Mapping transcriptomic signatures downstream of mitochondrial superoxide dismutase SOD2 in OVCA433 multicellular aggregates.

During metastatic spread, tumor cells adapt to evade anchorage-independent cell death by upregulating mitochondrial antioxidant systems. We previously showed that ovarian cancer cells upregulate mitochondrial manganese superoxide dismutase (SOD2) following detachment and multicellular aggregate (MCA) formation. SOD2 scavenges mitochondrial superoxide and manipulates cellular hydrogen peroxide levels, both functions necessary for metastasis. Here, we investigated SOD2's metastatic function by assessing its transcriptomic effects in OVCA433 ovarian cancer cells cultured in low-attachment conditions that induce MCAs, mimicking anchorage-independent states in malignant ascites. SOD2 siRNA-mediated knockdown effects in MCAs were compared to adherent culture conditions. RNA sequencing and pathway analysis revealed that SOD2 lies upstream of pro-metastatic pathways, including PI3K/AKT signaling. Notably, in MCAs, cytokine and immune cell signaling pathways were more significantly enriched following SOD2 knockdown. We previously identified SIRT3 as an important SOD2 activity regulator in MCAs. While SIRT3 knockdown resulted in minor transcriptional changes, we identified that FOXO3 and ELF4 transcription factors, important for stress response and immune regulation, are downregulated by both SIRT3 and SOD2 knockdown. Comparing SOD2 knockdown transcriptional changes to the Cancer Genome Atlas, we found SOD2 expression strongly associates with pro-tumorigenic immune signaling in serous ovarian cancer specimens, including genes identified downstream of SOD2 from our siRNA screen. Moreover, SOD2 expression correlated with signatures related to pro-tumorigenic neutrophil and T-regulatory cell populations. Our data suggest SOD2 positively regulates pro-metastatic pathways, and those identified in MCAs more closely reflect gene expression profiles associated with SOD2 expression in patient tumors.