Abstract 550: Akt2 regulates mitophagy in breast cancer cell lines.

Abstract The PI3K-Akt signaling pathway is involved in the regulation of cell growth, proliferation, metabolism, and death by apoptosis and autophagy. There are three isoforms of Akt (1, 2, and 3) that share over 70% sequence identity and two activating phosphorylation sites. The activation of Akt by insulin or insulin-like growth factor (IGF-1) results in rapid phosphorylation of two amino acid residues that are conserved among all three Akt isoforms. Phosphorylation of threonine 308 (Thr308) leads to roughly 100-fold higher activity, while phosphorylation at serine 474 (Ser474) potentiates the activity another 10-fold. The two phosphorylation events are independent of one another and while phosphoinositide-dependent kinase 1 (PDK1) is accepted as the activating kinase for Thr308, the kinase that phosphorylates Ser474 has only recently been confirmed as the target of rapamycin in complex with Rictor and Sin1 (mTORC2). Among the three Akt isoforms, Akt2 is a particularly promising breast cancer therapeutic target, as its ablation most profoundly affects the long-term cell survival rate. Ablation of Akt2 induces autophagy, which increases cancer cell survival and resistance in response to chemotherapy. In this work, we examined the possibility that Akt2 may specifically regulate autophagic destruction of the mitochondria (mitophagy) via the mitophagy activator PINK1. This point of regulation may represent an attractive therapeutic target to decrease autophagy and increase cancer cell death. Insulin signaling promotes the phosphorylation of Akt by mTORC2 via the PTEN-induced putative kinase (PINK1). PINK1 is a serine/threonine mitochondrial kinase that has been linked to familial Parkinson's disease. PINK1 was originally classified in a screen for transcriptional upregulation of genes upon overexpression of PTEN in two endometrial cancer cell lines, but in the same publication high levels of PINK1 mRNA were present in ovarian tumours that were lacking PTEN. No further investigations into this relationship have been described and it remains unclear as to the relationship between PTEN, Akt, and PINK1, and how regulation occurs at the transcriptional or posttranslational level. Normally PINK1 regulates mitochondrial quality control by targeting defective mitochondria for autophagy (referred to as mitophagy) via the ubiquitin E3 ligase Parkin. We have identified an interaction between Akt2 and the major regulator of mitophagy (PINK1). We suggest that Akt2 normally inhibits mitophagy, and that inhibition of Akt leads to selective autophagic destruction of the mitochondria. We next wanted to determine whether Akt2 activation is required for its mitochondrial localization. Mitochondria isolations were performed using magnetic labelling of the mitochondria with Anti-TOM22 microbeads. The results suggest that the key activating residues of Akt2 may not be required for mitochondrial localization. Citation Format: Alison C. Douglas, James Knockleby, Hoyun Lee. Akt2 regulates mitophagy in breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2013-550