Identifikation epigenetisch regulierter Gene in Gliomen

In der vorliegenden Arbeit wurde die Beteiligung de novo methylierter Gene auf die Entstehung von Gliomen untersucht. Gliome sind die häufigsten intrakranialen Neoplasien beim Menschen. Mit einer Inzidenz von 7 Fällen pro 100.000 Einwohnern machen sie über 60% aller Hirntumore aus. Aufgrund ihrer Fähigkeit das umliegende gesunde Gewebe zu infiltrieren, stellt die neurochirurgische Entfernung dieser Tumoren keinen befriedigenden Therapieansatz dar. In den letzten Jahren konnten viele strukturell-genomische Aberrationen und größere chromosomale Bereiche die maßgeblich an der Tumorentstehung und Progession beteiligt sind, identifiziert werden. Dennoch sind die Identität und die Mechanismen der Regulation der Gene, die in Gliomen für diese Fähigkeit verantwortlich sind weitgehend ungeklärt. In der vorliegenden Arbeit wurde die de novo Methylierung der DNA niedriggradiger Tumoren mit Hilfe verschiedener Techniken untersucht. Um Kandidatengene zu identifizieren, wurde die DNA von niedriggradigen Tumoren zusammen mit Normalgewebe-DNA auf CpG-Insel Microarrays hybridisiert. Die Ergebnisse dieser Hybridisierungen führten zu einer Liste von Kandidatengenen, die in niedriggradigen Tumoren methyliert vorliegen. In der vorliegenden Arbeit wurden ausgewählte Kandidaten mit tumorbiologisch relevanter Funktion näher charakterisiert. Hierzu wurden die identifizierten Kandidatengene Bisulfit-sequenziert um die Methylierung quantitativ erfassen zu können. Für die sequenzierten Tumorgenome wurde mit Hilfe der kompetitiven RT-PCR überprüft, in wieweit die Transkription der Gene von der de novo Methylierung reguliert ist. Für alle untersuchten Gene wurde auf diese Weise eine Reduktion der Transkriptmenge im Tumor festgestellt. Durch die Kultivierung von primären Gliomzellen mit 5-Azacytidin wurde in der vorliegenden Arbeit gezeigt, dass eine Demethylierung des Genoms zur Wiederherstellung der ursprünglichen Transkriptionsmenge bei den untersuchten Genen führte. Um größere Tumorkollektive schnell und zuverlässig auf den Methylierungszustand von Kandidatengenen untersuchen zu können, wurde das bestehende Protokoll des Methylierungs-Target-Arrays (MTA) dahingehend modifiziert, dass die Signaldetektion statt über eine radioaktive Markierung der DNA durch den Einbau von digUTP stattfindet. Diese modifizierte Form des MTA ermöglicht eine leichtere Handhabung des Untersuchungskollektivs und eine relativ einfach durchzuführende Hybridisierung mit genspezifischen Sonden. Da viele Gene, die mit Hilfe der DMH identifiziert wurden, an der Zellmotilität beteiligt sind und darüber hinaus die Fähigkeit umgebendes Gewebe zu invadieren charakteristisch für Gliome ist, wurden aus frischen Tumorgewebestücken von Patienten mit einem Glioblastom WHO Grad IV Gliomzellen in Kultur genommen. Diese Zellen ermöglichten weitere in vitro Untersuchungen der Methylierung in frühen Passagen. Um nach charakteristischen Veränderungen der Methylierung, die mit der Fähigkeit zur Migration einhergehen zu suchen, wurde mit den kultivierten Gliomzellen Migrationsassays durchgeführt, die zur Identifikation weiterer Gene die mit dem Erwerb der Migrationsfähigkeit methyliert werden, führte. Die in dieser Arbeit gewonnenen Erkenntnisse führten zu einem tieferen Verständnis der Bedeutung von methylierungsabhängig regulierten Genen in der Entstehung astrozytärer Neoplasien. Ein Teil der Ergebnisse liefert darüber hinaus wichtige Informationen zum Verständnis der Beteiligung methylierter Gene beim Erwerb charakteristischer Eigenschaften von Gliomzellen, wie die ausgeprägte Fähigkeit gesundes Gewebe zu invadieren. Da die heute eingesetzten demethylierenden Chemotherapeutika aufgrund ihres Wirkmechansmus zu starken Nebenwirkungen führen, wurde in dieser Arbeit in Kooperation mit Dr. Frank Lyko (DKFZ, Heidelberg) ein neues, potentielles Chemotherapeutikum auf seine demethylierende Eigenschaften getestet. Zusammen mit den gewonnen Erkenntnissen über die Beteiligung von DNA-Hypermethylierung an der Tumorentstehung verspricht dieser Wirkstoff eine spezifischere Demethylierung von hypermethylierten Genen, als es heute zugelassene Medikamente vermögen

Cancer stem cells in solid tumors: elusive or illusive?

During the past years in vivo transplantation experiments and in vitro colony-forming assays indicated that tumors arise only from rare cells. These cells were shown to bear self-renewal capacities and the ability to recapitulate all cell types within an individual tumor. Due to their phenotypic resemblance to normal stem cells, the term "cancer stem cells" is used. However, some pieces of the puzzle are missing: (a) a stringent definition of cancer stem cells in solid tumors (b) specific markers that only target cells that meet the criteria for a cancer stem cell in a certain type of tumor. These missing parts started an ongoing debate about which is the best method to identify and characterize cancer stem cells, or even if their mere existence is just an artifact caused by the experimental procedures. Recent findings query the cancer stem cell hypothesis for solid tumors itself since it was shown in xenograft transplantation experiments that under appropriate conditions tumor-initiating cells are not rare

in Vitro and in Vivo Models to Decipher Intra-tumor Heterogeneity

Recent advances in next-generation sequencing and other omics technologies capable to map cell fate provide increasing evidence on the crucial role of intra-tumor heterogeneity (ITH) for cancer progression. The different facets of ITH, from genomic to microenvironmental heterogeneity and the hierarchical cellular architecture originating from the cancer stem cell compartment, contribute to the range of tumor phenotypes. Decoding these complex data resulting from the analysis of tumor tissue complexity poses a challenge for developing novel therapeutic strategies that can counteract tumor evolution and cellular plasticity. To achieve this aim, the development of in vitro and in vivo cancer models that resemble the complexity of ITH is crucial in understanding the interplay of cells and their (micro)environment and, consequently, in testing the efficacy of new targeted treatments and novel strategies of tailoring combinations of treatments to the individual composition of the tumor. This challenging approach may be an important cornerstone in overcoming the development of pharmaco-resistances during multiple lines of treatment. In this paper, we report the latest advances in patient-derived 3D (PD3D) cell cultures and patient-derived tumor xenografts (PDX) as in vitro and in vivo models that can retain the genetic and phenotypic heterogeneity of the tumor tissue

Glioblastoma cells express functional cell membrane receptors activated by daily used medical drugs

PURPOSE: Calcium ions are highly versatile spacial and temporal intracellular signals of non-excitable cells and have an important impact on nearly every aspect of cellular life controlling cell growth, metabolism, fluid secretion, information processing, transcription, apoptosis, and motility. Neurons and glia respond to stimuli, including neurotransmitters, neuromodulators, and hormones, which increase the intracellular calcium concentration. The function of intracellular calcium in gliomas is unknown. Lots of daily used drugs may act via receptors that can be linked to the intracellular calcium system and therefore could influence glioma biology. METHODS: Glioma cells were loaded with the calcium ion sensitive dye Fura 2-AM. Subsequently, cells were stimulated with 25 different medical drugs for 30 s. The increase of free intracellular calcium ions was measured and calculated by a microscope–camera–computer-unit. RESULTS: Except for the buffer solution HEPES that served as negative control and for the cortisol derivative dexamethasone, all other 24 tested drugs induced a rise of intracellular calcium ions. The cellular calcium responses were classified into seven functional groups. The tested substances activated several types of calcium channels and receptors. CONCLUSIONS: Our study impressively demonstrates that medical drugs are potent inducers of intracellular calcium signals. Totally unexpected, the results show a high amount of functional cellular receptors and channels on glioma cells, which could be responsible for certain biological effects like migration and cell growth. This calcium imaging study proves the usability of the calcium imaging as a screening system for functional receptors on human glioma cells

A RAS-Independent Biomarker Panel to Reliably Predict Response to MEK Inhibition in Colorectal Cancer

Background: In colorectal cancer (CRC), mutations of genes associated with the TGF-β/BMP signaling pathway, particularly affecting SMAD4, are known to correlate with decreased overall survival and it is assumed that this signaling axis plays a key role in chemoresistance. Methods: Using CRISPR technology on syngeneic patient-derived organoids (PDOs), we investigated the role of a loss-of-function of SMAD4 in sensitivity to MEK-inhibitors. CRISPR-engineered SMAD4R361H PDOs were subjected to drug screening, RNA-Sequencing, and multiplex protein profiling (DigiWest®). Initial observations were validated on an additional set of 62 PDOs with known mutational status. Results: We show that loss-of-function of SMAD4 renders PDOs sensitive to MEK-inhibitors. Multiomics analyses indicate that disruption of the BMP branch within the TGF-β/BMP pathway is the pivotal mechanism of increased drug sensitivity. Further investigation led to the identification of the SFAB-signature (SMAD4, FBXW7, ARID1A, or BMPR2), coherently predicting sensitivity towards MEK-inhibitors, independent of both RAS and BRAF status. Conclusion: We identified a novel mutational signature that reliably predicts sensitivity towards MEK-inhibitors, regardless of the RAS and BRAF status. This finding poses a significant step towards better-tailored cancer therapies guided by the use of molecular biomarkers

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

Aim Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. Methods Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipi‑ tation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. Results We identifed IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was signif‑ cantly associated with resistance to selumetinib, geftinib, and regorafenib in PDOs and to 5-fuorouracil and oxalipl‑ atin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabiliza‑ tion of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidifcation rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confrmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. Conclusions IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mito‑ chondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance

Age-related transcriptional changes in gene expression in different organs of mice support the metabolic stability theory of aging

Individual differences in the rate of aging are determined by the efficiency with which an organism transforms resources into metabolic energy thus maintaining the homeostatic condition of its cells and tissues. This observation has been integrated with analytical studies of the metabolic process to derive the following principle: The metabolic stability of regulatory networks, that is the ability of cells to maintain stable concentrations of reactive oxygen species (ROS) and other critical metabolites is the prime determinant of life span. The metabolic stability of a regulatory network is determined by the diversity of the metabolic pathways or the degree of connectivity of genes in the network. These properties can be empirically evaluated in terms of transcriptional changes in gene expression. We use microarrays to investigate the age-dependence of transcriptional changes of genes in the insulin signaling, oxidative phosphorylation and glutathione metabolism pathways in mice. Our studies delineate age and tissue specific patterns of transcriptional changes which are consistent with the metabolic stability–longevity principle. This study, in addition, rejects the free radical hypothesis which postulates that the production rate of ROS, and not its stability, determines life span

The molecular basis of genistein-induced mitotic arrest and exit of self-renewal in embryonal carcinoma and primary cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Genistein is an isoflavonoid present in soybeans that exhibits anti-carcinogenic properties. The issue of genistein as a potential anti-cancer drug has been addressed in some papers, but comprehensive genomic analysis to elucidate the molecular mechanisms underlying the effect elicited by genistein on cancer cells have not been performed on primary cancer cells, but rather on transformed cell lines. In the present study, we treated primary glioblastoma, rhabdomyosarcoma, hepatocellular carcinoma and human embryonic carcinoma cells (NCCIT) with μ-molar concentrations of genistein and assessed mitotic index, cell morphology, global gene expression, and specific cell-cycle regulating genes. We compared the expression profiles of NCCIT cells with that of the cancer cell lines in order to identify common genistein-dependent transcriptional changes and accompanying signaling cascades.</p> <p>Methods</p> <p>We treated primary cancer cells and NCCIT cells with 50 μM genistein for 48 h. Thereafter, we compared the mitotic index of treated versus untreated cells and investigated the protein expression of key regulatory self renewal factors as OCT4, SOX2 and NANOG. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools.</p> <p>Results</p> <p>We found that cancer cells treated with genistein undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes, <it>PBK</it>, <it>BUB1</it>, and <it>CDC20 </it>as determined by microarray-analysis and verified by Real-Time PCR. In contrast, human NCCIT cells showed over-expression of <it>GADD45 A </it>and <it>G </it>(growth arrest- and DNA-damage-inducible proteins 45A and G), as well as down-regulation of OCT4, and NANOG protein. Furthermore, genistein induced the expression of apoptotic and anti-migratory proteins p53 and p38 in all cell lines. Genistein also up-regulated steady-state levels of both <it>CYCLIN A </it>and <it>B</it>.</p> <p>Conclusion</p> <p>The results of the present study, together with the results of earlier studies show that genistein targets genes involved in the progression of the M-phase of the cell cycle. In this respect it is of particular interest that this conclusion cannot be drawn from comparison of the individual genes found differentially regulated in the datasets, but by the rather global view of the pathways influenced by genistein treatment.</p