Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jan 26, 2020; 12(1): 87-99
Published online Jan 26, 2020. doi: 10.4252/wjsc.v12.i1.87
HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling
Jie Bai, Wei-Bin Chen, Xiao-Yu Zhang, Xiao-Ning Kang, Li-Jun Jin, Hui Zhang, Zun-Yi Wang
Jie Bai, Xiao-Yu Zhang, Li-Jun Jin, Zun-Yi Wang, Thyroid and Breast Deptartment III, Cangzhou Central Hospital, Cangzhou 061001, Hebei Province, China
Wei-Bin Chen, Department of Radiology, North China University of Science and Technology Affiliated Hospital, Tangshan 063000, Hebei Province, China
Xiao-Ning Kang, Department of Second Ultrasound, Cangzhou Central Hospital, Cangzhou 061001, Hebei Province, China
Author contributions: Bai J and Wang ZY conceived and designed the study; Bai J, Chen WB, and Zhang XY performed the experiments; Zhang XY and Kang XN acquired the patients’ data; Bai J, Kang XN, Jin LJ, and Zhang H analyzed the data and drafted the report; Wang ZY supervised the study; all authors reviewed and revised the manuscript critically and approved the final version to be published.
Institutional review board statement: The study was approved by the Ethics Committee of Cangzhou Central Hospital.
Conflict-of-interest statement: The authors report no conflicts of interest in this work.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Zun-Yi Wang, MD, Doctor, Department of Third Surgical Oncology, Cangzhou Central Hospital, No. 16, West Xinhua Road, Cangzhou 061001, Hebei Province, China.
Received: June 15, 2019
Peer-review started: June 18, 2019
First decision: July 31, 2019
Revised: August 12, 2019
Accepted: October 14, 2019
Article in press: October 14, 2019
Published online: January 26, 2020

Breast cancer is a common malignant tumor that seriously threatens women’s health. Breast cancer stem cell (CSC)-like cell population may be the main factor for breast cancer metastasis. Therefore, targeted therapy for CSCs has great potential significance. Hypoxia-inducible factor is a transcription factor widely expressed in tumors. Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxia-inducible factor-2α (HIF-2α). However, the specific mechanism remains unclear and further research is necessary.


To investigate the effect of HIF-2α down-regulation on stem cell markers, microsphere formation, and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism.


Immunohistochemistry was used to detect the expression of HIF-2α and CD44 in triple-negative breast cancer (TNBC) and non-TNBC tissues. Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2α and CD44 in MDA-MB-231 cells and MCF-7 cells. HIF-2α was silenced by RNA interference, and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR. Further, flow cytometry, TdT-mediated X-dUTP nick end labeling, and mammosphere formation assays were used to evaluate the effect of HIF-2α on CSCs and apoptosis. The possible mechanisms were analyzed by Western blot.


The results of immunohistochemistry showed that HIF-2α was highly expressed in both TNBC and non-TNBC, while the expression of CD44 in different molecular types of breast cancer cells was different. In in vitro experiments, it was found that HIF-2α and CD44 were expressed almost in the same cell. Compared with hypoxia + negative-sequence control, HIF-2α small interfering ribonucleic acid transfection can lower the expression of HIF-2α and CD44 mRNA(P < 0.05), increase the percentage of apoptotic cells (P < 0.05), and resulted in a reduction of CD44+/CD24 population (P < 0.05) and mammosphere formation (P < 0.05) in hypoxic MDA-MB-231 cells. Western blot analysis revealed that phosphorylated protein-serine-threonine kinase (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) levels in MDA-MB-231 decreased significantly after HIF-2α silencing (P < 0.05).


Down-regulation of HIF-2α expression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis, and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.

Keywords: Breast cancer, Hypoxia-inducible factor-2α, Cancer stem cells, CD44

Core tip: Cancer stem cells (CSCs) play an important role in tumor formation, growth, invasion, metastasis, and recurrence. Hypoxia can promote the differentiation of various tumor cells, enable cells to acquire stem cell characteristics, and enhance tumor cell invasion and tumorigenicity. In the long-term exposure of tumors to hypoxia, the major regulatory factor is hypoxia-inducible factor-2α (HIF-2α), which can promote the malignant biological behavior of tumors by activating its downstream target genes. Studies have shown that the effect of HIF-2α on tumor cells may be related to CD44, a marker for breast CSCs. In this study, breast cancer cell line MCF-7 and basal breast cancer cell line MDA-MB-231 were utilized to investigate the relationship between HIF-2α and CD44 gene expression and the regulatory effect of HIF-2α on CD44.