Gens identifiziert. Unter hypoxischen Bedingungen bewirkt die Bindung von HIF-2 an das Enhancerelement eine WT1-Expression in Kelly-Neuroblastomzellen. Dieser neu entdeckte molekulare Mechanismus spielt möglicherweise in der Pathophysiologie von Neuroblastomen eine Rolle.Oxygen shortage (hypoxia) favors aggressive tumor growth. This is particularly relevant to neuroblastoma, the most common extracranial solid tumor in childhood. Neuroblastoma arises from neural crest-derived cells in the peripheral sympathetic nervous system. High levels of hypoxia-inducible factor (HIF) 2α in neuroblastoma correlate with an undifferentiated tumor phenotype and poor clinical outcome. Hypoxiainducible factors constitute a family of heterodimeric transcription factors that play a pivotal role in the adaptation of normal and tumor tissues to low oxygen conditions. Recent studies have shown that high expression levels of the Wilms tumor gene 1 (WT1) in neuroblastoma are associated with an aggressive tumor growth and poor prognosis. WT1 was initially identified as a tumor suppressor gene preventing the formation of childhood tumors of the kidney (Wilms tumor, nephroblastoma). However, WT1 is highly expressed in various types of cancer suggesting that it may have oncogenic properties in certain tissues. It was the purpose of this study to test whether WT1 expression in neuroblastoma cells is regulated by oxygen and, if so, to analyze the underlying molecular mechanism. Exposure of Kelly neuroblastoma cells to low ambient oxygen did indeed stimulate expression of the WT1 gene. Silencing of HIF-2α by RNA interference and knockout of HIF-2α using CRISPR/Cas9 genome editing significantly reduced WT1 levels in hypoxic Kelly cells. In contrast, inactivation of HIF-1α had no significant effect on WT1 expression in hypoxic Kelly cells. In silico analyses revealed a HIF binding motif within a cluster of DNase hypersensitivity in intron 3 of the WT1 gene. Binding of HIF-2 to this sequence was experimentally proven by chromatin immunoprecipitation (ChIP) and electromobility shift assay. A 256 bp DNA sequence encompassing the identified HIF binding site conferred oxygen sensitivity to otherwise hypoxia resistant WT1 and SV40 promoter constructs in transiently transfected Kelly cells. In functional assays, WT1 was found to enhance the migratory capacity of SK-NA-S neuroblastoma cells. R2 data analysis of cohorts of neuroblastoma patients showed that HIF-2α and WT1 are independently associated with increased mortality. In summary, these results identify an oxygen sensitive enhancer in intron 3 of the WT1 gene. Binding of HIF-2 to this newly discovered enhancer element stimulates WT1 expression in hypoxic Kelly neuroblastoma cells. It is suggested that this novel regulatory mechanism might play a role in the pathophysiology of neuroblastoma

Recent advances in urinary peptide and proteomic biomarkers in chronic kidney disease : a systematic review

Biomarker development, improvement, and clinical implementation in the context of kidney disease have been a central focus of biomedical research for decades. To this point, only serum creatinine and urinary albumin excretion are well-accepted biomarkers in kidney disease. With their known blind spot in the early stages of kidney impairment and their diagnostic limitations, there is a need for better and more specific biomarkers. With the rise in large-scale analyses of the thousands of peptides in serum or urine samples using mass spectrometry techniques, hopes for biomarker development are high. Advances in proteomic research have led to the discovery of an increasing amount of potential proteomic biomarkers and the identification of candidate biomarkers for clinical implementation in the context of kidney disease management. In this review that strictly follows the PRISMA guidelines, we focus on urinary peptide and especially peptidomic biomarkers emerging from recent research and underline the role of those with the highest potential for clinical implementation. The Web of Science database (all databases) was searched on 17 October 2022, using the search terms “marker *” OR biomarker * AND “renal disease” OR “kidney disease” AND “proteome *” OR “peptid *” AND “urin *”. English, full-text, original articles on humans published within the last 5 years were included, which had been cited at least five times per year. Studies based on animal models, renal transplant studies, metabolite studies, studies on miRNA, and studies on exosomal vesicles were excluded, focusing on urinary peptide biomarkers. The described search led to the identification of 3668 articles and the application of inclusion and exclusion criteria, as well as abstract and consecutive full-text analyses of three independent authors to reach a final number of 62 studies for this manuscript. The 62 manuscripts encompassed eight established single peptide biomarkers and several proteomic classifiers, including CKD273 and IgAN237. This review provides a summary of the recent evidence on single peptide urinary biomarkers in CKD, while emphasizing the increasing role of proteomic biomarker research with new research on established and new proteomic biomarkers. Lessons learned from the last 5 years in this review might encourage future studies, hopefully resulting in the routine clinical applicability of new biomarkers

Recent Advances in Urinary Peptide and Proteomic Biomarkers in Chronic Kidney Disease: A Systematic Review

Biomarker development, improvement, and clinical implementation in the context of kidney disease have been a central focus of biomedical research for decades. To this point, only serum creatinine and urinary albumin excretion are well-accepted biomarkers in kidney disease. With their known blind spot in the early stages of kidney impairment and their diagnostic limitations, there is a need for better and more specific biomarkers. With the rise in large-scale analyses of the thousands of peptides in serum or urine samples using mass spectrometry techniques, hopes for biomarker development are high. Advances in proteomic research have led to the discovery of an increasing amount of potential proteomic biomarkers and the identification of candidate biomarkers for clinical implementation in the context of kidney disease management. In this review that strictly follows the PRISMA guidelines, we focus on urinary peptide and especially peptidomic biomarkers emerging from recent research and underline the role of those with the highest potential for clinical implementation. The Web of Science database (all databases) was searched on 17 October 2022, using the search terms “marker *” OR biomarker * AND “renal disease” OR “kidney disease” AND “proteome *” OR “peptid *” AND “urin *”. English, full-text, original articles on humans published within the last 5 years were included, which had been cited at least five times per year. Studies based on animal models, renal transplant studies, metabolite studies, studies on miRNA, and studies on exosomal vesicles were excluded, focusing on urinary peptide biomarkers. The described search led to the identification of 3668 articles and the application of inclusion and exclusion criteria, as well as abstract and consecutive full-text analyses of three independent authors to reach a final number of 62 studies for this manuscript. The 62 manuscripts encompassed eight established single peptide biomarkers and several proteomic classifiers, including CKD273 and IgAN237. This review provides a summary of the recent evidence on single peptide urinary biomarkers in CKD, while emphasizing the increasing role of proteomic biomarker research with new research on established and new proteomic biomarkers. Lessons learned from the last 5 years in this review might encourage future studies, hopefully resulting in the routine clinical applicability of new biomarkers

A Novel Urinary Proteomics Classifier for Non-Invasive Evaluation of Interstitial Fibrosis and Tubular Atrophy in Chronic Kidney Disease

Non-invasive urinary peptide biomarkers are able to detect and predict chronic kidney disease (CKD). Moreover, specific urinary peptides enable discrimination of different CKD etiologies and offer an interesting alternative to invasive kidney biopsy, which cannot always be performed. The aim of this study was to define a urinary peptide classifier using mass spectrometry technology to predict the degree of renal interstitial fibrosis and tubular atrophy (IFTA) in CKD patients. The urinary peptide profiles of 435 patients enrolled in this study were analyzed using capillary electrophoresis coupled with mass spectrometry (CE-MS). Urine samples were collected on the day of the diagnostic kidney biopsy. The proteomics data were divided into a training (n = 200) and a test (n = 235) cohort. The fibrosis group was defined as IFTA ≥ 15% and no fibrosis as IFTA < 10%. Statistical comparison of the mass spectrometry data enabled identification of 29 urinary peptides with differential occurrence in samples with and without fibrosis. Several collagen fragments and peptide fragments of fetuin-A and others were combined into a peptidomic classifier. The classifier separated fibrosis from non-fibrosis patients in an independent test set (n = 186) with area under the curve (AUC) of 0.84 (95% CI: 0.779 to 0.889). A significant correlation of IFTA and FPP_BH29 scores could be observed Rho = 0.5, p < 0.0001. We identified a peptidomic classifier for renal fibrosis containing 29 peptide fragments corresponding to 13 different proteins. Urinary proteomics analysis can serve as a non-invasive tool to evaluate the degree of renal fibrosis, in contrast to kidney biopsy, which allows repeated measurements during the disease course

Differentiating primary and secondary FSGS using non-invasive urine biomarkers

Background: Focal segmental glomerulosclerosis (FSGS) is divided into genetic, primary (p), uncertain cause, and secondary (s) forms. The subclasses differ in management and prognosis with differentiation often being challenging. We aimed to identify specific urine proteins/peptides discriminating between biopsy-proven pFSGS and sFSGS. Methods: 63 urine samples were collected in two different centers (19 pFSGS and 44 sFSGS) prior to biopsy. Samples were analyzed using capillary electrophoresis coupled mass spectrometry. For biomarker definition, datasets of age-/sex-matched normal controls (NC, n = 98) and patients with other chronic kidney diseases (CKDs, n = 100) were extracted from the urinary proteome database. Independent specificity assessment was performed in additional data of NC (n = 110) and CKD (n = 170). Results: Proteomics data from patients with pFSGS were first compared to NC (n = 98). This resulted in 1179 biomarker (P &amp;lt; 0.05) candidates. Then, the pFSGS group was compared to sFSGS, and in a third step, pFSGS data were compared to data from different CKD etiologies (n = 100). Finally, 93 biomarkers were identified and combined in a classifier, pFSGS93. Total cross-validation of this classifier resulted in an area under the receiving operating curve of 0.95. The specificity investigated in an independent set of NC and CKD of other etiologies was 99.1% for NC and 94.7% for CKD, respectively. The defined biomarkers are largely fragments of different collagens (49%). Conclusion: A urine peptide-based classifier that selectively detects pFSGS could be developed. Specificity of 95%-99% could be assessed in independent samples. Sensitivity must be confirmed in independent cohorts before routine clinical application

Assessment and Risk Prediction of Chronic Kidney Disease and Kidney Fibrosis Using Non-Invasive Biomarkers

Effective management of chronic kidney disease (CKD), a major health problem worldwide, requires accurate and timely diagnosis, prognosis of progression, assessment of therapeutic efficacy, and, ideally, prediction of drug response. Multiple biomarkers and algorithms for evaluating specific aspects of CKD have been proposed in the literature, many of which are based on a small number of samples. Based on the evidence presented in relevant studies, a comprehensive overview of the different biomarkers applicable for clinical implementation is lacking. This review aims to compile information on the non-invasive diagnostic, prognostic, and predictive biomarkers currently available for the management of CKD and provide guidance on the application of these biomarkers. We specifically focus on biomarkers that have demonstrated added value in prospective studies or those based on prospectively collected samples including at least 100 subjects. Published data demonstrate that several valid non-invasive biomarkers of potential value in the management of CKD are currently available.Bundesministerium für Bildung und Forschung (BMBF; Federal Ministry of Education and Research)European Unio

Place cells on a maze encode routes rather than destinations

Hippocampal place cells fire at different rates when a rodent runs through a given location on its way to different destinations. However, it is unclear whether such firing represents the animal's intended destination or the execution of a specific trajectory. To distinguish between these possibilities, Lister Hooded rats (n=8) were trained to navigate from a start box to three goal locations via four partially overlapping routes. Two of these led to the same goal location. Of the cells that fired on these two routes, 95.8% showed route-dependent firing (firing on only one route), whereas only two cells (4.2%) showed goal-dependent firing (firing similarly on both routes). In addition, route-dependent place cells over-represented the less discriminable routes, and place cells in general over-represented the start location. These results indicate that place cell firing on overlapping routes reflects the animal's route, not its goals, and that this firing may aid spatial discrimination

Microangiopathy in multiple myeloma: a case of carfilzomib-induced secondary thrombotic microangiopathy successfully treated with plasma exchange and complement inhibition

Abstract Background Thrombotic microangiopathy (TMA) is a potentially organ and life-threatening condition affecting patients with multiple myeloma (MM). Cases of proteasome inhibitor-induced TMA and specifically carfilzomib-induced TMA have been rarely reported and standards for diagnostic workup and treatment are not available. Case presentation We describe a case of a male MM patient under salvage therapy including proteasome inhibitor carfilzomib following chemotherapy and autologous stem cell transplantation. The patient then developed acute kidney injury with clinical and laboratory signs of TMA. Hemodialysis became necessary and treatment with plasma exchange was initiated followed by therapy with C5 complement inhibitor eculizumab which led to amelioration of kidney function and hemolysis parameters. Conclusion We report a patient with suspected proteasome inhibitor-induced secondary thrombotic microangiopathy that has been successfully treated with plasma exchange and eculizumab, a monoclonal antibody targeting complement factor C5

First-line treatment with FOLFOXIRI for advanced pancreatic cancer in clinical practice: Patients' outcome and analysis of prognostic factors

FOLFIRINOX is a standard first-line treatment for advanced pancreatic cancer (aPC). The Gruppo Oncologico Nord Ovest (GONO) FOLFOXIRI regimen demonstrated efficacy in metastatic colorectal cancer. We aimed to evaluate activity and tolerability of FOLFOXIRI regimen in patients with aPC and to explore putative prognostic factors. One hundred thirty-seven consecutive aPC patients were treated with FOLFOXIRI in our institution between 2008 and 2014. Clinical, laboratory and pathological data were collected and their association with activity, progression free survival (PFS) and overall survival (OS) was investigated. After a median follow up of 30 months, median PFS and OS were 8.0 months (95% CI 6.19–9.81) and 12 months (95% CI 9.75–14.25), respectively. Response rate was 38.6%, while disease-control rate 72.2%. At multivariate analysis liver metastases (p = 0.019; Hazard Ratio, HR, 0.59, 95% Confidence Interval, CI, 0.380.96), Eastern Cooperative Oncology Group (ECOG) performance status (PS) 1 (p = 0.001; HR 2.26, 95%CI 1.42–3.59) and neutrophil-lymphocyte ratio (NLR)> 4 (p= 0.002; HR: 2.42; 95% CI 1.38–4.25) were associated with poorer OS. We categorized 119 pts with complete available data as good-risk (0 factors, 38 pts), intermediate-risk (1 factor, 49 pts) and poor-risk (≥2 factors, 32 pts). Median OS for these three groups were 17.6, 11.1 and 7.4 months, respectively (p < 0.001). FOLFOXIRI is active and feasible in aPC. Prognosis of aPC pts treated with FOLFOXIRI is influenced by easily available factors: our analysis revealed ECOG PS, liver metastases and NLR as the most important predictors of survival. These factors could be helpful for treatment decision and clinical trial design