MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer

www.nature.com/bjc ARTICLE Cancer Metabolism MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer Jessica C. Casciano1, Caroline Perry1,2, Adam J. Cohen-Nowak1, Katelyn D. Miller1, Johan Vande Voorde3, Qifeng Zhang4, Susan Chalmers5, Mairi E. Sandison5,6, Qin Liu1, Ann Hedley3, Tony McBryan3,7, Hsin-Yao Tang1, Nicole Gorman1, Thomas Beer1, David W. Speicher1, Peter D. Adams8, Xuefeng Liu9, Richard Schlegel9, John G. McCarron5, Michael J. O. Wakelam4, Eyal Gottlieb3,10, Andrew V. Kossenkov1 and Zachary T. Schug1 BACKGROUND: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. METHODS: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. RESULTS: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. CONCLUSION: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype. British Journal of Cancer (2020) 122:868–884; https://doi.org/10.1038/s41416-019-0711-3 BACKGROUND A recent study analysed the copy number and gene expression changes in over 2000 breast tumours.1,2 One of the most commonly and highly amplified genes in breast cancer is MYC.1,3 MYC is a transcription factor that activates genes involved in cell cycle regulation, cell growth, protein synthesis, mitochondrial function, and metabolism. MYC amplification occurs in ~25% of all breast cancers and occurs more frequently in triple negative breast cancer (TNBC) (up to 50%).4,5 MYC gene amplification is associated with risk of relapse, poor prognosis, and death.6,7 MYC is a known regulator of metabolic reprogramming in cancer.8,9 A recent study reported that TNBC cells with high expression of MYC have high rates of fatty acid β-oxidation (FAO).10 They further demonstrated that pharmacological inhibition of FAO with etomoxir impairs the growth of TNBC patientderived xenografts that have high MYC expression but not those with low MYC expression, suggesting that targeting FAO may be a viable treatment option for TNBC patients with high expression of MYC.10 Unfortunately, the safety of etomoxir has been called into question during clinical trials due to reports of liver toxicity and a recent study showed that etomoxir can cause severe oxidative stress in an off-target (non-CPT1A) dependent manner.11,12 There is therefore unmet need for better, safer inhibitors of FAO. Other targets associated with FAO have emerged more recently. For instance, FAO has been shown to promote SRC activation and metastasis in TNBC,13 while a separate study showed that CUBdomain containing protein 1 (CDCP1) inhibits acyl-CoA synthetases which then stimulates FAO in TNBC cell lines.14 However, the role of CDCP1 in breast is controversial with conflicting reports.15,16 The goal of our study was not to further prove the importance of FAO in TNBC, but to elucidate the mechanistic link between MYC and the activation of FAO and to identify new actionable targets for blocking FAO in TNBC. We used human mammary epithelial (HME) cells that express oncogenic levels of MYC as a model system to cleanly delineate how MYC leads to enhanced 1 The Wistar Institute, Molecular and Cellular Oncogenesis, 3601 Spruce Street, Philadelphia, PA 19104, USA; 2Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; 3The Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; 4The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK; 5Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, SIPBS Building, 161 Cathedral Street, Glasgow G4 0RE, UK; 6Department of Biomedical Engineering, University of Strathclyde, Wolfson Centre, 106 Rottenrow, Glasgow G4 0NW, UK; 7Institute of Cancer Sciences, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G61 1BD, UK; 8Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA; 9Center for Cell Reprogramming, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3900 Reservoir Road, Washington D.C. 20057, USA and 10The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, 1 Efron St. Bat Galim, 3525433 Haifa, Israel Correspondence: Zachary T. Schug () Received: 10 June 2019 Revised: 22 November 2019 Accepted: 19 December 2019 Published online: 16 January 2020 © The Author(s) 2020 Published by Springer Nature on behalf of Cancer Research UK MYC regulates fatty acid metabolism through a multigenic program in. . . JC Casciano et al. 1234567890();,: FAO. Our results indicate that MYC alters calcium (Ca2+) signalling that then promotes FAO by activating a Ca2+-CAMKK2-AMPK signalling axis. We also identified an important role for fatty acid transporter CD36, which mediates fatty acid uptake in TNBC. MYC HME cells exhibited a 10-fold increase in cell migration compared to isogenic cell lines expressing telomerase reverse transcriptase (TERT) or HER2. The migration of MYC HME cells could be inhibited by targeting CD36, CAMKK2, or FAO suggesting that uptake and oxidation of fatty acids may be an important metabolic pathway for supporting the energy demanding process of cell migration. We find that CD36 and other fatty acid metabolism genes (i.e. LPL, PDK4, FABP4) are highly co-expressed in TNBC, particularly in the claudin-low subtype of TNBC where their high expression predicts poor survival. Collectively, our studies helped to identify targets that could be considered as new options for blocking FAO in TNBC and suggests that targeting FAO may be most beneficial for claudin-low TNBC patients. METHODS Cell culture, siRNA transfection, and lentiviral transduction HME cells were cultured in 1× HMEC Basal Serum-Free Medium (Thermo Fisher Scientific) supplemented with the HMEC Supplement Kit (Thermo Fisher Scientific) and 1× penicillin–streptomycin (Corning). MDA-MB-468 (ATCC), T47D (ATCC), BT474 (ATCC), MDAMB-231 (ATCC), Cal120 ( (...truncated)