Published online Jan 26, 2020. doi: 10.4252/wjsc.v12.i1.87
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.
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.