IDDF2024-ABS-0263 PET117 promotes ferroptosis in colorectal cancer cells through regulation of mitochondrial metabolism

<h3>Background</h3> The precise involvement of mitochondria in ferroptosis in CRC cells remains unclear. We previously demonstrated that PET117 is a significant mitochondrial protein, while its deletion induces mitochondrial dysfunction. Our proteomic analysis found that PET117 was associated with ferroptosis. Hence, we hypothesize that PET117 regulates ferroptosis in CRC cells by modulating mitochondrial metabolism. <h3>Methods</h3> The study aims to elucidate the role of PET117 in CRC ferroptosis using cell, mouse and colorectal tumor models and investigate PET117’s regulation of CRC mitochondrial function through approaches such as seahorse analysis and metabolomics. Additionally, we will identify PET117-interacting proteins and elucidate their regulatory mechanisms by integrating multi-omics detection of tumor tissue and gene-edited cells. <h3>Results</h3> The Cancer Genome Atlas (TCGA) analysis unveiled an up-regulation of <i>PET117</i> mRNA in CRC tissues compared to normal colorectal tissues. According to proteomic analysis of control and <i>PET117</i>-KO cells, we found that loss of <i>PET117</i> altered mitochondrial proteome and was associated with ferroptosis. Importantly, compared with the control group, loss of <i>PET117</i> inhibited erastin or RSL3-induced ferroptosis, while overexpression of PET117 in <i>PET117</i>-KO cells increased sensitivity to erastin or RSL3-induced ferroptosis. In addition, using DCFH-DA fluorescent probe to label ROS in RSL3-treated control or <i>PET117</i>-KO cells, the flow cytometry results showed that RSL3 significantly increased ROS in RSL3-induced ferroptosis in control cells, interestingly, the ROS did not significantly change in <i>PET117</i>-KO cells. Meanwhile, the loss of <i>PET117</i> didn’t affect cell apoptosis or autophagy. Next, RNA-seq analysis showed that the absence of <i>PET117</i> altered the expression of ferroptosis-related genes. Finally, we conducted a phosphorylated proteomic analysis of control and <i>PET117</i>-KO cells, we found that the loss of <i>PET117</i> reconstructed the cellular phosphorylation network signal. We integrated Uniprot database, PhosphoSitePlus database, and String database, and analyzed the detected phosphorylated proteins with known ferroptosis pathway proteins, and draw phosphorylation networks. The results showed that PET117 regulates phosphorylation in the STAT3-TP53 signaling pathway. <h3>Conclusions</h3> Our study reveals that PET117 is significantly upregulated in CRC and exerts a pro-ferroptotic effect. PET117 may serve as a potential target for CRC treatment.