Mitochondrial E3 Ubiquitin Ligase MUL1 reduces proliferation and increases cell death in metastatic osteosarcoma cells; Abstracts of the ECTS 2024 Congress

Osteosarcoma is a highly aggressive and metastatic bone cancer. Identification of new therapeutic targets is important for effective treatments. A possible strategy leverages the inhibition of the hypoxia-inducible factor HIF-1α, upregulated in metastatic osteosarcoma. This study aims to evaluate the effects of HIF-1α downregulation by the mitochondrial E3 ubiquitin ligase MUL1 and the cofactor protein UBXN7 pathway in osteosarcoma cells. Firstly, we observed that MUL1 protein levels inversely correlate with tumor aggressiveness; it was expressed in the osteoblast-like osteosarcoma Saos-2 cells, while barely detected in the highly aggressive and metastatic 143B cells [Arbitrary Unit (AU), Saos-2: 0.86±0.15, 143B: 0.24±0.07, p<0.01); accordingly, the expression of UBXN7 (AU, Saos-2: 0.14±0.04, 143B: 1.06±0.05, p<0.01) and HIF-1α (AU, Saos-2:0.12±0.03, 143B:0.90±0.17, p<0.01) was higher in 143B cells. MUL1 overexpression in 143B cells causes a 50% reduction of UBXN7 (AU, pEmpty-143B: 1.01±0.08 pMUL1-143B: 0.50±0.03, p<0.0001) and HIF-1α protein levels (pEmpty-143B: 0.79±0.10, pMUL1143B: 0.43±0.07, p<0.008). Most importantly, MUL1 induction in 143B cells reduces the proliferative rate of 143B cells (AU, pEmtpy-143B: 1.01±0.04 , pMUL1-143B: 0.79±0.02, p<0.001) coupled with induction of apoptosis, as shown by the increase of Annexin V positive cells (AU, pEmtpy-143B: 1.01±0.02, pMUL1-143B: 1.36±0.01, p<0.01) and of the cleaved isoforms of PARP (AU, pEmpty-143B: 1.01±0.05 pMUL1-143B: 2.65±0.06, p<0.02) and Caspase 3 (AU, pEmpty-143B: 1.01±0.05 pMUL1143B: 3.36±0.09, p<0.04). Induction of MUL1 treatment with Ucf-101, an inhibitor of the upstream mediator of MUL1 HTRA2, achieves similar results. Moreover, overexpression of an inactive MUL1 mutant fails to induce HIF-1α downregulation and cell death. Our results show that the MUL1/UBXN7 pathway is dysregulated in metastatic osteosarcoma cells, and that restoring MUL1 levels in that scenario results not only in HIF-1α downregulation, but also in reduced cell proliferation and increased cell death.