1. Volume 18, Issue 8, Pages 1946-1957 (February 2017)
Chemotherapy-Induced Ca2+ Release Stimulates Breast Cancer Stem Cell Enrichment 
Haiquan Lu, Ivan Chen, Larissa A. Shimoda, Youngrok Park, Chuanzhao Zhang, Linh Tran, Huimin Zhang, Gregg L. Semenza 
Cell Reports 
Volume 18, Issue 8, Pages (February 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1946-1957DOI: (10.1016/j.celrep.2017.02.001)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1
Chemotherapy Induces GSTO1 Expression in a HIF-Dependent Manner
(A) Kaplan-Meier analysis of relapse-free survival was performed based on clinical and molecular data from 3,554 breast cancer (BC) patients. The patients were stratified by GSTO1 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.
(B and C) BC cell lines were treated with vehicle (V), paclitaxel (P), or carboplatin (C) for 3 days at the IC50 of the drugs. RT-qPCR (B) and immunoblot (C) assays of GSTO1 mRNA and protein expression were performed. In (B), results were normalized to vehicle-treated MCF-7 cells (mean ± SEM; n = 3). ∗∗p < 0.01, ∗∗∗p < versus vehicle in each cell line (one-way ANOVA). In (C), the experiment was repeated three times; a representative blot is shown.
(D) mRNA levels of four GST family members in each of 1,215 human BCs from TCGA database were compared with the HIF signature using Pearson’s correlation test. For each comparison, Pearson’s correlation coefficient (R) and p value are shown.
(E) MDA-MB-231 subclones were treated for 3 days, and GSTO1 mRNA expression was analyzed by RT-qPCR (mean ± SEM; n = 3). ∗∗∗p < versus NTC-V; ###p < versus NTC-P or NTC-C; ns, not significant (two-way ANOVA).
(F) MDA-MB-231 subclones were treated with vehicle (–) or carboplatin (+) for 3 days, and immunoblot assays were performed.
(G) MDA-MB-231 cells were treated with V, P, or C, either alone or in combination with 100 nM digoxin (D) for 3 days, and RT-qPCR was performed to analyze GSTO1 mRNA expression (mean ± SEM; n = 3). ∗∗p < 0.01, ∗∗∗p < versus V; ###p < versus P or C (one-way ANOVA).
(H) MDA-MB-231 cells were treated without (–) or with carboplatin (+) for 3 days, and chromatin immunoprecipitation (ChIP) assays were performed using immunoglobulin G (IgG) or antibody (Ab) against HIF-1α, HIF-2α, or HIF-1β. Primers flanking binding sites were used for qPCR, and results were normalized to carboplatin (–), IgG (mean ± SEM; n = 4). ∗∗p < 0.01, ∗∗∗p < versus carboplatin (–) (Student’s t test). The nucleotide sequences encompassing HIF binding sites (red) within intron 2 of the GSTO1 gene are shown.
See also Figure S1.
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4. Figure 2
Carboplatin-Induced BCSC Enrichment Is Blocked by GSTO1 Knockdown
(A and B) MDA-MB-231 cells were cultured on standard polystyrene tissue culture plates (Adherent) or ultra-low adherence plates (Sphere) for 7 days and harvested for analysis of GSTO1 and GSTP1 mRNA (A) and GSTO1 protein (B) expression. Results were normalized to Adherent (mean ± SEM; n = 3). ∗∗p < 0.01 versus Adherent; ns, not significant (Student’s t test).
(C) MDA-MB-231 subclones transfected with vector encoding non-targeting control shRNA (NTC) or either of two different shRNAs targeting GSTO1 (#1 and #2) were treated with vehicle [C(–)] or carboplatin [C(+)], and the percentage of ALDH+ [% ALDH (+)] cells was determined (mean ± SEM; n = 3). ∗∗∗p < versus NTC-C(–); ###p < versus NTC-C(+) (two-way ANOVA).
(D) MDA-MB-231 subclones transfected with NTC or GSTO1 shRNA vector were treated with vehicle [C(–)] or carboplatin [C(+)] for 3 days. The number of primary (1°) and secondary (2°) mammospheres per 1,000 cells seeded was calculated (mean ± SEM; n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus NTC-C(–); ###p < versus NTC-C(+) (two-way ANOVA). Bar, 200 μm.
(E) MDA-MB-231 subclones transfected with NTC or GSTO1 shRNA vector were treated with vehicle [C(–)] or carboplatin [C(+)] for 3 days, and RT-qPCR assays were performed (mean ± SEM; n = 3). ∗∗p < 0.01, ∗∗∗p < versus NTC-C(–); ###p < versus NTC-C(+) (two-way ANOVA).
(F) MDA-MB-231 subclones transfected with NTC or GSTO1 shRNA vector were treated with vehicle (–) or carboplatin (+) for 3 days, and immunoblot assays were performed.
See also Figure S2.
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5. Figure 3
GSTO1 Interacts with RYR1 and Regulates Cytosolic Calcium Levels and BCSC Phenotype
(A) MDA-MB-231 cells were treated with 100 μM carboplatin, 10 μM CRID3, or both for 3 days, and immunoblot assays were performed.
(B and C) MDA-MB-231 cells were transfected with empty pcDNA3 vector or vector encoding wild-type (WT) or mutant (C32A) GSTO1. In (B), the percentage of ALDH (+) cells was determined 4 days after transfection (mean ± SEM; n = 3). ∗∗p < 0.01, ∗∗∗p < versus pcDNA3 (one-way ANOVA). In (C), immunoblot assays were performed 3 days after transfection.
(D and E) MDA-MB-231 cells were treated with vehicle (–) or carboplatin (+) for 3 days (D), or transfected with empty vector, or vector expressing GSTO1 (WT or C32A), for 3 days (E). Immunoprecipitation (IP) was performed using IgG, anti-RYR1 (left panel) or anti-GSTO1 (right panel) Ab. Immunoblot assays were performed with Ab against GSTO1 or RYR1. WCL, aliquot of whole-cell lysate reserved prior to IP.
(F) MDA-MB-231 subclones were treated as indicated for 3 days, and cytosolic Ca2+ levels were measured (mean ± SEM; n = 6). ∗p < 0.05, ∗∗p < 0.01 versus NTC-C(–); ##p < 0.01 versus NTC-C(+) (two-way ANOVA).
(G) MDA-MB-231 cells were treated with 100 μM carboplatin, 1 μM BAPTA-AM, or both for 4 days, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ∗p < 0.05, ∗∗∗p < versus control; ###p < versus carboplatin (+), BAPTA-AM (–) group (one-way ANOVA).
(H) MDA-MB-231 subclones transfected with vector encoding NTC shRNA or either of two different shRNAs targeting RYR1 (#1 and #2) were treated as indicated for 4 days, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ∗∗p < 0.01, ∗∗∗p < versus NTC-C(–); ###p < versus NTC-C(+) (one-way ANOVA).
(I) MDA-MB-231 subclones transfected with NTC or RYR1 shRNA vector were treated as indicated for 3 days, and RT-qPCR assays were performed (mean ± SEM; n = 3). ∗∗p < 0.01, ∗∗∗p < versus NTC-C(–); ###p < versus NTC-C(+) (two-way ANOVA).
(J) MDA-MB-231 cells were treated with dantrolene, alone or in combination with carboplatin for 4 days, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus C(–), dantrolene 0 μM; ###p < versus C(+), dantrolene 0 μM (one-way ANOVA).
(K) MDA-MB-231 cells were treated with 4-chloro-m-cresol, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ∗∗∗p < versus 4-chloro-m-cresol 0 μM (one-way ANOVA).
See also Figure S3.
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6. Figure 4
Knockdown of GSTO1 or RYR1 Expression Inhibits Tumor Initiation, Metastasis, and Recurrence
(A) 1,000 cells of each MDA-MB-231 subclone were implanted into the mammary fat pad (MFP). The number of mice with tumors after 10 weeks and p value (Fisher’s exact test) are shown.
(B) 2 × 106 cells of each MDA-MB-231 subclone were implanted into the MFP. When tumor volume reached 200 mm3, lungs were fixed under inflation, and sections were stained with H&E to identify metastases (arrows). Bar, 2 mm.
(C) 2 × 106 cells of each MDA-MB-231 subclone were implanted into the MFP. When tumors were palpable, mice were treated with 20 mg/kg of carboplatin every 5 days until tumors were no longer palpable. The number of days for tumor formation, remission, and recurrence are reported (mean ± SEM). ∗p < 0.05, ∗∗p < 0.01 versus shNTC (one-way ANOVA).
See also Figure S4.
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7. Figure 5
GSTO1-Dependent PYK2 → SRC → STAT3 Signaling Is Required for Carboplatin-Induced BCSC Enrichment
(A) MDA-MB-231 subclones transfected with NTC or GSTO1 shRNA vector were treated with vehicle (–) or 100 μM carboplatin (+) for 3 days, and immunoblot assays were performed.
(B and C) MDA-MB-231 cells were treated as indicated with 100 nM STAT3 inhibitor VII, 100 μM carboplatin, 500 ng/mL IL-6 neutralizing Ab (NAb), or control IgG for 4 days, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ∗p < 0.05, ∗∗∗p < versus carboplatin (–); ###p < versus carboplatin (+) (one-way ANOVA).
(D and E) Immunoblot assays were performed with lysates of MDA-MB-231 cells that were treated for 3 days as indicated.
(F) MDA-MB-231 cells were treated without or with STAT3 inhibitor VII (left panel) or transfected with empty vector (pcDNA3), WT, or C32A GSTO1 expression vector (right panel) for 3 days, and ChIP assays were performed using IgG or STAT3 Ab. qPCR was performed using primers flanking a STAT3 site in the KLF4 gene (mean ± SEM; n = 3). The nucleotide sequence encompassing the binding site (blue), 5′ to the KLF4 gene, is shown. Left panel: ∗∗∗p < versus inhibitor VII (–), IgG; ###p < versus inhibitor VII (–), STAT3 Ab. Right panel: ∗∗∗p < versus pcDNA3, IgG; #p < 0.05, ##p < 0.01 versus pcDNA3, STAT3 Ab (two-way ANOVA).
(G) MDA-MB-231 cells were treated with carboplatin (C), alone or in combination with 10 μM W-7, 2.5 μM PF , or 2.5 μM PP-1, for 4 days, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ∗∗∗p < versus control; ###p < versus C (one-way ANOVA).
(H) MDA-MB-231 cells were treated with carboplatin, alone or in combination with PF or PP-1 for 3 days, and immunoblot assays were performed.
(I) MDA-MB-231 cells were cultured on standard (Adherent) or ultra-low adherence (Sphere) plates for 7 days and harvested for immunoblot assays.
(J) MDA-MB-231 cells were transfected with empty pcDNA3 vector or pcDNA3 encoding GSTO1 (WT or C32A). Immunoblot assays were performed 3 days after transfection.
(K) MDA-MB-231 subclones were treated as indicated and immunoblot assays were performed.
See also Figure S5.
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8. Figure 6
Carboplatin Induces HIF-Regulated GSTO1 Expression, which Promotes Pluripotency Factor Expression and BCSC Enrichment In Vivo
(A–E) 2 × 106 cells of MDA-MB-231 subclones were implanted into SCID mice. When tumor volume reached 200 mm3 (day 0), mice were grouped randomly and treated with saline [C(–)] or 20 mg/kg of carboplatin [C(+)] on days 0, 5, and 10. Tumor volumes were measured every 2–3 days (A). Tumors samples were harvested on day 13 for Aldefluor (B), mammosphere (C), and immunoblot (D and E) assays. Densitometric analysis of immunoblots in (D) was performed to generate the data (mean ± SEM, n = 3) shown in (E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus NTC, C(–); #p < 0.05, ##p < 0.01, ###p < versus NTC, C(+) (two-way ANOVA).
(F) Carboplatin induces HIF-dependent GSTO1 expression, increased cytosolic Ca2+, and a kinase cascade, leading to pluripotency factor expression and BCSC enrichment.
See also Figure S6.
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9. Figure 7
Digoxin Blocks Carboplatin-Induced Pluripotency Factor Expression and BCSC Enrichment In Vivo
(A–D) 2 × 106 MDA-MB-231 cells were implanted into the MFP. When tumor volume reached 200 mm3 (day 0), mice were randomized and treated with saline (Vehicle; V), carboplatin (C; 20 mg/kg, days 0, 5, and 10), digoxin (D; 2 mg/kg, days 1–13), or carboplatin and digoxin (C and D). Tumor volumes were measured every 2–3 days (A). Tumors were harvested on day 13 for Aldefluor (B), mammosphere (C), and RT-qPCR (D) assays. Data are shown as mean ± SEM (n = 4). In (A), ∗∗p < 0.01 versus C; ∗∗∗p < versus D. In (B)–(D), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus V; #p < 0.05, ##p < 0.01, ###p < versus C (one-way ANOVA).
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