Abstract P1-05-03: Phosphorylation of KAP1 Serine 473 regulates mitochondrial dynamics through miR-20b under metabolic stress in breast cancer

Cancer has been considered a metabolic disease in recent years. Compared to normal cells, cancer cells rewire metabolic pathways to sustain their outgrowth. While growing fast, cancer cells in the core region of a solid tumor encounter metabolic stress induced by hypoxia and insufficient nutrition, and mitochondrial dynamics has a profound effect on the survival of cancer cells. However, how mitochondrial dynamics affects cancer cell survival under metabolic stress has not been fully elucidated. Kruppel-associated box-associated protein-1 (KAP1), also known as TRIM28, is a transcriptional co-regulator participating in multiple cellular physiological processes. The post-translational modifications of KAP1 play an essential role in regulating its function. We have previously demonstrated that KAP1 mRNA is highly expressed in concomitant with the upregulation of genes involving in glycolysis and oxidative phosphorylation in breast cancer. Furthermore, KAP1 is phosphorylated at Serine 473 (pS473) upon the induction of nutrient stresses via reactive oxygen species (ROS)-p38 MAPK signaling in breast cancer cells. The S473 phosphorylation of KAP1 reduces the expression of mitochondrial outer membrane protein mitofusin 2 (MFN2), which prevents the hyperfusion of mitochondria and the overproduction of ROS under nutrient deprivation. However, the mechanism of how KAP1 regulates the expression of MFN2 remains unclear. We have engineered a KAP1-deficient MDA-MB-231 cell line and re-expressed KAP1 S473 wild-type (WT), S473A (phosphorylation-defective) or S473D (phosphorylation-mimic) mutants. By analyzing the mRNA expression profiles from these cell lines, several microRNAs (miRNAs) were shown to be upregulated in the KAP1 S473D-expressing cells, which implied that pS473-KAP1 may regulate MFN2 expression through miRNA. Using several miRNA target prediction algorithms, miR-20b was predicted to target the 3’-untranslated region (UTR) of MFN2 mRNA, and the sequence of target site was conserved through evolution. By performing MFN2 3’UTR dual luciferase reporter assays, we have confirmed that MFN2 3’UTRwas targeted by miR-20b and regulated by the phosphorylation status at S473 of KAP1. Also, miR-20b expression was upregulated upon glucose deprivation and both MFN2 mRNA and protein levels declined in breast cancercells overexpressed with miR-20b mimics. In addition, KAP1 S473D, rather than S473A-expressing cells produced more miR-20b under glucose depletion condition and MFN2 expression levels were inversely correlated with miR-20b expression. Moreover, the analysis of The Cancer Genome Atlas (TCGA) breast tumor datasets indicated that there was higher miR-20b but lower MFN2 expression in advanced breast tumors compared with the primary tumors and normal tissues. In conclusion, these results suggested that the induction of KAP1 S473 phosphorylation under nutrition deprivation inhibited MFN2 by upregulating miR-20b, which prevented mitochondrial hyperfusion and consequently benefited breast cancer cell survival in response to metabolic stress. Citation Format: Yu-Shu Liu, Chun-Ting Cheng, Ching Ouyang, Ge-Jyun Liu, David Kong Ann, Ching-Ying Kuo. Phosphorylation of KAP1 Serine 473 regulates mitochondrial dynamics through miR-20b under metabolic stress in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-03.