Die Regulation der CCL5-Expression in der Monozytendifferenzierung durch post-translationale Modifikation des Y-Box Binding Protein-1 (YB-1)

The infiltration of immune cells into the site of inflammation constitutes a key event during inflammatory processes. The migration is, for example, induced by monocytes/macrophages that secrete chemotactic cytokines, like CCL5/RANTES. The gene regulation of CCL5 is controlled by the Y-box binding protein-1. From cell culture analyses it is known, that the protein mediates a differential CCL5 expression in monocytes and macrophages: In monocytic cells YB-1 transactivates the CCL5 gene, whereas it represses the CCL5 transcription in macrophages. In this work, the molecular mechanisms of these contrary effects during the differentiation process from monocytes to macrophages where analyzed in vitro. It could be shown, that in the course of monocyte differentiation YB-1 is phosphorylated by the kinases AKT and RSK, leading to an enhanced binding affinity to the CCL5 promotor. Consequently, a significantly increased expression of the chemokine was shown on mRNA and protein levels. In the course of differentiation this phosphorylation was reversed. Moreover, using an endotoxin-mediated model of nephritis, the transient YB-1 phosphorylation could be confirmed in in the kidney in vivo. By means of co-immunoprecipitations, a direct physical interaction between the phosphatase calcineurin and YB-1 could be shown, suggesting a calcineurin-mediated dephosphorylation of the protein. Furthermore, the inhibition of the phosphatase by cyclosporine A lead to an enhanced phosphorylation of YB-1 in the nuclei of renal cells in vivo. Different cell lines overexpressing the phosphatase, showed a decreased amount of phosphorylated YB-1, resulting in a reduced binding of YB-1 to the CCL5 promotor in monocytes. Consequently, the expression of the chemokine was reduced. In previous work, a high mobility protein-DNA-complex at the YB-1 binding site within the CCL5 promoter could be detected in macrophages, but not in monocytic cells. It was suggested that this complex mediates the repressive effect of YB-1 on the CCL5 gene transcription in macrophages. In this work, calcineurin could be identified as a component of this complex by supershift analyses, confirming its influence on the phosphorylation state of YB-1. These results point to a dual role of YB-1 in inflammatory processes: At the onset of inflammation it promotes immune cell infiltration by upregulating CCL5. Later on, it is involved in the downregulation of inflammation by repression of CCL5 in differentiated macrophages

Advance of theragnosis biomarkers in lung cancer: from clinical to molecular pathology and biology

One distinct molecular subtype of non-small cell lung cancer (NSCLC) is defined by rearrangement of the anaplastic lymphoma kinase (ALK). The increasing knowledge over the last years has enabled the continuous improvement of ALK inhibitors; however, resistance in these patients remains a major concern. In this review, we summarize recent findings in ALK+-adenocarcinoma of the lung, highlighting the role of TP53 mutations in this specific cancer type and suggest new diagnostic strategies for the future, in order to improve patient's outcome

Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing

The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. Venous blood from healthy donors was collected in tubes from Streck, Roche, and Qiagen. Samples were spiked with artificially fragmented EGFR T790M-mutated DNA and stored for different periods of time or spiked with different DNA amounts before plasma preparation. Extracted ctDNA was analyzed by amplification-refractory mutation system PCR. Mutant DNA, spiked into blood samples from healthy donors at quantities of 1 or 3 ng, was still reliably detectable in all samples after 7 days. EGFR T790M could be detected when spiking was performed with an amount of artificial ctDNA of 0.5 ng when tubes from Roche and Qiagen were used. Blood collection tubes from Roche and Qiagen are highly suitable for ctDNA stabilization and subsequent liquid biopsy testing. Even low ctDNA concentrations allow the detection of somatic mutations

Clinical impact of PD-L1 and PD-1 expression in squamous cell cancer of the vulva

PurposeSquamous cell carcinoma of the vulva (SQCV) is the fifth most common cancer in women and accounts for about 5% of all genital cancers in women. The PD-L1 signaling pathway is activated in many malignant neoplasms and its blockade enhances anti-cancer immunity. The aim of our study was to examine the protein expression of PD-L1 and PD-1 in squamous cell cancer of the vulva, its correlations with clinicopathologic features and prognostic value.MethodsPatients with SQCV treated in one institution were used for the analyses. PD-L1 immunohistochemistry was performed on 4 mu m-thick section of the respective FFPE tissue blocks using the 28-8 antibody. PD-L1 scoring was performed separately for tumour cells (TC) and tumour associated immune cells. DNA was extracted to determine HPV status. Kaplan-Meier estimates for disease-free-survival and overall-survival were calculated and compared by log-rank test.ResultsPD-L1 expression in tumour cells could be observed in 32.9% of the patients. The expression of PD-L1 in peritumoural immune cells was confirmed in 91.4% of the patients. A significant correlation between PD-L1 expression in tumour cells and tumour stage was detected (p=0.007). PD-L1 expression was independent from HPV status. Using the log-rank test we could not prove any significant differences in disease-free survival (p=0.434) and overall survival (p=0.858). Regression analysis showed that nodal status is a predictive factor of survival (p<0.001).ConclusionThe present study showed that a relevant amount of patients with squamous cell cancer of the vulva express PD-L1 in both, tumour cells and tumour-associated immune cells. Furthermore, the significant correlation of PD-L1 expression in TCs with tumour stage indicated the clinical impact of PD-L1 expression during tumour development. These data indicate that SQCV might be amenable to immune checkpoint-inhibition and constitute a rational for the future clinical trials

The nucleic acid binding protein YB-1-controlled expression of CXCL-1 modulates kidney damage in liver fibrosis

Acute kidney injury is a common complication of advanced liver disease and increased mortality of these patients. Here, we analyzed the role of Y-box protein-1 (YB-1), a nucleic acid binding protein, in the bile duct ligation model of liver fibrosis and monitored liver and subsequent kidney damage. Following bile duct ligation, both serum levels of liver enzymes and expression of hepatic extracellular matrix components such as type I collagen were significantly reduced in mice with half-maximal YB-1 expression (Yb1(-/-)) as compared to their wild-type littermates. By contrast, expression of the chemokine CXCL1 was significantly augmented in these Yb1(-/-) mice. YB-1 was identified as a potent transcriptional repressor of the Cxc/1 gene. Precision-cut kidney slices from Yb1(-/-) mice revealed higher expression of the CXCL1 receptor CXCR2 as well as enhanced responsivity to CXCL1 compared to those from wild-type mice. Increased CXCL1 content in Yb1(-/-) mice led to pronounced bile duct ligation-induced damage of the kidneys monitored as parameters of tubular epithelial injury and immune cell infiltration. Pharmacological blockade of CXCR2 as well as application of an inhibitory anti-CXCL1 antibody significantly mitigated early systemic effects on the kidneys following bile duct ligation whereas it had only a modest impact on hepatic inflammation and function. Thus, our analyses provide direct evidence that YB-1 crucially contributes to hepatic fibrosis and modulates liver-kidney crosstalk by maintaining tight control over chemokine CXCL1 expression