Abstract 1414: miRNA-5096 induces ferroptosis by targeting SLC7A11 in breast cancer

Resistance to radiation and chemotherapy is a major obstacle in cancer treatment and one of the mechanisms by which cancer cells acquire radio-and chemo-resistance is by over-expressing a trans-membrane amino acid transporter protein, SLC7A11. It confers radio- and chemoresistance in bladder, breast, kidney, colon, liver, ovary and prostate cancers, and it is overexpressed in 70% of cancers as well as in all the NCI-60 panel cell lines. SLC7A11 imports cysteine into the cell which is the rate-limiting step of glutathione synthesis and downregulation of SLC7A11 has been reported to induce ferroptosis. Ferroptosis is an iron and ROS-dependent form of cell death that relies on metabolic enzymes to produce ROS. The objective of this study is to demonstrate the use of microRNAs to specifically down-regulate SLC7A11 as a potential approach in future to re-sensitize cancer cells to radiation and chemotherapy. We first performed a survival analysis of SLC7A11 with the help of ULCAN online software and found that overexpression of SLC7A11 leads to poor patient survival in different cancers. Using bioinformatics tools TargetScan and miRanda, we found that miRNA-5096 has two putative binding sites in the 3'UTR of SLC7A11. We determined the expression level of miR-5096 in breast cancer tissues and cell lines by qRT-PCR and the results indicate that the expression of miR-5096 is significantly decreased in human breast cancer specimens and cell lines. We overexpressed miR-5096 in breast cancer cell lines and observed the downregulation of SLC7A11. Using dual-luciferase reporter assays, we confirmed the prediction that SLC7A11 is a direct target of miR-5096. Next, we investigated the effect of miR-5096 overexpression on the cell viability with the Erastin (ferroptosis inducer) and ferrostatin (ferroptosis inhibitor), ROS, and hydroxyl radical levels. We observed that overexpression of mir-5096 alone, and together with Errastin significantly decreases the cell viability in a synergistic manner while Ferrrostatin rescued cell death. The ROS and hydroxyl ion levels were also found to be elevated by overexpression of miR-5096. Necrostatin (necroptosis inhibitor) failed to rescue miR-5096 overexpression driven cell death suggesting cell death is not due to necroptosis. To explore the molecular mechanism of miR-5096 in ferroptosis, we analyzed lipid peroxidation, which is important for signaling events in triggering ferroptosis. The lipid ROS generation was assessed by flow cy