Analysis of the transforming and growth suppressive activities of the PAX3-FKHR oncoprotein

Abstract

The 2;13 chromosomal translocation occurs in most cases of the cancer alveolar rhabdomyosarcoma (ARMS), and juxtaposes the genes encoding the PAX3 and FKHR transcription factors. The resulting chimeric protein PAX3-FKHR is a potent transcriptional activator, and is hypothesized to function as a dominant acting oncogene. To investigate its biological function, PAX3-FKHR was transduced into three immortalized murine cell lines in either a constitutive or inducible manner. These cells only tolerate expression of low PAX3-FKHR levels, which is sufficient for transformation in NIH3T3 cells. In contrast, higher PAX3-FKHR levels, which are comparable to the endogenous level expressed in ARMS cells, result in growth suppression. To determine as to which PAX3 functional domains are needed for growth suppression and transformation, inactivating mutations were introduced into the paired box and homeodomain of PAX3-FKHR. In these experiments, the homeodomain is necessary for transformation, but not growth suppression; whereas the paired box is not required for transformation but mediates growth suppression. In summary, our findings demonstrate that the transforming and growth suppressive activities of PAX3-FKHR are dominant at different activity levels and are mediated by distinct functional domains. These findings are consistent with the hypothesis that distinct expression pathways are operative in these opposing phenotypic end points.

FOXOs, cancer and regulation of apoptosis

“…The main evidence supporting this notion is that the Pax3-FOXO1 fusion protein is a more potent transcription activator than normal Pax3 protein in increasing the transcription of Pax3 target gene (Xia et al, 2002). The fusion protein is able to induce oncogenic transformation in cell culture (Scheidler et al, 1996;Kempf and Vogt, 1999;Xia and Barr, 2004), suggesting that the fusion protein may function as an oncogenic transcription factor by enhancing activation of normal PAX3 target genes. However, in transgenic or knock-in murine models, expression of PAX3-FOXO1 fusion proteins fails to induce tumors (Anderson et al, 2001;Lagutina et al, 2002;Relaix et al, 2003;Keller et al, 2004b).…”

Investigation of downstream target genes of PAX3c, PAX3e and PAX3g isoforms in melanocytes by microarray analysis

“…[9][10][11] Potential downstream targets of PAX3/Pax3 have been identified, including Met, a proto-oncogene 12 ; Waardenburg's syndrome type II associated gene, MITF 13 and the pigmentation gene, Trp1. 14 Other targets such as STX, TIMP3, NF-kB, NCAM, MyoD, Dep-1 and Mbp gene have also been reported.…”

Analysis of genetic events that modulate the oncogenic and growth suppressive activities of the PAX3-FKHR fusion oncoprotein

“…The PAX3-FKHR-ER fragment was subcloned into the pBabe-puro retroviral vector, and G48S and N269A mutations were introduced using corresponding primers 8 and the Quik-change site-directed mutagenesis kit (Stratagene). To generate the PAX3-FKHR-pBabe construct, a stop codon was introduced into the 3 0 end of PAX3-FKHR in PAX3-FKHR-ER-pBabe.…”

“…Based on these abnormal features, these fusion proteins act as aberrant transcription factors that alter the pattern of downstream gene expression within the cell and contribute to tumorigenesis, as evidenced by transforming activity in the NIH3T3 soft agar assay. [6][7][8] To further study the phenotypic consequences of fusion protein expression, PAX3-FKHR was introduced into NIH3T3 and other murine cells. 8 These experiments were conducted using the retroviral vector pK1, which contains a strong promoter, expressing the test and puromycin resistance genes as a single transcript with an internal ribosome entry site to permit translation of two proteins.…”

“…[6][7][8] To further study the phenotypic consequences of fusion protein expression, PAX3-FKHR was introduced into NIH3T3 and other murine cells. 8 These experiments were conducted using the retroviral vector pK1, which contains a strong promoter, expressing the test and puromycin resistance genes as a single transcript with an internal ribosome entry site to permit translation of two proteins. In contrast to control cells that progressively grew, cells expressing PAX3-FKHR diminished in number over a 6-day period at which point a population of cells started to grow.…”