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

Transcription

1 hemotherapy triggers HIF-1 dependent glutathione synthesis and opper helation that indues the breast aner stem ell phenotype Haiquan Lu a, ebangshu Samanta a, Lisha Xiang a, Huimin Zhang a, Hongxia Hu a, Ivan hen a, John W. ullen a, and Gregg L. Semenza a,b,1 a Johns Hopkins Institute for ell Engineering and MKusik-Nathans Institute of Geneti Mediine, The Johns Hopkins University Shool of Mediine, altimore, M 1; and b epartments of Pediatris, Mediine, Onology, Radiation Onology, and iologial hemistry, The Johns Hopkins University Shool of Mediine, altimore, M 1 ontributed by Gregg L. Semenza, July 9, 1 (sent for review May 11, 1; reviewed by Agnes Görlah and Maria d. M. Vivano) Triple negative breast aner (TN) aounts for 1 1% of all breast aner but is responsible for a disproportionate share of morbidity and mortality beause of its aggressive harateristis and lak of targeted therapies. hemotherapy indues enrihment of breast aner stem ells (Ss), whih are responsible for tumor reurrene and metastasis. Here, we demonstrate that hemotherapy indues the expression of the ystine transporter and the regulatory subunit of glutamate-ysteine ligase () in a hypoxia-induible fator (HIF)-1 dependent manner, leading to inreased intraellular glutathione levels, whih inhibit mitogenativated protein kinase kinase (MEK) ativity through opper helation. Loss of MEK- signaling auses nulear transloation and transriptional ativation of the gene enoding the pluripoteny fator, whih is required for enrihment of Ss. Inhibition of,,, or bloks hemotherapy-indued enrihment of Ss and impairs tumor initiation. These results suggest that, in ombination with hemotherapy, targeting Ss by inhibiting HIF-1 regulated glutathione synthesis may improve outome in TN. hypoxia-induible fator palitaxel pluripoteny fators hemotherapy resistane tumor-initiating ells Triple negative breast aner (TN), whih is defined as breast aner that laks expression of the estrogen reeptor (ER), progesterone reeptor, and human epidermal growth fator reeptor (HER), aounts for 1 1% of all breast arinomas (1). TNs are more aggressive than other subtypes of breast aner, as exemplified by the inreased risk of reurrene, metastasis, and patient mortality (). Unlike ER + breast aner, whih an be treated with an ER antagonist suh as tamoxifen, or HER + breast aner, whih an be treated with the anti-her antibody trastuzumab, patients with TN do not benefit from targeted therapy. urrent systemi treatment options for TN are limited to ytotoxi hemotherapy, whih may redue tumor bulk initially, but the majority of patients have residual disease or early relapse with a median survival of only 13 mo (3). Therefore, better understanding of hemotherapy resistane mehanisms and better treatment options are urgently needed. Tumor heterogeneity is a key harateristi of breast aner (). reast aner stem ells (Ss) are a small population of aner ells that possess an infinite proliferative potential and the apaity to initiate tumors lonally (). Ss play a ritial role in the metastati proess, whih is the major ause of death for breast aner patients, beause only Ss are apable of forming linially relevant metastases at a seondary site (). In addition, Ss have inreased resistane to hemotherapy through expression of drug transporter proteins, expression of survival fators, and other mehanisms (7). Ss are enrihed in response to hemotherapy, further potentiating the risk of tumor reurrene and metastasis (8 1). The enrihment of Ss an be measured by the Aldefluor assay, whih is based on the high aldehyde dehydrogenase ativity (ALH) in Ss (11), or by the mammosphere assay, whih is based on the ability of Ss to generate multiellular spheroids in suspension ulture (1). Similar to other solid tumors, most breast aners develop regions of intratumoral hypoxia beause of the imbalane between the delivery and onsumption of O (13). Hypoxiainduible fators (HIFs) are transription fators that serve as master regulators of ellular responses to hypoxia (1) and are assoiated with hemotherapy resistane in breast aner (1, 1). hemotherapy indues HIF-dependent expression of interleukin (IL)- and IL-8, whih promote the S phenotype, and of multidrug resistane protein 1 (MR-1), whih mediates hemotherapy efflux from Ss, suh that the perentage of Ss is inreased following treatment with palitaxel or gemitabine (1). oadministration of digoxin, whih inhibits HIF-1α protein aumulation, bloked hemotherapy-indued expression of IL-, IL-8, and MR-1, and bloked S enrihment (1). aner stem ells require an inreased antioxidant apaity to prevent oxidative stress and to maintain stemness and the apaity for tumor initiation (1, 17). The major antioxidant within ells is glutathione, a tripeptide that is synthesized from ysteine, glutamate, and glyine (18). ystine is taken up by ells primarily through the x(-) system, whih is a heterodimeri protein onsisting of a transporter subunit (, enoded by the SL7A11 gene) and a regulatory subunit (Fh, also known as 98, enoded by the SL3A gene) (19). In ells, ystine is redued to ysteine and reats with glutamate to form γ-glutamylysteine in Signifiane We demonstrate that glutathione biosynthesis is ontrolled by hypoxia-induible fator 1 and is ritial for hemotherapyindued enrihment of breast aner stem ells, making it an attrative therapeuti target in triple-negative breast aner, whih is the only subset of breast aners for whih there is no available targeted therapy. We also delineate a moleular mehanism in whih glutathione funtions as a signaling moleule to ativate the breast aner stem ell phenotype, establishing ross-talk between aner metabolism and signal transdution. We also demonstrate that mitogen-ativated protein kinase kinase (MEK)- inhibitors and opper helators have the ountertherapeuti effet of induing breast aner stem ell enrihment. Author ontributions: H.L. and G.L.S. designed researh; H.L.,.S., L.X., H.Z., H.H., I.., and J.W.. performed researh; H.L. and G.L.S. analyzed data; and H.L. and G.L.S. wrote the paper. Reviewers: A.G., Tehnishe Universitat Munhen; and M.d.M.V., I biogune. The authors delare no onflit of interest. 1 To whom orrespondene should be addressed. gsemenza@jhmi.edu. This artile ontains supporting information online at 173/pnas /-/Supplemental. E E9 PNAS Published online July 3, 1

2 Fig. 1. hemotherapy indues glutathione synthesis in a HIF-1 dependent manner. (A) MA-M- 31 ells were treated with vehile (V), 1 nm palitaxel (P), 1 nm gemitabine (G), or 1 μm arboplatin () for 7 h. Aliquots of total ellular RNA were subjeted to RT and qpr to analyze (SL7A11) and mrna expression. The results were normalized to ells treated with vehile (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. vehile. () MA-M-31 sublones, whih were stably transfeted with an expression vetor enoding nontargeting ontrol (NT) short hairpin RNA (shrna), or vetor enoding shrna against or, were treated without (-) or with (+) 1 nm palitaxel (Pa) for 7 h, and immunoblot assays were performed to analyze and protein expression. () MA-M-31 sublones were treated without or with 1 nm Pa for 7 h, glutathione levels were measured, and the results were normalized to NT Pa (-) (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. NT Pa (-); P <.1 vs. NT. () MA-M-31 sublones, whih were stably transfeted with NT vetor or vetor enoding shrna against HIF-1α or HIF-α, were treated without or with Pa for 7 h. RT-qPR was performed to assay and mrna levels (mean ± SEM; n = 3). P <.1, P <.1 vs. NT Pa (-); # P <., P <.1 vs. NT ; ns, not signifiant. (E) MA-M-31 ells were implanted into the mammary fat pad of female SI mie. When tumor volume reahed mm 3, mie were randomly assigned to treatment with saline (vehile, V), Pa (P; 1 mg/kg on days and 1), digoxin (; mg/kg on days 1 1), or Pa and digoxin (P + ). Tumors were harvested on day 1 for RT-qPR analysis of and mrna. *P <., P <.1 vs. vehile; ## P <.1, P <.1 vs. Pa. (F) MA-M-31 ells were E F inubated at % or 1% O for 1 h, and hip assays were performed by using IgG or antibodies against HIF-1α or HIF-1β. Primers flanking andidate binding sites in the SL7A11 and genes were used for qpr, and the results were normalized to ells exposed to % O and immunopreipitated with anti-hif-1α (mean ± SEM; n = 3). P <.1, P <.1 vs. % O. The nuleotide sequenes surrounding the HIF-1 binding sites (olored fonts) within intron 3 of SL7A11 and the -flanking region of are shown. MEIAL SIENES PNAS PLUS a reation atalyzed by glutamate ysteine ligase (GL), whih onsists of a atalyti subunit, GL, and a modifier subunit,. Glyine is added to γ-glutamylysteine by the enzyme glutathione synthetase (GSS) to form glutathione. The glutathione synthesis pathway has been shown to promote aner initiation and progression, and targeting this pathway by inhibiting or GL has shown some promise in inhibiting tumor growth in ombination with hemotherapy in mouse models of breast aner (, 1), although the underlying moleular mehanisms have not been fully delineated. eause hemotherapy indues oxidative stress, it has been assumed that the glutathione synthesis pathway promotes hemotherapy resistane through its antioxidant effets (17). Here, we demonstrate that in TN, glutathione synthesis is indued by hemotherapy in a HIF- 1 dependent manner, resulting in inreased intraellular glutathione levels, whih ativate expression of pluripoteny fators that diretly speify the S phenotype. Moreover, rather than solely funtioning in its traditional role as an antioxidant, glutathione indues the S phenotype by helating opper and, thereby, inhibiting mitogen-ativated protein kinase kinase (MEK)- signaling. Results hemotherapy Indues HIF-1 ependent Glutathione iosynthesis. We hypothesized that hemotherapy indues glutathione synthesis in breast aner ells to protet against oxidative stress. Palitaxel, gemitabine, and arboplatin are all Food and rug Administration-approved hemotherapy drugs that are used for the treatment of TN. We treated two TN ell lines, MA- M-31 and SUM-19, with palitaxel, gemitabine, or arboplatin for 7 h at the onentration of drug that inhibited growth by % (I ). Eah of these hemotherapeuti agents inreased and mrna levels in both ell lines as determined by reverse transription (RT) and quantitative real-time PR (qpr) (Fig. 1A and Fig. S1A). In MA-M-31 ells transfeted with a nontargeting ontrol (NT) short hairpin RNA (shrna), palitaxel treatment inreased and protein levels (Fig. 1), and intraellular total glutathione levels (Fig. 1). Knokdown of or expression by speifi shrnas (Fig. 1) bloked glutathione indution in response to palitaxel treatment (Fig. 1), indiating that both and are required for palitaxel-indued glutathione synthesis. Gene expression data from 1,1 human breast aners in the aner Genome Atlas (TGA) database were analyzed to ompare the expression patterns of and mrna in different moleular subtypes of breast aner (asal, HER-enrihed, Luminal A, Luminal, and Normal-like) that are based on a - mrna (PAM) signature () (Fig. S1). ompared with other breast aner subtypes, and mrna levels were signifiantly inreased in basal breast aners (Fig. S1), in whih hypoxia-induible fator (HIF)-target gene expression is also inreased (1). A detailed omparison of and mrna levels with those of XR3, L1AM, NIP3, PLO1, PHA1, PHA, VEGFA, SLA1, and A9 mrna, whih are all HIFregulated gene produts in TN ells, showed a signifiant orrelation of and expression with six of nine and eight of Lu et al. PNAS Published online July 3, 1 E1

3 nine HIF-target genes, respetively (Fig. S1). Exposure of MA- M-31 ells to palitaxel, gemitabine, or arboplatin at I indued HIF-1α protein expression (Fig. S1E). In addition, and mrna levels were indued in most breast aner ell lines when they were exposed to 1% O for h (Fig. S1F). In MA-M-31 ells, and mrna levels were signifiantly indued by either palitaxel or hypoxia alone, although no additive effet was observed by the ombined treatment (Fig. S1G). These data suggest that SL7A11 and gene expression are regulated by HIFs. To test this hypothesis, we analyzed MA-M-31 sublones that were stably transfeted with an expression vetor enoding shrna targeting HIF-1α or HIF-α, and found that knokdown of HIF-1α, but not HIF-α, dereased and mrna basal levels and bloked their indution in response to palitaxel treatment (Fig. 1). Knokdown of HIF-1α, but not HIF-α, also abrogated and indution in response to hypoxia (Fig. S1H). Pharmaologial inhibition of HIF-1α expression by treatment of ells with the HIF-1 inhibitor digoxin (1, 3) also bloked expression of and mrna in SUM-19 ells exposed to palitaxel, gemitabine, or arboplatin (Fig. S1A). Taken together, these data indiate that expression of the SL7A11 and genes is regulated by HIF-1, but not HIF-. We also implanted MA-M-31 ells into the mammary fat pad of female severe ombined immunodefiieny (SI) mie and treated the mie with palitaxel, either alone or in ombination with digoxin. igoxin treatment dereased and mrna levels, and bloked their indution by palitaxel (Fig. 1E), demonstrating that palitaxel treatment indues and mrna expression in a HIF-1 dependent manner in vivo. To investigate whether HIF-1 diretly regulates SL7A11 and expression, genomi NA sequenes were searhed for mathes to the onsensus HIF-1 binding-site sequene -(A/G)GTG-3 and andidate sites were evaluated by hromatin immunopreipitation (hip) assays performed in MA- M-31 ells. Hypoxia indued the binding of HIF-1α and HIF-1β to sites loated in the third intron of SL7A11 andinthe -flanking region of (Fig. 1F). Taken together, these data indiate that hemotherapy drugs indue HIF-1 dependent ativation of SL7A11 and transription. Inhibition of Glutathione Synthesis loks Palitaxel-Indued S Enrihment. We reently demonstrated that palitaxel treatment inreases the perentage of Ss in a HIF-dependent manner (1). and expression in breast aner ell lines is orrelated with expression of, an important S marker (, ). To test the role of the glutathione synthesis pathway in palitaxel-indued S enrihment, MA-M-31 ells were sorted into an ALH + population, whih is highly enrihed for Ss, and an ALH population, whih is depleted of Ss (11). and mrna levels were inreased four- to fivefold in ALH + ells ompared with ALH ells (Fig. A). We also enrihed Ss from MA-M-31 and SUM-19 ells through mammosphere formation (1) and found 3- to 1-fold inreased and mrna levels in primary and seondary mammospheres ompared with ells ultured in monolayer (Fig. ). To test the role of and in hemotherapy-indued S enrihment, MA-M-31 sublones with shrnamediated knokdown of or expression were exposed to palitaxel for 7 h and mammosphere assays were performed as a measure of Ss. Palitaxel treatment of the NT sublone signifiantly inreased the number of mammosphere-forming ells, whereas knokdown of or expression dereased the number of mammosphere-forming ells and abrogated the indution by palitaxel in both primary and seondary mammosphere assays (Fig. and ). Aldefluor assays revealed that palitaxel treatment of the NT sublone signifiantly inreased the perentage of ALH + ells, and this effet was attenuated by knokdown of or expression (Fig. E and Fig. S). Pharmaologi inhibition was also used to investigate the role of the glutathione synthesis pathway in Ss. SUM-19 ells were treated with palitaxel, either alone or in ombination with the HIF-1 inhibitor digoxin, inhibitor sulfasalazine (SSA) (1), or GL inhibitor buthionine sulphoximine (SO) () for 7 h. Palitaxel inreased the number of mammosphere-forming ells, whereas administration of digoxin, SSA, or SO dereased the number of mammosphere-forming ells and abolished the effet of palitaxel treatment (Fig. F and G). oadministration of digoxin, SSA, or SO also abrogated the inreased perentage of ALH + ells indued by palitaxel treatment (Fig. H). To further onfirm the role of glutathione synthesis in Ss, we exposed MA-M-31 ells to mm glutathione monoethyl ester (E), a ell membrane-permeable form of glutathione, for d. E treatment alone was suffiient to inrease the perentage of ALH + ells, even in HIF-1α,, or knokdown sublones (Fig. I). Taken together, these data demonstrate that inreased glutathione levels are both neessary and suffiient for indution of the S phenotype. Palitaxel Indues the Expression of Pluripoteny Fators. To delineate the mehanism through whih palitaxel-indued glutathione synthesis regulates Ss, we analyzed mrna samples from ALH and ALH + populations sorted from MA-M- 31 ells and found that the pluripoteny fators and were markedly overexpressed in ALH + ompared with ALH ells (Fig. 3A). Inreased expression of and was observed previously in MF-7 ells that were seleted for resistane to tamoxifen (). and were also overexpressed in S-enrihed mammosphere ultures of MA-M-31 ells (Fig. 3, Upper). mrna was not expressed in SUM-19 ells; however, and another pluripoteny fator,, were overexpressed in SUM-19 mammosphere ultures (Fig. 3, Lower). Palitaxel treatment of MA-M-31 ells indued the expression of and, whih was ompletely abrogated by knokdown of HIF-1α,, or ; in ontrast, expression was not affeted by either palitaxel or knokdown of HIF-1α,, or (Fig. 3). In SUM-19 ells, palitaxel treatment inreased the expression of and (Fig. 3), whereas was not detetable. The indution of and by palitaxel was ompletely abolished by otreatment with the HIF-1 inhibitor digoxin, inhibitor SSA, or GL inhibitor SO (Fig. 3). To diretly demonstrate the effet of glutathione on pluripoteny fator expression, we treated MA-M-31 sublones with E and found that addition of glutathione was suffiient to indue expression of and, even in sublones with knokdown of HIF-1α,, or (Fig. 3E). Similarly, treatment of SUM-19 ells with E indued the expression of and, even in the presene of digoxin, SSA, or SO (Fig. 3F). Thus, E treatment phenoopied the ell type-speifi pluripoteny fator expression that was indued by hemotherapy. β-meraptoethanol redues extraellular ystine to ysteine, whih is taken up by alanineserine-ysteine transporters, thereby bypassing geneti or pharmaologi inhibition of. Addition of β-meraptoethanol to ell ulture media partially resued the inhibition of and aused by knokdown, but not HIF-1α or knokdown, in MA-M-31 ells (Fig. 3E), and partially resued the inhibition of and aused by SSA, but not digoxin or SO, in SUM-19 ells (Fig. 3F). Although palitaxel treatment indued the expression of a different ombination of pluripoteny fators in different TN ell lines, expression was indued in both MA-M-31 E Lu et al.

4 MA-M-31 SUM-19 MA-M-31 E 1 1 mrna mrna mrna 1 ALH(-) ALH(+) Pa (-) shrna: NT #1 # 1 1 Pa (-) * * shrna: NT #1 # mrna mrna mrna ALH(-) ALH(+) F G 1 Mammospheres Pa (-) 1 8 * Mammospheres shrna: NT #1 # #1 # Pa(-) Pa (-) 1 1 shrna: NT #1 # V ig SSA SO V ig SSA SO Pa P + P + S P + Mammospheres 1 1 * #1 # V ig SSA SO Pa P + P + S P + Mammospheres shrna: NT #1 # H I 3 Pa (-) * #1 # V ig SSA SO Pa P + P + S P + E (-) 1 E (+) shrna: NT HIF-1 MEIAL SIENES PNAS PLUS Fig.. Palitaxel-indued S enrihment is bloked by inhibition of glutathione synthesis. (A) MA-M-31 ells were sorted into aldehyde dehydrogenase negative [ALH (-)] and positive [ALH (+)] populations by flow ytometry. RT-qPR was performed to analyze the expression of (SL7A11) and mrna, and the results were normalized to ALH (-) ells (mean ± SEM; n = 3). P <.1 vs. ALH (-). () MA-M-31 (Upper) and SUM-19 (Lower) ells were ultured on ultra-low adherene plates for 7 d to generate primary mammospheres (1 sphere). Half of the mammospheres were harvested for RT-qPR analyses, and the other half of the mammospheres were dissoiated and ultured for 7 d to generate seondary mammospheres ( sphere), whih were harvested for RT-qPR analyses. Results were normalized to MA-M-31 and SUM-19 adherent ells (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. adherent ells. ( and ) MA-M-31 sublones were treated without or with 1 nm palitaxel (Pa) for 7 h. The number of 1 and mammospheres per 1, ells initially seeded was alulated (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. NT Pa (-); P <.1 vs. NT. (E) MA-M-31 sublones were treated without or with 1 nm Pa for d, and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. NT Pa (-); P <.1 vs. NT. (F and G) SUM-19 ells were treated without or with nm Pa for 7 h, in ombination with vehile (V), 1 nm digoxin (ig), 1 μm sulfasalazine (SSA), or 1 μm buthionine sulphoximine (SO). Primary and seondary mammospheres were ounted (mean ± SEM; n = 3). P <.1, P <.1 vs. vehile; P <.1 vs. Pa. (H) SUM-19 ells were treated with V, ig, SSA, SO, or Pa alone, or Pa in ombination with ig (P + ), SSA (P + S), or SO (P + ) for d, and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). P <.1 vs. V; P <.1 vs. Pa. (I) MA-M-31 sublones were treated without or with mm glutathione monoethyl ester (E) for d and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). P <.1, P <.1 vs. E (-) in NT group; P <.1 vs. E (-) in the same sublone. (Sale bars: μm.) and SUM-19. To test whether is required for S maintenane, we generated stable -knokdown sublones of MA-M-31 ells. Knokdown of dramatially dereased the perentage of ALH + ells and bloked the enrihment of ALH + ells indued by palitaxel (Fig. 3G). Unlike HIF-1α,, or knokdown (Fig. I), the effet of knokdown on Ss ould not be resued by addition of E (Fig. 3H), onfirming that is downstream of glutathione in the regulation of the S phenotype. In addition, knokdown of in MA-M-31 ells dramatially dereased expression of and mrna (Fig. S3A), whih suggested that funtions as the master pluripoteny fator that speifies the S phenotype in these ells. We also analyzed survival data from breast aner patients that reeived hemotherapy by stratifying them aording to mrna levels in the primary tumor. mrna levels above the median were assoiated with dereased patient survival in the ER (Fig. 3I), but not in the ER + (Fig. S3), ohort. These results are onsistent with the overexpression of SL7A11/ and mrna in basal breast aners (Fig. S1), the majority of whih are ER (7). Palitaxel-Indued Glutathione Synthesis Ativates NANOG Transription by. Next, we investigated how glutathione regulates pluripoteny fator expression in TN. The FoxO Lu et al. PNAS Published online July 3, 1 E3

5 F MA-M-31 MA-M-31 SUM-19 mrna mrna mrna V ALH(-) ALH(+) SUM-19 mrna mrna mrna ig SSA SO ALH(-) ALH(+) 3 * 1 G 1 shrna: Pa HIF-1 shrna: NT #1 # HIF-1 NT #1 # NT #1 # NT #1 # V Pa Pa (-) SUM-19 ig P + SSA P + S SO P + H 1 1 MA-M-31 E shrna: NT #1 # MA-M-31 shrna: NT HIF-1 E (-) E (+) I Probability (RFS) Gene: ohort: ER (-) + hemo N = 11 Low High HR = 1.79 (P =.3) 1 1 Time (months) Fig. 3. Palitaxel-indued expression of pluripoteny fators mediates S enrihment. (A) MA-M-31 ells were sorted into ALH (-) and ALH (+) populations by flow ytometry. RT-qPR was performed to analyze the expression of and mrna, and the results were normalized to ALH (-) ells (mean ± SEM; n = 3). P <.1 vs. ALH (-). () Primary and seondary mammospheres from MA-M-31 (Upper) and SUM-19 (Lower) ells were harvested for RT-qPR analyses. The results were normalized to adherent ells (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. adherent. () MA-M-31 sublones were treated without or with 1 nm palitaxel (Pa) for 7 h, and immunoblot assays were performed. () SUM-19 ells were treated with vehile (V), nm palitaxel (Pa), 1 nm digoxin (ig), 1 μm sulfasalazine (SSA), 1 μm buthionine sulphoximine (SO), or Pa ombined with ig (P + ), SSA (P + S), or SO (P + ) for 7 h, and immunoblot assays were performed. (E) MA-M-31 sublones were treated with μm β-meraptoethanol (β) or mm glutathione monoethyl ester () for 7 h and immunoblot assays were performed. (F) SUM-19 ells were treated with ig, SSA, or SO, in ombination with vehile, β, or for 7 h, and immunoblot assays were performed. (G and H) MA-M-31 sublones stably transfeted with NT or shrna vetor were treated without or with Pa (G), and without or with glutathione monoethyl ester (E) (H), and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). P <.1 vs. NT Pa (-) or E (-); P <.1 vs. NT or E (+). (I) Kaplan Meier analysis of relapsefree survival (RFS) was performed based on linial and moleular data from 11 ER (-) breast aner patients who reeived hemotherapy. The patients were stratified aording to mrna levels in the primary tumor. High, mrna levels greater than the median; low, mrna levels less than the median. The P value (log-rank test) and hazard ratio (HR) are shown. family of transription fators has been impliated in the maintenane of somati stem ells and several types of aner stem ells (8 3). We first analyzed the orrelation between survival of breast aner patients who reeived hemotherapy and the expression of FoxO1 and, two FoxO family members that are expressed in human breast aners. mrna levels above the median were signifiantly assoiated with dereased patient survival, with an even larger survival differene when only patients with ER breast aner were analyzed (Fig. SA, Upper). In ontrast, FoxO1 mrna levels were not signifiantly assoiated with patient survival (Fig. SA, Lower). Neither FoxO1 nor mrna or protein levels were affeted by hanges in glutathione levels resulting from palitaxel treatment or knokdown of HIF-1α,,or(Fig. S and ). However, E treatment indued nulear transloation of (Fig. A), whih is required for its transriptional ativity. Palitaxel treatment also indued nulear transloation of (but not FoxO1) in the MA-M-31 NT sublone, whereas knokdown of HIF-1α,, or was assoiated with loss of nulear loalization (Fig. ). To test the hypothesis that glutathione regulates pluripoteny fator expression and the S phenotype through ativation of, we generated knokdown sublones of MA- M-31 ells and found that knokdown of ompletely abrogated the indution of and expression that was indued by treatment with palitaxel (Fig. ) or E (Fig. ). knokdown also bloked the enrihment of ALH + ells in response to palitaxel (Fig. E) or E (Fig. F). Taken together, these results establish that is required for indution of the S phenotype. To investigate whether diretly regulates NANOG gene expression, we searhed the NANOG genomi NA sequene for mathes to the onsensus FoxO binding-site sequene (31) and performed hip assays in MA-M-31 ells, whih revealed that bound to a site loated 3. kb to the transription start site and immunopreipitation of this NA sequene by anti- antibody was signifiantly redued in knokdown sublones ompared with the NT sublone (Fig. G), validating the speifiity of the antibody. Taken together, these data indiate that palitaxel-indued synthesis of glutathione promotes E Lu et al.

6 E FoxO1 -tubulin Histone H shrna: NT #1 # E Nuleus ytosol Nuleus ytosol shrna: HIF-1 HIF-1 NT #1 # NT #1 # Palitaxel FoxO1 -tubulin Histone H3 E Pa (-) 1 Pa (-) 1 E (-) E (+) 1 shrna: NT #1 # shrna: NT #1 # F G NANOG Enrihment shrna: NT #1 # Palitaxel kb ttgttta shnt sh #1 sh # ontrol IgG NANOG ## Ab Fig.. Palitaxel promotes nulear transloation of to ativate NANOG transription. (A and ) MA-M-31 ells were treated without or with mm E (A), or 1 nm palitaxel (), for 7 h. ytosoli and nulear lysates were prepared, and immunoblot assays were performed. ( and ) MA-M-31 sublones expressing NT or a shrna vetor were treated without or with palitaxel (), or E () for 7 h, and immunoblot assays were performed. (E and F) MA-M-31 NT and shrna sublones were treated without or with palitaxel (Pa, E), or E (F), and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). P <.1, P <.1 vs. NT Pa (-) or E (-); P <.1 vs. NT or E (+). (G) hromatin immunopreipitation (hip) was performed by using IgG or antibodies in NT and shrna sublones. qpr was performed using primers that flanked the andidate binding site, and the results were normalized to NT hip with IgG (mean ± SEM; n = 3). The nuleotide sequene of the binding site, whih is loated 3. kb to the NANOG transription start site, is shown. P <.1 vs. NT with IgG; ## P <.1, P <.1 vs. NT with antibody. MEIAL SIENES PNAS PLUS nulear transloation of, whih ativates expression of the pluripoteny fator. Glutathione Ativates by helating opper To Inhibit MEK Ativity. Glutathione is the most abundant antioxidant within mammalian ells, and its role as an antioxidant is well understood. We therefore investigated whether glutathione promotes nulear transloation by dereasing intraellular oxidative stress. We exposed MA-M-31 ells to different onentrations of the ROS savenger manganese (III) tetrakis(1-methyl--pyridyl)porphyrin (MnTMPyP) and found that at 1 μm, MnTMPyP had an effet on mitohondrial superoxide levels that was omparable to mm E (Fig. A), without hanging intraellular glutathione levels (Fig. ). Surprisingly, 1 μm MnTMPyP failed to indue nulear transloation of, or expression of and in MA-M-31 ells, whih were observed when ells were treated with mm E (Fig. and ). These data raised the intriguing possibility that glutathione was ating as a signaling moleule, rather than as an antioxidant, to regulate ativity. phosphorylates on serine residues S9, S3, and S and indues its nulear exlusion, leading to loss of transriptional ativity (3). We found that glutathione treatment inhibited phosphorylation of at T/Y and at S9 (Fig. ), suggesting that glutathione may ativate by inhibition of signaling. To test this hypothesis, MA-M-31 sublones were treated with mm E for 7 h. Knokdown of HIF-1α,, or, whih inhibited glutathione synthesis and dereased intraellular glutathione levels, also promoted phosphorylation of and (Fig. E). E treatment not only inhibited and phosphorylation in the NT sublone, but also reversed the ativation of signaling in the HIF-1α,, and knokdown sublones (Fig. E). In ontrast, palitaxel treatment inhibited and phosphorylation in the NT sublone, but not in the HIF-1α,, or knokdown sublone (Fig. S). These results were onsistent with the failure of palitaxel to inrease intraellular glutathione levels when the HIF-1 regulated glutathione synthesis pathway was bloked (Fig. 1). The phosphorylation of MEK1 was not affeted by hanges in glutathione levels (Fig. E). Pharmaologial inhibition of signaling by the MEK1 inhibitor U1 preisely phenoopied the effet of glutathione on inhibition of phosphorylation and indution of pluripoteny fators in MA-M-31 and SUM-19 ells (Fig. F and Fig. S), and enrihment of the ALH + population in MA-M-31 ells (Fig. G). Taken together, these data indiate that glutathione promotes ativity through inhibition of MEK1- signaling. Glutathione is a known helator of metal ions (33). Reently, it has been reported that opper is a required ofator for the kinase ativity of MEK1 (3, 3). We hypothesized that glutathione inhibits MEK1 ativity through helating opper. MA- M-31 ells were treated with E for 7 h, in the absene or presene of uso. E treatment inhibited phosphorylation and inreased expression of and, whih were partially reversed by opper supplementation in a dose-dependent manner (Fig. H). oadministration of uso with E also dereased the perentage of glutathioneindued ALH + ells dose-dependently (Fig. I). Treatment of MA-M-31 ells with the opper helator tetrathiomolybdate (TTM) or trientine for 7 h phenoopied the inhibition of phosphorylation and ativation of and by glutathione (Fig. J and K). In addition, both TTM and trientine inreased the perentage of ALH + ells (Fig. L). Taken together, these results indiate that glutathione helates opper and, thereby, inhibits MEK1 ativity, leading to dephosphorylation and nulear transloation of. Lu et al. PNAS Published online July 3, 1 E

7 E I MitoSox Red (+) ells mm MEE E uso (µm) 8 ns MnTMPyP (µm) J ns p- pk1 MEK1 Glutathione Levels 3 1 TTM (µm). 1 ontrol shrna: HIF-1 NT #1 # NT #1 # NT #1 # E p- pk1 MEK1 p- Pa ns E MnTMPyP ( mm) (1 µm) K F U1 p- p- p- pk1 MEK1 Nuleus ytosol E E MnTMPyP MnTMPyP p- -tubulin Histone H3 - + Trientine (µm) 1 G 8 U1 L H E uso (µm) 8 8 p- pk1 MEK E TTM (µm) p- Trientine (µm) Fig.. Glutathione ativates and indues pluripoteny fator expression through inhibition of MEK1 ativity by helating opper. (A) MA-M-31 ells were treated with E or MnTMPyP for 7 h. The perentage of ells positive for MitoSox Red staining was determined by flow ytometry, and the results were normalized to vehile ontrol () (mean ± SEM; n = 3). P <.1, P <.1 vs. E; ns, not signifiant. () MA-M-31 ells were treated with E or MnTMPyP for 7 h. Glutathione levels were measured, and the results were normalized to ontrol (mean ± SEM; n = 3). P <.1 vs. ontrol; ns, not signifiant. () ytosoli and nulear lysates were prepared from MA-M-31 ells treated with E or MnTMPyP, and immunoblot assays were performed. () MA-M-31 ells were treated with E or MnTMPyP, and immunoblot assays were performed. (E) MA-M-31 sublones were treated without or with E, and immunoblot assays were performed. (F and G) MA-M-31 ells were treated with. μm U1 (+) or vehile (-) for 7 h and immunoblot assays were performed (F), or the perentage of ALH (+) ells was determined by flow ytometry (G; mean ± SEM; n = 3). P <.1 vs. ontrol. (H) MA-M-31 ells were treated with mm E, either alone or in ombination with uso for 7 h, and immunoblot assays were performed. (I) MA-M-31 ells were treated with E, either alone or in ombination with uso, and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). P <.1 vs. ontrol; P <.1 vs. E alone. (J) MA-M-31 ells were treated with tetrathiomolybdate (TTM), palitaxel (Pa), or E () for 7 h, and immunoblot assays were performed. (K) MA-M-31 ells were treated with trientine for 7 h, and immunoblot assays were performed. (L) MA-M-31 ells were treated with TTM, trientine, or E, and the perentage of ALH (+) ells was determined (mean ± SEM; n = 3). P <.1 vs. ontrol. Palitaxel Promotes S Enrihment by Indution of Glutathione Synthesis in Vivo. To investigate whether palitaxel indues S enrihment in vivo through the moleular mehanisms that wehaddelineatedinellulture,ma-m-31nt,, or knokdown ells were implanted in the mammary fat pad of SI mie. When the tumors reahed a volume of mm 3, the mie were treated with 1 mg/kg palitaxel by i.p. injetion every d for three doses. Tumors were harvested 3 d after the last dose for analysis of glutathione levels (Fig. A), ALH + ells (Fig. ), and expression of mrna (Fig. S7A) and protein (Fig. and Fig. S7). Palitaxel indued and mrna and protein expression, and inreased intraellular glutathione levels, whih were bloked by knokdown of or. Palitaxel also inhibited phosphorylation of and, whereas knokdown of or, whih dereased glutathione levels, inreased the phosphorylation of and. In addition, palitaxel treatment indued expression of and mrna and protein, and inreased the perentage of ALH + ells, whih were abrogated by knokdown of or (Fig. and and Fig. S7). To test the role of,, and in determining the tumor initiation potential of breast aner ells in vivo, we injeted 1, MA-M-31 NT,,, or knokdown ells into the mammary fat pad of female SI mie. NT sublone ells formed tumors in all nine mie injeted after d, whereas,, and knokdown sublone ells all showed signifiantly dereased tumor initiating apaity (Fig. ), supporting their role in the maintenane of Ss. Taken together, these data support our model that hemotherapy indued glutathione synthesis through HIF-1 mediated SL7A11 and gene expression. Inreased glutathione levels inhibited MEK1- signaling and phosphorylation, leading to nulear transloation E Lu et al.

8 Glutathione levels. shrna: NT #1 #. * # Pa (-) # * ## ## Pa (-) #1 # ns * * * shrna: NT #1 # #1 # shnt sh sh Pa (-) Pa (-) Pa (-) p- p- pk1 MEK1 of, whih transriptionally ativated NANOG gene expression to promote the S phenotype (Fig. 7). isussion Ss are resistant to ytotoxi hemotherapy and play a major role in breast aner relapse and metastasis. Reent studies have suggested that hemotherapy inreases the perentage of Ss (8 1) and that HIFs are required for this proess (1). However, the moleular mehanisms through whih HIFs ontribute to S enrihment in response to hemotherapy are only beginning to be eluidated. In the present study, we have demonstrated that HIF-1 regulates Ss by inreasing the synthesis of glutathione for use as a signaling moleule. Our previous study impliated hemotherapy-indued and HIF-1 dependent Injeted sublone Tumor formation Tumor initiation apaity shnt sh sh sh 9/9 (1%) 3/9 (33.3%) P =.9 /8 (%) P =.9 1/8 (1.%) P =. Fig.. Palitaxel indues synthesis of glutathione, whih inhibits MEK1 ativity, inreases ativity and pluripoteny fator expression and promotes tumor initiation. (A ), MA-M-31 sublones were implanted into SI mie. When tumor volume reahed mm 3 (day ), mie were randomly assigned to treatment with i.p. injetions of saline [Pa (-)] or 1 mg/kg palitaxel [] on days,, and 1. Tumors were harvested on day 13, and samples were analyzed for intraellular total glutathione levels (A), perentage of ALH (+) ells (), and protein expression as indiated () (mean ± SEM; n = 3). *P <., P <.1, P <.1 vs. NT Pa (-); # P <., ## P <.1, P <.1 vs. NT ; ns, not signifiant. () One thousand ells of eah MA-M-31 sublone were implanted into the mammary fat pad of female SI mie. The number of mie that developed palpable tumors after d in eah group was reported, and Fisher s exat test was performed to determine statistial signifiane vs. NT group. expression of IL-, IL-8, and MR1 as a mehanism underlying the differential survival, relative to bulk aner ells, of Ss (1). In ontrast, the present study has delineated a mehanism that results in an ative indution of the S phenotype mediated by inreased expression of pluripoteny fators. aner ells are metabolially ative, under inreased oxidative stress, and require robust antioxidant apaity to maintain viability (3). Glutathione is the major endogenous antioxidant produed by mammalian ells to detoxify free radials. However, the role of glutathione in tumor initiation, aner progression, and hemotherapy resistane has been ontroversial for many years, perhaps beause glutathione may play both protetive and pathogeni roles with respet to aner (18, 37). Reently it has been shown that inhibition of or GL by treatment with MEIAL SIENES PNAS PLUS Fig. 7. Pluripoteny fator expression and the S phenotype are indued through HIF-1 dependent glutathione synthesis in response to hemotherapy. hemotherapy treatment indues HIF-1 dependent SL7A11 and transriptional ativation, leading to inreased glutathione synthesis. Glutathione helates opper, whih is a required ofator for MEK1, leading to inativation of MEK1- signaling, dephosphorylation, and nulear transloation, leading to transriptional ativation of NANOG, whih enodes a pluripoteny fator that speifies the S phenotype. Lu et al. PNAS Published online July 3, 1 E7

9 SSA or SO, respetively, potentiates the antianer effet of hemotherapy on breast aner ell lines (38, 39). Our results demonstrate that targeting the glutathione synthesis pathway also dereases the number of Ss, whih are required for breast aner reurrene and metastasis. Thus, inhibiting the glutathione synthesis pathway in ombination with hemotherapy may improve patient outome in TN. In addition, we demonstrate that administration of glutathione is suffiient to enrih Ss, whih may serve as a aveat to the use of glutathione as a dietary supplement (1). Although novel roles for glutathione in aner metabolism have been desribed reently (), they have been related to its role as an antioxidant. Our studies revealed that glutathione indues the S phenotype through a moleular mehanism that is independent of its antioxidant role. Glutathione also funtions as a helator to protet ells from damage aused by heavy metal ions (33). Although exess opper is toxi to ells, opper is indispensable for ells under physiologial onditions and ontrols many important biohemial proesses by regulating protein struture, atalyti ativity, and protein protein interations (1). It has been reported reently that opper is an obligate ofator for MEK1 ativity (3). In this study, we demonstrate that glutathione inhibits phosphorylation through opper helation, whih is phenoopied by two other opper helators, tetrathiomolybdate and trientine. opper has been onsidered as a target for aner therapy (), but our results suggest it may not be a good target beause opper helators may enrih for aner stem ells, whereas opper ionophores will inrease aner ell proliferation (3), neither of whih is desired. The inhibition of phosphorylation by glutathione is onsistent with the effet of ATN-, a speifi opper helator that is under linial trials, whih also inhibits phosphorylation (). The regulatory role of glutathione in aner ell signaling highlights the importane of glutathione in balaning proliferation, stem ell maintenane, and redox homeostasis. The Ras-RafK- signal transdution pathway plays a ritial role in mediating fundamental biologial proesses, inluding ell survival, proliferation, and migration (3). This pathway is ommonly ativated in many types of aner, making it a rational target for aner therapeutis. Several MEK inhibitors have been tested in prelinial and linial studies in ombination with hemotherapy. However, reent studies showed that signaling is important for promoting stem ell differentiation and antagonizing self-renewal (, ), findings that are onsistent with our results. In the urrent study, we demonstrate that inhibition of MEK1 ativity by U1 at a low dose (. μm) but for a relatively long time (7 h), promotes nulear transloation and ativates transriptional ativity, leading to the expression of pluripoteny fators and enrihment of Ss in TN. The negative orrelation between phosphorylation and pluripoteny fator expression in our study is also onsistent with the inhibitory effet of on pluripoteny fators that was observed in embryoni stem ells (7, 8). These observations raise the onern that although inhibition of MEK1- signaling pathway may derease primary breast tumor growth, it might enrih for Ss, thereby inreasing the risk of aner reurrene and/or metastasis. More detailed studies are required to evaluate MEK- inhibitors for their potential ountertherapeuti effets on Ss. The reported roles of are also ontraditory in different types of aner and in different phases of aner progression, whih is likely due to the fat that regulates diverse gene expression programs involved in ell division, survival, metabolism, and redox homeostasis (9). Our analysis revealed that the survival of breast aner patients who reeived hemotherapy was negatively orrelated with expression of in the primary tumor: Higher expression predited dereased survival, supporting the role of in promoting hemotherapy resistane and tumor reurrene in breast aner. In summary, we have delineated a signaling pathway through whih HIF-1 mediated glutathione synthesis ontributes to hemotherapy-indued aner stem ell enrihment in TN, whih an be targeted by inhibiting HIF-1α,, or ativity or expression. Glutathione plays an important role in regulating the expression of and other pluripoteny fators that determine the S phenotype, through its ativity as a signaling moleule rather than exlusively as an antioxidant. In addition, we have demonstrated that glutathione, MEK1- inhibitors, and opper helators promote the S phenotype, indiating that aution should be exerised regarding their use as aner therapeutis. oadministration of a HIF-1 inhibitor, suh as digoxin (1, 3, ) or ganetespib (1), with ytotoxi hemotherapy may be partiularly effetive beause of the multiple mehanisms by whih HIF-1 promotes Ss and other aspets of aner progression (). Methods ell ulture and Reagents. MA-M-31 and SUM-19 ells were maintained as desribed (1) at 37 in a % O, 9% air inubator (% O ). Hypoxi ells were plaed in a modular inubator hamber (illups-rothenberg) flushed with a gas mixture ontaining 1% O,%O, and 9% N. Palitaxel, gemitabine, arboplatin, digoxin, SSA, SO, β-meraptoethanol, TTM, trientine, and uso were purhased from Sigma-Aldrih. MnTMPyP and U1 were purhased from EM Millipore and Life Tehnologies. Lentivirus Transdution. Lentiviral vetors enoding shrna targeting HIF-1α and HIF-α were desribed (). plko.1-puro lentiviral vetors enoding shrna targeting,,, and were purhased from Sigma-Aldrih, and shrna sequenes are shown in Table S1. Lentiviruses were pakaged, and MA-M-31 ells were transdued and subjeted to puromyin seletion as desribed (). RT-qPR. Total RNA was extrated, NA was synthesized, and qpr analysis was performed as desribed (). PR primer sequenes are shown in Table S. hip. MA-M-31 ells were ross-linked in 3.7% formaldehyde for 1 min, quenhed in.1 M glyine for min, and lysed with SS lysis buffer. hromatin was sheared by soniation, and lysates were preleared with salmon sperm NA/protein A agarose slurry (Millipore) for 1 h and inubated with IgG or antibodies against HIF-1α (Santa ruz), HIF-1β, or (Novus iologials) in the presene of protein A agarose beads overnight. After sequential washes of the agarose beads, NA was eluted in 1% SS with.1 M NaHO 3, and ross-links were reversed by addition of. M Nal. NA was purified by phenol-hloroform extration and ethanol preipitation, and analyzed by qpr (Table S3). Immunoblot Assay. Whole-ell lysates were prepared in modified RIPA buffer, and proteins were separated by SS/PAGE, blotted onto nitroellulose membranes, and probed with primary antibodies (Table S) followed by HRP-onjugated seondary antibodies (GE Healthare). The hemiluminesent signal was deteted by using EL Plus (GE Healthare). reast aner Stem ell Assays. Aldefluor assays were performed aording to manufaturer s instrutions (Stem ell Tehnologies) as desribed (1). For ell sorting, the top and bottom % of Aldefluor-expressing ells were olleted by flow ytometry and onsidered as ALH + and ALH, respetively. Mammosphere assays were performed as desribed (1). Subellular Frationation. ells were resuspended in 1 mm Hepes, 1. mm Mgl, 1 mm Kl,. mm TT,.% Igepal, and protease inhibitor mixture (Rohe), inubated on ie for 1 min, and entrifuged for 1 min at 1, g. The supernatant was reserved as the ytosoli fration. The pellet was resuspended in mm Hepes, 1. mm Mgl,. mm ETA,. mm TT, % (vol/vol) glyerol and protease inhibitor mixture, homogenized with a oune homogenizer (3 strokes), inubated on ie for 3 min, and entrifuged to ollet supernatant as the nulear fration. Glutathione Assay. ultured ells were trypsinized, olleted by entrifugation, resuspended in % (wt/vol) -sulfosaliyli aid, subjeted to three E8 Lu et al.

10 freeze-and-thaw yles, and entrifuged to remove debris. Tumor tissues were frozen in liquid nitrogen, ground with a mortar and pestle, resuspended in % -sulfosaliyli aid, homogenized in a oune homogenizer, and entrifuged to remove debris. Supernatants were analyzed by using a glutathione assay kit (Sigma-Aldrih) based on the enzymati reation of glutathione with, -dithiobis--nitrobenzoi aid. Absorbane was quantified with a spetrophotometer at the wavelength of nm. Orthotopi Transplantation. Protools were approved by the Johns Hopkins University Animal are and Use ommittee and were in aordane with the NIH Guide for the are and Use of Laboratory Animals. MA-M-31 sublones 1. Foulkes W, Smith IE, Reis-Filho JS (1) Triple-negative breast aner. N Engl J Med 33(): ent R, et al. (7) Triple-negative breast aner: linial features and patterns of reurrene. lin aner Res 13(1 Pt 1): André F, Zielinski (1) Optimal strategies for the treatment of metastati triplenegative breast aner with urrently approved agents. Ann Onol 3(Suppl ): vi vi1.. Polyak K (11) Heterogeneity in breast aner. J lin Invest 11(1): Pee S, et al. (1) iologial and moleular heterogeneity of breast aners orrelates with their aner stem ell ontent. ell 1(1): 73.. Oskarsson T, atlle E, Massagué J (1) Metastati stem ells: Soures, nihes, and vital pathways. ell Stem ell 1(3): ean M, Fojo T, ates S () Tumour stem ells and drug resistane. Nat Rev aner (): Li X, et al. (8) Intrinsi resistane of tumorigeni breast aner ells to hemotherapy. J Natl aner Inst 1(9): hola NE, et al. (13) TGF-β inhibition enhanes hemotherapy ation against triplenegative breast aner. J lin Invest 13(3): Samanta, Gilkes M, haturvedi P, Xiang L, Semenza GL (1) Hypoxia-induible fators are required for hemotherapy resistane of breast aner stem ells. Pro Natl Aad Si USA 111():E9 E Ginestier, et al. (7) ALH1 is a marker of normal and malignant human mammary stem ells and a preditor of poor linial outome. ell Stem ell 1(): ontu G, et al. (3) In vitro propagation and transriptional profiling of human mammary stem/progenitor ells. Genes ev 17(1): Vaupel P, Mayer A, riest S, Hökel M () Hypoxia in breast aner: Role of blood flow, oxygen diffusion distanes, and anemia in the development of oxygen depletion. Adv Exp Med iol : Semenza GL (1) Hypoxia-induible fators in physiology and mediine. ell 18(3): Flamant L, Notte A, Ninane N, Raes M, Mihiels (1) Anti-apoptoti role of HIF-1 and AP-1 in palitaxel exposed breast aner ells under hypoxia. Mol aner 9: Suda T, Takubo K, Semenza GL (11) Metaboli regulation of hematopoieti stem ells in the hypoxi nihe. ell Stem ell 9(): Perales-lemente E, Folmes, Terzi A (1) Metaboli regulation of redox status in stem ells. Antioxid Redox Signal 1(11): Traverso N, et al. (13) Role of glutathione in aner progression and hemoresistane. Oxid Med ell Longev 13: Okuno S, et al. (3) Role of ystine transport in intraellular glutathione level and isplatin resistane in human ovarian aner ell lines. r J aner 88(): Timmerman LA, et al. (13) Glutamine sensitivity analysis identifies the antiporter as a ommon triple-negative breast tumor therapeuti target. aner ell ():. 1. Harris IS, et al. (1) Glutathione and thioredoxin antioxidant pathways synergize to drive aner initiation and progression. aner ell 7():11.. Parker JS, et al. (9) Supervised risk preditor of breast aner based on intrinsi subtypes. J lin Onol 7(8): Zhang H, et al. (8) igoxin and other ardia glyosides inhibit HIF-1alpha synthesis and blok tumor growth. Pro Natl Aad Si USA 1(): Sheridan, et al. () +/- breast aner ells exhibit enhaned invasive properties: An early step neessary for metastasis. reast aner Res 8():R9.. Qu Y, et al. (11) Thioredoxin-like regulates human aner ell growth and metastasis via redox homeostasis and NF-κ signaling. J lin Invest 11(1):1.. Piva M, et al. (1) promotes tamoxifen resistane in breast aner ells. EMO Mol Med (1): aner Genome Atlas Network (1) omprehensive moleular portraits of human breast tumours. Nature 9(718):1 7. were injeted into the mammary fat pad of - to 7-wk-old female SI mie in a 1:1 suspension of Matrigel ( iosienes) in PS. AKNOWLEGMENTS. We thank Karen Padgett of Novus iologials for providing antibodies against,, HIF-1β,,,,,, phospho-, MEK1, and phosphok1. This work was supported by Impat Award W81XWH-1-1- from the reast aner Researh Program of the epartment of efense. G.L.S. is an Amerian aner Soiety Researh Professor and the. Mihael Armstrong Professor at the Johns Hopkins University Shool of Mediine. L.X. was supported by a hina Sholarship ounil grant and National Natural Siene Foundation of hina Grant Miyamoto K, et al. (7) Foxo3a is essential for maintenane of the hematopoieti stem ell pool. ell Stem ell 1(1): Naka K, et al. (1) TGF-beta-FOXO signalling maintains leukaemia-initiating ells in hroni myeloid leukaemia. Nature 3(781): Zhang X, et al. (11) FOXO1 is an essential regulator of pluripoteny in human embryoni stem ells. Nat ell iol 13(9): Furuyama T, Nakazawa T, Nakano I, Mori N () Identifiation of the differential distribution patterns of mrnas and onsensus binding sequenes for mouse AF-1 homologues. iohem J 39(Pt ): Yang JY, et al. (8) promotes tumorigenesis by inhibiting FOXO3a via MM- mediated degradation. Nat ell iol 1(): Freedman JH, iriolo MR, Peisah J (1989) The role of glutathione in opper metabolism and toxiity. J iol hem (1): Turski ML, et al. (1) A novel role for opper in Ras/mitogen-ativated protein kinase signaling. Mol ell iol 3(7): rady, et al. (1) opper is required for onogeni RAF signalling and tumorigenesis. Nature 9(71): Trahootham, et al. () Seletive killing of onogenially transformed ells through a ROS-mediated mehanism by beta-phenylethyl isothioyanate. aner ell 1(3): alendiran GK, abur R, Fraser () The role of glutathione in aner. ell iohem Funt (): Lo M, Ling V, Wang YZ, Gout PW (8) The x- ystine/glutamate antiporter: A mediator of panreati aner growth with a role in drug resistane. r J aner 99(3): Narang VS, Pauletti GM, Gout PW, ukley J, ukley AR (7) Sulfasalazineindued redution of glutathione levels in breast aner ells: Enhanement of growth-inhibitory ativity of oxorubiin. hemotherapy 3(3): Sullivan L, et al. (13) The proto-onometabolite fumarate binds glutathione to amplify ROS-dependent signaling. Mol ell 1(): Kim E, Nevitt T, Thiele J (8) Mehanisms for opper aquisition, distribution and regulation. Nat hem iol (3): Gupte A, Mumper RJ (9) Elevated opper and oxidative stress in aner ells as a target for aner treatment. aner Treat Rev 3(1):3. 3. Ishida S, Andreux P, Poitry-Yamate, Auwerx J, Hanahan (13) ioavailable opper modulates oxidative phosphorylation and growth of tumors. Pro Natl Aad Si USA 11(8): Juarez J, et al. () opper binding by tetrathiomolybdate attenuates angiogenesis and tumor ell proliferation through the inhibition of superoxide dismutase 1. lin aner Res 1(1): ooth A, Trudeau T, Gomez, Luia MS, Gutierrez-Hartmann A (1) Persistent / MAPK ativation promotes latotrope differentiation and diminishes tumorigeni phenotype. Mol Endorinol 8(1): urdon T, Smith A, Savatier P () Signalling, ell yle and pluripoteny in embryoni stem ells. Trends ell iol 1(9): Hamilton W, rikman JM (1) Erk signaling suppresses embryoni stem ell selfrenewal to speify endoderm. ell Reports 9(): Leith HG, et al. (1) Embryoni germ ells from mie and rats exhibit properties onsistent with a generi pluripotent ground state. evelopment 137(1): Eijkelenboom A, urgering M (13) FOXOs: Signalling integrators for homeostasis maintenane. Nat Rev Mol ell iol 1(): Zhang H, et al. (1) HIF-1-dependent expression of angiopoietin-like and L1AM mediates vasular metastasis of hypoxi breast aner ells to the lungs. Onogene 31(1): Xiang L, et al. (1) Ganetespib bloks HIF-1 ativity and inhibits tumor growth, vasularization, stem ell maintenane, invasion, and metastasis in orthotopi mouse models of triple-negative breast aner. J Mol Med (erl) 9(): Semenza GL (1) The hypoxi tumor miroenvironment: A driving fore for breast aner progression. iohim iophys Ata pii:s17-889(1): MEIAL SIENES PNAS PLUS Lu et al. PNAS Published online July 3, 1 E9