Epigenetic Inactivation of Cell-Cycle Regulators in Lung Cancer

Table of Contents

Projektleiter und Institution

Prof. Dr. Andrea Tannapfel, Universitätsklinik Leipzig, Institut für Pathologie

Prof. Dr. Adrian Gillissen, Robert-Koch-Klinik, Klinikum „St. Georg”, Leipzig

Prof. Dr. Dr. Wataru Yasui, Hiroshima University, Department of Molecular Pathology

Stipendiatin

Cornelia Köhler

DNA methylation is a crucial component of the epigenetic control of gene activity through the regulation of chromatin state. It has been found that DNA methylation is required for protecting chromosomes and it is involved in recognition of parental DNA strands during mismatch repair [1]. DNA methylation also plays a very important role in regulating the spatial distribution of gene expression [2]. A number of factors involved in this process, such as methyl-binding proteins and histone-deacetylases have been identified [3]. Similar to other somatic genome alterations present in cancer cells, including gene deletions and gene mutations, DNA methylation changes often affect gene function; methylation of CpG dinucleotides clustered into CpG islands encompassing the transcriptional regulatory region of genes has been associated with transcriptional “silencing“ of many critical genes in cancer cells. Unlike other somatic genome alterations in cancer cells, however, DNA methylation changes typically do not disrupt DNA sequence. For this reason, somatic changes in DNA methylation in cancer cells are thought to be potentially reversible “epigenetic“ genome lesions, rather than irreversible “genetic“ genome alterations [4].

If suitable CpG markers are identified that are selectively hypermethylated in lung cancer (LC) but not in normal epithelium. They could be potential markers useful in classifying LC and defining subgroups of patients, as well as lend themselves to diagnosis of occult disease or recurrence. The detection of methylation of particular sets of genes could help select patients that respond well to certain drugs. Importantly, these studies will interact with preclinical and early clinical studies underway for compounds that reverse methylation-induced transcriptional silencing and thus have tremendous potential for translation to the clinic [5].

To additionally define epigenetic modifications and order of epigenomic events at CpG islands on a global scale, a microarray system that combines gene expression, DNA methylation, and DNA-protein interaction analyses comparing results from lung cancer tissue with specimens from healthy bronchi/lung tissue will be employed in the network as platform technique [6].

Our study represents a genomic approach that is capable of dissecting the complex hierarchy of transcriptional controls orchestrated by the epigenomic machinery. This integrated microarray system allows for both the identification of individual genes and a systematic analysis of the relationship among the epigenetic machinery, promoter targets, and downstream responses regulated by the epigenome.

We plan to generate array data in the lab of Professor Yasui, Hiroshima, Japan, which is an internationally accepted and well-known experts in the field of methylation/acetylation.

He is willing to generate the biological as well as bioinformatory data of our project. Within the network, the applicant is collecting tumour specimen of patients with LC. The analysis will take place in the Hiroshima lab.

Data validation and candidate methylation screening will be the second part of the project and take place in Leipzig (Institute of Pathology and St. George Medical Center, Robert-Koch-Hospital).

The objectives will be at follows:

• Identification of epigenitically altered genes for targeted therapy of lung cancer.

• Screening for possible methylation targets in Hiroshima/Japan using microarrays and sequencing. Implementing these methods in Leipzig and subsequent biological assessment in lung cancer specimens.

• Identification of methylated genes in lung cancer responsible for tumorgenesis with the intent of therapeutic intervention by reversing methylation, thereby reactivating the silenced gene.

• Comparing these data with healthy bronchopulmonary tissue.

The program has three integral parts which take place in Japan and Leipzig. The applicant has to learn the microarray technique in the Yasui lab, using tumour specimen from Germany (tumour bank Robert-Koch-Hospital).

All patients with bronchial carcinoma (NSCLC and SCLC) will be included as far as tumour tissue can be obtained and written consent of voluntary participation is obtained. We expect about 70 % NSCLC and 30 % SCLC. Using the tumour bank of the Robert-Koch-Hospital 500 patients may be included over time once specimen collection and documentation of these patients are already in place. Once the NSCLC is expected to be the majority the patients will be statistically divided in three groups: a) NSCLC (Stadium I-IIIA), b) NSCLC (Stadium IIIB-IV), c) SCLC.

The identified candidate genes will be assessed in a large series of tumour. The biological relevance (is the identified gene a tumour suppressor?) will be analysed in the Institute of Pathology. The identified gene will be transfected into a cell culture system to be able analyse possible effects on cell cycle and marker expression.

The project was designed as a collaboration project to obtain data from Leipzig's patients using the Japanese chip technology and biostatistics (Abb. [1]). The project as described above does already exist for gastrointestinal tumors. Thus, lung tumor specimen will be newly included in an already existing international scientific network collaboration effort. Therefore, the network will be expanded by a new organ tumour (lung cancer), using an already existing methodological platform. The advances of these complex techniques will be transferred to both German partners. The pre-existing network will be further expanded covering pulmonary oncology as well.

Fig. 1 Project description (see text for details) in which the scholarship will be embedded.

Literatur

• 1 Esteller M. DNA methylation and cancer therapy: new developments and expectations.  Curr Opin Oncol. 2005 Jan;  17 (1) 55-60
• 2 Das P M, Singal R. DNA methylation and cancer.  J Clin Oncol. 2004 Nov 15;  22 (22) 4632-4642
• 3 Khan A U, Krishnamurthy S. Histone modifications as key regulators of transcription.  Front Biosci. 2005 Jan 1;  10 866-872 Print 2005 Jan 1
• 4 Mockler T C, Ecker J R. Applications of DNA tiling arrays for whole-genome analysis.  Genomics. 2005 Jan;  85 (1) 1-15
• 5 Steensel B van, Henikoff S. Epigenomic profiling using microarrays.  Biotechniques. 2003 Aug;  35 (2) 346-350, 352-354 356-357
• 6 Shi H, Maier S, Nimmrich I. et al . Oligonucleotide-based microarray for DNA methylation analysis: principles and applications.  J Cell Biochem. 2003 Jan 1;  88 (1) 138-143