Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion

Abstract

Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression.

HOXD8/DIAPH2-AS1 epigenetically regulates PAX3 and impairs HTR-8/SVneo cell function under hypoxia

“…The data from the GSE44667 database showed high methylation levels of the PAX3 gene in the placenta of patients with PE [10]. This result is consistent with the low expression of PAX3 in the same patients, which was identified in our previous study [3]. Therefore, this evidence suggest that high methylation levels of PAX3 gene are an important cause of low expression.…”

“…However, PAX3 is abnormally down-regulated in the placenta of PE patients, and a low expression of PAX3 is associated with impaired proliferation and invasion of trophoblast cells under hypoxia. This suggests that decreased expression of PAX3 is closely related to PE pathogenesis [3]. Previous studies have used high-throughput genomic and epigenomic technologies to identify higher methylation levels in the promoter region of PAX3 gene in the placenta of PE patients, which has provided an important clue for elucidating the down-regulation mechanism of PAX3 .…”

“…Our previous study identified that abnormal down-regulation of PAX3 expression plays a critical role in the occurrence and development of PE. Trophoblasts with a low PAX3 expression show inhibited proliferative and invasive capacities [3], whereas the invasion of the foetally derived extravillous trophoblasts (EVTs) toward the myometrium layer of the uterine wall is a key step in placenta development [4,5]. An impaired invasion of the trophoblasts is likely to cause defective arterial remodeling, resulting in inadequate blood flow in the placenta [6].…”

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.…”