Autophagy Regulates Cancer Stem Cell Properties in Triple Negative Breast Cancer Via miR-181a-Mediated Regulation of ATG5/ATG2B

Background: Cancer stemness, miRNA, and autophagy flux are known to contribute to the maintenance of cancers; however, determination of the precise interactions among autophagy, miRNAs, and cancer stemness requires further investigation. Methods: Western blot, immunocytochemistry, immunofluorescence staining, quantitative real-time PCR were performed to detect gene expression. Tumorsphere formation assay, Aldefluor assay, limiting dilution assay were carried out to measure cancer stemness. Acridine orange and Cyto-ID® staining were used to detect autophagy vacuoles. Findings: Autophagy flux was inhibited in triple-negative breast cancer (TNBC) cancer stem cells (CSCs). Moreover, miRNA-181a (miR-181a) is upregulated both in TNBC CSCs and patients. ATG5 and ATG2B participate in the early formation of autophagosomes and were revealed as targets of miR-181a. Inhibition of miR-181a expression led to attenuation of cancer stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. Interpretation: It could be suggested that luminal and triple-negative breast cancer require distinct therapeutic approaches because of their different expressions of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. Autophagy inhibits cancer stemness through the miR-181a-regulated mechanism in TNBC. Promoting tumor-suppressive autophagy using curcumin may be a potential method for the treatment of TNBC. Funding: This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (2019R1A2B5B03069738, NRF- 2016R1A5A1011974). Declaration of Interest: All the authors declared no competing interests. Ethical Approval: This study was approved by the 235 Institutional Review Board of Seoul National University Hospital (IRB number: 1704-236 019-843). A breast cancer tissue array (BC081120f, US Biomax Inc, USA).