aromatase acetylation patterns and altered activity in ... · reports of the effect of lysine...

Oncogenes and Tumor Suppressors

Aromatase Acetylation Patterns and AlteredActivity in Response to Sirtuin InhibitionDeborah Molehin1, Isabel Castro-Piedras1, Monica Sharma1, Souad R. Sennoune2,Daphne Arena1, Pulak R. Manna1, and Kevin Pruitt1

Abstract

Aromatase, a cytochrome P450 member, is a key enzymeinvolved in estrogen biosynthesis and is dysregulated in themajority of breast cancers. Studies have shown that lysinedeacetylase inhibitors (KDI) decrease aromatase expression incancer cells, yet many unknowns remain regarding the mech-anism by which this occurs. However, advances have beenmade to clarify factors involved in the transcriptional regula-tion of the aromatase gene (CYP19A1). Yet, despite aromatasebeing a primary target for breast cancer therapy, its posttrans-lational regulation has been virtually unexplored. Acetylationis a posttranslational modification (PTM) known to alter theactivity and stability of many oncoproteins, and given therole of KDIs in regulating aromatase expression, we postulatethat aromatase acetylation acts as a novel posttranslationalregulatory mechanism that impacts aromatase expressionand/or activity in breast cancer. Liquid chromatography–

tandem mass spectrometry (LC-MS/MS) analysis revealedthat aromatase is basally acetylated on several lysine residues(108, 169, 242, 262, 334, 352, and 354) in MCF-7 cells, andtreatment with a SIRT-1 inhibitor induced additional acetyla-tion (376, 390, 440, and 448). These acetylated lysine residuesare in regions critical for aromatase activity. Site-directedmutagenesis and overexpression studies demonstrated thatK108R/Q or K440R/Q mutations significantly altered aroma-tase activity in breast cancer cells without altering its subcel-lular localization.

Implications: These findings demonstrate a novel posttrans-lational regulation of aromatase and uncover novel anticancereffects of deacetylase inhibitors, thus providing new insight forongoing development of deacetylase inhibitors as cancertherapeutics. Mol Cancer Res; 16(10); 1530–42. �2018 AACR.

IntroductionBreast cancer in women represents 15% of all new cancer cases

in the United States. Although a decline in death rates has beenobserved among women with breast cancer in the United States,reports from a Surveillance, Epidemiology and End Results pro-grams (SEER) database predict that approximately 266,120 per-sons will be diagnosed with breast cancer and 40,920 deaths willoccur in 2018 (1). Estrogen receptor–dependent breast cancers(ERþ) constitute about 70%of breast cancer cases andpatients areadministered anti-hormonal therapies such as selective estrogenreceptor modulators (SERM) or aromatase inhibitors (AI; ref. 2),with the aim of inhibiting intratumoral estrogen biosynthesis,which drives tumor progression (3, 4).

Aromatase belongs to the cytochrome P450 superfamily andis involved in steroidogenesis. Although it is critical in humandevelopment and a wide range of physiological processes(5, 6), high expression of aromatase has been reported in

tumors relative to nonneoplastic cells (5, 7–9) and linked withmetastatic ER-dependent breast tumor cells (10, 11). Aroma-tase is an important therapeutic target in the treatment ofpostmenopausal breast cancer (12) and endometriosis (13).Aromatase overexpression in majority of breast cancers leadsto chronic intratumoral increases in estrogens, which canimpact the tumor microenvironment (14, 15). Interestingly,postmenopausal women with the highest quintile of plasma17-b-estradiol (E2) present with a significantly higher rate ofbreast cancer within 10 years compared with the lowest quintile(16, 17). Emerging data demonstrate that estrogen's impactextends well beyond ERa-mediated signaling (18) and engagesboth ERa-dependent and ERa-independent signaling (17–22).However, even though the relative contribution of estrogens tobreast cancer exerting their impact via ERa, ERb, or unchar-acterized estrogen receptors (GPER-1/GPR30) remains unclear,it is clear that aromatase plays a critical role in the generation ofthe estrogen regardless of the specific signaling pathway that isactivated downstream (18, 23, 24).

Posttranslational modifications impact the stability, localiza-tion, and functionality of many proteins in cells including cancercells (25, 26). With respect to aromatase protein, little is knownabout how aromatase is modified posttranslationally and thepossible implications of these modifications. Recent studies haveshown that deacetylase inhibition sensitizes AI-resistant tumorsoverexpressing aromatase (27, 28). However, posttranslationalregulation of aromatasewas not investigated in these studies. Alsorecently, a phase I clinical trial of panobinostat in combinationwith letrozole in postmenopausal women proved efficacious inthe treatment of metastatic breast cancer (29). Based on recent

1Department of Immunology and Molecular Microbiology, Texas Tech UniversityHealth Sciences Center, Lubbock, Texas. 2Cell Physiology and MolecularBiophysics, Texas Tech University Health Sciences Center, Lubbock, Texas.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Kevin Pruitt, Texas Tech University Health SciencesCenter, 3601 4th Street, Lubbock, Texas 79430-6591. Phone: 806-743-2523;Fax: 806-743-2334; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0047

�2018 American Association for Cancer Research.

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reports of the effect of lysine deacetylases (KDAC) on aromataseenzyme in breast cancer (30, 31), this present study aims tounderstand the mechanism by which the KDAC, Sirtuin-1(SIRT-1), posttranslationally regulate aromatase proteins and toevaluate the functional implications of acetylation marks onaromatase activity and estrogen biosynthesis.

Materials and MethodsCell culture

Cell lines were obtained from the ATCC. MDA-MB 231(HTB-26), JEG-3 (HTB-36), and HEK293 (CRL-1573) cells werecultured in D-MEM, while T47D cells (HTB-133) andMCF-7 cells(HTB-22) were cultured in RPMI-1640 and MEM supplementedwith 0.07% to 0.1% insulin respectively (Sigma-Aldrich). Allculture media were supplemented with 10% fetal bovine serumand 1% penicillin/streptomycin (Invitrogen). Cell lines wereobtained between 2014 and 2017, immediately frozen down inliquid nitrogen; hence, cells were cultured for less than 6months.For enzyme inhibition studies, cells were treated with dimethylsulfoxide (DMSO) vehicle control or varying concentrations ofSIRT-1 inhibitor IV (566325; Millipore) as noted in the figurelegends.

Endpoint polymerase chain reactionTotal RNA was isolated from cells using a Pure-link RNA mini

kit (Invitrogen), and 2 mg of RNA was reverse transcribed withMoloney murine leukemia virus Reverse Transcriptase (Pro-mega). Intron-spanning primers were designed for each specifictarget DNA and gene expressionmeasured by endpoint PCR usingJumpStart RedTaq (Sigma-Aldrich). Human aromatase forwardand reverse primers used were 50-GGCAGTGCCTGCAACTACTA-30, 50-GTCACCTCCTCCAACCTGTC-30 and b-actin were 50-GGACTTCGAGCAAGAGATGG-30, 50-AGCACTGTGTTGGCGTA-CAG-30. Veriti 96-well thermal cycler (Applied Biosystems) andGel DOC EZ imager (Bio-Rad) were used for PCR analyses.

Western blotsProtein samples were extracted from cells. Antibodies used

include: aromatase (ab124776; Abcam), V5-tag (ab27671;Abcam), Calnexin (ab22595; Abcam), Gapdh, and b-actin (SC-47724 and -47778 Santa Cruz Biotechnology). Proteins trans-ferred onto polyvinylidene fluoride (PVDF) membranes (Milli-pore) were blotted with specific primary antibodies and horse-radish peroxidase-conjugated secondary antibodies. Immuno-blots were visualized by enhanced chemiluminescence on pre-mium X-ray films (Phenix Research). Densitometric analyses ofimages were performed using Image Processing and Analysis inJava (ImageJ) software from three independent experiments.

Enzyme-linked immunosorbent assayCells seeded in charcoal stripped serum (Thermo Fisher)media

for 24 hours were treated with DMSO or different dosesof inhibitor IV for 4 hours, starting with half its IC50 value(63 nmol/L determined against SIRT-1 enzymes purified frommammalian cell; ref. 32). Cells were supplemented with/without10 nmol/L Androstenedione (Sigma-Aldrich). Supernatant cul-turemediawere evaluated for estrogen production using estradiolELISA Kit (Cayman Chemical) following the manufacturer's pro-tocol at 412 nm absorbance using an Infinite M100 PRO Qua-druple monochromator microplate reader (Tecan). Standard

curve and estradiol concentrationswere determinedwithCaymanELISA competitive analysis tool.

ImmunoprecipitationCells were seeded and harvested after 48 hours in either

acetylation lysis buffer (acKIP; 1% Triton X-100, 0.5% NP-40,150 mmol/L NaCl, 50 mmol/L Tris (pH 7.4), 10% glycerol, withcomplete protease inhibitors (Invitrogen), 1 mmol/L TrichostatinA (TSA) and 1mmol/L nicotinamide] for acetylation studies or inradio-immunoprecipitation assay (RIPA) lysis buffer (pH 8.0)with protease inhibitors for ubiquitination, sumoylation, andmethylation analysis. Protein concentrationwas quantified by theBCA method (Thermo Fisher) and 2 mg protein was subjected toimmunoprecipitation using either 1.5 mg of normalmouse/rabbitIgG (control) or acetyl-lysine antibody (05-515, Millipore) oranti-aromatase antibody (ab124776; Abcam), anti-Sumo-1(ab11672; Abcam), anti-pan methyl lysine (ab7315; Abcam),anti-dimethyl lysine (ab23366; Abcam) and anti-ubiquitin(STI200;Millipore). Protein–antibody complexes were incubatedwith precleared Protein-G dynabeads (Invitrogen) on variablespeed nutator (Fisher). Immune complexeswere subjected to heatby boiling for 15 minutes and analyzed by polyacrylamide gelelectrophoresis using 4% to 12% bolt gel systems (Invitrogen).

Liquid chromatography/electrospray tandem massspectrometry (LC-MS/MS)

MCF-7 cellswere seeded and incubated at 37�Cwith either 20%or 2.5% O2. Cells were treated when they reached 70% to 80%confluence with either DMSO or 32 nmol/L inhibitor IV for 10minutes. Cells were lysed with acKIP buffer, and protein extractswere incubated with 4 mg anti-aromatase antibody (ab124776;Abcam) overnight. Protein-G dynabeads were added to the anti-body–protein complex and incubated for 2 hours. Immunopre-cipitates were processed and shipped to Applied Biomics Inc.(Hayward) for acetylation site identification by LC-MS/MS massspectrometry.

V5-Aromatase pcDNA3.1þ construct generationThe full-length CYP19A1 gene was obtained by a phusion high-

fidelity polymerase (New England Biolabs) PCR. The templateplasmid PLX304-V5-Arom was purchased from Harvard plasmiddepository (HsCD00418838). Synthetic nucleotide primers (Sup-plementary Table S1) used were designed with V5-epitope tageither at the amino (�NH2) or carboxyl (�COOH) terminus. The1.5-kb DNA product was subcloned into NheI/Not1 restrictionenzyme sites of pcDNA3.1þ mammalian expression plasmid(Thermo Fisher) using T4 DNA ligase (Invitrogen). MAX effi-ciency competent cells (Sbtl2; Thermo Fisher) were transformedwith ligation mixture, and plated on LB-ampicillin agar at 37�Covernight. Bacteria colonies were screened and plasmids extractedby the QIAprep spin miniprep Kit (Qiagen). Plasmids isolatedwere subjected to NheI and Not1 restriction digest, and fragmentswere resolvedon1%agarose gel (Bio-Rad) in 1X TAEbuffer. Sangerplasmid sequencing was used to confirm the DNA sequences.

Small interfering RNA (siRNA)-mediated knockdown andexpression plasmid transfections

MCF-7 cells were seeded 24 hours prior to transfection with50 nmol/L of nontarget control siRNA (D-001210-01-05; Dhar-macon) or SIRT-1 siGenome smartpool siRNA (M-003540-01;Dharmacon; Supplementary Table S2), using RNAi MAX lipofec-tamine reagent (Thermo Fisher) for 48 hours to study the effect

Acetylation Regulates Estrogen Synthesis

www.aacrjournals.org Mol Cancer Res; 16(10) October 2018 1531




of SIRT-1 downregulation. To investigate the effect of aromataseoverexpression, cells were transfected with 1 mg/well emptyvector or with N-V5 or C-V5 tagged aromatase plasmid usingLipofectamine P3000 reagent and opti-MEM reduced serummedia (Thermo Fisher) at 37�C following the manufacturer'sinstructions. Protein or total RNA samples were extracted after48 hours of transfection (HEK293) or 24 hours from other cells.Stable transfectants were selected with 900 mg/mL NeomycinG418 (Sigma-Aldrich) for HEK293 cells and 600 mg/mL forMCF-7 cells.

Site-directed mutagenesispcDNA3.1-V5-Aromatase plasmids were subjected to site-

directed mutagenesis with nucleotide primers (SupplementaryTable S3) designed using the quikchange mutagenesis primerdesign tool and kit (Agilent Technologies) following the manu-facturer's protocol. Plasmids were transformed into XL10 Goldultra-competent cells and plated on ampicillin impregnated LB-agar overnight at 37�C. Bacteria colonies were screened andplasmids purified using miniprep kit. Sanger plasmid sequencingwas done to validate the mutations.

ImmunofluorescenceCells were seeded onto coverslips (12 mm) in a 60-mm tissue

culture dish. The cells were washed with PBS and fixed with 4%paraformaldehyde and quenched with 50 mmol/L ammoniumchloride (NH4Cl) in PBS. Nonspecific binding was blockedwith 5%bovine serum albumin in PBS (blocking buffer), followedby a 1-hour incubation of anti V5-epitope tag (Abcam: ab27671;1/5,000) and anti calnexin (Abcam; ab22595; 1/250) antibody.Secondary antibodies anti rabbit Alexa Fluor 568 (1/300) or antimouse Alexa Fluor 647 (1/5000) were incubated for 1 hour in thedark at room temperature. The samples were mounted with pro-long gold antifademounting solution with DAPI (Thermo Fisher).Images were captured using a Nikon T-1E laser scanning confocalmicroscopy, with a 60� objective, and analyzedwithNIS software.

Endoplasmic reticulum fractionationStably transfected HEK293 cells were plated and allowed to

reach 80% confluence. Cells were trypsinized, washed with 1�PBS, and pelleted at 600 � g for 5 minutes. Fractionation wascarried out using an endoplasmic reticulum Isolation Kit(ER0100; Sigma-Aldrich) following the manufacturer's protocol.Protein fractions were quantified and analyzed by Western blots.

In silico analysisAromatase protein sequence was obtained from National Cen-

ter for Biotechnology Information (NCBI; NP_112503.1). Mul-tiple sequence alignment of aromatase protein across differentorganisms was performed using Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/). The structure and acetylat-ed lysine residues on aromatase were modeled using the ProteinHomology analogY Recognition Engine version 2 (Phyre2; http://www.sbg.bio.ic.ac.uk/phyre2), and the PredictProtein software(https://www.predictprotein.org/). ModPred tool was used topredict and analyze concurrently several PTM modifications onaromatase protein (33).

Statistical analysisUnpaired Student t tests and two-way analysis of variance

(ANOVA) were performed using GraphPad Prism software, to

assess whether differences observed in the various experimentswere significant. All results are expressed as mean � SEM andconsidered statistically significant at P < 0.05.

ResultsAromatase is expressed in multiple cancer cell lines andenzymatic activity is regulated by SIRT-1 inhibition

In order to assess the short-term impact of lysine deacetylaseinhibitor (KDI)-mediated regulation of aromatase, we firstestablished the basal expression of aromatase across threecancer cell lines. JEG3 cells were included in this study becauseof their high expression of aromatase protein. Reverse transcrip-tase polymerase chain reaction (RT-PCR) analysis using intron-spanning primers and Western blot analysis using aromatase-specific antibodies revealed expression of aromatase in all threecell lines examined (Supplementary Fig. S1A and S1B). Previousstudies revealed that decreased aromatase protein levels haddirect bearing on aromatase activity when cells were treated withSIRT-1 inhibitor for 24 hours. We decided to investigate whethera SIRT-1 inhibitor had a direct effect on aromatase activity bytreating the cells for a shorter duration. The effect of a preclinicalSIRT-1-specific KDI (inhibitor IV) on aromatase activity in MCF-7 breast cancer cell line showed a statistically significant dose-dependent reduction in 17-b estradiol (E2) levels, without adecrease in aromatase protein levels, 4 hours post KDI admin-istration (Fig. 1A). siRNA-mediated SIRT-1 knockdown resultedin a 65% and 45% downregulation of SIRT-1 and aromataseprotein levels, respectively. SIRT-1 downregulation led to astatistically significant decrease (P ¼ 0.02) in estradiol levelsin MCF-7 cells (Fig. 1B).

Aromatase protein is basally acetylated and KDI furtherinduces acetylation in breast cancer and placentalchoriocarcinoma cell lines

To investigate the role of aromatase acetylation in differentcancer context, we decided to include MDA-MB 231 (231) andJEG3 cell lines in the analysis. Immunoprecipitation andWesternblot analyses using acetyl-lysine specific monoclonal antibodiesshowed that aromatase is acetylated in all cancer cell lines tested(Fig. 2A). The lowest basal acetylation signal was observed inMCF-7 cellswhen comparedwith theother two cell lines (Fig. 2B).Time-dependent effect of inhibitor IV on aromatase protein inMCF-7 cell lines showed that induced acetylation signal peakedat 10 minutes posttreatment and started to diminish over time(Fig. 2C). We observed a significant induction of aromataseacetylation in MCF-7 (P ¼ 0.02), but variable induction inMDA-MB-231 and JEG3 cells when compared with the untreatedcell lines (Fig. 2d, and e).

Aromatase is acetylated at key lysine residues upontreatment with inhibitor IV at atmospheric andphysiological O2

To further probe aromatase acetylation inducedbyKDI inMCF-7 breast cancer cells, protein extracts were subjected to proteolyticanalysis by LC-tandem mass spectrometry to identify acetylatedlysine residues on aromatase protein. Results from LC-MS/MS were consistent with immunoprecipitation results whichshowed that aromatase is basally acetylated and further inducedby inhibitor IV (Supplementary Fig. S2 and SupplementaryTable S4). A total of 11 lysine residues were acetylated and

Molehin et al.

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http://www.ebi.ac.uk/Tools/msa/clustalo/

http://www.ebi.ac.uk/Tools/msa/clustalo/

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http://www.sbg.bio.ic.ac.uk/phyre2

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according to structural andmutational studies (34) located in theb-sheets, helical, and loop region connecting the transmembraneregion of the protein (Table 1). We further subjected the cells tophysiological oxygen conditions (2.5% O2) in which the inhibi-tors are likely to be eliciting their effects in vivo to determine theeffect of reduced oxygen tension on acetylation of aromatase.Notably, at physiologic oxygen, we observed acetylation of adifferent lysine residue (Lys 440) in comparison with those fromthe atmospheric oxygen incubation conditions.

Solvent accessibility prediction of acetylated aromatase resi-dues with PredictProtein software (35) revealed that amajority ofthe residues are buried (55%), exposed (18%), or intermediate(27%), suggesting that most of the acetylated residues may belocated on the luminal side of the endoplasmic reticulum.Because lysine acetylation neutralizes the positive charge onunmodified lysine, this posttranslational modification on aro-

matase could be playing a significant role in the regulation ofproperties of the domains in which it occurs.

Homology search revealed a total of 6 conserved lysine residuesof the 11 acetylated residues among P450 aromatase of differentorganisms analyzed; however, we only show the sequence align-ment data for K108 and K440, because these lysines were furtheranalyzed in this study (Fig. 3A). The proximity of the acetylatedlysine residues to the enzyme catalytic site and substrate bindingregion (pocket region) was analyzed based on the crystal structureof humanCYP19A1 (RCSB PDB ID: 3S7S). Results showed that ofall the conserved lysine residues, only Lys 440 had a þ3 aminoacids (aa) closeness to the catalytic site, which is fundamental inthe conversion of androgen substrate to estrogen by aromataseenzyme,whereas Lys 108, 354, 390, 440, and 448 had a proximityof �10 aa to the substrate binding region critical to the accessi-bility of the androgen substrate. Overall, we observed a repeatedoccurrence in the acetylation of Lys 108,which has been shownbyother studies to be involved in the association of aromatase andcytochrome P450.

Aromatase overexpression increased estradiol levels withandrogen substrate in transiently transfected MCF-7 cells

To determine the effect of aromatase overexpression onestradiol levels, aromatase gene was cloned into pcDNA3.1(þ) vector with V5-tag either at the N- or C-terminus. MCF-7cells transfected with N-V5-aromatase plasmids over a course oftime showed peak V5-tag expression at 24 hours (Fig. 3b);however, the highest V5-tag expression was observed at 48hours in HEK293 cells (Supplementary Fig. S1C). Therefore,further transfection experiments were done at those times forthe different cell lines. Estradiol quantification in MCF-7 cellstransfected with aromatase constructs showed minimal differ-ence between empty vector and V5-aromatase transfected cells(Supplementary Fig. S1D). Upon treatment with 10 nmol/Landrostenedione (A4) substrate, there was a marked differencein estradiol levels (at least >100-fold) between the empty vectorand V5-aromatase transfected cells (both N-and C-V5 taggedaromatase) with a significant difference observed after 2 days ofincubation (Supplementary Fig. S1E).

K108 and K440 mutations altered aromatase activity in breastcancer cell lines

Acetylation has been shown by many studies to affect thefunction of an enzyme. We wanted to determine if the ability ofaromatase to convert androgen to estrogen is regulated byspecific lysines that are acetylated. Lysine 108 is conservedacross species, and according to structural analysis, is locatedin the substrate binding region. We decided to determine itsrole in regulating cancer cell production of estrogens usinglysine point mutants. Because K440 has a proximity of 3 aa tothe heme-binding region, we reasoned that any alteration ofthis residue might influence aromatase activity. In order to testthe role of these lysines, we generated K108R/K440R andK108Q/K440Q aromatase point mutants and transfectedMCF-7, T47D, and MDA-MB 231 cells with either of theplasmids. As a substitute, arginine (R) and glutamine (Q) werechosen because lysine acetylation neutralizes the positivecharge, and although, these residues are similar in length tolysine, R maintains its charge while Q is neutral. Overexpres-sion of N-V5-Aromatase constructs was confirmed by expres-sion of mRNA (Figs. 3C, 4B, and 5B), and protein (Fig. 3D, 4C,

Figure 1.

Sirtuin deacetylases regulate aromatase activity. A, Aromatase activity andprotein levels with increasing Sirtuin deacetylase inhibitor concentrations(32 nmol/L–5 mmol/L) when MCF-7 cells were treated for 4 hours. B, siRNA-mediated inhibition of SIRT-1 in MCF-7 cells decrease aromatase protein andactivity when cells were treated for 48 hours. Data represented as mean� SEM.�� , P ¼ 0.0008; �, P < 0.05 of three different experiments.






and 5C) for WT and all four point mutants in MCF-7, T47D,and MDA-MB 231 cells.

In MCF-7 cells, there was no change in E2 levels with theexpression of K108R when compared with the WT; however,there was a significant increase (P ¼ 0.0172) in E2 levels withK108Qmutant (Fig. 3E).On the other hand, a significant decrease(P¼0.0159) in E2 levelswas observedwithK440Rmutant and anincrease (P ¼ 0.0124) with K440Q mutants when matched withtheWT (Fig. 3F). In T47D cells, another ER-positive cell line, K108Rmutant caused a significant increase (P < 0.0001) in E2 levelswhereasK108Qsignificantly reduced (P¼0.0182)E2 levels relativeto the WT (Fig. 4D). Similarly, a significant increase (P < 0.0001)in E2 levels was observed with K440R mutant while a decrease

(P ¼ 0.0004) was seen with K440Qmutants when matched to theWT (Fig. 4E). In triple negative breast cancerMDA-MB 231 cells, weobserved a significant increase (P < 0.0001) in E2 levels with K108Rmutant and a decrease (P ¼ 0.0122) with K108Q relative to theWT (Fig. 5D). A similar trend was observed with K440R mutant(P ¼ 0.0117) but no difference in E2 was observed with K440Qwhen compared with the WT (Fig. 5E).

K108/K440mutations does not change subcellular localizationof aromatase protein

Acetylation has been reported to change subcellular location ofsome proteins. Here, immunofluorescence staining detected N-V5-Aromatase WT, K108R, K108Q, K440R, and K440Q in the

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Figure 2.

Aromatase protein is basally acetylated and induced by SIRT-1 inhibitor IV in cancer cells. A, Basal aromatase protein acetylation pattern in MCF-7,MDA-MB 231, and JEG3 cells with acetyl-lysine antibodies #05-515 and #9681. B, Densitometric analysis of the acetylated aromatase (Kac) relative to totalaromatase images from #05-515 pulldown assays. C, Time course of aromatase acetylation induced by inhibitor IV in MCF-7 cells. D, Induction ofaromatase acetylation with 32 nmol/L inhibitor IV in MCF-7, MDA-MB 231, and JEG3 cells when treated for 10 minutes. E, Densitometric analysis of inducedaromatase acetylation. Data represented as mean � SEM. � , P < 0.05 of three different experiments.

Molehin et al.





endoplasmic reticulum of stably transfected HEK293 cells(Fig. 6A). Endoplasmic reticulum fractionation also confirmedthe protein expression of N-V5-aromatase constructs (Fig. 6B).Colocalization studies revealed an overlap of the overexpressedaromatase N-V5-Aromatase WT and mutant proteins with cal-nexin, an endoplasmic reticulum marker protein in stably trans-fected HEK293 cells (Supplementary Fig. S3). This demonstratesthat these point mutants localize to the endoplasmic reticulum,and these specific mutations do not cause a relocation of theprotein to another subcellular compartment in these cells.

Aromatase protein might be regulated by otherposttranslational modifications

Wepredicted thatwith acetylationmimetics, aromatase activitywould decrease, andwith deacetylationmimetics, E2 levelswouldeither increase or remain unchanged. We observed a contrarytrend inMCF-7 cells and this prompted us to probe other possiblelysine PTMs that might be working in concert with acetylation toregulate aromatase. In silico analysis using ModPred tool revealedthat other lysine PTMs might be involved in the regulation ofaromatase protein expression at K108 and K440 locations (Table2). In addition, immunoprecipitation and Western blot analysesshow for the first time that aromatase protein is basally sumoy-lated,methylated, and possibly ubiquitinated inMCF-7 cells (Fig.7a). Next, we decided to investigate whether any of this PTMsplayed a role in the phenotype observed when K108R, K108Q,K440R, or K440Qwere transfected intoMCF-7 cells.We subjectedstably transfected MCF-7 cells to immunoprecipitation usingantibodies specific to sumoylated, methylated, and ubiquitinatedproteins. Interestingly, results show evidence that the N-V5-Aro-matase WT and mutant proteins were ubiquitinated (Fig. 7b),sumoylated (Fig. 7c), and methylated (Fig. 7d) in MCF-7 cells.

DiscussionNumerous biological systems depend on the proper balance

of androgens and estrogens. Aromatase uniquely has thecapacity to deplete androgen levels, while simultaneouslyboosting estrogen reservoirs. This critical conversion is thefoundation for diverse biological processes and because ofthe impact of hormonal imbalances on numerous cell types, itis perhaps not surprising that aromatase is frequently elevatedin tumors and drives hormone-dependent breast and endo-metrial tumor progression. Because of its contribution tomultiple facets of tumor progression, numerous efforts haveled to the generation of AIs that are frequently used in theclinic. In fact, clinical trials demonstrated higher benefit in

patients treated with aromatase inhibitors compared withtamoxifen (36), and recommendations have been made toincorporate an AI to reduce the risk of breast cancer recurrence(37). Although AI therapy is effective against hormone-depen-dent cancers, the problem of resistance to endocrine therapy isthe leading cause of cancer death. Despite the clear importanceof aromatase in normal and pathophysiologic settings, muchremains unknown about how it is regulated. Our findings hereprovide additional insight into its posttranslational regulationbeyond phosphorylation. Our study was prompted by twoearlier seminal observations. First, it was shown that treatmentof MCF-7 cells for 24 hours with the clinical lysine deacetylaseinhibitor, panobinostat, led to a reduction in aromatasemRNA (30). Panobinostat inhibits class I/II deacetylases, butnot the class III sirtuin deacetylases. Second, we reported thatinhibition of sirtuin-1/2 over the same time frame and longerled to decreased aromatase mRNA and protein (31). Both ofthese studies focused on the impact of higher doses of KDACinhibitors on the transcriptional regulation of CYP19A1, yetthe short-term impact of these agents was not explored. Here,we show that aromatase is also regulated by sirtuin deacety-lases, and its enzymatic activity altered when cells were treatedwith low doses of SIRT-1 inhibitor IV over a short time frame,which suggested that a posttranslational regulation of aroma-tase may account for this change and we reasoned that acet-ylation could be the key. Based on a series of acetyl-lysineimmunoprecipitation, Western blotting and LC-MS/MS anal-yses, we have demonstrated that aromatase undergoes proteinacetylation as another regulatory mechanism.

This study provides the first report of aromatase acetylationas a novel posttranslational modification and has generated thevery first map of 11 acetylation sites that are either basallypresent or induced by a preclinical SIRT-1 inhibitor IV. Six ofthese lysines were found to be conserved across the analyzedorganisms, suggesting that these lysine residues play a crucialrole in the aromatase function. Other lysine residues that didnot appear conserved among homologues but specific tohuman aromatase might be implicative of adaptive plasticityin humans. K108, located in the substrate binding region, hasbeen shown to be key in the interaction between aromatase andits obligatory binding partner NADPH cytochrome P450 reduc-tase (38), which essentially transfers electrons to the hemegroup of aromatase, during estrogen biosynthesis from andro-gen substrate, in a process known as aromatization. We foundK108 to be basally acetylated and deacetylation of K108 willerase the positive charge as will substitution of K with Q. Lysine440 is situated 3 aa away from the catalytic site buried in the

Table 1. Structural location of novel acetylated lysine residues and predicted functional role of domains in aromatase protein

20% O2 2.5% O2

Lysine Basal Induced Basal Induced Structural location Functional predictions

K108 þ þ þ Helix B Substrate bindingK169 þ þ þ þ Helix D Structural domainK242 þ þ þ þ Helix G Conformation of the active siteK262 þ Helix G Structural domainK334 þ þ þ þ Helix J Substrate bindingK352 þ þ Loop region Substrate bindingK354 þ þ Helix K Substrate bindingK376 þ þ Major b-sheet b3 Substrate bindingK390 þ Loop region Substrate bindingK440 þ Helix L Heme bindingK448 þ Helix L Heme binding






heme-binding region of aromatase protein, where aromatiza-tion takes place (39).

Historically, histone or lysine deacetylases have been asso-ciated with formation of heterochromatin and the epigeneticrepression of tumor suppressor genes (40). However, newevidence demonstrates that acetylation directly modulates theactivity of nonhistone oncoproteins (41, 42), and tumorsuppressors (43, 44). With the recent approval of the firstdeacetylase inhibitors for cancer treatment (45), and their

phase I testing for breast cancer (29), it is important to fullycharacterize additional mechanisms of tumor regression.

Herein, functional analysis of K108 and K440 residues showeda decrease in aromatase activity with K108Q and K440Qmutantswhen compared with WT in T47D and MDA-MB-231 breastcancer cells. Moreover, with K108R and K440Rmutants, E2 levelseither remained unchanged or increased as predicted in these celllines. This suggests that these lysine residues are important in thefunction of aromatase to convert androgen to estrogen and that

Bacteria ----VRTWKERR TTVQDARDYLRRV---Fish VHHVLKNGNYTS PRACIG-KHMAMVMMKBird VFHVMKHWNYVS PRGCVG-KFIAMVMMKHumans MFHIMKHNHYSS PRGCAG-KYIAMVMMKMouse MFHVMKHSHYIS PRGCAG-KYIAMVMMKRat MVHVMKHSNYIS PRSCAG-KYIAMVMMK

Substrate 108

Heme440

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b-ActinTotal arom

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A B

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EVWTK440RK440Q

*

*EVWTK108RK108Q

2,0001,8001,6001,4001,2001000

20151050E

stra

dio

l lev

els

(pg

/mL

) *FE

Figure 3.

Functional importance of conserved residues on aromatase activity by mutational studies in MCF-7 cells. A, Multiple alignment of aromatase protein sequenceacross different organisms was performed using Clustal Omega software. The acetylated lysine residues (K) at positions 108 in the substrate binding regionand 440 in the heme binding domain are conserved (red). B, Time course of N-V5–tagged aromataseWT construct expression in MCF-7 cells. C, Transcript levels ofV5-aromatase WT and mutant constructs and loading control beta actin in MCF-7 cells after 24 hours. D, Expression of N-terminally tagged V5-aromataseconstructs, endogenous aromatase (Total aromatase), and endogenous control Gapdh in MCF-7 cells untreated or treated with androstenedione for 2 days (A4–2days). E, Estradiol levels of empty vector (EV), V5-aromatase WT and K108R/K108Q-transfected cells treated with/without 10 nmol/L androstenedione for 2 days(A4–2 days). F, Estradiol levels of empty vector (EV), V5-aromataseWT and K440R/K440Q-transfected cells with/without 10 nmol/L androstenedione after 2 days(A4–2 days). Data are representative of three independent experiments carried out in triplicates with mean � SEM. � , P < 0.05.

Molehin et al.





acetylation is a novel regulator of aromatase activity at thesesites in these cells. Contrary to the observed trend in these celllines, E2 levels increased with K108Q and K440Q mutants anddecreased with K440R mutants in MCF-7 cells, this perhaps isindicative of other possible posttranslational modifications in acell-type–specific manner regulating aromatase activity. Immu-

noprecipitation analysis revealed that sumoylation, methylation,and possibly ubiquitination are another set of PTMs thatmight beregulating endogenous aromatase. Here, ectopically overex-pressed aromatase WT, K108, and K440 mutants were sumoy-lated, methylated, and to a lesser extent ubiquitinated. Cross-talkbetween PTMs are increasingly reported on many proteins to

A

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******

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Figure 4.

Aromatase constructs expressed in T47D cells alter aromatase activity. A, Time course of N-V5-tagged aromatase WT construct expression in T47D cells.B, Transcript levels of V5-aromatase WT and mutant constructs and loading control beta actin in T47D cells after 24 hours. C, Expression of N-terminally taggedV5-aromatase constructs, endogenous aromatase (Total aromatase) and endogenous control Gapdh in T47D cells untreated or treated with androstenedionefor 2 days (A4–2 days). D, Estradiol levels of empty vector (EV), V5-aromatase WT and K108R/K108Q-transfected cells treated with/without 10 nmol/Landrostenedione for 2 days (A4–2days). E, Estradiol levels of empty vector (EV), V5-aromatase WT and K440R/K440Q-transfected cells with/without10 nmol/L androstenedione after 2 days (A4–2 days). Data are representative of three independent experiments carried out in triplicates with mean � SEM.�� , P < 0.0001; � , P < 0.05.






generate a PTM code, which can be read by specific effectors topositively/negatively regulate downstream processes. Perhaps,these modifications might be playing an inhibitory role in thecase of K440R, while positively regulating aromatase activity inK108Q and K440Qmutants in MCF-7 cells. Alternatively, if thesespecific lysines undergo methylation or sumoylation modifica-tions, then changing it to Q or another residue could prevent thismodification from taking place.

Arginine residues are capable of more electrostatic interactionswhen matched with lysine residues owing to the presence of the

guanidinium group on arginine, which allows for stronger ionicinteractions, hydrogen bonds, and salt bridge formations (46).One would imagine that substituting a positively charged residuewith a neutral residue would have significant implications on thefunction of an enzyme. Also, the change in lysine residue toarginine at position 440 could subject K440R to methylation,which could have led to decreased aromatase protein expressionand subsequently decreased activity. Additional studies are need-ed to provide insight into the dynamics and complexity of PTMcross-talk on aromatase in MCF-7 cells.

EV N-V

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Figure 5.

Aromatase constructs expressed in MDA-MB-231 cells alter aromatase activity. A, Time course of N-V5–tagged aromatase WT construct expression in 231 cells.B, Transcript levels of V5-aromatase WT and mutant constructs and loading control beta actin in 231 cells after 24 hours. C, Expression of N-terminallytagged V5-aromatase constructs, endogenous aromatase (Total aromatase) and endogenous control Gapdh in 231 cells untreated or treated with androstenedionefor 2 days (A4–2 days). D, Estradiol levels of empty vector (EV), V5-aromatase WT and K108R/K108Q transfected cells treated with/without 10 nmol/Landrostenedione for 2 days (A4–2 days). E, Estradiol levels of empty vector (EV), V5-aromatase WT and K440R/K440Q transfected cells with/without 10 nmol/Landrostenedione after 2 days (A4–2 days). Data are representative of three independent experiments carried out in triplicates with mean � SEM. �� , P < 0.0001;� , P < 0.05.

Molehin et al.





Here, we show that in T47D and MDA-MB-231 cells, K108Qand K440Q reduced ability of aromatase enzyme to convertandrogens to estrogens. Mutational studies by Hong and collea-gues (38) showed that K108Q decreased aromatase activity.Although they did not investigate the regulation of aromatase byacetylation, they observed a marked decline in aromatase activitywith K108Qmutants in Chinese Hamster Ovary (CHO) cells andconcluded that the substrate binding to the enzyme was unhin-dered. However the binding of aromatase to its partner was

altered; hence, a reduced electron transfer and ultimatelydecreased enzymatic activity. In this study, we did not assess theimpact of this mutation on the interaction between aromataseand reductase. However, our results are consistent with the effectof K108Q mutation on aromatase activity as seen in Hong'sstudy (38). Overall, we demonstrate that alteration of K108and K440 residues decreased aromatase activity in T47D andMDA-MB 231 cells.

Recent studies demonstrated that combination therapiesincluding KDIs are effective in sensitizing AI-resistant tumors andbreast cancer cells (27, 47). Previously, we showed that inhibitionof sirtuin deacetylases caused reduced aromatase protein (31).Wenow demonstrate that at significantly lower doses, SIRT-1 inhi-bition impacts aromatase acetylation which concomitantly influ-ences estrogen biosynthesis by regulating aromatase activity.Finally, there is evidence that aromatase (48, 49) and SIRT-1(50, 51) are regulated in an oxygen dependent manner and mayprovide meaning to the difference in some acetylation sites underphysiologic versus atmospheric O2 culture conditions. Overall,we now know that aromatase is regulated at yet another level andtreatment with specific KDIs may impact both its transcription

N-V5-AROMEV DAPI/AROMDAPI/EV

N-V5-K108R N-V5-K108QDAPI/K108R DAPI/K108Q

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Figure 6.

K108/K440 mutations do not change the subcellularlocalization of aromatase. A, Representativefluorescence images of N-V5-aromatase (magenta) instably transfected HEK293 cells with empty vector (EV),N-V5-aromatase (N-V5-AROM), K108R mutant (N-V5-K108R), K108Q mutant (N-V5-K108Q), K440R mutant(N-V5-K440R), and K440Q mutant (N-V5-K440Q)proteins are shown. Merge of nuclear staining withDAPI (blue) and N-V5-aromatase (magenta) proteins isshown as DAPI/AROM for each of the mutant. B,Endoplasmic reticulum fractions (ERec extract) and totalcell extract of HEK293 cells stably transfected withWT and mutant aromatase constructs showing V5-aromatase, calnexin, and Gapdh endogenous control.

Table 2. K108 and K440 residues are subject to other PTMs basally and whensubstituted with arginine residues

Residue PTM modification ModPred score Confidence

K108 Ubiquitination 0.54 lowAcetylation 0.53 low

K108R ADP-ribosylation 0.53 lowK108Q Not modifiedK440 Acetylated 0.91 High

Ubiquitinated 0.61 lowK440R Methylation 0.79 MediumK440Q Not modified






and acetylation. Thus, understanding the gaps in our knowledgeregarding an important but poorly understood posttranslationalaromatase regulation could help identify novel therapeuticvulnerabilities.

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

Authors' ContributionsConception and design: D. Molehin, P.R. Manna, K. PruittDevelopment of methodology: D. Molehin, I. Castro-Piedras, P.R. Manna, K.PruittAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Molehin, M. Sharma, S.R. Sennoune, D. ArenaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Molehin, P.R. Manna, K. PruittWriting, review, and/or revision of themanuscript:D.Molehin, I. Castro-Piedras,M. Sharma, S.R. Sennoune, D. Arena, P.R. Manna, K. Pruitt

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Molehin, K. PruittStudy supervision: K. Pruitt

AcknowledgmentsThis research was funded by the National Institutes of Health (NIH)

grant [CA155223 to K. Pruitt] and Cancer Prevention Research Institute ofTexas (CPRIT) Recruit of Rising Stars Award to K. Pruitt. We would liketo thank Edgar G. Martinez for his technical support.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 17, 2018; revised May 7, 2018; accepted June 11, 2018;published first June 19, 2018.

A

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Figure 7.

Endogenous and exogenous V5 aromatase are modified by PTMs. A, Basal aromatase (Arom) protein ubiquitination, sumoylation, and methylationpattern and light chain (IgG-Lc) in MCF-7 cells. B, Ubiquitinated V5-aromatase (Ub-V5), light chain (IgG-Lc), total V5-aromatase (V5-arom), and Gapdhendogenous control (gapdh) in stably transfected MCF-7 cells. C, Sumoylated V5-aromatase (Sumo-V5), light chain (IgG-Lc), total V5-aromatase(V5-arom) and Gapdh in stably transfected MCF-7 cells. D, Pan-methylated V5-aromatase (Kme-V5), light chain (IgG-Lc), total V5-aromatase (V5-arom),and Gapdh in stably transfected MCF-7 cells.

Molehin et al.





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Molehin et al.




2018;16:1530-1542. Published OnlineFirst June 19, 2018.Mol Cancer Res Deborah Molehin, Isabel Castro-Piedras, Monica Sharma, et al. Sirtuin InhibitionAromatase Acetylation Patterns and Altered Activity in Response to

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