Metabolic vulnerabilities of rhabdomyosarcoma: insights into the role of ASS1

Abstract

[eng] Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma (STS) in children and adolescents. The Alveolar subtype (ARMS) is driven by PAX3-FOXO1, a fusion protein originated from a chromosomal translocation. PAX3-FOXO1 is considered a key player in ARMS initiation and progression, due to its enhanced transcriptional activity, that leads to the upregulation of downstream targets genes involved in ARMS tumorigenesis and metastasis. Several studies have focused on the epigenetic and transcriptional profiling of PAX3-FOXO1 expressing cells, but very little is known about the metabolic landscape of these tumors. Although during the last decades, considerable advancements have been done in understanding ARMS pathogenesis, the outcome for children with metastatic or recurrent disease remains poor. Metabolic reprogramming is considered one of the main hallmarks of cancer. To sustain enhanced cell proliferation and build new biomass in a frequently nutrient-poor microenvironment, many metabolic requirements must be satisfied by tumor cells. This is achieved through the activation of gene expression and protein regulation programs, that involve, among others, deregulation of glucose and amino acids uptake and utilization. The main objective of our research project is to analyze the effects of PAX3-FOXO1 expression on RMS metabolic rewiring. Starting from transcriptomic analysis of publicly available data, we identified the Argininosuccinate Synthase 1 (ASS1), an enzyme of the urea cycle involved in the de novo synthesis of arginine, as a putative transcriptional target of PAX3-FOXO1. It has been described that ASS1 plays a role in enhancing tumor progression and invasion in different cancer types. Despite all the evidence that describe other STS as ASS1-deficient, we found that RMS cells overexpress ASS1. Using our cellular models of fusion protein silencing in ARMS cell lines, we confirmed that PAX3-FOXO1 downregulation results in ASS1 mRNA levels decrease, suggesting that the fusion protein could regulate ASS1 transcription. Moreover, through metabolomics experiments, we demonstrated that PAX3-FOXO1 ectopic introduction in fusion-negative RMS cells induced a modulation of the urea cycle intermediates relative levels, that reflected ASS1 upregulation upon the fusion protein expression. To better address its role in ARMS pathogenesis, we silenced ASS1 expression in fusion-positive RMS cells and characterized their phenotype. CRISPR-Cas9-mediated ASS1 knockdown impaired ARMS migratory capability in vitro without affecting cell proliferation, even under arginine starvation, suggesting that this amino acid metabolic rewiring could be promoting the metastatic phenotype of ARMS. Furthermore, in vivo tumorigenesis resulted to be notably delayed in mice injected with ASS1-silenced ARMS cells, confirming the oncogenic role of the enzyme in these cancers. Finally, to describe the molecular mechanism underlying ASS1- mediated ARMS progression and spreading we performed a microarray to study the transcriptional signature induced by ASS1 knockdown. We were able to identify a possible molecular mediator of the phenotype observed in ASS1-deficient cells: the elongation factor EEF1A2, extensively described in literature to display oncogenic activity in cancer, resulted to be transcriptionally downregulated upon ASS1 silencing. In accordance with the evidence that describe its activation as downstream EEF1A2 oncogenic signaling, AKT phosphorylation was also found to decrease, together with the activation of S6K1 and S6 downstream effectors. Further experiments are needed to better describe the upstream signaling involved and the precise mechanism that links ASS1 to EEF1A2 downregulation. In conclusion, our work suggests that urea cycle enzyme ASS1 is highly expressed in ARMS and its upregulation, driven by PAX3-FOXO1 fusion protein, could be involved in sustaining these cancers tumorigenesis and metastatic process, through the induction of EEF1A2-mediated AKT activation. Thus, the inhibition of this pathway could represent a promising tool to target RMS metabolism, hopefully leading to the potential development of more effective targeted therapies for the treatment of these aggressive pediatric cancers.