Effective Targeting of Estrogen Receptor Negative Breast ... · Small Molecule Therapeutics Effective Targeting of Estrogen Receptor– Negative Breast Cancers with the Protein Kinase

Small Molecule Therapeutics

Effective Targeting of Estrogen Receptor–Negative Breast Cancers with the ProteinKinase D Inhibitor CRT0066101Sahra Borges1, Edith A. Perez1,2, E. Aubrey Thompson1, Derek C. Radisky1,Xochiquetzal J. Geiger3, and Peter Storz1

Abstract

Invasive ductal carcinomas (IDC) of the breast are associatedwith altered expression of hormone receptors (HR), amplifi-cation or overexpression of HER2, or a triple-negative pheno-type. The most aggressive cases of IDC are characterized by ahigh proliferation rate, a great propensity to metastasize, andtheir ability to resist to standard chemotherapy, hormonetherapy, or HER2-targeted therapy. Using progression tissuemicroarrays, we here demonstrate that the serine/threoninekinase protein kinase D3 (PKD3) is highly upregulated inestrogen receptor (ER)–negative (ER�) tumors. We identifydirect binding of the ER to the PRKD3 gene promoter as a

mechanism of inhibition of PKD3 expression. Loss of ERresults in upregulation of PKD3, leading to all hallmarks ofaggressive IDC, including increased cell proliferation, migra-tion, and invasion. This identifies ER� breast cancers as idealfor treatment with the PKD inhibitor CRT0066101. We showthat similar to a knockdown of PKD3, treatment with thisinhibitor targets all tumorigenic processes in vitro anddecreases growth of primary tumors and metastasis in vivo.Our data strongly support the development of PKD inhibitorsfor clinical use for ER� breast cancers, including the triple-negative phenotype. Mol Cancer Ther; 14(6); 1–11. �2015 AACR.

IntroductionInvasive ductal carcinomas (IDC) of the breast are among the

most aggressive types of cancer affecting women. IDCs with theworst outcome are associated either with loss of expression ofhormone receptors, estrogen receptor (ER) and progesteronereceptor (PR), overexpression, or amplification of the humanepidermal growth factor receptor-2 (HER2/ErbB2), or a triple-negative phenotype [lack of expression of HER2 and bothhormone receptors (HR]. The most aggressive cases of IDC arecharacterized by a high proliferation rate, a great propensity tometastasize, and their ability to resist to standard chemother-apy, hormone therapy, or HER2-directed agents such as tras-tuzumab (1, 2). Particularly, triple-negative breast cancers(TNBC), which represent approximately 20% of all cases, aredifficult to treat, because molecular targets are lacking (3, 4).Cytotoxic radio- and chemotherapy currently are the onlyoptions for patients with TNBC (5). However, the rate ofrecurrence is high after such treatment (6). The high resistanceto therapy has been partially attributed to tumors that show

mesenchymal and stem cell features, for which no specifictargeted therapies are available (7–9). Thus, identification oftissue biomarkers that may lead to novel therapeutic strategiesis of utmost importance.

The protein kinaseD (PKD) familymembers PKD1, PKD2, andPKD3 have been implicated in the progression of breast cancer.PKD1 contributes to breast cancer cell proliferation (10), butinhibits the invasive phenotype (11, 12). This is mediated byinhibition of epithelial-to-mesenchymal transition (EMT)through phosphorylation of Snail (13–16), downregulation ofthe expression of several matrix metalloproteinases (MMP), andnegative-regulation of F-actin reorganization at the leading edgeat multiple levels (12, 13, 17, 18). Consequently, PRKD1 (encod-ing PKD1) is silenced by hypermethylation in themost aggressivebreast cancers, including the TNBC subtype (11, 19). In contrast toPKD1, the two other isoforms PKD2 and PKD3, in breast cancercell lines seem to drive all aspects of oncogenic transformation,including cell proliferation, migration, invasion, and chemore-sistance (20–22). Similar opposing functions in breast cancerhave been described for other kinases such asmembers of the Akt/PKB kinase family (23, 24). However, how subtypes of the samekinase family, which recognize the same substrate phosphoryla-tion motif, can have opposite cellular functions remains unclear.On the basis of the recent studies for PKD enzymes, it seems that anumber of different parameters, such as their relative level ofexpression or activity, their cellular localization, and/or theirability to form complexes, can differentially influence cellularphenotypes (25).

Using progression tissue microarrays (TMA), here we dem-onstrate that a switch toward the isoform PKD3 is associatedwith the aggressiveness of breast cancer. Although PKD1 isdownregulated and PKD2 is expressed homogeneously at low

1Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida.2Department of Hematology/Oncology, Mayo Clinic, Jacksonville,Florida. 3Laboratory Medicine and Pathology, Mayo Clinic, Jackson-ville, Florida.

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

Corresponding Author: Peter Storz, Mayo Clinic, Griffin Room 306, 4500 SanPablo Road, Jacksonville, FL 32224. Phone: 904-953-6909; Fax: 904-953-0277;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0945

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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levels in different breast cancer subtypes as well as in normaltissue, PKD3 is highly upregulated in ER-negative (ER�) tumors.We identify estrogen-dependent signaling as the mechanismof inhibition of PKD3 expression in ER-expressing ductal cancercells. Loss of ER results in upregulation of PKD3, leading toincreased cell proliferation, migration, and invasion. These dataidentify ER� breast cancers as ideal cancers for treatment withthe PKD inhibitor CRT0066101, because they express little orno PKD1 and high levels of PKD3. We show that, similar to aknockdown of PKD3, treatment with this inhibitor targetsmost tumorigenic processes in vitro, and also decreases growthof primary tumors and prevents metastasis in vivo. Thus, becauseit can be given orally, it may be developed for treatmentof PKD1-negative breast cancers, including the triple-negativephenotype.

Materials and MethodsCell lines, antibodies, and reagents

The MDA-MB-231-luc2 cell line was obtained from PerkinEl-mer in May 2009. All other cells lines were obtained from theAmerican Type Culture Collection in August 2008. All cell lineswere not further authenticated. MCF-7, MDA-MB-231, andT47D cells were maintained in Dulbecco's Modified EagleMedium (DMEM) with 10% fetal bovine serum (FBS). MDA-MB-468 and HCC1954 were maintained in RPMI-1640 with10% FBS. BT20 cells were maintained in Eagle's minimal essen-tial medium with 10% FBS, 2 mmol/L L-glutamine, 1.5 g/Lsodium bicarbonate, 0.1 mmol/L nonessential amino acids(NEAA), and 1 mmol/L sodium pyruvate. MCF-10A cells weremaintained in DMEM/Ham's F-10 medium (50:50 v/v) with 5%horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100ng/mL cholera toxin, 10 mg/mL insulin, and 1% penicillin/streptomycin. NEAAs were obtained from Mediatech, EGF fromPeproTech, and insulin and hydrocortisone from Sigma-Aldrich.Anti-b-actin antibody was obtained from Sigma-Aldrich; anti-Ki67 from Dako; anti-cleaved PARP, anti-cleaved caspase-3, andanti-MMP9 from Cell Signaling Technology; anti-COX-2 fromCayman Chemical; anti-smooth muscle actin (SMA), anti-GFP,and anti-cyclin D1 from Abcam; anti-Snail from Abgent; anti-vimentin (for Western blotting) from Santa Cruz Biotechnology;and anti-N-cadherin and anti-vimentin [for immunohistochem-istry (IHC)] from Epitomics. The rabbit polyclonal antibodyspecific for PKD2 was from Upstate Biotechnology and therabbit polyclonal antibody specific for PKD3 used for immu-noblotting was from Bethyl Laboratories. The mouse monoclo-nal antibody specific for PKD3 (H00023683-M01) used forIHC was from Abnova. The mouse monoclonal antibody spe-cific for PKD1 was raised by Creative Biolabs/Creative Dynamicsand is further described in ref. (11). Secondary horseradishperoxidase (HRP)–linked antibodies were obtained from RocheApplied Science. Luciferin was obtained from Gold Biotechnol-ogy. Fulvestrant and b-estradiol (E2) were purchased fromSigma-Aldrich. The PKD-specific inhibitor CRT0066101 wasobtained from Cancer Research Technology.

Lentiviral shRNA expression and shRNA constructsSpecific lentiviral expression constructs for short hairpin RNA

(shRNA) targeting human PKD3 were purchased from Sigma-Aldrich (MISSION shRNA Plasmid DNA). Constructs used wereNM_005813.x-3393s1c1 (labeled as PKD3-shRNA#1) andNM_005813.x-2494s1c1 (labeled as PKD3-shRNA#2). Lentivirus

was produced in HEK293FT cells using the ViraPower LentiviralExpression System (Life Technologies). MDA-MB-231 cellswere infected with PKD3-shRNA lentivirus to generate stable celllines. After infection, cell pools were selected using puromycin(1 mg/mL) for 15 days.

Plasmids and transfectionsTo generate a PKD3 promoter-luciferase reporter, the human

PRKD3 promoter region (�1000 to þ3) was cloned in pGL3plasmid from Promega via BglII and XhoI restriction sites, using50-TTTTTTGTCCCTTCTGTTTTTGAT-30 and 50-GACGGAAAGAAA-TTAGAAAATTTT-30 as primers. The pRL-CMV-Renilla luciferaseplasmid was from Promega. The ERa (pEGFP-C1-ERa; #28230)expression plasmid was from Addgene. The pSuper-PKD2-shRNAplasmid was obtained by cloning the oligonucleotides 50-GAT-CCCCGTTCCCTGAGTGTGGCTTCTTCAAGAGAGAAGCCACAC-TCAGGGAACTTTTTGGAAA-30 and 50-AGCTTTTCCAAAAAGTT-CCCTGAGTGTGGCTTCTCTCTTGAAGAAGCCACACTCAGGGA-ACGGG-30 into pSuper. GenJet from SignaGen was used fortransfection of breast cancer cells.

Cell lysates and Western blot analysisCells were washed twice with ice-cold phosphate-buffered

saline (PBS; 140 mmol/L NaCl, 2.7 mmol/L KCl, 8 mmol/LNa2HPO4, 1.5 mmol/L KH2PO4, pH 7.2) and lysed with BufferA (50 mmol/L Tris–HCl, pH 7.4, 1% Triton X-100, 150 mmol/LNaCl, 5 mmol/L EDTA, pH 7.4) plus Protease Inhibitor Cocktail(Sigma-Aldrich). Lysates were used for Western blot analysis asdescribed previously (12).

ImmunofluorescenceCells were seeded in 8-well ibiTreat m-Slides (ibidi) and treated

as indicated. Before fixation with 4% paraformaldehyde (20min-utes, 4�C), cells were washed twice with PBS. Fixed cells werewashed three times in PBS, permeabilizedwith 0.1%Triton X-100in PBS (2 minutes, room temperature), and then blocked withblocking solution [3% bovine serum albumin (BSA) and 0.05%Tween 20 in PBS] for 30minutes at room temperature. F-actinwasstained with Alexa Fluor 633–Phalloidin (Life Technologies),nuclei with 40,6-diamidino-2-phenylindole dihydrochloride(DAPI; Sigma-Aldrich) in blocking solution. After extensivewashes with PBS, cells were mounted in ibidi mounting medium(ibidi). Samples were examined using an IX81 DSU SpinningDisc Confocal from Olympus with a 40� objective.

Proliferation, migration, and invasion assaysTranswell migration and invasion assays were performed as

described previously (12). Briefly, Transwell chambers were leftuncoated (migration assay) or coated with Matrigel (2 mg/well;BD Biosciences), dried overnight, and rehydrated for 1 hour with40 mL of tissue culture media. Cells were harvested, washed oncewith media containing 1% BSA, and resuspended in mediacontaining 0.1% BSA. Then, 100,000 cells were seeded per Trans-well insert. NIH-3T3 conditioned medium served as a chemoat-tractant in the lower chamber. Remaining cells were used tocontrol the expression of genes of interest by Western blot anal-ysis. After 16 hours, cells on top of the Transwell insert wereremoved and cells that hadmigrated/invaded to the lower surfaceof the filters were fixed in 4% paraformaldehyde, stained withDAPI, and counted. For impedance-based real-time chemotacticassays, cells were seeded onto an E-Plate (for proliferation assays)

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Mol Cancer Ther; 14(6) June 2015 Molecular Cancer TherapeuticsOF2




or a CIM-Plate 16 Transwell (for migration/invasion assays) fromRoche Applied Science. After attachment, cell migration or inva-sion (coating of top well with 2 mg of Matrigel) toward NIH-3T3conditioned media was continuously monitored in real time forthe indicated times using the xCELLigence RTCA DP Instrumentfrom Roche Applied Science.

Patient samples, TMAs, and IHCTissue samples were initially collected with the approval of the

Mayo Clinic Institutional Review Board (IRB) under protocolMC0033. Written informed consent for the use of these tissuesin research was obtained from all participants. Generation of theTMA was performed under protocol 09-001642. Therefore, allunique patient identifiers and confidential data were removedand tissue samples were de-identified. The Mayo Clinic IRBassessed the protocol 09-001642 as minimal risk and waived theneed for further consent. All data were analyzed anonymously.TMAs were deparaffinized (1 hour at 60�C), dewaxed in xylene(five times for 4 minutes), and gradually rehydrated with ethanol(100%, 95%, and 75%, twice with each concentration for 3minutes). The rehydrated TMA sections were rinsed in water andsubjected to hematoxylin and eosin (H&E) staining or to antigenretrieval in citrate buffer (pH 6.0) as previously described (26).Slides were treated with 3% hydrogen peroxide (5 minutes) toreduce endogenous peroxidase activity and washed with PBScontaining 0.5% Tween 20 (PBS–Tween 20). Proteins of interestwere detected using indicated specific antibodies diluted in PBS–Tween 20 and visualized using the EnVisionþDual Link LabelledPolymer Kit following the manufacturer's instructions (Dako).Images were captured using the Aperio ScanScope slide scanner(Aperio).

Orthotopic tumor models and treatmentAnimal experiments were performed under protocols A43213

and A17313 approved by the Mayo Clinic Institutional AnimalCare and Use Committee (IACUC). Female nonobese diabeticsevere combined immunodeficiency (NOD scid) mice wereanesthetized, and 500,000 cells washed three times in PBS andmixed with 30 mL of complete Matrigel (BD Biosciences) wereinjected into the fourth mammary gland on the right side ofeach animal. As indicated, cell lines used were MDA-MB-231.Luc (MDA-MB-231-luc2 from PerkinElmer; additionally expres-sing luciferase), MDA-MB-231.Luc stably expressing controlshRNA (scr-shRNA), or two different shRNAs specifically target-ing PKD3 (PKD3-shRNA#1 and PKD3-shRNA#2). For studieswith CRT0066101, mice were treated orally with 80 mg/kgCRT0066101 diluted in a 5% dextrose saline solution (Sig-ma-Aldrich) or 5% dextrose saline solution alone (control)every other day starting 14 days after cell injection. Body weightand tumor volume (caliper measurement) were determinedonce per week. The presence of metastases was detected usingthe IVIS Spectrum Imaging System (PerkinElmer). At the end-point, primary tumors and sites of metastasis were removed andanalyzed as indicated.

Luciferase reporter assayCells were transfected with PRKD3 promoter-luciferase re-

porter (2 mg), Renilla luciferase reporter (0.1 mg), and pEGFP-ERa expression construct (2 mg) in 6-well plates, as indicated.Twenty-four hours after transfection, cells were washed twicewith ice-cold PBS, scraped in 250-mL Passive Lysis Buffer

(Promega), and centrifuged (13,000 rpm, 10 minutes, 4�C).Assays for luciferase activity were performed according to thePromega Luciferase assay protocol and measured using a Ver-itas luminometer (Symantec). Luciferase activity of the PRKD3promoter-luciferase reporter was normalized to Renilla luci-ferase activity. Expression of proteins was controlled by West-ern blot analysis.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) assays were per-

formed using the Imprint Chromatin Immunoprecipitation Kitfrom Sigma-Aldrich according to the manufacturer's protocol.Five micrograms of primary antibody (anti-GFP, Abcam) orrabbit IgG control was used for ChIPs. Immunoprecipitates wereanalyzed by PCR using the primer sets 50-TGACAATGCCTGT-CAGCTTC-30 and 50-AAACGCGAATGTGACCCTAC-30 to amplifya 243 bp fragment (ERa site 1) or 50-TGATAGACACGCTCGC-GACT-30 and 50-TGCCGGGAGCTGTAGTTCCT-30 to amplify a199-bp fragment (ERa site 2) of the human PRKD3 promoter.

BioinformaticsEvaluation of expression of PRKD3 in annotated breast cancer

datasets was performed using the Nextbio server (www.nextbio.com; ref. 27). KM Plotter data were obtained using the currentrelease of Kaplan–Meier Plotter [www.kmplot.com; (28); 2012version, n ¼ 2,978], interrogating using Affymetrix ID:"211084_x_at," survival set at distant metastasis-free survival,auto select best cutoff set at checked, follow-up threshold set atall, and array quality control set at exclude biased arrays.

Statistical analysisGraphPad Prism version 4.0c software (GraphPad Software)

was used for all statistical analyses. Statistical significance (P <0.05) was determined using a two-tailed Student t test andstandard deviations.

ResultsPKD3 is highly expressed in ER� breast cancers and correlateswith aggressiveness

Loss of gene expression of PRKD1 (encoding for PKD1) is amarker for aggressive breast cancer (11, 17). Using isoform-specific antibodies, we determined the expression pattern ofPKD1 and the two oncogenic versions of this kinase family,PKD2 and PKD3, in normal breast (n ¼ 60 samples) and TNBC(n ¼ 40 samples). Whereas PKD1 is the main isoform expressedin normal breast, TNBC show an isoform switch toward expres-sion of PKD3 (Fig. 1A). PKD2 generally was weakly expressed,but is also slightly downregulated in TNBC. In order to evaluatewhether increased PKD3 expression is indeed linked to thetriple-negative phenotype, we evaluated annotated clinicalbreast cancer datasets from 13 different studies. In all studies,triple-negative biopsy samples showed significantly increasedexpression of PRKD3 as compared with triple-positive (ERþ/PRþ/HER2þ) biopsies (Supplementary Table S1). Using TheGene Set Analysis Cell Lines module of GOBO (29, 30), wethen analyzed a panel of 51 breast cancer cell lines and alsofound a reverse correlation of PRKD3 gene expression betweenbasal and luminal breast cancer types (Fig. 1B, left). A similarreverse correlation was found between TNBC and HR-positive

Targeting of ER� Breast Cancer with CRT0066101

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cell lines (Fig. 1B, right), indicating that loss of HR expressionand increased PRKD3 expression may be linked. To test this, weanalyzed ER-positive (ERþ; n ¼ 44) and ER� (n ¼ 41) patienttumors for PKD3 expression and confirmed that increased PKD3expression is linked to loss of ER expression (Fig. 1C andSupplementary Fig. S1). The negative correlation between ERand PKD3 was bolstered by meta-analysis of 24 publishedstudies in which we found that PRKD3 shows significantlyhigher expression in ER� versus ERþ breast cancers (Supple-mentary Table S2). We next assessed the association of PRKD3expression with prognosis in breast cancer patients, and foundthat elevated PRKD3 expression levels in ER� tumors (n ¼1,353) were associated with significantly decreased distantmetastasis-free survival (Fig. 1D).

ERa regulates PKD3 expression levels in breast cancer cellsTo determine whether ERa could directly affect PRKD3

promoter activity, we performed luciferase reporter assays usinga PRKD3 promoter-luciferase gene reporter. Reintroductionof ERa into MDA-MB-231 (ER�/PR�/HER2�) cells led to asignificant decrease (approximately 40%) of PRKD3 gene pro-moter activity (Fig. 2A). These data were confirmed with BT20,another TNBC cell line. BT20 cells, when transfected with ERa,showed an approximately 75% decrease of PRKD3 expressionand this was further decreased, when media were supplement-ed with E2 (Fig. 2B). We analyzed the PRKD3 promotersequence and identified two potential ER-binding sites, 780bases (ERa site 1) or 318 bases (ERa site 2), upstream of thetranscription start site (Fig. 2C). To test whether ERa directly

HER2–, ER+ HER2+, ER+ HER2+, ER–

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Figure 1.PKD3 is highly expressed in ER� breast cancers and correlates with aggressiveness. A, TMA slides containing human TNBC (n ¼ 40) and normal humanbreast tissue samples (n ¼ 60) were analyzed for PKD1, PKD2, and PKD3 expression using isoform-specific antibodies. Scale bar, 100 mm. Statisticalanalysis of PKD1, PKD2, and PKD3 intensity was performed using Aperio positive pixel count algorithm in the Imagescope software (Aperio). B, analysisof PRKD3 gene expression in breast cancer cells lines using the Gene Set Analysis Cell Lines module of GOBO. Cell lines were grouped into Basal A(n ¼ 12), Basal B (n ¼ 14), or Luminal (n ¼ 25) subtypes (left), or TNBC (n ¼ 25) and HR-positive (n ¼ 15) subtypes (right). C, TMA slidescontaining histologically confirmed IDC of indicated subtypes were analyzed for PKD3 expression using an isoform-specific antibody. Scale bar, 100 mm.Statistical analysis of PKD3 intensity in ERþ or ER� groups (n ¼ 44 for ERþ; n ¼ 41 for ER�) was performed using Aperio positive pixel countalgorithm in the Imagescope software (Aperio). D, the Kaplan–Meier plot depicting the impact of levels of PRKD3 gene expression on the metastasis-freesurvival of patients (n ¼ 1,353) with ER� breast cancer. In A and C, P values were acquired with the Student t test using Prism v5 software. � , P < 0.05,statistical significance. In A and C, representative pictures from each group are depicted.

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binds to the PKD3 promoter at these sites, we performed ChIPand confirmed the direct binding of ERa to both of the twopredicted ER-binding sites (Fig. 2D). Next, we determined

whether presence of ERa translates to a decrease in PKD3protein expression. Therefore, we reintroduced ERa in triple-negative MDA-MB-231 and BT20 cells, as well as HCC1954

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Figure 2.ERa directly regulates PKD3 expression in breast cancer cells. A, MDA-MB-231 cells were transfected with PRKD3 promoter-luciferase, Renilla luciferasereporters and vector control or GFP-tagged ERa, as indicated. Forty-eight hours after transfection, reporter gene luciferase assays were performed.� , P < 0.05, statistical significance. Cell lysates were also analyzed by Western blot analysis for expression of ERa (anti-GFP), or b-actin (anti-b-actin)as a loading control. B, BT20 cells were cultivated in phenol-red–free media and transfected with PRKD3 promoter-luciferase and Renilla luciferasereporters and vector control or ERa, as indicated. Forty-eight hours after transfection, BT20 cells were stimulated with 10 nmol/L E2 or vehicle (control)for 6 hours and reporter gene luciferase assays were performed. �, P < 0.05, statistical significance as compared with unstimulated vector control; #, statisticalsignificance as compared with unstimulated ERa-transfected cells. Cell lysates were also analyzed by Western blot analysis for expression of ERa (anti-GFP),or b-actin (anti-b-actin) as a loading control. C, schematic representation of potential ERa-binding sites (in grey italic) in the PRKD3 promoter region �1000to �0. D, chromatin IP. MDA-MB-231 cells were transfected with vector control or GFP-tagged ERa. After cross-linking, the ERa–DNA complexes wereimmunoprecipitated using an anti-GFP antibody. Precipitates were analyzed by PCR for the ERa-bound PRKD3 promoter. A PCR for the PRKD3 promoterusing the input DNA served as a control (input control). E, ER� cell lines MDA-MB-231, BT20, and HCC1954 were transfected with vector control or GFP-taggedERa. Forty-eight hours after transfection, cell lysates were analyzed by Western blot analysis for the expression of PKD3 and ERa (anti-GFP).Staining for b-actin (anti-b-actin) served as a loading control. F, the ERþ cell line T47D was treated with 100 nmol/L fulvestrant or DMSO (control) for24 hours and cell lysates were analyzed by Western blot analysis for the expression of PKD3 or b-actin (anti-b-actin).






(ER�/PR�/HER2þ) cells. As expected, the presence of ERa ledto a significant decrease in PKD3 expression, and this wasindependent of the HER2 amplification status (Fig. 2E). Even-tually, we treated the ERþ cell line T47D with fulvestrant, acompound that leads to nuclear export and degradation ofER (31, 32). As expected, treatment of T47D cells with fulves-trant increased PKD3 expression further indicating a role forER as a regulator of PKD3 expression (Fig. 2F).

The knockdown of PKD3 decreases cancer cell proliferation,migration, and invasion in vitro and in vivo

To test the impact of PKD3 on breast cancer cell behavior, weused the invasive breast cancer cells MDA-MB-231 (ER�, PR�,

HER2�), which express high levels of PKD3, low levels of PKD2,and no PKD1 (Supplementary Fig. S2; Supplementary Table S3;ref. 11). A knockdown of basal PKD3 expression using twodifferent PKD3-specific shRNA sequences significantly decreasedMDA-MB-231 cell numbers over a time period of 60 hours(Fig. 3A and B and Supplementary Fig. S3). Similar results wereobserved in another ER� cell line, HCC1954, which show asimilar PKD expression pattern (Supplementary Fig. S2; datanot shown). Next, we tested the role of PKD3 on the invasivephenotype. The knockdown of PKD3 in MDA-MB-231 cells ledto a dramatic decrease in directed cell migration (Fig. 3C andSupplementary Fig. S4), and similar effects were observed forcell invasion through extracellular matrix (Fig. 3D). This confirms

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Figure 3.Knockdownof PKD3 decreases cancer cellproliferation, migration, and invasion invitro and in vivo. A–E, MDA-MB-231 cellswere infected with lentivirus harboringcontrol shRNA (scr-shRNA) or twodifferent shRNAs specifically targetingPKD3 (PKD3-shRNA#1 and PKD3-shRNA#2). A, 48 hours after initialinfection, a fraction of the cells was lysedand PKD3 knockdown was verified byWestern blot analysis. B, cellswere seededin E-plates and numbers of attachedcells were continuously monitored in realtime for 60 hours using an xCELLigenceRTCA DP instrument. Error bars (gray)represent three experiments. C, cellswere seeded in CIM-plates and (afterattachment) directed cell migration wascontinuously monitored in real time for10 hours using an xCELLigence RTCA DPinstrument. Error bars (gray) representthreeexperiments. D, cellswere seededonMatrigel-coated Transwell filters andTranswell invasion assayswere performedas described in Materials and Methods. E,cells were fixed and stained with DAPI(blue) and phalloidin (red). Bars, 10 mm.Representative pictures from each groupare depicted. The cell area (graph) wasmeasured and analyzed using the ImageJsoftware. In B–E, the results presented arethe mean�SEM. P values were calculatedusing a two-tailed Student t test.� , P < 0.05, statistical significance. F andG,MDA-MB-231.Luc cells stably expressingcontrol shRNA (scr-shRNA) or twodifferent shRNAs specifically targetingPKD3 (PKD3-shRNA#1 and PKD3-shRNA#2) were injected into the mfp offemale NOD scid mice. In F, the presenceof metastasis in lymph nodes and lungswas detected by immunostaining with anantibody specific for human vimentin(detects human cancer cells).Representative pictures from each groupare depicted. Bars, 200 mm for lymphnodes; 1 mm for lungs. In G, the number ofpulmonary metastases per mm2, or theaverage size of pulmonary metastases inmm2 was quantified from five fields eachlung. � , P < 0.05, statistical significance.

Borges et al.





that PKD3 is the major isoform driving motility (and prolifera-tion) in these cells. Interestingly, along with a decreased motility,we noticed a dramatic increase in cell spreading and altered F-actin organization when PKD3 was knocked down (Fig. 3E). Totest whether the knockdown of PKD3 can affect breast tumormetastasis in vivo, we orthotopically implantedMDA-MB-231.Luccells either stably expressing scrambled shRNA control or twodifferent specific shRNA sequences for PKD3 into the mammaryfat pad (mfp) of female NOD scid mice. To exclude that PKD3effects on cell proliferation affect results onmetastasis, endpointsof the experiment were set for each individual mouse at a primarytumors size of 700 � 100 mm3. At the endpoint, tumors andtissues of potential sites of metastasis were extracted. As a control,primary tumors were analyzed by IHC with a monoclonal anti-body specific for PKD3 (Supplementary Fig. S5A). As predicted byour in vitro experiments, primary tumor growth was significantlyslowerwhenPKD3 expressionwas decreased (Supplementary Fig.S5A). Analysis of expression of Ki67 and cleaved caspase-3 stain-ing indicated that thiswas due to adecrease in cell proliferation, aswell as an increase in cell death (Supplementary Fig. S5B andS5C). Interestingly, PKD3-shRNA tumors showed reduced localinvasion when compared with control (scr-shRNA) tumors (Sup-plementary Fig. S5D). Next, we determined metastasis to distantorgans by immunohistochemical staining for human vimentin asa marker for human cancer cells. We found that PKD3 down-regulation dramatically decreased cancer cells infiltration tolymph nodes and lungs (Fig. 3F). Furthermore, mice implantedwith PKD3-shRNA cells had significantly fewer and smallermetastases to their lungs compared with controls (scr-shRNA; Fig. 3G). Taken together, our data indicate that PKD3plays an important role in breast tumor growth, progression, andmetastasis.

The PKD inhibitor CRT0066101 decreases cancer cellproliferation, migration, and invasion in vitro

Next, we tested the impact of PKD3 inhibition on cell pro-liferation and the invasive phenotype. Because PKD3 is upregu-lated and as such can be targeted in ER� breast cancer indepen-dently of the HER2 status, we decided to test two different celllines, MDA-MB-231 as a model for TNBC and HCC1954 as amodel for ER�, HER2þ breast cancer. We used the pan-PKDinhibitor CRT0066101, which has been shown to have antican-cer activity in pancreatic, prostate, and colorectal cancer cells (33,34). CRT0066101 induced a significant decrease in cell prolif-eration in both cell lines (Fig. 4A and D and Supplementary Fig.S6). In a similar fashion to PKD3 depletion, inhibition of PKDactivity with CRT0066101 also blocked directed cell migration(Fig. 4B and E and Supplementary Fig. S7) and invasion (Fig. 4Cand F). Similar as observed with the PKD3 knockdown in Fig. 3E,along with a decreased motility, we noticed increased spreadingand altered F-actin organization of cells that were treated withCRT0066101 (Fig. 4G and H). Overall, data obtained with PKD3knockdown and CRT0066101 were similar, although both celllines also express PKD2 that may have similar functions as PKD3in regard to regulation of cell migration and invasion (Supple-mentary Fig. S8).

CRT0066101 decreases primary tumor size, local invasiveness,and metastasis in vivo

Next, we tested whether CRT0066101 can be used as an effi-cient strategy for the treatment of tumor growth andmetastasis of

ER� cancers in vivo. Therefore, we orthotopically implantedMDA-MB-231 cells into the mfp of female NOD scid mice. After estab-lishment of primary tumors (day 14 after cell implantation),mice were treated with 80 mg/kg CRT0066101 (oral administra-tion, everyotherday)or vehicle control. At the endpoint (10weeksafter cell implantation), primary tumors and tissues of potentialsites ofmetastasis were extracted. As in a previous studymodelingpancreatic cancer (33), no toxicity was detected at this dosage ofCRT0066101 and no significant changes in body weight ordamage in tissue was observed (not shown). Treatment of micewith CRT0066101 did result in a significant decrease of primarytumor size and weight (Fig. 5A and Supplementary Fig. S9),associated with an approximately 50% decrease in tumor cellproliferation (Fig. 5B), and an increase of apoptosis (Fig. 5C). Ofnote, the effects of CRT0066101 on tumor cell viability andproliferation that were observed in vivo were in line with effectsobserved in vitro (Fig. 4). Additional analysis of tumor edges aswell as the connection of tumor cells to the normal adjacentmouse mammary tissue showed a reduced local invasion intumors treated with the PKD inhibitor (Fig. 5D). This decreasein invasiveness correlated with decreased expression of COX-2(Fig. 5E), which previously was associated with local invasion ofbreast cancer cells, as well as metastasis to the lungs (35). Indeed,IVIS imaging of animals indicated that CRT0066101 may affectmetastasis to distant organs (Fig. 6A). Immunohistochemicalanalysis for human cancer cells (IHC for anti-human vimentin)at the endpoint indicated a dramatic decrease of infiltration oftumor cells into lymph nodes in CRT0066101-treated mice (Fig.6B). Similarly, lungmetastaseswere fewer innumbers and smallerin size (Fig. 6B–D). Metastases in the lungs of the treated miceshowed a significant lower expression of Ki67, indicating that thedecrease in average size ofmetastasesmay be due to CRT0066101effects on the ability of tumor cells to proliferate in their newenvironment (Fig. 6E).

DiscussionSilencing of PKD1 and increased expression of the oncogenic

versions of this kinase family, PKD2 and PKD3, has beendescribed to contribute to progression of several epithelial can-cers, including breast cancer (11, 13, 16, 17, 20–22), gastric cancer(36), pancreatic cancer (37, 38), colorectal cancer (34), andprostate cancer (14, 39–42). We here show that the switch fromPKD1 to PKD3 expression defines the transition to an aggressivebreast tumor phenotype. PKD1 previously had been shown tomaintain the epithelial phenotype bypreventing EMT (13, 14, 16)and to negatively regulate cell migration (12), invasion (17), andmetastatic progression of breast cancer (11, 19). Consequently, ininvasive breast cancers, PKD1 expression is downregulated bypromoter hypermethylation (11, 36). The signaling mechanismsby which other PKD isoforms are (up)regulated at the transcrip-tional levels have not been identified so far.

Analysis of cell lines, TMAs from patient samples (Fig. 1), andannotated clinical breast cancer datasets showed significantlyhigher PRKD3 gene expression in ER� or TNBC biopsies (Sup-plementary Table S1). A detailed analysis suggested that increasedPKD3 expression is mainly due to loss of ER expression and notdependent on the HER2 status of tumors (Fig. 1C and Supple-mentary Table S2). This led to the questions whether the ER couldbe a direct negative regulator of PRKD3 expression; or whetherobserved reverse expression between both molecules is






correlative. By reexpressing ER in ER� cell lines (Fig. 2E) orinhibiting ER expression in ERþ T47D cells (Fig. 2F), we clearlydemonstrate that PKD3 repression depends on ER activity. As amechanism of regulation, we demonstrate that ER decreasesPKD3 expression through direct binding to the PRKD3 promoterat two different ER-binding sites (Fig. 2C and D). Thus, our datademonstrate a direct negative regulation of a gene by this receptor.Although ER is mostly known for its positive effect on genetranscription, some studies have demonstrated that it can alsoact as a repressor of gene expression (43, 44). For example, ER canrepress a cytochrome P450-encoding gene (CYP1A1) by targetingDnmt3B DNA methyltransferase (44).

PKD3 has been implicated in all aspects of tumor formationand progression, such as mediating proliferation, survival, andinvasiveness, in different cancers (20, 22, 39, 45). However,relatively little is known about the molecular mechanisms bywhich PKD3may drive carcinogenesis. PKD3 previously has been

shown to mediate activation of Akt, leading to prolonged acti-vation of extracellular signal–regulated kinase (ERK) 1/2 (39).Furthermore, S6 kinase 1 (S6K1), a member of the mammaliantarget of rapamycin complex 1 signaling cascade (mTORC1), wasidentified as a downstream target of PKD3 tomediate its effect oncell proliferation in TNBC cell lines (22). Another target involvedmaybe theG-protein–coupled receptor kinase-interacting protein1 (GIT1), a key mediator of PKD3-induced cell spreading andproliferation (46).

Besides describing a previously unknown regulation of PKD3expression that can be linked to aggressiveness of breast cancers,we also tested the use of PKD inhibitors in breast cancers thatmainly express PKD3. Our data and previous work implicate thatideal targets to test the efficacy of PKD inhibitors would be ER� ortriple-negative, invasive breast cancers, in which upregulation ofPKD3 is accompanied by the loss of PKD1 expression.CRT0066101 is a selective and potent PKD inhibitor that targets

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The PKD inhibitor CRT0066101decreases the cell proliferation and theinvasive phenotype of ER� breast cancercells. A and D, indicated cell lines wereseeded in E-plates and (afterattachment) treated with 2.5 mmol/LCRT0066101 or DMSO (control).Numbers of attached cells werecontinuously monitored in real time forindicated times using an xCELLigenceRTCA DP instrument. Error bars (gray)represent three experiments. B, MDA-MB-231 cells were seeded in CIM-platesand treatedwith 2.5mmol/LCRT0066101orDMSO(control). After attachment, cellmigrationwas continuouslymonitored inreal time for indicated time using anxCELLigence RTCA DP instrument. Errorbars (gray) represent three experiments.C and F, indicated cell lines were seededon Matrigel-coated Transwell filters andtreated with 2.5 mmol/L CRT0066101 orDMSO (control). Transwell invasionassays were performed as described inMaterials and Methods. E, HCC1954 wereseeded on Transwell filters and treatedwith 2.5 mmol/L CRT0066101 or DMSO(control). Transwell migration assayswere performed as described inMaterials and Methods. G and H,indicated cell lines were treated with2.5 mmol/L CRT0066101 or DMSO(control) for 16 hours. Cells were fixedand stained with DAPI (blue) andphalloidin (red). Bar, 10 mm.Representative pictures fromeachgroupare depicted. The cell areawasmeasuredand analyzed using the ImageJ software.In A–H, the results presented are themean � SEM. P values were calculatedusing a two-tailed Student t test.� , P < 0.05, statistical significance.

Borges et al.





all three isoforms in a low nanomolar range (i.e., IC50 for PKD3is 2 nmol/L). In a previous work, it has been shown to be activein vivo in orthotopic animal models for pancreatic cancer andcolorectal cancer (33, 34). Because it can be orally administer-ed and has no side effects in mice, when used at doses thatinhibit PKD (33, 34), it is an inhibitor that could be developedfor clinical use.

Our data not only show that CRT0066101 can block all aspectsof the tumor phenotype in PKD1-negative/PKD3-positive breastcancer cells in vitro (Fig. 4), but also demonstrate in vivo relevanceby showing that CRT0066101 significantly inhibits primarytumor growth, local invasion, and metastasis to distant organsin vivo (Figs. 5 and 6). It is also important to note that the sameevents were obtained with specific knockdown of PKD3 showingthat this kinase is the main target in ER� cancer cells (Fig. 3).Although our in vitro data using shRNA or CRT0066101 clearlydemonstrate effects on cell proliferation,migration, and invasion,it is possible that additional tumorigenic functions are affected by

knockdown or inhibition of PKD3 in vivo. For example, PKDsignaling previously has been implicated in angiogenesis(38, 47, 48) and it is very possible that the decrease in size ofthe primary tumors that we observed in response to CRT0066101treatment is partly due to blocking of angiogenesis.

It should be noted that for breast cancers that undergo a switchfrom PKD1 to PKD3, two strategies are possible, either to reex-press PKD1, or to inhibit PKD3 (discussed in ref. 49). We recentlyhave tested the first strategy, and shown that reverting the epige-netic silencing of PKD1with theDNAmethyltransferase inhibitordecitabine can dramatically reduce the invasive and metastaticpotential of triple-negative orthotopic tumors in vivo (11, 19).What is still ill-defined is how PKD1, once it is reexpressed ininvasive breast cancers, can exert a protective effect and antagonizePKD3 functions. Because the ESR1 gene promoters also can besilenced by DNA methylation (50), the simplest explanation forthis may be that decitabine treatment also upregulates ER, whichthen decreases PKD3 expression.

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Figure 5.CRT0066101 inhibits tumor growth andlocal tumor cell invasion in vivo. A, MDA-MB-231.Luc cells were injected into themfp of female NOD scid mice. Afterestablishment of primary tumors, atweek 2, mice were treated orally with 80mg/kg CRT0066101 or vehicle everyother day for an additional period of 8weeks. Tumor growth was continuouslymonitored using the IVIS Spectrumimaging system. Left, a representativepicture of mice from each group (n ¼ 6per group) at week 7. At the endpoint(week 10), tumor volume and weightwere measured. � , P < 0.05, statisticalsignificance. B and C, primary tumors ofdifferent treatment groups were stainedby IHC for the expression of Ki67 (B) andcleaved caspase-3 (C). Bars, 50 mm. D,samples of primary tumor were stainedwith H&E. Bar, 200 mm. Areas wheretumors connect with mouse mammarygland tissue were enhanced. E, primarytumors were stained by IHC for theexpression of COX2. Bars, 50 mm. In B,C, and E, statistical analysis wasperformed using Aperio positive pixelcount algorithm in the Imagescopesoftware. P values were acquired withthe Student t test using Prism v5software. �, P < 0.05, statisticalsignificance. In B–E, representativepictures from each group are depicted.






In conclusion, our study provides a rationale that supports theuse of PKD inhibitors such as CRT0066101 for treatment ofpatients diagnosed with ER� or TNBC. Key for treatment withPKD inhibitors is a downregulation of PKD1 and upregulationof the oncogenic version PKD3 (or PKD2). This requires devel-oping reliable techniques that could be used in clinical settingsto determine PKD1, 2, and 3 expression status before treatmentdecisions are made.

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

Authors' ContributionsConception and design: S. Borges, E.A. Perez, E.A. Thompson, P. StorzAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.A. Perez, E.A. Thompson, X.J. GeigerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Borges, E.A. Perez, E.A. Thompson, D.C. Radisky,P. StorzWriting, review, and/or revision of the manuscript: S. Borges, E.A. Perez,D.C. Radisky, X.J. Geiger, P. Storz

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.A. PerezStudy supervision: P. Storz

AcknowledgmentsThe authors thank members of the Storz laboratory for helpful discussions

and the Luther and Susie Harrison Foundation for their support.

Grant SupportThis work was supported by the NIH (GM086435 and CA140182) to

P. Storz, the Bankhead-Coley Program of the Florida Department of Health(1BG11) to P. Storz, the Donna Foundation (26.2 with Donna) to E.A. Perezand E.A. Thompson, and the Mayo Clinic Breast Cancer SPORE (CA116201)to P. Storz and D.C. Radisky.

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 October 31, 2014; revised April 2, 2015; accepted April 2, 2015;published OnlineFirst April 7, 2015.

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Published OnlineFirst April 7, 2015.Mol Cancer Ther Sahra Borges, Edith A. Perez, E. Aubrey Thompson, et al. Cancers with the Protein Kinase D Inhibitor CRT0066101

Negative Breast−Effective Targeting of Estrogen Receptor

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