Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells

// Chuanzhao Zhang 1,2 , Wanqing Iris Zhi 3 , Haiquan Lu 1,9 , Debangshu Samanta 1,9 , Ivan Chen 1,9 , Edward Gabrielson 3,4 and Gregg L. Semenza 1,3,5,6,7,8,9 1 Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2 Department of General Surgery, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China 3 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 4 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 5 Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA 6 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA 7 Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 8 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 9 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA Correspondence to: Gregg L. Semenza, email: // Keywords : breast cancer stem cells; hypoxia; metastasis; N 6 -methyladenosine; pluripotency factors Received : August 02, 2016 Accepted : August 12, 2016 Published : August 31, 2016 Abstract Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N 6 -methyladenosine (m 6 A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m 6 A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.