Christine Sers

A genome-wide survey of RAS transformation targets.

An important aspect of multi-step tumorigenesis is the mutational activation of genes of the RAS family, particularly in sporadic cancers of the pancreas, colon, lung and myeloid system. RAS genes encode small GTP-binding proteins that affect gene expression in a global way by acting as major switches in signal transduction processes, coupling extracellular signals with transcription factors. Oncogenic forms of RAS are locked in their active state and transduce signals essential for transformation, angiogenesis, invasion and metastasis via downstream pathways involving the RAF/MEK/ERK cascade of cytoplasmic kinases, the small GTP-binding proteins RAC and RHO, phosphatidylinositol 3-kinase and others. We have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, to contrast differential gene expression profiles in immortalized, non-tumorigenic rat embryo fibroblasts and in HRAS- transformed cells. Sequence and expression analysis of more than 1,200 subtracted cDNA fragments revealed transcriptional stimulation or repression of 104 ESTs, 45 novel sequences and 244 known genes in HRAS- transformed cells compared with normal cells. Furthermore, we identified common and distinct targets in cells transformed by mutant HRAS, KRAS and NRAS, as well as 61 putative target genes controlled by the RAF/MEK/ERK pathway in reverted cells treated with the MEK-specific inhibitor PD 98059.

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling

Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady‐state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf‐1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post‐translational mechanism although variation of Erk levels occurs on a timescale of days.

KRAS Genotyping of Paraffin-Embedded Colorectal Cancer Tissue in Routine Diagnostics: Comparison of Methods and Impact of Histology

KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.

An Alu Element–Associated Hypermethylation Variant of the POMC Gene Is Associated with Childhood Obesity

The individual risk for common diseases not only depends on genetic but also on epigenetic polymorphisms. To assess the role of epigenetic variations in the individual risk for obesity, we have determined the methylation status of two CpG islands at the POMC locus in obese and normal-weight children. We found a hypermethylation variant targeting individual CpGs at the intron2–exon3 boundary of the POMC gene by bisulphite sequencing that was significantly associated with obesity. POMC exon3 hypermethylation interferes with binding of the transcription enhancer P300 and reduces expression of the POMC transcript. Since intron2 contains Alu elements that are known to influence methylation in their genomic vicinity, the exon3 methylation variant seems to result from an Alu element–triggered default state of methylation boundary definition. Exon3 hypermethylation in the POMC locus represents the first identified DNA methylation variant that is associated with the individual risk for obesity.

Personalized medicine and development of targeted therapies: The upcoming challenge for diagnostic molecular pathology. A review.

Due to continuous technical developments and new insights into the high complexity of many diseases, molecular pathology is a rapidly growing field gaining center stage in the clinical management of tumors as well as in the pharmaceutical development of new anti-cancer drugs. The application of novel compounds in clinical trials has revealed promising results; however, the current diagnostic procedures available for determining which patients will primarily benefit from rational tumor therapy are insufficient. To read a patient’s tissue as “deeply” as possible, in the future, gaining information on the morphology and on genetic, proteomic, and epigenetic alterations will be the upcoming task of surgical pathologists experienced in molecular diagnostics to provide the clinicians with information relevant for an individualized medicine. Among the different high-throughput technologies, DNA microarrays are now the first array approach close to enter routine diagnostics. Technically advanced and well-established microarray platforms can nowadays be evaluated by distinct bioinformatic tools capable of identifying both novel genes associated with disease development and clusters of genes predicting clinical outcome of an individual tumor. The automatic, highly parallel analysis of proteins and complex proteins lysates for early detection of cancers such as breast, prostate and ovary as proteomic patterns in the serum also appears at the horizon. In addition, an improved analysis of tumor samples via antibody or reverse-phase protein arrays is likely to provide the pathologist in the future with information about activated oncogenic signaling pathways and other cell functions, such as drug response or the potential to metastasize. While expression microarrays and proteomic analysis rely on relatively unstable material incompatible with paraffin-embedded tissue samples, an investigation of DNA methylation using specialized high-throughput platforms has revealed the potential of being used in future diagnostics. Each of these approaches on its own might not suffice to extract all information required for an efficient individualized diagnostics. Therefore, a “multiplex approach” combining the different biological levels DNA, RNA, and protein, may be necessary to functionally classify malignant tumors. This appears to become a major challenge for diagnostic pathologists.

A Helminth Immunomodulator Exploits Host Signaling Events to Regulate Cytokine Production in Macrophages

Parasitic worms alter their hosts immune system to diminish the inflammatory responses directed against them, using very efficient immunomodulating molecules. We have previously shown that the helminth immunomodulator cystatin (AvCystatin) profoundly reduces the progression of inflammatory diseases via modulation of macrophages. Here we elucidate the signaling events in macrophages triggered by AvCystatin. Labeled AvCystatin was predominantly taken up by macrophages and subsequently induced the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38. IL-10 expression induced by AvCystatin in macrophages was tyrosine kinase sensitive and dependent on activation of both MAP kinases, in clear contrast to expression of IL-12/23p40. In addition, phosphorylation of the transcription factors CREB and STAT3 was induced by AvCystatin and regulated by phospho-ERK. Chemical inhibition of phosphoinositide 3-kinase (PI3K) reduced AvCystatin-induced cytokine release; however, AKT, the downstream target of PI3K, was not activated following AvCystatin exposure. To characterize signaling elements involved in alteration of the macrophage phenotype we applied mathematical modeling. Experimental testing of the in silico generated hypotheses identified dual specificity phosphatase (DUSP) 1 and 2, as regulators in AvCystatin triggered macrophages in vitro and in vivo. In particular, DUSP1 was subsequently found to be responsible for regulation of ERK- and p38-phosphorylation and controlled the IL-10 expression in macrophages by AvCystatin. Thus, we show that AvCystatin exploits activation and deactivation pathways of MAP kinases to induce regulatory macrophages. This study provides insights into molecular mechanisms of macrophage manipulation by parasites and highlights the utility of mathematical modeling for the elucidation of regulatory circuits of immune cells.

Network quantification of EGFR signaling unveils potential for targeted combination therapy

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small‐molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model‐based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR‐dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF‐ as well as KRAS‐mutated tumor cells, which we confirmed using a xenograft model.

EGFR mutation detection in NSCLC—assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC

EGFR mutation testing in non-small cell lung cancer (NSCLC) is a novel and important molecular pathological diagnostic assay that is predictive of response to anti-epidermal growth factor receptor (EGFR) therapy. A comprehensive compilation of a large number of EGFR mutation analyses of the German Panel for Mutation Analyses in NSCLC demonstrates (a) a higher than previously reported mutation frequency outside the conventionally tested exons 19 and 21 and (b) an overall superiority of sequencing based assays over mutation-specific PCR. The implications for future diagnostic EGFR mutation testing are discussed.

HMGA2 gene is a promising target for ovarian cancer silencing therapy.

Ovarian cancer is one of the most lethal gynecological malignancies and the small success rate of routine therapeutic methods justifies efforts to develop new approaches. Evaluation of targets for effective inhibition of ovarian cancer cell growth should precipitate clinical application of gene silencing therapy. In our previous work, we showed upregulation of HMGA2 gene expression as a result of Ras‐induced rat ovarian surface epithelial cell transformation. This gene codes the HMGA2 protein, a member of the high‐mobility group AT‐hook (HMGA) family of nonhistone chromatin proteins. Genome‐wide studies revealed upregulation of the HMGA2 gene in human ovarian carcinomas. Herein we have evaluated over‐expression of the HMGA2 gene, relevant to ovarian cancer, in subsets of human specimens and cell lines by in situ RNA hybridization and RT‐PCR. Transient silencing of HMGA2 gene by means of siRNA inhibited proliferation of those ovarian cancer cells, which over‐express this gene initially. Growth suppression was mediated by cell‐cycle arrest. Stable silencing of highly expressed HMGA2 gene by shRNAi in A27/80, Ovcar‐3 and OAW‐42 ovarian cancer cell lines resulted in growth inhibition because of G1 arrest and increase of apoptosis as well. The tumor growth inhibition effect of HMGA2 silencing for Ovcar‐3 cells was validated in vivo. Our findings revealed that the HMGA2 gene represents a promising target for gene silencing therapy in ovarian cancer.

Transcriptional basis of KRAS oncogene-mediated cellular transformation in ovarian epithelial cells

To understand the relationship between oncogenic signaling and the reprogramming of gene expression, we performed transcriptional profiling in rat ovarian surface epithelial cells (ROSE), in which neoplastic transformation is driven by a mutated KRAS oncogene. We identified >200 genes whose expression was elevated or reduced following permanent KRAS expression. Deregulated KRAS-responsive genes encode transcriptional regulators, signaling effectors, proteases, extracellular matrix and adhesion proteins, transformation-suppressing proteins and negative growth regulators. Many of them have not been previously identified in cells expressing oncogenic RAS genes or in other well-studied models of oncogenic signaling. The number of critical genes related to the execution of anchorage-independent proliferation and epithelial–mesenchymal transition was narrowed down to 79 by selectively inhibiting the mitogen-activated protein kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K) pathways. Blocking MAPK/ERK-signaling caused reversion to the normal epithelial phenotype in conjunction with the reversal of deregulated target transcription to pretransformation levels. In addition, silencing of the overexpressed transcriptional regulator Fra-1 by RNA interference resulted in growth reduction, suggesting that this factor partially contributes to, but is not sufficient for the proliferative capacity of KRAS-transformed epithelial cells.