Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects

Abstract

While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

Keywords: Pax3-FOXO1; alveolar rhabdomyosarcoma; aneuploidy; myogenesis; phosphorylation.

Figure 1. The expression of PAX3-FOXO1 promotes aneuploidy and chromosomal structural abnormalities

Expression of A. PAX3, PAX3-FOXO1 or B. PAX3-FOXO1 phosphomutants. Total extracts were made from stably transduced cells and protein was determined using an antibody specific for PAX3, as described in the Materials and Methods. Representative picture of a metaphase chromosome analysis for C. cells stably transduced with empty vector, PAX3 or PAX3-FOXO1 or D. individual PAX3-FOXO1 phosphomutants. The closed arrows indicate representative sister chromatid dissociation, the open arrow indicate representative telomere association, and the dotted arrow indicate a representative double minute.

Figure 2. Stably transduced primary myoblasts (vector, PAX3, wild-type PAX3-FOXO1, or PAX3-FOXO1 phosphomutant) were plated, grown for four days, growth determined using the CCK-8 cell counting kit and plotted as a function of time (A) from which doubling times were determined, as described in the Materials and Methods

Error bars represent the standard deviation from three independent determinations and P-values were computed using non-parametric two-way ANOVA analyses comparing each treatment condition to the empty vector negative control. (**P = 0.005, ****P < 0.0001).

Figure 3. Quantitative RT-PCR analysis for aneuploidy mRNA (A), proliferation mRNA (B) and miRNA (C)

Total RNA was isolated from stably transduced cells [empty vector (white), PAX3 (stippled), PAX3-FOXO1 (black), or the phosphomutants S201A (light gray), S205A (medium gray), or S209A (hashed gray). Quantitative RT-PCR was performed using primers specific for the indicated mRNA A. and B. or microRNA C., as described in the Materials and Methods. Error bars represent the standard deviation from three independent determinations and P-values were computed using non-parametric two-way ANOVA analyses. The asterisk indicates statistical comparisons between empty vector and each sample (*P = 0.001, **P < 0.0001). The hash-tap indicates comparisons between wild-type PAX3-FOXO1 and the sample (#P = 0.001).

Figure 4. Model describing the role of PAX3-FOXO1 in the development of ARMS and how it informs potential therapy development