loss of brca1 in the cells of origin of ovarian cancer ... · glycolysis. furthermore, when these...

Research Article

Loss of BRCA1 in the Cells of Origin of OvarianCancer Induces Glycolysis: A Window ofOpportunity for Ovarian CancerChemopreventionTatsuyuki Chiyoda1, Peter C. Hart1, Mark A. Eckert1, Stephanie M. McGregor2,Ricardo R. Lastra2, Ryuji Hamamoto3, Yusuke Nakamura3, S. Diane Yamada1,Olufunmilayo I. Olopade3, Ernst Lengyel1, and Iris L. Romero1

Abstract

Mutations in the breast cancer susceptibility gene 1(BRCA1) are associated with an increased risk of developingepithelial ovarian cancer. However, beyond the role of BRCA1in DNA repair, little is known about other mechanisms bywhich BRCA1 impairment promotes carcinogenesis. Giventhat altered metabolism is now recognized as important inthe initiation and progression of cancer, we asked whether theloss of BRCA1 changes metabolism in the cells of origin ofovarian cancer. The findings show that silencing BRCA1 inovarian surface epithelial and fallopian tube cells increasedglycolysis. Furthermore, when these cells were transfected withplasmids carrying deleterious BRCA1 mutations (5382insC or

the P1749R), there was an increase in hexokinase-2 (HK2), akey glycolytic enzyme. This effect was mediated by MYCand the STAT3. To target the metabolic phenotype inducedby loss of BRCA1, a drug-repurposing approach was used andaspirin was identified as an agent that counteracted theincrease in HK2 and the increase in glycolysis induced byBRCA1 impairment. Evidence from this study indicates thatthe tumor suppressor functions of BRCA1 extend beyondDNA repair to include metabolic endpoints and identifiesaspirin as an ovarian cancer chemopreventive agent capableof reversing the metabolic derangements caused by loss ofBRCA1. Cancer Prev Res; 10(4); 255–66. �2017 AACR.

IntroductionBreast cancer susceptibility gene1 (BRCA1)was cloned in1994.

Since then, the relationship between germline mutations in thisgene and a predisposition to ovarian cancer have become clear.Carriers of a BRCA1mutation have up to a 46% risk of developingovarian cancer by age 70 (1); in contrast, the general populationlifetime risk of ovarian cancer is only 1.4% (2). Unfortunately,screening for ovarian cancer is ineffective (3); therefore, to preventovarian cancer, clinical guidelines recommend that BRCA1muta-tion carriers have their fallopian tubes and ovaries removedbetween the ages of 35 and 40 years (4). To date, nonsurgicalstrategies for ovarian cancer prevention have been limited to theuse of oral contraceptive pills, which reduce ovarian cancer risk by50% with five years of use (5).

Given that neither of these options is optimal for all patients,increased understanding of the functions of BRCA1 mightelucidate new approaches for ovarian cancer prevention. Incells lacking BRCA1, double-stranded DNA breaks are repairedby error-prone nonhomologous end joining, leading to chro-mosomal instability (6). Impairment of homologous recom-bination is one means by which BRCA1 loss promotes carci-nogenesis; however, there are other functions of BRCA1 that areless understood (7). Just as our understanding of cancer hasmoved beyond a focus on the fidelity of the genome, so mayour understanding of BRCA1.

Metabolism is one of the newest frontiers in cancer biology. Theobservation that cancer cells have altered metabolism dates backto the 1920s, when the German Nobel laureate, Otto Warburg,reported that cancer cells preferentially utilize the inefficientsystem of glycolysis to produce ATP, in contrast to normal cells,which use highly efficient mitochondrial respiration (8). Thereliance of cancer cells on aerobic glycolysis, even in the presenceof oxygen, is now termed the "Warburg effect" and the reprogram-ing of metabolism in cancer cells has been recognized as ahallmark of cancer (9, 10). In pancreatic cancer, the Warburgeffect has been shown to be an early event in carcinogenesis,induced by activation of oncogenic Kras (11). Others have shownthat suppression of the Warburg effect with a liver-X-receptor(LXR) inverse agonist inhibits growth of colon cancer (12). More-over, metabolic alterations in the tumor microenvironment, aswell as the intrinsic metabolism of the cancer cell, can contributeto carcinogenesis (13). In ovarian cancer, we have reported that

1Department of Obstetrics and Gynecology/Section of Gynecologic Oncology,The University of Chicago, Chicago, Illinois. 2Department of Pathology, TheUniversity of Chicago, Chicago, Illinois. 3Department of Medicine, Section ofHematology/Oncology, The University of Chicago, Chicago, Illinois.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Iris L. Romero, The University of Chicago, 5841 SouthMarylandAve, MC2050, Chicago, IL 60637. Phone: 773-702-6722; Fax: 773-702-5411; E-mail: [email protected].

doi: 10.1158/1940-6207.CAPR-16-0281

�2016 American Association for Cancer Research.

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metabolic alterations in cancer cells allow them to scavengeenergysubstrates from adjacent adipocytes to fuel tumor growth (14).

The role of BRCA1 in metabolism is unknown and, with theexception of a limited number of studies, has not been investi-gated. In vitro, metabolite profiling of breast cancer cell linesindicated that mutations in BRCA1 reprogram metabolism(15). In MCF7 breast cancer cells, BRCA1 suppresses lipid syn-thesis through binding to phosphorylated acetyl coenzyme Acarboxylase alpha (ACCa; ref. 16). In a study of BRCA1mutationcarriers, Friebel and colleagues showed that weight loss wasassociated with a reduced risk of breast cancer (17). While thedata are limited, in combination, given these studies (15–17) andwith the important role of glycolysis in tumorigenesis, we rea-soned that BRCA1 impairment could induce metabolic changesthat promote the initiation of cancer. We, therefore, investigatedwhether the loss of BRCA1 alters the metabolic phenotype in thecells that are thought to be the origin of ovarian cancer, andwhether thesemetabolic changes could be exploited as ameans ofovarian cancer prevention.

Materials and MethodsPatient samples and primary fallopian tube isolation

Tissue specimens were collected under protocols approved bythe Institutional Review Board at the University of Chicago(Chicago, IL) with written consent from the patients. Primaryfallopian tube secretory epithelial cells (FTEC) were isolated andcultured as described previously (18). To isolate fallopian tubestromal cells (FTSC), fallopian tube tissue was minced withscissors and digested on an orbital shaker with hyaluronidase(Worthington Biochemical; 100 U) and collagenase type 3(Worthington Biochemical; 1,000 U) in 20-mL PBS for 6 hoursat 37�C. FTSCs were plated in DMEM supplemented with 10%FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, nonessentialamino acids, and vitamins.

Reagents, plasmids, and cell linesThe IOSE397 cell line was provided by Dr. Nelly Auersperg

(University of British Columbia) and the fallopian tube cells(FT33-TAg and FT33-MYC; ref. 18) were provided by Dr. RonnyDrapkin (University of Pennsylvania). The TYK-nu was from Dr.Gottfried Konecny (UCLA Medical Center). The HeyA8 ovariancancer cell line was provided by Dr. Gordon Mills (MD AndersonCancer Center). The CAOV3, Kuramochi, and SNU119 ovariancancer cell lines were purchased from ATCC, the Japanese Col-lection of Research Bioresources Cell Bank, and the Korean CellLine Bank, respectively. The MCF7 and HCC1937 breast cancercell lineswere purchased fromATCC.All cell lineswere genotypedto confirm their authenticity (IDEXX Bioresearch short tandemrepeatmarker profiling every 3months; all cell lines last validatedJanuary–July 2016).

AllStars negative control siRNA and siBRCA1 oligos wereobtained from Qiagen. Hs_BRCA1_2 FlexiTube siRNA is designa-ted siBRCA1 no. 1 and Hs_BRCA1_25 FlexiTube siRNA is desig-nated siBRCA1no. 2; siBRCA1no. 2 is used unless otherwise noted.pBABE puro HA BRCA1 was a gift from Stephen Elledge (Addgeneplasmid # 14999). pcDNA BRCA1, pcDNA BRCA1 5382insC, andpcDNA BRCA1 P1749R were confirmed by sequencing.

Cell viability assaysCells were plated in 96-well plates in quintuplicate overnight

and subsequently treated as indicated. Cell viability was deter-

mined via aMTT assay with 0.5mg/mL thiazolyl blue tetrazoliumbromide (Sigma-Aldrich), as described previously (19).

ImmunoblottingFor Western blots, lysates were prepared and immunoblotting

was performed as described previously (20). Coimmunoprecipi-tation was performed with rabbit polyclonal MYC antibody [c-MYC antibody (9E10), Santa Cruz Biotechnology) or mousemonoclonal HA antibody (16B12, Covance) using protein Gagarose (Roche). Intensity of bands were quantified using theImageJ software (NIH, Bethesda, MD).

Metabolic assaysGlycolysis, glycolytic capacity (extracellular acidification rate,

ECAR), and oxidative phosphorylation (OXPHOS) were mea-sured with the Seahorse Extracellular Flux (XF-96) analyzer (Sea-horse Bioscience) as described previously (20). The glucoseuptake assay was performed as described previously (19). Briefly,cells were transfected with siRNA and 48 hours after transfection,glucose uptake was determined using a fluorescently labeledglucose analogue, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG; Cayman Chemical Company),according to the manufacturer's instructions. Relative fluores-cence was normalized to protein concentration.

Fatty acid oxidation was assessed using a previously describedmethod (14), with minor modifications. Briefly, cells were cul-tured in media containing [9,10(n)-3H] palmitic acid. 3H2Osecreted into themediumwas quantified by scintillation countingand normalized to the cellular protein content in each well. Tomeasure NADPH/NADPþ 48 hours after siRNA transfection,NADPH and NADPþ were measured using NADP/NADPH-GloAssay (Promega) according to the manufacturer's protocol.

IHCSamples were formalin-fixed, paraffin-embedded, sectioned,

and mounted on slides. For IHC, slides were deparaffinized andincubated with anti-HK2 (Cell Signaling Technology) at 1:200dilution. Antigen retrieval was performed with 0.01 mol/Lsodium citrate in a rice cooker for 30 minutes. Slides werestained using the VECTASTAIN Elite ABC Kit (Vector Labora-tories) and counterstained with hematoxylin. Intensity of stain-ing was determined by gynecologic pathologists (S.M. McGre-gor and R.R. Lastra).

Promoter luciferase assayA dual luciferase assay was performed according to the man-

ufacture's protocol (Promega). 293T cells were transfected withBRCA1 siRNA and 24 hours later cotransfected with Firefly lucif-erase and pRL-SV40 using Lipofectamine 2000 (Invitrogen). Thedual luciferase assay was done 48 hours after BRCA1 siRNAtransfection and 24 hours after luciferase construct transfection.All experiments were performed in triplicate and normalized toRenilla luciferase activity.

Chromatin immunoprecipitationFragmented chromatin was immunoprecipitated using a chro-

matin immunoprecipitation (ChIP) Assay Kit (EMD Millipore).293T cells transfected with pBabe or pBabeHA BRCA1were cross-linked by formaldehyde. Fragmented chromatin was immuno-precipitated with rabbit polyclonal HA (Y-11; Santa Cruz Bio-technology) or rabbit IgG (Santa Cruz Biotechnology). The qPCR

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BRCA1 mutations increase glycolysis in IOSE and fallopian tube (FT) cells. A, Glycolysis. IOSE cells were transfected with plasmids carrying deleterious BRCA1mutations (P1749R or 5382insC) and the glycolytic response to pharmacologic inhibitors of metabolism (1, 2, and 3) was determined using a Seahorse SF96Extracellular Flux Analyzer. ECAR for 3 groups are shown. Bar graph represents glycolysis and glycolytic capacity. B, Glycolysis. ECAR measured in IOSEcells with BRCA1 silenced using siRNA. C, Glycolysis. ECAR measured in immortalized fallopian tube cells transfected with a BRCA1 siRNA. 1, glucose 10 mmol/L; 2,oligomycin 1 mmol/L; 3, 2-deoxyglucose 100 mmol/L treatments; n ¼ 5/group. Cont, control. D, Glucose uptake assay. The uptake of fluorescently labeleddeoxyglucose analogue (2-NBDG)wasmeasured byfluorescent plate reader in IOSE and fallopian tube cells transfectedwithBRCA1 siRNA. y-axis, fluorescence unitsnormalized by protein concentration; n ¼ 3. P values were calculated using an unpaired t test. Data in A–D represent mean value � SD. � , P < 0.05; �� , P < 0.01;�� , P < 0.001; or �� , P < 0.0001 between the indicated values calculated using an unpaired t test; N.S., not significant.

BRCA1 Impairment Upregulates Glycolysis

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was performed using SYBR Select Master Mix (Applied Biosys-tems). The primer sequences for the ChIP assay were describedpreviously (21).

Statistical analysisSpecific analyses performed for each assessment are described

in the figure legends. In all analyses, data were evaluated using atwo-tailed t test or Fisher exact test; P < 0.05 was consideredstatistically significant (GraphPad).

ResultsMutations in BRCA1 increase aerobic glycolysis in ovariansurface epithelial and fallopian tube cells

To determine whether the mutations in BRCA1 alter metabo-lism, glycolysis was measured in noncancerous immortalizedovarian surface epithelial (IOSE) cells transfected with plasmidscarrying deleterious BRCA1 mutations, 5382insC or P1749R,mutations associated with increased risk of breast and ovariancancer (22). The results showed increased glycolytic activity(glycolysis and glycolytic capacity) in IOSE cells transfected witheither of the BRCA1 mutations when compared with mock-transfected cells (Fig. 1A). Glycolytic reserve was not significantlyincreased (average glycolytic reserve: mock, 25.0 mpH/min;BRCA1 P1749R, 30.6 mpH/min; and BRCA1 5382insC, 31.9mpH/min; P > 0.05). To confirm that loss of BRCA1 increasesglycolytic activity, BRCA1 was silenced in IOSE cells using siRNAand efficiency of the knockdown was confirmed (SupplementaryFig. S1A and S1B). The results showed that IOSE cells with BRCA1silenced had increased aerobic glycolysis as compared with con-trol-transfected cells (Fig. 1B; Supplementary Fig. S2). Recentstudies suggest that ovarian cancer may actually originate fromthe fallopian tube (23, 24), rather than from ovarian surfaceepithelial cells; therefore, the next group of experiments testedwhether loss of BRCA1 in immortalized fallopian tube cellsincreased aerobic glycolysis. As with IOSE cells, immortalizednoncancerous fallopian tube cells with BRCA1 knockdown hadelevated glycolytic activity (Fig. 1C). Moreover, glucose uptakewas increased in both IOSE and fallopian tube cells with BRCA1knockdown (Fig. 1D).

BRCA1 loss increases a key regulator of glycolysis, hexokinase-2To understand how loss of BRCA1 increase glycolysis, a screen

of several kinases in the glycolytic pathwaywas performed includ-ing knownmajor glycolytic enzymes in cancer cells, Hexokinase-2(HK2) andpyruvate kinasemuscle isoenzyme type 2 (PKM2). Thefindings show that knockdown of BRCA1 increased HK2 proteinand mRNA levels in IOSE and immortalized fallopian tube cells,whereas other regulators of glycolysis, including HIF1a and HK1,were not significantly changed (Fig. 2A and B; Supplementary Fig.S3). HK2 expression was also higher in cells transfected withplasmids carrying either the 5382insC or the P1749R deleteriousBRCA1 mutations when compared with mock-transfected cells(Fig. 2C). Next, we tested whether reexpressing BRCA1 wouldreverse the increase in HK2 expression induced by BRCA1 loss. Inboth IOSE (left) and fallopian tube cells (right), the increasedHK2expression induced by loss of BRCA1 was reversed when BRCA1was reconstituted (Fig. 2D; lane 3 vs. 4).

The p53 mutation in the immortalized IOSE and fallopiantube cells could alter metabolism (25); therefore, nonimmor-talized primary human FTECs and FTSCs were also tested.Consistent with the findings in immortalized cells, both pri-

mary human fallopian tube cell types transfected with BRCA1siRNA demonstrated higher HK2 expression compared withcontrol-transfected cells (Fig. 2E). The next experiments wereaimed at testing whether, like in noncancerous IOSE andfallopian tube cells, loss of BRCA1 induces metabolic changesin ovarian cancer cells. Three cell lines that represent high-gradeserous ovarian cancer (TYK-nu, SNU119, and Kuramochi) wereselected: all have wild-type BRCA1 (26). As the case with theIOSE and fallopian tube cells, knockdown of BRCA1 in theTYK-nu and SNU119 ovarian cancer cell lines resulted inincreased HK2 expression (Fig. 2F). In the Kuramochi ovariancancer cell line, baseline HK2 expression was very high and notincreased further by BRCA1 knockdown (Supplementary Fig.S4A) and HK2 mRNA levels were increased with one of thesiRNA BRCA1 transfections (Supplementary Fig. S4B). Overall,these findings indicate that in both noncancerous IOSE andfallopian tube cells and in ovarian cancer cell lines BRCA1 lossincreases HK2 expression.

Loss of BRCA1 has diverse metabolic consequencesTo determine whether the loss of BRCA1 induces changes

beyond glycolysis, several other metabolic pathways were evalu-ated. Fatty acid oxidation activity was noted to be lower in IOSEand fallopian tube cells with a BRCA1 knockdown comparedwithcontrol transfected cells (Fig. 3A). Consistent with this finding,the mRNA levels of carnitine palmitoyl transferase 1 (CPT1), therate-limiting enzyme of fatty acid oxidation, were also decreasedin IOSE and fallopian tube cells with a BRCA1 knockdown(Fig. 3B). Evaluation of fatty acid synthase (FASN) after BRCA1silencing showed a slight increase in FASN mRNA levels in IOSEcells and no change in FASN mRNA in fallopian tube cells(Fig. 3C). Consistent with our finding that BRCA1 appears tohave a minimal impact on fatty acid synthesis, silencing BRCA1did not change phosphorylated ACC or AMPK, an ACC sub-strate (Supplementary Fig. S3). Cellular NADPH productionwas also evaluated in IOSE and fallopian tube cells withsilenced BRCA1, and NADPH was found to be elevated in cellswith BRCA1 knockdown (Fig. 3D). Two measures of mitochon-drial OXPHOS, basal and maximal respiration, were elevated inthe fallopian tube cells with BRCA1 knockdown, but not inIOSE cells (Fig. 3E). Together, these findings indicate thatsilencing BRCA1 increases glycolysis, reduces fatty acid oxida-tion, increases NADPH production, and upregulates OXPHOS,providing multiple metabolic pathways by which loss ofBRCA1 could increase ATP and anabolic substrate productionin IOSE and fallopian tube cells.

BRCA1 impairment activates HK2 via MYC and STAT3Having identified glycolysis as the primary metabolic pathway

altered by BRCA1 loss, the next experiments tested the molecularmechanism mediating this effect. Because BRCA1 is known toregulate transcription through chromatin remodeling by histonedeacetylase (HDAC1andHDAC2; ref. 27), the possibility of directtranscriptional regulation of HK2 by BRCA1 was investigated.ChIP assays performed in HEK293T cells transfected with non-mutated BRCA1 indicated that the BRCA1 protein did not bind tothe HK2 promoter (Fig. 4A) and co-immunoprecipitation assaysdemonstrated no direct binding of the BRCA1 and HK2 proteins(Supplementary Fig. S5). Next, HK2 promoter activity was eval-uated using luciferase assays. The results show increased HK2promoter activity in cells with BRCA1 silenced compared with

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BRCA1 impairment upregulates the glycolytic enzyme HK2.A, Immunoblot for glycolytic enzymes. IOSE and fallopian tube cells were transfected with BRCA1 siRNAand the expression of kinases involved in glycolysis were measured. B, qRT-PCR. IOSE and fallopian tube cells were transfected with BRCA1 siRNA and the mRNAlevels ofBRCA1, HK2, andHIF1Aweremeasured. Results expressed as relative quantity over GAPDH control gene. n¼ 3C, Immunoblot for HK2 and BRCA1. 293T cellswere transfected with pcDNA mock, BRCA1 P1749R, or BRCA1 5382insC plasmids and Western blotting analysis for designated protein was completed48 hours after transfection. D, Immunoblot for HK2 and BRCA1. BRCA1 was silenced in IOSE or fallopian tube cells by transfection of 30-untranslated region (UTR)targetedBRCA1 siRNA then BRCA1was reconstituted by transfecting cellswithBRCA1 (pBabe-BRCA1). Control ismock (pBabe). Immunoblotting for BRCA1 andHK2was performed after BRCA1 silencing with siRNA and again after restoration of BRCA1 with pBabe-BRCA1 transfection. The results show that silencingBRCA1 increased HK2 expression (lane 1 vs. 3) and that restoration of BRCA1 rescues HK2 upregulation induced by BRCA1 knockdown (lane 3 vs. 4). Note, exogenousBRCA1 is not entirely abolished by the siBRCA1 used here because the siRNA targets 3'-UTR. E, Immunoblot for HK2 and BRCA1 in primary human fallopian tube cells.Primary human FTECs, passage ¼ 0 and FTSCs, passage ¼ 1 were extracted from a patient and BRCA1 was silenced using siRNA. Western blotting for designatedprotein was completed 48 hours after transfection. F, Immunoblot in ovarian cancer cell lines. Two different epithelial ovarian cancer cell lines (TYK-nu andSNU119) were transfected with two different BRCA1 siRNAs and the expression of HK2 and BRCA1 was determined by immunoblotting. Data in "B" represent meanvalue � SD; �� , P < 0.01; �� , P < 0.001; or �� , P < 0.0001 between the indicated values calculated using an unpaired t test. N. S.; not significant. HIF1a, hypoxia-inducible factor 1-alpha, PDHK1, pyruvate dehydrogenase kinase type 1.






Figure 3.

BRCA1 knockdown induces diverse metabolic changes in IOSE and fallopian tube cells. A, Fatty acid oxidation assay. IOSE and fallopian tube cells were transfectedwith two differentBRCA1 siRNAs and [9,10(n)-3H] palmitate oxidation converted to 3H2Owas quantified by scintillation counting. (negative control: etomoxir), n¼ 3.B, qRT-PCR. IOSE and fallopian tube cellswere transfectedwith two different BRCA1 siRNAs. mRNA expression of the rate-limiting fatty acid oxidation enzyme, CPT1was measured. Results expressed as relative quantity over GAPDH control gene. n ¼ 3. C, qRT-PCR. IOSE and fallopian tube cells were transfected with twodifferent BRCA1 siRNAs. mRNA expression of the FASNwhich catalyzes fatty acid synthesiswasmeasured. n¼ 3.D,NADPH andNADHþmeasurements. NADPH andNADPþ were measured using NADP/NADPH-Glo Assay (Promega) in IOSE and fallopian tube cells after BRCA1 knockdown. n ¼ 4. E, Mitochondrial function.OXPHOS was evaluated by determining the oxygen consumption rate (OCR) in IOSE and fallopian tube cells with BRCA1 knockdown using a Seahorse SF96Extracellular Flux Analyzer. Bar graph represents basal respiration andmaximal respiration. 1, oligomycin 1 mmol/L; 2, FCCP 0.5 mmol/L; 3, antimycin A 1 mmol/L plusrotenone 1 mmol/L treatment; n¼ 5. Data in A–E represent mean value� SD. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; or �� , P < 0.0001 between the indicated valuescalculated using an unpaired t test; N.S., not significant.

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Figure 4.

BRCA1 impairment upregulates HK2 via MYC and STAT3.A, ChIP. To test for binding of BRCA1 to theHK2 promoter, HEK293T cells were transfected with BRCA1 andChIP assays with qRT-PCR were performed using an anti-HA antibody (or rabbit IgG as a specificity control). Relative quantitation of input DNA (notimmunoprecipitated) is shown. Primerswere selected from across theHK2 promoter (schematic) B,HK2 promoter luciferase assay. A luciferase reporter gene assaywas used to evaluate activity following silencing BRCA1 in HEK293T. BRCA1 was silenced in HEK293T cells followed by cotransfection with a HK2 gene promoterluciferase vector and a Renilla luciferase vector to normalize transfection efficiency. Relative luciferase activity (RLU) was calculated; n ¼ 3. C, qRT-PCR.IOSE and fallopian tube cells were transfected with two different BRCA1 siRNAs and mRNA levels ofMYCwere measured; n¼ 3. D, Immunoblot. IOSE and fallopiantube cellswere transfectedwithBRCA1 siRNAand the expressionofBRCA1,MYC, andpSTAT3measured.E, Immunoblot. HEK293Tcellswere transfectedwith pcDNAmock, BRCA1 P1749R, or BRCA1 5382insC plasmids and Western blotting analysis for the indicated proteins completed. F, Coimmunoprecipitation of STAT3 withBRCA1. HEK293T cells were transiently transfected with HA-BRCA1–expressing construct or empty vector. Proteins were precipitated by anti-HA antibody andexamined by immunoblot using Abs against STAT3 and BRCA1. � , immunoprecipitation. G,HK2 promoter assay. BRCA1 was silenced in HEK293T cells andluciferase reporter gene assaywasused tomeasureHK2promoter activity using aHK2promoter construct after treatmentwith designateddoses of a STAT3 inhibitor(Stattic), a MYC inhibitor (10058-F4), or a HIF1a inhibitor (PX-478) for 24 hours. n ¼ 3. Data in A–C, G represent mean value � SD. � , P < 0.05; �� , P < 0.01;�� , P < 0.0001 between the indicated values using an unpaired t test; N.S., not significant.






control cells (Fig. 4B). Together, these findings indicate thatsilencing BRCA1 upregulates HK2 activity via a transcriptionfactor, rather than through the direct binding of BRCA1 to theHK2 promoter or through direct interactions between the BRCA1and HK2 proteins.

The transcriptional regulation of HK2 is primarily driven byHIF1a (28), MYC (21), and STAT3 (29). HIF1a was not upre-gulated by BRCA1 knockdown (Fig. 2A and B), but IOSE andfallopian tube cells with BRCA1 silenced did have increasedMYCmRNA levels (Fig. 4C) and increased protein levels of MYC andphosphorylated STAT3 (pSTAT3; Fig. 4D). MYC and pSTAT3were also increased in HEK293T cells transfected with plasmidscarrying deleterious BRCA1 mutations (5382insC or P1749R)compared with mock-transfected cells (Fig. 4E). Similar find-ings were noted in breast cancer cell lines (Supplementary Fig.S6A and S6B). Coimmunoprecipitation experiments showedbinding of BRCA1 to STAT3 (Fig. 4F) and others have alsoreported binding of MYC to BRCA1 (30). Consistent with thefinding that MYC and STAT3 activate HK2 in BRCA1 knock-down cells, HK2 promoter activity induced by knockdown ofBRCA1 was partially reversed by either a STAT3 (Stattic) or aMYC (10058-F4) inhibitor, whereas a HIF1a inhibitor (PX-478) had no effect (Fig. 4G).

Aspirin counteracts the increase in glycolysis induced byBRCA1 mutations

The metabolic phenotype induced by BRCA1 loss may rep-resent a window of opportunity for chemoprevention; there-fore, we sought to identify agents that could reverse thisphenotype by decreasing HK2 expression and glycolysis. Usinga drug repurposing approach, several drugs that are used fornoncancer indications and are reported to be protective againstovarian cancer were tested (Fig. 5A; refs. 20, 31–33). IOSE cellswere treated with the candidate chemopreventive agents andglycolysis, as well as the expression of HK2, MYC, pSTAT3, andHIF1a, measured. Two agents, aspirin and luteolin, induced adose-dependent decrease in the protein levels of HK2 andreduced MYC expression (Fig. 5B). Glycolytic activity wasdecreased in IOSE and immortalized fallopian tube cells treatedwith either aspirin or luteolin compared with control-treatedcells (Fig. 5C and D). Two other drugs also suppressed glycol-ysis, but these agents either did not change HK2 expression(lovastatin) or increased it (resveratrol; Supplementary Fig. S7Aand S7B) and were not evaluated further.

HK2 has been reported to be necessary for oncogenic trans-formation (34); therefore, suppressing HK2may protect againstovarian cancer. Consistent with this, treatment with an HK2inhibitor [bromopyruvic acid (3-BP); ref. 35] induced dose-dependent toxicity in MYC-transformed fallopian tube secre-tory cells (FT33-MYC) and in the ovarian cancer cell lines, TYK-nu and Kuramochi (Fig. 6A). As 3-BP is not approved for use bythe FDA, aspirin was also evaluated. Luteolin was not testedbecause the agent upregulated HK2 at low concentration (datanot shown). In the ovarian cancer cell lines, aspirin treatmentresulted in a dose–dependent reduction in HK2 and MYCexpression, with no effect on HK1 or pSTAT3 (Fig. 6B). InIOSE and fallopian tube cells with BRCA1 silenced, aspirinpartially reversed the HK2 upregulation induced by loss ofBRCA1 (Fig. 6C). To confirm in patient samples that HK2 ispresent in the target tissue, benign fallopian tubes were stainedfor HK2 expression by IHC. Uniformly high HK2 expression

was noted with 12 of 14 (85.7%) of the benign fallopian tubesdemonstrating strong HK2 staining in the secretory cells, butnot in the ciliated cells (Fig. 6D). Through this drug screen,aspirin emerged as a potential chemopreventive agent that cancounteract the HK2 activation induced by BRCA1 loss inovarian surface epithelial, fallopian tube, and ovarian cancercells (Fig. 6E).

DiscussionThere is increasing evidence that metabolic derangements

facilitate cancer growth (10) and it is well-known that mutationsin BRCA1 significantly increase the risk of ovarian cancer. In thisstudy, to identify novel means by which BRCA1 loss promotescarcinogenesis, we asked whether the loss of BRCA1 inducesprotumorigenic metabolic changes in the two cell types thoughtto be the origin of ovarian cancer (fallopian and ovarian surfaceepithelial cells). Themain finding is that loss of BRCA1 in ovariansurface epithelial and fallopian tube cells increases glycolysisthrough HK2 activation. With the goal of improving care forpatients who carry BRCA1 mutations, we next asked whether themetabolic changes induced by loss of BRCA1 could be reversed bychemopreventive agents. Using a drug-repurposing approach, wefound that aspirin reverses themetabolic derangements caused byloss of BRCA1.

The experiments presented here show that glycolytic activity issignificantly increased in ovarian surface epithelial cells that aretransfected with either the 5382insC or the P1749R deleteriousBRCA1 mutation (Fig. 1) and in fallopian tube cells with BRCA1silenced. To the best of our knowledge, this is the first reportdescribing metabolic changes induced by BRCA1 mutations innoncancerous cells of origin of ovarian cancer. In fact, little isknown about BRCA1's role in metabolism in normal tissue. Incardiomyocytes, loss of BRCA1 has been reported to reduceglucose levels and fatty acid oxidation (36) and in skeletalmuscle,BRCA1 loss reduces fatty acid synthesis (37). Menendez andcolleagues use targeted metabolomics to show that breast epi-thelial cells transfected with a BRCA1 mutation (185delAG)undergo metabolic reprograming to increase anabolic processes,including the tricarboxylic acid cycle and lipogenesis (38). Ourfindings add to these studies by demonstrating in ovarian cancerthat loss of BRCA1 decreases fatty acid oxidation and increasesNADPH andMYC expression, indicating that BRCA1 impairmentmay also upregulate the pentose phosphate pathway and gluta-minolysis. Themost prominent finding in the IOSE and fallopiantube cells is that when BRCA1 is silenced glycolytic activity issignificantly increased.

To understand how loss of BRCA1 increases glycolytic activity,we evaluated several kinases in the glycolytic pathway and foundthat enhanced HK2 activity was central to the process. ElevatedHK2 expression was noted in ovarian surface, fallopian, ovariancancer, and breast cancer cells when BRCA1 was silenced. Wefound that silencing BRCA1 induces the MYC and STAT3 tran-scription factors, resulting in increased HK2 promoter activity.This finding adds to those of others who have shown that highlevels ofHK2 expression are needed for tumor initiation (34), andthat HK2-mediated glycolysis is required for tumorigenesisin Pten-/p53- mouse embryonic fibroblasts (39). Analysis ofpatient samples demonstrates uniformly high HK2 expression infallopian tubes, specifically in the secretory cells. Previously, HK2expression has been reported to be limited to embryonic tissue,

Chiyoda et al.





skeletal muscle, and adipose (40). Our results suggest that highbasal HK2 levels combined with loss of BRCA1 in fallopian tubecells can further push these cells of origin of ovarian cancer into astate of metabolic derangement.

To target the metabolic phenotype induced by loss of BRCA1,we tested drugs that are already FDA approved for noncancerindications and identified aspirin and luteolin as agents thatreduced both HK2 and glycolysis. Luteolin is a natural flavonoid

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Aspirin and luteolin suppress HK2 and glycolysis in IOSE and fallopian tube cells. A, Drugs tested. FDA-approved drugs tested for repurposing for ovariancancer chemoprevention targeted against HK2 and glycolysis. B, Immunoblot. IOSE cells treated with indicated concentration of aspirin or luteolin for 24 hours andprobed for HK2, MYC, pSTAT3, STAT3, HIF1a, and actin. Bars represent mean optical density (OD) for each concentration of aspirin (0–1 mmol/L) or luteolin(0–10 mmol/L). C, Glycolysis. ECAR measured in IOSE cells treated with indicated concentration of aspirin or luteolin for 24 hours. 1, glucose 10 mmol/L; 2,oligomycin 1 mmol/L; 3, 2-deoxyglucose 100 mmol/L treatments. n ¼ 5. D, Glycolysis. Fallopian tube cells were treated with aspirin (4 mmol/L) or luteolin(2 mmol/L) for 24 hours and ECAR was measured by Seahorse SF96; n ¼ 5. Data in C and D represent mean value � SD; �, P < 0.05; ��, P < 0.01; or �� , P < 0.001between the indicated values calculated using an unpaired t test; N.S., not significant.






in several fruits and vegetables with reported activity againstcancer (32, 41). In our study, luteolin reduced glycolysis bysuppressing MYC-dependent HK2 expression but only at supra-physiologic concentrations. In contrast, aspirin, at physiologicallyrelevant concentrations (2mmol/L; ref. 42), efficiently suppressed

both HK2 expression and glycolysis. The in vitro findings supportthat assertion that suppression of glycolysis by aspirinmayprotectagainst ovarian cancer; however, these findings are limited by thelack of confirmation in an animal model. Because of the limita-tions of animal models ovarian cancer (43), testing aspirin as a

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Aspirin suppresses HK2 in ovarian cancercells and reverses BRCA1 knockdown–induced HK2 upregulation in IOSE orfallopian tube cells.A,Proliferation. Aftertreatment with a HK2 inhibitor (3-BP)cell survivalwasmeasured byMTT assay.IC50s: FT33-MYC (MYC-transformedfallopian tube secretory cells), 6.02mmol/L; TYK-nu, 4.88 mmol/L; andKuramochi, 26.54 mmol/L. n ¼ 5.B, Immunoblot. Ovarian cancer cell linestreated with indicated concentration ofaspirin for 48 hours and probed for HK2,HK1, MYC, pSTAT3, STAT3, and actin.Bars represent mean OD normalized toactin � SD for each concentration ofaspirin (0–2 mmol/L). C, Immunoblot.Fallopian tube or IOSE cells weretransfected with BRCA1, treated withaspirin (fallopian tube 2 mmol/L, IOSE:0.5mmol/L) and probed for HK2, BRCA1,and actin. Bars represent mean ODnormalized to actin� SD for control andsiBRCA1 transfected cells treated withDMSO or aspirin. D, IHC image.Representative HK2 staining pattern infallopian tube demonstrate secretorycells with high HK2 expression (whitearrows) compared with surroundingciliated and stroma cell (black arrows).Bars, 200 mm. E, Model. High basal HK2expression in fallopian tube secretorycells is intensified by loss of BRCA1. As achemopreventive strategy, aspirininhibits the HK2 activity induced by lossof BRCA1 restoring the metabolic ofphenotype of fallopian tube cells to thatof cells with functional BRCA1.

Chiyoda et al.





chemopreventive agent in a murine model was beyond the scopeof the study. A protective effect of aspirin in ovarian cancer hasbeen suggested in epidemiologic studies (44, 45). A recent anal-ysis of the Iowa's Women's Health Study found that use of aspirinwas associated with a decrease in incidence of ovarian cancer andother aspirin-sensitive cancers [HR, 0.87; 95% confidence inter-val, 0.72–1.06; ref. 46]. Similar findings were reported in ananalysis of the Danish Cancer Registry (47). For colorectal cancerprevention, theUSPreventive Service Task Force is recommending81 mg of aspirin daily (48), based on two large randomizedclinical trials (49, 50). For ovarian cancer, precedent already existsfor the development of therapeutics directed against the patho-logic effects of BRCA1 impairment, the most prominent examplebeing PARP inhibitors (51). Aspirin's ability to suppress HK2 andglycolysis induced by loss of BRCA1 provides a rationale forrepurposing aspirin as a chemoprevention in BRCA1 mutationcarriers.

Historically, the evaluation of BRCA1 has largely focused on itsrole in DNA repair. Less is known about the other functions ofBRCA1, including any effects on metabolism. Given that aerobicglycolysis and other metabolic alterations are vital for initiationand growth of cancer (52), the increase in glycolysis in fallopiantube and OSE cells induced by loss of BRCA1 reported here is anew mechanism by which BRCA1 impairment promotes ovariancancer growth. The loss of BRCA1 may metabolically prime thecells of origin of ovarian cancer for oncogenic transformation andprovide a window of opportunity for primary cancer preventionwith agents such as aspirin.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Chiyoda, E. Lengyel, I.L. Romero

Development of methodology: T. Chiyoda, R. Hamamoto, I.L. RomeroAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Chiyoda, P.C. Hart, M.A. Eckert, R.R. Lastra,S.D. Yamada, I.L. RomeroAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Chiyoda, P.C. Hart, R. Hamamoto, E. Lengyel,I.L. RomeroWriting, review, and/or revision of the manuscript: T. Chiyoda, P.C. Hart,R. Hamamoto, O.I. Olopade, E. Lengyel, I.L. RomeroAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): O.I. Olopade, E. Lengyel, I.L. RomeroStudy supervision: Y. Nakamura, E. Lengyel, I.L. Romero

AcknowledgmentsWe thank Gail Isenberg for critically reviewing the manuscript.

Grant SupportThis work was supported by the NIH through the University of Chicago

Cancer Center Support grant (CA014599) and the Mayo Clinic Ovarian CancerSPORE grant (P50CA136393-06A1). I.L. Romero is supported by grants fromtheNIH, Eunice Kennedy ShriverNational Institute ofChildHealth andHumanDevelopment (2K12HD000849-26), the American Board of Obstetrics andGynecology, and a StandUpToCancer-OvarianCancer Research FundAlliance-National Ovarian Cancer Coalition Dream Team Translational Research Grant(SU2C-AACR-DT16-15). StandUp ToCancer is a program of the EntertainmentIndustry Foundation. Research grants are administered by the American Asso-ciation for Cancer Research, the scientific partner of SU2C. E. Lengyel issupported by grants from the National Cancer Institute (5R01CA111882-07and 1R01CA169604-01A1). T. Chiyoda is a recipient of Japan Society for thePromotion of Science Fellowship for Research Abroad and Kanae FoundationFellowships for Research Abroad.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 2, 2016; revised January 6, 2017; accepted February 27,2017; published OnlineFirst March 6, 2017.

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2017;10:255-266. Published OnlineFirst March 6, 2017.Cancer Prev Res Tatsuyuki Chiyoda, Peter C. Hart, Mark A. Eckert, et al. ChemopreventionGlycolysis: A Window of Opportunity for Ovarian Cancer Loss of BRCA1 in the Cells of Origin of Ovarian Cancer Induces

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loss of brca1 in the cells of origin of ovarian cancer ... · glycolysis. furthermore, when these...

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