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1.  Mutated KRAS Results in Overexpression of DUSP4, a MAP-Kinase Phosphatase, and SMYD3, a Histone Methyltransferase, in Rectal Carcinomas 
Genes, chromosomes & cancer  2010;49(11):1024-1034.
Mutations of the KRAS oncogene are predictive for resistance to treatment with antibodies against the epithelial growth factor receptor in patients with colorectal cancer. Overcoming this therapeutic dilemma could potentially be achieved by the introduction of drugs that inhibit signaling pathways that are activated by KRAS mutations. To identify comprehensively such signaling pathways we profiled pretreatment biopsies and normal mucosa from 65 patients with locally advanced rectal cancer - 30 of which carried mutated KRAS - using global gene expression microarrays. By comparing all tumor tissues exclusively to matched normal mucosa, we could improve assay sensitivity, and identified a total of 22,297 features that were differentially expressed (adjusted P-value <0.05) between normal mucosa and cancer, including several novel potential rectal cancer genes. We then used this comprehensive description of the rectal cancer transcriptome as the baseline for identifying KRAS-dependent alterations. The presence of activating KRAS mutations is significantly correlated to an upregulation of 13 genes (adjusted P-value <0.05), among them DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase. Inhibition of the expression of both genes has previously been shown using the MEK1-inhibitor PD98059 and the antibacterial compound Novobiocin, respectively. These findings suggest a potential approach to overcome resistance to treatment with antibodies against the epithelial growth factor receptor in patients with KRAS-mutant rectal carcinomas.
PMCID: PMC3535184  PMID: 20725992
2.  Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy 
Carcinogenesis  2011;32(12):1824-1831.
A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G2/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.
PMCID: PMC3254167  PMID: 21983179
3.  Extrinsic and intrinsic regulation of DOR/TP53INP2 expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues 
DOR/TP53INP2 acts both at the chromosomal level as a nuclear co-factor e.g. for the thyroid hormone receptor and at the extrachromosomal level as an organizing factor of the autophagosome. In a previous study, DOR was shown to be down-regulated in skeletal muscle of obese diabetic Zucker fa/fa rats.
To identify sites of differential DOR expression in metabolically active tissues, we measured differences in DOR expression in white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (SM) and heart muscle (HM) by qPCR. To assess whether DOR expression is influenced in the short term by nutritional factors, NMRI mice were fed different fat rich diets (fat diet, FD: 18% or high fat diet, HFD: 80% fat) for one week and DOR expression was compared to NMRI mice fed a control diet (normal diet, ND: 3.3% fat). Additionally, DOR expression was measured in young (45 days old) and adult (100 days old) genetically obese (DU6/DU6i) mice and compared to control (DUKs/DUKsi) animals.
ANOVA results demonstrate a significant influence of diet, tissue type and sex on DOR expression in adipose and muscle tissues of FD and HFD mice. In SM, DOR expression was higher in HFD than in FD male mice. In WAT, DOR expression was increased compared to BAT in male FD and HFD mice. In contrast, expression levels in female mice were higher in BAT for both dietary conditions.
DOR expression levels in all tissues of 100 days old genetically obese animals were mainly influenced by sex. In HM, DOR expression was higher in male than female animals.
DOR expression varies under the influence of dietary fat content, tissue type and sex. We identified target tissues for further studies to analyze the specific function of DOR in obesity. DOR might be part of a defense mechanism against fat storage in high fat diets or obesity.
PMCID: PMC3497704  PMID: 22995226
DOR/TP53INP2; High fat diet; Genetically induced obesity; Fat tissue; Muscle tissue
4.  Cohesin is required for expression of the estrogen receptor-alpha (ESR1) gene 
In conjunction with posttranslational chromatin modifications, proper arrangement of higher order chromatin structure appears to be important for controlling transcription in the nucleus. Recent genome-wide studies have shown that the Estrogen Receptor-alpha (ERα), encoded by the ESR1 gene, nucleates tissue-specific long-range chromosomal interactions in collaboration with the cohesin complex. Furthermore, the Mediator complex not only regulates ERα activity, but also interacts with the cohesin complex to facilitate long-range chromosomal interactions. However, whether the cohesin and Mediator complexes function together to contribute to estrogen-regulated gene transcription remains unknown.
In this study we show that depletion of the cohesin subunit SMC3 or the Mediator subunit MED12 significantly impairs the ERα-regulated transcriptome. Surprisingly, SMC3 depletion appears to elicit this effect indirectly by rapidly decreasing ESR1 transcription and ERα protein levels. Moreover, we provide evidence that both SMC3 and MED12 colocalize on the ESR1 gene and are mutually required for their own occupancy as well as for RNAPII occupancy across the ESR1 gene. Finally, we show that extended proteasome inhibition decreases the mRNA expression of cohesin subunits which accompanies a decrease in ESR1 mRNA and ERα protein levels as well as estrogen-regulated transcription.
These results identify the ESR1 gene as a cohesin/Mediator-dependent gene and indicate that this regulation may potentially be exploited for the treatment of estrogen-dependent breast cancer.
PMCID: PMC3488477  PMID: 22913342
Estrogen receptor; Cohesin; Mediator; Chromatin
5.  Gender-Specific Acute Organ Toxicity during Intensified Preoperative Radiochemotherapy for Rectal Cancer 
The Oncologist  2011;16(5):621-631.
Patients with locally advanced rectal cancer (cUICC stages II/III) are typically treated with preoperative 5-fluorouracil–based (5-FU–based) radiochemotherapy. However, trials are currently being conducted to improve the complete remission rates and the systemic control by combining 5-FU with oxaliplatin in order to identify the subgroups of rectal cancer patients at risk for high-grade toxicity. The results indicate that there are basic clinical parameters, such as gender and body mass index, that may be potential markers for generating individual risk profiles of radiochemotherapy-induced toxicity.
Learning Objectives
After completing this course, the reader will be able to: Describe present strategies of treatment of locally advanced rectal cancer and ongoing clinical trials, including neoadjuvant radiochemotherapy with 50.4 Gy and concomitant 5-FU +/− oxaliplatin.Define the basic clinical parameters, with special emphasis on gender and BMI, correlating with radiochemotherapy-associated side effects in rectal cancer patients and differences in severity of toxicity.
This article is available for continuing medical education credit at
Patients with locally advanced rectal cancer (cUICC stages II/III) are typically treated with preoperative 5-fluorouracil–based (5-FU–based) radiochemotherapy (RCT). However, trials are currently being conducted to improve the complete remission rates and the systemic control by combining 5-FU with oxaliplatin. The primary objective was to identify the subgroups of rectal cancer patients who were at risk for high-grade toxicity.
All 196 patients who were included in the present study were treated with 50.4 Gy and chemotherapy that included either 5-FU (n = 115) or 5-FU+oxaliplatin (n = 81). The preoperative RCT was followed by a total mesorectal excision and adjuvant chemotherapy. Acute toxicity was monitored weekly and a toxicity grade ≥3 (Common Toxicity Criteria) for a skin reaction, cystitis, proctitis, or enteritis was defined as high-grade acute organ toxicity. After RCT with 5-FU+oxaliplatin, complete tumor remission was achieved in 13.6% of the patients and in 11.3% after RCT with 5-FU alone.
Complete irradiation dosages of 50.4 Gy were given to 99% (5-FU) and 95% (5-FU+oxaliplatin) of the patients. Concomitant chemotherapy was fully administered in 95% of the patients treated with 5-FU compared with the 84% of patients treated with 5-FU+oxaliplatin.
A significantly higher proportion of acute organ toxicity was found in the patients who were treated with 5-FU+oxaliplatin compared with those who were treated with 5-FU. Additionally, women with a low body mass index were at the highest risk for acute organ toxicity.
These results suggest that there are basic clinical parameters, such as gender and body mass index, that may be potential markers for generating individual risk profiles of RCT-induced toxicity.
PMCID: PMC3228181  PMID: 21558132
Rectal cancer; Gender effect; Radiochemotherapy; Toxicity; Body mass index
6.  A new analysis approach of epidermal growth factor receptor pathway activation patterns provides insights into cetuximab resistance mechanisms in head and neck cancer 
BMC Medicine  2012;10:43.
The pathways downstream of the epidermal growth factor receptor (EGFR) have often been implicated to play crucial roles in the development and progression of various cancer types. Different authors have proposed models in cell lines in which they study the modes of pathway activities after perturbation experiments. It is prudent to believe that a better understanding of these pathway activation patterns might lead to novel treatment concepts for cancer patients or at least allow a better stratification of patient collectives into different risk groups or into groups that might respond to different treatments. Traditionally, such analyses focused on the individual players of the pathways. More recently in the field of systems biology, a plethora of approaches that take a more holistic view on the signaling pathways and their downstream transcriptional targets has been developed. Fertig et al. have recently developed a new method to identify patterns and biological process activity from transcriptomics data, and they demonstrate the utility of this methodology to analyze gene expression activity downstream of the EGFR in head and neck squamous cell carcinoma to study cetuximab resistance. Please see related article:
PMCID: PMC3366885  PMID: 22548923
HNSCC; EGFR; cetuximab; drug resistance; matrix factorization; GSEA; pathway signature
7.  Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells 
Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting.
We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes.
We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.
PMCID: PMC3347988  PMID: 22436005
8.  A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets 
Genes that are highly overexpressed in tumor cells can be required for tumor cell survival, and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi-mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRC) and matched normal mucosa. In order to identify credible models for in-depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or shRNAs, i.e., HMGA1, TACSTD2, RRM2, RPS2, and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA-mediated silencing. Exploration of these RNAi-specific gene expression signatures allowed the identification of the functional space in which the five genes operate, and showed enrichment for cancer specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas we could recapitulate these defined RNAi signatures, therefore establishing the biologically relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1, and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.
PMCID: PMC3008507  PMID: 20473941
Colorectal cancer; RNAi; functional genomics; systems biology; therapeutic targets
9.  Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells 
Molecular Cancer  2012;11:1.
Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.
A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.
We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.
PMCID: PMC3281783  PMID: 22216762
CASP8AP2; FLASH; RNAi screening; RNAi analysis; siRNA; replication-dependent histone transcripts
10.  Thymidylate Synthase as a Prognostic Biomarker for Locally Advanced Rectal Cancer after multimodal Treatment 
Annals of Surgical Oncology  2011;18(9):2442-2452.
For years, 5-fluorouracil (5-FU) has been the backbone of radiochemotherapy (RCT) of locally advanced rectal cancer. Its main target, thymidylate synthase (TS), is speculated to be an important biomarker for response prediction and long-term prognosis. In this study, we analyzed TS expression in the rectal cancer tissue of 208 patients to evaluate its predictive/prognostic potential.
All patients included were diagnosed with locally advanced adenocarcinoma of the rectum (UICC II and III) and were treated within randomized clinical trials of the German Rectal Cancer Study Group. Preoperative RCT (50.4 Gy and concomitant either 5-FU or 5-FU and oxaliplatin) was administered in 167 patients followed by surgical resection with total mesorectal excision (TME). Another 41 patients received postoperative RCT. TS levels and further clinicopathological parameters were assessed in univariate and multivariate analyses. Additionally, a TS gene polymorphism was analyzed with respect to the intratumoral protein levels.
Low TS expression in pretreatment biopsies correlated with impaired patient survival (p = 0.015). Analysis of a 28-bp repeat revealed a correlation between the *3/*3 genotype and high TS expression in pretherapeutic biopsies. In this study, a correlation of TS expression and grade of RCT-induced tumor regression was not found. Histopathological examination confirmed a complete tumor remission in 16 patients (9.6%).
Analyses of the resection specimen indicated an unfavorable prognosis for patients with low intratumoral TS expression in case of detected lymph node metastases (p = 0.04).
TS can serve as a prognostic biomarker indicating an unfavorable prognosis for patients with low TS expression.
Electronic supplementary material
The online version of this article (doi:10.1245/s10434-011-1608-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3162628  PMID: 21347782
11.  Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification 
Proteome Science  2010;8:36.
Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level.
A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins.
Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range.
PMCID: PMC2908584  PMID: 20569466
12.  Preoperative Chemoradiotherapy Does Not Necessarily Reduce Lymph Node Retrieval in Rectal Cancer Specimens—Results from a Prospective Evaluation with Extensive Pathological Work-up 
Preoperative chemoradiotherapy (CRT) is supposed not only to reduce lymph node metastases but also lymph node recovery in rectal cancer specimens. The objective of this prospective study was to determine the effects of chemoradiation on mesorectal lymph node retrieval under terms of a meticulous histopathological evaluation.
Specimens from 64 consecutive patients with stage II/III rectal cancer receiving preoperative 5-FU-based CRT were investigated. All patients were treated within the German Rectal Cancer Trial CAO/ARO/AIO-04. After surgery (including quality assessed total mesorectal excision), extensive pathological diagnostics was performed with embedding and microscopic evaluation of the whole mesorectal soft tissue compartment.
A total number of 2,021 lymph nodes were recovered (31.6 per specimen) within pathological work-up. There was no significant correlation between the number of retrieved nodes and patient- as well as tumor-dependent parameters. Lymph node size constantly amounted for less than 0.5 cm. Twenty patients (31.3%) had persistent nodal metastases. A considerable incidence of residual micrometastatic involvement in lymph nodes <0.3 cm (in 9.4% of all patients) was detected by extensive pathologic work-up.
Reliable nodal staging with high numbers of detected nodes was feasible after neoadjuvant CRT. Micrometastases frequently occur in small lymph nodes detected by microscopic evaluation.
PMCID: PMC2793396  PMID: 19830503
Locally advanced rectal cancer; Preoperative chemoradiotherapy; Total mesorectal excision; Pathologic diagnostics
13.  Molecular networks involved in mouse cerebral corticogenesis and spatio-temporal regulation of Sox4 and Sox11 novel antisense transcripts revealed by transcriptome profiling 
Genome Biology  2009;10(10):R104.
SAGE analysis reveals spatiotemporally regulated transcripts and overlapping sense and antisense transcripts that are important for mouse cerebral cortex development
Development of the cerebral cortex requires highly specific spatio-temporal regulation of gene expression. It is proposed that transcriptome profiling of the cerebral cortex at various developmental time points or regions will reveal candidate genes and associated molecular pathways involved in cerebral corticogenesis.
Serial analysis of gene expression (SAGE) libraries were constructed from C57BL/6 mouse cerebral cortices of age embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4 to 6 months. Hierarchical clustering analysis of 561 differentially expressed transcripts showed regionalized, stage-specific and co-regulated expression profiles. SAGE expression profiles of 70 differentially expressed transcripts were validated using quantitative RT-PCR assays. Ingenuity pathway analyses of validated differentially expressed transcripts demonstrated that these transcripts possess distinctive functional properties related to various stages of cerebral corticogenesis and human neurological disorders. Genomic clustering analysis of the differentially expressed transcripts identified two highly transcribed genomic loci, Sox4 and Sox11, during embryonic cerebral corticogenesis. These loci feature unusual overlapping sense and antisense transcripts with alternative polyadenylation sites and differential expression. The Sox4 and Sox11 antisense transcripts were highly expressed in the brain compared to other mouse organs and are differentially expressed in both the proliferating and differentiating neural stem/progenitor cells and P19 (embryonal carcinoma) cells.
We report validated gene expression profiles that have implications for understanding the associations between differentially expressed transcripts, novel targets and related disorders pertaining to cerebral corticogenesis. The study reports, for the first time, spatio-temporally regulated Sox4 and Sox11 antisense transcripts in the brain, neural stem/progenitor cells and P19 cells, suggesting they have an important role in cerebral corticogenesis and neuronal/glial cell differentiation.
PMCID: PMC2784319  PMID: 19799774
14.  Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance 
In breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer.
First, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab), demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation.
In this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not have potential for treatment of de novo trastuzumab resistant cells. Instead, c-MYC was identified as a novel potential target protein in breast cancer cells.
PMCID: PMC2652436  PMID: 19118495
15.  Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas 
Genome Biology  2008;9(10):R150.
Differences in MYCN/c-MYC target gene expression are associated with distinct neuroblastoma subtypes and clinical outcome.
Amplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes.
We defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis.
High MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression.
PMCID: PMC2760877  PMID: 18851746
16.  Integrative analysis of RUNX1 downstream pathways and target genes 
BMC Genomics  2008;9:363.
The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.
Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.
This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.
PMCID: PMC2529319  PMID: 18671852
17.  Estimating large-scale signaling networks through nested effect models with intervention effects from microarray data 
Bioinformatics  2008;24(22):2650-2656.
Motivation: Targeted interventions using RNA interference in combination with the measurement of secondary effects with DNA microarrays can be used to computationally reverse engineer features of upstream non-transcriptional signaling cascades based on the nested structure of effects.
Results: We extend previous work by Markowetz et al., who proposed a statistical framework to score different network hypotheses. Our extensions go in several directions: we show how prior assumptions on the network structure can be incorporated into the scoring scheme by defining appropriate prior distributions on the network structure as well as on hyperparameters. An approach called module networks is introduced to scale up the original approach, which is limited to around 5 genes, to infer large-scale networks of more than 30 genes. Instead of the data discretization step needed in the original framework, we propose the usage of a beta-uniform mixture distribution on the P-value profile, resulting from differential gene expression calculation, to quantify effects. Extensive simulations on artificial data and application of our module network approach to infer the signaling network between 13 genes in the ER-α pathway in human MCF-7 breast cancer cells show that our approach gives sensible results. Using a bootstrapping and a jackknife approach, this reconstruction is found to be statistically stable.
Availability: The proposed method is available within the Bioconductor R-package nem.
PMCID: PMC2579711  PMID: 18227117
18.  Extending pathways based on gene lists using InterPro domain signatures 
BMC Bioinformatics  2008;9:3.
High-throughput technologies like functional screens and gene expression analysis produce extended lists of candidate genes. Gene-Set Enrichment Analysis is a commonly used and well established technique to test for the statistically significant over-representation of particular pathways. A shortcoming of this method is however, that most genes that are investigated in the experiments have very sparse functional or pathway annotation and therefore cannot be the target of such an analysis. The approach presented here aims to assign lists of genes with limited annotation to previously described functional gene collections or pathways. This works by comparing InterPro domain signatures of the candidate gene lists with domain signatures of gene sets derived from known classifications, e.g. KEGG pathways.
In order to validate our approach, we designed a simulation study. Based on all pathways available in the KEGG database, we create test gene lists by randomly selecting pathway genes, removing these genes from the known pathways and adding variable amounts of noise in the form of genes not annotated to the pathway. We show that we can recover pathway memberships based on the simulated gene lists with high accuracy. We further demonstrate the applicability of our approach on a biological example.
Results based on simulation and data analysis show that domain based pathway enrichment analysis is a very sensitive method to test for enrichment of pathways in sparsely annotated lists of genes. An R based software package domainsignatures, to routinely perform this analysis on the results of high-throughput screening, is available via Bioconductor.
PMCID: PMC2245903  PMID: 18177498
19.  Large scale statistical inference of signaling pathways from RNAi and microarray data 
BMC Bioinformatics  2007;8:386.
The advent of RNA interference techniques enables the selective silencing of biologically interesting genes in an efficient way. In combination with DNA microarray technology this enables researchers to gain insights into signaling pathways by observing downstream effects of individual knock-downs on gene expression. These secondary effects can be used to computationally reverse engineer features of the upstream signaling pathway.
In this paper we address this challenging problem by extending previous work by Markowetz et al., who proposed a statistical framework to score networks hypotheses in a Bayesian manner. Our extensions go in three directions: First, we introduce a way to omit the data discretization step needed in the original framework via a calculation based on p-values instead. Second, we show how prior assumptions on the network structure can be incorporated into the scoring scheme using regularization techniques. Third and most important, we propose methods to scale up the original approach, which is limited to around 5 genes, to large scale networks.
Comparisons of these methods on artificial data are conducted. Our proposed module network is employed to infer the signaling network between 13 genes in the ER-α pathway in human MCF-7 breast cancer cells. Using a bootstrapping approach this reconstruction can be found with good statistical stability.
The code for the module network inference method is available in the latest version of the R-package nem, which can be obtained from the Bioconductor homepage.
PMCID: PMC2241646  PMID: 17937790

Results 1-19 (19)