Taking Aim at Important Targets in Sarcoma
First photo shows Rhabdomyosarcoma undergoing mitosis, producing two daughter cells. Second photo shows a young patient’s eye that has developed rhabdomyosarcoma of the orbit. (Photo credit: Online Journal of Ophthalmology: www.onjoph.com)
Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, a cancer of the body’s connective or supportive tissues such as muscle, cartilage, or fat. The two major classifications of RMS include the embryonal subtype, which accounts for approximately three-quarters of children diagnosed with RMS, and the more aggressive alveolar (ARMS) subtype, which has a five-year survival rate of less than 30 percent.
Poor prognosis in ARMS cases is associated with a chromosomal translocation, which results in the expression of a fusion protein composed of pieces of two distinct transcription factors, PAX3 and FKHR. Transcription factors recognize and bind specific DNA sequences, helping to recruit the cellular protein machinery required for gene expression. The PAX3-FKHR fusion contains the DNA binding domain of PAX3 and the machinery recruiting domain of FKHR, suggesting that PAX3 and the cancer-causing fusion protein recognize the same DNA sequences. PAX3 is thought to regulate genes important for embryonic and muscle development, but few specific genes have been identified. These genes controlled by PAX3 and, presumably the PAX3-FKHR fusion, are likely critical for promoting ARMS and could provide therapeutic targets to treat the disease.
To determine the PAX3/PAX3-FKHR target genes, Liang Cao, Ph.D., working with Paul Meltzer, M.D., Ph.D. in the Genetics Branch and their colleagues, decided to look throughout the genome of RMS cells for DNA sequences bound by the PAX3-FKHR fusion protein. The researchers found approximately 1200 potential binding sites in cells expressing the fusion protein. The specificity of the assay was supported by the identification of a known PAX3 site in the MYF5 gene, confirming that the PAX3-FKHR fusion binds sequences in ARMS that PAX3 regulates during development.
The majority of the identified PAX3-FKHR binding sites were found a significant distance from the start site of a gene or within introns, non-coding regions within human genes, both of which are valid locations for functional regulatory sites. Over 1000 genes overlapped with or were near the identified fusion binding sites, and, in agreement with the normal role of PAX3, most play a role in development. The scientists then demonstrated a correlation between the genes induced in 160 ARMS tumor samples and genes near the binding sites identified in the screen. Likewise, introduction of the fusion protein into RMS cells stimulated the expression of genes containing the identified binding sites, verifying these genes as bona fide PAX3-FKHR targets.
Many of these newly identified PAX3-FKHR regulated genes were previously implicated in the development of a variety of cancers. The fibroblast growth factor receptor 4 (FGFR4), which had two fusion protein binding sites, is associated with poor survival in ARMS patients and is over-expressed in pancreatic and head and neck cancers. MET, which is up-regulated in RMS tumors and has PAX3 regulatory sites within its introns, codes for a growth factor that promotes cancer cell proliferation, invasiveness, and metastasis. The researchers also found that PAX3-FKHR could regulate other transcription factors like MYCN that promotes the growth of RMS tumors. Likewise, the fusion protein could control the expression of the insulin-like growth factor 1 receptor (IGF1R), a potential target for antibody therapy.
In addition to identifying the genes regulated by PAX3/PAX3-FKHR, the scientists were able to study the mechanism used by PAX3 to control the expression of its target genes. PAX3 is known to be a weak transcription factor and might require other proteins to promote gene expression. The researchers found sequences called E-box motifs that co-localized with many PAX3 sites throughout the genome. E-boxes are bound by the MYF family of transcription factors, suggesting that PAX3 and MYF proteins collaborate to regulate the target genes.
This study was the first genome-wide study to identify genes controlled by the PAX3 transcription factor and the PAX3-FKHR fusion in ARMS. The results also implicate E-box proteins in the regulation of many of these target genes. The roles that the PAX3-regulated genes play in normal muscle development and in cancer initiation require further investigation. Additionally, the identified PAX3 target genes may serve as potential therapeutic targets since current treatments are unable to inhibit the PAX3-FKHR fusion directly.Summary Posted: Sun, 08/01/2010
Cancer Res. 2010 Aug 15;70(16):6497-508. Reviewed by Jennifer Crawford, PhD PubMed Link