CuO-NPs suppress esophageal squamous cell carcinoma by altering oxidant-dependent energy metabolism, leading to mitophagy.

Nanoparticles coupled with metals hold great promise for biomedical applications. While previous studies have shown the cytotoxic effects of copper oxide nanoparticles (CuO-NPs) on esophageal squamous cell carcinoma (ESCC), the underlying mechanisms are unclear. Here, we demonstrate that CuO-NPs accumulate around mitochondria in esophageal cells, substantially inducing reactive oxygen species (ROS), which activates NRF2 in ESCC cells compared to normal esophageal keratinocytes. Transcriptomic analysis reveals that NRF2-downstream genes play crucial roles in both antioxidant activity and energy metabolism, which activate the protein kinase AMPK and inhibit AMPK downstream target mTORC1, ultimately leading to mitochondrial dysfunction and cell death in ESCC cells. We show that the critical mitophagy regulators, PINK1 and Parkin, are significantly upregulated in CuO-NP-treated ESCC cells. Since the transcription factor TFEB is a mTORC1 downstream target that has been shown to directly control PINK1/Parkin expression, we find that the mTORC1-TFEB-PINK1-Parkin (mTTPP) axis is involved in CuO-NP-induced cytotoxicity in ESCC cells. CuO-NPs inhibit mTORC1-mediated TFEB phosphorylation, thereby inducing TFEB nuclear translocation that increases PINK1/Parkin expression and activates the PINK1/Parkin-mediated mitophagy pathway. Ablation of the mTTPP axis by RNA interference attenuates the cytotoxicity induced by CuO-NPs in ESCC cells. The tumor-killing effects of CuO-NPs observed in vitro are also evident in the subcutaneous ESCC xenograft model in vivo. Taken together, our results suggest that CuO-NPs have potential for ESCC treatment and warrant further preclinical study.