Modellierung der MYCN-getriebenen Tumorgenese in vitro und in transgenen Mausmodellen

Das Ziel dieser Dissertation war, MYCN abhängige und –unabhängige Prozesse im Rahmen von Tumorentstehung und Tumortherapie in vitro und in vivo zu modellieren. Beim Neuroblastom, dem häufigsten soliden, extrakraniellen Tumor des Kindesalters, ist im Rahmen von Tumorentstehung und Tumortherapie, in 20% der Tumore eine Amplifikation des Onkogens MYCN beschrieben. MYCN Amplifikation ist mit aggressivem Tumorwachstum und einer schlechten Prognose für den Patienten assoziiert. Bei normalem MYCN Status ist eine Risikoeinschätzung schwierig. Analysen von mRNA Profilen primärer Neuroblastome führten in dieser Arbeit zur Identifikation von JARID1C als MYCN unabhängigem prognostischen Marker. JARID1C ist eine Histondemethylase, welche tri- und dimethyliertes H3K4 erkennt und die Methylreste entfernt. In allen getesteten Neuroblastomzelllinien führte eine Inhibition der JARID1C Funktion zu einem Anstieg apoptotischer und dem Abfall proliferativer Zellen. Hierzu wurde JARID1C-spezifische siRNA, und Pbit, einen kürzlich identifizierten JARID1C Inhibitor eingesetzt. Die JARID1C Expression wurde als essentiell für das Überleben von Neuroblastomzellen unabhängig vom MYCN Status nachgewiesen. Durch die Kreuzung transgener Mäuse mit konditional aktivierbarem MYCN (Lsl-MYCN) und Mäusen, die gewebespezifisch die Cre-Rekombinase exprimieren, wurde MYCN gezielt in diesen Zellen herauf reguliert. In doppelt-transgenen GFAP-Cre;Lsl-MYCN Mäusen kam es zu einer konditionaler Überexpression von MYCN in pankreatischen α-Zellen, wodurch sich Glukagonome entwickelten. Neben Glukagonomen bildeten einige Tiere auch Hypophysenadenome aus. Tiere mit pankreatischen Tumoren zeigten eine positive Glukagonreaktion im Tumorgewebe sowie erhöhte Werte von Plasma-Glukagon, während Insulinwerte unverändert blieben. Die Tumore zeigten keine chromosomalen Aberrationen und wenige signifikant auf mRNA Ebene regulierte Gene. Aus einem Glukagonom wurden Zelllinien etabliert, die auf eine MYCN Inhibition durch den Brd4 Inhibitor JQ1 und den Aurorakinase A Inhibitor MLN 8237 sensibel reagierten. Das Tumorwachstum von Xenografts dieser Zellen wurde durch beide Inhibitoren signifikant reduziert. Serielle Transplantationen dieser Zelllinien führten zu einer erhöhten Resistenz gegen MYCN-gerichtete Therapien. Somit wurde in dieser Arbeit erstmals ein transgenes Modell für Glukagonome und Hypophysenadenome entwickelt. Neben dem Einsatz zur Überprüfung MYCN-gerichteter Therapien kann dieses Tiermodell auch zur Untersuchung der Biologie neuroendokriner Tumore genutzt werden.This thesis was aiming to model MYCN dependent and independent processes in vitro and in vivo. In neuroblastoma, the most common, extracranial childhood malignancy, the oncogene MYCN is amplified in 20% of tumors. MYCN Amplification is associated with aggressive progression and poor outcome. Risk assessment in patients with normal MYCN status remains difficult. Analyses of mRNA expression profiles from primary neuroblastomas resulted in the identification of JARID1C as a prognostic marker for aggressive neuroblastoma independent of the MYCN amplification status. JARID1C is a histone demethylase, detecting and removing trimethyl and dimethyl residues of lysines at the position 4 of histone 3 (H3K4). The JARID1C expression is essential for neuroblastoma cell survival. An inhibition of JARID1C resulted in an increased fraction of apoptotic cells and a decreased number of proliferative cells in all cell lines tested. For the inhibition of JARID1C function, JARID1C specific siRNA and Pbit, a recently discovered small-molecule inhibitor, was applied. Cross-breeding of transgenic mice with conditional expression of MYCN (Lsl-MYCN) with mice expressing tissue-specific Cre recombinase, MYCN was upregulated in these cells. In double-transgenic GFAP-Cre;Lsl-NMYC mice, this led to a conditional overexpression of MYCN in pancreatic α-cells, resulting in the development of glucagonoma. Apart from glucagonoma, some animals developed pituitary adenoma. Animals with pancreatic tumors showed a positive reaction for glucagon in tumor tissue and increased levels of blood glucagon, while insulin levels remained unchanged. The tumors showed only a few significantly regulated mRNAs and no chromosomal aberrations. Cell lines established from a glucagonoma responded to targeted inhibition of MYCN using the Brd4 inhibitor JQ1 and the Aurora kinase A inhibitor MLN 8237. Growth of xenografted tumor cells was also significantly decreased upon inhibitor treatment, while serial transplantation induced resistance against JQ1 and MLN 8237.This thesis presents the first transgenic mouse model for glucagonoma and pituitary adenoma. Besides using the animal model for further investigantions of MYCN-directed therapies, it may also foster the understanding of the biology of neuroendocrine tumors

Robust selection of cancer survival signatures from high-throughput genomic data using two-fold subsampling

Identifying relevant signatures for clinical patient outcome is a fundamental task in high-throughput studies. Signatures, composed of features such as mRNAs, miRNAs, SNPs or other molecular variables, are often non-overlapping, even though they have been identified from similar experiments considering samples with the same type of disease. The lack of a consensus is mostly due to the fact that sample sizes are far smaller than the numbers of candidate features to be considered, and therefore signature selection suffers from large variation. We propose a robust signature selection method that enhances the selection stability of penalized regression algorithms for predicting survival risk. Our method is based on an aggregation of multiple, possibly unstable, signatures obtained with the preconditioned lasso algorithm applied to random (internal) subsamples of a given cohort data, where the aggregated signature is shrunken by a simple thresholding strategy. The resulting method, RS-PL, is conceptually simple and easy to apply, relying on parameters automatically tuned by cross validation. Robust signature selection using RS-PL operates within an (external) subsampling framework to estimate the selection probabilities of features in multiple trials of RS-PL. These probabilities are used for identifying reliable features to be included in a signature. Our method was evaluated on microarray data sets from neuroblastoma, lung adenocarcinoma, and breast cancer patients, extracting robust and relevant signatures for predicting survival risk. Signatures obtained by our method achieved high prediction performance and robustness, consistently over the three data sets. Genes with high selection probability in our robust signatures have been reported as cancer-relevant. The ordering of predictor coefficients associated with signatures was well-preserved across multiple trials of RS-PL, demonstrating the capability of our method for identifying a transferable consensus signature. The software is available as an R package rsig at CRAN (http://cran.r-project.org)

Providing Information by Resource- Constrained Data Analysis

The Collaborative Research Center SFB 876 (Providing Information by Resource-Constrained Data Analysis) brings together the research fields of data analysis (Data Mining, Knowledge Discovery in Data Bases, Machine Learning, Statistics) and embedded systems and enhances their methods such that information from distributed, dynamic masses of data becomes available anytime and anywhere. The research center approaches these problems with new algorithms respecting the resource constraints in the different scenarios. This Technical Report presents the work of the members of the integrated graduate school

Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis

The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels

Inflammation-induced acute phase response in skeletal muscle and critical illness myopathy.

OBJECTIVES: Systemic inflammation is a major risk factor for critical-illness myopathy (CIM) but its pathogenic role in muscle is uncertain. We observed that interleukin 6 (IL-6) and serum amyloid A1 (SAA1) expression was upregulated in muscle of critically ill patients. To test the relevance of these responses we assessed inflammation and acute-phase response at early and late time points in muscle of patients at risk for CIM. DESIGN: Prospective observational clinical study and prospective animal trial. SETTING: Two intensive care units (ICU) and research laboratory. PATIENTS/SUBJECTS: 33 patients with Sequential Organ Failure Assessment scores ≥ 8 on 3 consecutive days within 5 days in ICU were investigated. A subgroup analysis of 12 patients with, and 18 patients without CIM (non-CIM) was performed. Two consecutive biopsies from vastus lateralis were obtained at median days 5 and 15, early and late time points. Controls were 5 healthy subjects undergoing elective orthopedic surgery. A septic mouse model and cultured myoblasts were used for mechanistic analyses. MEASUREMENTS AND MAIN RESULTS: Early SAA1 expression was significantly higher in skeletal muscle of CIM compared to non-CIM patients. Immunohistochemistry showed SAA1 accumulations in muscle of CIM patients at the early time point, which resolved later. SAA1 expression was induced by IL-6 and tumor necrosis factor-alpha in human and mouse myocytes in vitro. Inflammation-induced muscular SAA1 accumulation was reproduced in a sepsis mouse model. CONCLUSIONS: Skeletal muscle contributes to general inflammation and acute-phase response in CIM patients. Muscular SAA1 could be important for CIM pathogenesis. TRIAL REGISTRATION: ISRCTN77569430

Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells

Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCNdriven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies

A robust signature obtained with RS-PL from the breast cancer data set (<i>π</i> = 0.0.85).

<p>For ZCCHC24, two transcripts (with probeset IDs 212419_at and 212413_at) were chosen.</p><p>A robust signature obtained with RS-PL from the breast cancer data set (<i>π</i> = 0.0.85).</p

Characteristic of patients before/after GNUSE filtering (adenocarcinoma).

<p>Microarrays with median GNUSE scores>1.0 and with no overall survival time annotation were discarded (NA: not available).</p><p>Characteristic of patients before/after GNUSE filtering (adenocarcinoma).</p

Characteristic of patients before/after GNUSE filtering (breast cancer).

<p>Microarrays with median GNUSE scores>1.0 and with no overall survival time annotation were discarded (NA: not available).</p><p>Characteristic of patients before/after GNUSE filtering (breast cancer).</p

A robust signature obtained with RS-PL from the lung adenocarcinoma data set (<i>π</i> = 0.85).

<p>*It was reported to the contrary that CYP4B1 was normally expressed in lung cancer patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108818#pone.0108818-Czerwinski1" target="_blank">[58]</a>. If the relevance of features was unclear or unknown, it was marked with hyphens.</p><p>A robust signature obtained with RS-PL from the lung adenocarcinoma data set (<i>π</i> = 0.85).</p