CuET inhibits Ewing sarcoma of bone progression through modulation of the ESM1-MAPK/ERK signaling axis

Ewing's sarcoma of bone is the second most common primary malignant bone tumor in children and adolescents. Although multimodal therapy has improved 5-year survival to 60-70%, outcomes for relapsed or metastatic disease remain poor (<30%). Notably, copper(II) diethyldithiocarbamate (CuET), a copper-ligand complex generated through the in vivo metabolism of the FDA-approved anti-alcoholism drug disulfiram, has been reported to exhibit antitumor activity across a range of solid malignancies, including colorectal and breast cancers; however, its biological relevance and therapeutic potential in Ewing sarcoma remain unclear. This study investigates the copper-diethyldithiocarbamate complex (CuET), the active metabolite of disulfiram‑copper complexes, to elucidate its role in the pathogenesis and therapeutic mechanisms of bone Ewing sarcoma.

This study employed a multidimensional strategy to evaluate the anti-tumor efficacy of CuET against bone Ewing sarcoma and its mechanism. We first compared the effectiveness of DDTC, Cu²⁺, and CuET in TC32 cells and xenograft models. Using RNA-seq, Western blot, and bioinformatics, we identified ESM1 as a key biomarker. Its function was then validated in ESM1-knockdown/overexpression models through in vitro and in vivo assays. Finally, KEGG enrichment and experimental verification revealed the involvement of the MAPK/ERK pathway.

CuET showed significantly stronger anti-tumor efficacy than DDTC or Cu²⁺ alone, effectively suppressing tumor growth both in vitro and in vivo (p < .01). We identified ESM1 as a central dual-functional regulator in Ewing sarcoma, with its expression elevated at both transcriptional and protein levels. Functionally, ESM1 knockdown inhibited proliferation and sensitized cells to CuET, while its overexpression accelerated tumor growth and induced treatment resistance. Mechanistically, KEGG analysis implicated the MAPK/ERK pathway, which was subsequently confirmed to be the primary route through which ESM1 promotes tumor aggressiveness and CuET resistance.

This study demonstrates that CuET exhibits markedly enhanced anti-tumor activity against Ewing sarcoma compared with its precursor compounds, DDTC and copper ions. Furthermore, we identify ESM1 as a potential regulatory factor that dually governs sarcoma proliferation and confers resistance to CuET, primarily through activation of the MAPK/ERK signaling cascade. Significantly, targeting the ESM1-MAPK/ERK axis synergistically enhances the therapeutic efficacy of CuET treatment. In summary, this research establishes a foundation for future investigations and novel therapeutic strategies in the field of Ewing's sarcoma of bone.