Subtractive gene expression profiling of articular cartilage and mesenchymal stem cells: serpins as cartilage-relevant differentiation markers

SummaryObjectiveMesenchymal stem cells (MSCs) are a population of cells broadly discussed to support cartilage repair. The differentiation of MSCs into articular chondrocytes is, however, still poorly understood on the molecular level. The aim of this study was to perform an almost genome-wide screen for genes differentially expressed between cartilage and MSCs and to extract new markers useful to define chondrocyte differentiation stages.MethodsGene expression profiles of MSCs (n=8) and articular cartilage from OA patients (n=7) were compared on a 30,000 cDNA-fragment array and differentially expressed genes were extracted by subtraction. Expression of selected genes was assessed during in vitro chondrogenic differentiation of MSCs and during dedifferentiation of expanded chondrocytes using quantitative and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Protein secretion was measured by enzyme-linked immunosorbent assay.ResultsEighty-seven genes were differentially expressed between MSCs and cartilage with a more than three-fold difference. Sixty-seven of them were higher expressed in cartilage and among them 15 genes were previously not detected in cartilage. Differential expression was confirmed for 69% of 26 reanalysed genes by RT-PCR. The profiles of three unknown transcripts and six protease-related molecules were characterised during differentiation. SERPINA1 and SERPINA3 mRNA expression correlated with chondrogenic differentiation of MSCs and dedifferentiation of chondrocytes, and SERPINA1 protein levels in culture supernatants could be correlated alike.ConclusionscDNA-array analysis identified SERPINA1 and A3 as new differentiation-relevant genes for cartilage. Since SERPINA1 secretion correlated with both chondrogenesis of MSCs and dedifferentiation during chondrocyte expansion, it represents an attractive marker for refinement of chondrocyte differentiation

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

<p>Abstract</p> <p>Background</p> <p>We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.</p> <p>Results</p> <p>Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.</p> <p>Conclusion</p> <p>From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.</p

The landscape of viral associations in human cancers

Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, for which whole-genome and—for a subset—whole-transcriptome sequencing data from 2,658 cancers across 38 tumor types was aggregated, we systematically investigated potential viral pathogens using a consensus approach that integrated three independent pipelines. Viruses were detected in 382 genome and 68 transcriptome datasets. We found a high prevalence of known tumor-associated viruses such as Epstein–Barr virus (EBV), hepatitis B virus (HBV) and human papilloma virus (HPV; for example, HPV16 or HPV18). The study revealed significant exclusivity of HPV and driver mutations in head-and-neck cancer and the association of HPV with APOBEC mutational signatures, which suggests that impaired antiviral defense is a driving force in cervical, bladder and head-and-neck carcinoma. For HBV, HPV16, HPV18 and adeno-associated virus-2 (AAV2), viral integration was associated with local variations in genomic copy numbers. Integrations at the TERT promoter were associated with high telomerase expression evidently activating this tumor-driving process. High levels of endogenous retrovirus (ERV1) expression were linked to a worse survival outcome in patients with kidney cancer

Recommendations for a practical implementation of circulating tumor DNA mutation testing in metastatic non-small-cell lung cancer

BACKGROUND: Liquid biopsy (LB) is a rapidly evolving diagnostic tool for precision oncology that has recently found its way into routine practice as an adjunct to tissue biopsy (TB). The concept of LB refers to any tumor-derived material, such as circulating tumor DNA (ctDNA) or circulating tumor cells that are detectable in blood. An LB is not limited to the blood and may include other fluids such as cerebrospinal fluid, pleural effusion, and urine, among others. PATIENTS AND METHODS: The objective of this paper, devised by international experts from various disciplines, is to review current challenges as well as state-of-the-art applications of ctDNA mutation testing in metastatic non-small-cell lung cancer (NSCLC). We consider pragmatic scenarios for the use of ctDNA from blood plasma to identify actionable targets for therapy selection in NSCLCs. RESULTS: Clinical scenarios where ctDNA mutation testing may be implemented in clinical practice include complementary tissue and LB testing to provide the full picture of patients’ actual predictive profiles to identify resistance mechanism (i.e. secondary mutations), and ctDNA mutation testing to assist when a patient has a discordant clinical history and is suspected of showing intertumor or intratumor heterogeneity. ctDNA mutation testing may provide interesting insights into possible targets that may have been missed on the TB. Complementary ctDNA LB testing also provides an option if the tumor location is hard to biopsy or if an insufficient sample was taken. These clinical use cases highlight practical scenarios where ctDNA LB may be considered as a complementary tool to TB analysis. CONCLUSIONS: Proper implementation of ctDNA LB testing in routine clinical practice is envisioned in the near future. As the clinical evidence of utility expands, the use of LB alongside tissue sample analysis may occur in the patient cases detailed here

Genome-Wide DNA Methylation Profiling in Early Stage I Lung Adenocarcinoma Reveals Predictive Aberrant Methylation in the Promoter Region of the Long Noncoding RNA PLUT: An Exploratory Study

Introduction: Surgical procedure is the treatment of choice in early stage I lung adenocarcinoma. However, a considerable number of patients experience recurrence within the first 2 years after complete resection. Suitable prognostic biomarkers that identify patients at high risk of recurrence (who may probably benefit from adjuvant treatment) are still not available. This study aimed at identifying methylation markers for early recurrence that may become important tools for the development of new treatment modalities. Methods: Genome-wide DNA methylation profiling was performed on 30 stage I lung adenocarcinomas, comparing 14 patients with early metastatic recurrence with 16 patients with a long-term relapse-free survival period using methylated-CpG-immunoprecipitation followed by high-throughput next-generation sequencing. The differentially methylated regions between the two subgroups were validated for their prognostic value in two independent cohorts using the MassCLEAVE assay, a high-resolution quantitative methylation analysis. Results: Unsupervised clustering of patients in the discovery cohort on the basis of differentially methylated regions identified patients with shorter relapse-free survival (hazard ratio: 2.23; 95% confidence interval: 0.66-7.53; p = 0.03). In two validation cohorts, promoter hypermethylation of the long noncoding RNA PLUT was significantly associated with shorter relapse-free survival (hazard ratio: 0.54; 95% confidence interval: 0.31-0.93; p < 0.026) and could be reported as an independent prognostic factor in the multivariate Cox regression analysis. Conclusions: Promoter hypermethylation of the long noncoding RNA PLUT is predictive in patients with early stage I adenocarcinoma at high risk for early recurrence. Further studies are needed to validate its role in carcinogenesis and its use as a biomarker to facilitate patient selection and risk stratification

倫理創成研究の動向 : シュレーダー＝フレチェットの『リスクと合理性』から

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies

Author Correction: The landscape of viral associations in human cancers

Correction to: Nature Genetics https://doi.org/10.1038/s41588-019-0558-9, published online 05 February 2020

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis