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

Abstract

Alveolar rhabdomyosarcoma (ARMS) is associated with chromosomal translocations that generate PAX3-FKHR and PAX7-FKHR fusion oncoproteins. Based on studies demonstrating that high PAX3-FKHR expression causes growth suppression, the hypothesis is proposed that, during ARMS tumorigenesis, the translocations cause low oncoprotein expression and are followed by collaborating events that block growth suppression pathways and permit upregulation of oncoprotein expression. To investigate oncogenic function at low expression levels, PAX3-FKHR was introduced into NIH3T3 cells in the pBabe retroviral vector. Compared to high expression systems, PAX3-FKHR expression from pBabe was lower and did not suppress growth, but showed transforming activity in the soft agar assay. As a possible collaborating event, PAX3-FKHR paired box mutations were previously shown in high expression systems to reverse growth suppressive effects. In the low expression system, the paired box mutation enhanced transformation in soft agar and focus formation assays. Although these mutations are candidate collaborating events, sequencing of paired box regions in ARMS tumors did not identify mutations. Finally, genes from known genetic alterations in ARMS were introduced, alone or combined, into NIH3T3 cells with high PAX3-FKHR expression and did not rescue growth suppression. In summary, these studies provide a model for an event in ARMS tumorigenesis that enhances PAX3-FKHR oncogenicity and abrogates growth suppression, but do not demonstrate a known event occurring in ARMS tumors that fulfills these criteria. Alveolar rhabdomyosarcoma (ARMS) is an aggressive softtissue tumor associated with the skeletal muscle lineage that occurs mainly in children. 1 Cytogenetic analyses revealed that recurrent chromosomal translocations, usually t(2;13) (q35;q14) and less frequently t(1;13)(p36;q14), occur in ARMS. These translocations generate chimeric genes that encode fusion proteins with the N-terminal portion of PAX3 or PAX7 fused in-frame to the C-terminal region of FKHR. 2 These fusion products are more highly expressed in ARMS cells, 3 are more potent transcription factors, 4 and are expressed exclusively in the nucleus 5 compared to the corresponding wild-type (WT) products. 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. In contrast to control cells that progressively grew, cells expressing PAX3-FKHR diminished in number over a 6-day period at which po...

Identification of PAX3‐FKHR‐regulated genes differentially expressed between alveolar and embryonal rhabdomyosarcoma: Focus on MYCN as a biologically relevant target

“…Instead, we detected dramatic changes in neural development genes. This pattern may indicate that expression of (Xia et al, 2007), incubated in Iscove's medium (Gibco) containing 10% FBS with 0.35% agar (Agar Noble, Difco Laboratories, Detroit, MI), and seeded on top of 0.7% agar in the same medium. Cells were then incubated with the indicated 4-OHT concentration, and colonies were scored after 3 weeks.…”

“…B. For the focus formation assay, 10 3 cells were transduced with the indicated retroviral constructs (Xia et al, 2007), mixed with 10 6 nontransduced NIH3T3 cells in 100-mm dishes, and treated with the indicated concentrations of 4-OHT. After 2 weeks, three plates were stained and counted for each condition, and results were expressed as the mean (6SD).…”

“…In this experiment, the retroviral vector was pBabe, which expresses lower levels of P3FK/ER and, unlike the retroviral vector pK1, does not result in growth suppression (Xia et al, 2007). Transforming activity was assessed in two independent assays: soft agar colony formation and focus formation (Fig.…”

CDK4 Amplification Reduces Sensitivity to CDK4/6 Inhibition in Fusion-Positive Rhabdomyosarcoma

“…Human CDK4 or CDK6 were PCR amplified from the CDK4-MigR1 retroviral vector (25) or the CDK6-pCMV6-XL6 vector (Origene), respectively, subcloned into the AgeI and MluI sites of pINDUCER10, and sequence verified. Cells were incubated with doxycycline (Sigma) for 24 h to induce CDK4 or CDK6 expression.…”

Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates

“…Stable transfection and transduction of PC12 cells with tau constructs or HTRA1 were carried out as described (36,41).…”