2006 Grant Recipient - Dr. Frederic G. Barr, University of Pennsylvania School of Medicine

The Joanna McAfee Childhood Cancer Foundation announces its grant recipients for 2006. Dr. Frederic G. Barr of the University of Pennsylvania School of Medicine in Pennsylvania, PA was awarded a $9000 grant to continue his work on “The Role Of Fusion Oncoproteins And Collaborating Events In Alveolar Rhabdomyosarcoma”

 

Dr. Frederic G. Barr

University of Pennsylvania, School of Medicine

505C Stellar-Chance

422 Curie Blvd.

Pennsylvania, PA 19104-6082

(215)898-0884

(215)898-4227 (fax)

www.med.upenn.edu/camb/faculty/cgc/barr.html

 

OVERVIEW OF THE RESEARCH PROJECT

Two major research goals in my laboratory are to investigate the function of the PAX3-FKHR and PAX7-FKHR fusion oncoproteins in the pediatric soft tissue cancer alveolar rhabdomyosarcoma (ARMS) and to define and characterize additional genetic events that collaborate with the fusion proteins in the pathogenesis of ARMS.

Previous cytogenetic studies showed that the majority of ARMS cases have a characteristic translocation involving chromosomes 2 and 13, and a smaller subset have a translocation involving chromosomes 1 and 13. By using available genomic reagents to map the regions on chromosomes 2 and 13 in which the translocation breaks occur, my research group established that the 2;13 translocation consistently breaks the PAX3 gene on chromosome 2 and the FKHR gene on chromosome 13 to generate a PAX3-FKHR fusion gene. Similarly, the 1;13 translocation breaks the related PAX7 gene on chromosome 1 along with FKHR to generate a PAX7-FKHR fusion gene. These fusion genes are expressed as PAX3-FKHR and PAX7-FKHR fusion transcripts, which are translated into PAX3-FKHR and PAX7-FKHR fusion proteins, combining domains from the PAX3 (or PAX7) and FKHR proteins to generate novel functions in the tumor cell. Wild-type PAX3, PAX7, and FKHR are transcription factors, and PAX3-FKHR and PAX7-FKHR are aberrant transcription factors that bind to DNA and activate a novel set of genes to generate a unique gene expression program.

In addition to the finding of chromosomal translocations, cytogenetic studies have frequently shown evidence of gene amplification in ARMS, in which a region of the genome is present in multiple copies, usually on extra-chromosomal elements. Using a technology called comparative genomic hybridization to survey copy number changes throughout the genome, previous studies demonstrated frequent sites of high-level copy number gain on specific regions of chromosome 2 and 12. Additional sites of highlevel copy number gain were also seen on two regions of chromosome 13 and one region of chromosome 1. Based on these findings, the hypothesis is proposed that one or more genes are present in these regions of high copy number gain. Furthermore, as a result of the increased number of gene copies, the corresponding protein products of these genes are expressed at higher levels in these cells, resulting in a higher level of function than when the genes are present at normal copy number.

To investigate the function of PAX3-FKHR and PAX7-FKHR, we are interested in developing a human cell culture system that is relevant to the etiology of ARMS. Since ARMS is a cancer related to the skeletal muscle lineage, we decided to specifically work with a human skeletal muscle precursor cell, termed a myoblast. Since the fusion gene cannot convert myoblasts into cancerous cells alone, a major goal of these studies is to determine a rationale combination of genes that will collaborate with the fusion gene to transform these myoblasts into cells with oncogenic properties. Recent studies have defined properties that must be overcome to transform human cells, and thus model oncogenes that have been shown to overcome these properties in other cell types will be the starting point for these studies. To investigate the acquisition of oncogenic (tumor-like) properties in cell culture, we will assay for two classic features of transformed cells in culture - focus formation, which results when a cell divides irrespective of the inhibitory effects of neighboring cells, and soft agar colony formation, which results when a cell divides without the need for normal extracellular requirements for anchorage. In addition, we will also assay the ability of these oncogenes to inhibit the cells response to stimuli that normally cause the cells to stop dividing and differentiate into more mature muscle cells. Once cells expressing various combinations of fusion gene and collaborating genes have been analyzed by these assays, the gene expression pattern responsible for these phenotypes will be investigated by expression profiling studies. In particular, to investigate downstream consequences of the PAX3-FKHR or PAX7-FKHR fusion genes, we will compare cells expressing the collaborating genes with or without the fusion gene. In these studies, mRNA will be isolated from the various cell populations and analyzed on microarrays to determine the expression levels of numerous genes throughout the genome. By comparing differences in gene expression, the genes responsible for these phenotypes can be potentially identified and then further investigated in directed experiments.

To identify actual collaborating genes in ARMS, we are utilizing a technique similar to comparative genomic hybridization in which copy number is quantified in tumor DNA on a microarray containing 50,000 separate genomic markers. Such technology permits the regions of copy number gain to be localized with very high precision, and thus comparison of similar regions of copy number gain among multiple tumors will permit a minimal region of copy number gain to be localized. When this genomic information is compared to the maps of human genes (derived from the Human Genome Project), the genes potentially involved by the amplification event can be determined. Furthermore, we have performed expression profiling on many of these tumors and thus have information on the expression status of most of the genes in the genome. This information will thus permit us to determine which amplified genes are actually overexpressed at the mRNA level, thus further implicating these genes as true targets of the amplification process. Candidate genes will be further investigated by developing quantitative PCR and RT-PCR assays to measure copy number and expression of the genes by an independent method and thus to verify the change in expression in tumors with and without amplification. The oncogenic role of specific genes will finally be tested at the functional level by cell culture assays described above with and without PAX3-FKHR. Finally, the clinical utility of these genes will be investigated by studies of copy number or expression in patients on Childrens Oncology Group Soft Tissue Sarcoma Committee clinical trials. In particular, expression or copy number will be compared with outcome measures to determine the utility of these amplified genes as clinical markers.

Though part of the functional and mapping studies described in this proposal are funded by two National Institutes of Health grants, it is important to emphasize that both grants are funded for less than the requested amounts. Furthermore, the approved budget for the annual renewal of these grants is being reduced each year due to the current budgetary constraints at the National Institutes of Health. The budget of one funded grant was initially reduced by 10% and the budget of the other funded grant was initially reduced by 23%. During the last annual renewal of each grant, there was an additional reduction of 2.35% for each grant budget, and these annual reductions are likely to continue to occur in the future. Therefore, there is a widening gap between the available funds and the needed funds to perform the optimal level of research. Funding from sources of support such as the Joanna McAfee Childhood Cancer Foundation is thus truly needed to bridge these gaps for this research effort on rhabdomyosarcoma.