Figure 1.

Chemotherapy induces breast cancer stem cell enrichment through repression of GSTM1/2/3/4. (A) TNBC cell lines were treated with carboplatin at the dose of IC₅₀ for 72 h and RT-qPCR was performed for expression of glutathione S-transferases. (B) MDA-MB-231 cells were treated with vehicle (V), 100 μM carboplatin (C), 10 nM paclitaxel (P), or 10 nM gemcitabine (G) for 72 h and immunoblot assays were performed to analyze GSTM protein expression. (C) MDA-MB-231 cells were implanted into the mammary fat pad of female SCID mice and mice were treated with vehicle or carboplatin (20 mg/kg on days 0, 5, 10). Tumors were harvested on day 13 for RT-qPCR (mean ± SEM; n = 5); ^∗P < 0.05, ^∗∗∗P < 0.001 vs. V. (D) MMTV-PyMT transgenic mice were treated with vehicle or carboplatin (20 mg/kg on days 0, 5, 10) and tumors were harvested on day 13 for RT-qPCR (mean ± SEM; n = 5); ^∗P < 0.05, ^∗∗P < 0.01 vs. V. (E, F) MDA-MB-231 subclones were treated with carboplatin at indicated doses for 72 h and cell counting kit-8 (CCK-8) assay was performed (mean ± SEM; n = 3). (G) The correlation of protein sequences was analyzed. (H) TNBC cell lines were cultured on standard polystyrene tissue culture plates (adherent) or ultra-low adherence plates (sphere) for 7 days and RT-qPCR was performed. (I) MDA-MB-231 cells were cultured on standard polystyrene tissue culture plates or ultra-low adherence plates for 7 days and immunoblot assays were performed. (J) MDA-MB-231 cells were sorted into ALDH-negative (−) and ALDH-positive (+) populations by flow cytometry and RT-qPCR was performed (mean ± SEM; n = 3); ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. ALDH (−). (K–S) The percentage of ALDH⁺ cells (L, O, R; mean ± SEM; n = 3) and the number of mammospheres per 1000 cells seeded (M, P, S; mean ± SEM; n = 6) were determined in MDA-MB-231 subclones. The representative results of the Aldefluor assay were shown (K, N, Q). ^∗∗∗P < 0.001 vs. NTC; ^###P < 0.001 vs. shGSTM3 in (L, M), ^###P < 0.001 vs. shGSTM1/2 in (R, S); ns, not significant. (T) SCID mice were injected with indicated numbers of MDA-MB-231 subclone cells and tumor-initiating cell frequency (TCF) with 95% confidence intervals (CI) was calculated. (U) 2 × 10⁶ MDA-MB-231 subclone cells were implanted into SCID mice. When tumors became palpable, mice were treated with 20 mg/kg carboplatin every 5 days until tumors were no longer palpable. Kaplan–Meier survival curves of tumor-free (left), tumor-bearing (center), and recurrence-free (right) were plotted and P values of log-rank tests are shown (n = 10). (V) Expression of GSTM1/2/3/4 and ten mRNAs encoded by HIF target genes are shown for primary breast cancers accessed from the TCGA database. (W) MDA-MB-231 subclones were treated with vehicle or 100 μM carboplatin for 72 h and RT-qPCR was performed (mean ± SEM; n = 3); ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. NTC V; ^###P < 0.001 vs. NTC C; ns, not significant. (X) MDA-MB-231 cells were implanted into the mammary fat pad of female SCID mice and mice were treated with vehicle, carboplatin (20 mg/kg on days 0, 5, 10), digoxin (2 mg/kg on days 1–13), or carboplatin and digoxin. Tumors were harvested on day 13 for RT-qPCR (mean ± SEM; n = 5); ^∗P < 0.05, ^∗∗∗P < 0.001 vs. V; ^###P < 0.001 vs. C; ns, not significant. (Y) Pearson's test was performed to analyze the correlation of GSTM1/2/3/4 mRNA expression with mRNAsi in TCGA samples. (Z) Kaplan–Meier analyses of relapse-free survival were performed based on clinical and molecular data from 4,929 breast cancer patients. The patients were stratified by GSTM1/2/3/4 mRNA levels in the primary tumor, which were greater (red) or less (black) than the median level. The hazard ratio (HR) and P value (log-rank test) are shown.