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1.  FirebrowseR: an R client to the Broad Institute’s Firehose Pipeline 
With its Firebrowse service (http://firebrowse.org/) the Broad Institute is making large-scale multi-platform omics data analysis results publicly available through a Representational State Transfer (REST) Application Programmable Interface (API). Querying this database through an API client from an arbitrary programming environment is an essential task, allowing other developers and researchers to focus on their analysis and avoid data wrangling. Hence, as a first result, we developed a workflow to automatically generate, test and deploy such clients for rapid response to API changes. Its underlying infrastructure, a combination of free and publicly available web services, facilitates the development of API clients. It decouples changes in server software from the client software by reacting to changes in the RESTful service and removing direct dependencies on a specific implementation of an API. As a second result, FirebrowseR, an R client to the Broad Institute’s RESTful Firehose Pipeline, is provided as a working example, which is built by the means of the presented workflow. The package’s features are demonstrated by an example analysis of cancer gene expression data.
Database URL: https://github.com/mariodeng/
doi:10.1093/database/baw160
PMCID: PMC5216271  PMID: 28062517
2.  Identification of aberrant tRNA-halves expression patterns in clear cell renal cell carcinoma 
Scientific Reports  2016;6:37158.
Small non-coding RNAs (sncRNA; <200 nt) regulate various cellular processes and modify gene expression. Under nutritional, biological or physiochemical stress some mature sncRNAs (e.g. tRNAs) are cleaved into halves (30–50 nt) and smaller fragments (18–22 nt); the significance and functional role of these tRNA fragments is unknown, but their existence has been linked to carcinogenesis. We used small RNA sequencing to determine the expression of sncRNAs. Subsequently the findings were validated for miR-122-5p, miR-142-3p and 5'tRNA4-Val-AAC using qPCR. We identified differential expression of 132 miRNAs (upregulated: 61, downregulated: 71) and 32 tRNAs (upregulated: 13, downregulated: 19). Read length analysis showed that miRNAs mapped in the 20–24 nt fraction, whereas tRNA reads mapped in the 30–36 nt fraction instead the expected size of 73–95 nt thereby indicating cleavage of tRNAs. Overexpression of miR-122-5p and miR-142-3p as well as downregulation of 5'tRNA4-Val-AAC was validated in an independent cohort of 118 ccRCC and 74 normal renal tissues. Furthermore, staging and grading was inversely correlated with the 5'tRNA4-Val-AAC expression. Serum levels of miR-122-5p, miR-142-3p and 5'tRNA4-Val-AAC did not differ in ccRCC and control subjects. In conclusion, 5′ cleavage of tRNAs occurs in ccRCC, but the exact functional implication of tRNA-halve deregulation remains to be clarified.
doi:10.1038/srep37158
PMCID: PMC5121638  PMID: 27883021
3.  FGFR1 expression levels predict BGJ398-sensitivity of FGFR1-dependent head and neck squamous cell cancers 
Background
FGFR1 copy number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors and patient-derived xenografts (PDXs) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398.
Methods
FGFR1 status, expression levels and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n=353) were assessed for FGFR1 CNG and mRNA levels and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n=39) were submitted to FGFR1 copy number detection and mRNA assays to identify putative FGFR1-dependent tumors.
Results
Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. 31% of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The non-amplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398.
Conclusion
FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts TKI sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC.
doi:10.1158/1078-0432.CCR-14-3357
PMCID: PMC4592392  PMID: 26015511
FGFR1; HNSCC; BGJ398; small molecule inhibitor; targeted therapy
4.  The activation of OR51E1 causes growth suppression of human prostate cancer cells 
Oncotarget  2016;7(30):48231-48249.
The development of prostate cancer (PCa) is regulated by the androgen-dependent activity of the androgen receptor (AR). Androgen-deprivation therapy (ADT) is therefore the gold standard treatment to suppress malignant progression of PCa. Nevertheless, due to the development of castration resistance, recurrence of disease after initial response to ADT is a major obstacle to successful treatment. As G-protein coupled receptors play a fundamental role in PCa physiology, they might represent promising alternative or combinatorial targets for advanced diseases. Here, we verified gene expression of the olfactory receptors (ORs) OR51E1 [prostate-specific G-protein coupled receptor 2 (PSGR2)] and OR51E2 (PSGR) in human PCa tissue by RNA-Seq analysis and RT-PCR and elucidated the subcellular localization of both receptor proteins in human prostate tissue. The OR51E1 agonist nonanoic acid (NA) leads to the phosphorylation of various protein kinases and growth suppression of the PCa cell line LNCaP. Furthermore, treatment with NA causes reduction of androgen-mediated AR target gene expression. Interestingly, NA induces cellular senescence, which coincides with reduced E2F1 mRNA levels. In contrast, treatment with the structurally related compound 1-nonanol or the OR2AG1 agonist amyl butyrate, neither of which activates OR51E1, did not lead to reduced cell growth or an induction of cellular senescence. However, decanoic acid, another OR51E1 agonist, also induces cellular senescence. Thus, our results suggest the involvement of OR51E1 in growth processes of PCa cells and its impact on AR-mediated signaling. These findings provide novel evidences to support the functional importance of ORs in PCa pathogenesis.
doi:10.18632/oncotarget.10197
PMCID: PMC5217014  PMID: 27374083
OR51E1; proliferation; prostate cancer; cellular senescence; androgen receptor
5.  Integrative genome analyses identify key somatic driver mutations of small cell lung cancer 
Peifer, Martin | Fernández-Cuesta, Lynnette | Sos, Martin L | George, Julie | Seidel, Danila | Kasper, Lawryn H | Plenker, Dennis | Leenders, Frauke | Sun, Ruping | Zander, Thomas | Menon, Roopika | Koker, Mirjam | Dahmen, Ilona | Müller, Christian | Di Cerbo, Vincenzo | Schildhaus, Hans-Ulrich | Altmüller, Janine | Baessmann, Ingelore | Becker, Christian | de Wilde, Bram | Vandesompele, Jo | Böhm, Diana | Ansén, Sascha | Gabler, Franziska | Wilkening, Ines | Heynck, Stefanie | Heuckmann, Johannes M | Lu, Xin | Carter, Scott L | Cibulskis, Kristian | Banerji, Shantanu | Getz, Gad | Park, Kwon-Sik | Rauh, Daniel | Grütter, Christian | Fischer, Matthias | Pasqualucci, Laura | Wright, Gavin | Wainer, Zoe | Russell, Prudence | Petersen, Iver | Chen, Yuan | Stoelben, Erich | Ludwig, Corinna | Schnabel, Philipp | Hoffmann, Hans | Muley, Thomas | Brockmann, Michael | Engel-Riedel, Walburga | Muscarella, Lucia A | Fazio, Vito M | Groen, Harry | Timens, Wim | Sietsma, Hannie | Thunnissen, Erik | Smit, Egbert | Heideman, Daniëlle AM | Snijders, Peter JF | Cappuzzo, Federico | Ligorio, Claudia | Damiani, Stefania | Field, John | Solberg, Steinar | Brustugun, Odd Terje | Lund-Iversen, Marius | Sänger, Jörg | Clement, Joachim H | Soltermann, Alex | Moch, Holger | Weder, Walter | Solomon, Benjamin | Soria, Jean-Charles | Validire, Pierre | Besse, Benjamin | Brambilla, Elisabeth | Brambilla, Christian | Lantuejoul, Sylvie | Lorimier, Philippe | Schneider, Peter M | Hallek, Michael | Pao, William | Meyerson, Matthew | Sage, Julien | Shendure, Jay | Schneider, Robert | Büttner, Reinhard | Wolf, Jürgen | Nürnberg, Peter | Perner, Sven | Heukamp, Lukas C | Brindle, Paul K | Haas, Stefan | Thomas, Roman K
Nature genetics  2012;44(10):1104-1110.
Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor survival1–3. We sequenced 29 SCLC exomes, two genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million basepairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in histone-modifying genes, CREBBP, EP300, and MLL. Furthermore, we observed mutations in PTEN, in SLIT2, and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from p53/Rb1-deficient mice4. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genome alterations, and provides a generalizable framework for identification of biologically relevant genes in the context of high mutational background.
doi:10.1038/ng.2396
PMCID: PMC4915822  PMID: 22941188
small-cell lung cancer; cancer genome; integrated analysis
6.  Spatial niche formation but not malignant progression is a driving force for intratumoural heterogeneity 
Nature Communications  2016;7:ncomms11845.
Intratumoural heterogeneity (ITH) is a major cause of cancer-associated lethality. Extensive genomic ITH has previously been reported in clear cell renal cell carcinoma (ccRCC). Here we address the question whether ITH increases with malignant progression and can hence be exploited as a prognostic marker. Unexpectedly, precision quantitative image analysis reveals that the degree of functional ITH is virtually identical between primary ccRCCs of the lowest stage and advanced, metastatic tumours. Functional ITH was found to show a stage-independent topological pattern with peak proliferative and signalling activities almost exclusively in the tumour periphery. Exome sequencing of matching peripheral and central primary tumour specimens reveals various region-specific mutations. However, these mutations cannot directly explain the zonal pattern suggesting a role of microenvironmental factors in shaping functional ITH. In conclusion, our results indicate that ITH is an early and general characteristic of malignant growth rather than a consequence of malignant progression.
It has been increasingly recognised that tumours are not made up of a homogeneous population of cells. Here, the authors show heterogeneous expression of five protein markers in renal cell cancer and demonstrate that the progression of the tumour does not influence the degree of heterogeneity in the tumour.
doi:10.1038/ncomms11845
PMCID: PMC4910022  PMID: 27291893
7.  MERTK as a novel therapeutic target in head and neck cancer 
Oncotarget  2016;7(22):32678-32694.
Although head and neck cancer (HNSCC) is the sixth most common tumor entity worldwide therapy options remain limited leading to 5-year survival rates of only 50 %. MERTK is a promising therapeutic target in several tumor entities, however, its role in HNSCC has not been described yet. The aim of our study was to investigate the biological significance of MERTK and to evaluate its potential as a novel therapeutic target in this dismal tumor entity. In two large HNSCC cohorts (n=537 and n=520) we found that MERTK is overexpressed in one third of patients. In-vitro, MERTK overexpression led to increased proliferation, migration and invasion whereas MERTK inhibition with the small molecule inhibitor UNC1062 or MERTK knockdown reduced cell motility via the small GTPase RhoA.
Taken together, we are the first to show that MERTK is frequently overexpressed in HNSCC and plays an important role in tumor cell motility. It might therefore be a potential target for selected patients suffering from this dismal tumor entity.
doi:10.18632/oncotarget.8724
PMCID: PMC5078043  PMID: 27081701
head and neck cancer; MERTK; targeted therapy
8.  Telomerase activation by genomic rearrangements in high-risk neuroblastoma 
Nature  2015;526(7575):700-704.
Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2–4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.
doi:10.1038/nature14980
PMCID: PMC4881306  PMID: 26466568
9.  Comprehensive genomic profiles of small cell lung cancer 
George, Julie | Lim, Jing Shan | Jang, Se Jin | Cun, Yupeng | Ozretić, Luka | Kong, Gu | Leenders, Frauke | Lu, Xin | Fernández-Cuesta, Lynnette | Bosco, Graziella | Müller, Christian | Dahmen, Ilona | Jahchan, Nadine S. | Park, Kwon-Sik | Yang, Dian | Karnezis, Anthony N. | Vaka, Dedeepya | Torres, Angela | Wang, Maia Segura | Korbel, Jan O. | Menon, Roopika | Chun, Sung-Min | Kim, Deokhoon | Wilkerson, Matt | Hayes, Neil | Engelmann, David | Pützer, Brigitte | Bos, Marc | Michels, Sebastian | Vlasic, Ignacija | Seidel, Danila | Pinther, Berit | Schaub, Philipp | Becker, Christian | Altmüller, Janine | Yokota, Jun | Kohno, Takashi | Iwakawa, Reika | Tsuta, Koji | Noguchi, Masayuki | Muley, Thomas | Hoffmann, Hans | Schnabel, Philipp A. | Petersen, Iver | Chen, Yuan | Soltermann, Alex | Tischler, Verena | Choi, Chang-min | Kim, Yong-Hee | Massion, Pierre P. | Zou, Yong | Jovanovic, Dragana | Kontic, Milica | Wright, Gavin M. | Russell, Prudence A. | Solomon, Benjamin | Koch, Ina | Lindner, Michael | Muscarella, Lucia A. | la Torre, Annamaria | Field, John K. | Jakopovic, Marko | Knezevic, Jelena | Castaños-Vélez, Esmeralda | Roz, Luca | Pastorino, Ugo | Brustugun, Odd-Terje | Lund-Iversen, Marius | Thunnissen, Erik | Köhler, Jens | Schuler, Martin | Botling, Johan | Sandelin, Martin | Sanchez-Cespedes, Montserrat | Salvesen, Helga B. | Achter, Viktor | Lang, Ulrich | Bogus, Magdalena | Schneider, Peter M. | Zander, Thomas | Ansén, Sascha | Hallek, Michael | Wolf, Jürgen | Vingron, Martin | Yatabe, Yasushi | Travis, William D. | Nürnberg, Peter | Reinhardt, Christian | Perner, Sven | Heukamp, Lukas | Büttner, Reinhard | Haas, Stefan A. | Brambilla, Elisabeth | Peifer, Martin | Sage, Julien | Thomas, Roman K.
Nature  2015;524(7563):47-53.
We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.
doi:10.1038/nature14664
PMCID: PMC4861069  PMID: 26168399
10.  Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types 
Oncotarget  2016;7(17):23043-23055.
The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.
We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.
doi:10.18632/oncotarget.8469
PMCID: PMC5029609  PMID: 27050271
transcriptional profile; mediator complex; cancer; oncomine; MED8; Pathology Section
11.  NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas 
International Journal of Cancer  2015;138(4):927-938.
Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non‐small cell tumors and secondary transitions from non‐small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of “small cell‐ness” based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT‐signaling in the context of mutual bi‐allelic RB1 and TP53 lesions. Additionaly, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH‐ASCL1‐RB‐p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon‐based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.
What's new?
Using next generation sequencing and establishing features of ‘small cell‐ness’, we identified a NOTCH‐ASCL1‐RB1‐TP53 signaling axis driving small cell cancers. In contrast to the previously described bi‐allelic RB1/TP53 loss in neuroendocrine stem cells as origin of primary small cell neuroendocrine cancers, the NOTCH‐ASCL1 mediated signaling defines an alternative pathway driving secondary small cell neuroendocrine cancers arising from non‐small cell cancers. Moreover, we show a preclinical rational for therapeutically testing WNT‐inhibitors in small cell cancers.
doi:10.1002/ijc.29835
PMCID: PMC4832386  PMID: 26340530
Lung cancer; small cell lung cancer; achaete‐scute homolog 1; neurogenic locus notch homolog; retinoblastoma protein
12.  Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity 
Scientific Reports  2016;6:24146.
Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility.
doi:10.1038/srep24146
PMCID: PMC4823793  PMID: 27052161
13.  Web-TCGA: an online platform for integrated analysis of molecular cancer data sets 
BMC Bioinformatics  2016;17:72.
Background
The Cancer Genome Atlas (TCGA) is a pool of molecular data sets publicly accessible and freely available to cancer researchers anywhere around the world. However, wide spread use is limited since an advanced knowledge of statistics and statistical software is required.
Results
In order to improve accessibility we created Web-TCGA, a web based, freely accessible online tool, which can also be run in a private instance, for integrated analysis of molecular cancer data sets provided by TCGA. In contrast to already available tools, Web-TCGA utilizes different methods for analysis and visualization of TCGA data, allowing users to generate global molecular profiles across different cancer entities simultaneously. In addition to global molecular profiles, Web-TCGA offers highly detailed gene and tumor entity centric analysis by providing interactive tables and views.
Conclusions
As a supplement to other already available tools, such as cBioPortal (Sci Signal 6:pl1, 2013, Cancer Discov 2:401–4, 2012), Web-TCGA is offering an analysis service, which does not require any installation or configuration, for molecular data sets available at the TCGA. Individual processing requests (queries) are generated by the user for mutation, methylation, expression and copy number variation (CNV) analyses. The user can focus analyses on results from single genes and cancer entities or perform a global analysis (multiple cancer entities and genes simultaneously).
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-016-0917-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-016-0917-9
PMCID: PMC4744375  PMID: 26852330
TCGA; Cancer genomics; Statistics; Web application; Genomic data
14.  Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma 
Molecular Cancer  2016;15:10.
Background
Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies.
Methods
In order to provide better understanding of the molecular biology of ccRCC and to identify potential diagnostic/prognostic biomarker and therapeutic targets, we utilized a microarray to profile mRNA expression of corresponding normal and malignant renal tissues. Real-time PCR, Western Blot and immunohistochemistry were applied to study the expression of candidate biomarkers. ccRCC cell lines were treated with sertraline to inhibit the dopamine transporter SLC6A3.
Results
Differential expression of fourteen mRNAs, yet not studied in ccRCC in depth, was confirmed using qPCR (upregulation: SLC6A3, NPTX2, TNFAIP6, NDUFA4L2, ENPP3, FABP6, SPINK13; downregulation: FXYD4, SLC12A1, KNG1, NPHS2, SLC13A3, GCGR, PLG). Up-/downregulation was also confirmed for FXYD4, KNG1, NPTX2 and SLC12A1 by Western Blot on the protein level. In contrast to the mRNA expression, protein expression of the dopamine transporter SLC6A3 was lower in ccRCC compared to normal renal tissue. Immunohistochemistry indicated that this decrease was due to higher concentrations of SLC6A3 in the proximal tubules. Immunohistochemical analyses further demonstrated that high SLC6A3 expression in ccRCC tissue was correlated with a shorter period of recurrence-free survival following surgery. Treatment of ccRCC cells with the SLC6A3 inhibitor sertraline induced dose-dependent cell-death.
Conclusion
Our study identified several novel biomarkers with diagnostic potential and further investigations on sertraline as therapeutic agent in ccRCC patients are warranted.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-016-0495-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12943-016-0495-5
PMCID: PMC4736613  PMID: 26831905
Renal cell carcinoma; SLC6A3; Expression profiling; Biomarker; Microarray; Sertraline
15.  Molecular and functional interactions between AKT and SOX2 in breast carcinoma 
Oncotarget  2015;6(41):43540-43556.
The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC.
PMCID: PMC4791249  PMID: 26498353
SOX2; AKT; breast carcinoma; cancer stem cells; clonogenicity
16.  Prognostic Significance and Functional Role of CEP57 in Prostate Cancer1 
Translational Oncology  2015;8(6):487-496.
We have recently shown that centrosomal protein 57 (CEP57) is overexpressed in a subset of human prostate cancers. CEP57 is involved in intracellular transport processes, and its overexpression causes mitotic defects as well as abnormal microtubule nucleation and bundling. In the present study, we further characterized the prognostic and functional role of CEP57 in prostate cancer. Unexpectedly, we found that high CEP57 expression is an independent prognostic factor for a more favorable biochemical recurrence-free survival in two large patient cohorts. To reconcile this finding with the ability of CEP57 to cause cell division errors and thus potentially promote malignant progression, we hypothesized that alterations of microtubule-associated transport processes, in particular nuclear translocation of the androgen receptor (AR), may play a role in our finding. However, CEP57 overexpression and microtubule bundling had, surprisingly, no effect on the nuclear translocation of the AR. Instead, we found a significant increase of cells with disarranged microtubules and a cellular morphology suggestive of a cytokinesis defect. Because mitotic dysfunction leads to a reduced daughter cell formation, it can explain the survival benefit of patients with increased CEP57 expression. In contrast, we show that a reduced expression of CEP57 is associated with malignant growth and metastasis. Taken together, our findings underscore that high CEP57 expression is associated with mitotic impairment and less aggressive tumor behavior. Because the CEP57-induced microtubule stabilization had no detectable effect on AR nuclear translocation, our results furthermore suggest that microtubule-targeting therapeutics used in advanced prostate cancer such as docetaxel may have modes of action that are at least in part independent of AR transport inhibition.
doi:10.1016/j.tranon.2015.11.004
PMCID: PMC4700294  PMID: 26692530
17.  Adaptive responses of androgen receptor signaling in castration-resistant prostate cancer 
Oncotarget  2015;6(34):35542-35555.
Prostate Cancer (PCa) is an important age-related disease being the most common cancer malignancy and the second leading cause of cancer mortality in men in Western countries. Initially, PCa progression is androgen receptor (AR)- and androgen-dependent. Eventually advanced PCa reaches the stage of Castration-Resistant Prostate Cancer (CRPC), but remains dependent on AR, which indicates the importance of AR activity also for CRPC. Here, we discuss various pathways that influence the AR activity in CRPC, which indicates an adaptation of the AR signaling in PCa to overcome the treatment of PCa. The adaptation pathways include interferences of the normal regulation of the AR protein level, the expression of AR variants, the crosstalk of the AR with cytokine tyrosine kinases, the Src-Akt-, the MAPK-signaling pathways and AR corepressors. Furthermore, we summarize the current treatment options with regard to the underlying molecular basis of the common adaptation processes of AR signaling that may arise after the treatment with AR antagonists, androgen deprivation therapy (ADT) as well as for CRPC, and point towards novel therapeutic strategies. The understanding of individualized adaptation processes in PCa will lead to individualized treatment options in the future.
PMCID: PMC4742123  PMID: 26325261
androgen receptor; prostate cancer
18.  Non-Amplified FGFR1 is a Growth Driver in Malignant Pleural Mesothelioma 
Molecular cancer research : MCR  2014;12(10):1460-1469.
Malignant pleural mesothelioma (MPM) is associated with asbestos exposure and is a cancer that has not been significantly impacted by small molecule-based targeted therapeutics. Previously, we demonstrated the existence of functional subsets of lung cancer and head and neck squamous cell carcinoma (HNSCC) cell lines in which fibroblast growth factor receptor (FGFR) autocrine signaling functions as a non-mutated growth pathway. In a panel of pleural mesothelioma cell lines, FGFR1 and FGF2 were co-expressed in 3 of 7 cell lines and were significantly associated with sensitivity to the FGFR-active tyrosine kinase inhibitor (TKI), ponatinib, both in vitro and in vivo using orthotopically propagated xenografts. Furthermore, RNAi-mediated silencing confirmed the requirement for FGFR1 in specific mesothelioma cells and sensitivity to the FGF ligand trap, FP-1039, validated the requirement for autocrine FGFs. None of the FGFR1-dependent mesothelioma cells exhibited increased FGFR1 gene copy number, based on a FISH assay, indicating that increased FGFR1 transcript and protein expression were not mediated by gene amplification. Elevated FGFR1 mRNA was detected in a subset of primary MPM clinical specimens and like MPM cells, none harbored increased FGFR1 gene copy number. These results indicate that autocrine signaling through FGFR1 represents a targetable therapeutic pathway in MPM and that biomarkers distinct from increased FGFR1 gene copy number such as FGFR1 mRNA would be required to identify MPM patients bearing tumors driven by FGFR1 activity.
Implications
FGFR1 is a viable therapeutic target in a subset of malignant pleural mesotheliomas, but FGFR TKI-responsive tumors will need to be selected by a biomarker distinct from increased FGFR1 gene copy number, possibly FGFR1 mRNA or protein levels.
doi:10.1158/1541-7786.MCR-14-0038
PMCID: PMC4201974  PMID: 24966347
19.  Prevention and Early Detection of Prostate Cancer 
The Lancet. Oncology  2014;15(11):e484-e492.
Prostate cancer is one of the most common cancers in men and the global burden of this disease is rising. Lifestyle modifications like smoking cessation, exercise and weight control offer opportunities to decrease the risk of developing prostate cancer. Early detection of prostate cancer by PSA screening remains controversial; yet, changes in PSA threshold, frequency of screening, and addition of other biomarkers have potential to minimise overdiagnosis associated with PSA screening. Several new biomarkers appear promising in individuals with elevated PSA levels or those diagnosed with prostate cancer, these are likely to guide in separating individuals who can be spared of aggressive treatment from those who need it. Several pharmacological agents like 5α-reductase inhibitors, aspirin etc. have a potential to prevent development of prostate cancer. In this review, we discuss the current evidence and research questions regarding prevention, early detection of prostate cancer and management of men either at high risk of prostate cancer or diagnosed with low-grade prostate cancer.
doi:10.1016/S1470-2045(14)70211-6
PMCID: PMC4203149  PMID: 25281467
prostate cancer; prevention; risk factors; PSA; screening
20.  Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors 
Diagnostic Pathology  2015;10:165.
Background
Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes.
Methods
Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software.
Results
In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis.
Conclusions
In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15 expression in the preinvasive precursor cells, which may provide diagnostic value to distinguish between benign and pre-malignant testicular specimen, and may indicate a role for MED15 in carcinogenesis in TGCT.
doi:10.1186/s13000-015-0398-6
PMCID: PMC4573996  PMID: 26377566
21.  The long non-coding RNA lnc-ZNF180-2 is a prognostic biomarker in patients with clear cell renal cell carcinoma 
American Journal of Cancer Research  2015;5(9):2799-2807.
Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies. Long non-coding RNAs (lncRNA) regulate various cellular functions and have been implicated in ccRCC pathogenesis. In order to decipher the molecular biology of this tumor and to identify potential prognostic biomarkers and therapeutic targets, we re-evaluated published lncRNA expression profiling data. An expression profile of 49 lncRNAs allowed discrimination of localized and advanced ccRCC. The expression profile of six lncRNAs transcripts (lnc-ACO1625, lnc-CYP4A22-2/3, lnc-PEAK1.1-1, lnc-PCYOX1L, lnc-VCAN-1, lnc-ZNF180-2) with potential prognostic interest were validated in a cohort of 50 normal renal, 57 localized ccRCC and 45 advanced ccRCC tissues. lnc-ZNF180-2 levels were similar in localized ccRCC and normal renal tissue, but we observed a significant increase of lnc-ZNF180-2 expression in advanced ccRCC tissue. Furthermore, lnc-ZNF180-2 expression levels were an independent predictor of progression-free survival, cancer-specific survival and overall survival in ccRCC patients. We also observed that lnc-CYP4A22-2/3 expression levels allowed discrimination of ccRCC and normal renal tissue. In conclusion, lncRNAs are involved in renal carcinogenesis, and quantification of lnc-ZNF180-2 may be useful for the prediction of ccRCC patients outcome following nephrectomy.
PMCID: PMC4633906  PMID: 26609485
Renal cell carcinoma; long non-coding RNA; lncRNA; lnc-ZNF180-2; biomarker; prognosis
22.  NDUFA4 expression in clear cell renal cell carcinoma is predictive for cancer-specific survival 
American Journal of Cancer Research  2015;5(9):2816-2822.
Like other cancers, renal cell carcinoma (RCC) derives the essential energy for proliferation and survival from high rates of glycolysis rather than from oxidative phosphorylation of the mitochondrial respiration pathway. NDUFA4 (NADH Dehydrogenase (Ubiquinone) 1 Alpha Subcomplex, 4) is encoding a protein belonging to the respiratory chain of mitochondria. For a better understanding of the tumor biology and for identification of a potential new biomarker, we analyzed the regulation of NDUFA4 in RCC compared to normal tissue cells. Downregulation of NDUFA4 mRNA and protein was detected in RCC compared to normal renal tissues in quantitative real-time PCR as well as in western blot and immunohistochemical staining. Histological analysis revealed higher NDUFA4 expression in the distal tubules compared to the proximal tubules and the loop of Henle. A higher molecular weight of the NDUFA4 protein was discovered in RCC samples, possibly indicating a posttranslational modification. Moreover, NDUFA4 protein expression was predictive for cancer-specific survival. Our analysis revealed a potential new biomarker, but future studies are warranted to investigate the prognostic value of NDUF4A expression.
PMCID: PMC4633908  PMID: 26609487
Renal cell carcinoma; NDFUA4; biomarker; mitochondria; electron transport chain
23.  STAT3 regulated ARF expression suppresses prostate cancer metastasis 
Nature Communications  2015;6:7736.
Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19ARF as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF–Mdm2–p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14ARF expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.
IL6-STAT3 signaling is activated in prostate cancer, however inhibiting this pathway has not lead to a survival advantage in patients. Here, Pencik et al. show that loss of the IL6-STAT3 axis in mice and humans leads to metastasis due to loss of ARF, unravelling STAT3 and ARF as potential prognostic markers in prostate cancer.
doi:10.1038/ncomms8736
PMCID: PMC4525303  PMID: 26198641
24.  AIM2 Drives Joint Inflammation in a Self-DNA Triggered Model of Chronic Polyarthritis 
PLoS ONE  2015;10(6):e0131702.
Mice lacking DNase II display a polyarthritis-like disease phenotype that is driven by translocation of self-DNA into the cytoplasm of phagocytic cells, where it is sensed by pattern recognition receptors. While pro-inflammatory gene expression is non-redundantly linked to the presence of STING in these mice, the contribution of the inflammasome pathway has not been explored. To this end, we studied the role of the DNA-sensing inflammasome receptor AIM2 in this self-DNA driven disease model. Arthritis-prone mice lacking AIM2 displayed strongly decreased signs of joint inflammation and associated histopathological findings. This was paralleled with a reduction of caspase-1 activation and pro-inflammatory cytokine production in diseased joints. Interestingly, systemic signs of inflammation that are associated with the lack of DNase II were not dependent on AIM2. Taken together, these data suggest a tissue-specific role for the AIM2 inflammasome as a sensor for endogenous DNA species in the course of a ligand-dependent autoinflammatory condition.
doi:10.1371/journal.pone.0131702
PMCID: PMC4482750  PMID: 26114879
25.  FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity Across All Lung Cancer Histologies 
Purpose
FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous-cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically-relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer.
Experimental Design
Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray comprised of resected lung tumors was submitted to FGFR1 GCN and mRNA analyses and the results were validated with TCGA lung cancer data.
Results
14/58 cell lines exhibited ponatinib sensitivity (IC50 values ≤ 50 nM) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations.
Conclusions
FGFR1 dependency is frequent across various lung cancer histologies and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types.
doi:10.1158/1078-0432.CCR-13-3060
PMCID: PMC4062100  PMID: 24771645

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