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.

Acquisition of an oncogenic fusion protein is sufficient to globally alter the landscape of miRNA expression to inhibit myogenic differentiation

“…Interestingly the presence of PAX3-FOXO1 promoted more robust changes in miRNA expression at 30 minutes with 72/82 miRNA (89%) having ≥1.3-fold increases or decreases in expression, with nearly half of these miRNA changing over 2.5-fold (Figure 2 ). The results described here are part of a larger genomics study and as such the observed changes in miRNA expression were previously validated by qRT-PCR and published by us [ 19 , 20 ].…”

“…The transduced cells were selected with puromycin; selected cells were harvested from three independent transductions and pooled, resulting in a single mixed population for each individual construct, which removes the potential for variability that may occur from clonal effects. The observed level of PAX3-FOXO1 expression is equivalent to that of the fusion protein in ARMS tumor cell lines [ 19 , 20 ] and is therefore directly relevant to the role of the oncogenic fusion protein in ARMS.…”

“…We previously proposed the use of a multi-faceted regimen that targets genetic, molecular, or biological characteristics of ARMS. This proposed regimen included LiCl to target phosphorylation of PAX3-FOXO1, chloroquine to target the aneuploid state, and Cixutumumab to target growth factor related proliferation [ 20 ]. However, the present work argues against the inclusion of Cixutumumab.…”

“…The inhibition or replacement of microRNAs are growing in interest for use as potential cancer therapies [ 35 ], in particular as potential targets for differentiation therapy in solid tumors such as ARMS [ 7 ]. When combined with LiCl and chloroquine, this multi-faceted regimen would inhibit the activity of PAX3-FOXO1 [ 36 ], promote the death of aneuploid cells, which is induced by the presence of the oncogenic fusion protein [ 20 ], and potentially reactivate the differentiation process, thereby attacking ARMS tumor cells on multiple different biological fronts. Experiments are ongoing to test this proposed multi-faceted treatment regimen.…”

Developing cancer therapies – think global

“…In a recent publication we identified a known oncogenic protein to be one such driving mutation [1]. Alveolar Rhabdomyosarcoma (ARMS), an aggressive pediatric muscle tumor, is primarily characterized by the somatically acquired t(2;13)(q35;q14) balanced chromosomal translocation, which generates the oncogenic fusion protein PAX3-FOXO1.…”