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1.  Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data 
Genome Biology  2015;16(1):7.
Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0558-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0558-0
PMCID: PMC4300615  PMID: 25650807
2.  Frequent mutations in chromatin-remodeling genes in pulmonary carcinoids 
Nature communications  2014;5:3518.
Pulmonary carcinoids are rare neuroendocrine tumors of the lung. The molecular alterations underlying the pathogenesis of these tumors have not been systematically studied so far. Here we perform gene copy number analysis (n=54), genome/exome (n=44) and transcriptome (n=69) sequencing of pulmonary carcinoids and observe frequent mutations in chromatin-remodeling genes. Covalent histone modifiers and subunits of the SWI/SNF complex are mutated in 40% and 22.2% of the cases respectively, with MEN1, PSIP1 and ARID1A being recurrently affected. In contrast to small-cell lung cancer and large-cell neuroendocrine tumors, TP53 and RB1 mutations are rare events, suggesting that pulmonary carcinoids are not early progenitor lesions of the highly aggressive lung neuroendocrine tumors but arise through independent cellular mechanisms. These data also suggest that inactivation of chromatin remodeling genes is sufficient to drive transformation in pulmonary carcinoids.
doi:10.1038/ncomms4518
PMCID: PMC4132974  PMID: 24670920
3.  Supraphysiological androgen levels induce cellular senescence in human prostate cancer cells through the Src-Akt pathway 
Molecular Cancer  2014;13(1):214.
Background
Prostate cancer (PCa) is the second leading cause of cancer mortality of men in Western countries. The androgen receptor (AR) and AR-agonists (androgens) are required for the development and progression of the normal prostate as well as PCa. However, it is discussed that in addition to their tumor promoting activity, androgens may also exhibit tumor suppressive effects. A biphasic growth response to androgens a growth-promoting and -inhibition has been observed that suggests that administration of supraphysiological androgen levels mediates growth reduction in AR expressing PCa cells.
Methods
Detection of senescence markers, three dimensional interphase fluorescence in situ hybridization (3D-iFISH), qRT-PCR, Western blotting, detection of GFP fusions, prostatectomy, ex vivo culturing.
Results
Here, we describe that supraphysiological levels of androgens induce cell cycle arrest and markers of cellular senescence in human PCa cells, which may in part explain the growth inhibitory role of androgens. The expression of the senescence associated beta galactosidase is observed by treatment with the natural androgen DHT or the less metabolized synthetic androgen R1881. The induction of senescence marker was detected in human PCa cell lines as well as in human primary PCa tissue derived from prostatectomy treated ex vivo. Using interphase FISH (iFISH) suggests that the androgen-induced cellular senescence is associated with localizing the genomic E2F1 locus to senescence associated heterochromatic foci. Analysis of different signaling pathways in LNCaP cells suggest that the p16-Rb-E2F1 pathway is essential for the induction of cellular senescence since treatment with siRNA directed against p16 reduces the level of androgen-induced cellular senescence. Based on the rapid induction of androgen-mediated cellular senescence we identified the Src-PI3K-Akt-signaling pathway and autophagy being in part involved in androgen regulation.
Conclusions
Taken together, our data suggest that AR-agonists at supraphysiological levels mediate induction of cellular senescence in human PCa cells, which may have a protective anti-cancer role. These results provide also new insights for understanding androgen-mediated regulation of PCa growth.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-214) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-214
PMCID: PMC4171558  PMID: 25216853
Nuclear receptor; Non-genomic signaling; Tumor suppression; Cellular senescence; Autophagy
4.  SIAH2 antagonizes TYK2-STAT3 signaling in lung carcinoma cells 
Oncotarget  2014;5(10):3184-3196.
The Janus tyrosine kinases JAK1-3 and tyrosine kinase-2 (TYK2) are frequently hyperactivated in tumors. In lung cancers JAK1 and JAK2 induce oncogenic signaling through STAT3. A putative role of TYK2 in these tumors has not been reported. Here, we show a previously not recognized TYK2-STAT3 signaling node in lung cancer cells. We reveal that the E3 ubiquitin ligase seven-in-absentia-2 (SIAH2) accelerates the proteasomal degradation of TYK2. This mechanism consequently suppresses the activation of STAT3. In agreement with these data the analysis of primary non-small-cell lung cancer (NSCLC) samples from three patient cohorts revealed that compared to lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) show significantly higher levels of SIAH2 and reduced STAT3 phosphorylation levels. Thus, SIAH2 is a novel molecular marker for SCC. We further demonstrate that an activation of the oncologically relevant transcription factor p53 in lung cancer cells induces SIAH2, depletes TYK2, and abrogates the tyrosine phosphorylation of STAT1 and STAT3. This mechanism appears to be different from the inhibition of phosphorylated JAKs through the suppressor of cytokine signaling (SOCS) proteins. Our study may help to identify molecular mechanisms affecting lung carcinogenesis and potential therapeutic targets.
PMCID: PMC4102802  PMID: 24833526
lung cancer; SIAH2; STAT3; TYK2; UBCH8
5.  Frequent and Focal FGFR1 Amplification Associates With Therapeutically Tractable FGFR1 Dependency in Squamous-cell Lung Cancer 
Science translational medicine  2010;2(62):62ra93.
Lung cancer remains one of the leading causes for cancer-related death in developed countries. In lung adenocarcinomas, EGFR mutations and EML4-ALK fusions are associated with response to EGFR and ALK inhibition. By contrast, therapeutically exploitable genetic alterations have been lacking in squamous-cell lung cancer. We conducted a systematic search for alterations that are therapeutically amenable and performed high-resolution gene-copy number analyses in a set of 232 lung cancer specimens. We identified frequent and focal FGFR1 amplification in squamous-cell lung cancer (n=155), but not in other lung cancer subtypes, and confirmed its presence in an independent cohort of squamous-cell lung cancer samples employing FISH (22% of cases). Using cell-based screening with the FGFR inhibitor (PD173074) in a large (n=83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth (p=0.0002) and induced apoptosis (p=0.008) specifically in those lung cancer cells carrying amplified FGFR1. We validated the dependency on FGFR1 of FGFR1-amplified cell lines by knockdown of FGFR1 and by ectopic expression of a resistance allele of FGFR1 (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Focal FGFR1 amplification is common in squamous-cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.
doi:10.1126/scitranslmed.3001451
PMCID: PMC3990281  PMID: 21160078
6.  Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer 
Oncogene  2009;29(10):1421-1430.
DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover novel such alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adaptor protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to EGF-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contributes to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlighting a role of adapter proteins as primary genetic drivers of tumorigenesis.
doi:10.1038/onc.2009.437
PMCID: PMC3320568  PMID: 19966867
CRKL; lung cancer; DNA amplification; genomic profiling; adapter protein
7.  Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer 
Cancer discovery  2011;1(1):78-89.
While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials.
doi:10.1158/2159-8274.CD-11-0005
PMCID: PMC3274752  PMID: 22328973
Squamous cell lung cancer; DDR2; dasatinib; tyrosine kinase inhibitors; lung cancer genomics
8.  Gender and ploidy in cancer survival 
Cellular Oncology (Dordrecht)  2011;34(3):199-208.
Background
Females carry a better prognosis than men for many cancer types. We hypothesized that chromosomal changes, in particular numerical alterations of the sex chromosomes or the presence of near-triploidy may contribute to these gender differences.
Methods
To characterize the influence of gender a literature search was performed for survival data of 27 tumor types. All entities were categorized by the strength of evidence for differences in survival between females and males. To test our hypothesis the Mitelman database of chromosomal alterations was evaluated for the major tumor types occurring in both women and men. Numerical gonosome alterations were documented and mean chromosome numbers were converted into histograms to provide insight into the ploidy level of 37 cancer types.
Results
In general, a survival advantage of women could be shown for most, but not all cancer types. In addition, 36.859 karyograms were analyzed. Numerical gonosome alterations were more frequent in males than females indicating a potential link with gender differences in survival. Neartriploidy was a common phenomenon in many cancer types suggesting that it represents a metastable condition of the cancer genome. It was not related to gender differences in survival. However, the extent of triploidy and aneuploidy was associated with poor prognosis in carcinomas. There was no single case in the Mitelman database with normal chromosome number (n = 46) that did not carry at least one structural or numerical aberration.
Conclusions
Our study highlights the importance of chromosomal changes in tumor formation and progression. In addition, it suggests potential associations with gender specific differences in survival.
Electronic supplementary material
The online version of this article (doi:10.1007/s13402-011-0013-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s13402-011-0013-0
PMCID: PMC3149121  PMID: 21424817
Gender; Aneuploidy; Chromosomal chaos; Prognosis
9.  Immunoprofiles of 11 Biomarkers Using Tissue Microarrays Identify Prognostic Subgroups in Colorectal Cancer1 
Neoplasia (New York, N.Y.)  2005;7(8):741-747.
Abstract
BACKGROUND AND AIMS: Genomewide expression profiling has identified a number of genes expressed at higher levels in colorectal cancer (CRC) than in normal tissues. Our objectives in this study were: 1) to test whether genes were also distinct on the protein level; 2) to evaluate these biomarkers in a series of well-characterized CRCs; and 3) to apply hierarchical cluster analysis to the immunohistochemical data. METHODS: Tissue microarrays (TMAs) comprising 351 CRC specimens from 270 patients were constructed to evaluate the genes Adam10, CyclinD1, AnnexinII, NFKB, Casein-kinase-2-beta (CK2B), YB-1, P32, Rad51, c-fos, IGFBP4, and Connexin26 (Cx26). In total, 3,797 samples were analyzed. RESULTS: Unsupervised hierarchical clustering discovered subgroups of CRC that differed by tumor stage and survival. Kaplan-Meier analysis showed that reduced Cx26 expression was significantly associated with shorter patient survival and higher tumor grade (G1/G2 vs G3, P = .02), and Adam10 expression with a higher tumor stage (pT1/2 vs pT3/4, P = .04). CONCLUSIONS: Our study highlights the potential of TMAs for a higher-dimensional analysis by evaluating serial sections of the same tissue core (three-dimensional TMA analysis). In addition, it endorses the use of immunohistochemistry supplemented by hierarchical clustering for the identification of tumor subgroups with diagnostic and prognostic signatures.
PMCID: PMC1501883  PMID: 16207476
Connexin26 (Cx26); Adam10; colorectal cancer (CRC); hierarchical clustering; tissue microarray (TMA)
10.  Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma 
BMC Cancer  2005;5:77.
Background
Chromosomal Comparative Genomic Hybridization (CGH) has been applied to all stages of cervical carcinoma progression, defining a specific pattern of chromosomal imbalances in this tumor. However, given its limited spatial resolution, chromosomal CGH has offered only general information regarding the possible genetic targets of DNA copy number changes.
Methods
In order to further define specific DNA copy number changes in cervical cancer, we analyzed 20 cervical samples (3 pre-malignant lesions, 10 invasive tumors, and 7 cell lines), using the GenoSensor microarray CGH system to define particular genetic targets that suffer copy number changes.
Results
The most common DNA gains detected by array CGH in the invasive samples were located at the RBP1-RBP2 (3q21-q22) genes, the sub-telomeric clone C84C11/T3 (5ptel), D5S23 (5p15.2) and the DAB2 gene (5p13) in 58.8% of the samples. The most common losses were found at the FHIT gene (3p14.2) in 47% of the samples, followed by deletions at D8S504 (8p23.3), CTDP1-SHGC- 145820 (18qtel), KIT (4q11-q12), D1S427-FAF1 (1p32.3), D9S325 (9qtel), EIF4E (eukaryotic translation initiation factor 4E, 4q24), RB1 (13q14), and DXS7132 (Xq12) present in 5/17 (29.4%) of the samples.
Conclusion
Our results confirm the presence of a specific pattern of chromosomal imbalances in cervical carcinoma and define specific targets that are suffering DNA copy number changes in this neoplasm.
doi:10.1186/1471-2407-5-77
PMCID: PMC1186020  PMID: 16004614
11.  Chromosomal Alterations during Lymphatic and Liver Metastasis Formation of Colorectal Cancer1 
Neoplasia (New York, N.Y.)  2004;6(1):23-28.
Abstract
Comparative genomic hybridization (CGH) was used to screen colorectal carcinomas for chromosomal aberrations that are associated with metastatic phenotype. In total, 63 tumor specimens from 40 patients were investigated, comprising 30 primary tumors, 22 systemic metastases (12 liver, 6 brain, and 4 abdominal wall metastases) and 11 lymph node tumors. Using statistical analysis and histograms to evaluate the chromosomal imbalances, overrepresentations were detected most frequently at 20q11.2–20q13.2, 7q11.1–7q12, 13q11.2–13q14, 16p12, 19p13, 9q34, and 19q13.1–19q13.2. Deletions were prominent at 18q12–18q23, 4q27–4q28, 4p14, 5q21, 1p21–1p22, 21q21, 6q16–6q21, 3p12, 8p22–8p23, 9p21, 11q22, and 14q13–14q21. Hematogenous metastases showed more alterations than lymph node tumors, particularly more deletions at 1p, 3, 4, 5q, 10q, 14, and 21q21 and gains at 1q, 7p, 12qter, 13, 16, and 22q. Comparing liver metastases with their corresponding primary tumors, particularly deletions at 2q, 5q, 8p, 9p, 10q, and 21q21 and gains at 1q, 11, 12qter, 17q12–q21, 19, and 22q were more often observed. The analysis suggested that the different pathways of tumor dissemination are reflected by a nonrandom accumulation of chromosomal alterations with specific changes being responsible for the different characteristics of the metastatic phenotype.
PMCID: PMC1508628  PMID: 15068668
CGH; colorectal cancer; metastasis; lymph node metastases; liver metastases

Results 1-11 (11)