CYP1A1 Regulates Breast Cancer Proliferation and Survival

Published OnlineFirst April 10, 2013; DOI: 10.1158/1541-7786.MCR-12-0675

Molecular Cancer Signal Transduction Research

Mariangellys Rodriguez1 and David A. Potter1

Abstract Cytochrome P450-1A1 (CYP1A1) is an extrahepatic phase I metabolizing enzyme whose expression is suppressed under physiologic conditions but can be induced by substrates via the aryl hydrocarbon receptor (AhR). Recent studies have shown that the majority of breast cancer tumors constitutively express CYP1A1. These ﬁndings led us to test the hypothesis that CYP1A1 promotes breast cancer progression by evaluating the effects of CYP1A1 knockdown on the proliferation and survival of the MCF7 and MDA-MB-231 lines. Independently of estrogen receptor status, CYP1A1 knockdown decreased colony formation, decreased cell

proliferation, blocked the cell cycle at G0-G1 associated with reduction of cyclin D1, and increased apoptosis associated with reduction of survivin. CYP1A1 knockdown markedly increased phosphorylation of AMP- activated protein kinase (AMPK) and decreased phosphorylation of AKT, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and 70-kDa ribosomal protein S6 kinase (P70S6K). AMPK inhibition by compound C partially abrogated the proapoptotic effects of CYP1A1 knockdown, suggesting that effects of CYP1A1 knockdown are mediated in part through AMPK signaling. Consistent with CYP1A1 knockdown, pharmacologic reduction of CYP1A1 levels by the phytopolyphenol carnosol also correlated with impaired proliferation and induced AMPK phosphorylation. These results indicate that reduction of basal CYP1A1 expression is critical for inhibition of proliferation, which is not affected by a-naphthoﬂavone-mediated inhibition of CYP1A1 activity nor modulated by AhR silencing. This study supports the notion that CYP1A1 promotes breast cancer proliferation and survival, at least in part, through suppression of AMPK signaling and that reduction of CYP1A1 levels is a potential strategy for breast cancer therapeutics. Mol Cancer Res; 11(7); 780–92. 2013 AACR.

Introduction confirmation of protein expression (6). However, little is Cytochrome P450 1A1 (CYP1A1) is an extrahepatic known about the roles of CYP1A1 in cancer biology in the phase I enzyme that metabolizes endogenous and xenobiotic absence of xenobiotic exposure. substrates. Prior studies implicating CYP1A1 in cancer have Under physiologic conditions, CYP1A1 expression is dealt primarily with the metabolic activation of procarcino- suppressed but it is induced in the presence of substrates gens including polycyclic aromatic hydrocarbons and estra- via the transcriptional regulation of the aryl hydrocarbon diol (1–4). Other studies have focused on associations receptor (AhR; ref. 4). In recent years, studies have shown that breast tumors constitutively express CYP1A1 (7, 8). between CYP1A1 single-nucleotide polymorphisms (SNP) fi and increased cancer incidence. For example, the A2455G Murray and colleagues pro led the expression levels of 21 CYPs in 170 breast tumors from patients who had not allele has been implicated with increased breast cancer risk in fi Caucasian populations (4, 5). Nonetheless, lack of associa- received prior adjuvant treatment. This pro ling revealed tions between SNPs and cancer has been reported, and these that CYP1A1 was expressed in about 90% of breast tumors. Even so, the degree of CYP1A1 expression varied among discrepancies have been attributed to ethnic variations a between populations, variation in sample size, and lack of tumors and did not correlate with estrogen receptor (ER)- levels, tumor grade, or clinical outcome (7). Vinothini and colleagues reported on the expression levels of various Authors' Affiliation: 1Division of Hematology, Oncology, and Transplan- xenobiotic-metabolizing enzymes, including CYP1A1, in tation, Department of Medicine, The Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 60 breast tumors from newly diagnosed patients who had not received prior adjuvant therapy. Their study showed that Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). CYP1A1 levels were: (i) elevated in tumors compared with adjacent breast tissue, (ii) higher in premenopausal com- Corresponding Author: David A. Potter, Division of Hematology, Oncol- ogy and Transplantation, Department of Medicine, University of Minnesota, pared with postmenopausal patients, and (iii) positively 420 Delaware St. SE, MMC 480, Minneapolis, MN 55455. Phone: 612-625- correlated with tumor grade (8). These studies provide 8933; Fax: 612-625-6919; E-mail: [email protected] evidence that CYP1A1 is expressed to varying degrees in doi: 10.1158/1541-7786.MCR-12-0675 breast tumors and may be associated with cancer biology. 2013 American Association for Cancer Research. Moreover, because of its ubiquitous expression in breast

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CYP1A1 Regulates Breast Cancer Cell Proliferation

cancer and its ability to metabolize xenobiotics, interest phosphatase inhibitors are from Sigma and Roche. Human has been shown to exploit CYP1A1 activity for breast fibronectin (#356008) and the apoptosis detection kit are cancer therapeutics (9–12). Nonetheless, little is known from BD Pharmingen. Anti-phospho-p70S6 kinase about the activities of CYP1A1 that may participate in (Thr421/Ser424) is from Millipore. Antibodies raised breast cancer progression. Therefore, better understanding against total and phosphorylated forms of the following the function of CYP1A1 in breast cancer will assist in the proteins were obtained from Cell Signaling Technology: development of targeted therapy and improve treatment mTOR (p-Ser2448/clone D9C2), ERK1/2 (p-Thr202/ strategies. Tyr204), Akt (total clone 40D4 and p-Ser473), LKB1 The purpose of this study was to determine the biologic (total), AMPK (total clone F6 and p-Thr172/clone functions and roles in signal transduction of CYP1A1 in 40H9), P70S6K (total). Anti-CDK4 is from Santa Cruz breast cancer cells. Toward this goal, we used siRNA to Biothecnology. Anti-survivin antibody is from R&D Sys- knock down CYP1A1 in the breast cancer lines MCF7 (ER- tems. Anti-CYP1A1 (ab3568) was obtained from Abcam. þ positive; ER ) and MDA-MB-231 (triple negative; ER/ Anti-GAPDH antibody is from Research Diagnostics/Fitz- PR/HER2). We determined that CYP1A1 knockdown gerald Industries. The ECL Western blotting detection decreases proliferation, decreases colony formation, blocks reagent was purchased from GE Healthcare/Biocompare. – the cell cycle at G0 G1 associated with reduction of cyclin D1, and increases apoptosis associated with reduction of Cell lines survivin. CYP1A1 silencing reduces proliferation and sur- The lines MDA-MB-231 and MCF7 were purchased vival, at least in part, through increased phosphorylation of from American Type Culture Collection and grown at 37C AMP-activated protein kinase (AMPK) and reduced phos- under 5% CO2. Unless otherwise indicated, cells were phorylation of AKT, extracellular signal-regulated kinases grown and maintained in complete medium (MCF7: MEM, (ERK)-1and2,and70kDaribosomalproteinS6kinase 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin, (P70S6K). Consistent with CYP1A1siRNA results, phar- and 1% HEPES; MDA-MB-231: MEM, 10% FBS, 1% macologic reduction of CYP1A1 levels by carnosol also L-glutamine, 1% penicillin/streptomycin, 1% sodium pyru- impairs proliferation and induces AMPK phosphoryla- vate, and 2 mg/mL insulin). Because antibiotics reduce tion. Together, these results implicate CYP1A1 in cell transfection efficiency, for all siRNA transfection experi- proliferation and survival pathways. In addition, modu- ments cells were passaged once in antibiotic-free medium lation studies of CYP1A1 activity via 2,3,7,8-tethachlor- (AFM) before plating for the experiments and maintained in odibenzo-p-dioxin (TCDD, inducer) and a-naphthofla- this medium throughout the duration of the experiment. vone (inhibitor) indicate that reduction of CYP1A1 MCF7: 10% charcoal/dextran-stripped serum, phenol red- expression levels is critical for these biologic effects and free MEM, 1% HEPES, and 1% L-glutamine; MDA-MB- cannot be substituted by modulation of its activity alone. 231: 10% FBS, phenol red- free MEM, 1% sodium pyru- The evidence presented here suggests that CYP1A1 silenc- vate, 1% L-glutamine, and 2 mg/mL insulin). ing is a potential therapeutic strategy for breast cancer independent of ER status. siRNA transfection To knock down CYP1A1 or AhR levels, cells were Materials and Methods transfected with a pool of siRNA designed by Dharmacon Chemicals and reagents (sequences below). Cells were seeded at 30% confluence on Minimum essential medium (MEM) and cell culture 6-well plate and incubated overnight. Before transfection, supplements are from GIBCO/Invitrogen/Life Technolo- cells were washed twice with phenol red-free MEM and 0.8 gies. Charcoal/dextran-stripped serum and FBS from mL of OptiMEM was added to each well. A transfection HyClone. ON-TARGETplus CYP1A1- and NTsiRNA solution of 60 nmol/L siRNA suspended in 0.3% oligofec- pools (sequences below) are from Thermo Scientific Dhar- tamine/OptiMEM was gently added to the cells (200 mL/ macon. Oligofectamine, Opti-MEM, SuperScript III First- well). After overnight incubation, AFM medium was added Strand synthesis system, and SYBR GreenER qPCR Super- to obtain 10% serum medium. For transfection quality Mixes for iCycler are from Invitrogen/Life Technology. All purposes, a separate well was transfected with siGLO green primers were purchased from Integrated DNA Technolo- transfection indicator # D-001630-01. Sequences: human gies. The QiaShredder columns, RNeasy Mini Kit, Quan- CYP1A1siRNA ON-TARGETplus SMARTpool #L- tiTect Reverse Transcription Kit, and RNase-A are from 004790-00-0005: UCGACAAGGUGUUAAGUGA, AAA- Qiagen. The Wright–Giemsa was purchased from Thermo UGCAGCUGCGCUCUUA, CUACAGGUAUGUGGUG- Fisher Scientific. Carnosol was purchased from Cayman GUA, GAACUGCUUAGCCUAGUCA. ON-TARGET- Chemical and dissolved in dimethyl sulfoxide (DMSO) to plus Human AhRsiRNA SMART pool # L-004990-00- a final concentration of 40 mmol/L. Anti-actin antibody 0005: GCAAGUUAAUGGCAUGUUU, GAACUCAAGC- (HHF-35), MG-132, and compound C are from Calbio- UGUAUGGUA, GCACGAGAGGCUCAGGUUA, GCAA- chem/EMD Millipore. The tetrazolium salt, propidium CAAGAUGAGUCUAUU. Nontargeting siRNA ON-TAR- iodide, NP-40, cell culture grade DMSO HYBRI-MAX, GETplus #1 D-001810-10: UGGUUUACAUGUCGAC- ethoxyresorufin, a-naphthoflavone, and sodium citrate are UAA, UGGUUUACAUGUUGUGUGA, UGGUUUACA- from Sigma. TCDD is from AccuStandard. Protease and UGUUUUCUGA, UGGUUUACAUGUUUUCCUA.

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Quantitative RT-PCR maliemide, and 40 mmol/L MG-132) and processed accord- To evaluate CYP1A1 mRNA knockdown efficiency, total ing to standard Western blot analysis. GAPDH was used as RNA was collected in lysis buffer (RLT) and b-mercaptoetha- loading control and relative protein amounts were quantified nol, passed through a QiaShredder column, and purified by densitometry of X-ray film exposures using an Alpha- using the RNeasy Mini Kit according to the manufacturer's Innotec densitometer. protocol. First-strand cDNA was made using the QuantiTect Reverse Transcription Kit according to the manufacturer's MTT cell proliferation assay To assess viability, cells were incubated with 0.1 mg/mL of protocol. The quantitative reverse transcriptase PCR (RT- PCR) was prepared using SYBR GreenER qPCR SuperMixes tetrazolium MTT for 2 hours at 37 C under 5% CO2. The for iCycler and conducted at following temperatures: 50C supernatant was replaced with DMSO. Proliferating cells for 2 minutes, 95C for 8.5 minutes, 40 cycles of 95C for 15 reduce the yellow tetrazolium into DMSO-soluble purple C seconds, and 60 C for 1 minute. The comparative t value formazan, which was read in a BioTek plate reader at an method was used for data analysis (13). Glyceraldehyde-3- absorbance of 540 nm. phosphate dehydrogenase (GAPDH) was used for normal- ization. Primer sequences: CYP1A1 forward: 50-GCT GAC Anchorage-dependent clonogenic assay 0 0 To assess cell survival, 200 transfected cells were seeded TTC ATC CCT ATT CTT CG-3 , reverse: 5 :TTT TGT fi AGT GCT CCT TGA CCA TCT-30; GAPDH forward: 50- onto bronectin-coated 6-well plates and incubated for 14 0 0 days until visible colonies were observed. Colonies were fixed GGG AAG GTG AAG GTC GGA GT-3 , reverse: 5 -GAG – TTA AAA GCA GCC CTG GTG A-30. and stained with Wright Giemsa and total colony number was counted. Plate preparation: 1 mL fibronectin (10 mg/mL Semiquantitative RT-PCR in PBS) incubated overnight at 4 C, then blocked with 3% To evaluate siRNA specificity for CYP1A1 compared with bovine serum albumin in PBS for 1 hour at room temper- other CYPs, total mRNA was collected and purified as ature, and rinsed with PBS before use. described above. The cDNA was prepared using the First Cell-cycle assay Strand Synthesis Kit according to the manufacturer's pro- To evaluate the cell-cycle distribution, cellular DNA tocol (Invitrogen). Semiquantitative RT-PCR analysis of content was measured by propidium iodide staining. CYP1A1, CYP1A2, and CYP1B1 was conducted using Because MCF7 cells form clumps in the presence of ethanol, SuperScript III Reverse Transcriptase (Invitrogen) and Taq 2 different methods were used for fixing and staining. MDA- Master Mix Kit according to manufacturer's protocol (Qia- MB-231 method: cells were collected by trypsinization, gen) in a PX2 Thermal Cycler (Thermo; 94 C for 2 minutes, washed with ice-cold PBS, resuspended in 200 mL of ice- 50 cycles: 94 C/30 seconds, 55 C/30 seconds, 72 C/45 cold 70% ethanol, incubated overnight at 4 C, centrifuged, seconds, 72 C for 10 minutes, and 4 C for 5 minutes). The washed with PBS, and finally resuspended in 0.5 mL of PCR products were resolved in a 1.5% agarose gel and Nicoletti buffer (50 mg/mL propidium iodide, 0.1% sodium GAPDH was used to normalize samples. Primer sequences 0 0 citrate, 0.1% Triton X-100, and 1 mg/mL RNase-A in PBS). [Gene (amplicon size), F ¼ forward primer (5 -3 ), R ¼ 0 0 MCF7 method: cells were washed twice with PBS over ice, reverse primer (5 -3 )]: GAPDH (609 bp), F: CAC AGT scraped off the plate with 1 mL of PBS, collected and CCA TGC CAT CAC TGC, R: GGT CTA CAT GGC CYP1A1 centrifuged for 5 minutes, resuspended in 0.5 mL of staining AAC TGT GAG; (294 bp), F: CTT CAT CCT solution (0.1% sodium citrate, 50 mg/mL propidium iodide, GGA GAC CTT CC, R: AAG ACC TCC CAG CGG GCA 0.01% NP-40, and 1 mg/mL Rnase-A in water), and A; CYP1A2 (371 bp), F: CAA TCA GGT GGT GGT GTC CYP1B1 incubated overnight at 4 C. Following the respective stain- AG, R: GCT CCT GGA CTG TTT TCT GC; ing methods, propidium iodide content was profiled using a (350 bp), F: TCA ACC GCA ACT TCA GCA ACT T, R: FACScalibur flow cytometer (BD Bioscience). Cell-cycle ATA GGG CAG GTT GGG CTG GTC A. distribution was determined using the Watson Pragmatic algorithm in the FlowJo software. Western blot analysis To evaluate protein levels, cell extracts were prepared Apoptosis assay using radioimmunoprecipitation assay extraction buffer (50 To evaluate cell death, cells were collected by trypsiniza- mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% NP-40, tion, washed with PBS, and stained with propidium iodide 0.5% sodium deoxycholate, 0.1% SDS, 2 mmol/L EDTA, and Annexin-V/Fluorescein isothiocyanate (FITC) for 15 pH 8, 2 mmol/L EGTA pH 8, 1 mmol/L DL-dithiothreitol minutes at room temperature. Stained cells were evaluated fl m (DTT), 1 mmol/L phenylmethylsulfonyl uoride, 5 mol/L using a FACScalibur flow cytometer (BD Bioscience) and phenylarsine oxide (PAO), 25 mmol/L NaF, 2 mmol/L the results were analyzed with FlowJo software (Tree Star). Na3VO4, 100 mmol/L leupeptin, 2 mmol/L pepstatin, 2.8 mmol/L Tosyl-L-phenylalanyl-chloromethane (TPCK), Ethoxyresorufin-O-deethylase assay 2.7 mmol/L Tosyl-L-lysyl-chloromethane hydrochloride To assess CYP1A1 activity, an ethoxyresorufin-O-deethy- (TLCK), 4.17 mmol/L 4-(2-aminoethyl)-benzenesulfonyl lase (EROD) assay adapted from refs. (14, 15) was used. Cells fluoride hydrochloride (AEBSF), 4.17 mmol/L chymostatin, were plated onto 6-well plates, treated as desired, washed 2 2 mmol/L aprotinin, 2 mmol/L antipain, 5 mmol/L N-ethyl- times with 1 PBS, then incubated at 37C for 60 minutes

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CYP1A1 Regulates Breast Cancer Cell Proliferation

with 2 mmol/L ethoxyresorufin in buffer (50 mmol/L Tris, metastatic hormone therapy refractory and triple-negative fi 0.1 mol/L NaCl, 6.25 mmol/L MgCl2, pH 7.8, warmed to breast cancer. To achieve the goal of de ning the role of 37C). A 100 mL aliquot of the assay supernatant was CYP1A1 in breast cancer growth, pooled siRNA was used to transferred to a black-wall clear bottom 96-well plate, and knock down CYP1A1 mRNA and protein. To validate the fluorescence was read in a BioTek fluorescence plate reader at knockdown efficiency, CYP1A1 mRNA and protein levels excitation l(530 nm)/emission l (590 nm). When necessary, were measured by quantitative RT-PCR and by Western blot fluorescent counts were normalized to total cell number. analysis, respectively. Significant reductions of CYP1A1 mRNA and protein levels were observed between 48 and Statistical analysis 120 hours. CYP1A1 mRNA levels were reduced by 53% (P ¼ All experiments were reproducible and carried out at least 0.002) in the MCF7 line and by 70% (P ¼ 6 10 6)in in triplicate. Statistical significance (P values) was calculated MDA-MB-231 line (Fig. 1A, top). CYP1A1 protein levels using 2-tailed Student t test. Equality of variance was were reduced by 64% (P ¼ 0.02) in the MCF7 line and by determined by F test. 52% (P ¼ 0.04) in the MDA-MB-231 line (Fig. 1B). Because CYP1A1 shares 73% identity with CYP1A2 and 41% iden- Results tity with CYP1B1, we also evaluated by semiquantitative RT- CYP1A1 knockdown impairs cell proliferation and PCR whether CYP1A1siRNA alters the mRNA levels of these survival CYP1 family members. No change was observed in the levels The purpose of this study was to understand the biologic of these CYPs indicating that the biologic effects observed are roles of CYP1A1 in MCF7 and MDA-MB-231 breast cancer fi þ speci cally due to reduction of CYP1A1 (Fig. 1A, bottom). lines. We chose ER and triple-negative breast cancer because To investigate the functional roles of CYP1A1 in breast there is a significant unmet need for new treatments for cancer, we determined the effects of CYP1A1 knockdown

A B MCF7 MDA-MB-231 NTsiRNA CYP1A1siRNA 0.6 1.8 1.20 0.4 1.2 0.80 * * * CYP1A1 CYP1A1 0.2 0.6 0.40 ** CYP1A1 mRNA CYP1A1 Relative to GAPDH Relative Relative to GAPDH Relative Figure 1. CYP1A1 knockdown to GAPDH Relative 0.00 0.0 0.0 MCF7 MDA-MB-231 impairs breast cancer cell NTsiRNA 1A1siRNA NTsiRNA 1A1siRNA proliferation and survival. The Primer: GAPDH 1A1 1A2 1B1 efﬁciency of CYP1A1siRNA NTsi 1A1si NTsi 1A1si NTsiRNA knockdown in MCF7 and MDA-MB- CYP1A1 CYP1A1 231 cells was evaluated by 1A1siRNA GAPDH GAPDH

quantitative RT-PCR (A, top) MCF7 and by Western blot analysis (B). NTsiRNA To evaluate siRNA speciﬁcity semiquantitative RT-PCR was 1A1siRNA M-231 conducted with the indicated primers and the products were run in 1.5% agarose gel (representative samples C MCF7 MDA-MB-231 NTsiRNA CYP1A1siRNA shown in A, bottom). C, proliferation NTsiRNA CYP1A1siRNA 120 experiments for lines MCF7 and 120 * MDA-MB-231 after transfection with * CYP1A1siRNA for the indicated 80 80 ** times. D, clonogenicity was ** ** ** evaluated by transfection for

% Growth 40 % Growth 40 48 hours followed by seeding of 200 transfected cells onto 0 ﬁbronectin-coated 6-well plates. 0 48 h 72 h 96 h 48 h 72 h 96 h Visible colonies were stained and counted after 14 days of culture. D MCF7 MDA-MB-231 n ¼ P < P < ( 3; , 0.05; , 0.001) 300 300

200 200

100 100 * Colony number Colony Colony number Colony ** 0 0 NTsiRNA CYP1A1siRNA NTsiRNA CYP1A1siRNA

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on 2 "hallmarks of cancer:" uncontrolled cell proliferation proliferation, we investigated the impact of siRNA-mediated and loss of inhibition of cell death. First, we examined the knockdown of AhR on basal CYP1A1 expression and cell effects of CYP1A1 knockdown on the ability of MCF7 and proliferation. Although AhRsiRNA reduces AhR levels by at MDA-MB-231 cells to proliferate. After transfection for 48, least 70% (P < 0.05) in both lines, the levels of CYP1A1 72, and 96 hours, viable cells were measured by MTT assay. remain unaffected (Supplementary Fig. S1A). Therefore, CYP1A1siRNA reduces the proliferation of both lines. At 96 this system allows us to distinguish AhR-specific effects. As hours of transfection with CYP1A1siRNA, proliferation was assessed by MTT assay, the proliferation of these lines is not reduced by approximately 40% for the MCF7 and MDA- impaired by AhR knockdown (Supplementary Fig. S1B), MB-231 lines (Fig. 1C; P < 0.001). suggesting that the effects of CYP1A1 knockdown on cell To determine the effects of CYP1A1 knockdown on cell proliferation and survival (Fig. 1) may be independent of the survival,weconductedaclonogenicassaytocountthe AhR expression status of these lines. cells able to survive and proliferate to form visible colonies. MCF7 and MDA-MB-231 cells were transfected for 48 CYP1A1 knockdown blocks the cell cycle hours, seeded at low density onto fibronectin-coated The ability of cells to proliferate depends on the balance plates, and colonies were counted after 2 weeks of culture. between cell growth, division, and death. For this reason, we Compared with NTsiRNA control, CYP1A1siRNA investigated the effect of CYP1A1 knockdown on the ability inhibited colony formation of MCF7 line by 82% (P of MCF7 and MDA-MB-231 lines to progress through the ¼ 4 10 6) and of MDA-MB-231 line by 56% (P ¼ cell cycle. To achieve this goal, cells were transfected for 48 0.03; Fig. 1D). Together these results indicate that hours, permeabilized, stained with propidium iodide, and CYP1A1 silencing impairs proliferation and survival of cell-cycle distribution was analyzed by flow cytometry. The – breast cancer cells. G0 G1 populations of MCF7 and MDA-MB-231 lines The AhR localizes to the cytosol of MCF7 cells and increased by 37% (P ¼ 0.001) and 20% (P ¼ 0.003) translocates to the nucleus upon ligand activation. In con- respectively, consistent with a block in the cell cycle at – trast, MDA-MB-231 cells display primarily nuclear AhR G0 G1 (Fig. 2A). – expression. In both lines, nuclear AhR is responsible for the Because the cyclin D1/CDK4 complex regulates the G1 S inducible transcriptional regulation of CYP1A1. Given the transition of the cell cycle (16), we investigated the effects of important role of AhR in the regulation of CYP1A1, it is CYP1A1 knock down on these regulatory proteins. Cyclin P ¼ possible that the biologic functions of CYP1A1 may be D1 levels were reduced by 74% ( 0.001) in MCF7 line affected by or be dependent on the AhR status. To determine and by 36% (P ¼ 0.001) in MDA-MB-231 line (Fig. 2B). whether AhR is required for the roles of CYP1A1 on cell CDK4 levels were not significantly reduced in both lines,

A MCF7 MDA-MB-231 NTsiRNA CYP1A1siRNA NTsiRNA CYP1A1siRNA

600 600 1,500 1,500

400 400 1,000 1,000

200 200 500 500 Figure 2. CYP1A1 knockdown blocks the cell cycle. A, cell-cycle ﬂ 0 0 0 0 distribution was assessed by ow 0 200 400 600 800 1k 0 200 400 600 800 1k 0 200 400 600 800 1k 0 200 400 600 800 1k cytometry of MDA-MB-231 and

Cell number MCF7 cells transfected for 48 DNA Content hours and stained with propidium iodide. B, cyclin D1 and CDK4 MCF7 G –G SG–M MDA-MB-231 G0–G1 SG2–M 0 1 2 proteins were visualized by NTsiRNA 40.01 28.79 34.55 NTsiRNA 49.36 28.03 16.40 Western blot analysis. Fold CYP1A1siRNA 63.51 18.22 22.11 CYP1A1siRNA 62.01 18.60 12.65 − − changes are expressed relative to % Difference 37% −58% −56% % Difference 20% 51% 30% P CYP1A1siRNA knockdown (n ¼ 3; P 0.001* 0.011* 0.009* 0.003* 0.001* 0.002* P values shown; , statistical signiﬁcance). B MCF7 MDA-MB-231 MCF7 MDA231 NTsi 1A1si NTsi 1A1si Fold P Fold P change change Cyclin D1

Cyclin D1 0.26 0.001* 0.64 0.001* GAPDH CDK4 0.51 0.136 0.75 0.162 CDK4

GAPDH

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CYP1A1 Regulates Breast Cancer Cell Proliferation

although a trend toward reduction is observed. These results was associated with an 80% reduction of survivin (P ¼ suggest that CYP1A1 knockdown may block the cell cycle, at 0.003) in MCF7 line and a 56% reduction (P ¼ 0.04) in least in part, through downregulation of cyclin D1. MDA-MB-231 line (Fig. 3B).

CYP1A1 knockdown increases cell death CYP1A1 knockdown inhibits the ERK1/2 and AKT To further understand the antiproliferative effects of pathways CYP1A1 silencing, apoptosis was measured by ﬂow cyto- Because the MAP–ERK kinase (MEK)/ERK and phos- metry. In this assay, cells were transfected for 48 hours, phoinositide 3-kinase (PI3K)/AKT signaling pathways pro- stained with propidium iodide and Annexin-V/FITC, and mote the growth of breast cancer (19–22), we determined analyzed by ﬂow cytometry. CYP1A1 silencing of the MCF7 whether CYP1A1 knockdown inhibits ERK1/2 and/or AKT line correlated with a 50% increase in the early (bottom phosphorylation in these lines (Fig. 4). CYP1A1 knockdown right) and late (top right quadrant) apoptotic populations reduced phosphorylation of ERK in both lines, most notably (Fig. 3A; P < 0.05). CYP1A1 silencing of the MDA-MB-231 by 45% (P ¼ 0.001) in the MCF7 line (Fig. 4). CYP1A1- line correlated with a 50% increase in the late apoptotic knockdown resulted in reduction of AKT phosphorylation population (Fig. 3A; P ¼ 0.006). by 45% (P ¼ 0.02) in MCF7 line and by 65% in MDA-MB- Survivin is an antiapoptotic protein of the inhibitor of 231 line (P ¼ 0.004; Fig. 4). These reductions in phos- apoptosis family whose importance in breast cancer, includ- phorylation correlate with downstream inhibition of the ing MCF7 and MDA-MB-231 lines, has been established protein synthesis regulator P70S6K (23, 24). Phosphoryla- (17, 18). Therefore, to further understand the role of tion of P70S6K was reduced by 53% (P ¼ 0.001) in the CYP1A1 in apoptosis, we measured the effects of CYP1A1 MCF7 line and by 37% (P ¼ 0.03) in the MDA-MB-231 knockdown on the levels of survivin. Consistent with line (Fig. 4). These results implicate CYP1A1 upstream of increased apoptosis, we observed that CYP1A1 knockdown the ERK and PI3K/AKT pathways.

A MCF7 MDA-MB-231 NTsiRNA CYP1A1siRNA NTsiRNA CYP1A1siRNA PI

AnnexinV-FITC

MCF7 Early Late Necrotic Viable MDA-MB-231 Early Late Necrotic Viable NTsiRNA 12.40 7.68 32.73 47.23 NTsiRNA 3.71 1.47 2.58 92.23 CYP1A1siRNA 24.97 14.90 22.77 37.37 CYP1A1siRNA 5.47 3.07 2.92 88.53 % Difference 50% 48% −44% −26% % Difference 32% 52% 12% −4% P 0.043* 0.004* 0.154 0.067 P 0.142 0.006* 0.401 0.070

B MCF7 MDA-MB-231 MCF7 MDA231 NTsi 1A1si NTsi 1A1si Fold P Fold P Survivin change change Survivin 0.19 0.003* 0.44 0.041* GAPDH

Figure 3. CYP1A1 knockdown increases cell death. A, apoptosis was assessed by ﬂow cytometry of MDA-MB-231 or MCF7 cells transfected for 48 hours and stained with propidium iodide and Annexin V-FITC. B, survivin protein was visualized by Western blot analysis. Fold changes are expressed relative to CYP1A1siRNA knockdown (n ¼ 3; P values shown; , statistical signiﬁcance). Legend: Early, early apoptotic population in the bottom right quadrant; Late, late apoptotic population in the top right quadrant; Necrotic, necrotic population in the top left quadrant; Viable, alive population in the bottom left quadrant.

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7FCM 132-BM-ADM MCF7 MDA-MB-231 NTsi 1A1si NTsi 1A1si Fold P Fold P change change Phospho AKT p-AKT 0.55 0.02* 0.37 0.004* t-AKT 1.61 0.04* 1.27 0.41 Total AKT Fractional-p 0.39 0.01* 0.33 0.02* p-ERK1/2 0.55 0.001* 0.71 0.02* GAPDH Figure 4. CYP1A1 knockdown t-ERK1/2 1.22 0.18 1.01 0.93 inhibits ERK1/2 and AKT and Fractional-p 0.45 0.002* 0.71 0.05* Phospho ERK1/2 activates AMPK. The effects of p-AMPK 5.64 0.01* 2.42 0.02* CYP1A1siRNA on downstream signaling were evaluated by Total ERK1/2 t-AMPK 1.00 0.99 0.98 0.97 Western blot analysis of cells Fractional-p 5.62 0.003* 3.30 0.01* transfected for 72 to 120 hours. p-P70S6k 0.47 0.001* 0.63 0.03* GAPDH Fold changes are expressed t-P70S6K 0.96 0.88 0.84 0.37 relative to CYP1A1siRNA Phospho AMPK Fractional-p 0.57 0.02* 0.72 0.02* knockdown (n ¼ 3; P values shown; , statistical signiﬁcance). Total AMPK Fractional phosphorylation results are included (Fractional-p ¼ GAPDH phosphorylated/total protein).

Phospho P70S6K

Total P70S6K

GAPDH

CYP1A1 knockdown induces the AMPK pathway CYP1A1siRNA, followed by treatment for 24 hours with Although reduction of P70S6K phosphorylation caused either vehicle (DMSO) or the AMPK inhibitor compound C by CYP1A1 silencing could be related to loss of ERK1/2 and (6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl- PI3K/AKT signaling, this inhibition could also be due to pyyrazolo[1,5-a] pyrimidine; from now on abbreviated as activation of AMPK, a major regulator of cellular bioener- CC). The reverse order of treatment (i.e., 24 hours treatment getics and metabolic tumor suppressor (25). CYP1A1 with DMSO or CC followed by NT/CYP1A1siRNA trans- knockdown increases phosphorylation of AMPK 5.6-fold fection) was also conducted, leading to similar results. (P ¼ 0.01) in the MCF7 line and 2.4-fold (P ¼ 0.02) in Following treatment, apoptosis was measured by flow cyto- MDA-MB-231 line (Fig. 4). The activation of AMPK metry, and the percentage of overall cell death was calculated phosphorylation by CYP1A1 silencing in conjunction with as follows: SDead ¼ % early apoptotic þ% late apoptotic concomitant downregulation of ERK1/2 and AKT phos- þ% necrotic populations. It should be noted that because phorylation is consistent with known cross-talk with AMPK compound C induces cell death in MCF7 line (ref. 30; and (26–28) and potentially places CYP1A1 as a candidate confirmed in Fig. 5), the results are interpreted only within upstream regulator of these kinases. The levels of LKB1, a the context of treatment (i.e., DMSO or CC) as shown in the known regulator of AMPK (29), were determined by West- diagram on Fig. 5. Therefore, within the context of vehicle ern blot analysis in the MCF7 line. Although an initial treatment CYP1A1siRNA increases cell death [Fig. 5; marginal reduction of LKB1 by CYP1A1siRNA was SDead (NTsiþDMSO) ¼ 27% vs. SDead observed (48 hours transfection fold change ¼ 0.70, P ¼ (1A1siþDMSO) ¼ 44%; P ¼ 0.01)]. This proapoptotic 0.047), this reduction was not sustained at longer time effect of CYP1A1siRNA in the presence of vehicle is con- points (72–96 hours; 96 hours transfection fold change ¼ sistent with our results in Fig. 3 and confirms that these 1.01, P ¼ 0.98). This suggests that LKB1-dependent and effects are due to CYP1A1siRNA and not vehicle treatment. LKB1-independent mechanisms of AMPK activation may Within the context of CC treatment, 2 potential out- be involved. A possible model summarizing these mechan- comes could be expected depending on whether AMPK is isms is presented in Fig. 7. necessary for the proapoptotic effects of CYP1A1siRNA (Fig. 5, Diagram): (i) if AMPK is necessary, then inhibition AMPK inhibition partially abrogates CYP1A1siRNA- of AMPK would prevent CYP1A1siRNA-induced apoptosis mediated apoptosis and thus less cell death would be observed with "1A1si þ We hypothesized that if AMPK signaling is necessary for CC" treatment compared with "NTsi þ CC" treatment, (ii) the biologic effects of CYP1A1 knockdown, then blocking on the other hand, if AMPK in not necessary, then inhibition AMPK activation should abrogate these effects. To test this of AMPK would not abrogate the proapoptotic effects of hypothesis, MCF7 cells were transfected for 24 hours with CYP1A1siRNA but would instead add to the proapoptotic

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NTsiRNA + DMSO NTsiRNA + CC 1A1siRNA + DMSO 1A1siRNA + CC

siRNA → Treatment Σ Dead Early Late Necrotic Viable NTsi → DMSO 27.3 13.2 9.8 4.2 72.7 NTsi → Comp C 71.7** 3.1** 49.2** 19.4* 28.3** 1A1si → DMSO 43.7 5.5 20.1 18.2 55.6 1A1si → Comp C 46.4 6.7 25.5 14.3 53.6

Diagram of potential outcomes:

DMSO Basal cell death NTsiRNA CC AMPK Cell death induced by CC

DMSO Cell death induced by 1A1si 1A1siRNA CC AMPK Outcome 1: ↔ or ↓ cell death than 1A1si + DMSO

Outcome 2: ↑↑ cell death due to CC plus 1A1siRNA proapoptotic effects

Figure 5. AMPK inhibition partially abrogates CYP1A1siRNA-mediated apoptosis. To support the hypothesis that the proapoptotic effects of CYP1A1 siRNA require AMPK, MCF7 cells were transfected for 24 hours and then treated with 10 mmol/L of the AMPK inhibitor compound C for 24 hours. Cells were collected, stained with propidium iodide, and Annexin V-FITC, and analyzed by flow cytometry. A diagram of expected outcomes is included, outcome 1 was observed in our experiments. (n ¼ 3; , P < 0.01; , P < 0.001; comparison between DMSO and compound C treatment). effects of CYP1A1siRNA, thus resulting in an increase in cell cancer lines (31–33). Treatment with carnosol inhibits the death when treated with "1A1si þ CC" compared with proliferation of the MCF7 and MDA-MB-231 lines exhi- þ fi m "NTsi CC". Our results agree with the rst scenario, 72% biting IC50 values of approximately 40 mol/L for both lines total cell death is observed with "NTsi þ CC," whereas 46% (Fig 6A; P < 0.001). We carried out time-course experiments total cell death is observed in "1A1si þ CC" (Fig. 5; P ¼ to investigate early effects (i.e., 2–12 hours) of carnosol 0.003). This reduction of cell death observed for CYP1A1 treatment on CYP1A1 expression and determined that the knockdown cells treated with the AMPK inhibitor (CC) optimal time for CYP1A1 reduction is 8 hours (Fig. 6B; P < indicates that AMPK is necessary for the effects of CYP1A1- 0.05). Carnosol treatment reduces AhR levels in both lines siRNA and agrees with our findings showing that CYP1A1- by more than 50% (Fig. 6B; P < 0.05). In agreement with siRNA induces AMPK signaling (Fig. 4). Together, these CYP1A1siRNA results (Fig. 4), treatment of the MCF7 and results suggest that AMPK phosphorylation may be MDA-MB-231 lines with 40 mmol/L carnosol for 8 hours repressed by CYP1A1 and reduction of CYP1A1 levels results in the activation of AMPK (Fig. 6B; P < 0.01). This promotes AMPK phosphorylation. In this manner, AMPK suggests that CYP1A1 reduction, whether by siRNA or may be required for the effects of CYP1A1siRNA on cell carnosol, is associated with inhibition of proliferation medi- death (Fig. 7). ated, in part, through activation of AMPK signaling. Because carnosol inhibits both AhR and CYP1A1 levels, Carnosol impairs proliferation, in part, via reduction of we tested whether the antiproliferative effects of carnosol CYP1A1 and activation of AMPK were mediated through AhR-dependent mechanisms. As To further test whether CYP1A1 signals, in part, through previously shown, knockdown of AhR does not reduce basal AMPK, we sought a pharmacologic approach to reduce CYP1A1 levels (Supplementary Fig. S1A), suggesting that CYP1A1 levels (31). Carnosol inhibits the AhR, a transcrip- carnosol inhibits basal CYP1A1 through an AhR-indepen- tion factor that regulates the inducible and basal expression dent mechanism. Therefore, to better understand these of CYP1 family members including CYP1A1 (31). Carnosol mechanisms, we tested whether AhR is required for the has been shown to reduce basal CYP1A1 expression in therapeutic effects of carnosol. If so, we would expect AhR premalignant tongue and bronchial lines and in prostate knockdown to shift the dose–response curve of carnosol.

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A MCF7 MDA-MB-231 DMSO Carnosol DMSO Carnosol ** 100.0 100.0

75.0 ** 75.0 ** ** ** 50.0 50.0 Figure 6. The AhR inhibitor carnosol impairs proliferation, in part, via % Growth % Growth 25.0 ** 25.0 ** ** reduction of CYP1A1 and ** activation of AMPK. Carnosol 0.0 0.0 treatment was used as an 60403020100 100 60403020100 100 independent method to reduce μ μ Carnosol ( mol/L) Carnosol ( mol/L) CYP1A1 levels and evaluate downstream signaling. A, the IC50 B MCF7 MDA-MB-231 MCF7 MDA-MB-231 of carnosol was determined by DMSO + − + − Fold P Fold P MTT assay. B, cell extracts were Carnosol − + − + change change collected after treatment with 40 AhR mmol/L carnosol for 8 hours, the CYP1A1 0.72 0.03* 0.40 2.5E-4** indicated proteins were evaluated GAPDH AhR 0.34 0.03* 0.43 2.2E-4** by Western blot analysis. Fold p-AMPK 4.57 8.5E-5** 5.20 0.01* changes are expressed relative to CYP1A1 t-AMPK 0.78 0.10 0.77 0.56 CYP1A1siRNA knockdown (n ¼ 3; P < P < GAPDH , 0.05; , 0.001).

p-AMPK

Total AMPK

GAPDH

Treatment of AhR knockdown cells with carnosol does not sured the effects of inhibiting CYP1A10s activity on cell result in a shift of the dose–response curve (Supplementary proliferation. Cells were treated with 1 mmol/L of Fig. S1C), further supporting that carnosol's effects may be a-naphthoflavone, an inhibitor of CYP1A1 activity that independent of AhR. Therefore, together these results sug- does not affect CYP1A1 levels (Supplementary Fig. S2B and gest that carnosol's antiproliferative effects are primarily due data not shown). Inhibition of CYP1A1 activity by to CYP1A1 reduction, AMPK activation, and potentially a-naphthoflavone does not affect cell proliferation (Supple- other yet unidentified mechanisms. mentary Fig. S2C), supporting the hypothesis that reduction of CYP1A1 levels, but not its activity, is required for CYP1A1 targeting: expression versus activity impairment of proliferation and survival (Fig. 1). To this point, we have focused on the impact of CYP1A1 Second, the effects of inducing CYP1A1 activity on cell levels in the biology of breast cancer cells. Nonetheless, for proliferation were determined. Cells were treated with the therapeutic development purposes, we should distinguish CYP1A1 inducer TCDD, which results in increased between the expression levels and the enzymatic activity of CYP1A1 levels and activity. Treatment with 5 to CYP1A1. To address this issue, we tested whether inhibition 20 nmol/L TCDD induces CYP1A1 activity, but does and/or induction of CYP1A1 activity affects breast cancer not stimulate cell proliferation (10 nmol/L shown in cell proliferation. Supplementary Fig. S2D–S2E). It is noteworthy that the First, the effects of inhibiting CYP1A1 activity on cell induction of CYP1A1 activity is the greatest in the MCF7 proliferation were determined. To achieve this we first line, when compared with MDA-MB-231, which is likely evaluated whether CYP1A1siRNA affects the activity of attributable to differences in AhR status in these lines CYP1A1. To test this, cells were transfected with CYP1A1- (Supplementary Fig. S2D). These results suggest that siRNA for 48 hours, and TCDD-induced CYP1A1 activity breast cancer lines, MCF7 and MDA-MB-231, have an was measured by EROD assay. CYP1A1 knockdown optimal amount of CYP1A1 protein and further increas- reduces CYP1A1 EROD activity by 38% (P ¼ 0.04) in ing its level or activity may not enhance the proliferative MCF7 line, but it is unaffected (P ¼ 0.52) in MDA-MB- capability of these lines. 231 line (Supplementary Fig. S2A). These results led us to Together, our findings suggest that the development of hypothesize that, although reduction of CYP1A1 levels is therapeutic strategies to target CYP1A1 should consider necessary for impaired proliferation (Fig. 1), this effect on the expression levels of the protein and not just its proliferation may not be dependent on the inhibition of activity. Nonetheless, it remains to be determined whether CYP1A1 activity. To further test this hypothesis, we mea- CYP1A10s enzymatic activity or a yet unidentified function

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Figure 7. Possible mechanisms of action of CYP1A1 knockdown. CYP1A1 knockdown by siRNA results in the dual inactivation ERK and AKT (1). Inactivation of ERK could presumably remove the suppressive pressure that this exerts on LKB1(2), thereby allowing activation of AMPK(3). LKB1-independent mechanisms may also be possible. In addition, inhibition of AKT could result in the release of its suppressive effect on AMPK(4). Alternatively, inhibition of AKT by CYP1A1siRNA could result in the release of the TSC1/2 complex from suppression(5) , followed by release of Ras homolog enriched in brain (RHEB)(6), and subsequent inhibition of mTOR signaling(7). Alternatively, AMPK could directly activate TSC2 (5) and inhibit RAPTOR (7), thereby inhibiting mTOR signaling(8). In this manner CYP1A1siRNA treatment results in cell-cycle arrest and increased apoptosis(9). In summary, we propose that CYP1A1siRNA- mediated inhibition of ERK1/2 and/or AKT results in AMPK activation, thereby inhibiting the mTOR/P70S6K pathway and thus resulting in cellular death.

(s) is mechanistically responsible for the proliferative and metabolism of xenobiotics, but also has its own role in survival cell signaling identiﬁed in the present studies. breast cancer progression. The PI3K/AKT and the MEK/ERK pathways are critical Discussion for breast cancer progression (19–22). Knockdown of CYP1A1 in breast cancer biology CYP1A1 correlates with decreased phosphorylation of AKT Recent studies showing that CYP1A1 is expressed in (Ser473) and ERK1/2 (Thr202/Tyr204). CYP1A1 silencing breast tumors (7, 8) led us to investigate the functional also correlates with induction of AMPK tumor suppressor roles of CYP1A1 in the proliferation, survival, and signal via phosphorylation of Thr172 in the catalytic subunit a. transduction of breast cancer cells. Although CYP1A1 has The AKT oncogene promotes proliferation via inhibition of been extensively studied in context of extrahepatic drug the TSC1/TSC2 complex upstream of the mTOR/P70S6K metabolism, little is known about its roles in cancer pathway (34). In contrast, AMPK activates the TSC1/TSC2 progression and cancer cell signal transduction in the complex thereby inhibiting protein synthesis and growth absence of xenobiotics. In this study, we provide evidence (35, 36). In this manner, AKT and AMPK signaling con- that CYP1A1 silencing impairs proliferation and survival, verge on P70S6K to regulate cell proliferation (24, 37). inpart,throughactivationofAMPKphosphorylationand The effects of CYP1A1 knockdown on cell proliferation reduction of AKT, ERK, and P70S6K signaling. These correlate with cell-cycle arrest and increased apoptosis. Our results mean that CYP1A1 is not only involved in the results indicate that cyclin D1 is suppressed by CYP1A1

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– þ knockdown and correlates with a decrease in G1 S cell-cycle would be more effective in ER compared with triple- progression. In contrast, in the absence of TCDD induction, negative breast cancer or effective in both remain to be AhR knockdown does not seem to significantly affect basal determined. CYP1A1 expression (Supplementary Fig. S1A), cyclin D1,or The widespread expression of CYP1A1 in breast cancer has fi – cell-cycle pro le (38). AKT and ERK1/2 promote G1 S been exploited as a strategy to activate prodrug compounds to transition of the cell cycle by stabilizing cyclin D1, whereas cytotoxic intratumoral metabolites. For example, drugs of the AMPK activation inhibits this transition by decreasing cyclin 2-(4-aminophenyl)benzothiazole class such as 5F 203 (Phor- – fl D1 levels, (39 42). In addition, AKT and ERK1/2 regulate tress; ref. 47) and amino avone drugs (48) exhibit potent apoptosis through the antiapoptotic protein survivin (43, antitumor properties in xenograft models and have been 44). Other feedback regulations between AKT, ERK1/2, moved forward to clinical trials (9, 12, 47, 49). The proposed and AMPK have also been described (26–28, 45). Therefore, mechanism of action is that these agents induce and are based on these previous findings, the effects of CYP1A1 activated by CYP1A1 into electrophilic metabolites that silencing on proliferation, cell cycle, and apoptosis are bind to and damage DNA, thus resulting in tumor growth consistent with inhibition of AKT, ERK1/2, and P70S6K arrest. Of considerable interest, the benzothiazoles and þ and activation of AMPK signaling. Moreover, our results aminoflavone seem to be active in ER but not triple- showing that AMPK inhibition by compound C abrogates negative breast cancer (10, 47). Our results suggest that an CYP1A1siRNA-mediated apoptosis further suggest that alternative approach to exploit CYP1A1 expression in breast CYP1A1 signals through AMPK. cancer through reduction of basal levels may extend the range In light of previous findings and the evidence presented in of CYP1A1-targeted approaches to triple-negative breast this study, we propose a model where CYP1A1 silencing cancer. inhibits AKT and ERK phosphorylation, thereby activating Consistent with observations in other cell types (31, AMPK signaling (Fig. 7; steps 1–4). AMPK activation and 32, 50), our results indicate that carnosol treatment inhibits concurrent loss of AKT signaling result in the inhibition of breast cancer cell proliferation. This inhibition of prolifer- mTOR/P70S6K signaling (Fig. 7; steps 5–8), which con- ation in breast cancer lines is associated with reduced sequently decreases synthesis of proliferative and prosurvival CYP1A1 expression and activation of AMPK, which has proteins such as cyclin D1 and survivin (Fig. 7; step 9). These not been previously described in breast cancer. Although the findings differ from other cytochrome P450 enzymes, such activation of AMPK phosphorylation by carnosol may be as CYP3A4, which may act primarily through activation of part of a generalized stress response, these results provide new – STAT3 and regulation of the G2 M checkpoint (46). Our mechanistic information that carnosol is likely to affect model, however, has limitations because it remains to be bioenergetics in breast cancer in addition to exhibiting determined whether CYP1A1 affects these signaling path- antioxidant properties. Our results suggest that the relevant ways through its enzymatic activity or by other as yet target of carnosol in breast cancer may be reduction of unidentified functions. The results presented suggest that CYP1A1 levels, resulting in AMPK phosphorylation, rather CYP1A1 expression is critical for these biologic functions than modulation of AhR. The studies presented here suggest and the roles of CYP1A1 in cancer cell growth may not be that reduction of CYP1A1 levels is a potential therapeutic abrogated by inhibition of its measurable (EROD) enzy- strategy for breast cancer and that carnosol may be an matic activity alone. Moreover, it is possible that CYP1A1 approach to actualize this strategy. may be carrying functions distinct from its canonical met- In summary, we show that the basal level of CYP1A1, abolic functions. Novel hypotheses about nonenzymatic independent of measurable enzymatic (EROD) activity and and enzymatic functions of CYP1A1 in cancer cell growth AhR status, is important for breast cancer proliferation and remain to be investigated. survival. The identification of widespread expression of CYP1A1 in breast cancer suggests that its therapeutic poten- Clinical impact tial could be exploited either by induction followed by Inhibition of the PI3K/AKT and MEK/ERK pathways metabolism of prodrugs to DNA-damaging agents (9, 11, correlating with CYP1A1 silencing is important because 47) or by a new approach of CYP1A1 reduction. Our study cross-talk between these pathways displays synergistic effects suggests that strategies that directly lower CYP1A1 levels when combined inhibition is used for cancer therapeutics using RNA silencing or carnosol may be a potential new (23). The involvement of CYP1A1 in both pathways sug- approach for breast cancer therapeutics. gests that CYP1A1 may be a promising target for cancer therapeutics. Furthermore, because CYP1A1 silencing sig- Disclosure of Potential Conflicts of Interest þ fl nificantly inhibits proliferation and survival of ER and No potential con icts of interest were disclosed. triple-negative breast cancer lines, the results presented in this study may have therapeutic implications for breast Authors' Contributions cancer independent of ER status. The effect of CYP1A1 Conception and design: M. Rodriguez, D.A. Potter þ Development of methodology: M. Rodriguez, D.A. Potter knockdown on cell death appears to be greater in ER Acquisition of data (provided animals, acquired and managed patients, provided MCF7 line than in triple-negative MDA-MB-231 line, facilities, etc.): M. Rodriguez, D.A. Potter Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- whereas both lines appear to be strongly inhibited at the tational analysis): M. Rodriguez, D.A. Potter – G1 S checkpoint. Whether strategies to inhibit CYP1A1 Writing, review, and/or revision of the manuscript: M. Rodriguez, D.A. Potter

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Administrative, technical, or material support (i.e., reporting or organizing data, Grant Support constructing databases): M. Rodriguez, D.A. Potter This study was supported by National Institute of Health grants F31CA144084- Study supervision: D.A. Potter 02, T32 CA009138-38, and R01CA113570, and by the University of Minnesota OVPR Grant-in-Aid, University of Minnesota Department of Medicine Research Acknowledgments Allocation, Masonic Cancer Center Support Grant P30CA077598, and the Randy We would like to thank the expert advice of Drs. Chee-Onn Leong, Tracey D. Shaver Foundation and Community Fund. Bradshaw, Robert Harris, John Falck, Bruce Hammock, Ian Blair, Narayan Avadhani, The costs of publication of this article were defrayed in part by the payment of page Douglas Yee, Philip McGlave, Ameeta Kelekar, Ranjana Mitra, Zhijun Guo, and charges. This article must therefore be hereby marked advertisement in accordance with Monica Milani. We also appreciate Drs. James McCarthy, Carol Lange, Kaylee 18 U.S.C. Section 1734 solely to indicate this fact. Schwertfeger for helpful discussions and serving on the thesis committee of MR. We also thank Dr. Deepali Sachdev and Dr. Elizabeth Wattenberg for kindly providing the cell cycle and quantitative RT-PCR protocols, respectively. Thanks to Dr. Kaylee Received December 4, 2012; revised March 19, 2013; accepted April 2, 2013; Schwertfeger for critical reading of the manuscript. published OnlineFirst April 10, 2013.

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792 Mol Cancer Res; 11(7) July 2013 Molecular Cancer Research

CYP1A1 Regulates Breast Cancer Proliferation and Survival

Mariangellys Rodriguez and David A. Potter

Mol Cancer Res 2013;11:780-792. Published OnlineFirst April 10, 2013.

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