Characterization of a novel amplicon in 8q in hepatoblastoma

The proposed project represents an effort to elucidate the molecular mechanisms that are involved in the pathogenesis of the rare but highly malignant childhood tumour hepatoblastoma (HB). In the past decades the identification of genes that are involved in frequent genetic aberrations that are associated with a specific or a broad variety of neoplasias, has uncovered a series of oncogenes and tumour suppressor genes. The detection of such genes rendered information that are used in clinical settings to provide patient prognosis information as it is exemplified for the MYCN gene amplification in neuroblastoma, and, further, may provide new potentials for identifying avenues for therapeutic intervention. Recently a comparative genomic hybridization (CGH) analysis of a large series of HBs showed that DNA copy number gain of chromosome 8 material is associated with a poor outcome for the patients and that the critical amplified region on chromosome 8 is located in 8q11-13. The identification of a gene with prognostic relevance within this critical region may be one of the potential benefits of the proposed studies. Therefore, based on the results of the CGH analysis we have subjected a hepatoblastoma primary tumour with amplification of this region to 2-dimensional DNA electrophoresis of genomic restriction fragments. By this means we were able to uncover at least four genes that are included in the amplicon of this tumour. Three of them, a tyrosine kinase, a serinehreonine kinase and a zinc finger protein have a strong oncogenic potential and may contribute to the malignancy of HB. Therefore, one of the major aims of the proposed project will be to study their role in HB. Moreover, the identified sequences will serve as entry-clones to fine map the minimal critical region of the amplicon in 8q12-13 in a large series of HBs. By this means we expect to identify all genes located in this region, whose overrepresentation and consequent overexpression may confer the poor prognosis for HB patients. We further propose to characterize pathways by which the identified genes contribute to the malignancy of HB. Our recent results of studies performed for hepatoblastoma, and other childhood cancers show that the combined application of genomic comparative hybridisation and 2-dimensional electrophoresis of genomic restriction fragments represent one of the most powerful tools to rapidly identify genes affected by genetic imbalances. The enormous progress that has been made within the last few years in uncovering the human genome and the rapidly increasing amount of available sequence information has contributed to the even greater effectiveness of this approach. Since comparative genomic hybridisation as well as other cytogenetic techniques have uncovered regions that are likely to harbor genes of oncogenic potential in a large number of other pediatric and adult tumours, there is a great interest to apply similar approaches as proposed here for hepatoblastoma to these tumour types. Therefore, the performance of the here proposed project will enable us to accumulate knowledge on how to most efficiently exploit the available data of the human genome which will facilitate further anticipated projects at our institute.

An oncogene is a gene that when expressed (produced) abnormally contributes to transforming a normal cell into a cancer cell. Under normal conditions, oncogenes play an important role in regulation of various cellular processes such as cell growth and death, differentiation, etc. However, due to mutations the gene might become hyper-active and subsequently the fragile balance between regulatory factors in the cell is destroyed, which leads to carcinogenesis. Gene amplification, in other words an increase of the copy number of the particular oncogene per cell, is a specific mechanism that can lead to abnormal over-production of the protein product encoded by this gene. The over-production of these proteins provides the cell a selective advantage, meaning the cell has better viability or the ability to reproduce itself and survive even under for the normal cell unsuitable conditions which is one of the characteristics of the cancer cells and a feature important for tumour growth. The detection of oncogenes that are hyper-activated in specific tumours is important to better understand the mechanisms of carcinogenesis of a specific tumour entity. The hope and aim are that this information may in the future be used in clinical settings to provide prognosis information or even may open up new strategies for therapeutic intervention. To identify potential oncogenes, we studied within the frame of our project an amplified region (amplicon) on the long arm of chromosome 8q in hepatoblastoma (HB), because copy number gain of this region in the rare but highly malignant childhood tumour HB has been associated with poor outcome for the patients. Our results showed that compared to healthy liver samples, an oncogene called PLAG1 is over-expressed in HB cells, indicating indeed the miss-regulation of this gene. However, over-expression of PLAG1 has been observed also in the patients that did not show the evidence of 8q amplification. It is known that PLAG1 is able to up-regulate the function of another gene, IGF2, which is a protein with growth-promoting activity which supports the possible involvement of PLAG1 in cancer development. However, there are still many questions concerning both proteins, their function and cooperation within the cell that need to be answered before the final conclusion about their role in HB can be drawn. During the characterisation of HB amplicon we established a highly efficient methodical repertoire in our laboratory that can be used to find amplified genes also in other tumour entities. Therefore, we implemented these methods also for the characterisation of an amplicon of the long arm of chromosome 11 (11q) in acute myeloid leukaemia (AML) patients. The AML patients we studied are characterized multiple copies of the MLL gene in their leukaemic cells as demonstrated in our institute. This gene has been known to be involved in leukemogenesis due to other chromosomal rearrangements and it is frequently contained in the 11q amplicon. Thus, it was the MLL gene that has been believed to be the main gene triggering the amplification on chromosome 11q observed in AML. However, in our study we have identified a highly complex nature of the 11q-amplicon has and the involvement also of genes from other parts of chromosome 11q. We could show that two additional regions are frequently co-amplified with MLL gene. One of them is located more proximal in chromosome 11, in 11q13.5. A few genes with potential oncogenic function are located within this region, thus the characterisation of their possible involvement in leukaemia is and will be a subject of our present and future study.