Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
DKFZ Logo
















 


























.
.

Archive      Epigenetic Analyses
..
..
Finished Projects
Current Projects
   - Common cancer biomarker GHSR    - Correction of experimental biases    - NGFN epigenetics platform     - Initial profiling

   - microRNA-192 promoter in PDAC    - Epigenetically regulated miR-375    - Carcinoma drug resistance


  ...
 







..
..

FINISHED PROJECT:
GHSR DNA hypermethylation: a common epigenetic alteration of high diagnostic value in many cancers

           logo NGFN                logo DAAD

Diagnosis
Identification of a single molecular trait that is determinant of common malignancies may serve as a powerful diagnostic supplement to cancer type-specific markers. Substantial hypermethylation at the promoter and first exon of growth hormone secretagouge receptor (GHSR) was found to be characteristic of seven studied malignancies with very high sensitivity and specificity. Discrimination of breast or pancreatic cancer from healthy tissue samples exhibited 100% specificity and sensitivity, for example.
...

Figure to the right: Typical ROC diagrames are shown for five particular cancer entities. Also the overall accuracy for all studied cancers is presented. The AUC value indicates the respetive degree of diagnostic accuracy.

Early detection
Moreover, differential methylation was observed for ductal carcinoma in situ (DCIS), for instance, which is considered an early stage breast cancer that may progress to invasive cancer. GHSR gene methylation could therefore be particularly attractive for early detection, which is a key factor in cancer control.









Field defect in sporadic cancers
The increased methylation of the signature CpGs was also present in normal-appearing tissues collected from cancer patients, but not in samples obtained from healthy donors. This suggests the involvement of DNA methylation in a “field defect”. Field defect (or field cancerization) refers clinically to the existence of pre-neoplastic alterations in cells of a tissue that are associated with local recurrences. From a molecular point of view, this phenomenon has been explained by genetic abnormalities in patients with familial cancers. However, our data propose a contribution of epigenetic alterations to the field defect in sporadic cancers. GHSR methylation could thus possibly act as a marker of treatment success and recurrence.
...
Figure to the left: To visualise differences in the degree of methylation in breast samples, correspondence analysis (CA) was used. In the projection plot, each sample is depicted as a coloured square and CpG sites that exhibited the most significant differential methylation levels are represented as black dots. All co-localise with the cancer samples at the right side of the plot, indicating that the highest methylation level is found in cancer. In contrast, the healthy samples are located to the left, in the opposite direction off the centroid, indicating that the CpGs are at the lowest level of methylation in these samples. Likewise, based on the localisation of normal-appearing tissues of cancer patients and benign samples along the horizontal axis (first principal component; i.e., the direction along which the samples show the largest variation), it can be seen that an intermediate methylation load existed in these samples


Amini et al. (2019) J. Cell. Physiol., in press.  pdf icon
Jandaghi et al. (2015) Cell Cycle 14, 689.  pdf icon

.
Moskalev et al. (2015) Oncotarget 6, 4418.  pdf icon
Botla et al. (2012) Breat Cancer Res. Treat. 136, 705.  pdf icon
..
..








..
..
FINISHED PROJECT:
Early epigenetic down-regulation of microRNA-192 expression promotes pancreatic cancer progression
logo NGFN

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.
rr
Botla et al. (2016) Cancer Res. 76, 4149. 
pdf icon

Methylation patterns of miR-192 promoter

Down-regulation of miR-192 in chronic pancreatitis (CP) and PDAC patient samples compared to normal pancreas (NP) is epigenetically regulated. (left) Pooled bisulfite sequencing of the CpG island located 3.5 kb upstream of miR-192  revealed an increase in its methylation status in PDAC and CP samples compared to NP (boxed region in the heatmap). (right) Hypermethylation inversely correlated with expression of miR-192 in CP and PDAC.
.

..




.

...
FINISHED PROJECT:
Correction of PCR-bias in quantitative DNA-methylation studies

          logo NGFN                logo DAAD

PCR amplification of bisulfite-treated DNA is a processing step that is common to many currently used methods of quantitative methylation analysis. Preferential amplification of unmethylated alleles – known as PCR-bias – may significantly affect the accuracy of quantification. To date, reported processes for avoiding PCR-bias relied on an optimisation of PCR conditions. Although shown to be effective for particular genes, the implementation is time-consuming and labour-intensive, especially if multiple loci are analysed, thus the usefulness remains contradictory.
...
We developed an effective method of correcting biased methylation data. As opposed to refining experimental conditions, we approached the correction of amplification bias by accepting its occurrence during experimentation but adjusting the initial amplification result by a comparison to calibration data and the application of regression curves for deriving correction factors. As few as three calibration samples (0, 50, 100% methylation) are sufficient to define methylation degrees accurately. Two types of regression – hyperbolic and cubic polynomial – were checked. The correction process based on cubic polynomial regression was found to be superior overall.
...
The method is applicable irrespective of the locus that is interrogated or the number of sites analysed. Based on curve-fitting, the method is not influenced by the type of bias – preferential recovery of methylated or unmethylated alleles – and works equally well for both. Furthermore, any bias that was additionally introduced by the analysis procedure, for example a sequencing readout, could be compensated by the very process. The method is also automatable for high-throughput analyses.

...
Figure legend: The degree of bias introduced by PCR-amplification is shown for three gene promoters.The apparent degree of methylation observed after amplification (y axis) was plotted as a function of the actual methylation (x axis). The red line indicates the regression curve fitted to the data. Without correction (left) there was partly a substantial deviation between real and observed methylation, as indicated by the distance between the red line and the expected diagonal. The deviation basically disappeared upon correction (right).


Kapsner et al. (2021) Int. J. Cancer 149, 1150-1165. pdf icon
Moskalev et al. (2011) Nucleic Acids Res. 39, e77.   pdf icon
  











...

 
FINISHED PROJECT:
Epigenetically de-regulated miR-375 is involved in a positive feedback loop with Estrogen Receptor alpha in breast cancer cells

     logo NGFN                  logo BMBF

Breast cancer is the leading cause of cancer death in women worldwide. Although it is a heterogeneous disease, two-thirds of breast cancers share the common feature of being dependent on the presence and interaction of estrogen with the nuclear Estrogen Receptor α (ERα) protein. Approximately 70% of invasive breast cancers express ERα in actively proliferating cells.
...
It has become evident that ERα is up-regulated in luminal mammary epithelial cells during early stages of tumorigenesis and its overexpression is an important stimulatory factor for proliferation of mammary cells, leading to cell division and eventually to tumor development. The obvious role of ERα signalling in orchestrating the expression of genes involved in growth-related pathways, has established ERα as an important therapeutic target in breast cancer treatment. However, our understanding of the molecular mechanisms underlying deregulation of this signaling pathway is scarce.
...
We identified a high expression of microRNA 375 (miR-375) in ERα-positive cell lines. miR-375 overexpression is mainly caused by the loss of epigenetic marks, such as H3K9me2 and local DNA hypomethylation, which, in turn, triggers the dissociation of the transcriptional repressor CTCF from the promoter and enables interactions of ERα with regulatory regions of miR-375. Inhibition of miR-375 in ERα positive MCF-7 cells results in reduced ERα activation and cell proliferation.
...
A combination of expression profiling from tumour samples and miRNA target prediction identified RASD1 as a potential miR-375 target. Our findings show that miR-375 regulates RASD1 through targeting its 3' UTR. In addition, we demonstrated that RASD1 negatively regulates ERα expression. Our data indicate the existence of a positive regulation between ERα and miR-375 and suggest new strategies for the treatment of ER-positive invasive breast tumours.
...
Further analyses are under way to add more regulative mechanisms involved in the overall process.

de Soza Rocha Simonini et al. (2010) Cancer Res. 70, 9175-9184.  pdf icon












FINISHED PROJECT:
Systematic Methological Platform Epigenetics
logo NGFN

locations of project partnersAs part of the National German Research Network (NGFN), we applied microarray and sequencing technology toward a genome-wide and at the same time high-resolution analysis of DNA methylation patterns.
...
The group assembled in this platform had established the means for (1) the sodium bisulfite modification of DNA samples for the generation of methylation dependent polymorphisms, (2) the detection of the resulting single-base polymorphisms on complex oligonucleotide microarrays or by sequencing processes and (3) bioinformatics platforms for subsequent data analysis, with a special focus on combining the epigenetic information with transcript profiles and clinical information. Also, clinical material with corresponding clinico-pathological information was available for the analyses performed within the platform.
...
Infrastructure was provided for collaborations within the NGFN-network and beyond. Different systems were provided, which met the requirements of the respective project. By comparisons between them, also standards were defined that allow comparability of data across platforms and various areas of analysis. Also for this purpose, the close connection and integration of the consortium with international partners was crucial. The platform combined groups with complementary but nevertheless sufficiently overlapping expertise, competence and capability. Parallel to data production and a biological, functionally oriented interpretation, the resulting information was used for biomedical applications. In addition, technical developments were performed in order to improve quality and reliability.
...
The results from the platform's activities were: (1) definition and establishment of standards for epigenetic analyses on microarray systems; evaluation and comparison to other systems via external networking; (2) establishment of genome-wide microarrays for a global analysis of disease-relevant variations in methylation; (3) identification of disease-specific markers or patterns for diagnosis and prognosis; (4) identification of methylation patterns that are indicative for the action of drugs (clinical and commercial utilisation by the respective platform- and NGFN-partners). In terms of tumour entities, there was a focus on chronic lymphocytic leukemia, melanoma, pancreatic and breast cancer.
...
Specific results were a process for the correction of experimental biases during the measurement of methylation, methylation patterns closely associated with the occurance of pancreatic cancer as well as the elucidation of functional mechanisms in breast and pancreatic cancer.


de Soza Rocha Simonini et al. (2010) Cancer Res. 11, 162-167. pdf icon
Moskalev et al. (2011) Nucleic Acids Res. 39, e77.  pdf icon
Moskalev et al. (2012) Genes Chrom. Cancer 51, 105-110. pdf icon






 
Jörg Hoheisel (coordinator) DKFZ, Heidelberg ... Methodical analysis of the methylation sites in chromosome 21:




Frank Lyko DKFZ, Heidelberg




Hermann-Josef Gröne DKFZ, Heidelberg
.. Jörn Walter Universität Saarbrücken



Andreas Waha Universität Bonn

Albert Jeltsch Internationale Universität Bremen



Matthias Schuster Epigenomics, Berlin

Richard Reinhard Max-Planck-Institut für Molekulare Genetik, Berlin



Peer Stähler febit biotech, Heidelberg

Matthias Platzer Leibniz Institut für Altersforschung, Jena





...









FINISHED PROJECT:
High definition profiles of drug-resistent breast and ovarian carcinoma

                        logo BMBF

Acquired drug resistance represents a frequent obstacle which hampers efficient chemotherapy of cancers. Hence, treatment via standardised chemotherapy fails in a large fraction of patients, making additional therapy regimens necessary. In order to minimise side-effects caused by ineffective chemotherapy, prediction of individual patients’ response to therapeutic agents before the commencement of treatment would be desirable. The contribution of aberrant DNA methylation to the development of drug resistant tumour cells has gained increasing attention over the past decades. Hence, the objective of this study was to characterise DNA methylation changes which arise from treatment of tumour cells with the chemotherapeutic drug doxorubicin.
...
DNA methylation levels from CpG islands (CGIs) linked to twenty-eight genes were analysed, whose expression levels had previously been shown to contribute to resistance against DNA double strand break inducing drugs or tumor progression in different cancer types. High-definition DNA methylation profiles which consisted of methylation levels from 800 CpG sites mapping to CGIs around the transcription start sites of the selected genes were determined. In order to investigate the influence of CGI methylation on the expression of associated genes, their mRNA levels were investigated via qRT-PCR. It was shown that the employed method is suitable for providing highly accurate methylation profiles, comparable to those obtained via clone sequencing, the gold standard for high-definition DNA methylation studies.
...
In breast carcinoma cells with acquired resistance against the double strand break inducing drug doxorubicin, changes in methylation of specific cytosines from CGIs linked to thirteen genes were detected. Moreover, similarities between methylation profiles obtained from breast and ovarian carcinoma cell lines with acquired doxorubicin resistance were found. The expression levels of a subset of analysed genes were shown to be linked to the methylation levels of the analyzed CGIs.
...
Our results provide detailed DNA methylation information from two separate model systems for acquired doxorubicin resistance and suggest the occurrence of similar methylation changes in both systems upon exposure to the drug. This collaborative effort is being continued with the aim of stratification of patients prior to treatment dicisions.

Böttcher et al. (2010) PloS ONE 5, e11002.   pdf icon
 
methylation differences in gene promoters











FINISHED PROJECT:
High-resolution epigenetic profiling
logo DFG     

The basis for the methodological NGFN platform was laid in an early collaborative projects with Frank Lyko (Division of Epigenetics, DKFZ) as well as the company Epigenomics in Berlin, funded by the Deutsche Forschungsgemeinschaft.
...
In a pilot study, we studied the CpG island in the promoter region of the tumour suppressor gene p16. In total, 876 oligonucleotide probes of 21 nucleotides in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines.
...
In addition, we developed alternative modes and techniques for on-chip detection. For example, primer extension reactions were developed, which reduced significantly the number of oligonucleotides needed for an analysis and concomitantly increased the accuracy of base calling, since the process combines binding to a complementary oligonucleotide with the specificity of the polymerase.

For this purpose, we used and modified in collaboration the APEX system established in the group of Andres Metspaly (Tartu University, Estonia). In this process, the chip-bound primers hybridise to the genomic sequence directly adjacent to the base of interest. Upon an incubation with labelled dideoxynucleotides, the fitting nucleotide is incorporated by the polymerase and can be detected by means of the specific fluorophore.



Beier et al. (2004) BIOforum 05/04, 31-33.

Mund et al. (2005) Nucleic Acids Res. 33, e73.

Beier et al. (2005) Exocrine Pancreas Cancer (Gress, T.M. et al., eds.), Solvay, 254-260.


Beier et al. (2007) Adv. Biochem. Engineer/Biotechno 104, Volume: Analytics of Protein-DNA Interactions (Seitz, H., ed.), Springer Verlag, 1-11.


correlation data
...









                       Overview
DKFZ-logo
..
DKFZ Homepage
Top of Page