Abstract 3328: MYC and MCL1 cooperatively promote chemotherapy-resistant cancer stem cells through regulation of mitochondrial biogenesis and oxidative phosphorylation
Authors: Lee Kyung-min , Giltnane Jennifer , Balko Justin , Arteaga Carlos 
Published: July 2016

Abstract
Triple negative breast cancer is the most virulent subtype of this malignancy. Although initially responsive to cytotoxic chemotherapy, most patients with TNBC eventually develop drug-resistant disease. We previously reported co-amplification of MYC and MCL1 in a cohort of TNBCs after neoadjuvant chemotherapy (Balko et al. Cancer Discov. 2014). In addition, MYC and MCL1 are overexpressed in paclitaxel-resistant compared to paclitaxel-sensitive TNBC cells. We hypothesized that MYC and MCL1 play a role in chemotherapy resistance. TNBC cell lines with claudin-low gene expression exhibited the highest levels of MYC and MCL1 mRNAs. This molecular subtype of breast cancer is enriched for cancer stem cells (CSCs) and exhibits resistance to neoadjuvant chemotherapy. Knockdown of both MYC and MCL1 by siRNA in SUM159PT and MDA-MB-436 TNBC cells significantly reduced mammosphere formation and CSC markers such as ALDH and CD44high/CD24low. Conversely, overexpression of ectopic MYC and MCL1 in MDA-MB-468 cells increased mammosphere formation and CSC markers. It has been reported that mitochondrial oxidative phosphorylation (OXPHOS) is enhanced in CSCs. Thus, we investigated a role for MYC and MCL1 in mitochondrial OXPHOS using Seahorse XFe extracellular flux analyzer. CSCs with high expression of MYC and MCL1 sorted by flow cytometry exhibited increased mitochondrial OXPHOS compared to non-CSCs. Transfection of MYC and MCL1 expression vectors into MDA-MB-468 cells enhanced mitochondrial OXPHOS activity. Conversely, knockdown of MYC and MCL1 in SUM159PT and MDA-MB-436 cells reduced mitochondrial OXPHOS activity. MYC mediated these effects by promoting mitochondrial biogenesis whereas MCL1 potentiated mitochondrial OXPHOS via localization into the mitochondrial matrix, independent of its ability to associate with BH3-domain containing anti-apoptotic proteins (BAD, BCL2, BCL-XL). Deletion of the mitochondrial target sequence (MTS) of MCL1 impaired its ability to increase mitochondrial OXPHOS and mammosphere formation. Reactive oxygen species (ROS), which are produced in CSCs as a by-product of activated mitochondrial OXPHOS, were abrogated by MYC and MCL1 siRNA. To determine the effect of ROS on CSCs, we treated SUM159PT and MDA-MB-436 cells with hydrogen peroxide (H2O2). Treatment with H2O2 enriched CSCs, which showed up-regulation of several cancer stem cell genes such as Nanog and IL-8. Enrichment of CSCs by H2O2 was diminished by n-acetylcysteine (NAC), an antioxidant. These data suggest that MYC and MCL1 co-amplification confers drug resistance to TNBC via mitochondrial OXPHOS-mediated enrichment of CSCs. Moreover, MYC and MCL1 stimulate H2O2 production, which potentiates CSCs. Thus, targeting mitochondrial OXPHOS and H2O2 may be an effect strategy to delay or reverse resistance to chemotherapy in MYC and MCL1 amplified TNBC.
Citation Format: Kyung-min Lee, Jennifer Giltnane, Justin Balko, Carlos Arteaga. MYC and MCL1 cooperatively promote chemotherapy-resistant cancer stem cells through regulation of mitochondrial biogenesis and oxidative phosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3328.