Cancer Biology and Translational Studies PPARg Ligand–induced Annexin A1 Expression Determines Chemotherapy Response via Deubiquitination of Death Domain Kinase RIP in Triple-negative Breast Cancers Luxi Chen 1,2,3 , Yi Yuan 1 , Shreya Kar 1,2 , Madhu M. Kanchi 1 , Suruchi Arora 4 , Ji E. Kim 1 , Pei F. Koh 1,2 , Einas Yousef 5,6 , Ramar P. Samy 4 , Muthu K. Shanmugam 2 , Tuan Z. Tan 1 , Sung W. Shin 7 , Frank Arfuso 8 , Han M. Shen 4,9 , Henry Yang 1 , Boon C. Goh 1,2,10,11 , Joo I. Park 7 , Louis Gaboury 5 , Peter E. Lobie 1,2,11,12 , Gautam Sethi 2,13 , Lina H.K. Lim 4,9,14 , and Alan P. Kumar 1,2,11,15,16 Abstract Metastatic breast cancer is still incurable so far; new specifically targeted and more effective therapies for triple-negative breast cancer (TNBC) are required in the clinic. In this study, our clinical data have established that basal and claudin-low subtypes of breast cancer (TNBC types) express significantly higher levels of Annexin A1 (ANXA1) with poor survival outcomes. Using human cancer cell lines that model the TNBC subtype, we observed a strong positive correlation between expression of ANXA1 and PPARg . A similar correlation between these two markers was also established in our clinical breast cancer patients' specimens. To establish a link between these two markers in TNBC, we show de novo expression of ANXA1 is induced by activation of PPARg both in vitro and in vivo and it has a predictive value in determining chemosensitivity to PPARg ligands. Mechanistically, we show for the first time PPARg -induced ANXA1 protein directly interacts with receptor interacting protein-1 (RIP1), promoting its deubi- quitination and thereby activating the caspase-8–dependent death pathway. We further identified this underlying mechanism also involved a PPARg -induced ANXA1-dependent autoubiquiti- nation of cIAP1, the direct E3 ligase of RIP1, shifting cIAP1 toward proteosomal degradation. Collectively, our study provides first insight for the suitability of using drug-induced expression of ANXA1 as a new player in RIP1-induced death machinery in TNBCs, presenting itself both as an inclusion criterion for patient selection and surrogate marker for drug response in future PPARg chemotherapy trials. Mol Cancer Ther; 16(11); 2528–42. Ó2017 AACR. Introduction In the past two decades, the risk of breast cancer has been increasing worldwide, as well in Asia (Japan, Korea and Taiwan; refs. 1–3). The rate of breast cancer incidence in Singapore has also become one of the highest in the world with 5.7% per year increase in premenopausal and 3.9% per year of postmenopausal women (4). Breast cancers can be classified into several classes, with each representing unique molecular or genetic characteristics. Triple-negative breast cancer (TNBC), which is estrogen receptor (ER), progesterone receptor (PR), and C-erb B2 receptor (ERBB2R)-negative (5), is known to be heterogeneous in nature of disease. Anthracycline–taxane regimens remain the current standard for TNBC patients who tend to have a higher risk of relapse and worse overall survival rates with mixed outcomes (6, 7). However, the natural heterogeneity of TNBC has made it difficult to evaluate the true clinical value of the tested drugs, making the cohorts' selection based on specific biomarkers associated with distinct TNBC subgroups a prerequisite (8, 9). A "Genome-first Approach" concept has been introduced where patients will be prestratified and assigned to clinical trials designed to address the thera- peutic hypotheses, based upon analysis of individual tumor profiles (8, 9). 1 Cancer Science Institute of Singapore, National University of Singapore, Sin- gapore. 2 Department of Pharmacology, National University of Singapore, Sin- gapore. 3 Department of Chemistry and Biochemistry, School of Natural Sciences & Mathematics, The University of Texas at Dallas, Texas. 4 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 5 Institute for Research in Immunology and Cancer, Universite de Montreal, Montreal, Quebec, Canada. 6 Department of Histology, Faculty of Medicine, Menoufia University, Menoufia, Egypt. 7 Department of Biochemistry, Dong-A University, College of Medicine, Busan, South Korea. 8 Stem Cell and Cancer Biology Laboratory, School of Biomedical Sciences, Curtin Health Inno- vation Research Institute, Curtin University, Perth WA, Australia. 9 NUS Graduate School for Integrative Sciences and Engineering, National University of Singa- pore, Singapore. 10 Department of Haematology-Oncology, National University Health System, Singapore. 11 National University Cancer Institute, National Uni- versity Health System, Singapore. 12 Tsinghua Berkeley Shenzhen Institute and Division of Life Science and Health, Tsinghua University Graduate School, Shenzhen, P.R. China. 13 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA, Australia. 14 NUS Immunology Program, National University of Singapore, Singapore. 15 Curtin Medical School, Faculty of Health Sciences, Curtin University, Perth WA, Australia. 16 Department of Biological Sciences, University of North Texas, Denton, Texas. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Authors: Alan Prem Kumar, Centre for Translational Medicine (CeTM), National University of Singapore, 14 Medical Drive, Singapore 117599, Singapore. Phone: 656-516-5456; Fax: 656-873-9664; E-mail: csiapk@nus.edu.sg; and Lina H.K. Lim, lina_lim@nuhs.edu.sg doi: 10.1158/1535-7163.MCT-16-0739 Ó2017 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 16(11) November 2017 2528 on May 6, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst August 15, 2017; DOI: 10.1158/1535-7163.MCT-16-0739