Role Of G1P3-Induced Mitochondrial Reactive Oxygen Species In Cancer Cell Migration

Breast cancer remains the second leading cause of cancer-related deaths among U.S women with metastasis accounting for ˃ 90% of those deaths. G1P3 (IFI6/ISG6-16) is an interferon-stimulated gene encoding a 13 kDa protein with pleiotropic functions. We and others characterized G1P3 as an anti-apoptotic protein located in the mitochondria with a role in cancer progression and metastasis. In accordance with its mitochondrial location, ectopically expressed G1P3 in breast cancer cells (MCF-7G1P3), resulted in elevated levels of mitochondrial reactive species (mtROS) and remodeled actin, potentiating augmented migration. Both scavenging of mtROS and down-regulation of G1P3 expression significantly reduced the migration of MCF-7G1P3 cells. Comparative gene expression analysis identified the upregulation of Caveolin 1 (CAV1), a regulator of wound healing, by 4-fold (p ≤ 0.05) in MCF-7G1P3 cells. Knock-down of CAV1 with small interfering RNA (siRNA) resulted in reduced migration (p ≤ 0.05) and filopodia formation (p ≤ 0.01) in MCF-7G1P3 cells compared to MCF-7Vector cells. Considering filopodia is the predominant migratory structure in MCF-7G1P3 cells and CDC42 is a regulator of filopodia formation and can mediate cell motility, its role in cell migration was assessed. Relative to MCF-7Vector cells, suppression of CDC42 expression significantly reduced migration in MCF-7G1P3 cells (p ≤ 0.05). In G-Lisa assays, MCF-7G1P3 cells had increased GTP-bound CDC42 compared to MCF-7Vector cells which could be reversed with knockdown of Caveolin 1 and G1P3, as well as scavenging of mtROS with MitoTEMPO. In agreement with G1P3’s pleiotropic functions, in confocal microscopy studies, G1P3-FLAG co-occurred with mitochondria as well as organelles of the endomembrane system, including trans-Golgi, lysosomes, endoplasmic reticulum and endosomes. In particular, 52% of G1P3-FLAG co-occurred with RAB5 positive endosomes. RAB5 has been reported to locate to the mitochondria during events of cell stress. In MCF-7G1P3 cells, RAB5 co-occurred with mitochondria ~1.9-fold more than MCF-7Vector cells suggesting that RAB5 may play a role in conferring cell survival in MCF-7G1P3 cells. In conclusion, this study demonstrates that elevated mtROS generated in MCF-7G1P3 cells can alter gene expression to mediate formation of actin structures and augmented migration and metastatic potential in breast cancer cells.