Fig 1: HIF-2a activates UPRER via regulating SOD2-mtROS axis.A The mRNA level of SOD2 and SOD1 were detected in HIF-2a OE MCF7 cells and HIF-2a KD MCF7 MS cells. B The correlation between HIF-2a and SOD2 was detected in mRNA level from TCGA database (n = 1169). C The mRNA expression of SOD2 was compared in CD44+CD24- and non CD44+CD24- patients from TCGA database (n = 1169). D The levels of mtROS were detected in MCF7 and MCF7 MS cells, HIF-2a OE MCF7 cells and HIF-2a KD MCF7 MS cells. E The mRNA level of SOD2 and mtROS level were measured in SOD2-overexpressing and HIF-2a-silencing (SOD2 OE + HIF-2a KD) MCF7 MS cells, under 1% O2. F The mRNA level of SOD2 and mtROS level were measured in HIF-2a KD MCF7 MS cells cultured with mitoTEMPOL (100 µM) for 48 h, under 1% O2. G The self-renewal ability rescued in HIF-2a KD MCF7 MS cells cultured mitoTEMPOL (100 µM) for 48 h, compared with HIF-2a KD MCF7 MS cells, under 1% O2. Scale bar, 250 µm. H The cells viability rate was detected in HIF-2a KD MCF7 MS cells cultured with mitoTEMPOL (100 µM) for 48 h, under 1% O2. I The expression level of GRP78 was detected in HIF-2a KD MCF7 MS cells cultured with mitoTEMPOL (100 µM) for 48 h, under 1% O2. NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, compared to MCF7 cells/Ctrl/shCtrl/shHIF-2a; Student’s t test, two-way ANOVA test, Mann–Whitney U analysis, Pearson correlation analysis. Error bars, mean ± SD (n = 3).
Fig 2: Silencing HIF-2a suppresses the early tumorigenesis and increases the sensitivities of PTX via SOD2-mtROS-PDI/GRP78-UPRER in vivo.A The diagram showed the time of tumor formation in BALB/c (nu/nu) mice transplanted with control or HIF-2a KD MCF7 MS cells (1 × 105). 15 days after the inoculation, the mice were intraperitoneally injected with or without PTX (5 mg/kg) once every other day till to the 31th day (n = 5). The other mice (n = 5) were observed survival till the 120th day. B Small animal imaging showed the expression of green fluorescent protein (GFP) in the xenografted mice of each group. n = 5. C The tumor weights were measured in each group after sacrifice of xenograft mice at the 31th days. n = 5. Two-way ANOVA test. D The growth curves of tumor volumes were measured in xenograft mice were measured every other day. n = 5. Two-way ANOVA test. E The survival of the mice in each group (left) were analyzed by Kaplan Meier-plotter curve. Median survival times (MST). Hazard ratio (HR). n = 5. Two-way ANOVA test. F The PTX accumulation were measured in the HIF-2a KD xenografted tissue by HPLC-MS. n = 3. Student’s t test. G The proportion of CD44+CD24- cell in the HIF-2a KD xenografted tumor were detected by flow cytometry. H The protein expressions of HIF-2a, GRP78, P-IRE1, IRE1, XBP1s, p-PERK, PERK, ATF6 and P-gp were detected in the HIF-2a KD xenografted tissue. I The mRNA expression of SOD2 was detected in the HIF-2a KD xenografted tissue. n = 3. Student’s t test. J The level of mtROS and PDI were detected in the HIF-2a KD xenografted tissue. Scale bar, 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, compared to shCtrl.
Fig 3: YQ-0629 targets HIF-2a to suppress stem trait of BCSCs and synergy the sensitization to PTX in vitro.A The chemical structure of YQ-0629 and docking conformation showed the interaction of the YQ-0629 with the active site of HIF-2a through MOE software (left). YQ-0629 and HIF-2a PAS-B domain was confirmed direct binding by surface plasmon resonance (SPR)-based Biacore assay (right). B The expression level of HIF-2a was detected in the nucleus (N) and cytoplasm (C) of MCF7 MS cells cultured with YQ-0629 (10 µM) alone, PTX (3 nM) alone, or YQ-0629 (10 µM) combined with PTX (3 nM) for 72 h. C The expression and location of HIF-2a were detected in MCF7 MS cells cultured with YQ-0629 (10 µM) alone, PTX (3 nM) alone, or YQ-0629 (10 µM) combined with PTX (3 nM) for 72 h by immunofluorescence staining. Scale bar, 10 µm. D The cell viability rate of MCF7 MS cells cultured with different concentrations of YQ-0629 for 24–96 hours were determined by CCK-8 assay (left). The IC50 values of PTX and synergic index (R) were calculated in MCF7 MS cells cultured with indicated dose of YQ-0629 for 72 h (right). n = 3. Student’s t test. E The self-renewal ability was detected in MCF7 MS cells cultured with YQ-0629 (10 µM) alone, PTX (3 nM) alone, or YQ-0629 (10 µM) combined with PTX (3 nM) for 72 h. n = 3. Synergic index (R). Two-way ANOVA test. Scale bar, 250 µm. F–H The mRNA expression of SOD2 and mtROS level, the expressions level of PDI, GRP78, and P-gp were detected in the MCF7 MS cells cultured with indicated dose of PTX and YQ-0629 for 72 h. n = 3. Two-way ANOVA test. **P < 0.01, **P < 0.01, ***P < 0.001, compared to MCF7 MS cells/MCF7 MS cells + PTX treatment.
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