Chemotherapy triggers HIF-1–dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype

Chemotherapy Induces HIF-1–Dependent Glutathione Biosynthesis.

We hypothesized that chemotherapy induces glutathione synthesis in breast cancer cells to protect against oxidative stress. Paclitaxel, gemcitabine, and carboplatin are all Food and Drug Administration-approved chemotherapy drugs that are used for the treatment of TNBC. We treated two TNBC cell lines, MDA-MB-231 and SUM-149, with paclitaxel, gemcitabine, or carboplatin for 72 h at the concentration of drug that inhibited growth by 50% (IC₅₀). Each of these chemotherapeutic agents increased xCT and GCLM mRNA levels in both cell lines as determined by reverse transcription (RT) and quantitative real-time PCR (qPCR) (Fig. 1A and Fig. S1A). In MDA-MB-231 cells transfected with a nontargeting control (NTC) short hairpin RNA (shRNA), paclitaxel treatment increased xCT and GCLM protein levels (Fig. 1B), and intracellular total glutathione levels (Fig. 1C). Knockdown of xCT or GCLM expression by specific shRNAs (Fig. 1B) blocked glutathione induction in response to paclitaxel treatment (Fig. 1C), indicating that both xCT and GCLM are required for paclitaxel-induced glutathione synthesis.

Gene expression data from 1,215 human breast cancers in the Cancer Genome Atlas (TCGA) database were analyzed to compare the expression patterns of xCT and GCLM mRNA in different molecular subtypes of breast cancer (Basal, HER2-enriched, Luminal A, Luminal B, and Normal-like) that are based on a 50-mRNA (PAM50) signature (22) (Fig. S1B). Compared with other breast cancer subtypes, xCT and GCLM mRNA levels were significantly increased in basal breast cancers (Fig. S1C), in which hypoxia-inducible factor (HIF)-target gene expression is also increased (10). A detailed comparison of xCT and GCLM mRNA levels with those of CXCR3, L1CAM, BNIP3, PLOD1, P4HA1, P4HA2, VEGFA, SLC2A1, and CA9 mRNA, which are all HIF-regulated gene products in TNBC cells, showed a significant correlation of xCT and GCLM expression with six of nine and eight of nine HIF-target genes, respectively (Fig. S1D). Exposure of MDA-MB-231 cells to paclitaxel, gemcitabine, or carboplatin at IC₅₀ induced HIF-1α protein expression (Fig. S1E). In addition, xCT and GCLM mRNA levels were induced in most breast cancer cell lines when they were exposed to 1% O₂ for 24 h (Fig. S1F). In MDA-MB-231 cells, xCT and GCLM mRNA levels were significantly induced by either paclitaxel or hypoxia alone, although no additive effect was observed by the combined treatment (Fig. S1G). These data suggest that SLC7A11 and GCLM gene expression are regulated by HIFs.

To test this hypothesis, we analyzed MDA-MB-231 subclones that were stably transfected with an expression vector encoding shRNA targeting HIF-1α or HIF-2α, and found that knockdown of HIF-1α, but not HIF-2α, decreased xCT and GCLM mRNA basal levels and blocked their induction in response to paclitaxel treatment (Fig. 1D). Knockdown of HIF-1α, but not HIF-2α, also abrogated xCT and GCLM induction in response to hypoxia (Fig. S1H). Pharmacological inhibition of HIF-1α expression by treatment of cells with the HIF-1 inhibitor digoxin (10, 23) also blocked expression of xCT and GCLM mRNA in SUM-149 cells exposed to paclitaxel, gemcitabine, or carboplatin (Fig. S1A). Taken together, these data indicate that expression of the SLC7A11 and GCLM genes is regulated by HIF-1, but not HIF-2. We also implanted MDA-MB-231 cells into the mammary fat pad of female severe combined immunodeficiency (SCID) mice and treated the mice with paclitaxel, either alone or in combination with digoxin. Digoxin treatment decreased xCT and GCLM mRNA levels, and blocked their induction by paclitaxel (Fig. 1E), demonstrating that paclitaxel treatment induces xCT and GCLM mRNA expression in a HIF-1–dependent manner in vivo.

To investigate whether HIF-1 directly regulates SLC7A11 and GCLM expression, genomic DNA sequences were searched for matches to the consensus HIF-1 binding-site sequence 5′-(A/G)CGTG-3′ and candidate sites were evaluated by chromatin immunoprecipitation (ChIP) assays performed in MDA-MB-231 cells. Hypoxia induced the binding of HIF-1α and HIF-1β to sites located in the third intron of SLC7A11 and in the 5′-flanking region of GCLM (Fig. 1F). Taken together, these data indicate that chemotherapy drugs induce HIF-1–dependent activation of SLC7A11 and GCLM transcription.

Inhibition of Glutathione Synthesis Blocks Paclitaxel-Induced BCSC Enrichment.

We recently demonstrated that paclitaxel treatment increases the percentage of BCSCs in a HIF-dependent manner (10). xCT and GCLM expression in breast cancer cell lines is correlated with expression of CD44, an important BCSC marker (20, 24). To test the role of the glutathione synthesis pathway in paclitaxel-induced BCSC enrichment, MDA-MB-231 cells were sorted into an ALDH⁺ population, which is highly enriched for BCSCs, and an ALDH⁻ population, which is depleted of BCSCs (11). xCT and GCLM mRNA levels were increased four- to fivefold in ALDH⁺ cells compared with ALDH⁻ cells (Fig. 2A). We also enriched BCSCs from MDA-MB-231 and SUM-149 cells through mammosphere formation (12) and found 3- to 10-fold increased xCT and GCLM mRNA levels in primary and secondary mammospheres compared with cells cultured in monolayer (Fig. 2B).

To test the role of xCT and GCLM in chemotherapy-induced BCSC enrichment, MDA-MB-231 subclones with shRNA-mediated knockdown of xCT or GCLM expression were exposed to paclitaxel for 72 h and mammosphere assays were performed as a measure of BCSCs. Paclitaxel treatment of the NTC subclone significantly increased the number of mammosphere-forming cells, whereas knockdown of xCT or GCLM expression decreased the number of mammosphere-forming cells and abrogated the induction by paclitaxel in both primary and secondary mammosphere assays (Fig. 2 C and D). Aldefluor assays revealed that paclitaxel treatment of the NTC subclone significantly increased the percentage of ALDH⁺ cells, and this effect was attenuated by knockdown of xCT or GCLM expression (Fig. 2E and Fig. S2).

Pharmacologic inhibition was also used to investigate the role of the glutathione synthesis pathway in BCSCs. SUM-149 cells were treated with paclitaxel, either alone or in combination with the HIF-1 inhibitor digoxin, xCT inhibitor sulfasalazine (SSA) (21), or GCL inhibitor buthionine sulphoximine (BSO) (25) for 72 h. Paclitaxel increased the number of mammosphere-forming cells, whereas administration of digoxin, SSA, or BSO decreased the number of mammosphere-forming cells and abolished the effect of paclitaxel treatment (Fig. 2 F and G). Coadministration of digoxin, SSA, or BSO also abrogated the increased percentage of ALDH⁺ cells induced by paclitaxel treatment (Fig. 2H). To further confirm the role of glutathione synthesis in BCSCs, we exposed MDA-MB-231 cells to 2 mM glutathione monoethyl ester (GSH-MEE), a cell membrane-permeable form of glutathione, for 4 d. GSH-MEE treatment alone was sufficient to increase the percentage of ALDH⁺ cells, even in HIF-1α, xCT, or GCLM knockdown subclones (Fig. 2I). Taken together, these data demonstrate that increased glutathione levels are both necessary and sufficient for induction of the BCSC phenotype.

Paclitaxel Induces the Expression of Pluripotency Factors.

To delineate the mechanism through which paclitaxel-induced glutathione synthesis regulates BCSCs, we analyzed mRNA samples from ALDH⁻ and ALDH⁺ populations sorted from MDA-MB-231 cells and found that the pluripotency factors Nanog and Sox2 were markedly overexpressed in ALDH⁺ compared with ALDH⁻ cells (Fig. 3A). Increased expression of Nanog and Sox2 was observed previously in MCF-7 cells that were selected for resistance to tamoxifen (26). Nanog and Sox2 were also overexpressed in BCSC-enriched mammosphere cultures of MDA-MB-231 cells (Fig. 3B, Upper). Sox2 mRNA was not expressed in SUM-149 cells; however, Nanog and another pluripotency factor, Oct4, were overexpressed in SUM-149 mammosphere cultures (Fig. 3B, Lower). Paclitaxel treatment of MDA-MB-231 cells induced the expression of Nanog and Sox2, which was completely abrogated by knockdown of HIF-1α, xCT, or GCLM; in contrast, Oct4 expression was not affected by either paclitaxel or knockdown of HIF-1α, xCT, or GCLM (Fig. 3C). In SUM-149 cells, paclitaxel treatment increased the expression of Nanog and Oct4 (Fig. 3D), whereas Sox2 was not detectable. The induction of Nanog and Oct4 by paclitaxel was completely abolished by cotreatment with the HIF-1 inhibitor digoxin, xCT inhibitor SSA, or GCL inhibitor BSO (Fig. 3D).

To directly demonstrate the effect of glutathione on pluripotency factor expression, we treated MDA-MB-231 subclones with GSH-MEE and found that addition of glutathione was sufficient to induce expression of Nanog and Sox2, even in subclones with knockdown of HIF-1α, xCT, or GCLM (Fig. 3E). Similarly, treatment of SUM-149 cells with GSH-MEE induced the expression of Nanog and Oct4, even in the presence of digoxin, SSA, or BSO (Fig. 3F). Thus, GSH-MEE treatment phenocopied the cell type-specific pluripotency factor expression that was induced by chemotherapy. β-mercaptoethanol reduces extracellular cystine to cysteine, which is taken up by alanine-serine-cysteine transporters, thereby bypassing genetic or pharmacologic inhibition of xCT. Addition of β-mercaptoethanol to cell culture media partially rescued the inhibition of Nanog and Sox2 caused by xCT knockdown, but not HIF-1α or GCLM knockdown, in MDA-MB-231 cells (Fig. 3E), and partially rescued the inhibition of Nanog and Oct4 caused by SSA, but not digoxin or BSO, in SUM-149 cells (Fig. 3F).

Although paclitaxel treatment induced the expression of a different combination of pluripotency factors in different TNBC cell lines, Nanog expression was induced in both MDA-MB-231 and SUM-149. To test whether Nanog is required for BCSC maintenance, we generated stable Nanog-knockdown subclones of MDA-MB-231 cells. Knockdown of Nanog dramatically decreased the percentage of ALDH⁺ cells and blocked the enrichment of ALDH⁺ cells induced by paclitaxel (Fig. 3G). Unlike HIF-1α, xCT, or GCLM knockdown (Fig. 2I), the effect of Nanog knockdown on BCSCs could not be rescued by addition of GSH-MEE (Fig. 3H), confirming that Nanog is downstream of glutathione in the regulation of the BCSC phenotype. In addition, knockdown of Nanog in MDA-MB-231 cells dramatically decreased expression of Sox2 and Oct4 mRNA (Fig. S3A), which suggested that Nanog functions as the master pluripotency factor that specifies the BCSC phenotype in these cells. We also analyzed survival data from breast cancer patients that received chemotherapy by stratifying them according to Nanog mRNA levels in the primary tumor. Nanog mRNA levels above the median were associated with decreased patient survival in the ER⁻ (Fig. 3I), but not in the ER⁺ (Fig. S3B), cohort. These results are consistent with the overexpression of SLC7A11/xCT and GCLM mRNA in basal breast cancers (Fig. S1C), the majority of which are ER⁻ (27).

Paclitaxel-Induced Glutathione Synthesis Activates NANOG Transcription by FoxO3.

Next, we investigated how glutathione regulates pluripotency factor expression in TNBC. The FoxO family of transcription factors has been implicated in the maintenance of somatic stem cells and several types of cancer stem cells (28–30). We first analyzed the correlation between survival of breast cancer patients who received chemotherapy and the expression of FoxO1 and FoxO3, two FoxO family members that are expressed in human breast cancers. FoxO3 mRNA levels above the median were significantly associated with decreased patient survival, with an even larger survival difference when only patients with ER⁻ breast cancer were analyzed (Fig. S4A, Upper). In contrast, FoxO1 mRNA levels were not significantly associated with patient survival (Fig. S4A, Lower). Neither FoxO1 nor FoxO3 mRNA or protein levels were affected by changes in glutathione levels resulting from paclitaxel treatment or knockdown of HIF-1α, xCT, or GCLM (Fig. S4 B and C). However, GSH-MEE treatment induced nuclear translocation of FoxO3 (Fig. 4A), which is required for its transcriptional activity. Paclitaxel treatment also induced nuclear translocation of FoxO3 (but not FoxO1) in the MDA-MB-231 NTC subclone, whereas knockdown of HIF-1α, xCT, or GCLM was associated with loss of FoxO3 nuclear localization (Fig. 4B).

To test the hypothesis that glutathione regulates pluripotency factor expression and the BCSC phenotype through activation of FoxO3, we generated FoxO3 knockdown subclones of MDA-MB-231 cells and found that knockdown of FoxO3 completely abrogated the induction of Nanog and Sox2 expression that was induced by treatment with paclitaxel (Fig. 4C) or GSH-MEE (Fig. 4D). FoxO3 knockdown also blocked the enrichment of ALDH⁺ cells in response to paclitaxel (Fig. 4E) or GSH-MEE (Fig. 4F). Taken together, these results establish that FoxO3 is required for induction of the BCSC phenotype.

To investigate whether FoxO3 directly regulates NANOG gene expression, we searched the NANOG genomic DNA sequence for matches to the consensus FoxO binding-site sequence (31) and performed ChIP assays in MDA-MB-231 cells, which revealed that FoxO3 bound to a site located 3.6 kb 5′ to the transcription start site and immunoprecipitation of this DNA sequence by anti-FoxO3 antibody was significantly reduced in FoxO3 knockdown subclones compared with the NTC subclone (Fig. 4G), validating the specificity of the FoxO3 antibody. Taken together, these data indicate that paclitaxel-induced synthesis of glutathione promotes nuclear translocation of FoxO3, which activates expression of the pluripotency factor Nanog.

Glutathione Activates FoxO3 by Chelating Copper To Inhibit MEK Activity.

Glutathione is the most abundant antioxidant within mammalian cells, and its role as an antioxidant is well understood. We therefore investigated whether glutathione promotes FoxO3 nuclear translocation by decreasing intracellular oxidative stress. We exposed MDA-MB-231 cells to different concentrations of the ROS scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and found that at 100 μM, MnTMPyP had an effect on mitochondrial superoxide levels that was comparable to 2 mM GSH-MEE (Fig. 5A), without changing intracellular glutathione levels (Fig. 5B). Surprisingly, 100 μM MnTMPyP failed to induce nuclear translocation of FoxO3, or expression of Nanog and Sox2 in MDA-MB-231 cells, which were observed when cells were treated with 2 mM GSH-MEE (Fig. 5 C and D). These data raised the intriguing possibility that glutathione was acting as a signaling molecule, rather than as an antioxidant, to regulate FoxO3 activity.

ERK phosphorylates FoxO3 on serine residues S294, S344, and S425 and induces its nuclear exclusion, leading to loss of FoxO3 transcriptional activity (32). We found that glutathione treatment inhibited phosphorylation of ERK at T202/Y204 and FoxO3 at S294 (Fig. 5D), suggesting that glutathione may activate FoxO3 by inhibition of ERK signaling. To test this hypothesis, MDA-MB-231 subclones were treated with 2 mM GSH-MEE for 72 h. Knockdown of HIF-1α, xCT, or GCLM, which inhibited glutathione synthesis and decreased intracellular glutathione levels, also promoted phosphorylation of ERK and FoxO3 (Fig. 5E). GSH-MEE treatment not only inhibited ERK and FoxO3 phosphorylation in the NTC subclone, but also reversed the activation of ERK signaling in the HIF-1α, xCT, and GCLM knockdown subclones (Fig. 5E). In contrast, paclitaxel treatment inhibited ERK and FoxO3 phosphorylation in the NTC subclone, but not in the HIF-1α, xCT, or GCLM knockdown subclone (Fig. S5). These results were consistent with the failure of paclitaxel to increase intracellular glutathione levels when the HIF-1–regulated glutathione synthesis pathway was blocked (Fig. 1C). The phosphorylation of MEK1 was not affected by changes in glutathione levels (Fig. 5E). Pharmacological inhibition of ERK signaling by the MEK1 inhibitor U0126 precisely phenocopied the effect of glutathione on inhibition of FoxO3 phosphorylation and induction of pluripotency factors in MDA-MB-231 and SUM-149 cells (Fig. 5F and Fig. S6), and enrichment of the ALDH⁺ population in MDA-MB-231 cells (Fig. 5G). Taken together, these data indicate that glutathione promotes FoxO3 activity through inhibition of MEK1-ERK signaling.

Glutathione is a known chelator of metal ions (33). Recently, it has been reported that copper is a required cofactor for the kinase activity of MEK1 (34, 35). We hypothesized that glutathione inhibits MEK1 activity through chelating copper. MDA-MB-231 cells were treated with GSH-MEE for 72 h, in the absence or presence of CuSO₄. GSH-MEE treatment inhibited ERK phosphorylation and increased expression of Nanog and Sox2, which were partially reversed by copper supplementation in a dose-dependent manner (Fig. 5H). Coadministration of CuSO₄ with GSH-MEE also decreased the percentage of glutathione-induced ALDH⁺ cells dose-dependently (Fig. 5I). Treatment of MDA-MB-231 cells with the copper chelator tetrathiomolybdate (TTM) or trientine for 72 h phenocopied the inhibition of ERK phosphorylation and activation of Nanog and Sox2 by glutathione (Fig. 5 J and K). In addition, both TTM and trientine increased the percentage of ALDH⁺ cells (Fig. 5L). Taken together, these results indicate that glutathione chelates copper and, thereby, inhibits MEK1 activity, leading to dephosphorylation and nuclear translocation of FoxO3.

Paclitaxel Promotes BCSC Enrichment by Induction of Glutathione Synthesis in Vivo.

To investigate whether paclitaxel induces BCSC enrichment in vivo through the molecular mechanisms that we had delineated in cell culture, MDA-MB-231 NTC, xCT, or GCLM knockdown cells were implanted in the mammary fat pad of SCID mice. When the tumors reached a volume of 200 mm³, the mice were treated with 10 mg/kg paclitaxel by i.p. injection every 5 d for three doses. Tumors were harvested 3 d after the last dose for analysis of glutathione levels (Fig. 6A), ALDH⁺ cells (Fig. 6B), and expression of mRNA (Fig. S7A) and protein (Fig. 6C and Fig. S7B). Paclitaxel induced xCT and GCLM mRNA and protein expression, and increased intracellular glutathione levels, which were blocked by knockdown of xCT or GCLM. Paclitaxel also inhibited phosphorylation of ERK and FoxO3, whereas knockdown of xCT or GCLM, which decreased glutathione levels, increased the phosphorylation of ERK and FoxO3. In addition, paclitaxel treatment induced expression of Nanog and Sox2 mRNA and protein, and increased the percentage of ALDH⁺ cells, which were abrogated by knockdown of xCT or GCLM (Fig. 6 B and C and Fig. S7).

To test the role of xCT, GCLM, and FoxO3 in determining the tumor initiation potential of breast cancer cells in vivo, we injected 1,000 MDA-MB-231 NTC, xCT, GCLM, or FoxO3 knockdown cells into the mammary fat pad of female SCID mice. NTC subclone cells formed tumors in all nine mice injected after 65 d, whereas xCT, GCLM, and FoxO3 knockdown subclone cells all showed significantly decreased tumor initiating capacity (Fig. 6D), supporting their role in the maintenance of BCSCs. Taken together, these data support our model that chemotherapy induced glutathione synthesis through HIF-1–mediated SLC7A11 and GCLM gene expression. Increased glutathione levels inhibited MEK1-ERK signaling and FoxO3 phosphorylation, leading to nuclear translocation of FoxO3, which transcriptionally activated NANOG gene expression to promote the BCSC phenotype (Fig. 7).

Cell Culture and Reagents.

MDA-MB-231 and SUM-149 cells were maintained as described (10) at 37 °C in a 5% CO₂, 95% air incubator (20% O₂). Hypoxic cells were placed in a modular incubator chamber (Billups-Rothenberg) flushed with a gas mixture containing 1% O₂, 5% CO₂, and 94% N₂. Paclitaxel, gemcitabine, carboplatin, digoxin, SSA, BSO, β-mercaptoethanol, TTM, trientine, and CuSO₄ were purchased from Sigma-Aldrich. MnTMPyP and U0126 were purchased from EMD Millipore and Life Technologies.

Lentivirus Transduction.

Lentiviral vectors encoding shRNA targeting HIF-1α and HIF-2α were described (50). pLKO.1-puro lentiviral vectors encoding shRNA targeting xCT, GCLM, Nanog, and FoxO3 were purchased from Sigma-Aldrich, and shRNA sequences are shown in Table S1. Lentiviruses were packaged, and MDA-MB-231 cells were transduced and subjected to puromycin selection as described (50).

RT-qPCR.

Total RNA was extracted, cDNA was synthesized, and qPCR analysis was performed as described (50). PCR primer sequences are shown in Table S2.

ChIP.

MDA-MB-231 cells were cross-linked in 3.7% formaldehyde for 15 min, quenched in 0.125 M glycine for 5 min, and lysed with SDS lysis buffer. Chromatin was sheared by sonication, and lysates were precleared with salmon sperm DNA/protein A agarose slurry (Millipore) for 1 h and incubated with IgG or antibodies against HIF-1α (Santa Cruz), HIF-1β, or FoxO3 (Novus Biologicals) in the presence of protein A agarose beads overnight. After sequential washes of the agarose beads, DNA was eluted in 1% SDS with 0.1 M NaHCO₃, and cross-links were reversed by addition of 0.2 M NaCl. DNA was purified by phenol-chloroform extraction and ethanol precipitation, and analyzed by qPCR (Table S3).

Immunoblot Assay.

Whole-cell lysates were prepared in modified RIPA buffer, and proteins were separated by SDS/PAGE, blotted onto nitrocellulose membranes, and probed with primary antibodies (Table S4) followed by HRP-conjugated secondary antibodies (GE Healthcare). The chemiluminescent signal was detected by using ECL Plus (GE Healthcare).

Breast Cancer Stem Cell Assays.

Aldefluor assays were performed according to manufacturer’s instructions (Stem Cell Technologies) as described (10). For cell sorting, the top and bottom 5% of Aldefluor-expressing cells were collected by flow cytometry and considered as ALDH⁺ and ALDH⁻, respectively. Mammosphere assays were performed as described (10).

Subcellular Fractionation.

Cells were resuspended in 10 mM Hepes, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.05% Igepal, and protease inhibitor mixture (Roche), incubated on ice for 15 min, and centrifuged for 10 min at 1,000 × g. The supernatant was reserved as the cytosolic fraction. The pellet was resuspended in 5 mM Hepes, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 26% (vol/vol) glycerol and protease inhibitor mixture, homogenized with a Dounce homogenizer (30 strokes), incubated on ice for 30 min, and centrifuged to collect supernatant as the nuclear fraction.

Glutathione Assay.

Cultured cells were trypsinized, collected by centrifugation, resuspended in 5% (wt/vol) 5-sulfosalicylic acid, subjected to three freeze-and-thaw cycles, and centrifuged to remove debris. Tumor tissues were frozen in liquid nitrogen, ground with a mortar and pestle, resuspended in 5% 5-sulfosalicylic acid, homogenized in a Dounce homogenizer, and centrifuged to remove debris. Supernatants were analyzed by using a glutathione assay kit (Sigma-Aldrich) based on the enzymatic reaction of glutathione with 5,5′-dithiobis-2-nitrobenzoic acid. Absorbance was quantified with a spectrophotometer at the wavelength of 405 nm.

Orthotopic Transplantation.

Protocols were approved by the Johns Hopkins University Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. MDA-MB-231 subclones were injected into the mammary fat pad of 5- to 7-wk-old female SCID mice in a 1:1 suspension of Matrigel (BD Biosciences) in PBS.