Article

GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell and Pathogenicity

Graphical Abstract Authors Sharon M. Louie, Elizabeth A. Grossman, Lisa A. Crawford, ..., Andrei Goga, Eranthie Weerapana, Daniel K. Nomura

Correspondence [email protected]

In Brief Using a reactivity-based chemoproteomic platform, Louie et al. have identified GSTP1 as a triple-negative breast cancer target that, when inhibited, impairs breast cancer pathogenicity through inhibiting GAPDH activity and downstream metabolism and signaling pathways.

Highlights d We used chemoproteomics to profile metabolic drivers of breast cancer d GSTP1 is a novel triple-negative breast cancer-specific target d GSTP1 inhibition impairs triple-negative breast cancer pathogenicity d GSTP1 inhibition impairs GAPDH activity to affect metabolism and signaling

Louie et al., 2016, Cell Chemical Biology 23, 1–12 May 19, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.chembiol.2016.03.017 Please cite this article in press as: Louie et al., GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.03.017 Cell Chemical Biology Article

GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity

Sharon M. Louie,1 Elizabeth A. Grossman,1 Lisa A. Crawford,2 Lucky Ding,1 Roman Camarda,3 Tucker R. Huffman,1 David K. Miyamoto,1 Andrei Goga,3 Eranthie Weerapana,2 and Daniel K. Nomura1,* 1Departments of Chemistry and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA 2Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA 3Department of Cell and Tissue Biology and Medicine, University of California, San Francisco, San Francisco, CA 94143, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2016.03.017

SUMMARY subtypes that are correlated with heightened malignancy and poor prognosis remain poorly understood. Breast cancers possess fundamentally altered meta- Mortality from breast cancer is almost always attributed to bolism that fuels their pathogenicity. While many metastatic spread of the disease to other organs, thus pre- metabolic drivers of breast cancers have been iden- cluding resection as a treatment method. Unfortunately, con- tified, the metabolic pathways that mediate breast ventional chemotherapy fails to eradicate most human cancers, cancer malignancy and poor prognosis are less including aggressive breast cancers. Studies over the past well understood. Here, we used a reactivity-based decade have uncovered certain breast cancer types and cell types that are associated with poor prognosis, such as chemoproteomic platform to profile metabolic en- estrogen/progesterone/HER2 receptor-negative (triple-nega- zymes that are enriched in breast cancer cell types tive) breast cancers (TNBCs) or cancer stem/precursor cells linked to poor prognosis, including triple-negative that possess self-renewing and tumor-initiating capabilities, breast cancer (TNBC) cells and breast cancer cells epithelial-to-mesenchymal transition (EMT), poor prognosis, that have undergone an epithelial-mesenchymal and chemotherapy resistance within breast tumors (Dawson transition-like state of heightened malignancy. We et al., 2009; Dietze et al., 2015; Polyak and Weinberg, 2009). identified glutathione S- Pi 1 (GSTP1) as While eliminating these breast cancer types is critical in combat- a novel TNBC target that controls cancer pathoge- ting breast cancer, there are currently few to no therapies that nicity by regulating glycolytic and lipid metabolism, target this malignant population of breast cancer cells. energetics, and oncogenic signaling pathways In this study, we used a reactivity-based chemoproteomic plat- through a protein interaction that activates glyceral- form to identify metabolic that are heightened in TNBC cells or upon induction of an EMT-like programming of height- dehyde-3-phosphate dehydrogenase activity. We ened malignancy in breast cancer cells. Through this profiling show that genetic or pharmacological inactivation effort, we identified glutathione S-transferase Pi 1 (GSTP1) as a of GSTP1 impairs cell survival and tumorigenesis critical metabolic driver that is heightened specifically in TNBCs in TNBC cells. We put forth GSTP1 inhibitors as a to control multiple critical nodes in cancer metabolism and novel therapeutic strategy for combatting TNBCs signaling pathways to drive breast cancer pathogenicity. through impairing key cancer metabolism and signaling pathways. RESULTS

Profiling Dysregulated Metabolic Enzymes in TNBC INTRODUCTION Cells and CDH1 Knockdown Breast Cancer Cells To identify metabolic drivers of breast cancer pathogenicity in Breast cancers possess fundamentally altered metabolism that aggressive breast cancer cell types associated with malignancy drives their pathogenic features. Since Otto Warburg’s seminal and poor prognosis, we used a reactivity-based chemical prote- discovery in the 1920s that cancer cells have heightened glucose omic strategy to map cysteine and lysine reactivity in TNBC cells uptake and aerobic , recent studies have identified and breast cancer cells with EMT-like features (Figure 1; Table many other biochemical alterations in cancer cells, including S1). Both TNBC cells and breast cancer cells that have under- heightened glutamine-dependent anaplerosis and de novo lipid gone EMT have been linked to heightened aggressiveness and biosynthesis, which serve as metabolic platforms for breast poor prognosis. Specifically, we wanted to (1) identify TNBC- cancer cells to generate biomass for cell division and metabo- specific metabolic targets by comparing a panel of lites that modulate cancer cell signaling, epigenetics, and path- four non-TNBC and five TNBC cell lines, and (2) identify upregu- ogenicity (Benjamin et al., 2012; Cantor and Sabatini, 2012; lated enzyme targets in MCF7 breast cancer cells upon knock- Pavlova and Thompson, 2016). While targeting dysregulated down of CDH1, a critical mediator of EMT and cell-cell adhesion. metabolism is a promising strategy for breast cancer treatment, We knocked down CDH1 in MCF7 cells with short-hairpin (sh) the metabolic pathways that drive pathogenicity in breast cancer oligonucleotides (shCDH1 cells) to induce an EMT-like state.

Cell Chemical Biology 23, 1–12, May 19, 2016 ª 2016 Elsevier Ltd. 1 E SP yieratvt nTB el rmec niiulcl ieadcmie vrgdseta onsfo o-NCadTB cells. TNBC and non-TNBC from counts spectral averaged combined and (D). line cells Heatmap cell respectively. MCF7 individual counting. protein, shCDH1 each spectral the and from of (C) by cells enrichment cells quantified TNBC lower TNBC in and and in reactivity higher LC-MS/MS targets lysine indicate enzyme by GSTP1 blue metabolic (E) analyzed and light upregulated shControl and and Significantly and dark trypsinized D) (A) where and were lines protein, (C proteins each cell for enriched TNBC respe levels and and probes, relative iodoacetamide-alkyne non-TNBC, enriched, represent cysteine-reactive cells, avidin and epithelial dichlorotriazine-alkyne Cells were mammary lysine-reactive Cancer proteins non-transformed the Breast with MCF10A labeled (B) Knockdown of cells CDH1 panel cancer and breast a Cells MCF7 of shCDH1 TNBC profiling in Chemoproteomic Targets B) Enzyme and Metabolic (A Dysregulated Profiling 1. Figure 2 laect hsatcei rs s oi ta. SP saDie fTil-eaieBes acrCl eaoimadPtoeiiy elChemica Cell Pathogenicity, and Metabolism Cell Cancer Breast Triple-Negative of Driver a Is GSTP1 al., et Louie http://dx.doi.org/10.1016/j.chembiol.2016.03.017 as: (2016), press in Biology article this cite Please elCeia Biology Chemical Cell ABC FG GSTP1 actin lysine reactivityinnon-TNBC HSD17B10 ALDH16A1 ALDH18A1 PAFAH1B3 GNPNAT1 PRPSAP1 MTHFD1L ALOX15B ALDH4A1 ALDH6A1 PTPLAD1 ALDH5A1 ALDH9A1 ALDH1A1 ALDH3A2 ALDH1A3 ALDH7A1 LDHAL6B ALDH1B1 HSD17B4 AKR1B10 ALDH1L2 SLC27A2 HMGCS2 HMGCS1 SULT1A1 COMTD1 BCKDHA BCKDHB SAMHD1 C21orf33 PIP4K2C CKMT1A PCYOX1 PDXDC1 L2HGDH MTHFD2 MTHFD1 MTHFD1 C11orf54 MBOAT7 GNPTAB SUCLG2 CPPED1 SUCLG1 DHCR24 NDUFS2 DHTKD1 NDUFS1 NDUFS3 NDUFB8 NDUFV1 SACM1L ACADVL PTGES3 PTGES3 NDUFA9 PTGES2 FAHD2A AKR1C1 ABHD12 AKR1C3 GALNT7 AKR7A2 AKR1A1 PNPLA6 PNPLA2 AKR1B1 DPYSL2 PAPSS2 TALDO1 AGPAT9 AGPAT1 SPTLC2 IMPDH2 HIBADH LPCAT1 LPCAT2 GRHPR ACADM PHGDH HMGCL ISYNA1 C12orf5 INPP4B GAPDH HMOX2 MCCC2 MCCC1 HADHA PGAM1 GANAB GANAB HADHB COASY NAMPT ACACA ALDOC GSTM3 MGST1 DHCR7 MGST3 GSTM1 BCKDK UGGT1 ALDOA ACOX3 ACOX1 DEGS1 DECR1 SHMT1 DHRS2 NCEH1 PCMT1 DHRS7 SHMT2 GSTO1 NSDHL ECHS1 PDHA1 GLUD1 ACAD9 ACOT2 GLUD2 ACOT9 ACOT7 PTGR2 OXCT1 ACOT8 ETFDH EPHX1 ENPP1 PLOD3 PLOD1 GSTP1 ACAA2 PRPS2 GSTK1 PLCG2 ACSS2 ACAA1 PLOD2 MAT2A HPRT1 MAT2B CPT1A SGPL1 GALK1 ALDH2 HSDL2 CCBL2 FAHD1 ACSF2 PNPT1 PCYT2 GFPT1 GSTZ1 BLVRA RDH11 ACSL5 ACSL1 PLCB3 P4HA2 P4HA1 ACSL4 ACSL3 ACAT2 ACAT1 FLAD1 FDFT1 PSAT1 GMDS MMAB UGCG OGDH LTA4H IDH3A MAOA IDH3B GMPS GGCX PYGM UGDH COMT SORD GCLM QDPR CMAS OGFR GAMT GAMT PI4KA NQO1 DCXR PGM1 HADH MDH1 MDH2 DERA SDHA AGPS PYGB NANS NME1 BLMH PFKM DPYD NME3 PDPR DHPS SDHB PDHB AHCY MPST TYMP CMBL GART MCAT HEXA G6PD PSPH ACO1 SOD2 ENO3 ADSS UGP2 ACO2 GPD2 PDXK ENO1 ENO2 PKM2 FDXR GOT1 NMT1 GOT2 GCAT MTAP LDHA GYS1 PGK2 OAS3 GALE PGLS PGK1 GLO1 BDH1 GPX1 ADH5 CBR1 LDHB ECH1 CTPS FDPS PYGL APRT ILVBL FASN ACP6 CPS1 SCP2 PKLR PCK2 PFKP ADSL PLAA ASS1 ALG2 LDLR PFAS DLST ACLY ABAT PLD3 FBP1 FAR1 PPA2 PPA1 PFKL PFKL DLAT PECI IDH2 IDH1 GGH GSR GNE MVK PGD OGT GSS PGP CKB ADK ME2 CBS ESD CTH PNP DUT NNT GLS DLD DBT FTO OAT CA2 HK2 HK1 FAH AK2 LSS TKT IDI1 MPI GPI DCI IVD PC CS

MCF10A metabolicenzymes metabolic enzymes low relative o-NCTNBC non-TNBC MCF7 levels

MCF10A T47D cells and TNBC o-NCTNBC non-TNBC MCF7 ZR751

23 T47D MDA-MB-361

–2 a 9 2016 19, May 1–12, , ZR751 231MFP MDA-MB-361 HCC1143 231MFP HCC38 HCC1143 HCC70 HCC38 MDA-MB-468 HCC70 MDA-MB-468 GSTP1 proteinexpression

relative expression in MCF7shCDH1cells 1,000 1,500 2,000 500 MC cysteine reactivity 0 TNBC ALDH18A1 PAFAH1B1 HSD17B10 high relative MTHFD1L ALDH9A1 ALDH1A2 ALDH7A1 ALDH1B1 ALDH7A1 ALDH5A1 PRPSAP PTPLAD1 MTHFD1 HMGCS1 CKMT1A non- ACADSB TXNRD2 IMPDH2 TXNDC5 PTGES2 PAPSS1 HADHA PHGDH GAPDH HADHB MCCC2 GSTM3 PCMT1 SHMT2 ASMTL PDHA1 ACAD9 OXCT1 GSTO1 ACAA2 ALDH2 NAT10 SOAT1 HSDL2 USP9X ACAT2 ACAT1 F PRDX1 MDH2 MDH1 PYCR2 GLRX3 GSPT1 PLCG1 GSTP1 PSAT1 ECHS1 GFPT1 GMPS CMAS PDHB PFKM SDHA GOT2 ACO2 SORD RPN1 CBR1 AGPS LDHB ASNS FASN ADSL PPAT DLAT PGLS SCP2 ACLY PFAS CTPS ACLY IDH1 PFKP OAT 10A PGD ESD CKB TKT GLS

MC FH levels MDA-MB-36T47DF7 2 Z R75 shControl

TNBC 231MFP1 HCC1431 shCDH1 HCC3 MDA-MB- HCC70 8 46 8 D E

spectral counts spectral counts 100 20 40 60 80 infcnl peuae eaoi enzyme targets metabolic upregulated significantly

relativeexpression 20 40 60 80 200 400 600 0 LDHB 0 0 non-TNBC spectral counts enzyme metabolic upregulated significantly 100 150 200 250 PFK * GSTP1 GSTP1 lysine reactivity in TNBC cells TNBC reactivity in lysine GSTP1

MC 50 TNBC 0

AGPSM * TNBC non- F10A

MCF7 * DLAT * * PSAT1

MD T47D * A-MZR75 *

GLS in MCF7shCDH1cells TNBC B 1 ALDH PC targets inTNBCcells 231MFP * -361 ALDH1A2

HCC143 * * 2 shCDH1 shControl

primary humanbreasttumors GSTP1

HCC38 * MDA HCC7 PLCG1 ACSL *

GSTP1 expressionin -M

log fold-change B-4680 PPAT * 3 (TCGA database)

2 * FDR 2.69x10 (TNBC vs CTPS ** SOAT1 receptor-positive) PSA

ADSL * 0.0 0.5 1.0 1.5 2.0 2.5 spectral counts T1 100 PFAS * ACADSB *** 20 40 60 80 non-TNBC 0 TNBC non- * lgn otne nnx page) next on continued (legend

TNB * PHGCK ASS1 TNBCC ALDH1B1B * -42 DH * *

ASNS * SORD

* AC AT2 ACAT1OAT * * tvl.Probe- ctively. * l s Please cite this article in press as: Louie et al., GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.03.017

These cells show upregulation of the mesenchymal marker vi- ures 1E and 1F), shown both by dichlorotriazine-alkyne reactivity mentin and concordant increases in serum-free cell survival, and western blotting. We also show that GSTP1 expression is proliferation, and migration (Figure S1), consistent with EMT- significantly heightened in TNBC compared with receptor-posi- like characteristics. tive primary human breast tumors (Figure 1G). Overall, our data We labeled cell lysates from these two breast cancer models indicate that GSTP1 may represent a TNBC-specific target. with previously validated lysine-reactive dichlorotriazine-alkyne or cysteine-reactive iodoacetamide-alkyne reactivity-based Genetic or Pharmacological Inactivation of GSTP1 probes to tag proteins bearing functional lysines or cysteines, Impairs TNBC Pathogenicity respectively, for subsequent attachment of a biotin handle by To investigate the role of GSTP1 in TNBC pathogenicity, we click chemistry, enrichment, and analysis by mass spectrometry generated two knockdown lines of GSTP1 in TNBC 231MFP (MS), using an adapted method established by Weerapana and cells (shGSTP1-1 and shGSTP1-2) (Figure 2A). We showed co-workers (Figures 1A and 1B; Table S1)(Shannon et al., that GSTP1 knockdown impairs serum-free cell survival in 2014; Weerapana et al., 2010). We chose to profile cysteine 231MFP cells without affecting cell proliferation (Figure 2B). and lysine reactivity in this study, since these amino acids GSTP1 knockdown also impaired in vivo 231MFP breast tumor are important mediators of enzyme activity or function through xenograft growth in immune-deficient mice (Figure 2C). nucleophilic and redox catalysis, allosteric regulation, metal Intrigued by these results, we next tested whether pharmaco- binding, and structural stabilization across a wide range of logical inhibition of GSTP1 with a highly selective GSTP1 inhibitor protein classes (Pace and Weerapana, 2013; Shannon and LAS17 also impaired breast cancer pathogenicity. LAS17 was Weerapana, 2015). Upon proteomic analysis of probe-enriched recently developed as a highly potent, selective, and irreversible protein targets, we filtered these proteins for metabolic enzyme dichlorotriazine GSTP1 inhibitor that targets an active-site tyro- targets that were significantly upregulated (p < 0.01, >5-fold) in sine (Figure 2D) (Crawford and Weerapana, 2016). In addition TNBC cells or shCDH1 MCF7 cells compared with non-TNBC to its property as a GSTP1 inhibitor, LAS17 also bears a bio- or shControl counterparts, respectively (Figures 1C and 1D; orthogonal alkyne handle, which can be coupled with copper- Table S1). GSTP1 was the most significantly upregulated target catalyzed click chemistry (Martell and Weerapana, 2014)to in TNBC cells that was also significantly heightened in shCDH1 append a fluorophore-azide or biotin-azide analytical handle cells, so we focused on investigating the pathogenic role of for subsequent fluorescent or proteomics-based detection this target in breast cancer (Figures 1C and 1D). of GSTP1, respectively (Figures 2D and 2E). LAS17 inhibits Many human cancers, including breast, lung, colon, and GSTP1 activity in vitro with a 50% inhibitory concentration ovarian cancers, have been shown to express high levels of (IC50) value of 0.5 mM(Figure 2F). GSTP1, and its expression has been correlated with both disease LAS17 treatment in 231MFP breast cancer cells recapitulated progression and resistance to chemotherapeutic drugs (La- the serum-free cell survival impairments observed with genetic borde, 2010). GSTP1 inhibitors have been studied in the context inactivation of GSTP1 (Figure 2G). Daily administration of of cancer for many years as adjuvant therapies with chemo- LAS17 (20 mg/kg intraperitoneally, once per day) significantly therapy agents to prevent their detoxification by cancer cells, impaired 231MFP breast tumor xenograft growth in immune- with the aim of improving chemotherapy efficacy (Tew and Town- deficient mice when treatment was initiated 2 days after subcu- send, 2011; Townsend and Tew, 2003). Yet the majority of taneous injection of cells, and LAS17 even slowed tumor growth chemotherapeutic drugs have a relatively weak affinity for when initiated 16 days after tumor implantation (Figure 2H), with GSTP1 compared with other GST enzymes. This discrepancy be- no observable toxicity and no weight change (Figure S2A). tween expression of GSTP1 and its correlation with the develop- LAS17 had no effect on serum-free survival of shControl MCF7 ment of multidrug resistance suggests additional roles for GSTP1 non-TNBC cells that do not express GSTP1, but significantly on influencing metabolic and signaling pathways in cancer cells impaired the survival of shCDH1 MCF7 breast cancer cells (Fig- (Laborde, 2010). GSTP1 inhibitors have not yet been investigated ure 2I). While GSTP1 inhibition selectively impaired survival in for their potential as stand-alone therapies toward thwarting shCDH1 cells, LAS17 treatment in shCDH1 cells did not rescue breast cancer. We thus focused our attention on further investi- vimentin expression or impair proliferation, indicating that gating the role of GSTP1 in driving breast cancer pathogenicity. GSTP1 is not a critical driver of the EMT process (data not shown). LAS17 treatment also impaired serum-free cell survival GSTP1 Is a TNBC-Specific Target in additional TNBC lines HCC38, HCC70, and HCC1143 (Fig- We next investigated the expression pattern of GSTP1 in different ure 2J). We also show that GSTP1 knockdown in HCC38 cells types of breast cancer cell lines and primary human breast tu- impairs cell survival (Figures S2B and S2C). We thus show that mors. Interestingly, we show that GSTP1 is not expressed in pharmacological GSTP1 inactivation impairs breast cancer any of the non-TNBC cells (MCF7, T47D, ZR751, and MDA- pathogenicity in culture and in vivo in a seemingly TNBC-specific MB-361), but is highly expressed across all of the TNBC cells manner, suggesting that GSTP1 inhibitors may be promising (231MFP, HCC1143, HCC38, HCC70, and MDA-MB-468) (Fig- therapeutics for treating TNBCs.

(F) GSTP1 expression was measured across a panel of MCF10A non-transformed mammary epithelial cells, non-TNBC, and TNBC cell lines by western blotting. Western blotting image shown is representative of n = 3/group. GSTP1 expression was normalized to actin loading control and quantified by densitometry. (G) GSTP1 expression in receptor-positive and TNBC primary human breast tumors from The Cancer Genome Atlas (TCGA) database. FDR, false-discovery rate. Data in (C–G) are presented as mean ± SEM, n = 3–5/group. Significance is presented as *p < 0.05 compared with non-TNBC or shControl cells. Detailed data from the chemoproteomic study in (A–D) are provided in Table S1. Characterization of shCDH1 cells is shown in Figure S1.

Cell Chemical Biology 23, 1–12, May 19, 2016 3 Please cite this article in press as: Louie et al., GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.03.017

ABCGSTP1 knockdown 231MFP TNBC 231MFP TNBC in 231MFP TNBC cells pathogenicity in vitro pathogenicity in vivo GSTP1 expression GSTP1 expression proliferation survival tumor xenograft growth 1.5 1.5

qPCR Western blotting ) 1,500 3 l 1.5 1.5 1.0 1.0 shControl 2 ** 1,000 shGSTP1-1 1.0 1.0 0.5 surviva 0.5 proliferation 0.5 shControlshGSTP1-1shGSTP1- 0.5 0.0 0.0 500 expression

GSTP1 expression ** ** 0.0 0.0 tumor volume (mm * actin 0 * * * * shControl shControl 0 102030 shGSTP1-1shGSTP1-2 shGSTP1-1shGSTP1-2 days shControl shControl shGSTP1-1shGSTP1-2 shGSTP1-1shGSTP1-2 DGHEFLAS17 labeling LAS17 inhibition 231MFP TNBC tumor xenograft GSTP1 inhibitor of shCDH1 cells of GSTP1 activity pathogenicity in vitro dichlorotriazine MCF7 100 survival tumor xenograft growth electrophile 1.5 600 Cl N Cl 75 ) 3 l control a 1.0 m shCont NN shCDH1 LAS17 (day 2) O 50 viv 400

ur LAS17 (day 16) th (m N IC50 s 0.5 O * w

μ ro % inhibition 25 0.5 M * * 0.0 200 * * alkyne * 0 mor g * * u -3 -2 -1 0 1 2 t * binding group 10 10 10 10 10 10 control LAS17 0 *** * GSTP1 > concentration (μM) 0102030 LAS-17 days GSTP1 inhibitor IJsensitivity in shCDH1 cells GSTP1 inhibitor effects upon TNBC cells

MCF7 survival HCC38 survival HCC70 survival HCC1143 survival 2.0 1.5 1.5 1.5 * l l 1.5 1.0 1.0 * 1.0 va 1.0 val

# * urvi * survi survival s surviva 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 LAS17 - + - +

control LAS17 control LAS17 control LAS17

trol

shCDH1 shCon

Figure 2. The Effect of Genetic and Pharmacological Inactivation of GSTP1 on Breast Cancer Pathogenicity (A) GSTP1 was knocked down in 231MFP TNBC cells using two independent shRNA, and expression was confirmed by both qPCR and western blotting. Western blotting image is representative of n = 3/group and protein expression was quantified by densitometry. (B) GSTP1 knockdown in 231MFP cells shows impaired serum-free cell survival, with no change in cell proliferation 48 hr after seeding. Survival and proliferation were assessed using Hoechst stain. (C) shGSTP1 231MFP cells show impaired tumor growth in SCID mice compared with shControl cells. (D) Selective and irreversible dichlorotriazine GSTP1 inhibitor LAS17 bearing a dichlorotriazine electrophile, GSTP1 binding group, and an alkyne handle for subsequent click chemistry. (E) LAS17 labeling of shControl and shCDH1 MCF7 cells showing heightened GSTP1 in shCDH1 cells. Cell lysates were labeled with LAS17 for 30 min prior to click chemistry with rhodamine-azide. Proteomes were separated on SDS-PAGE and visualized by in-gel fluorescence. (F) LAS17 inhibits GSTP1 activity with a 50% inhibitory concentration (IC50) of 0.5 mM. GSTP1 activity was assessed by pre-incubating vehicle DMSO or LAS17 with pure GSTP1 protein for 30 min prior to measuring the rate of conjugation of glutathione to CDNB and quantifying the rate of increase in the absorption of the reaction product, glutathione-DNB conjugate. (G) Serum-free survival 48 hr after seeding in 231MFP cells treated with DMSO vehicle or LAS17 (10 mM), determined by Hoechst staining. (H) 231MFP tumor xenograft growth in SCID mice upon once-per-day daily treatment with vehicle (18:1:1 PBS/ethanol/PEG 40) or LAS17 (20 mg/kg, intra- peritoneally) initiated either 2 or 16 days after subcutaneous injection of 2 3 106 231MFP cells. (I) Serum-free cell survival in shControl and shCDH1 MCF7 cells treated with DMSO vehicle or LAS17 treatment (10 mM) in shControl and shCDH1 MCF7 cells, determined by WST-1 assay. (J) Serum-free cell survival 48 hr after seeding in HCC38, HCC70, and HCC1143 cells treated with DMSO vehicle or LAS17 (10 mM), determined by Hoechst staining. Data in (A–C), (F), and (G–J) are presented as mean ± SEM, n = 3/group in (A), n = 5/group in (B), n = 8/group in (C), n = 3/group in (F), n = 5/group in (G), n = 8/group in (H), and n = 5/group in (I) and (J). Significance is presented as *p < 0.05 compared with shControl and #p < 0.05 compared to shCDH1 vehicle-treated control. Body weights and further effects of GSTP1 inhibition upon TNBC pathogenicity are shown in Figure S2.

Functional Metabolomic Profiling Reveals GSTP1 studies have shown that GSTP1 may directly affect cancer Control Over Glycolytic Metabolism, Energetics, and pathogenicity through protein-protein interactions with the Macromolecular Building Blocks MAPK family protein JNK, leading to JNK phosphorylation and We next wanted to understand the mechanisms through which activation (Tew and Townsend, 2011; Townsend and Tew, GSTP1 was controlling breast cancer pathogenicity. Previous 2003). However, GSTP1 genetic or pharmacological inactivation

4 Cell Chemical Biology 23, 1–12, May 19, 2016 Please cite this article in press as: Louie et al., GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.03.017

did not lead to JNK activation compared with treatment with the gested heightened phosphorylation and activity of AMP JNK activator anisomycin in 231MFP cells (Figure S3A). We also (AMPK) and also phosphorylation and inhibition of the down- postulated that GSTP1 could modulate glutathione levels and stream substrate acetyl coenzyme A (CoA) carboxylase (ACC). oxidative stress to cause cell-survival impairments. However, Inhibited ACC would presumably lead to decreased malonyl genetic or pharmacological inactivation of GSTP1 did not CoA levels and derepression of CPT1, and thus the observed in- change oxidative stress levels or reduced oxidized glutathione crease in AC levels. We show that GSTP1 knockdown in both (GSH/GSSG) ratios under both basal and menadione-induced shGSTP1 cells and LAS17 treatment in 231MFP cells result in oxidative stress conditions compared with controls (Figures increased levels of phosphorylated AMPK and ACC (Figures S3B–S3D). 4A and 4B). To identify alternative mechanisms, we next performed func- Activation of AMPK has been previously shown to impair tional metabolomic profiling to comprehensively map metabolic breast cancer pathogenicity, partly by inhibiting mammalian alterations conferred by genetic or pharmacological inactivation target of rapamycin (mTOR) activity and downstream protein of GSTP1 in 231MFP breast cancer cells (Figures 3A–3C and synthesis (Dowling et al., 2007). Consistent with the role of S4A–S4F; Table S2). We used a targeted single-reaction AMPK in driving the GSTP1 inhibition-mediated breast cancer monitoring (SRM)-based liquid chromatography-tandem MS pathogenicity impairments, we show that the cell-survival im- (LC-MS/MS) approach to comparatively measure the levels of pairments conferred by GSTP1 knockdown are partially rescued 200 metabolites encompassing glycolytic, tricarboxylic acid upon treatment with the AMPK inhibitor dorsomorphin (Fig- (TCA) cycle, amino acid, nucleotide, and lipid metabolism. We ure 4C). We also show that the levels of phosphorylated S6, also used an untargeted LC-MS approach in which we collected downstream of mTOR and S6 kinase, are lower in shGSTP1- mass spectra from mass-to-charge ratios (m/z) of 100–1,200 and and LAS17-treated 231MFP breast cancer cells compared with analyzed the resulting features by XCMSOnline (Gowda et al., shControl or vehicle-treated control cells, respectively (Fig- 2014) to identify metabolites that were altered upon GSTP1 inac- ure 4D). Consistent with the role of impaired mTOR signaling tivation. We subsequently analyzed any changing m/z by in GSTP1 inhibition-mediated survival defects, treatment of METLIN to identify candidate metabolites, used targeted SRM- 231MFP cells with the mTOR inhibitor Torin 1 alone causes sur- based LC-MS/MS to quantify the levels of these metabolites, vival impairments, but co-treatment of 231MFP cells with Torin 1 and added these data to our total targeted analyses. Through and LAS17 does not cause additional survival impairments these approaches we identified several metabolites that were beyond those observed with LAS17 treatment alone (Figure 4E). commonly changing between both shGSTP1 231MFP lines, including lowered levels of , ATP, nucleotides, diacy- GSTP1 Interacts with and Activates GAPDH Activity to lated phospholipids, and alkylacyl ether lipids, and increased Influence Glycolytic Metabolism levels of acyl carnitines (ACs), ceramides, and lysophospholipids We next wanted to investigate the mechanism through which (Figures 3A–3C; Table S2). LAS17 treatment in 231MFP cells GSTP1 controlled glycolytic metabolism. GSTP1 has been previ- also showed reduced levels of ATP, lactic acid, purine nucleo- ously shown to control signaling pathways through protein- tides, diacylated phospholipids, and alkylacyl ether lipids, and protein interactions. We thus generated a 231MFP cell line stably increased levels of ACs, ceramides, and lysophospholipids (Fig- overexpressing a FLAG-tagged GSTP1 and performed a ures S4B–S4F; Table S2). While there were many metabolomic pulldown study to identify GSTP1 interacting proteins through changes that were unique to either shGSTP1 or LAS17 treat- proteomic profiling (Figures S5A–S5C). We found that anti- ment, we attributed these differences to metabolic alterations FLAG pulldown in GSTP1-FLAG-overexpressing cell lysates that may manifest under chronic and stable knockdown versus significantly enriched seven proteins compared with mock- acute inhibition of GSTP1. Nonetheless, between the common infected control cell lysates (Figure S5C). Among these seven changes and even the uncommon changes in metabolite levels, proteins was glyceraldehyde-3-phosphate dehydrogenase we were able to identify overlapping metabolic pathways that (GAPDH), which was significantly enriched in GSTP1-FLAG-ex- were altered upon GSTP1 inhibition. pressing cells compared with mock-infected control 231MFP Upon mapping the observed metabolomic changes to meta- cells (Figure 5A). While we could not confirm this GSTP1 interac- bolic pathway maps, our data suggested that GSTP1 inactiva- tion with endogenous GAPDH, we showed that addition of exog- tion led to impairments in glycolytic metabolism, leading to enous pure and active GAPDH enzyme could lead to enrichment reduced lactic acid and ATP levels as well as reductions in the of GAPDH upon anti-FLAG pulldown in GSTP1-FLAG-over- levels of macromolecular building blocks, including purine nucle- expressing cell lysates compared with mock-infected control otides, fatty acids, diacylphospholipids, and alkylacyl ether lipids lysates (Figure 5B). (Figure 3C). Consistent with this premise, we observed signifi- We thus postulated that GSTP1 may interact with GAPDH to cant and time-dependent reductions in lactic acid secretion or influence its activity. Consistent with this hypothesis, we show glucose consumption upon GSTP1 knockdown in shGSTP1 that GSTP1 greatly activates GAPDH activity in vitro (Figure 5C). cells or upon LAS17 treatment in 231MFP cells, compared with This activation in GAPDH activity was independent of glutathione shControl or vehicle-treated controls, respectively (Figures 3D (GSH) or oxidized glutathione (GSSG), indicating that this activity and S4E), indicating reduced glycolytic metabolism. was not due to glutathione conjugation of a small-molecule metabolite or glutathionylation of GAPDH (Figure S5D). LAS17 it- GSTP1 Inhibition Impairs Oncogenic Signaling Pathways self does not inhibit GAPDH activity (Figure S5E). This GSTP1- The reduced levels of ATP and increased levels of ACs (C18:0 induced GAPDH activity was, however, partially suppressed by AC), a product of carnitine palmitoyltransferase 1 (CPT1), sug- LAS17 pre-treatment, indicating that LAS17 binding to GSTP1

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A

B

C D

Figure 3. Functional Metabolomic Profiling and Pathway Mapping of GSTP1 Inactivation in TNBC Cells (A) We performed untargeted and targeted SRM-based metabolomic profiling of shControl and shGSTP1 231MFP cancer cells. Shown is a heatmap of shGSTP1/ shControl ratios of all the metabolites that were measured by SRM analysis. (B) Shown are metabolites that were significantly (p < 0.05) changing in the same direction in both shGSTP1-1 and shGSTP1-2 cells compared with shControl cells. Highlighted in red are the metabolite changes that were mapped to metabolic pathways in (C). UMP, uridine monophosphate; PA, phosphatidic acid; PI, phosphatidylinositol; LPC, lysophosphatidylcholine; PAe, phosphatidic acid-ether; PIe, phosphatidylinositol-ether; LPCe, lyso- phosphatidylcholine-ether; LPCp, lysophosphatidylcholine-plasmalogen; MAGp, monoalkylglycerol-plasmalogen; FFA, free fatty acid; AC, acyl carnitine; NAE, N-acyl ethanolamine. (C) Pathway mapping of many of the metabolic changes observed in shGSTP1 231MFP cells indicates a primary impairment in glycolysis with resulting secondary impairments in nucleotide and lipid metabolism.

(legend continued on next page)

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ABp-AMPK and p-ACC levels in shGSTP1 231MFP cells p-AMPK and p-ACC levels in LAS17-treated 231MFP cells

2.5 * 3 shControlshGSTP1-1 2.0 * ** p-AMPK controlLAS17 controlLAS17 2 1.5 p-AMPK p-ACC total AMPK levels 1.0 total AMPK total ACC levels 1 p-ACC 0.5 actin actin total ACC 0.0 0 actin control LAS17 control LAS17

shControl shControl shGSTP1-1 shGSTP1-1 p-AMPK p-ACC p-AMPK p-ACC C AMPK inhibitor rescue D p-S6 levels in shGSTP1 and LAS17-treated E Torin 1 with LAS17 treatment 231MFP cells

1.5 1.5 1.5 # 1.0 1.0 1.0 * shControlshGSTP1-1controlLAS17 * * * p-S6 levels survival

0.5 0.5 * survival 0.5 * * total S6 0.0 0.0 0.0 AMPKi - + - + actin LAS17 - + - + control LAS17 Torin 1 --++

shControl shGSTP1-1 shControl shGSTP1-1

Figure 4. GSTP1 Inhibition Impairs Oncogenic Signaling Pathways (A and B) shGSTP1 (A) and LAS17-treated (B) 231MFP cells show increased levels of phosphorylated AMPK and ACC compared with shControl and DMSO vehicle-treated controls, respectively, as assessed by western blotting. (C) Serum-free cell survival impairments 48 hr post seeding in shControl and shGSTP1 231MFP cells treated with DMSO vehicle or AMPK inhibitor dorsomorphin (10 mM). (D) Phosphorylated S6 levels, a readout of mTOR activity, in shControl and shGSTP1 or DMSO vehicle or LAS17-treated (10 mM) 231MFP cells. (E) Serum-free cell survival 48 hr post seeding in 231MFP cells treated with DMSO vehicle or LAS17 (10 mM) and DMSO vehicle or mTOR inhibitor Torin 1 (250 nM). Western blotting images in (A), (B), and (D) are representative images from n = 3/group. Quantification was performed by normalizing to actin or total S6 loading controls by densitometry. Data in (A–E) are presented as mean ± SEM, n = 3–5/group. Significance is presented as *p < 0.05 compared with shControl or DMSO vehicle-treated controls, respectively in (A–E) and #p < 0.05 compared with DMSO vehicle-treated shGSTP1 in (C). at least partially disrupts the GSTP1-GAPDH protein-protein We also show that the impaired glycolytic metabolism down- interaction (Figure 5C). stream of GAPDH also affects other related downstream path- To complement our steady-state metabolomic profiling data ways, including lowered levels of [13C]TCA cycle and [13C] and further confirm our proposed mechanism that GSTP1 inhibi- glycine metabolites. This reduction of carbon into the TCA cycle tion indirectly inhibits glycolysis through impairing GAPDH ac- and particularly into [13C]citrate is also consistent with reduced tivity, we performed [13C]glucose isotopic tracing analysis in de novo lipogenic pools of [13C]palmitate levels (Figures 5D 231MFP cells to follow 13C into relevant metabolic pathways us- and 5E), and the reductions in the levels of phospholipid and ing SRM-based LC-MS/MS methods (Figures 5D and 5E). ether lipid levels (Figures 3B, 3C, and 5E). Our steady-state me- GSTP1 inhibition with LAS17 led to an accumulation of the tabolomic data also showed reduced levels of purine nucleo- GAPDH substrate [13C]glyceraldehyde-3-phosphate/dihydroxy- tides such as adenine and guanine, which is counter-indicative acetone phosphate (G3P/DHAP) and lowered [13C]glycolytic to heightened PPP observed from our tracing studies. However, intermediates downstream of GAPDH, which represent the detailed tracing analysis shows that the levels of [13C]adenine ATP-generating steps of glycolysis (Figures 5D and 5E). We AMP (m+7), derived from isotopic incorporation of glucose into also show an accumulation in the levels of [13C]glycolytic and PPP metabolites (m+5) and glycine (m+2), are lowered, whereas [13C]pentose phosphate pathway (PPP) metabolites upstream levels of [13C]AMP (m+5), derived only from glucose incorpora- of GAPDH (Figures 5D and 5E). Overall, these data are consistent tion into PPP metabolites, are elevated (Figure 5D). These data with an inhibition of GAPDH activity from GSTP1 inhibition. [13C] indicate that the observed reductions in nucleotide levels are Fructose-1,6-bisphosphate levels were reduced, indicating that likely due to the reduction in glycine necessary for de novo syn- GSTP1 may potentially also affect additional nodes in glycolysis. thesis of purines (Figures 3B, 3C, 5D, and 5E).

(D) Lactic acid secretion into the media in shControl and shGSTP1-1 231MFP cells or DMSO vehicle-treated or LAS17-treated (10 mM) 231MFP cells. Lactic acid was measured using a lactic acid measurement kit. Data in (B) and (D) are presented as mean ± SEM, n = 5/group. Significance in (D) is presented as *p < 0.05 compared with shControl or DMSO vehicle-treated controls. Detailed data, explanation of abbreviations, and SRM transitions are provided in Table S2. Effect of GSTP1 inhibition on JNK signaling and oxidative stress is shown in Figure S3, and metabolic characterization of LAS17-treated 231MFP cells is shown in Figure S4.

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A anti-FLAG-GSTP1 pulldown B anti-FLAG pulldown C GSTP1 activates GAPDH activity in 231MFP cells of GAPDH in 231MFP cells GAPDH activity GAPDH activity GSTP1 GAPDH 0.025 0.8 * # 800 * 40 * * 0.020 600 30 0.6 0.015 400 20 0.4 activity 0.010 200 10 eGFP GSTP1-FLAGeGFP GSTP1-FLAGeGFP GSTP1-FLAG activity 0.005 0.2 spectral counts spectral counts GAPDH(input) 0 0 0.000 0.0 0204060GAPDH ++ + eGFP eGFP GSTP1 -++ GAPDH(IP)IP) min LAS17 --+ GAPDH GSTP1+GAPDH GSTP1-FLAG GSTP1-FLAG GSTP1+GAPDH+LAS17

D [13C]glucose labeling of 231MFP cells E GSTP1 inhibition in TNBC cells impairs cancer cell metabolism and signaling Glu glycolytic metabolism pentose phosphate PPP purine pathway (PPP) G6P 2.5 8 nucleotides control s 2.0 * * LAS17 * control F6P 6 LAS17 1.5 diacyl- 4 F-1,6-BP phospholipids 1.0 LAS17 ** alkylacyl ** 2 relative levels relative level 0.5 * * G3P DHAP ether lipids 0.0 0 GSTP1 GAPDH serine glycine 1,3-BPG (m+6) (m+3) (m+5) (m+5) mTORS6K p-S6 lysophospholipids P P (m+4) ATP PEP F-6-P G-6-P (m+6) PPNG (m+6) 3-PG 3/2-PG (m+3) F-1,6-BP (m+6)1,3-BPG (m+3) pyruvate (m+3) ribose-5-P G3P/DHAP (m+3) ribulose-5- 2-PG p-AMPK p-ACC fatty acids erythrose-4- TCA cycle palmitate serine/glycine nucleotide PEP metabolism metabolism ATP malonyl CoA 1.5 control 1.5 control 1.5 2.5 control pyruvate lactic acid LAS17 control LAS17 s LAS17 LAS17 2.0 * 1.0 1.0 1.0 acetyl CoA citrate acetyl-CoA levels 1.5 ive level * * ive OAA citrate aconitate t 1.0 0.5 * 0.5 0.5 * ** isocitrate rela relative levels relat relative levels * 0.5 * malate TCA cycle 0.0 0.0 0.0 0.0 αKG fumarate succinate succinyl-CoA (m+4) (m+7) A (m+16) KG (m+5) OA AMP (m+5)AMP mitochondria citrate (m+6) serine (m+3) malate (m+4) glycine (m+2) fumarate (m+4)

C16:0 FFA

Figure 5. GSTP1 Interacts with and Activates GAPDH Activity to Influence Glycolytic Metabolism (A) We generated stable GFP-expressing control and GSTP1-FLAG-overexpressing 231MFP cells to identify GSTP1 interaction partners. Upon anti-FLAG pulldown of GFP-expressing control (EGFP) or GSTP1-FLAG-overexpressing 231MFP lysates and subsequent proteomic analysis of pulled-down proteins, we found enrichment of GSTP1 and GAPDH. (B) This GSTP1-GAPDH interaction was confirmed by incubation of pure and active GAPDH enzyme with GFP-expressing or GSTP1-FLAG-overexpressing 231MFP cell lysates, subsequent anti-FLAG pulldown, and western blotting of GAPDH. Shown are GAPDH blots for input and immunoprecipitation fractions. (C) Effect of co-incubating pure and active GSTP1 and GAPDH proteins individually or together on GAPDH activity, assessed by a GAPDH activity assay measuring NADH, a by-product of the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. GAPDH was treated with DMSO vehicle and GAPDH + GSTP1 was treated with DMSO vehicle or LAS17 (10 mM) 30 min prior to assessment of GAPDH activity. (D) Isotopic tracing of [13C]glucose into metabolic pathways in 231MFP cells. Cells were pre-treated with DMSO vehicle or LAS17 (10 mM) 1 hr prior to labeling of 231MFP cells with [13C]glucose (10 mM, 8 hr). Cell metabolomes were analyzed by SRM-based LC-MS/MS. (E) Model of mechanisms underlying GSTP1 control over breast cancer pathogenicity, based on steady-state metabolomics, isotopic tracing, and western blotting data. Data in (A) and (D) are presented as mean ± SEM, n = 3–5/group. Significance is presented as *p < 0.05 compared with EGFP or DMSO vehicle-treated controls, respectively. Data in (C) are presented as mean ± SEM, n = 3/group. Significance in (C) is presented as *p < 0.05 compared with GAPDH only and #p < 0.05 compared with the GAPDH and GSTP1 group. Further characterization of GSTP1 protein interactions and the role of GSTP1 in MCF10A cells are described in Figure S5.

While GSTP1 expression is heightened in TNBC compared inhibitor 2-deoxyglucose (2DG) to a degree comparable to with non-TNBC cells, we also showed that GSTP1 is ex- that of LAS17, but MCF10A cells show no survival impairment pressed in non-transformed MCF10A mammary epithelial upon 2DG treatment (Figure S5H). cells. We find that GSTP1 inhibition in MCF10A cells does not impair cell survival compared with significantly impaired DISCUSSION survival in 231MFP cells (Figure S5F). Interestingly, LAS17 treatment in MCF10A cells also impairs lactic acid secretion, Taken together, our data point to GSTP1 as a novel TNBC target indicating that glycolytic metabolism may also be impaired in that, when inactivated, impairs glycolytic metabolism through these cells (Figure S5G). We postulated that TNBC cells may a unique mechanism of disrupting GSTP1-induced activation be more addicted to glycolytic metabolism compared with of GAPDH, leading to lower levels of macromolecular building MCF10A cells. Consistent with this premise, 231MFP cell blocks (e.g., lipids and nucleotides) and ATP levels. This reduc- survival is significantly impaired by the glycolytic tion in energy levels in turn also leads to impaired oncogenic

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signaling through activation of AMPK and inhibition of mTOR GAPDH by GSTP1 was independent of GSSG and GSH, it signaling (Figure 5E). may still be possible that GAPDH activity and glycolytic meta- While our interpretations presented here are consistent with bolism may be regulated by GSTP1-mediated glutathionylation. the metabolomic and signaling changes that we observe with Furthermore, while GAPDH is not generally considered to be a GSTP1 inactivation in breast cancer cells, we expect that there rate-limiting step of glycolysis, a recent study has shown that are also additional mechanisms involved that may arise from GAPDH is rate limiting in cancer cells that possess aerobic the metabolomic changes we observed. For example, we glycolysis (Hildebrandt et al., 2015; Moellering and Cravatt, showed increased levels of ACs with GSTP1 inhibition, which 2013; Shestov et al., 2014). Our studies with GSTP1 provide we presume to be downstream of ACC inhibition and derepres- additional support for how GAPDH may act as a major regulatory sion of CPT1. These results could indicate that fatty acid hub for TNBC glycolytic activity. b-oxidation pathways may be activated upon GSTP1 inactiva- We show that pharmacological inactivation of GSTP1 over a tion. While we did not observe rescue of cell-survival impair- sustained period does not show any observable toxicity, and ments with the CPT1 inhibitor etomoxir (data not shown), the not only prevents breast tumor growth but even slows estab- observed increase in AC levels may play a role in other aspects lished breast tumor growth in mice. A highly potent GSTP1 inhib- of GSTP1-mediated effects (Carracedo et al., 2013). itor, ezatiostat (developed by Telik Inc.) has passed phase II GSTP1 has also been linked to many other functions in cancer clinical trials in patients for the treatment of myelodysplastic and other human pathologies and even in drug addiction. syndrome, indicating that GSTP1 inhibitors are likely to be well Beyond glutathionylation and detoxification functions, GSTP1 tolerated in humans (Mahadevan and Sutton, 2015). Beyond has been shown to possess chaperone functions, regulation of the many previously reported biochemical and therapeutic roles nitric oxide pathways, and control over various kinase signaling of GSTP1 (Grek et al., 2013; Tew and Townsend, 2011; Town- pathways (Zhang et al., 2014). For example, GSTP1 inhibits send and Tew, 2003), our study suggests that GSTP1 inhibitors JNK signaling and prevents downstream transcriptional activa- may also be promising stand-alone therapeutics for TNBCs. Our tion of cell-stress pathways. Under cellular stress conditions study also underscores the utility of using reactivity-based che- whereby reactive oxygen stress is heightened, GSTP1 has moproteomic platforms coupled with functional metabolomic been shown to dimerize into larger aggregates and preclude approaches to identify novel metabolic drivers and pathways binding to JNK, enabling JNK activation. In the context of underlying breast cancer malignancy. hematopoiesis, GSTP1 inhibition has been shown to play a cytoprotective role in both erythroid and lymphoid cells, and SIGNIFICANCE the GSTP1 inhibitor ezatiostat has been shown to be clinically effective for myelodysplastic syndrome (Mahadevan and Sutton, TNBCs and stem-like breast cancers that have undergone 2015; Zhang et al., 2014). While we report here that GSTP1 inhi- EMT have been linked to breast cancer malignancy and bition does not activate JNK signaling in our TNBC cells, GSTP1 poor prognosis. While these breast cancer cell types are may still directly regulate other signaling pathways through pro- particularly dangerous, there are few to no therapeutic tein interactions or glutathionylation-mediated pathways (Zhang strategies that specifically target these types of cells. et al., 2014). Here, we have used chemoproteomic profiling platforms to We also acknowledge that the apparent activation of GAPDH identify GSTP1 as a novel potential therapeutic target for activity by GSTP1 is only modestly reduced upon LAS17 treat- TNBCs. We show that GSTP1 interacts with GAPDH to acti- ment, indicating that there may be additional complexities vate its activity and that GSTP1 inactivation impairs glyco- involved in the mechanism underlying the GSTP1 induction of lytic metabolism after the GAPDH step in glycolysis to impair GAPDH activity. While our isotopic tracing data clearly indicate ATP generation, nucleotide and fatty acid metabolism, and that GSTP1 inhibition leads to an impairment in the ATP-gener- oncogenic signaling pathways. We show that GSTP1 inhibi- ating steps of glycolysis downstream of GAPDH, we do not yet tors show selective killing of TNBC cells over non-TNBC or understand the mechanism through which this occurs. GAPDH non-transformed mammary epithelial cells and that long- activity is dependent on a highly reactive catalytic cysteine that term GSTP1 inhibition in mice does not cause overt toxicity coordinates the interconversion between glyceraldehyde-3- or weight loss. Taking our findings together, we show that phosphate into 1,3-bisphosphoglycerate and is particularly sen- GSTP1 inhibitors may potentially be novel therapeutic sitive to oxidation by agents such as hydrogen peroxide or other agents to specifically target TNBCs. oxidants that can inhibit GAPDH activity (Hildebrandt et al., 2015). Reports have also shown that GAPDH can even be in- EXPERIMENTAL PROCEDURES hibited by its reactive 1,3-bisphosphoglycerate product on a hy- perreactive and functional lysine to inhibit its activity (Moellering Chemicals and Cravatt, 2013). It would be of future interest to investigate The AMPK inhibitor dorsomorphin dihydrochloride and the mTOR inhibitor whether GSTP1 interacts with GAPDH to protect hyperreactive Torin 1 were obtained from Tocris. LAS17 was synthesized as described pre- sites from being adducted and inhibited. GSTP1 has also been viously (Crawford and Weerapana, 2016). Menadione was obtained from Spectrum Chemical. shown to glutathionylate cysteines on proteins to post-transla- tionally regulate protein structure and function and protect Cell Culture proteins from degradation from sulfhydryl overoxidation or pro- The 231MFP cells were obtained from Prof. Benjamin Cravatt and were gener- teolysis (Grek et al., 2013). While we were not able to detect ated from explanted tumor xenografts of MDA-MB-231 cells. MCF7, MCF10A, GAPDH glutathionylation by GSTP1 and the activation of T47D, ZR751, MDA-MB-361, HCC1143, HCC38, HCC70, MDA-MB-468, and

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HEK293T cells were obtained from the American Type Culture Collection. Lactic Acid Secretion HEK293T cells were cultured in DMEM containing 10% (v/v) fetal bovine serum Lactic acid secretion from L-15 medium was measured by collecting medium  (FBS) and maintained at 37 C with 5% CO2. 231MFP, MDA-MB-361, and and performing a colorimetric lactic acid assay using a kit purchased from MDA-MB 468 cells were cultured in L15 medium containing 10% FBS and Abcam in accordance with the manufacturer’s protocol.  maintained at 37 C with 0% CO2. MCF10A cells were cultured in DMEM/ F12K medium containing 5% horse serum, 20 ng/ml epidermal growth factor, Glucose Consumption 100 ng/ml cholera toxin, 10 ng/ml insulin, and 500 ng/ml hydrocortisone, and Glucose consumption from RPMI medium was measured by collecting me-  maintained at 37 C with 5% CO2. MCF7, T47D, ZR751, HCC1143, HCC38, dium and performing a colorimetric glucose assay using a kit purchased and HCC70 cells were cultured in RPMI medium containing 10% FBS and from Abcam following the manufacturer’s protocol.  maintained at 37 C with 5% CO2. Western Blotting Expression by qPCR E-cadherin antibody was obtained from BD Biosciences. Vimentin antibody qPCR was performed using the manufacturer’s protocol for Fisher Maxima was obtained from Sigma. Antibodies to cyclophilin, GSTP1, b-actin, phos- SYBR Green with 10 mM primer concentrations or for Bio-Rad SsoAdvanced pho-AMPK a (Thr172), AMPK a, phospho-ACC (Ser79), ACC, phospho-S6, Universal Probes Supermix. Primer sequences for Fisher Maxima SYBR Green total S6, phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were obtained were derived from Primer Bank. Primer sequences for Bio-Rad SsoAdvanced from Cell Signaling Technology. FLAG antibody was obtained from Cayman Universal Probes Supermix were designed with Primer 3 Plus. Chemicals. Cells were lysed in lysis buffer (CST) containing both protease and phospha- Constructing Knockdown Cell Lines tase inhibitors. Proteins were resolved by electrophoresis on 4%–15% We used two independent short-hairpin oligonucleotides to knock down the Tris-glycine precast Mini-PROTEAN TGX gel (Bio-Rad) and transferred to expression of GSTP1 and one short-hairpin oligonucleotide to knock down nitrocellulose membranes using the iBlot system (Invitrogen). Blots were the expression of CDH1 using previously described methods (Benjamin blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20 et al., 2013). For generation of stable shRNA lines, lentiviral plasmids in the (TBST) solution for 1 hr at room temperature, washed in TBST, and probed pLKO.1 backbone containing shRNA (Sigma) against human GSTP1 were with primary antibody diluted in recommended diluent per manufacturer over- transfected into HEK293T cells using Fugene (Roche). Lentivirus was collected night at 4C. Following washes with TBST, the blots were incubated in the dark from filtered cultured medium and used to infect the target cancer cell line with with an IR-linked secondary antibody at room temperature for 1 hr. Blots were Polybrene. Target cells were selected over 3 days with 1 mg/ml puromycin. The visualized using an Odyssey Li-Cor scanner after additional washes. short-hairpin sequences used for generation of the GSTP1 knockdown lines were: shGSTP1-1, CCGGCGCTGACTACAACCTGCTGGACTCGAGTCCAGC Anti-FLAG Pulldown Studies AGGTTGTAGTCAGCGTTTTTG; shGSTP1-2, CCGGCCTCACCCTGTACCAG Pulldown studies were performed used Anti-FLAG M2 magnetic beads TCCAACTCGAGTTGGACTGGTACAGGGTGAGGTTTTTG. (Sigma) according to the manufacturer’s protocol. FLAG-tagged GSTP1-over- The short-hairpin sequence used for generation of the CDH1 knockdown expressing and GFP-overexpressing control cells were lysed in lysis buffer line was CCGGAGAAGGGTCTGTTCACGTATTCTCGAGAATACGTGAACAGA (CST), and 500 mg of lysate was incubated with 32 ml of anti-FLAG magnetic CCCTTCTTTTTTG. beads for 2 hr at 4C. Beads were collected and washed three times with The control shRNA was targeted against GFP with the target sequence Tris-buffered saline before elution with 4% SDS (w/v) in 120 mM Tris-HCl. GCAAGCTGACCCTGAAGTTCAT. Knockdown was confirmed by qPCR or Samples were heated to 95C for 3 min. Eluent was subsequently prepared western blotting. for proteomic profiling with a shotgun proteomic analysis protocol as described below. Constructing FLAG-Tagged GSTP1 Cells GSTP1 cDNA (Dharmacon) was subcloned into the pENTR4-FLAG vector Shotgun Proteomic Profiling of Anti-FLAG Pulldown (Addgene). This entry vector was recombined via an attL-attR (LR) reaction Pulldown products were precipitated in 20% trichloroacetic acid at À80C into a pLenti CMV puro DEST vector (Addgene). For generation of the FLAG- overnight and centrifuged at 10,000 3 g at 4C for 10 min to pellet protein. Pel- tagged GSTP1 line, the lentiviral plasmid containing FLAG-GSTP1 was trans- leted proteins were washed three times with 8 M urea in PBS. After solubiliza- fected into HEK293T cells using Fugene (Roche). Lentivirus was collected from tion, 30 ml of 0.2% ProteaseMAX Surfactant (Promega) was added and the filtered cultured medium and used to infect the target cancer cell line with resulting mixture was vortexed followed by the addition of 40 ml of 100 mM Polybrene. Target cells were selected over 3 days with 1 mg/ml puromycin. ammonium bicarbonate and 10 mM tris(2-carboxyethyl)phosphine (TCEP). Af- ter 30 min, 12.5 mM iodoacetamide was added and allowed to react for 30 min Cellular Phenotype Studies in the dark before adding 120 ml of PBS and 1.2 ml of 1% ProteaseMAX Surfac- Cell proliferation, serum-free cell survival, and migration assays were per- tant. The protein solution was vortexed, and 0.5 mg/ml sequencing-grade formed in a manner similar to previously described assays (Benjamin et al., trypsin (Promega) was added and allowed to react overnight at 37C. The pep- 2013, 2015). More details are provided in Supplemental Experimental tide solution was then centrifuged at 10,000 3 g before the supernatant was Procedures. subsequently analyzed by LC-MS/MS.

Tumor Xenograft Studies GAPDH Activity Assay Human tumor xenografts were established by transplanting cancer cells ectop- GAPDH activity was measured using a colorimetric kit purchased from ically into the flank of C.B17 severe combined immunodeficiency (SCID) mice BioVision and performed according to the manufacturer’s protocol. Active hu- (Taconic Farms) as previously described (Nomura et al., 2010). In brief, cells man GSTP1 and active human GAPDH full-length proteins were purchased were washed twice with PBS, trypsinized, and harvested in serum-containing from Abcam. Proteins were co-incubated at 37C for 1 hr before GAPDH ac- medium. Harvested cells were washed twice with serum-free medium and tivity was measured using the kit. resuspended at a concentration of 2.0 3 104 cells/ml, and 100 ml was injected. Tumors were measured every 2 days with calipers. Animal experiments were Oxidative Stress conducted in accordance with the guidelines of the Institutional Animal Care Oxidative stress was measured using CellROX Green Reagent (Invitrogen) and Use Committees of the University of California, Berkeley. according to the manufacturer’s protocol.

Metabolomic Profiling of Cancer Cells Cysteine and Lysine Reactivity Profiling Metabolomic analyses were conducted using previously described methods Cysteine and lysine reactivity profiling was performed based on an adapted (Benjamin et al., 2013, 2014, 2015; Long et al., 2011; Mulvihill et al., 2014). method that has been previously described (Medina-Cleghorn et al., 2015; More details are provided in Supplemental Experimental Procedures. Weerapana et al., 2010). Tryptic digests for proteomic profiling were analyzed

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using a Thermo LTQ-XL and quantified by spectral counting using previously the balance of structural and signaling lipids to fuel cancer pathogenicity. Proc. described methods (Nomura et al., 2010). More details are provided in Supple- Natl. Acad. Sci. USA 110, 14912–14917. mental Experimental Procedures. Benjamin, D.I., Louie, S.M., Mulvihill, M.M., Kohnz, R.A., Li, D.S., Chan, L.G., Sorrentino, A., Bandyopadhyay, S., Cozzo, A., Ohiri, A., et al. (2014). Inositol GSTP1 Activity Assay phosphate recycling regulates glycolytic and lipid metabolism that drives The IC50 of LAS17 was determined using a GSTP1 activity assay. 1-Chloro- cancer aggressiveness. ACS Chem. Biol. 9, 1340–1350. 2,4-dinitrobenzene (CDNB) was incubated with 200 mM reduced glutathione Benjamin, D.I., Li, D.S., Lowe, W., Heuer, T., Kemble, G., and Nomura, D.K. and 1 mg of active GSTP1 protein with 100, 10, 1, 0.1, 0.01, 0.001, and 0 mM (2015). Diacylglycerol metabolism and signaling is a driving force underlying LAS17. GSTP1 catalyzes the conjugation of glutathione to CDNB, generating FASN inhibitor sensitivity in cancer cells. ACS Chem. Biol. 10, 1616–1623. the reaction product glutathione-DNB conjugate, which absorbs at 340 nm. The rate of increase in the absorption of the product is proportional to Cantor, J.R., and Sabatini, D.M. (2012). Cancer cell metabolism: one hallmark, GSTP1 activity and was used to measure GSTP1 activity. many faces. Cancer Discov. 2, 881–898. Carracedo, A., Cantley, L.C., and Pandolfi, P.P. (2013). Cancer metabolism: In Vitro Labeling of GSTP1 with LAS17 fatty acid oxidation in the limelight. Nat. Rev. Cancer 13, 227–232. 50 mg of protein was incubated with 5 mM LAS17 for 30 min at room temperature. Crawford, L.A., and Weerapana, E. (2016). A tyrosine-reactive irreversible in- Following probe labeling, 25 mM rhodamine-azide, 1 mM TCEP, 100 mM tris(ben- hibitor for glutathione S-transferase Pi (GSTP1). Mol Biosyst. http://dx.doi. zyltriazoylmethyl)amine, and 1 mM Cu(II)SO4 were added and incubated for 1 hr org/10.1039/C6MB00250A.  at room temperature. Laemmli sample buffer was added, heated to 95 Cfor Dawson, S.J., Provenzano, E., and Caldas, C. (2009). Triple negative breast 5 min, and cooled to room temperature. Samples were loaded onto an SDS- cancers: clinical and prognostic implications. Eur. J. Cancer 45 (Suppl 1 ), PAGE gel and imaged by in-gel fluorescence using a Typhoon flatbed scanner. 27–40. Dietze, E.C., Sistrunk, C., Miranda-Carboni, G., O’Regan, R., and Seewaldt, SUPPLEMENTAL INFORMATION V.L. (2015). Triple-negative breast cancer in African-American women: dispar- ities versus biology. Nat. Rev. Cancer 15, 248–254. Supplemental Information includes Supplemental Experimental Procedures, Dowling, R.J.O., Zakikhani, M., Fantus, I.G., Pollak, M., and Sonenberg, N. five figures, and three tables and can be found with this article online at (2007). Metformin inhibits mammalian target of rapamycin-dependent transla- http://dx.doi.org/10.1016/j.chembiol.2016.03.017. tion initiation in breast cancer cells. Cancer Res. 67, 10804–10812. Gowda, H., Ivanisevic, J., Johnson, C.H., Kurczy, M.E., Benton, H.P., Rinehart, AUTHOR CONTRIBUTIONS D., Nguyen, T., Ray, J., Kuehl, J., Arevalo, B., et al. (2014). Interactive XCMS Online: simplifying advanced metabolomic data processing and subsequent D.K.N. and S.M.L. conceived the project, designed and performed experi- statistical analyses. Anal. Chem. 86, 6931–6939. ments, analyzed and interpreted data, and wrote the paper. E.A.G. designed and performed experiments, and analyzed and interpreted data. L.A.C. per- Grek, C.L., Zhang, J., Manevich, Y., Townsend, D.M., and Tew, K.D. (2013). formed synthetic efforts and provided GSTP1 pure protein, provided reagents, Causes and consequences of cysteine S-glutathionylation. J. Biol. Chem. and designed and performed experiments. L.D. designed and performed ex- 288, 26497–26504. periments, and analyzed and interpreted data. R.C. performed data analysis Hildebrandt, T., Knuesting, J., Berndt, C., Morgan, B., and Scheibe, R. (2015). and provided data. T.R.H. and D.K.M. performed synthetic efforts for probes Cytosolic thiol switches regulating basic cellular functions: GAPDH as an infor- used in the study and performed experiments. A.G. performed data analysis mation hub? Biol. Chem. 396, 523–537. and provided data. E.W. designed and performed experiments, wrote the pa- Laborde, E. (2010). Glutathione as mediators of signaling path- per, and provided reagents. ways involved in cell proliferation and cell death. Cell Death Differ. 17, 1373– 1380. ACKNOWLEDGMENTS Long, J.Z., Cisar, J.S., Milliken, D., Niessen, S., Wang, C., Trauger, S.A., Siuzdak, G., and Cravatt, B.F. (2011). Metabolomics annotates ABHD3 as a We thank the members of the Nomura Research Group for critical reading of physiologic regulator of medium-chain phospholipids. Nat. Chem. Biol. 7, the manuscript. This work was supported by grants from the NIH 763–765. (R01CA172667 for D.K.N., S.L., E.G., L.D., and R01CA170447 for A.G.), Amer- ican Cancer Society Research Scholar Award (RSG14-242-01-TBE for D.K.N., Mahadevan, D., and Sutton, G.R. (2015). Ezatiostat hydrochloride for the treat- S.L., E.G., and L.D.), DOD Breakthroughs Award (CDMRP W81XWH-15-1- ment of myelodysplastic syndromes. Expert Opin. Investig. Drugs 24, 0050 for D.K.N., S.L., E.G., and L.D.), DOD Era of Hope Award (CDMRP 725–733. W81XWH-12-1-0272 for A.G.), the Searle Scholar Foundation (D.K.N., S.L., Martell, J., and Weerapana, E. (2014). Applications of copper-catalyzed click E.G., and L.D.), the National Science Foundation Graduate Research Fellow- chemistry in activity-based protein profiling. Molecules 19, 1378–1393. ship (S.L.), Damon Runyon-Rachleff Innovator Award (DRR-18-12 for E.W. Medina-Cleghorn, D., Bateman, L.A., Ford, B., Heslin, A., Fisher, K.J., Dalvie, and L.A.C.), the Smith Family Foundation (E.W. and L.A.C.), Boston College E.D., and Nomura, D.K. (2015). Mapping proteome-wide targets of environ- (E.W. and L.A.C.), and the Diabetes, Endocrinology and Metabolism training mental chemicals using reactivity-based chemoproteomic platforms. Chem. grant (T32 DK007418 for R.C.). Biol. 22, 1394–1405. Moellering, R.E., and Cravatt, B.F. (2013). Functional lysine modification Received: October 23, 2015 by an intrinsically reactive primary glycolytic metabolite. Science 341, Revised: March 18, 2016 549–553. Accepted: March 20, 2016 Published: May 12, 2016 Mulvihill, M.M., Benjamin, D.I., Ji, X., Le Scolan, E., Louie, S.M., Shieh, A., Green, M., Narasimhalu, T., Morris, P.J., Luo, K., et al. (2014). 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Cell Chemical Biology 23, 1–12, May 19, 2016 11 Please cite this article in press as: Louie et al., GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.03.017

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