Julia Feichtinger

Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA‐methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA‐methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241–2253.

miR-199a and miR-497 Are Associated with Better Overall Survival due to Increased Chemosensitivity in Diffuse Large B-Cell Lymphoma Patients.

Micro-RNAs (miRNAs) are short non-coding single-stranded RNA molecules regulating gene expression at the post-transcriptional level. miRNAs are involved in cell development, differentiation, apoptosis, and proliferation. miRNAs can either function as tumor suppressor genes or oncogenes in various important pathways. The expression of specific miRNAs has been identified to correlate with tumor prognosis. For miRNA expression analysis real-time PCR on 81 samples was performed, including 63 diffuse large B-cell lymphoma (DLBCL, 15 of germinal center B-cell like subtype, 17 non germinal center B-cell, 23 transformed, and eight unclassified) and 18 controls, including nine peripheral B-cells, 5 germinal-center B-cells, four lymphadenitis samples, and 4 lymphoma cell lines (RI-1, SUDHL4, Karpas, U2932). Expression levels of a panel of 11 miRNAs that have been previously involved in other types of cancer (miR-15b_2, miR-16_1*, miR-16_2, miR-16_2*, miR-27a, miR-27a*, miR-98-1, miR-103a, miR-185, miR-199a, and miR-497) were measured and correlated with clinical data. Furthermore, cell lines, lacking miR-199a and miR-497 expression, were electroporated with the two respective miRNAs and treated with standard immunochemotherapy routinely used in patients with DLBCL, followed by functional analyses including cell count and apoptosis assays. Seven miRNAs (miR-16_1*, miR-16_2*, miR-27a, miR-103, miR-185, miR-199, and miR-497) were statistically significantly up-regulated in DLBCL compared to normal germinal cells. However, high expression of miR-497 or miR-199a was associated with better overall survival (p = 0.042 and p = 0.007). Overexpression of miR-199a and miR-497 led to a statistically significant decrease in viable cells in a dose-dependent fashion after exposure to rituximab and various chemotherapeutics relevant in multi-agent lymphoma therapy. Our data indicate that elevated miR-199a and miR-497 levels are associated with improved survival in aggressive lymphoma patients most likely by modifying drug sensitivity to immunochemotherapy. This functional impairment may serve as a potential novel therapeutic target in future treatment of patients with DLBCL.

Microarray Meta-Analysis: From Data to Expression to Biological Relationships

Since the introduction of microarray technology, it has become the workhorse for mRNA expression profiling. Its application ranges from investigating gene function, regulation, and co-expression, to clinical use in diagnosis and prognosis. Over the last decade, a large number of microarray experiments have become available in public repositories often addressing similar or related hypotheses. The large compendia of gene expression data provide the opportunity to conduct meta-analyses by combining data from various independent but related studies. Such data integration has the potential to enhance the reliability and generalizability of the results of individual microarray studies.

Meta-analysis of expression of l(3)mbt tumor-associated germline genes supports the model that a soma-to-germline transition is a hallmark of human cancers.

Evidence is starting to emerge indicating that tumorigenesis in metazoans involves a soma‐to‐germline transition, which may contribute to the acquisition of neoplastic characteristics. Here, we have meta‐analyzed gene expression profiles of the human orthologs of Drosophila melanogaster germline genes that are ectopically expressed in l(3)mbt brain tumors using gene expression datasets derived from a large cohort of human tumors. We find these germline genes, some of which drive oncogenesis in D. melanogaster, are similarly ectopically activated in a wide range of human cancers. Some of these genes normally have expression restricted to the germline, making them of particular clinical interest. Importantly, these analyses provide additional support to the emerging model that proposes a soma‐to‐germline transition is a general hallmark of a wide range of human tumors. This has implications for our understanding of human oncogenesis and the development of new therapeutic and biomarker targets with clinical potential.

NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer-specific survival in patients with aggressive B-cell lymphomas.

NR4A1 (Nur77) and NR4A3 (Nor-1) function as tumor suppressor genes as demonstrated by the rapid development of acute myeloid leukemia in the NR4A1 and NR4A3 knockout mouse. The aim of our study was to investigate NR4A1 and NR4A3 expression and function in lymphoid malignancies. We found a vastly reduced expression of NR4A1 and NR4A3 in chronic lymphocytic B-cell leukemia (71%), in follicular lymphoma (FL, 70%), and in diffuse large B-cell lymphoma (DLBCL, 74%). In aggressive lymphomas (DLBCL and FL grade 3), low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. To investigate the function of NR4A1 in lymphomas, we overexpressed NR4A1 in several lymphoma cell lines. Overexpression of NR4A1 led to a higher proportion of lymphoma cells undergoing apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stable lentiviral-transduced SuDHL4 lymphoma cell line harboring an inducible NR4A1 construct was further investigated in xenografts. Induction of NR4A1 abrogated tumor growth in the NSG mice, in contrast to vector controls, which formed massive tumors. Our data suggest that NR4A1 has proapoptotic functions in aggressive lymphoma cells and define NR4A1 as a novel gene with tumor suppressor properties involved in lymphomagenesis.

Resolving Tumor Heterogeneity: Genes Involved in Chordoma Cell Development Identified by Low-Template Analysis of Morphologically Distinct Cells

The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase ALG11 (695-fold) and the phosphatase subunit PPP2CB (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.

CancerMA: a web-based tool for automatic meta-analysis of public cancer microarray data

The identification of novel candidate markers is a key challenge in the development of cancer therapies. This can be facilitated by putting accessible and automated approaches analysing the current wealth of ‘omic’-scale data in the hands of researchers who are directly addressing biological questions. Data integration techniques and standardized, automated, high-throughput analyses are needed to manage the data available as well as to help narrow down the excessive number of target gene possibilities presented by modern databases and system-level resources. Here we present CancerMA, an online, integrated bioinformatic pipeline for automated identification of novel candidate cancer markers/targets; it operates by means of meta-analysing expression profiles of user-defined sets of biologically significant and related genes across a manually curated database of 80 publicly available cancer microarray datasets covering 13 cancer types. A simple-to-use web interface allows bioinformaticians and non-bioinformaticians alike to initiate new analyses as well as to view and retrieve the meta-analysis results. The functionality of CancerMA is shown by means of two validation datasets. Database URL: http://www.cancerma.org.uk

Cancer germline gene activation: friend or foe?

The human male germ line is passed on via the production of haploid sperm, which, upon fusion with the female gamete, the ovum, will form a diploid zygote that will ultimately become a genetically unique individual. Male gametogenesis occurs throughout the lifespan of adult males. Sperm production is restricted to the seminiferous tubules of the testis, and this process is continually fed by the mitotic proliferation of germline stem cells (GSCs). The GSCs are a sub-group of spermatagonial cells, which are found at the basal layer of the seminiferous tubules. Upon receipt of differentiation signals, spermatagonial cells will undergo differentiation, first maturing to primary spermatocytes and then ultimately through to fully differentiated spermatozoa. During this cellular differentiation, meiosis occurs, reducing the chromosomal content from diploid to haploid status and driving genetic variation.

Translin and Trax differentially regulate telomere-associated transcript homeostasis

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

Germline/meiotic genes in cancer: new dimensions

Intergenerational passage of genetic information in humans is dependent on a complex sexual program which is orchestrated by a large cohort of tissue-specific germline genes. These genes drive a range of germline-specific pathways including spermatogenesis in males and the reductional chromosome segregation of meiosis. In somatic tissues these genes are transcriptionally silenced, but can become activated in cancerous tissue.1-3 Initial interest in these genes was driven by the need to identify new cancer-specific biomarkers that could be used for diagnostics, prognostics and/or immunotherapeutics, and the first group to be discovered became widely known as the cancer/testis antigen (CTA) genes as they were expressed only in the testis and cancerous tissues, but not in healthy somatic cells. Latterly, many of these gene have also been referred to as the cancer germline genes.1,2 Despite the early interest in these genes as clinical biomarkers or targets, recent works have started to reveal another important cancer-associated role, one which has undergone remarkably limited scrutiny. It has emerged from various studies that germline genes can contribute to oncogenesis and can also contribute to tumor maintenance and drug resistance,1,2 potentially revealing this family of proteins as potent new drug targets. The genes that drive and maintain the male germline have a range of distinct functional roles, including germline cell maintenance in mitotically proliferating spermatagonial cell populations, cellular differentiation during spermatogenesis and the chromosomal reduction of meiosis. To date, many of the CTA genes that have been identified are encoded by the X chromosome and their expression is restricted to the spermatagonial germline cells, becoming transcriptionally inactive during the differentiation to meiotic spermatocytes. A recent screen for new cancer germline genes based on the human orthologues of mouse meiotic spermatocyte genes revealed a large number of autosomally encoded cancer germline genes that were transcriptionally activated in a wide range of cancer types.4,5 A number of these human genes have been shown to encode meiosis-specific functions, including the meiosis-specific inter sister chromatid cohesion protein gene RAD21L and the transcriptional activator / meiotic recombination hot spot regulator gene PRDM9.4,5 From this, it was speculated that the activation of such genes could drive oncogenesis and tumor evolution by interfering with normal equational mitotic chromosome segregation programmes, thus driving oncogenic genome instability.4,5 However, while germline genes have been implicated in a number of oncogenic processes, until recently no direct evidence had been offered to implicate meiosis-specific chromosome regulators in oncogenesis or tumor maintenance. This changed when it was discovered that the ALT telomere maintenance pathway used by some telomerease deficient cancer cells to maintain their telomeres was dependent upon 2 meiosis-specific factors, Hop2 and Mnd1, both of which normally drive the mechanism that biases meiotic recombination to establish the inter homolog connections required for correct reductional chromosome segregation during meiosis I.6 This discovery adds a new class of factors to the complex mix of genes that become activated during oncogenesis and highlights the need to explore the functional activity of other human meiotic genes that may contribute to cancer formation, maintenance and evolution.4,5 In addition, it places further importance on the study of basic molecular mechanisms of the meiotic program. Remarkably, given the emerging importance of germline genes and meiosis-specific genes in cancer, very little is known about their transcriptional regulation. Some studies imply that many of these genes are under a similar regulatory pathway, so that when this becomes dysregulated, functionally related groups of genes become active driving inappropriate functional germline/meiotic modules.3 The epigenetic regulation of previously well characterized CTA genes has been relatively well documented with all studied genes being controlled at some level by the methylation of DNA transcriptional regulatory regions. These genes are activated when cells are treated with the DNA methyltransferase inhibitor 5-AzaC. However, a new layer of complexity to this activation has been revealed by a recent study that demonstrated that not all human cancer germline genes are activated by inhibition of DNA methyltransferases, indicating a complex hierarchy of germline, and possibly meiotic, gene activation.7 In this study it was shown that not only did some cancer germline genes fail to become activated upon inhibition of DNA methyltransferase activity, a number were only transiently silenced during exposure to the demethylating agents while others remained more robustly activated following removal of the demethylation agent.7 Altogether, the finding that meiosis-specific factors can contribute to oncogenesis and that the normal somatic silencing of germline genes is controlled by unique, as yet uncharted factors, makes the study of meiotic and germline genes in cancer an emerging and important field, one which impinges directly on a range of clinical applications including diagnostics, patient stratification/prognostics and new therapeutic and drug targeting strategies.