How Ewing Sarcoma Led to Discoveries in Rhabdomyosarcoma

Researchers discovered that three childhood sarcomas appear to have overlapping targetable mechanisms involved in disease progression.

Rhabdomyosarcoma (RMS) and Ewing Sarcoma are among the most common types of cancer that develop in childhood. In recent years, new studies from researchers at the University of Colorado Denver Anschutz Medical Campus have contributed to the expansion of biomedical knowledge about how these diseases progress—paving a way for novel treatment strategies and therapeutics to inhibit the progression of these diseases. 

Introduction

RMS consists of two predominant and distinct disease subtypes: fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS). Fusion-positive RMS is the more aggressive subtype and shows a strong metastatic tendency to spread to other parts of the body. The sarcomagenesis of FP-RMS is driven by a genetic mutation that spawns the abnormal cancer-driving protein PAX3/FOXO1, or P3F. These P3F oncofusion genes use transcriptional and epigenetic mechanisms to drive FP-RMS pathogenesis. However, little was previously known about the mechanisms P3F uses for metastasis.

Recently, a team of dedicated researchers—Lays Martin Sobral, Marybeth Sechler, Janet K. Parrish, Tyler S. McCann, Kenneth L. Jones, Joshua C. Black, and Paul Jedlicka—endeavored to better understand the mechanisms and molecular pathways contributing to RMS progression, including the more aggressive FP-RMS subtype. In 2020, their influential research paper was published in Genes & Cancer and entitled, “KDM3A/Ets1/MCAM axis promotes growth and metastatic properties in Rhabdomyosarcoma.”

The Study

Researchers previously identified the histone demethylase epigenetic regulator KDM3A as a cancer-promoting gene in Ewing Sarcoma. Later, researchers found that Ewing Sarcoma cell migration and metastasis is promoted by KDM3A and its downstream target, the melanoma cell adhesion molecule (MCAM). In Ewing Sarcoma, MCAM expression is directly and indirectly regulated by KDM3A; MCAM is indirectly regulated by KDM3A through the transcription factor Ets1. Importantly, KDM3A had also been shown to be highly expressed in RMS.

“Our previous studies noted robust expression of KDM3A in RMS cell lines [7], prompting further investigation into its potential role(s), as a disease-promoting factor and candidate therapeutic target, in RMS.”

In this study, the researchers turned to investigating the molecular roles and mechanisms of KDM3A in RMS. The team began by first confirming that KDM3A is indeed highly expressed in RMS. Next, they carried out a stable shRNA-mediated depletion, or knockdown, of KDM3A in multiple FN-RMS and FP-RMS cell lines. Confirmed by immunoblotting, clonogenic assays and transendothelial invasion assays, they found that KDM3A promotes important properties of disease progression in both FN-RMS and FP-RMS: colony growth and transendothelial invasion. To define KDM3A-controlled gene sets, or transcriptomes, in FN-RMS and FP-RMS cells, the researchers used RNA-sequencing analysis. Gene Set Enrichment Analysis showed that KDM3A controls gene sets involved in cell proliferation, cell cycle, migration, epithelial-mesenchymal transition, and metastasis.

“Thus, consistent with the phenotypes observed in our functional studies, KDM3A positively controls expression of pro-growth and pro-metastatic genes in both FN-RMS and FP-RMS cells.”

Interestingly, the researchers found that their phenotypic and transcriptomic RMS results were very similar to their previous studies in Ewing Sarcoma. Therefore, the team searched for specific gene overlaps in KDM3A-controlled transcriptomes between rhabdomyosarcoma and Ewing Sarcoma. They discovered that KDM3A/Ets1/MCAM, the recently found disease-promoting axis in Ewing Sarcoma, was also shared by RMS. Notably, the team determined that, in FP-RMS, KDM3A did not appear to directly control Ets1 and MCAM expression.

“In summary, we show that the KDM3A/Ets1/MCAM molecular axis, which we have previously demonstrated to manifest tumor and metastasis promotional properties in Ewing Sarcoma, also plays potent disease-promoting roles in both FN-RMS and FP-RMS.”

Conclusion

Overall, the researchers found new parts of P3F machinery that enable rhabdomyosarcoma to spread to other parts of the body. They identified important roles of KDM3A and Ets1 in the aggressive properties and disease progression of both rhabdomyosarcoma subtypes, and especially FP-RMS. The targetable mechanisms in this axis, KDM3A and MCAM, could be used as therapeutic targets to treat fusion-positive RMS, fusion-negative RMS and Ewing Sarcoma. In continued RMS studies, the researchers would come to learn that the KDM3A/Ets1 epigenetic axis contributes to P3F-driven and independent disease-promoting gene expression in FP-RMS.

 “Our findings further suggest that KDM3A and MCAM, the pharmacologically targetable components of this axis merit further attention as potential new therapeutic targets in all three diseases.”

Click here to read the full research paper, published by Genes & Cancer.

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