Role of JNK in Mammary Gland Development and Breast...

Molecular and Cellular Pathobiology

Role of JNK in Mammary Gland Development andBreast Cancer

Cristina Cellurale1, Nomeda Girnius1, Feng Jiang1, Julie Cavanagh-Kyros1, Shaolei Lu2, David S. Garlick2,Arthur M. Mercurio2, and Roger J. Davis1

AbstractcJun NH2-terminal kinase (JNK) signaling has been implicated in the developmental morphogenesis of

epithelial organs. In this study, we employed a compound deletion of the murine Jnk1 and Jnk2 genes in themammary gland to evaluate the requirement for these ubiquitously expressed genes in breast development andtumorigenesis. JNK1/2 was not required for breast epithelial cell proliferation or motility. However, JNK1/2deficiency caused increased branching morphogenesis and defects in the clearance of lumenal epithelial cells. Inthe setting of breast cancer development, JNK1/2 deficiency significantly increased tumor formation. Together,thesefindings established that JNK signaling is required for normalmammary gland development and that it has asuppressive role in mammary tumorigenesis. Cancer Res; 72(2); 1–10. �2011 AACR.

Introduction

The formation of epithelial organs requires the coordinatedgrowth and movement of epithelial cell sheets. These devel-opmental processes are critically regulated by many mechan-isms, including cytokine and endocrine signal transductionpathways. One signaling pathway that has been implicated inepithelial organ morphogenesis is the cJun NH2-terminalkinase (JNK) signaling pathway (1, 2). Thus, genetic analysisof Drosophila shows that JNK is essential for the morphoge-netic epithelial cell movements that occur during dorsalclosure (3), thoracic closure (4), imaginal disc development(5), and formation of the egg dorsal appendages andmicropyle(6). Studies of mammalian development show that JNK isrequired for closure of the optic fissure (7), eyelid closure (8,9), and neural tube closure (10). Key molecular mechanismsthatmay underly these processes include a requirement of JNKfor paxillin phosphorylation and epithelial cell motility (11)and a requirement of JNK for actin polymerization-dependentcell protrusions at the leading edge of the epithelial cell sheet(12). An understanding of the role of JNK in these develop-mental processes is important because the mechanisms may

be relevant to both normal physiology and to disease states(1, 2).

The purpose of this studywas to testwhether JNK is requiredfor mammary gland development (13). Indeed, JNK may play acritical role in morphogenesis of the breast epithelium (14, 15).These authors report that the drug SP600125 inhibits both JNKactivity and lumenal clearance ofmammary epithelial cells (14,15). However, SP600125 exhibits poor selectivity for JNK (16). Itis therefore unclear whether JNK inhibition mediates theeffects of SP600125 onmorphogenesis of the breast epithelium.Moreover, detailed studies of breast epithelium development(17) indicate that this morphogenetic process differs substan-tially from other epithelial morphogenetic movements that areknown to be JNK dependent (1, 2). Thus, JNK is required forshape changes in the cells that form the leading edge of theepithelial cell sheet prior to coordinated cell movements (12).In contrast, elongating mammary epithelial cell ducts form amultilayer epithelium that moves coordinately without exten-sion of leading edge cells (17). Whether JNK contributes to thisprocess during mammary gland development is unclear.

The JNK protein kinase in mammals is encoded by 2ubiquitously expressed genes (Jnk1 and Jnk2) and by a thirdgene (Jnk3) that is selectively expressed in the brain (1). Wehave not detected developmental defects in mammary glanddevelopment in Jnk1�/� mice or Jnk2�/� mice (18). BecauseJNK1 and JNK2 display partially redundant functions (19, 20),we examined the effect of compound deficiency of JNK1 plusJNK2. Compound Jnk1�/� Jnk2�/� mice die during embryonicdevelopment (10). We therefore employed a conditional geneablation strategy. This experimental approach enabled exam-ination of the role of JNK in primary cultures of mammaryepithelial cells and mammary gland organoids in vitro. Fur-thermore, transplantation assays allowed analysis of the role ofJNK in mammary gland development in vivo. We report thatJNK is not required for mammary epithelial cell motility orformation of mammary epithelial cell ducts. However, JNK

Authors' Affiliations: 1Howard Hughes Medical Institute and Program inMolecular Medicine, and 2Department of Cancer Biology, University ofMassachusetts Medical School, Worcester, Massachusetts

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

Current address for F. Jiang: Aveo Pharmaceuticals, Cambridge, MA02139.

Corresponding Author:Roger J. Davis, HowardHughesMedical Institute,Program in Molecular Medicine, University of Massachusetts MedicalSchool, 373 Plantation Street, Worcester, MA 01605. Phone: 508-856-6054; Fax: 508-856-3210; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1628

�2011 American Association for Cancer Research.

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contributes to branching morphogenesis of the mammaryepithelium and is required for normal lumenal clearance ofepithelial cells. Moreover, studies of mammary carcinogenesisshow that JNK deficiency causes significantly increased breastcancer. Together, these observations indicate that JNK mayplay an important role in both mammary gland developmentand mammary carcinoma formation.

Materials and Methods

MiceWe have described Jnk2�/� mice (21) and mice with con-

ditional expression of Jnk1 (22). Nudemice (strainNU/J, Stock #002019), mice with conditional expression of KRasG12D (Ref. 23;strain B6.129S4-Krastm4Tyj/J, Stock # 008179), mice with con-ditional expression of Trp53 (Ref. 24; strain B6.129P2-Trp53tm1Brn/J, Stock # 008462), mice expressing 4-hydroxy-tamoxifen-stimulated Cre (25; strain B6;129-Gt(ROSA)26Sortm1(cre/ERT)Nat/J, Stock # 004847), and Villin-Cre mice(Ref. 26; strain B6.SJL-Tg(Vil-cre)997Gum/J, Stock # 004586)were obtained from the Jackson Laboratory. The mice used inthis study were backcrossed to the C57BL/6 strain (JacksonLaboratories) and were housed in a facility accredited by theAmerican Association for Laboratory Animal Care. The Insti-tutional Animal Care and Use Committee of the University ofMassachusetts approved all studies using animals.

Genotype analysisGenotype analysis was carried out by PCR using genomic

DNA as the template. The Jnk1þ (1.5 kb), Jnk1LoxP (1.1 kb), andJnk1D (0.4 kb) alleles were identified using the amplimers 50-CCTCAGGAAGAAAGGGCTTATTTC-30 and 50-GAACCACTG-TTCCAATTTCCATCC-30. The wild-type Jnk2 (400 bp) andknockout Jnk2 (270 bp) alleles were identified using the ampli-mers 50-GGAGCCCGATAGTATCGAGTTACC-30, 50-GTTAGA-CAATCCCAGAGGTTGTGTG-30, and 50-CCAGCTCATTCCTC-CACTCATG-30. The wild-type Trp53 (288 bp) and Trp53LoxP

(370 bp) alleles were identified using the amplimers 50-AGCA-CATAGGAGGCAGAGAC-30 and 50-CACAAAAACAGGT-TAAACCCAG-30. The Trp53D (612 bp) allele was identifiedusing the amplimers 50-CACAAAAACAGGTTAAACCCAG-30

and 50-GAAGACAGAAAAGGGGAGGG-30. The wild-type KRas(285 bp), KRasG12D (315 bp), and LoxP-Stop-LoxP-KRasG12D (600bp) alleles were identified using the amplimers 50-GGG-TAGGTGTTGGGATAGCTG-30 and 50-TCCGAATTCAGTGAC-TACAGATGTACAGAG-30. The Rosa26 (600 bp) and Rosa26-CreERT (300 bp) alleles were identified using the amplimers50-GCGAAGAGTTTGTCCTCAACC-30, 50-GGAGCGGGAGAA-ATGGATATG-30, and 50-AAAGTCGCTCTGAGTTGTTAT-30.The Villin-Cre allele (450 bp) was detected using the amplimers50-TTACTGACCGTACACCAAATTTGCCTGC-30 and 50-CCTG-GCAGCGATCGCTATTTTCCATGAGTG-30.

Cre-mediated recombinationMice were treated with tamoxifen (Sigma, T5648) to activate

Cre-mediated recombination in animals with an inducible Crerecombinase (strain B6; 129-Gt(ROSA)26Sortm1(cre/ERT)Nat/J).The tamoxifen was dissolved in sunflower seed oil (10 mg/mL).

Mice were treated by intraperitoneal injection with 1 mgtamoxifen each day for 3 consecutive days.

Mammary gland transplantation assaysTransplantation assays were carried out using procedures

described previously (27). Donor mice were euthanized and 1mm3 fragments of the 4th inguinal mammary glands wereremoved aseptically and stored in Dulbecco's modified Eaglemedium on ice. Host mice (3- to 4-week-old female nudemice)were anesthetized. The 4th inguinal mammary gland on oneside was excised, a small pocket was formed in the cleared fatpad proximal to the inguinal lymph node, and a fragment ofdonor mammary tissue was placed in the pocket. The sameprocedure was carried out on the contralateral side. Each hostmouse was transplanted with both Control and JNK-deficientmammary tissue.

Results

Isolation of JNK-deficient mammary epithelial cellsThe Jnk1 and Jnk2 genes are expressed in mammary epi-

thelial cells. We employed a conditional gene ablation strategyto create mice with deficiency of JNK1 plus JNK2. We foundthat Jnk1LoxP/LoxP Jnk2�/� CreERT mice were viable. These miceexpress a 4-hydroxy-tamoxifen-stimulated Cre recombinase.We treated the Jnk1LoxP/LoxP Jnk2�/� CreERT mice with tamox-ifen, isolated mammary tissue, and prepared primary mam-mary epithelial cell cultures (Supplementary Fig. S1). Genotypeanalysis of genomic DNA prepared from mammary tissue andcultured epithelial cells showed that tamoxifen caused abla-tion of the conditional Jnk1 gene (Fig. 1A). Immunoblot anal-ysis confirmed that JNK protein was not detected inmammarytissues or epithelial cells of Jnk1D/D Jnk2�/� mice (Fig. 1B).

To examine the effect of JNK deficiency on primary mam-mary epithelial cells, we compared cultures of cells preparedfrom Control mice (CreERT) and Jnk1LoxP/LoxP Jnk2�/� CreERT

mice following treatment with tamoxifen. The epithelial cellswere identified by immunofluorescence analysis by stainingwith antibodies to pan-cytokeratin and E-cadherin. We did notdetect an effect of JNK deficiency on the morphology ofprimary mammary epithelial cells (Fig. 1C).

Role of JNK in mammary epithelial cell proliferationIt is established that compound JNK deficiency in primary

mouse embryonic fibroblasts (MEF) causes p53-dependentsenescence (20, 22). Growth of Jnk1�/� Jnk2�/� MEF requiresloss-of-function of the p53 pathway (22). We therefore antic-ipated that Jnk1D/D Jnk2�/� primary mammary epithelial cellswould also exhibit reduced growth and senescence. However,themorphology of JNK-deficient epithelial cells was not typicalof senescent cells (Fig. 2A).Moreover, cell-cycle analysis byflowcytometry did not show a requirement of JNK for proliferation(Fig. 2B). Indeed, Jnk1D/D Jnk2�/� primary mammary epithelialcells incorporated significantly more BrdU than Control cells,indicating that JNK deficiency may increase epithelial cellproliferation (Fig. 2B). This conclusion was confirmed bymeasurement of cell proliferation (Fig. 2C). Together, thesedata show that the effect of JNK deficiency on the proliferation

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of MEF markedly differs from mammary epithelial cells. Thus,JNK is required for MEF proliferation, but is not required forproliferation of mammary epithelial cells.

It is unclear whether the failure of JNK-deficient mammaryepithelial cells to senesce reflects a specific role of JNK inmammary epithelial cells or whether this reflects a general roleof JNK in other epithelial cells. To address this question, weexamined the effect of JNK deficiency in intestinal epithelialcells using conditional gene ablation in vivo with Villin-Cre(Supplementary Fig. S2). Compound JNK deficiency caused nodetected defects in the proliferation of intestinal epithelial cells(Supplementary Fig. S2A) or colon tumor cells (SupplementaryFig. S2C and S2D). JNK deficiency causes increased expressionof p53 by MEF (20), but no increase in p53 expression wasdetected in JNK-deficient intestinal epithelial cells (Supple-mentary Fig. S2B). Moreover, ionizing radiation caused asimilar increase in p53 expression byControl and JNK-deficientintestinal epithelial cells. Together, these data indicate that thep53-dependent senescence of JNK-deficient MEF (20, 22)reflects a particular role of JNK in MEF, but not other celltypes (e.g., mammary and intestinal epithelial cells).

Role of JNK in mammary epithelial cell motilityIt is has been reported that JNK is required for actin

polymerization-dependent cell protrusions at the leading edgeof epithelial cell sheets duringmorphogeneticmovements (12).It has also been reported that paxillin phosphorylation by JNKis essential for epithelial cell movement (11). Together, thesedata indicate that JNK is a critical cellular component that isrequired for cell motility. To test this hypothesis, we preparedcultures of mammary epithelial cells from Control mice(CreERT) and Jnk1D/D Jnk2�/� CreERT mice. Comparison ofControl and JNK-deficient primary mammary epithelial cellsindicated that JNK is not required for cell motility. Indeed,studies using Boyden chambers coated with collagen showed

Figure 1. JNK-deficient mammary epithelial cells. A and B, Jnk1LoxP/LoxP

Jnk2�/� CreERT mice treated without or with tamoxifen in vivo were usedto prepare mammary gland extracts and primary mammary glandepithelial cells. Genomic DNA was examined by PCR to detect Jnk1LoxP

and Jnk1D alleles (A). The expression of JNK anda-tubulin was examinedby immunoblot analysis (B). C, primary cultures of CreERT (Control) andJnk1D/D Jnk2�/� CreERT mammary epithelial cells prepared fromtamoxifen-treatedmicewere examined by immunofluorescence analysisby probing with antibodies to pan-cytokeratin (red) and E-cadherin(green). DNA was stained with 40,6-diamidino-2-phenylindole (DAPI;blue). Representative images are shown.

Figure 2. Effect of JNK deficiency on mammary epithelial cell proliferation. A, primary cultures of CreERT (Control) and Jnk1D/D Jnk2�/� CreERT mammaryepithelial cells prepared from tamoxifen-treated mice were examined by phase contrast microscopy. Representative images are shown. B, the cellswere pulse labeledwith BrdU and examined by flow cytometry. The number of BrdU-positive cells (%) is presented (mean�SD; n¼ 5). Significant differencesbetween Control cells and JNK-deficient cells are indicated with an asterisk (P < 0.05). C, relative cell proliferation was measured using the WST-1assay (mean � SD; n ¼ 3). Significant differences between Control cells and JNK-deficient cells are indicated with an asterisk (P < 0.05).

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that JNK deficiency increasedmammary epithelial cell motility(Fig. 3A). Moreover, assays using Boyden chambers with aMatrigel layer showed that JNKdeficiency increasedmammaryepithelial cell invasion (Fig. 3B). Together, these data do notsupport a critical role for JNK as a positive regulator of primarymammary epithelial cell motility. This finding contrasts withprevious reports that JNK plays a key role in epithelial cellmotility (2, 11). We therefore examined mice with JNK defi-ciency in the intestinal epithelium (Supplementary Fig. S2).Epithelial cells formed in intestinal crypts migrate on thesurface of the villus to create the intestinal epithelium. Intes-tinal epithelium morphology was not disrupted by JNK defi-ciency (Supplementary Fig. S2), consistent with a nonessentialfunction of JNK in intestinal epithelial cell motility. Together,these data indicate that JNK is not essential for epithelial cellmotility.

Role of JNK in mammary branching morphogenesisBranching morphogenesis is an important aspect of mam-

mary gland development (28, 29). This process can be studiedin vitro using mammary organoid cultures in the presence ofFGF2 (17). We prepared cultures of mammary organoids fromControl mice (CreERT) and Jnk1D/D Jnk2�/� CreERT mice. Theorganoids are formed by bilayer structures with lumenalmammary epithelial cells and basal myoepithelial cells thatexpress smooth muscle actin (30, 31). Branching morphogen-esis is regulated by hormones/growth factors and by theinteraction of the lumenal epithelial cells with basal myoe-pithelial cells and the extracellular matrix (29). Branchingmorphogenesis was detected in cultures of Control andJnk1D/D Jnk2�/� mammary organoids (Fig. 4A). This observa-tion indicated that JNK is not required for branching morpho-genesis. However, quantitation of the branching indicated thatJNK-deficient organoids exhibited significantly greater branch-ing morphogenesis than Control organoids (Fig. 4B). Together,

these data show that JNK can influence mammary branchingmorphogenesis.

Effect of JNK deficiency onmammary epithelial cell geneexpression

It is established that the JNK signal transduction pathwaycan regulate gene expression (1). JNK-regulated gene expres-sion may therefore account for the effect of JNK deficiency onepithelial cell motility and invasion (Fig. 3) and branchingmorphogenesis (Fig. 4). We therefore examined the effect ofJNK deficiency on the expression of candidate genes that mayinfluence these processes (Supplementary Fig. S3).

Matrix metalloproteases (MMP) play key roles in mammarygland development (28). Thus, ADAM17 induces shedding ofthe EGF receptor ligand amphiregulin that can induce expres-sion of MMP2 (32). MMP2 (and its activator MMP14) canpromote ductal elongation (33), and both MMP3 and MMP9promote branchingmorphogenesis (33, 34).We found that JNKdeficiency caused significantly decreased expression ofMmp2,Mmp9, and Mmp14 mRNA (Supplementary Fig. S3A). Thesechanges do not account for the increased branching morpho-genesis caused by JNK deficiency. MMPs and ADAM17 arenegatively regulated by tissue inhibitors of metalloproteases(TIMP). Downregulated expression of TIMP1/2/3 could there-fore increase MMP and/or ADAM17 activity and thereforeinfluence branching morphogenesis (29). Indeed, a significantreduction in Timp1, Timp2, and Timp3 mRNA expression wascaused by JNK deficiency (Supplementary Fig. S3B). In con-trast, the expression of 2 other genes that are implicated inbranchingmorphogenesis (Tgfb1 and Sprouty2) was unaffectedby JNK deficiency (Supplementary Fig. S3B). Together, thesedata suggest that decreased TIMP expression may contributeto increased branching morphogenesis caused by JNKdeficiency.

The increasedmotility and invasion activity of JNK-deficientmammary epithelial cells detected in Boyden chamber assaysmay reflect altered integrin expression. We found decreasedexpression of a1, a5, a6, and b1 integrins and also decreasedexpression of the collagen receptor DDR1 in JNK-deficientmammary epithelial cells (Supplementary Fig. S3C). Thedecreased expression of integrin a5 protein was confirmedby immunofluorescence analysis (Supplementary Fig. S4).Previous studies have implicated integrin a2, integrin b1, andDDR1 in mammary gland development (29), but decreasedexpression of these proteins is not predicted to cause theincreased motility, invasion, and branching morphogenesiscaused by JNK deficiency (Figs. 3 and 4). The mechanism thataccounts for increased motility and invasion by JNK-deficientmammary epithelial cells is therefore unclear.

Effect of JNKdeficiency onmammary gland developmentWe employed transplantation assays to test the role of JNK

in mammary gland development using the 4th inguinal glandpair. Control (CreERT) tissue was transplanted in 1 clearedmammary gland of a female nude mouse and Jnk1D/D Jnk2�/�

CreERT tissue was transplanted in the contralateral gland of thesame recipient mouse. Analysis of mammary gland develop-ment at 8 weeks following transplantation showed that JNK

Figure 3. Effect of JNK deficiency on mammary epithelial cell motilityand invasion. A and B, primary cultures of CreERT (Control) and Jnk1D/D

Jnk2�/� CreERT mammary epithelial cells prepared from tamoxifen-treated mice were examined using Boyden chambers coated withCollagen I (A) or with a Matrigel layer (B). The relative number of cells thatmoved from the upper chamber to the lower chamber is presented (mean� SD; n ¼ 5). Significant differences between Control cells and JNK-deficient cells are indicated with an asterisk (�, P < 0.05; ��, P < 0.001).

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deficiency did not prevent the elaboration of mammary epi-thelial ducts (Fig. 5A). However, JNK deficiency caused anincrease in the number of branches (Fig. 5B and C). Thesedata indicate that JNK can influence mammary branchingmorphogenesis in vivo.To test whether JNK may alter the early time course of

mammary gland development, we examined mice at 2 weeksfollowing transplantation. This analysis showed the presenceof terminal end buds (TEB) in glands formed by Control andJNK-deficient tissue (Fig. 5D). No differences in proliferatingcells (PCNA-positive) or dying cells (TUNEL-positive) weredetected between Control and JNK-deficient mammary glands(Supplementary Fig. S5). Sections of TEBs formed by trans-plantation of Control tissue showed the presence of a lumenalspace (Fig. 5E). In contrast, this TEB lumenal space waspartially filled with cells in glands formed by transplantationof JNK-deficient tissue (Fig. 5E). These data indicate that JNKdeficiency disrupts TEB morphology during mammary glanddevelopment. Moreover, JNK deficiency increased the numberof branches detected at 2 weeks following transplantation(Fig. 5F).

Effect of JNK deficiency on mammary tumorigenesisJNK deficiency influences the proliferation, motility, inva-

sion activity, and branching morphogenesis in vitro (Figs. 2–4)and mammary gland development in vivo (Fig. 5). Thesechanges indicate that JNK may influence mammary tumordevelopment. To test this hypothesis, we examined the effect ofmutational activation of the endogenous KRas gene in vivo.

Transplantation assays were carried out using donor tissuefrom Control mice (KRasLSL-G12D/þ CreERT) and mice withconditional expression of JNK (KRasLSL-G12D/þ Jnk1LoxP/LoxP

Jnk2�/� CreERT). The recipient nude mice were transplantedwith both Control tissue and JNK-deficient tissue in the 4thinguinal gland pair. The transplanted mice were treated withtamoxifen at 2 weeks postsurgery to induce expression ofactivated Ras (KRasG12D) and to ablate the conditional Jnk1gene. No tumors were detected in these mice within 6 monthsof transplantation.

TheKRasG12D oncogenemay require a cooperatingmutationto efficiently induce breast cancer (35). We therefore examinedthe effect of p53 loss-of-function on KRasG12D-induced breastcancer. Transplantation assays were carried out using donortissue fromControlmice (KRasLSL-G12D/þTrp53LoxP/LoxP CreERT)and mice with conditional expression of JNK (KRasLSL-G12D/þ

Jnk1LoxP/LoxP Jnk2�/� Trp53LoxP/LoxP CreERT). These trans-plantedmice developedmammary carcinoma (Fig. 6). Analysisof tissue sections showed that both Control and JNK-deficienttumors were composed primarily of spindle-like cells (Fig. 6A)that stainedwith an antibody to the proliferationmarker PCNA(Fig. 6B). Genotype analysis confirmed Jnk1 gene disruption intumors obtained from mammary glands transplanted withJNK-deficient tissue (Fig. 6C). Kaplan–Meier analysis showedthat compound JNK deficiency caused a significant increase inthe number of mice with breast cancer (Fig. 6D). The meantumor volume at necropsy was 0.62 cm3 � 0.19 (mean � SD;n ¼ 7) and no significant difference between Control andJNK-deficient tumor volume was observed. The Control and

Figure 4. Effect of JNKdeficiency onmammary branchingmorphogenesis. A, primary organoidCreERT (Control) and Jnk1D/D Jnk2�/�CreERT cultures preparedfrom tamoxifen-treatedmice were examined by immunofluorescencemicroscopy by stainingwith an antibody to smoothmuscle actin (SMA, green) andwithphalloidin to stain F-actin (red). DNA was stained with DAPI (blue). Branching morphogenesis was initiated by treatment of the cultures with FGF2. Theorganoids were examined using a Leica TCSSP2 confocal microscope by acquiring 10 optical sections that were collapsed to a single image. Representativeimages are shown. B, the number of branches per organoid was measured (mean � SD, n ¼ 20). Significant differences between Control cells andJNK-deficient cells are indicated with an asterisk (�, P < 0.01).






JNK-deficient tumors were locally invasive (Fig. 7A), but nometastasis was detected. Tumor sections stained for markersof basal-like (cytokeratin 5) and nonbasal-like (cytokeratin 8)breast cancer showed that the tumors obtained representedmixed origins (basal and nonbasal) (Fig. 7B). However, theJNK-deficient tumors stained more efficiently for cytokeratin5 than Control tumors, indicating that JNK deficiency maypromote basal-like tumors in thisKRas/Trp53model ofmurinebreast cancer.

Discussion

Mice with defects in JNK expression have provided insightinto the biological function of the JNK signaling pathway(1, 36). However, the ubiquitously expressed Jnk1 and Jnk2genes have partially redundant functions (19, 20) and com-pound mutant Jnk1�/� Jnk2�/� mice die during mid-embryo-genesis (10). Studies of the effect of compound JNK deficiencyhave therefore largely focused on an analysis of MEF (20).

However, MEF are not representative of many differentiatedcell types. Progress has beenmade toward the creation of micewith conditional and chemical genetic Jnk alleles (19, 22) thatenable the analysis of cell types that are relevant to specificphysiologic processes (37). The focus of this study was toemploy conditional gene targeting to examine the role of JNKinmammary epithelial cells. We report that JNK contributes tomammary gland development. Importantly, the functions ofJNK in mammary epithelial cells differ from those previouslyidentified in MEF.

JNK is not essential for mammary epithelial cellproliferation or motility

Compound JNK deficiency in MEF causes increased p53expression and senescence (20, 22). In contrast, JNK-deficientmammary epithelial cells did not exhibit a defect in prolifer-ation in vitro (Fig. 2). Moreover, transplantation assays showedthat JNK-deficient cells retained sufficient proliferative capac-ity to form a mammary gland in vivo (Fig. 5). These data show

Figure 5. Effect of JNK deficiencyon mammary gland development.Transplantation assays werecarried out using mammary tissuefrom tamoxifen-treated femaledonor mice and female recipientnude mice. Control (CreERT) tissuewas transplanted in 1 clearedmammary gland and Jnk1D/D

Jnk2�/� CreERT tissue wastransplanted in the contralateralgland of the same recipient mouse.A–C, whole mount mammaryglands stained with carmine alumat 8 weeks followingtransplantation are shown (A andC). The number of branches perunit length (mm) of duct ispresented as themean� SD; n¼ 3(B). Statistically significantdifferences are indicated with anasterisk (�, P < 0.05). Scale bars:5 mm (A); 200 mm (C). D–F, TEBs inwhole mount mammary glandsstained with carmine alum at 2weeks following transplantationare shown (D). Stained sections ofthe end buds in whole mountmammary glands are presented(E). The number of branches perunit length (mm) of duct ispresented as themean� SD; n¼ 3(F). Statistically significantdifferences are indicated with anasterisk (�, P < 0.05). Scale bars:200 mm (D); 100 mm (E).

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that JNK is not essential for proliferation of mammary epithe-lial cells. This conclusion may apply to other epithelial cellsbecause studies of intestinal epithelial cells also showed thatJNK is not required for proliferation (Supplementary Fig. S2).Thus, the effect of JNK deficiency to engage the p53-mediatedsenescence pathway may represent a specialized response ofMEF to loss of JNK signaling.JNK signaling has been implicated in the regulation of cell

motility (2). The role of JNK may be mediated by JNK-depen-dent paxillin phosphorylation (11) and/or a requirement of JNKfor actin polymerization-dependent cell protrusions (12). Nev-ertheless, we did not detect defects in mammary epithelial cellmotility caused by JNK deficiency. Indeed, JNK deficiencycaused an increase in mammary epithelial cell motility inBoyden chamber assays (Fig. 3A). Similarly, JNK deficiencycaused increased invasion ofmammary epithelial cells througha Matrigel layer (Fig. 3B). Furthermore, JNK deficiency did notprevent the formation of mammary epithelial cell ducts or theintestinal epithelium in vivo (Fig. 5 and Supplementary Fig. S2).Together, these data show that JNK is not essential for epi-thelial cell motility. This conclusion may reflect a redundantrole of JNK for paxillin phosphorylation (11). Moreover, JNK-dependent leading edge cell protrusions may not be rate

limiting for motility (e.g., roles of adhesion and rear-enddetachment).

JNK is required for normal mammary glanddevelopment

Transplantation assays showed that JNK is not required forthe formation of a mammary gland in a virgin female mouse(Fig. 5). However, developmental defects were detected. Thus,examination of TEBs at 2 weeks following transplantationshowed that lumenal cell clearance found in Control TEBswas incomplete in JNK-deficient TEBs (Fig. 5E). Previousstudies have established that lumenal clearance is caused bycell death, partially mediated by apoptosis (38), that involvesthe BH3-only proteins Bim (15, 39) and Bmf (40). Interestingly,Bim and Bmf are targets of proapoptotic signaling by JNK (41,42). Loss of JNK signaling may lead to defects in Bim/Bmffunction and consequently failure of lumenal clearance (15).Nevertheless, it should be noted that the defect in TEB lumenalcell clearance caused by JNK deficiency was partial (Fig. 5E),suggesting the presence of redundant or compensatorymechanisms of lumenal cell clearance in the JNK-deficientmammary glands. This type of compensation has been noted instudies of Bim-deficient mammary glands (39).

Figure 6. Effect of JNK deficiency on mammary tumor development. Transplantation assays were carried out using mammary tissue from female donor miceand female recipient nude mice. Control (KRasLSL-G12D/þ Trp53LoxP/LoxP CreERT) tissue was transplanted in 1 cleared mammary gland and KRasLSL-G12D/þ

Trp53LoxP/LoxP Jnk1LoxP/LoxP Jnk2�/� CreERT tissue was transplanted in the contralateral gland of the same recipient mouse. The transplanted mice weretreated with tamoxifen at 2 weeks postsurgery. A, sections of transplanted breast mammary glands were stained with hematoxylin and eosin (H&E). Scalebar ¼ 50 mm. B, sections of breast tumors were stained with an antibody to PCNA (red). DNA was stained with DAPI. Scale bar ¼ 75 mm. C, genomic DNAisolated from Control and JNK-deficient breast tumors was examined by PCR using amplimers to detect the wild-type Jnk1 allele (1.5 kb), the Jnk1LoxP

allele (1.1 kb), and the ablated allele DJnk1 (0.4 kb). D, Kaplan–Meier analysis of tumor-free survival of transplanted mice (n ¼ 8). The development ofJNK-deficient tumors was significantly more rapid than Control tumors (P < 0.02; log-rank test).






JNK deficiency caused altered branching morphogenesis.Transplantation assays showed that JNK deficiency causedincreased branching of mammary ducts in vivo (Fig. 5B and F).This effect of JNK deficiency to cause increased branchingmorphogenesis was also observed in FGF2-stimulated orga-noid cultures in vitro (Fig. 4). Themechanism that accounts forincreased branching morphogenesis is unclear, but it is estab-lished that this process is regulated by hormones/growthfactors and by the interaction of the lumenal epithelial cellswith basal myoepithelial cells and the extracellular matrix (28,29). One potential role of JNK is represented by expression ofTIMP isoforms that inhibit MMPs. JNK deficiency causeddecreased expression of Timp1/2/3 (Supplementary Fig.S3B). Decreased TIMP activity may lead to increased activityof MMP3 and MMP9 that function, in part, to promotebranching morphogenesis (33, 34). This mechanism may con-tribute to the altered branching morphogenesis caused by JNKdeficiency.

JNK contributes to mammary tumor developmentThe effect of JNK deficiency to perturb normal mammary

gland development may be relevant to breast cancer. How-ever, the contribution of JNK to breast cancer is unclear (1).Mutations in JNK pathway genes (Jnk1, Jnk2, Mkk4, andMkk7) are detected in human cancers (43, 44), but it isunclear whether these mutations are causally related to

tumorigenesis (45). Insight into the potential function ofJNK has been obtained from murine studies of KRas-inducedlung cancer (46), carcinogen-induced hepatocellular carci-noma (47), and colon cancer (Supplementary Fig. S2) withtissue-specific compound ablation of the Jnk1 and Jnk2genes. These studies have shown an essential role for JNKin KRas-induced lung cancer (46), but no required role forJNK in carcinogen-induced colon cancer (SupplementaryFig. S2). In contrast, JNK plays a more complex role inhepatocellular carcinoma because JNK promotes an inflam-matory microenvironment to support tumor development,but acts in hepatocytes to reduce tumor development (47).Together, these data indicate that, in individual tumor types,JNK may play no role in tumor development or may con-tribute (positively or negatively) to tumor pathology.

Studies using single gene ablation (Jnk1or Jnk2) indicate thatJNK deficiency can increase mammary carcinoma in the Trp53BALB/cmousemodel (18).Moreover, JNK2deficiency shortenstumor latency and increases tumormultiplicity in a transgenicmouse model with expression of polyoma virus T antigen (48).These observations suggest that JNK may function to reducetumor development. However, JNK1 and JNK2 exhibit partiallyredundant functions (20, 22). Consequently, single gene abla-tion (Jnk1 or Jnk2) may not provide insight into the effects ofcompound JNK deficiency (37, 47, 49). Here, we show thatcompound JNK deficiency promotes tumor development in aKRas/Trp53 mouse model of breast cancer (Fig. 6). Furtherstudies will be required to confirm the protumorigenic effect ofJNK deficiency in a model of breast cancer that has directrelevance to human disease. Nevertheless, it is likely thatprotumorigenic effects of JNK deficiency reflect functionalroles for JNK pathway gene (Jnk1, Jnk2, Mkk4, and Mkk7)mutations that have been detected in human cancer (43, 44)and JNK-regulated genetic instability (48).

Small molecule inhibitors of JNK have been proposed to beuseful for the treatment of metabolic and inflammatory dis-orders in humans (50). The design of such therapies shouldtake into account the potential protumorigenic effects of JNKinhibition.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Tammy Barrett for expert technical assistance, and KathyGemme for administrative assistance.

Grant Support

These studies were supported by a grant from the NIH (R01-CA65861). R.J.Davis is an Investigator of the Howard Hughes Medical Institute and is also amember of the Diabetes and Endocrinology Research Center of the University ofMassachusetts Medical School that is funded by the NIH grant P30-DK32520. N.Girnius was supported by NIH training grant T32-CA130807.

The costs of publication of this article were defrayed in part 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 May 16, 2011; revised September 22, 2011; accepted November 21,2011; published OnlineFirst November 29, 2011.

Figure 7. JNK deficiency causes increased basal-like mammarycarcinogenesis. Transplantation assays were carried out using Controland JNK-deficient tissue (Fig. 6). A, sections showing the periphery ofControl and JNK-deficient mammary tumors were stained with H&E.Scale bar¼ 150 mm. B, sections of Control and JNK-deficient mammarytumors were stained with an antibody to cytokeratin 5 (green) andcytokeratin 8 (red). DNA was stained with DAPI. Scale bar ¼ 150 mm.

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Published OnlineFirst November 29, 2011.Cancer Res Cristina Cellurale, Nomeda Girnius, Feng Jiang, et al. CancerRole of JNK in Mammary Gland Development and Breast

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