Maria Fälth

Circulating miRNAs are correlated with tumor progression in prostate cancer

Circulating miRNAs have recently been indicated as practicable and promising biomarkers for noninvasive diagnosis in various tumor entities. However, cell‐free miRNAs have not been found to correlate with clinicopathological variables in epithelial carcinomas. To learn more about the potential clinical relevance of circulating miRNAs in prostate cancer, we screened 667 miRNAs in serum samples from patients with metastatic (n = 7) and localized prostate cancer (n = 14). Various miRNAs were highly abundant in the sera of patients with metastatic disease, and five upregulated miRNAs (miRNA‐375, miRNA‐9*, miRNA‐141, miRNA‐200b and miRNA‐516a‐3p) were selected for further validation. In the first validation study (n = 45), selected miRNAs were analyzed in a prospectively collected serum set taken from different prostate cancer risk groups. Most of the selected miRNAs were significantly correlated with adverse risk factors when different clinicopathological variables were analyzed. Circulating miRNA‐375 and miRNA‐141 turned out to be the most pronounced markers for high‐risk tumors. Their levels also correlated with high Gleason score or lymph‐node positive status in a second independent validation study (n = 71). In addition, the expression levels of miRNA‐375 and miRNA‐141 were monitored in 72 prostate tissue samples (36 tumor vs. 36 benign). Both miRNAs were highly expressed in all samples and significantly upregulated in the tumors compared to normal tissues. Overall, our observations suggest that miRNA‐375 and miRNA‐141 expression is enhanced in prostate cancer specimens and their release into the blood is further associated with advanced cancer disease.

Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.

BACKGROUND & AIMS Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type-related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.

SwePep, a Database Designed for Endogenous Peptides and Mass Spectrometry

A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.

TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-β signaling

BackgroundTMPRSS2-ERG gene fusions occur in about 50% of all prostate cancer cases and represent promising markers for molecular subtyping. Although TMPRSS2-ERG fusion seems to be a critical event in prostate cancer, the precise functional role in cancer development and progression is still unclear.MethodsWe studied large-scale gene expression profiles in 47 prostate tumor tissue samples and in 48 normal prostate tissue samples taken from the non-suspect area of clinical low-risk tumors using Affymetrix GeneChip Exon 1.0 ST microarrays.ResultsComparison of gene expression levels among TMPRSS2-ERG fusion-positive and negative tumors as well as benign samples demonstrated a distinct transcriptional program induced by the gene fusion event. Well-known biomarkers for prostate cancer detection like CRISP3 were found to be associated with the gene fusion status. WNT and TGF-β/BMP signaling pathways were significantly associated with genes upregulated in TMPRSS2-ERG fusion-positive tumors.ConclusionsThe TMPRSS2-ERG gene fusion results in the modulation of transcriptional patterns and cellular pathways with potential consequences for prostate cancer progression. Well-known biomarkers for prostate cancer detection were found to be associated with the gene fusion. Our results suggest that the fusion status should be considered in retrospective and future studies to assess biomarkers for prostate cancer detection, progression and targeted therapy.

Genome-wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with miR-26a Hypermethylation

UNLABELLED Prostate cancer is the second most common cancer among men worldwide. Alterations in the DNA methylation pattern can be one of the leading causes for prostate cancer formation. This study is the first high-throughput sequencing study investigating genome-wide DNA methylation patterns in a large cohort of 51 tumor and 53 benign prostate samples using methylated DNA immunoprecipitation sequencing. Comparative analyses identified more than 147,000 cancer-associated epigenetic alterations. In addition, global methylation patterns show significant differences based on the TMPRSS2-ERG rearrangement status. We propose the hypermethylation of miR-26a as an alternative pathway of ERG rearrangement-independent EZH2 activation. The observed increase in differential methylation events in fusion-negative tumors can explain the tumorigenic process in the absence of genomic rearrangements. SIGNIFICANCE In contrast to TMPRSS2-ERG -rearranged tumors, the pathomechanism for gene fusion-negative tumors is completely unclear. Using a sequencing-based approach, our work uncovers significant global epigenetic alterations in TMPRSS2-ERG gene fusion-negative tumors and provides a mechanistic explanation for the tumor formation process.

Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20–30% on average compared with raw data.

EZH2 Depletion Blocks the Proliferation of Colon Cancer Cells

The Enhancer of Zeste 2 (EZH2) protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi)-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vector-borne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer.

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.

l-DOPA-induced Dyskinesia is Associated with Regional Increase of Striatal Dynorphin Peptides as Elucidated by Imaging Mass Spectrometry

Opioid peptides are involved in various pathophysiological processes, including algesia, epilepsy, and drug dependence. A strong association between l-DOPA-induced dyskinesia (LID) and elevated prodynorphin mRNA levels has been established in both patients and in animal models of Parkinsons disease, but to date the endogenous prodynorphin peptide products have not been determined. Here, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) was used for characterization, localization, and relative quantification of striatal neuropeptides in a rat model of LID in Parkinsons disease. MALDI IMS has the unique advantage of high sensitivity and high molecular specificity, allowing comprehensive detection of multiple molecular species in a single tissue section. Indeed, several dynorphins and enkephalins could be detected in the present study, including dynorphin A(1–8), dynorphin B, α-neoendorphin, MetEnkRF, MetEnkRGL, PEnk (198–209, 219–229). IMS analysis revealed elevated levels of dynorphin B, α-neoendorphin, substance P, and PEnk (220–229) in the dorsolateral striatum of high-dyskinetic animals compared with low-dyskinetic and lesion-only control rats. Furthermore, the peak-intensities of the prodynorphin derived peptides, dynorphin B and α-neoendorphin, were strongly and positively correlated with LID severity. Interestingly, these LID associated dynorphin peptides are not those with high affinity to κ opioid receptors, but are known to bind and activate also μ- and Δ-opioid receptors. In addition, the peak intensities of a novel endogenous metabolite of α-neoendorphin lacking the N-terminal tyrosine correlated positively with dyskinesia severity. MALDI IMS of striatal sections from Pdyn knockout mice verified the identity of fully processed dynorphin peptides and the presence of endogenous des-tyrosine α-neoendorphin. Des-tyrosine dynorphins display reduced opioid receptor binding and this points to possible novel nonopioid receptor mediated changes in the striatum of dyskinetic rats. Because des-tyrosine dynorphins can only be detected by mass spectrometry, as no antibodies are available, these findings highlight the importance of MALDI IMS analysis for the study of molecular dynamics in neurological diseases.