Mouse Models of Overexpression Reveal Distinct Oncogenic … · Genome and Epigenome Mouse Models of Overexpression Reveal Distinct Oncogenic Roles for Different Type I Protein Arginine

Genome and Epigenome

Mouse Models of Overexpression Reveal DistinctOncogenic Roles for Different Type I ProteinArginine MethyltransferasesJianqiang Bao1, Alessandra Di Lorenzo1, Kevin Lin1,Yue Lu1,Yi Zhong1, Manu M. Sebastian1,William J. Muller2, Yanzhong Yang3, and Mark T. Bedford1

Abstract

Protein arginine methyltransferases (PRMT) are generallynotmutated in diseased states, but they are overexpressed in anumber of cancers, including breast cancer. To address thepossible roles of PRMT overexpression in mammary glandtumorigenesis, we generated Cre-activated PRMT1, CARM1,and PRMT6 overexpression mouse models. These threeenzymes are the primary type I PRMTs and are responsiblefor the majority of the asymmetric arginine methylationdeposited in the cells. Using either a keratin 5-Cre recombi-nase (K5-Cre) cross or anMMTV-NICmouse, we investigatedthe impact of PRMT overexpression alone or in the context ofa HER2-driven model of breast cancer, respectively. Theoverexpression of all three PRMTs induced hyper-branchingof the mammary glands and increased Ki-67 staining. Whencombined with the MMTV-NIC model, these in vivo experi-ments provided the first genetic evidence implicating elevat-ed levels of these three PRMTs in mammary gland tumori-genesis, albeit with variable degrees of tumor promotion and

latency. In addition, these mouse models provided valuabletools for exploring the biological roles and molecularmechanisms of PRMT overexpression in the mammarygland. For example, transcriptome analysis of purified mam-mary epithelial cells isolated from bigenic NIC-PRMT1Tg andNIC-PRMT6Tg mice revealed a deregulated PI3K–AKT path-way. In the future, these PRMTTg lines can be leveraged toinvestigate the roles of arginine methylation in other tissuesand tumor model systems using different tissue-specific Crecrosses, and they can also be used for testing the in vivoefficacy of small molecule inhibitors that target these PRMT.

Significance: These findings establish Cre-activated mousemodels of three different argininemethyltransferases, PRMT1,CARM1, and PRMT6, which are overexpressed in humancancers, providing a valuable tool for the study of PRMTfunction in tumorigenesis.

See related commentary by Watson and Bitler, p. 3

IntroductionIn mammals, arginine methylation is catalyzed by a family of

nine related protein arginine methyltransferases (PRMT); thisposttranslational modification is implicated in a multitude ofbiological processes, including cell growth, proliferation, differ-entiation, and transformation. The PRMTs are classed into threetypes, based on the chemical modification they deposit. Type Ienzymes generate asymmetrical di-methylarginine (ADMA)marks (PRMT1, 2, 3, 4, 6, 8); Type II enzymes deposit symmetricaldi-methylarginine (SDMA) marks (PRMT5 and 9); Type IIIenzymes catalyze the mono-methylarginine (MMA) mark(PRMT7; ref. 1). Most PRMTs methylate glycine/arginine-rich

(GAR)motifs, but the exceptions are PRMT4 (also called CARM1)that targets an ill-defined proline-rich motif in its substrates, andPRMT7 that recognizes a RXR motif.

Deregulated PRMT expression (particularly overexpression ofPRMT1, 4, 5, and 6) has been well documented in a number ofsolid and hematological malignancies (1–3). PRMT1 is themajortype I enzyme that is responsible for >85% of ADMA deposition(4, 5). PRMT1 is overexpressed in breast cancer tumor samples, ascompared to the adjacent normal tissue, and the degree ofexpression correlates with tumor grade (6, 7). PRMT1 is not onlyupregulated, but also aberrantly spliced in breast cancer, and theoverexpression of certain splice variants is tightly correlated withpoor disease prognosis (8). CARM1 displays elevated expressionlevels in grade 3 breast cancer tumors where it likely functions as acoactivator of the E2F1-regulated transcription (9). It also worksclosely with the AIB1 (amplified in breast cancer 1) in the contextof estrogen stimulated cell proliferation (10). Furthermore, ele-vated CARM1 activity promotes migration and metastasis ofbreast cancer cells in in vitro and in vivo models (11). Recently,in the context of ovarian cancer, it was found that CARM1 over-expression tumors are sensitive to EZH2 inhibitors (12), thusidentifying the first therapeutic vulnerability for CARM1-driventumors. PRMT5 and its cofactorMEP50 are essential for regulatingtranscriptional programs that promote invasive phenotypes inbreast carcinoma cells and other cancers (13). Finally, there are anumber of reports that support an association of high PRMT6

1Department of Epigenetics and Molecular Carcinogenesis, The University ofTexas MD Anderson Cancer Center, Smithville, Texas. 2Department of Biochem-istry, Rosalind and Morris Goodman Cancer Research Centre, McGill University,Montreal, Quebec, Canada. 3Department of Cancer Genetics and Epigenetics,Beckman Research Institute at City of Hope, Duarte, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Mark T. Bedford, MD Anderson Cancer Center, Smith-ville, TX 78957. Phone: 512 237 9539; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-1995

�2018 American Association for Cancer Research.

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expression levels with poor breast cancer prognosis. Two studiesfound that high levels of PRMT6 expression correlate with poorpatient prognosis (7, 14), and a third study reported a similarcorrelation when looking at PRMT6-dependent gene expressionsignatures (15).

The remaining five PRMTs have not been strongly linked tocancer. Indeed, PRMT2 has weak type I activity (16). PRMT3methylates GAR motifs, like PRMT1 and PRMT6, but its primarysubstrate is the ribosomal protein rpS2, and not histones or RNAbinding proteins (17). PRMT7 is the only type III enzyme, and it isalso the only PRMT to be mutated in a disease setting, namely inan intellectual disability syndrome called SBIDDA (18). PRMT8 isvery similar in primary sequence to PRMT1, but it is membrane-associated through an N-terminal myristoylation modificationand displays neuron-restricted expression (19). PRMT9 is a type IIenzyme that seems largely dedicated to methylating the SAP145splicing factor (20).

Here, we generated three independent transgenic mouse linesto address the effects of type I PRMT overexpression in different invivo settings. The forced overexpression of human PRMTs(PRMT1, CARM1, and PRMT6) was achieved by Cre-mediatedremoval of a floxed STOP cassette. Two independent Cre driverswere used. First, we crossed the three PRMT transgenic mice to aK5-Cre line,which induced PRMTexpression broadly in epithelialtissues including the basal cells of the mammary epithelium.Second, the three lines were crossed with the MMTV-NIC(Neu-IRES-Cre) mouse, which harbors Neu/ErbB2/HER2 underthe control of a MMTV promoter, has Cre recombinase coex-pressed with the oncogene, and provides a tight system for theanalysis of factors that synergize with HER2 (21).

K5-driven conditional overexpression of Prmt1, Carm1, andPrmt6 led to the hyperbranching of mammary ducts and epi-thelial hyperplasia at different time points with varied degree.Additionally, spontaneous mammary tumors were observed inthe aged Prmt1- and Carm1-overexpressing mammary glands.In the Neu-induced oncogenic context, overexpression of Prmt1and Prmt6 significantly accelerated mammary tumor onset,whereas Carm1 augmented the tumor progression exclusivelyupon tumor initiation. These data demonstrate that all threetype I PRMTs possess oncogenic activity that predisposed themouse mammary gland to tumor development, and supportthe therapeutic targeting of these PRMTs for the treatment ofpatients with breast cancer.

Materials and MethodsMice

The full-length cDNA sequences of open reading frame(ORF) for human PRMT1 (variant 1) and CARM1 were insertedin-frame into the multiple cloning site (MCS) of pCAG-floxedSTOP-3XFlag-MCS-IRES-GFP plasmid backbone. HumanPRMT6 ORF was inserted in-frame into the pCAG-floxedSTOP-3XFlag-MCS vector without GFP tagging. The constructedplasmids encoding all three PRMT ORFs were sequenced toascertain mutation-free, and PRMT protein overexpression wasverified through transient transfection into 293T cells with thePRMT plasmids and Cre plasmid, followed by Western blotanalysis using Flag antibody. To generate the PRMT overexpres-sion transgenic mice, the respective plasmids were introducedinto the pronucleus of the zygotes (FVB/Nhsd substrain back-ground) through microinjection. Founder pups were geno-

typed through PCR using the tail clip to examine the germlinetransmission. Primers are listed in Supplementary Table S1. Allmouse experiments were approved by the Institutional AnimalCare and Use Committee.

The inducible overexpression of Flag-tagged PRMTs in theepithelial cells of skin and mammary glands was initially con-firmed by crossing with keratin 5 promoter–driven Cre (K5-Cre)mouse and Western blot analysis of the bigenic K5-Prmt mousemodels (maleK5-Cremicewere crossedwith female PrmtTgmice).The K5 promoter is activated in utero (E13.5) in epithelial pre-cursor cells (22), and removal of the STOP cassette at this earlystage results in ectopic expression (driven by the CAGGS pro-moter) of the PRMTs in epithelial cells of the skin and mammaryglands in adults.

TransgenicNICmice [MMTV-NEU(NDL2-5)-IRES-CRE] weregenerated and characterized as previously described (21). Toeliminate the high variability of tumorigenic onset due togenetic background, NIC transgenic mice used in this studywere maintained in the inbred FVB/N background (23, 24).Tumor onset and progression was monitored by manual pal-pation bi-weekly starting from 10 weeks of age. Following thetumor onset by palpation, the mice were killed 6 weeks there-after, except when the tumor size reached the maximal burdenin length (1.5 cm) allowed by our protocol. Primary tumorvolume burden was monitored by caliper measurements onlive sedated mice twice a week for six consecutive weeks aftertumor onset, and the tumor volume was estimated with theformula: volume ¼ L � S2 � p/6 (mm3), where L was thelongest dimension whereas S represents the shortest dimen-sion. Tumor-free survival was analyzed using the Kaplan–Meierapproach, and the statistical significance was determined by alog-rank (Mantel–Cox) test using Prism software.

All mice were housed through 12-hour light/12-hour darkcycles with free access to food and water. All animal studies wereapproved by the Institutional Animal Care and Use Committee atUT MD Anderson Cancer Center.

Purification of mammary epithelial cellsMouse mammary epithelial cells (MEC) were purified follow-

ing the procedures published as previously described with minormodifications (25). Briefly, #3, #4, and #5mammary glands wereremoved from the mice, free of muscle tissue contamination. Thelymph lode from the #4 inguinal mammary gland was carefullyexcised under a dissection microscope. The mammary glandtissues were then manually minced to pieces (1 mm � 1 mm),followed by the digestion in Collagenase IV (1 mg/mL CollagenaseIVþDNase I in DMEM/F12) medium at 37�C for 2 hours. Afterwashing three times inDMEM/F12medium, enrichedMECswerefinally collected by pulse centrifuge.

Western blot analysisMice were anesthetized by CO2, and tissues were freshly har-

vested. Primary tissues were freshly collected and flash-frozen inliquid nitrogen and stored at �80�C. When ready, tissue lysateswere prepared on ice by an electronic homogenizer in the RIPAlysis buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1%Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 2 mmol/LEDTA plus 1Xprotease inhibitor; Roche). Protein concentrationsof the cleared tissue lysates were determined by Bradford assay(Thermal Fisher). Protein samples were separated in 10% to 12%fresh SDS-PAGE gels and semi-dry transferred to a 0.45 mmPVDF

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membrane. After blocking in 5% skim milk for 1 hour at roomtemperature, the blots were incubated with the primary antibo-dies as indicated at 4�C overnight. The next day, the blots wereincubated for 1 hour with the HRP-conjugated secondary anti-bodies (IgG, HþL; GE Healthcare), before being subjected tovisualization using the enhanced chemiluminescence (Amer-sham). HRP-conjugated anti-mouse secondary antibody (lightchain-specific; Jackson ImmunoResearch) was used in order toeliminate the nonspecific bands for mouse Flag antibody. Theprimary antibodies used were: PRMT1 (CST:E5A8F, 1:1,000 dilu-tion), CARM1 (Bethyl:A300-421A, 1:1,000 dilution), PRMT6(Bethyl: A300-929A, 1:1,000 dilution), mouse monoclonal FlagM2 antibody (Sigma, F3165: 1:5,000 dilution), rabbit Flag anti-body (Sigma: F7425, 1:1,000 dilution), phospho-AKT (pAKTS473;D9E; CST: 1:1,000 dilution), AKT (C67E7; CST: 1:1,000 dilution),CCND1 (ab134175; Abcam: 1:2,000 dilution), CCND2 (D52F9;CST: 1:500 dilution), PIK3R3 (GTX107779; GenTex: 1:500 dilu-tion), MAT2A (A304-279A; Bethyl: 1:500 dilution).

Mammary gland whole mount preparationMice were anesthetized by CO2, followed by cervical disloca-

tion. The inguinal mammary glands (#4 pair) were carefullydissected out, mounted on slides, and fixed in 10% formalinsolution for 24 hours. Mammary glands were stained in 0.2%Carmine alum solution overnight at room temperature, followedby a series of washing in gradient ethanol solution (70%, 95%,and 100%) for 1 hour each. Fat tissue was cleared in Xyleneovernight, andwholemountwas stored inmethyl salicylate bufferuntil imaging.

Quantification of mammary intraepithelial neoplastic lesionsThe number four inguinal tumor-free mammary glands from

the NIC and NIC-Prmt bitransgenic animals were freshly har-vested and fixed in 10% formalin solution. The designation andevaluation of mammary intraepithelial neoplasias (MIN) andadenocarcinomas is in accordance with Annapolis guidelines(26, 27).MINs are defined as the focal epithelial lesions harboringcells with atypical cytology and/or organization. Those cells arepotentially capable of undergoingmalignant transformation, andthus are linked to breast cancer. To identify MINs, hematoxylinand eosin stained sections (4mm thickness) of one inguinalmammary gland from each of five mice in each of the fourgenotypeswere analyzed, and total number ofMINswere countedfrom the entire section. The total number of MINs from inguinalmammary glands of animal cohorts were compared among fourgenotypes. Statistics were performed with unpaired two-tailedStudent t test.

RT-qPCRMECs were collected as described above. Total RNA was

extracted using TRIzol reagent following the manufacturer'smanual. Two micrograms of total RNA was employed forreverse-transcription using the first-strand cDNA Synthesis Kit(Invitrogen) for each sample in a total of 20 mL reactionvolume. Quantitative PCR was performed using 10 ng cDNAtemplate in biological triplicates using a SYBR Green-basedsystem (Bio-Rad). Data were normalized by the expressionlevels of actin and were analyzed using the DCt method. Primersare listed in Supplementary Table S1.

Hematoxylin and eosin stainingThe dorsal skin or mammary glands were dissected carefully

and fixed in 10% formalin solution for 48 hours at 4�C.Hematoxylin and eosin (H&E) staining on paraffin-embeddedsections was performed following the standard protocol asdescribed previously (28).

ImmunohistochemistryFreshly harvested tissues were immediately fixed in 10% for-

malin solution for 24 to 48 hours at room temperature. Paraffin-embedded sections with 10-mm thickness were deparaffinized inxylene and rehydrated through graded alcohols. Antigen retrievalwas performed in 10mmol/L citrate buffer (pH 6.0). Endogenousperoxidase activity was blocked with 3% H2O2 for 10 minutes.Standard IHC staining was applied using the primary antibodiesagainst K5 (1:500; Covance), K8 (1:200; Developmental StudiesHybridomaBank), K14 (1:500;Covance), ER (1:500; SantaCruz),PR (1:400; Santa Cruz), HER2 (1:500; Santa Cruz), Flag (1:5,000;Sigma), phospho-AKT (pAKTS473; D9E; 1:1,000; CST), and Ki67(1:250; Bethyl Lab). Following extensive washing, HRP-conjugat-ed secondary antibodies were incubated with the sections for 30minutes at room temperature. Signals were visualized usingTablet DAB Monitoring Staining Kit (Sigma). For quantificationof Ki-67 staining, slides were scanned at �20 using an AperioScanScope (Aperio). Algorithms within Spectrum software(Aperio) were used at the default settings to score staining, andthe Ki-67 index was determined as the percentage of cells withKi67-positive nuclear immunostaining among the total numberof cells. The slides with CK5, CK8, CK14 staining were scored forthe staining intensity defined ranging among 0 to 3, for a score as"0" (no staining), "1" (weakly stained), "2" (moderately stained),and "3" (strongly stained). The slides with ER, PR, and HER2staining were scored for intensity according to the criteria asdescribed before (29).

RNA-seqTotal RNAs purified from the MECs were treated with DNase I

from three biological replicates for each genotype. 1.5 mg of totalRNAs was exploited to generate RNA-seq libraries using theTruSeq RNA Library Prep Kit v2 (Illumina) following manufac-turer's protocols, and were sequenced using 2 � 75 bases paired-end protocol on a HiSeq 2000 instrument (Illumina). Note thatoneNIC sample and oneNIC-Prmt6Tg sample were subsequentlyremoved in the later analysis due to a quality problem. For othersamples that passed our quality control, 28 to 39 million pairs ofreads were generated per sample, and were mapped to the mousegenome (mm10) by TopHat (version 2.0.10). The overall map-ping rates were between 95% and 96%. Ninety-three percent offragments had both ends mapped to the mouse genome. To callthedifferentially expressed genes (DEG), thenumber of fragmentsfor each known gene from RefSeq database (UCSC GenomeBrowser on July 17, 2015) was enumerated using htseq-countfrom HTSeq package (version 0.6.0). Genes with less than 10fragments in all samples were removed before differential expres-sion analysis. The differential expression between conditions wasstatistically assessed by R/Bioconductor package DESeq (version1.18.0). Genes with FDR <0.05, fold change >2 and length >200bp were called as DEGs. To generate heatmaps, hierarchicalclustering was performed on the union of DEGs from any ofthree comparisons (NIC-Prmt1Tg vs. NIC, NIC-Carm1Tg vs. NICand NIC-Prmt6Tg vs. NIC) using the log2 ratio values of each

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sample to the average of NIC samples by hclust function in Rpackage. Euclidean distance and ward clustering parameters wereadopted to construct the dendrograms against genes and samples.The heatmaps were plotted by heatmap.2 function in R. RNA-seqdata were deposited in the gene expression omnibus database inNCBI (GEO number: GSE111624).

Statistical analysisAll experiments were biologically repeated at least three to six

times and data were illustrated as average � SD unless otherwisestated. Kaplan–Meier tumor-free curves were calculated using thePrism software (v7)with pairwise comparisons using the log-ranktest. For other statistical analyses, P values were calculated usingunpaired Student t test or by two-way ANOVA test, unlessotherwise stated. Statistic differences were deemed as signifi-cant if P < 0.05 (�) and very significant if P < 0.01 (��).

ResultsEstablishment of conditional PRMT-overexpressing mousemodels

Numerous studies have implicated PRMT1 (6–8), CARM1(9–11), and PRMT6 (7, 14, 15) overexpression in the promo-tion of breast cancer cell migration and metastasis, and in poor

disease prognosis. To specifically interrogate the roles of thesethree PRMTs in mammary gland tumorigenesis, we generatedthree conditional overexpression alleles, in which Flag-taggedORFs of three human PRMTs were inserted following a floxedSTOP cassette driven by the CAGGS promoter, which facilitatesrobust and ubiquitous expression (Fig. 1A). In the case of PRMT1,the v1 splice variant was used. All three conditional targetingconstructs were injected into the pronucleus of zygotes(FVB background). PCR-mediated genotyping identified twofounder lines for PRMT1 (PRMT1Tg), three founder lines forCARM1 (CARM1Tg), and two founder lines for PRMT6(PRMT6Tg). These lines were further crossed with WT mice (FVBbackground) to generate the F1 offspring in order to ensure thestable inheritance.

To direct the targeted overexpression of PRMTs in the murinemammary gland, we first used the widely studied bovine K5-cremodel, in which Cre faithfully recapitulates the in vivo keratin 5expression pattern in the basal cell layer of stratified epithelium ofthe mammary gland, thymus and skin (30). To verify the condi-tional expression of transgenic PRMTs in the epithelium, weinterbred male K5-cre mice with the F1 PRMTTg females togenerate bigenic K5-cre/PRMTþ/Tg F2 offspring (referred to asK5-PRMTTg hereafter). To confirm the induced PRMT overexpres-sion of the transgenic vector, we used the aFlag antibody to

Figure 1.

Generation and validation ofconditional transgenic overexpressionof PRMTs.A, Schematic diagramof thevector design strategy for conditionalPRMT overexpression. The full ORFs ofPRMT1 and CARM1 were cloneddownstream of 3XFlag and upstreamof IRES-GFP-PolyA cassette. Thedesign for PRMT6 was similar, but thisvector did not harbor an IRES-GFPcassette. A floxed STOP signalelement was placed downstream of aubiquitous, synthetic chicken b-actinpromoter (CAGGS). When crossedwith CRE-expressing mouse lines, theSTOP cassette will be removed, andthe CAG promoter will drive the highexpression of Flag-tagged PRMTsfusion proteins. B, Relative mRNAexpression of PRMT1, CARM1, andPRMT6 by qRT-PCR, using RNAisolated from the skin and purifiedMECs from PRMTTg and K5-PRMTTg

littermates. Data were calculated fromthree biological replicates. C,Westernblot analysis of PRMT1, CARM1, andPRMT6 expression in the purifiedMECs isolated from PRMTTg andK5-PRMTTg. Asterisks, nonspecificprotein bands. Triangles, expectedprotein bands. Note that CARM1displays a smear and not a sharp band,which is likely due to extensiveposttranslational modifications, likeglycosylation and O-GlcNAcylation, asreported previously (49, 50).

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Figure 2.

Phenotypic and histologic analyses of K5-PrmtTg mice. A,Whole mount images of the mammary glands by Carmine alum staining from the nulliparous females asindicated at different ages. Note the hyperbranching of the mammary ducts observed in the virgin bigenic K5-PRMTTg female mice, starting from differenttime points (left). Right, H&E staining of themammary duct epitheliumwith different genotypes as labeled. B, Left, H&E staining showing the MIN lesions in the non-palpable, nulliparous females of all three bigenic K5-PRMTTg models as indicated. Right, immunostaining of the epithelium by Ki-67 showing the proliferationstatus of the MIN lesions. Scale bar, 100 mm.






Figure 3.

Analyses of the tumors induced by HER2 and PRMT overexpression. A, Female PRMTTgmice were crossed with male MMTV-NIC (NEU-IRES-CRE) mice, in which theMMTV promoter drives the simultaneous expression of the activated NEU (NDL2-5) oncogene and the Cre enzyme in the same epithelial cells. NulliparouspureNIC andbigenicNIC-PRMTTg femaleswere selected for subsequent studies.B,Kaplan–Meier curves of tumor-free survival in the cohorts of virginNIC andbigenicNIC-PRMTTg females. T1/2 indicates the agewhen 50%ofmice developed tumors by palpation. TheP valueswere calculated by log-rank test.C,Histologic analyses byH&E staining and immunostaining using the Flag antibody on nonpalpable mammary epithelium. The average total number of MINs were counted from fivemice for each genotype. � , P < 0.05; Student t test. Scale bar, 60 mm. D, Histologic analyses by H&E staining and immunostaining of Flag antibody inparaffin-embedded sections of mammary tumors collected from the end-point mice. Simultaneous overexpression of individual PRMTs in the HER2-inducedmammary tumors led to the phenotypic HER2 characteristics showing the solid nodular growth with intermediate cells. Scale bar, 120 mm.

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perform Western blot analysis on skin and thymus tissues, andIHC andH&E staining on skin, from F2 females at 4 weeks of age.Using these approaches, we successfully validated the generationof Founder lines for PRMT1 (line #4: Supplementary Fig. S1A–S1C), CARM1 (lines #6 & #9: Supplementary Fig. S2A–S2C), andPRMT6 (line #4: Supplementary Fig. S3A–S3C), all of whichexhibit the expected overexpression of the Flag-tagged PRMTs inthe epithelial tissues of the bigenic K5- PRMTTg females, whereasFlag signal was not detectable in the single PRMTTg females.Consistent with Western blot results, the IHC staining revealedrobust transgenic tagged-PRMT signal in the basal layer of skinepidermis exclusively in the bigenic K5-PRMTTg females, but notin single PRMTTg females, for PRMT1 (Supplementary Fig. S1B),CARM1 (Supplementary Fig. S2B), and PRMT6 (SupplementaryFig. S3B), indicating that we have successfully generated condi-tional overexpression transgenic mouse models for PRMT1 (#4),CARM1 (#6 and #9), and PRMT6 (#4). The line #9 exhibits higherexpression levels of CARM1 than line #6, and we thus chose line#9 for the subsequent CARM1-related studies.

Overexpression of PRMTs in the mammary gland leads tohyperproliferative phenotypes

To determine the extent to which the three PRMTs are over-expressed in comparison to their respective endogenous geneexpression, we performed qPCR analyses on RNA isolated fromepidermis tissue of the skin (generated by dorsal skin scraping)and purified MECs. These samples were purified from femalemice at 6 weeks of age. The mRNA levels for three PRMTs werenearly three- to five-fold higher in mammary epithelium andskin of bigenic K5-PRMTTg females as compared with those inthe single PRMTTg females (Fig. 1B). To specifically explore theprotein overexpression levels of transgenic PRMTs relative toendogenous PRMTs, we carried out the Western analysis usingspecific PRMT antibodies on protein samples extracted fromMECs (Fig. 1C). PRMT1, CARM1, and PRMT6 protein levelswere clearly elevated in the respective bigenic K5-PRMTTg

mouse models (Fig. 1C).All threemodels of virgin bigenic K5-PRMTTg female pups grow

and develop normally, and are indistinguishable from the singlePRMTTg littermates when they are young. However, we observedover side-branching of themammary glands from the nulliparousK5-PRMT1Tg females starting at 4 months of age (Fig. 2A, left).H&E staining revealed the hyperplasia of the mammary glandepithelium (Fig. 2A, right). This phenomenon was also observedfor the K5-PRMT6Tg females when they reached 6 months old(Fig. 2A, bottom). In contrast, hyperbranching was not noticedin the mammary glands of K5-CARM1Tg females until theyreached �18 months (Fig. 2A, middle), a time point that issignificantly delayed when considering the hyperbranching onsetin the K5-PRMT1Tg and K5-PRMT6Tg females. The rapidly prolif-erating epithelial cells were evidenced by the Ki-67 staining,and overexpression eventually promoted the developmentof MIN in the aged bigenic K5-PRMTTg females of all threemodels (Fig. 2B). Quantification indicated that MIN lesions inK5-Prmt1Tg, K5-Carm1Tg and K5-Prmt6Tg displayed a Ki-67 indexof 63, 47, and 54, respectively. Moreover, we observed sponta-neous mammary tumors that occur at higher rates in the agedbigenic K5-PRMT1Tg (in 6 of 28 mice) and K5-CARM1Tg (in 8 of22 mice) females (with a median onset for both >20 months),when compared with the sporadic background incidence in thesingle PRMTTg female littermates (Supplementary Fig. S4A). An

increased incidence of spontaneous mammary gland tumors wasnot observed in the aged K5-PRMT6Tg bigenic line. To identify thehistological subtypes of those spontaneous tumors,we performedIHC using a panel of markers on three independent mammarygland tumors (only two are shown in Supplementary Fig. S4B)from the two lines that produced an increased incidence ofspontaneous mammary tumors. Based on IHC scoring, tumorsin the K5-CARM1Tg females display carcinoma feature of lumi-nal cell origin (K8þ/K5�), whereas K5-PRMT1Tg tumors exhibitboth the basal and luminal features (Supplementary Fig. S4C).In addition to the mammary gland tumors listed above, the K5-CARM1Tg aged females also presented with a high incidence ofspontaneous skin tumors (in 9/22 mice). These data suggestthat the three PRMTs play distinct roles in predisposing epi-thelial tissues to transformation.

Distinct oncogenic roles of PRMT1, PRMT6, and CARM1 in thecontext of the NIC mouse model

The long latency of tumor onset in the K5-PRMT1Tg and K5-CARM1Tg females and the incomplete penetrance of tumor for-mation implicate that overexpression of PRMT1 or CARM1 itselfmight not be a major breast cancer-initiating event, which isconsistent with the notion thatmammary tumorigenesis involvescooperation of oncogenic events (31, 32). Recent IHC studies onalmost 250 breast cancer samples revealed that CARM1 is over-expressed in both the HER2 subtype (69%) and TNBC subtype(57%; ref. 33), and the positive correlation between CARM1 andHER2 expression was also observed in a second large study (34).Furthermore, detailed examination of TCGA dataset demonstrat-ed that both CARM1 and PRMT1 genes exhibit higher mRNAexpression levels in the HER2 high-expressing breast tumor sam-ples as compared with the HER2 low-expressing tumors (Sup-plementary Fig. S5). We thus chose to investigate the effects ofmodulating PRMT levels in a HER2-drivenmodel of breast cancer(HER2 is also known as ERBB2/Neu; Fig. 3A). This modelexpresses the activated form of Neu under the control of a MMTVpromoter, and has Cre recombinase coexpressed with the onco-gene due to an internal ribosome entry sequence, thus the nameMMTV-NIC (Neu-IRES-Cre; ref. 21). This setup provides a tightsystem for the analysis of factors that cooperate or synergize withHER2. Importantly, by crossing the MMTV-NIC mouse with thedifferent PRMTTgmice, the same cells will overexpress both HER2and the PRMT of interest.

PRMTTg female mice were crossed with male MMTV-NIC mice,and cohorts of monogenic and bigenic females were retainedand aged (Fig. 3A). As consistently reported, nulliparous MMTV-NIC females developed multi-focal, signature nodular adenocar-cinomas in the mammary glands with 100% penetrance. Com-pared with the average tumor latency of MMTV-NIC females,bigenicNIC-PRMT1Tg andNIC-PRMT6Tg virgin females displayedaccelerated tumorigenic onset, with T50 being decreased by�2 weeks and �three weeks, respectively (Fig. 3B). In contrast,bigenic NIC-CARM1Tg females exhibited delayed tumorigenesiswith T50 being increased by�2 weeks, suggesting that these threePRMTs play distinct roles in the HER2-induced tumorigeniccontext (Fig. 3B). Histologic analyses revealed that the averagenumber of the MIN lesions is elevated in the mammary glands ofNIC-PRMT1Tg and NIC-PRMT6Tg females, but not in NIC-Carm1Tg females, as compared to their MMTV-NIC female litter-mates, respectively (Fig. 3C). All three models of nulliparousbigenic females eventually developed multifocal, nodular






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carcinoma (Fig. 3D, left). Interestingly, whereas �100% of thetumorigenic cells displayed the robust Flag-tag staining in theNIC-CARM1Tg and NIC-PRMT6Tg females, �45% of tumor cellsshowed Flag staining in theNIC-PRMT1Tg females (Fig. 3D, right).The underlyingmechanism that contributed to this phenomenonis not clear, but could be due to epigenetic silencing of the PRMT1transgenic locus after or during tumor development.

In addition, we followed up the earlier tumorigenic events bycounting the average number of focal lesions in the whole-mount mammary glands in the bigenic females prior to theformation of palpable mammary tumors. The NIC-PRMT1Tg

and NIC-PRMT6Tg females displayed a higher than averagenumbers of MINs, as compared with that of NIC females at�12 and �15 weeks of age, unlike NIC-CARM1Tg females(Supplementary Fig. S6A). Unexpectedly, the average numberof focal tumor nodules is higher in the NIC-CARM1Tg females,compared with the NIC-PRMT1Tg and NIC-PRMT6Tg females atthe experimental end-point (Supplementary Fig. S6B). Detailedrecording of the cumulative tumor volume showed that tumorsfrom NIC-CARM1Tg females are smaller than those from NICfemales at the onset of tumorigenesis, but rapidly caught upand exceeded the volume of tumors from NIC females after�24 weeks (Supplementary Fig. S6C). To explore whether thevariability of tumor growth behaviors was due to the variedprotein overexpression levels of the respective PRMTs, weperformed Western analysis with a Flag antibody that detectedthe ectopic expression level of all three PRMTs simultaneously.As shown in Supplementary Fig. S6D, ectopic PRMT expressionwas observed in all the tumor tested (two independent tumorsfor each line), and the levels of expression varied betweentumor samples. Ectopic PRMT6 expression was consistentlylower than the other two PRMTs. However, NIC-PRMT1Tg andNIC-PRMT6Tg females displayed similar dynamics of MIN andfocal tumor development, as compared with NIC-CARM1Tg

females. Thus, it is unlikely that the different tumor character-istics were due to the varied amounts of PRMTs overexpressed.Together, these data demonstrated that in the HER2-inducedtumorigenic context, both PRMT1 and PRMT6 were capable ofaccelerating the mammary tumorigenesis, whereas CARM1initially delayed tumorigenesis, but promoted tumor growthonce the tumor initiated.

PRMT1 and PRMT6 overexpression promoted the activity of thePI3K–AKT pathway

To begin to decipher themolecularmechanisms underlying theaccelerated mammary tumorigenic onset in the NIC-PRMT1Tg

and NIC-PRMT6Tg females described above, we carried outthe RNA-seq analyses using purified MECs. MECs were isolatedfrom virgin female glands at 12 weeks for NIC-PRMT1/6Tg and

15 weeks for NIC-CARM1Tg, when the mammary glands are stilltumor-free and pre-neoplastic (Supplementary Fig. S6A). Over1,000 genes displayed differential gene expression (DEG) in bothNIC-PRMT1Tg and NIC-PRMT6Tg female mammary epithelia, ascompared with NIC epithelia (Fig. 4A). In contrast, only �400DEGs were uncovered in the MECs isolated from NIC-CARM1Tg

female mammary glands (Fig. 4A).At the global level, hierarchical clustering heatmap demon-

strates that the DEGs pattern is very similar between NIC-PRMT1Tg and NIC-PRMT6Tg, which clearly grouped together,whereas the NIC-CARM1Tg DEGs pattern segregates (Fig. 4B).Kyoto Encyclopedia of Genes and Genomes pathway analysisof the DEGs from NIC-CARM1Tg mice did not identify anoncogenic pathway. However, pathway analysis of thePRMT1/6 signature revealed robust activation of the PI3K–AKTsignaling pathway, and also the ECM-receptor interaction andfocal adhesion pathways (Fig. 4C). The upregulated DEGs inthese three pathways overlap substantially. Furthermore, thesegenes were largely upregulated in the NIC-PRMT1Tg and NIC-PRMT6Tg females, as compared to the NIC-CARM1Tg females(Fig. 4D, left). We validated a number of the DEGs using qPCR,including Itga2b, Itga5, Itga7, Pik3r3, Ccnd2, and Creb5 (Fig. 4D,right). These data suggest that overexpression of PRMT1 andPRMT6 likely augments NIC-induced mammary tumorigenesisby promoting the PI3K–AKT pathway. Indeed, further Westernanalysis revealed that AKT phosphorylation levels were elevatedin these tumors (Fig. 5A). Also, protein level changes were seenfor PIK3R3 and CCND2 (Fig. 5A), which were deregulated atthe RNA level (Fig. 4D). Furthermore, in the bigenic NIC-PRMT1/6Tg mice we observed elevated levels of CCND1 protein(Fig. 5A), which is stabilized by active AKT signaling (35). IHCstaining of mammary tumors demonstrates elevated AKT phos-phorylation levels in bigenic NIC-PRMT1/6Tg mice (Fig. 5B).Recently, PRMT1 overexpression was shown to amplify theactivation of AKT pathway in colorectal cancer cell lines(36), but this is the first in vivo data supporting this link.

DiscussionTransgenic overexpression mouse models have long been uti-

lized to elucidate the biological roles and molecular mechanismsof proto-oncogenes and tumor suppressors during the mammarygland tumorigenesis (37–42). PRMT1, CARM1, and PRMT6are overexpressed in a number of cancers and their elevatedlevels often correlate with poor disease prognosis. TCGA datasetanalyses revealed thatPRMT1 andCARM1 aremarkedly amplifiedand overexpressed in human breast cancer samples, with PRMT6being upregulated to a less degree (Supplementary Fig. S7Aand S7B). Detailed analysis revealed that PRMT1 and CARM1

Figure 4.Transcriptome analyses identifies deregulation of the PI3K–AKT pathway. A, Scatter plots showing DEGs in pre-neoplastic mammary epithelium revealed byRNA-seq analyses between nulliparous NIC and bigenic NIC-PRMTTg females. Up, upregulated DEGs. Dn, downregulated DEGs. Cutoff: fold change > 2, FDR < 0.05.Black circles mark overexpressed PRMT1, CARM1, and PRMT6, respectively. B, Hierarchical clustering heatmap of the DEGs in the mammary epitheliumamong different biologic replicates of NIC-PRMTTg females. Red and green colors represent the log2 ratios of the expression values in the NIC-PRMTTg epitheliumrelative to those in NIC epithelium. Euclidean distance and ward clustering methods were used to construct the dendrograms against genes and samples.Note that the NIC-CARM1Tg samples segregated from the NIC-PRMT1Tg and NIC-PRMT6Tg samples, which clusteredwith each other. C, Kyoto Encyclopedia of GenesandGenomes pathway analysis showing the overrepresented pathways among the upregulatedDEGs in themammary epitheliumof NIC-PRMT1Tg andNIC-PRMT6Tg

females (left). The Venn diagram illustrates the overlapping genes among the top three hit pathways (right). D, Left, a heatmap displaying a list of representativeupregulated genes involved in the PI3K–AKT pathway. The bar graphs representing RT-qPCR validation of six representative genes in the PI3K–AKT pathway,demonstrating their upregulated expression levels in the mammary epithelium of NIC-PRMT1Tg and NIC-PRMT6Tg females, but not NIC-CARM1Tg females, ascompared with NIC females (right). �, P < 0.05; �� , P < 0.01; Student t test.






are prominently upregulated in the basal-like subtype of humanbreast cancers (Supplementary Fig. S7C). These data suggest thatthese PRMTs may have oncogenic activity on their own, or thatthey cooperate with oncogenic drives to promote tumorigenesis.In this study, we investigated the oncogenic potential of PRMT1,CARM1, and PRMT6 by the generation of independent induciblemouse models for each of these PRMTs, and activating them inepithelial tissue using K5-Cre and MMTV-NIC driver lines. Weprovide in vivo genetic evidence that PRMT1, CARM1, and PRMT6all promote cell proliferation and transformation, albeit to vary-ing degrees.

K5-driven targeted overexpression of PRMT1, CARM1, andPRMT6 elicited increased side-branching and hyperplasia of themammary gland (Fig. 2A). This phenotype was most obvious infairly young (4–6 month) K5-PRMT1Tg and K5-PRMT6Tg virginfemales with >85% penetrance, but also became evident in older(18 months) K5-CARM1Tg females with >80% penetrance. Inter-estingly, overexpression of PRMT6 did not result in an increase ofsporadic tumor incidence in either the mammary gland or skin inaged female mice (Supplementary Fig. S4A). However, overex-pression of PRMT1 and especially CARM1did induce a significantincrease in sporadic tumors in the bigenic K5-PRMTTg, as opposedto the uninduced PRMTTg littermates. Note that these sporadictumors required long latency before development (>18months).

PRMTs may collaborate with well-established oncogenicdrivers to promote the development of tumors. To investigatethis possibility, we crossed the three different PRMTTg mouselines to the MMTV-NIC mouse model. The MMTV-NIC mousemodel has been effectively used to study the interplay betweenHER2 and ShcA (21), PTEN (43), b-catenin (44), and noncod-ing RNAs (45), as well as in preclinical in vivo studies (46). Here

we find that in the context of HER2 oncogene-induced mam-mary tumors, PRMT1 and PRMT6 significantly acceleratedtumor growth. Surprisingly, in the same context CARM1 retardsthe timing of tumor initiation, but once a tumor is initiated, itdisplays accelerated growth (Supplementary Fig. S6C). Thus,CARM1 seems to function differently to PRMT1/6 in thissetting. Transcriptome analysis has shed some light on thesedifferences. Indeed, RNA-seq experiments performed on puri-fied MECs isolated from bigenic NIC-PRMTTg mice revealedthat PRMT1 and PRMT6 overexpression induced similarderegulated gene expression patterns, that were distinct fromthe altered transcriptome pattern seen in MECs from isolatedNIC-CARM1Tg. This clustering is perhaps not surprising con-sidering that PRMT1 and PRMT6 recognize similar substrates,which are not shared by CARM1. Pathway analysis of the DEGsfrom NIC-PRMT1/6Tg mice identified enhanced activation ofPI3K–AKT signaling. Further, Western analysis revealed thatAKT is phosphorylated in NIC-PRMT1/6Tg mammary tumorsamples, and that selected proteins in the PI3K–AKT pathwaydisplayed elevated protein levels (Fig. 5). This observation isconsistent with previous findings that PRMT1-mediated ERamethylation at arginine 260 plays a key role in nongenomicsignaling mediated by the estrogen receptor, in which ERamethylation facilitates the assembly of ERa/p85/PI3K/SRC/FAK complex in the cytoplasm and activates the downstreamPI3K–AKT pathway (47, 48). Also, PRMT1 overexpression wasshown to stimulate AKT phosphorylation in colorectal cancercell lines, and PRMT1 knockdown or small molecule inhibitionattenuates this pathway (36).

It should be noted that we did not observe a dramatic increasein arginine methylation levels in the different PRMT overexpres-sing tumors. The methyl-specific antibodies that we used maymiss substrates that display increased methylation. Thus, wecannot rule out the possibility that there may be nonenzymaticscaffolding roles for the PRMTs, which could contribute totumorigenesis.

Taken together, the three different transgenic PRMT mousemodels (PRMT1, CARM1, and PRMT6) that we have establishedand described here, provide compelling in vivo evidence foroncogenic activities of type I PRMTs. These data also provideimportant insights into the molecular determinants of mammarygland tumorigenesis driven by PRMT overexpression, and couldoffer a valuable resource for testing the efficacy of therapeuticsmall molecules that target these PRMTs.

Disclosure of Potential Conflicts of InterestM.T. Bedford is a cofounder of EpiCypher. No potential conflicts of interest

were disclosed by the other authors.

Database DepositionsDeep sequencing data have been submitted to the NCBI: Geo #GSE111624.

Authors' ContributionsConception and design: J. Bao, W.J. Muller, M.T. BedfordDevelopment of methodology: J. Bao, A. Di Lorenzo, Y. Yang, M.T. BedfordAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Bao, A. Di Lorenzo, M.M. Sebastian, Y. YangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Bao, K. Lin, Y. Lu, Y. Zhong, M.M. Sebastian,Y. Yang, M.T. BedfordWriting, review, and/or revision of the manuscript: J. Bao, M.M. Sebastian,M.T. Bedford

Figure 5.

Protein analyses identifies activation of the AKT pathway in NIC-PRMT1/6Tg

tumors. A, Immunoblotting analyses of mammary tumor samples derived fromdifferent NIC and biogenic NIC-PRMTs virgin females. A panel of proteinsinvolved in the PI3K–AKT pathway was tested on these lysates, as labeled.ACTIN and MAT2A served as a loading control. B, IHC staining on mammarytumors from the indicated genotypes using the pAKTS473-specific antibody.Nuclei were counterstained by hematoxylin. Positive signals were visualized asbrown. Scale bar, 50 mm.

Bao et al.





Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.M. Sebastian, Y. YangStudy supervision: M.T. Bedford

AcknowledgmentsThis work was supported by a NIH grant (GM126421) and a CPRIT grant

(RP110471) to M.T. Bedford. W.J. Muller is supported by CRC Chair inMolecular Oncology and CIHR Foundation grant.

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 June 28, 2018; revised September 3, 2018; accepted October 16,2018; published first October 23, 2018.

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Bao et al.




2019;79:21-32. Published OnlineFirst October 23, 2018.Cancer Res Jianqiang Bao, Alessandra Di Lorenzo, Kevin Lin, et al. for Different Type I Protein Arginine MethyltransferasesMouse Models of Overexpression Reveal Distinct Oncogenic Roles

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