Research / Clinical Summary

Karen Arden, PhD Adjunct Professor, Medicine Cancer Genetics Program Contact by Email

My research interests center around cancer genetics. A major focus has been the characterization of the function of the fusion gene associated with the t(2;13) found in the alveolar subtype of the skeletal muscle tumor rhabdomyosarcoma. This chromosomal translocation results in the fusion of the PAX3 gene at 2q35 to the FKHR gene at 13q14. Little is known about the role the PAX3-FKHR fusion gene plays in the etiology of rhabdomyosarcoma despite its unique and specific association with 80% of rhabdomyosarcomas of the alveolar subtype. We have produced transgenic mice in which PAX3-FKHR expression is driven by mouse Pax3 promoter/enhancer sequences. The resulting phenotype closely resembles that reported for the Pax3-defective Splotch mice suggesting that PAX3-FKHR may be interfering with the normal function of Pax3. We have tested this hypothesis by mating our PAX3-FKHR mice to Splotch mice thus reducing the levels of Pax3 resulting in an increase in phenotypic severity. Furthermore, interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal PAX3 functions, possibly as a prelude to transformation. However, in contrast to the fusion genes created as a result of similar types of chromosomal translocations in leukemias and lymphomas, PAX3-FKHR alone is not sufficient to elicit tumors as demonstrated by the absence of tumors in over 300 mice. It is possible that PAX3-FKHR may cooperate with other mutations such as p53 inactivation to cause transformation. Alternatively, the fusion gene may serve to cause developmental arrest creating an expanded pool of target cells to accumulate additional genetic lesions. Studies are currently underway to address these possibilities.

To further characterize the role of the fusion gene in rhabdomyosarcoma we have undertaken a detailed study of FKHR and two additional genes which make up the FKHR sub-family of forkhead genes. We had previously described this sub-family in humans and have now cloned and characterized the mouse orthologs of these genes, Fkhr1, Fkhr2, and Afxh . Our previous work demonstrated that the transcriptional activity Fkhr1 is subject to negative regulation by growth factors that activate Phosphatidylinositol-3 kinase (PI-3 kinase). Activation of the PI-3 kinase signal transduction pathway affects inactivation of Fkhr1 by two distinct mechanisms: inhibition of transcriptional activity and stimulation of active (CRM1-mediated) nuclear export. A key mediator for the effects of PI-3 kinase activation on the transcriptional activity of Fkhr1 is the serine/threonine kinase AKT (or Protein Kinase B, PKB). Our studies have shown that not only is Fkhr1 a direct target of AKT (Protein Kinase B, or PKB)-mediated phosphorylation, but also that this phosphorylation is instrumental in the functional inactivation of
Fkhr1.

In an effort to extend our biochemical and cell cultures studies we have generated a line of mice that carry a targeted (or null) allele of the Fkhr1. Embryos lacking functional Fkhr1 die at midgestation of cardiovascular insufficiency. A detailed analysis is currently underway. We have also generated lines of mice that carry null alleles of Fkhr2 and Afxh , the other two members of the FKHR sub-family of forkhead genes. We anticipate that studies, similar to those undertaken with the Fkhr1-/- animals, will provide insight into the roles of Fkhr2 and Afxh during development.

As part of a San Diego-based multi-institutional effort including the UCSD Cancer Center, the Veteran’s Administration Hospital, the Sidney Kimmel Cancer Center and the Ludwig Institute we are using Restriction Landmark Genomic Scanning (RLGS) to identify changes in DNA methylation in prostate tumors that will contribute to the molecular characterization of prostate cancer. RLGS provides both a quantitative genetic and epigenetic assessment of cytosine methylation in thousands of CpG islands in a single gel without requiring any prior knowledge of gene sequence. RLGS relies on the two-dimensional separation of radiolabeled genomic DNA into approximately 2,000 discrete fragments. Due to the selection of particular restriction enzymes to cut the DNA, each fragment is likely to contain gene sequences. The resulting RLGS profile displays both the copy number and methylation status of the CpG islands. After the identification of differences in RLGS profiles the fragments of interest can be cloned and subjected to sequence analysis. Tumor-specific changes in RLGS profiles have been previously identified for other tumor types (Costello et al., 2000). Comparing prostate tumor profiles will allow us to easily identify patterns of similarity between tumors as well as differences and establish markers that will contribute to the molecular classification of tumors.

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