Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
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  Transcript Studies
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        microRNA Functions
        Blood-based microRNA Diagnostics

mRNA Studies



      - Melanoma tumour-niche formation 
      - Signatures of human pathologies


      - Pancreatic cancer progression
      - Primary CNS lymphoma

 
      - Variations upon chemotherapy
      - Trypanosoma infection
Archive
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For the understanding of the complex regulative mechanisms and the investigation of  the cellular control management, a simultaneous analysis of the expression of all genes of an organism under various conditions and over time is indispensable. Our emphasis is on studying human cancer material and analyses particularly at the level of microRNA, with other RNA types also being worked at, although to a lesser extent. Information is gathered for enabling early diagnosis and accurate prognosis, the identification of potentially interesting avenues for therapy as well as the evaluation of the success of disease treatment. For this, the measurements of transcriptional variations are combined with the analysis of epigenetic modulations of the genomic DNA and actual protein expression. For particular microRNA molecules, their mode of action is studied in detail, elucidating the mechanisms by which their activity is transformed into function.









 

 
figure of melanoma in situ  
Melanoma microRNA trafficking controls tumour primary niche formation

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Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.
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Figure legend. Melanoma tumour in situ. The melanoma cells have not yet left the epidermis; green: fibroplasts; red: melanosomes; blue: DNA in cell nuclei.
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Dror, Sanders et al. (2016) Nature Cell Biol., in press.  (doi: 10.1038/ncb3399). 
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Early epigenetic down-regulation of microRNA-192 expression promotes pancreatic cancer progression logo NGFN
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Pancreatic ductal adenocarcinoma (PDAC) is characterized by very early metastasis, suggesting the hypothesis that metastasis-associated changes may occur prior to actual tumor formation. We identified miR-192 as an epigenetically regulated suppressor gene with predictive value in this disease. miR-192 was downregulated by promoter methylation in both PDAC and chronic pancreatitis (CP), the latter of which is a major risk factor for development of PDAC. Functional studies in vitro and in vivo in mouse models of PDAC showed that overexpression of miR-192 was sufficient to reduce cell proliferation and invasion. Mechanistic analyses correlated changes in miR-192 promoter methylation and expression with epithelial-mesenchymal transition (EMT). Cell proliferation and invasion were linked to altered expression of the miR-192 target gene SERPINE1 that is encoding the protein plasminogen activator inhibitor-1 (PAI-1), an established regulator of these properties in PDAC cells. Notably, our data suggested that invasive capacity was altered even before neoplastic transformation occurred, as triggered by miR-192 downregulation. Overall, our results highlighted a role for miR-192 in explaining the early metastatic behavior of PDAC and suggested its relevance as a target to develop for early diagnostics and therapy.
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Figure legend. Two cell lines, which express miR-192 at high level (CFPAC-1) or low level (MIAPaCa-2) were transfected with constructs that suppressed or increased miR-192 expression, respectively. Xenografted into mice, strong effects on tumour growth were observed.
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Botla et al. (2016) Cancer Res. 76, 4149-4159. 
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Blood-borne microRNA signatures in human pathologies logo NGFN
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Beyond studies that describe microRNAs frequently as markers for specific traits, we asked whether a general pattern for microRNAs across many diseases exists that could act as an intial, non-invasive means of diagnostics. In a multicenter study, we evaluated circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals from three different centers using qRT-PCR. We detected consistently deregulated profiles for particular diseases; pathway analysis confirmed disease association of the respective microRNAs. Overall, we discovered 34 miRNAs with strong disease association. In addition, a set of microRNAs was discovered  that act as rather stable markers, offering reasonable control microRNAs for future studies. Our study further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers.
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Figure legend. The balloon plot shows in how many diseases microRNAs are up- or down-regulated. The bubble size represents the number of microRNAs showing the respective distribution of up- and down-regulation. Orange bubbles represent microRNAs that are predominantly down-regulated, while blue bubbles stand for predominantly up-regulated microRNAs. The two green bubbles represent 9 microRNAs that were equally often up- and down-regulated in disease.
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Keller et al. (2014) BMC Med. 12, 224. 
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Keller et al. (2011) Nature Meth. 8, 841-843.  pdf icon

number of miRNA expression variations in diseases

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MicroRNAs in the blood of patients with primary CNS lymphoma (PCNSL) act as prognostic biomarkers
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Despite improved therapeutic regimens, primary CNS lymphoma (PCNSL) remains a therapeutic challenge. We characterized by next generation sequencing the microRNA fingerprints in the blood of PCNSL patients enrolled in a large randomized phase III study, comparing short-term to long-term survival. Twelve microRNAs were significantly deregulated between the two groups. The microRNAs miR-493-3p and miR-432-5p exhibited the most prominent differences. Furthermore, we identified short RNA molecules with putative microRNA function that had not been described before.
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Roth et al. (2015) Eur. J. Cancer 51, 382-390.







Boxplot presentation of the pancreas classification results

Diagnosis of pancreatic ductal adenocarcinoma and chronic pancreatitis by measurement of microRNA abundance in blood and tissue logo NGFN
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MicroRNAs can regulate hundreds of genes post-transcriptionally and appear to regulate virtually all cellular processes. Owing to these properties, microRNAs have a critical role not only in physiological but also in pathological processes. There is meanwhile a lot of evidence that miRNA profiles from body fluids, such as blood, and informative about the disease status of a blood donor.
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A detailed analysis of microRNA profiles was performed for pancreatic ductal adenocarcinoma. A solid diagnostic process could have substantial impact on the successful treatment of the disease, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analysed. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors.
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Thee blood test demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples. In addition, discrimination between cancer and chronic pancreatitis was achieved. Several miRNAs were identified that exhibited abundance variations in both tissue and blood samples.
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The results could have an immediate diagnostic value for the evaluation of tumour reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer.
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Bauer et al. (2012) PLoS ONE 7, e34151.  pdf icon





MicroRNA signatures of peripheral blood cells in humans infected with Trypanosoma brucei gambiense logo DAAD
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Human African Trypanosomiasis - sleeping sickness - still affects thousands of people a year. Control relies on diagnosis and treatment, and in the absence of this surveillance, the number of cases rapidly rebounds. Diagnosis relies on an antibody test and microscopy. The type of treatment depends on the disease stage: whether parasites have entered the central nervous system; and this can be determined only by lumber puncture and analysis of cerebrospinal fluid. The development of sensitive, simple and reliable tools for diagnosis and staging of human African Trypanosomiases would significantly ease field surveillance and enhance patient care. We investigated whether the patterns of miRNAs from peripheral blood could be used to decide whether patients are infected, or to determine the disease stage. We found that the miRNA patterns did differ between parasite-positive patients and uninfected controls with no immune reaction to trypanosomes. Also, people with immune reactions to trypanosomes, but no detectable parasites, sometimes showed patient-like profiles. The patterns were not reliable enough, however, to be used for diagnosis.
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Lueong et al. (2013) PLOS ONE 8, e74555.  pdf icon




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MicroRNA expression profiles in peripheral blood cells of rats infected with Trypanosoma congolese and Trypanosoma brucei subspecies
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To identify differentially regulated miRNAs during trypanosome infections, we analyzed the miRNA expression profiles of uninfected rats and animals infected with Trypanosoma congolense and different Trypanosoma brucei species. The potential target genes of the regulated miRNAs as well as their biological pathways and biological functions were investigated.

Simo et al. (2015) Microb. Infect. 17, 596-608. 
 
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MicroRNA variations in cells and their exosomes upon chemotherapy
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It has been convincingly proposed that exchange of molecules via exosomes is a means of eukaryotic intercellular communication, especially within the tumour microenvironment. However, there are no data on the alterations of exosomal molecular cargo upon pharmacological anticancer treatments. To approach this issue, we analysed the abundance of miRNAs and cancer-related proteins in exosomes secreted by Caco-2 (Cetuximab-responsive) and HCT-116 (Cetuximab-resistant) colorectal cancer cells before and after Cetuximab treatment. We also characterized both profiles in whole source cells.
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Cetuximab significantly altered the molecular cargo of exosomes from Caco-2: we detected increased abundance of miRNAs and proteins that activate cell proliferation and proinflammatory processes; simultaneously, we observed a decrease of miRNAs and proteins related to immune suppression. These changes did not overlap entirely with those in source cells, suggesting a Cetuximab-linked distribution bias.
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Molecular changes of a minor extent were also detected in exosomes from HCT-116. Transfection of exosomes from Cetuximab-treated Caco-2 into HCT-116 significantly increased HCT-116 viability; conversely, Caco-2 transfected with exosomes from treated HCT-116 did not show viability alterations. This suggests that the molecular phenotype of source cells is important for determining both the exosomal cargo as the biological effects of transferred exosomes.
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Gene Ontology analysis of networks that comprise targets of differentially expressed exosomal miRNAs and proteins demonstrated a significant involvement of biological processes related to proliferation control, inflammation, immune response, and apoptosis. Our data shed light on molecular mechanisms of intercellular communication in eukaryotes.

Ragusa et al. (2014) Oncoscience 1, 132-157pdf icon



Quantitative asymmetric distribution of miRNAs in colorectal cancer cells and exosomes. Relative quantities (RQ) of the miRNAs amounts as isolated from exosomes were compared to those from the source cells Caco-2. Values are shown as log10 of RQ.









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