Associate Professor Erasmus Medical Center Rotterdam, Zuid-Holland, Netherlands
Objective: The rarity and heterogeneity of soft tissue sarcomas hamper in-depth studies into their biology and complicate clinical trials, thus limiting the development and introduction of novel, more effective therapeutic treatments. It was recently demonstrated that cancer cells in a mesenchymal, therapy-resistant state are extremely sensitive to ferroptosis induction. Ferroptosis is a form of regulated cell death caused by the iron-dependent formation and accumulation of lipid hydroperoxides. The enzyme glutathione peroxidase 4 (GPX4) fulfils a key regulatory role in this cell death process as it converts lipid hydroperoxides into alcohol thereby preventing ferroptosis. It is examined whether soft tissue sarcomas in general, because of their mesenchymal origin, may also exhibit an increased sensitivity for ferroptosis induction.
Methods: A variety of sarcoma (n=9) and carcinoma (n=6) cell lines, representing distinct tumor types, were exposed to ferroptosis-inducing compounds like the GPX4 inhibitor ML210 and the system Xc- inhibitor erastin after which in vitro cytotoxicity assays were performed. The nature of cell death was evaluated by the use of ferroptosis-, apoptosis- and necrosis-inhibitors: ferrostatin-1, Z-VAD-fmk and necrostatin-1 and the level of oxidation of membrane lipids was determined using the BODIPY-C11 probe and FACS. The NRF2/KEAP1 oxidative stress response signaling pathway was examined by the CDDO methyl ester, a well-known potent activator of the oxidative stress response, and a luciferase reporter as proxy for NRF2 transcriptional activity. Total and reduced glutathione levels were measured. Protein and/or mRNA expression of GPX4, ACSL4, KEAP1 and specific NRF2 target genes (NQO1, GCLC) were determined by Western blot and RT-PCR. ACSL4 activity was inhibited by rosiglitazone.
Results: Sarcoma cell lines proved much more sensitive to ferroptosis induction by ML210 than carcinoma cell lines that failed to respond. Also, exposure to erastin, which indirectly affects glutathione levels and hence GPX4 activity, revealed a significantly enhanced sensitivity of sarcoma cells for ferroptosis. To better understand the molecular underpinnings of these findings GPX4 transcript levels were determined and found to be reduced in sarcoma cells compared to carcinoma cells. However, these transcript level differences were not reflected at a protein level implying that GPX4 levels do not underlie the observed sensitivity differences in ferroptosis induction. Further, these data confirm the dependency of sarcoma cells on GPX4 and imply that carcinoma cells have additional means to cope with oxidative stress caused by GPX4 inhibition. Next, the involvement of the NFR2/KEAP1 oxidative stress pathway, as a master regulator of the antioxidant response, was investigated. CDDO treatment and consequent activation of the oxidative stress response reduced the sensitivity of sarcoma cells for ML210 and erastin 3 – 6 fold. Subsequent investigations into the steady-state NRF2 activity the cell line panel demonstrated that, although NRF2 activity differed between cell lines, there was no significant difference between sarcoma and carcinoma cells. Likewise, no differences in total and reduced glutathione levels were detected. To assess the NRF2/KEAP1 pathway induction, a short-term (8 h) exposure to erastin was used. In both sarcoma- and carcinoma cells the NRF2 mediated antioxidant response was induced to a similar degree. Finally, acyl-CoA synthetase long chain 4 (ACSL4) expression was investigated as it is a known mediator of ferroptosis. It was shown that ACSL4 is expressed in both sarcoma- and carcinoma cells and that its inhibition by rosiglitazone makes most, but not all, sarcoma cell lines more resistant to ferroptosis induction by ML210.
Conclusion: This study clearly highlights a biochemical vulnerability of soft tissue sarcomas namely the sensitivity to ferroptosis induction which could be exploited to develop more effective therapeutic strategies for this class of rare mesenchymal tumors. The underlying molecular causes for the observed increased vulnerability for ferroptosis are not yet completely understood and warrant further investigations.
