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Autophagy, Cell Viability, and Chemoresistance Are Regulated By Published OnlineFirst May 21, 2018; DOI: 10.1158/1541-7786.MCR-17-0634 Cell Death and Survival Molecular Cancer Research Autophagy, Cell Viability, and Chemoresistance Are Regulated By miR-489 in Breast Cancer Mithil Soni1,2, Yogin Patel1,2, Eleni Markoutsa3, Chunfa Jie4, Shou Liu1,2, Peisheng Xu3, and Hexin Chen1,2 Abstract It is postulated that the complexity and heterogeneity in autophagy inhibition and LAPTM4B downregulation as cancer may hinder most efforts that target a single pathway. a major mechanism responsible for miR-489–mediated Thus, discovery of novel therapeutic agents targeting multi- doxorubicin sensitization. Finally, miR-489 and LAPTM4B ple pathways, such as miRNAs, holds promise for future levels were inversely correlated in human tumor clinical cancer therapy. One such miRNA, miR-489, is downregu- specimens, and more importantly, miR-489 expression lated in a majority of breast cancer cells and several drug- levels predict overall survival in patients with 8q22 ampli- resistant breast cancer cell lines, but its role and underlying fication (the region in which LAPTM4B resides). mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy Implications: These findings expand the understanding of as a novel pathway targeted by miR-489 and reports Unc-51 miR-489–mediated tumor suppression and chemosensitiza- like autophagy activating kinase 1 (ULK1) and lysosomal tion in and suggest a strategy for using miR-489 as a thera- protein transmembrane 4 beta (LAPTM4B) to be direct peutic sensitizer in a defined subgroup of resistant breast targets of miR-489. Furthermore, the data demonstrate cancer patients. Mol Cancer Res; 16(9); 1348–60. Ó2018 AACR. Introduction Since then, several groups have demonstrated its tumor-suppres- sive role in many different cancers including gastric cancer, lung Despite recent advances in chemotherapy and novel treatment cancer, ovarian cancer, hepatic cancer, osteosarcoma, and bladder strategies for breast cancer, the development of broad-range cancer (4–12). Remarkably, miR-489 has been reported to induce treatments has been restricted due to the discrepancy in transcrip- cell-cycle arrest and apoptosis, inhibit metastasis and epithelial- tional and genomic heterogeneity of the disease, and the com- to-mesenchymal transition in context-dependent manner. A thor- plexity of resistance mechanisms. Therefore, identification of a ough understanding of miR-489–mediated tumor suppression in single therapeutic agent targeting multiple oncogenic and pro- a specific cancer will be valuable in evaluating the possibility of survival pathways would provide a promising future cancer miRNA-489–based therapy. Hence, this study was aimed to therapy (1, 2). Because miRNAs target multiple pathways and identify novel pathways and molecular targets affected by control gene expression of their targets, they could offer consid- miR-489, which will lead to better understanding of miR-489– erable therapeutic options. miRNAs are small noncoding RNAs mediated tumor suppression. that posttranscriptionally regulate expression of their target genes Macroautophagy (referred to as autophagy now onwards) has (3). Because miRNAs regulate many genes, their dysregulation recently received great attention in the field of cancer and che- have been shown to cause various pathologic conditions includ- moresistance due to its prosurvival role under stressful condi- ing cancer. Identifying such dysregulated miRNAs and their targets tions. Autophagy is a highly conserved process by which cells might provide insights for the development of novel therapy. capture intracellular proteins, lipids, and organelles, and deliver Previously, we have established miR-489 as a tumor suppressor them to the lysosomal compartment for degradation (13). The miRNA in breast cancer by targeting HER2 signaling pathway (4). role of autophagy in cancer remains controversial, as it exhibits tumor-suppressive and tumor-promoting activity in context and – 1Department of Biological Science, University of South Carolina, Columbia, South molecular subtype dependent manner. For example, autophagy Carolina. 2Center for Colon Cancer Research, University of South Carolina, has been shown to inhibit tumor initiation and progression by Columbia, South Carolina. 3Department of Drug Discovery and Biomedical protecting cells from ROS-induced DNA damage and mutagen- Sciences, South Carolina College of Pharmacy, University of South Carolina, esis. Conversely, other studies reported that autophagy protects 4 Columbia, South Carolina. Master of Science in Biomedical Sciences Program, cancer cells from metabolic stress such as starvation or hypoxia Des Moines University, Des Moines, Iowa. (14). Because of its cytoprotective function, it enables cancer cells Note: Supplementary data for this article are available at Molecular Cancer to cope with cytotoxic or other stress induced by treatment. Research Online (http://mcr.aacrjournals.org/). Indeed, autophagy inhibition has been shown to reverse resis- Corresponding Author: Hexin Chen, Department of Biological Sciences, tance to several chemotherapeutic agents. Currently, chloroquine, University of South Carolina, 715 Sumter Street, PSC621, Columbia, SC 29205. an antimalarial drug, is under clinical trials for adjuvant therapy to Phone: 803-777-2928; Fax: 803-777-4002; E-mail: [email protected] reduce resistance via autophagy inhibition in various cancers. doi: 10.1158/1541-7786.MCR-17-0634 Many miRNAs have also been shown to sensitize cancer cells to Ó2018 American Association for Cancer Research. chemotherapy by inhibiting autophagy. For example, miR-200b 1348 Mol Cancer Res; 16(9) September 2018 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 21, 2018; DOI: 10.1158/1541-7786.MCR-17-0634 miR-489 Regulates Autophagy in Breast Cancer Cells has been shown to reverse autophagy-mediated resistance to cells were transfected with 9.3 nmol/L scr or mimic in complete docetaxel by targeting ATG12 (15), while miR-140-5p disrupts media or low serum in presence or absence of 3-MA, siATG5, or cancer stem cell growth in colorectal cancer via autophagy inhi- Bafilomycin A1 followed by cell viability assay using MTT bition (16, 17). Understanding the role of miRNA in autophagy- reagent and Western blot analysis to examine the effect on mediated cancer cell survival and resistance may provide detailed autophagy and apoptosis. To examine the effect of miR-489 on insight on potential miRNA-based therapies. doxorubicin-induced cytoprotective autophagy, MDA-MB-231 In this study, for the first time, we reported that miR-489 and HCC1954 cells were transfected with 28 nmol/L scr or inhibits autophagy by affecting multiple genes involved in the mimic with or without doxorubicin. Protein was isolated 72 process. We further report that miR-489 induced autophago- hours posttreatment and Western blot analysis was performed. some accumulation is partially responsible for its cytotoxic effect. Moreover, we found that miR-489 can also sensitize Chemosensitization assays breast cancer cells to doxorubicin via autophagy inhibition. To study doxorubicin localization, cells were treated with From the combination of our in vitro and in vivo studies, 28 nmol/L scr or mimic for 24 hours then treated with we report that miR-489 inhibits autophagy by targeting ULK1 0.5 mmol/L doxorubicin for 48 hours followed by confocal and LAPTM4B and sensitizes tumor cells to doxorubicin by microscopy. MDA-MB-231 cells were transfected with inhibiting doxorubicin-induced cytoprotective autophagy and 9.3 nmol/L scr or mimic for 24 hours and treated with indicated LAPTM4B expression. concentration of doxorubicin in presence or absence of 3-MA (5 mmol/L), siATG5 (50 nmol/L), or Bafilomycin A1 (50 nmol/L) Materials and Methods for 48 hours and cell proliferation was measured by MTT assay to examine role of autophagy inhibition in miR-489–mediated The detailed procedures about cell lines and reagents, plasmid doxorubicin sensitization. To assess lysosomal integrity, MDA- construction, cell culture, Western blot analysis, IHC, qRT-PCR, MB-231 cells were transfected with 28 nmol/L scr or mimic and luciferase assay, and MTT assay are described in detail in Supple- stained with Acridine orange (1 mg/mL) for 20 minutes followed mentary Experimental Procedures. All the primer sequences are by flow cytometry and confocal microscopy. listed in Supplementary Table S1. Microarray analysis miRNA-nanoparticles' preparation T47D cells were seeded in 6-well culture dish, treated with Liposomes were prepared as described elsewhere with slight fi fl 28 nmol/L scramble miRNA or miR-489 mimic for 72 hours. RNA modi cations (18, 19). Brie y, cationic liposomes consisting of was extracted with TRIzol reagent, followed by clean-up and DOTAP and cholesterol (2:1 molar ratio) were prepared using the fi fi DNase I treatment with QIAGEN RNeasy mini kit in accordance thin- lm hydration method. The lm was hydrated with nuclease with the prescribed protocol provided with the kit. Quality free water and sonicated using probe sonicator for 15 minutes. control was performed with Agilent Bioanalyser before perform- The lipid concentration adjusted at 10 mg/mL. For miRNA- m ing microarray. The data were normalized using the default nanoparticles' preparation, 231.4 L of cationic liposomes and m fi quantile normalization with R/bioconductor package lumi ver- 90 L of miRNA were
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