Ulf Schmitz

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.

Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

WEE1 kinase has been described as a major gate keeper at the G2 cell cycle checkpoint and to be involved in tumour progression in different malignant tumours. Here we analysed the expression levels of WEE1 in a series of melanoma patient samples and melanoma cell lines using immunoblotting, quantitative real-time PCR and immunohistochemistry. WEE1 expression was significantly downregulated in patient samples of metastatic origin as compared with primary melanomas and in melanoma cell lines of high aggressiveness as compared with cell lines of low aggressiveness. Moreover, there was an inverse correlation between the expression of WEE1 and WEE1-targeting microRNA miR-195. Further analyses showed that transfection of melanoma cell lines with miR-195 indeed reduced WEE1 mRNA and protein expression in these cells. Reporter gene analysis confirmed direct targeting of the WEE1 3′ untranslated region (3′UTR) by miR-195. Overexpression of miR-195 in SK-Mel-28 melanoma cells was accompanied by WEE1 reduction and significantly reduced stress-induced G2-M cell cycle arrest, which could be restored by stable overexpression of WEE1. Moreover, miR-195 overexpression and WEE1 knockdown, respectively, increased melanoma cell proliferation. miR-195 overexpression also enhanced migration and invasiveness of melanoma cells. Taken together, the present study shows that WEE1 expression in malignant melanoma is directly regulated by miR-195. miR-195-mediated downregulation of WEE1 in metastatic lesions may help to overcome cell cycle arrest under stress conditions in the local tissue microenvironment to allow unrestricted growth of tumour cells.

MicroRNA-regulated networks: the perfect storm for classical molecular biology, the ideal scenario for systems biology.

MicroRNAs (miRNAs) are involved in many regulatory pathways some of which are complex networks enriched in regulatory motifs like positive or negative feedback loops or coherent and incoherent feedforward loops. Their complexity makes the understanding of their regulation difficult and the interpretation of experimental data cumbersome. In this book chapter we claim that systems biology is the appropriate approach to investigate the regulation of these miRNA-regulated networks. Systems biology is an interdisciplinary approach by which biomedical questions on biochemical networks are addressed by integrating experiments with mathematical modelling and simulation. We here introduce the foundations of the systems biology approach, the basic theoretical and computational tools used to perform model-based analyses of miRNA-regulated networks and review the scientific literature in systems biology of miRNA regulation, with a focus on cancer.

Tumor-promoting role of signal transducer and activator of transcription (Stat)1 in late-stage melanoma growth

A large-scale gene expression study of melanoma metastases was performed to identify genes involved in late-stage tumor progression. Overall 248 genes, out of more than 47,000 tested, are differentially expressed when comparing peripheral areas (invasion front) with central tumor areas of melanoma metastases. As determined by gene ontology analysis, members of the STAT signaling pathway show significant enrichment. In particular, Stat1 is highly expressed in peripheral compared with central tumor areas. In line with this, stable knockdown of STAT1 in metastatic melanoma cells significantly impairs their migratory and invasive capacity in wounding and matrigel assays. Moreover, STAT1 knockdown affects the metastatic behavior of melanoma cells in a mouse model of melanoma metastasis. Taken together, these data suggest that Stat1 might play a role in late-stage melanoma progression. Interference with the Stat1 pathway could have therapeutic implications for late-stage melanoma patients.

Kinetic Modeling-Based Detection of Genetic Signatures that Provide Chemoresistance via the E2F1-p73/DNp73-miR-205 Network

Drug resistance is a major cause of deaths from cancer. E2F1 is a transcription factor involved in cell proliferation, apoptosis. and metastasis through an intricate regulatory network, which includes other transcription factors like p73 and cancer-related microRNAs like miR-205. To investigate the emergence of drug resistance, we developed a methodology that integrates experimental data with a network biology and kinetic modeling. Using a regulatory map developed to summarize knowledge on E2F1 and its interplay with p73/DNp73 and miR-205 in cancer drug responses, we derived a kinetic model that represents the network response to certain genotoxic and cytostatic anticancer drugs. By perturbing the model parameters, we simulated heterogeneous cell configurations referred to as in silico cell lines. These were used to detect genetic signatures characteristic for single or double drug resistance. We identified a signature composed of high E2F1 and low miR-205 expression that promotes resistance to genotoxic drugs. In this signature, downregulation of miR-205, can be mediated by an imbalance in the p73/DNp73 ratio or by dysregulation of other cancer-related regulators of miR-205 expression such as TGFβ-1 or TWIST1. In addition, we found that a genetic signature composed of high E2F1, low miR-205, and high ERBB3 can render tumor cells insensitive to both cytostatic and genotoxic drugs. Our model simulations also suggested that conventional genotoxic drug treatment favors selection of chemoresistant cells in genetically heterogeneous tumors, in a manner requiring dysregulation of incoherent feedforward loops that involve E2F1, p73/DNp73, and miR-205.

Cooperative gene regulation by microRNA pairs and their identification using a computational workflow

MicroRNAs (miRNAs) are an integral part of gene regulation at the post-transcriptional level. Recently, it has been shown that pairs of miRNAs can repress the translation of a target mRNA in a cooperative manner, which leads to an enhanced effectiveness and specificity in target repression. However, it remains unclear which miRNA pairs can synergize and which genes are target of cooperative miRNA regulation. In this paper, we present a computational workflow for the prediction and analysis of cooperating miRNAs and their mutual target genes, which we refer to as RNA triplexes. The workflow integrates methods of miRNA target prediction; triplex structure analysis; molecular dynamics simulations and mathematical modeling for a reliable prediction of functional RNA triplexes and target repression efficiency. In a case study we analyzed the human genome and identified several thousand targets of cooperative gene regulation. Our results suggest that miRNA cooperativity is a frequent mechanism for an enhanced target repression by pairs of miRNAs facilitating distinctive and fine-tuned target gene expression patterns. Human RNA triplexes predicted and characterized in this study are organized in a web resource at www.sbi.uni-rostock.de/triplexrna/.

E2F1 induces miR‐224/452 expression to drive EMT through TXNIP downregulation

Malignant melanoma is highly lethal due to its aggressive invasive properties and metastatic dissemination. The transcription factor E2F1 is crucial for melanoma progression through poorly understood mechanisms. Here, we show that the miR‐224/miR‐452 cluster is significantly increased in advanced melanoma and invasive/metastatic cell lines that express high levels of E2F1. miR‐224/miR‐452 expression is directly activated by E2F1 through transactivation of the GABRE gene. Ectopic expression of miR‐224/miR‐452 in less aggressive cells induces EMT and cytoskeletal rearrangements and enhances migration/invasion. Conversely, miR‐224/miR‐452 depletion in metastatic cells induces the reversal of EMT, inhibition of motility, loss of the invasive phenotype and an absence of lung metastases in mice. We identify the metastasis suppressor TXNIP as new target of miR‐224/miR‐452 that induces feedback inhibition of E2F1 and show that miR‐224/452‐mediated downregulation of TXNIP is essential for E2F1‐induced EMT and invasion. The E2F1‐miR‐224/452‐TXNIP axis constitutes a molecular signature that predicts patient survival and may help to set novel therapies.

A dynamic intron retention program in the mammalian megakaryocyte and erythrocyte lineages.

Intron retention (IR) is a form of alternative splicing that can impact mRNA levels through nonsense-mediated decay or by nuclear mRNA detention. A complex, dynamic IR pattern has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We globally assessed IR in primary maturing mammalian erythroid and megakaryocyte (MK) lineages as well as their common progenitor cells (MEPs). Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes with an overwhelming loss of IR in erythroid cells and MKs compared to MEPs. Moreover, complex IR patterns were seen throughout murine erythroid maturation. Similarly complex patterns were observed in human erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and MKs, and overlapping gene expression patterns, the MK IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. Importantly, our results suggest that IR serves to broadly regulate mRNA levels. These findings highlight the importance of this understudied form of alternative splicing in gene regulation and provide a useful resource for studies on gene expression in the MK and erythroid lineages.

Identification of immunogenic consensus T-cell epitopes in globally distributed influenza-A H1N1 neuraminidase

Antigenic drift is the ability of the swine influenza virus to undergo continuous and progressive changes in response to the host immune system. These changes dictate influenza vaccine updates annually to ensure inclusion of antigens of the most current strains. The identification of those peptides that stimulate T-cell responses, termed T-cell epitopes, is essential for the development of successful vaccines. In this study, the highly conserved and specific epitopes from neuraminidase of globally distributed H1N1 strains were predicted so that these potential vaccine candidates may escape with antigenic drift. A total of nine novel CD8(+) T-cell epitopes for MHC class-I and eight novel CD4(+) T-cell epitopes for MHC class-II alleles were proposed as novel epitope based vaccine candidates. Additionally, the epitope FSYKYGNGV was identified as a highly conserved, immunogenic and potential vaccine candidate, capable for generating both CD8(+) and CD4(+) responses.

MicroRNA–mRNA interactions in colorectal cancer and their role in tumor progression

MicroRNAs (miRNA/miR) play an important role in gene regulatory networks through targeting mRNAs. They are involved in diverse biological processes such as cell proliferation, differentiation, angiogenesis, and apoptosis. Due to their pivotal effects on multiple genes and pathways, dysregulated miRNAs have been reported to be associated with different diseases, including colorectal cancer (CRC). Recent evidence indicates that aberrant miRNA expression is tightly linked with the initiation and progression of CRC. To elucidate the influence of miRNA regulation in CRC, it is critical to identify dysregulated miRNAs, their target mRNA genes and their involvement in gene regulatory and signaling networks. Various experimental and computational studies have been conducted to decipher the function of miRNAs involved in CRC. Experimental studies that are used for this purpose can be classified into two categories: direct/individual and indirect/high‐throughput gene expression studies. Here we review miRNA target identification studies related to CRC with an emphasis on experimental data based on Luciferase reporter assays. Recent advances in determining the function of miRNAs and the signaling pathways they are involved in have also been summarized. The review helps bioinformaticians and biologists to find extensive information about downstream targets of dysregulated miRNAs, and their pro‐/anti‐CRC effects.