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1.  A global microRNA screen identifies regulators of the ErbB receptor signaling network 
Background
The growth factor heregulin (HRG) potently stimulates epithelial cell survival and proliferation through the binding of its cognate receptor ErbB3 (also known as HER3). ErbB3-dependent signal transmission relies on the dimerization partner ErbB2, a receptor tyrosine kinase that is frequently overexpressed and/or amplified in breast cancer cells. Substantial evidence suggests that deregulated ErbB3 expression also contributes to the transformed phenotype of breast cancer cells.
Results
By genome-wide screening, we identify 43 microRNAs (miRNAs) that specifically impact HRG-induced activation of the PI3K-Akt pathway. Bioinformatic analysis combined with experimental validation reveals a highly connected molecular miRNA-gene interaction network particularly for the negative screen hits. For selected miRNAs, namely miR-149, miR-148b, miR-326, and miR-520a-3p, we demonstrate the simultaneous downregulation of the ErbB3 receptor and multiple downstream signaling molecules, explaining their efficient dampening of HRG responses and ascribing to these miRNAs potential context-dependent tumor suppressive functions.
Conclusions
Given the contribution of HRG signaling and the PI3K-Akt pathway in particular to tumorigenesis, this study not only provides mechanistic insight into the function of miRNAs but also has implications for future clinical applications.
Electronic supplementary material
The online version of this article (doi:10.1186/s12964-015-0084-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12964-015-0084-z
PMCID: PMC4314810  PMID: 25630670
MicroRNA/miRNA; ErbB3/HER3; Heregulin; PI3K-Akt pathway; Breast cancer
2.  Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone 
Development (Cambridge, England)  2014;141(24):4740-4750.
Wnt/β-catenin and hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. Here, we use the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. We find that Wnt/β-catenin signaling acts upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. We determine the target gene sets of the Wnt/β-catenin and Hh pathways and find that the growth zone signaling center activates a much greater number of genes and that the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist. Furthermore, the Wnt pathway is required for hindgut development and we identify Tc-senseless as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function.
doi:10.1242/dev.112797
PMCID: PMC4299277  PMID: 25395458
Head; Growth zone; Segment addition zone; Wnt; Hedgehog; Gut; Senseless
3.  Inactivation of Patched1 induces Pdgfrα-positive Kit-negative GIST-like tumors in mice 
Gastroenterology  2012;144(1):134-144.e6.
Background and aims
A fraction of gastrointestinal stromal tumors (GIST) overexpress PDGFRA rather than KIT. Presently it is unknown if this reflects a complementary oncogenic potential of PDGFRA and KIT pathways, or heterogeneity in the cellular origin of GIST. Similarly unknown is the significance of activated Hedgehog (HH)/PATCHED1 (PTCH) signaling found in many GIST.
Methods
Mouse Ptch was conditionally inactivated using a Cre recombinase driven by the lysozyme M (LysM) promoter. Lineage tracing was done using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, Western blot and qRT-PCR. Cellular transformation was assessed by clonogenic assay.
Results
Inactivation of Ptch in LysM-expressing cells resulted in imatinib-responsive tumors of GIST-like localization and histology. In addition to activation of Hh signaling, the tumors showed overexpression and activation of Pdgfrα, but not of Kit. Lineage tracing revealed that LysM-expressing intestinal cells were Kit-negative. These cells were juxtapposed with Kit-positive interstitial cells of Cajal (ICC) and sometimes co-expressed Pdgfrα. In contrast to KIT, PDGFRA cooperated with HH signaling in cellular transformation.
Conclusions
Mutations in Ptch result in formation of Pdgfrα-positive Kit-negative GIST-like tumors in mice. These tumors may develop due to cooperativity between Hh and Pdgfrα pathways from Kit-negative cells in the intestine.
doi:10.1053/j.gastro.2012.09.061
PMCID: PMC4231777  PMID: 23041331
GIST; mouse model; Hedgehog and Pdgfrα signaling
4.  The regulatory toll-like receptor 4 genetic polymorphism rs11536889 is associated with renal, coagulation and hepatic organ failure in sepsis patients 
Background
Toll-like receptor 4 (TLR4), a lipopolysaccharide (LPS) receptor complex signal-transducing molecule, plays a crucial role in sensing LPS from gram-negative bacteria. TLR4 signaling pathway activation by LPS plays a major role in sepsis pathogenesis. A single nucleotide polymorphism, rs11536889, in the 3’-untranslated region of the TLR4 gene is thought to affect TLR4 translation. This study aimed to investigate whether organ failure in sepsis patients is related to the TLR4 rs11536889 genotype.
Methods
Adult Caucasian patients with sepsis from the intensive care unit of a university medical center were followed up for 90 days, and organ failure was recorded as the primary outcome variable. Blood samples were collected at enrollment for TLR4 rs11536889 genotyping. Sepsis-related organ failure assessment (SOFA) scores were quantified at sepsis onset and throughout the observational period to monitor organ failure.
Results
A total of 210 critically ill patients with sepsis were enrolled into this study. Wild-type GG was compared to GC/CC. During their stay in the intensive care unit, GG patients presented significantly higher SOFA scores than did C allele carriers (7.9 ± 4.5 and 6.8 ± 4.2, respectively; p = 0.0005). Analysis of organ-specific SOFA sub-scores revealed significant differences in three organ systems: renal, coagulation and hepatic (p = 0.0005, p = 0.0245 and p < 0.0001, respectively). Additionally, the rs11536889 polymorphism was associated with a higher incidence of gram-negative infections.
Conclusions
These results offer the first evidence that TLR4 rs11536889 is a useful marker of organ failure in patients with sepsis.
doi:10.1186/1479-5876-12-177
PMCID: PMC4085654  PMID: 24950711
Toll-like receptor 4; Single-nucleotide polymorphism (SNP); Intensive care unit; Organ failure marker; SOFA scores
5.  Dynamics of mRNA and polysomal abundance in early 3T3-L1 adipogenesis 
BMC Genomics  2014;15(1):381.
Background
Adipogenesis is a complex process, in which immature pre-adipocytes change morphology, micro-anatomy and physiology to become mature adipocytes. These store and accumulate fat and release diverse hormones. Massive changes in protein content and protein composition of the transforming cell take place within a short time-frame.
In a previous study we analyzed changes in the abundance of free and polysomal, i.e. ribosome bound, RNAs in the first hours of adipogenesis in the murine cell line 3T3-L1. Here we analyze changes of mRNA levels and their potential contribution to the changing protein pool by determination of mRNA levels and ribosome binding to mRNAs in 3T3-L1 cells stimulated for adipogenesis. We grouped mRNA species into categories with respect to up- or down-regulated transcription and translation and analyzed the groups regarding specific functionalities based on Gene Ontology (GO).
Results
A shift towards up-regulation of gene expression in early adipogenesis was detected. Genes up-regulated at the transcriptional (TC:up) and translational (TL:up) level (TC:up/TL:up) are very likely involved in control and logistics of translation. Many of them are known to contain a TOP motif. In the TC:up/TL:unchanged group we detected most of the metal binding proteins and metal transporters. In the TC:unchanged/TL:up group several factors of the olfactory receptor family were identified, while in TC:unchanged/TL:down methylation and repair genes are represented. In the TC:down/TL:up group we detected many signaling factors. The TC:down/TL:unchanged group mainly consists of regulatory factors.
Conclusions
Within the first hours of adipogenesis, changes in transcriptional and translational regulation take place. Notably, genes with a specific biological or molecular function tend to cluster in groups according to their transcriptional and translational regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-381) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-381
PMCID: PMC4039748  PMID: 24886538
Adipogenesis; Transcription; Translation; 3T3-L1 pre-adipocytes; Gene ontology; Gene enrichment; Differential expression; TOP motif
6.  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.
doi:10.1002/gcc.20811
PMCID: PMC3535184  PMID: 20725992
7.  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.
doi:10.1093/carcin/bgr222
PMCID: PMC3254167  PMID: 21983179
8.  Extrinsic and intrinsic regulation of DOR/TP53INP2 expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1743-7075-9-86
PMCID: PMC3497704  PMID: 22995226
DOR/TP53INP2; High fat diet; Genetically induced obesity; Fat tissue; Muscle tissue
9.  Cohesin is required for expression of the estrogen receptor-alpha (ESR1) gene 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1756-8935-5-13
PMCID: PMC3488477  PMID: 22913342
Estrogen receptor; Cohesin; Mediator; Chromatin
10.  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 CME.TheOncologist.com
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.
doi:10.1634/theoncologist.2010-0414
PMCID: PMC3228181  PMID: 21558132
Rectal cancer; Gender effect; Radiochemotherapy; Toxicity; Body mass index
11.  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: http://www.biomedcentral.com/1471-2164/13/160
doi:10.1186/1741-7015-10-43
PMCID: PMC3366885  PMID: 22548923
HNSCC; EGFR; cetuximab; drug resistance; matrix factorization; GSEA; pathway signature
12.  Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2199-13-9
PMCID: PMC3347988  PMID: 22436005
13.  A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets 
Summary/Abstract
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.
doi:10.1002/ijc.25453
PMCID: PMC3008507  PMID: 20473941
Colorectal cancer; RNAi; functional genomics; systems biology; therapeutic targets
14.  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.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1476-4598-11-1
PMCID: PMC3281783  PMID: 22216762
CASP8AP2; FLASH; RNAi screening; RNAi analysis; siRNA; replication-dependent histone transcripts
15.  Thymidylate Synthase as a Prognostic Biomarker for Locally Advanced Rectal Cancer after multimodal Treatment 
Annals of Surgical Oncology  2011;18(9):2442-2452.
Purpose
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1245/s10434-011-1608-4
PMCID: PMC3162628  PMID: 21347782
16.  Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification 
Proteome Science  2010;8:36.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1477-5956-8-36
PMCID: PMC2908584  PMID: 20569466
17.  Preoperative Chemoradiotherapy Does Not Necessarily Reduce Lymph Node Retrieval in Rectal Cancer Specimens—Results from a Prospective Evaluation with Extensive Pathological Work-up 
Purpose
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1007/s11605-009-1057-6
PMCID: PMC2793396  PMID: 19830503
Locally advanced rectal cancer; Preoperative chemoradiotherapy; Total mesorectal excision; Pathologic diagnostics
18.  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
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/gb-2009-10-10-r104
PMCID: PMC2784319  PMID: 19799774
19.  Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1752-0509-3-1
PMCID: PMC2652436  PMID: 19118495
20.  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.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/gb-2008-9-10-r150
PMCID: PMC2760877  PMID: 18851746
21.  Integrative analysis of RUNX1 downstream pathways and target genes 
BMC Genomics  2008;9:363.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2164-9-363
PMCID: PMC2529319  PMID: 18671852
22.  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.
Contact: h.froehlich@dkfz-heidelberg.de
doi:10.1093/bioinformatics/btm634
PMCID: PMC2579711  PMID: 18227117
23.  Extending pathways based on gene lists using InterPro domain signatures 
BMC Bioinformatics  2008;9:3.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2105-9-3
PMCID: PMC2245903  PMID: 18177498
24.  Large scale statistical inference of signaling pathways from RNAi and microarray data 
BMC Bioinformatics  2007;8:386.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2105-8-386
PMCID: PMC2241646  PMID: 17937790

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