Effects of the Oncoprotein PAX3-FOXO1 on Modulation of Exosomes Function and Protein Content: Implications on Oxidative Stress Protection and Enhanced Plasticity

Abstract

Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma classified into two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS). ARMS subtype is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 (P3F) that drives oncogenic cellular properties. To understand the role of the fusion oncoprotein in paracrine signaling, we focused on secreted exosomes, which have been demonstrated to contribute to metastasis in multiple tumor types. Advanced Proteomics-bioinformatics analysis of the protein cargo of exosomes isolated from C2C12 myoblasts transduced with P3F fusion gene revealed 52 deregulated proteins compared to control cells, with 26 enriched and 26 depleted proteins. Using both PANTHER gene classification and Ingenuity Pathway Analysis (IPA) software, we found that the main biological processes in which the 52 deregulated proteins are involved, include “catalytic activity,” “binding,” “metabolic process,” and “cellular process.” The pathways engaging the 26 enriched proteins include the “14-3-3 mediated signaling,” “cell cycle,” and “ERK5, VEGF, IGF1,and p70S6K signaling.” Furthermore, the main nodes in which deregulated exosome proteins and miRNAs intersected revealed pathways conferring protection from stress and promoting plasticity. Based on the bioinformatics analysis and the altered exosome proteome profile, we performed biochemical functional analysis to study the diverse properties of these exosomes where angiogenesis, stemness, and anti-oxidative stress properties were validated using different platforms. P3F-modulated exosomes activated ERK, 4-EBP1, and MMP-2 in recipient cells, and enhanced angiogenesis and stemness. In addition, P3F led to lower cellular reactive oxygen species levels and enhanced resistance against oxidative stress; and treatment of stromal cells with P3F-modulated exosomes also conferred protection against exogenous oxidative stress. Our findings highlight the role of P3F fusion protein in modulating exosome cargo to confer a protective effect on recipient cells against oxidative stress and to promote plasticity and survival, potentially contributing to the known aggressive phenotype of the fusion gene-positive subtype of RMS.

Translational studies of exosomes in sports medicine – a mini-review

“…Empirical investigations have corroborated that exosomal antioxidants, for instance, glutathione and superoxide dismutase, are capable of neutralizing excessive reactive species, ameliorating oxidative detriment ( 82 , 83 ). This plays an instrumental role in diminishing muscular fatigue post-exercise and decelerating cellular senescence.…”

“…In addition, exosomes can augment athletes’ antioxidative capabilities. During exercise, increased oxygen consumption leads to the production of reactive oxygen species (ROS), which can cause cellular damage ( 8 , 48 , 82 , 83 ). However, antioxidative enzymes and other molecules within exosomes can effectively scavenge these free radicals, protecting cells from damage and expediting the recovery process.…”

Crosstalk between Oxidative Stress and Exosomes

“…Oxidative Medicine and Cellular Longevity Surprisingly, cells pretreated with exosomes secreted from other cells under oxidative stress often become resistant to oxidative damage. Fahs et al suggested that the resistance to oxidative stress in mouse myoblasts could be transferred to recipient cells through exosomes [67]. Eldh et al reported that MC/9 cells pretreated with exosomes secreted from other MC/9 cells cultured under oxidative stress showed higher resistance to oxidative stress [68].…”

Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia

“…Increasing the ratio of GSH/oxidized GSH (GSSG) in patients with heart failure and acute myocardial infarction (MI) can reduce OS and improve the MI area and cardiac function [72][73][74][75]. This finding is Under pathological conditions, the exosomes released during OS carry antioxidant molecules, such as superoxide dismutase 1 (SOD1) and glutathione S-transferase (GST) [62,63], and defense molecules, such as glutathione peroxidase 1 (GPX1) [64], which can be absorbed by neighboring cells to enrich their cellular defense mechanisms; thus, these cells are already protected from OS induced by adverse environmental conditions. Therefore, exosomes can potentially transfer defense molecules from one cell to another [65].…”

“…They can modify gene expression and protein synthesis by inhibiting protein synthesis or initiating mRNA degradation to perform their functions at the post-transcriptional level. Moreover, circulating exosomes are considered as new biomarkers of disease performance and progression [ 57 , 62 , 63 ].…”