HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling

Abstract

Background: 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.

Aim: 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.

Methods: 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.

Results: 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).

Conclusion: 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; CD44; Cancer stem cells; Hypoxia-inducible factor-2α.

Figure 1

Expression of hypoxia-inducible factor-2α and CD44 in carcinoma tissues of triple-negative breast cancer and non-triple-negative breast cancer patients (×200). TNBC: Triple-negative breast cancer; HIF-2α: Hypoxia-inducible factor-2α.

Figure 2

Expression of hypoxia-inducible factor-2α and CD44 in MCF-7 and MDA-MB-231 cells (×200). The expression of HIF-2α and CD44 in MDA-MB-231 cells was significantly higher than that in MCF-7 cells. HIF-2α: Hypoxia-inducible factor-2α; DAPI : 4',6-diamidino-2-phenylindole.

Figure 3

Expression of HIF-2α and CD44 mRNAs in human breast cancer cells after transfection. ^aP < 0.05 vs normoxia; ^bP < 0.05 vs hypoxia + NC. HIF-2α: Hypoxia-inducible factor-2α; NC: Negative-sequence control.

Figure 4

HIF-2α silencing reduces the CD44⁺/CD24⁻ population in MDA-MB-231 cells. Flow cytometry of the stem cell-associated cell surface markers CD44 and CD24 in hypoxia and HIF-2α-siRNA treated cells. HIF-2α: Hypoxia-inducible factor-2α.

Figure 5

TUNEL assay of hypoxia-induced MDA-MB-231 cells after transfection (×200). Light green cells are TUNEL-positive staining cells. ^aP < 0.05 vs normoxia; ^bP < 0.01 vs hypoxia + NC. DAPI: 4',6-diamidino-2-phenylindole; NC: Negative-sequence control.

Figure 6

Quantification of mammosphere formation assay of MDA-MB-231 cells before and after transfection. Cells were cultured for 7-21 d under mammosphere culture conditions. ^bP < 0.01 vs hypoxia + NC. NC: Negative-sequence control.

Figure 7

Effect of HIF-2α on the PI3K/AKT signaling pathway. In MDA-MB-231 cells induced by hypoxia, HIF-2α knockdown reduced the AKT and mTOR phosphorylation levels; ^aP < 0.05 vs normoxia; ^bP < 0.05 vs hypoxia + NC. PI3K: Phosphoinosmde-3-kinase; AKT: Protein-serine-threonine kinase; mTOR: Mammalian target of rapamycin; p-AKT: Phosphorylated AKT; p-mTOR: Phosphorylated mTOR; NC: Negative-sequence control.