Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer … · Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer Growth and Predicts Poor Patient Survival Petra den Hollander1,

Therapeutics, Targets, and Chemical Biology

Phosphatase PTP4A3 Promotes Triple-NegativeBreast Cancer Growth and Predicts PoorPatient SurvivalPetra den Hollander1, Kathryn Rawls1, Anna Tsimelzon2, Jonathan Shepherd1,3,Abhijit Mazumdar1, Jamal Hill1, Suzanne A.W. Fuqua2, Jenny C. Chang4,C. Kent Osborne2, Susan G. Hilsenbeck2, Gordon B. Mills5, and Powel H. Brown1,6

Abstract

Triple-negative breast cancer (TNBC)has theworst prognosis ofall breast cancers, and women diagnosed with TNBC currentlylack targeted treatment options. To identify novel targets forTNBC, we evaluated phosphatase expression in breast tumorsand characterized their contributions to in vitro and in vivo growthof TNBC. Using Affymetrix microarray analysis of 102 breastcancers, we identified 146 phosphatases that were significantlydifferentially expressed in TNBC comparedwith estrogen receptor(ER)-positive tumors. Of these, 19 phosphatases were upregu-lated (0.66-fold; FDR ¼ 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressedphosphatases in four triple-negative and four ER-positive breastcancer lines using specific siRNAs and found that depletion of sixof these phosphatases significantly reduced growth and anchor-

age-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3(also known as PRL-3) demonstrated its requirement for G1–Scell-cycle progression in all breast cancer cells, but PTP4A3 reg-ulated apoptosis selectively in TNBC cells. In addition, PTP4A3inhibition reduced the growth of TNBC tumors in vivo. Moreover,in silico analysis revealed the PTP4A3 gene to be amplified in 29%of basal-like breast cancers, and high expression of PTP4A3 couldserve as an independent prognostic indicator for worse overallsurvival. Collectively, these studies define the importance ofphosphatase overexpression in TNBC and lay the foundationfor the development of new targeted therapies directed againstphosphatases or their respective signaling pathways for TNBCpatients. Cancer Res; 76(7); 1942–53. �2016 AACR.

IntroductionBreast cancer is the second leading cause of cancer-related

death in women (1). Targeted therapies can significantly reducerecurrence for estrogen receptor (ER)-positive (e.g., selectiveER modulators, aromatase inhibitors) and HER2-positive (e.g.,HER2-specific inhibitors) breast cancers (2–4). However, 15%to 20% of breast cancers do not express ER, progesteronereceptor (PR), or HER2 (5, 6). These triple-negative breastcancers (TNBC) are highly aggressive, have poor prognoses,lack targeted therapies (7–9), and are currently treated withtoxic, nonspecific chemotherapy drugs. Consequently, there is

an urgent need to identify more effective, less toxic therapeuticstrategies.

In addition to ER and HER2 signaling pathways, othergrowth regulatory pathways have been shown to regulategrowth and development of cancers. One of our major goalsis to identify signaling pathways and molecules critical forgrowth of TNBCs. Previously, we identified a set of kinasesoverexpressed in ER-negative compared with ER-positive breastcancers (10). Further analysis demonstrated that many of theseoverexpressed kinases are essential for the growth of ER-neg-ative breast cancer cell lines (10). Aberrant phosphorylationhas been implicated in many diseases, including cancer(11, 12), and regulate a number of signaling pathways, includ-ing those associated with growth, survival, metabolism, and thecell cycle (13–15).

In this study, we hypothesized that phosphatases are differen-tially expressed in triple-negative as compared with ER-positivetumors. Using transcriptional profiling data to evaluate phospha-tase expression in a breast cancer dataset, we identified two sets ofphosphatases (those highly expressed or underexpressed) inTNBC as compared with ER-positive tumors. Further analysis ofthe phosphatases upregulated in triple-negative compared withER-positive breast cancers, identified several phosphatases criticalfor anchorage-independent growth in TNBC cells. We selectedthose phosphatases (PTP4A3, PPAP2B, CDC25B, and TIMM50)that were found critical for the tumorigenicity of TNBC cells forfurther study. Of these, PTP4A3 (protein tyrosine phosphatasetype IVA, member 3, also known as, PRL-3) was required for

1Department of Clinical Cancer Prevention,TheUniversityof TexasMDAnderson Cancer Center, Houston, Texas. 2Lester and Sue SmithBreast Center, Baylor College of Medicine, Houston, Texas. 3Depart-ment of Molecular and Cellular Biology, Baylor College of Medicine,Houston, Texas. 4Methodist Cancer Center, The Methodist HospitalResearch Institute, Houston, Texas. 5Department of Systems Biology,The University of Texas MDAnderson Cancer Center, Houston, Texas.6Department of Breast Medical Oncology,The University of Texas MDAnderson Cancer Center, Houston, Texas.

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

Corresponding Author: Powel H. Brown, The University of Texas M.D. AndersonCancer Center, 1515 Holcombe Blvd, Unit 1360, Houston, TX 77030. Phone: 713-745-3672; Fax: 713-794-4679; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0673

�2016 American Association for Cancer Research.

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in vitro and in vivo TNBC cell growth. PTP4A3 has previously beenimplicated in the regulation of migration, invasion, and metas-tasis (16), but its role in regulating cancer cell growth is lessunderstood. PTP4A3 inhibition induces G1 arrest in breast cancercells, reduces growth and Ki67 expression in xenograft tumors,and loss of PTP4A3 in breast cancer cells induces apoptosisspecifically in TNBC cells. Our results demonstrate that phospha-tases represent promising targets for the treatment and preventionof TNBC, and provide the rationale for further investigationssupporting the development of inhibitors of phosphatases ortheir signaling pathways as viable strategies for women with orat high risk of TNBC.

Materials and MethodsStudy population and design

Tissue samples used for this study were obtained from theLester and Sue Smith Breast Cancer Tumor bank at Baylor Collegeof Medicine (Houston, TX). We studied 102 breast tumors fromthis bank (collected from sites in the United States and Europeaccording to institutionally approved guidelines) for the analysisof gene expression, DNA copy number, and protein change (GEOaccession number GSE76275). We used a subset of these tissuesamples, which are listed in Supplementary File - Tissue SampleIDs. All studies associated with this research were conducted withthe approval of the Baylor College of Medicine and University ofTexas MD Anderson Cancer Center Institutional Review Boards.Study demographics define the majority of the population to bepostmenopausal women, 97% Caucasian, and a median subjectage of 53 years (Supplementary Table S1). After collecting andfreezing tumors in liquid nitrogen, DNA, RNA, and proteincontent was individually isolated. In this cohort, 96% of tumorswere invasive ductal carcinomas, 35% presented no lymph nodeinvolvement, none were metastasic, and 75% of study partici-pants had tumors�2 cm3 at the time of diagnosis. The ER and PRstatus of each tissue sample was determined through immuno-histochemical analysis, and HER2 status was determined byfluorescent in situ hybridization, identifying 49 ER-positivetumors and 53 triple-negative tumors. Of the ER-positive breastcancers, 4 tumors were also HER2-positive (5%), and 42 PRpositive (41%; Supplementary Table S1).

Selection of genes for further studyIn total, 49 ER-positive and 53 triple-negative breast tumors

were profiled using the Affymetrix U133 Plus 2.0 gene expres-sion array. Data analysis was limited to 332 genes identified asphosphatase or phosphatase-interacting proteins. If a gene wasrepresented by more than one probeset on the U133 Plus2 chip,we used the most variable probeset. The list of genes includedin the analysis and gene identifiers are presented in Supple-mentary Table S3. Analysis (expression estimation and groupcomparison) was performed using BRB-ArrayTools developedby Dr. Richard Simon and the BRB-ArrayTools DevelopmentTeam (http://linus.nci.nih.gov/BRB-ArrayTools.html). dChipsoftware was used for preparing the clustering picture (http://www.hsph.harvard.edu/cli/complab/dchip). Expression wasestimated using Robust Multiarray Average (RMA) procedure.Criterion for group comparison was false discovery rate (FDR)¼ 0.05. Genes with �1.5-fold increased expression in triple-negative compared with ER-positive breast tumors and an FDR< 0.05 were selected for further study (Table 1).

Cell lines, reagents, and plasmidsAll cell lines were purchased from ATCC, and were DNA

fingerprinted by the Characterized Cell Line Core Facility atMD Anderson Cancer Center in 2012 to insure correct identity.MCF-7, MDA-MB-231, MDA-MB-468, and HS578T cell lineswere cultured in DMEM (Cellgro by Mediatech, Inc.), and ZR-75-1, HCC1143, T47D, and BT474 cell lines were cultured inRPMI1640 medium (Cellgro by Mediatech, Inc.). Growthmedia for all cell lines was supplemented with 10% FBS,penicillin (100 mg/mL), and streptomycin (100 mg/mL). ThesiRNA oligos were purchased from Sigma-Aldrich. A pool ofthree siRNA oligos was used at a final concentration of 30nmol/L using DharmaFECT1 Transfection Reagent (Dharma-con, Inc.), following the manufacturer's instructions. pGIPZlentiviral short hairpin RNAs (shRNA) for PTP4A3 (OpenBiosystems, Inc.) were subcloned into a pTRIPZ lentiviralexpression system (Open Biosystems, Inc.). The PTP4A3 over-expression vector and control vector (pCMV6) were commer-cially obtained and sequence-verified.

RNA preparation and qRT-PCRRNA was isolated using the RNeasy Kit (Qiagen). cDNA was

generated using random primers, and Superscript II ReverseTranscriptase (Invitrogen; Life Technologies). TaqMan assayswere designed and qRT-PCR was performed. Cyclophilin wasused as an endogenous control, and results were normalized tocyclophilin � SD.

Generation of stable cell linesLentiviral particles were generated by transfecting 293T cells

with lentiviral constructs and packaging plasmids encodingVSV-G, Gag, Pol, and Tat using FuGENE 6 Transfection Reagent(Roche Applied Science). Media overlaying the cells was har-vested at 24, 48, and 72 hours posttransfection, and filteredthrough a 0.45-mm MEC filter. Polyethylene glycol (PEG)precipitation was used to concentrate the virus. Stable cell linesexpressing inducible shRNAs were generated by lentiviral infec-tion using the pTRIPZ lentiviral expression system in thepresence of 4 mg/mL polybrene, followed by puromycin selec-tion. All pTRIPZ stable cell lines were maintained in media withTet-safe Serum (Clontech Laboratories, Inc.).

MDA-MB-231 and MCF-7 cell lines stably transfected withcontrol vector or PTP4A3 were generated using FuGENE 6 Trans-fection Reagent. After 48 hours, transfected cell were selected bythe addition of G418 (1 mg/mL). Colonies visible by eye wereexpanded and tested for expression of PTP4A3.

Growth assaysCells were transfected with 30 nmol/L final concentration of

siRNA. After 24 hours, cells were plated in triplicate in 48-wellplates. Cell proliferation was measured using the Countess Auto-mated Cell Counter (Invitrogen; Life Technologies) and Trypanblue staining. Cell count was assessed at days 4 and 6. Each datapoint was performed in triplicate, with results reported as averagepercentage � SD.

For the growth assays with PTP4A3 overexpression, cells wereplated in triplicate in 48-well plates. Two clones stably transfectedwith control vector and four clones stably transfected with CMV-PTP4A3 were used for the growth assay. Cells count was assessedat days 1, 2, and 4. Each data point was performed in triplicate,with results reported as average percentage � SD.

PTP4A3 Is Critical for Triple-Negative Breast Cancer Growth

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Anchorage-independent growth assaysCells were transfected with 30 nmol/L final concentration of

siRNA. Anchorage-independent growth assays were performedby plating cells 24 hours after siRNA transfection in 0.35%SeaPlaque GTG Agar (FMC Corp.) in the appropriate medium.Depending on the cell line, either 104 or 2 � 104 cells wereplated per well. Suspended cells were plated on a 0.7% agarbase in the same medium. Colonies were counted using theGelCount Colony Counter (Oxford Optronix) 2 and 3 weekspostplating. All experiments were performed in triplicate, andresults reported as average colony number � SD.

Apoptosis assaysApoptosis assays were performed using Annexin V staining

(eBioscience) following the manufacturer's instructions, andreplacing propidium iodide (PI) with DAPI staining. Cells

were plated on 6-well plates, transfected with siRNA, andharvested after 3 days, then stained and analyzed in tripli-cate using a Gallios Flow Cytometer (Beckman Coulter,Inc.). Results were reported as average percentage of eachpopulation � SD.

Cell-cycle analysisCells were plated on 6-well plates, transfected with siRNA, and

harvested after 4 days, at which time they were fixed, stained withPI, and analyzed in triplicate using a Gallios Flow Cytometer(Beckman Coulter, Inc.). Results are reported as the averagepercentage of each population � SD.

Immunofluorescence staining and microscopyCells were plated on coverslips in 6-well plates. The next day

cells were transfected with 30 nmol/L final concentration of

Table 1. Phosphatases differentially expressed �1.5-fold in triple-negative compared with ER-positive breast tumors (all FDR � 0.05)

Gene

Type of expression Name SymbolFoldchange FDR

Overexpressed Inositol(myo)-1(or 4)-monophosphatase 2 IMPA2 2.43 <0.001Protein tyrosine phosphatase-like (proline instead of catalytic arginine), mem A PTPLA 2.32 <0.001Protein phosphatase 1, regulatory (inhibitor) subunit 14B PPP1R14B 2.05 <0.001Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 PTPRZ1 2.01 0.007Protein tyrosine phosphatase type IVA, member 3 PTP4A3 1.95 <0.001Phosphatidic acid phosphatase type 2B PPAP2B 1.93 <0.001Protein tyrosine phosphatase, receptor type, F PTPRF 1.85 <0.001Cyclin-dependent kinase inhibitor 3 CDKN3 1.83 <0.001CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 1.80 <0.001Discs, large (Drosophila) homolog-associated protein 5 DLGAP5 1.74 0.007Nudix (nucleoside diphosphate linked moiety X)-type motif 11 NUDT11 1.68 <0.001Cell division cycle 25 homolog B (S. pombe) CDC25B 1.66 <0.001Phosphoserine phosphatase PSPH 1.63 0.034Phospholipase D family, member 3 PLD3 1.61 <0.001Cell division cycle 25 homolog A (S. pombe) CDC25A 1.59 <0.001Protein phosphatase 1, regulatory (inhibitor) subunit 11 PPP1R11 1.56 <0.001Nudix (nucleoside diphosphate linked moiety X)-type motif 5 NUDT5 1.55 <0.001Protein tyrosine phosphatase, nonreceptor type 14 PTPN14 1.53 <0.001Translocase of inner mitochondrial membrane 50 homolog (S. cerevisiae) TIMM50 1.52 <0.001

Underexpressed Inositol polyphosphate-4-phosphatase, type II, 105 kDa INPP4B 0.27 <0.001Protein phosphatase 1, regulatory (inhibitor) subunit 3C PPP1R3C 0.37 <0.001Dual specificity phosphatase 4 DUSP4 0.37 <0.001Nudix (nucleoside diphosphate linked moiety X)-type motif 12 NUDT12 0.38 <0.001Fructose-1,6-bisphosphatase 1 FBP1 0.38 <0.001Ectonucleotide pyrophosphatase/phosphodiesterase 1 ENPP1 0.39 <0.001Protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform PPP2R2C 0.41 <0.001Ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) ENPP4 0.43 <0.001Protein tyrosine phosphatase, receptor type, B PTPRB 0.48 <0.001Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase CILP 0.49 <0.001Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform PPM1A 0.51 <0.001Phosphatase, orphan 2 PHOSPHO2 0.51 <0.001Dual specificity phosphatase 5 DUSP5 0.55 <0.001Protein tyrosine phosphatase, receptor type, T PTPRT 0.55 <0.001Chromosome 12 open reading frame 51 C12orf51 0.58 <0.001Protein phosphatase 1H (PP2C domain containing) PPM1H 0.58 0.001Dual specificity phosphatase 28 DUSP28 0.59 <0.001Protein phosphatase 2, catalytic subunit, alpha isozyme PPP2CA 0.59 <0.001Protein phosphatase 1D magnesium-dependent, delta isoform PPM1D 0.59 <0.001Protein tyrosine phosphatase, receptor type, N polypeptide 2 PTPRN2 0.59 0.002Protein tyrosine phosphatase, nonreceptor type 13 PTPN13 0.60 <0.001Myotubularin related protein 9 MTMR9 0.61 <0.001SAC1 suppressor of actin mutations 1-like (yeast) SACM1L 0.60 <0.001Protein phospahatase 2, regulatory subunit B, alpha PPP2R2A 0.62 <0.001Multiple inositol polyphosphate histidine phosphatase, 1 MINPP1 0.62 <0.001FANCD2/FANCI-associated nuclease 1 FAN1 0.63 <0.001Protein phosphatase 6, catalytic subunit PPP6C 0.63 <0.001

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siRNA. After 48 hours, cells were washed in PBS, fixed for 20min-utes in 4% paraformaldehyde at room temperature, and blockedfor 1 hour at room temperature in 5% goat serum and 1% TritonX-100 in 1� PBS. Coverslips were incubated with primary anti-body [phospho-histone H3 (p-H3; 1:500) from Cell SignalingTechnology, andKi67 (Clone SP6) fromThermo Fisher Scientific]overnight at 4�C in 1% goat serum and 1% Triton X-100 in 1�PBS, incubated with secondary antibody (Alexa-488 goat anti-rabbit; 1:200) for 1 hour at room temperature, and thenmountedwith DAPI-containing mounting media (Vectashield). Fluores-cent images were capturedwith aNikonmicroscope. Experimentswere performed in triplicate.

IHC and staining analysisTumor sections of 5mmwere stained for either Ki67 (Clone SP6

from Thermo Fisher Scientific) or with cleaved caspase-3 (CellSignaling Technology). Antigen retrieval was performed in citratebuffer, and endogenous peroxidase was blocked with 3% hydro-gen peroxide for 10 minutes. Primary antibodies were incubatedovernight at 4�C, followed by incubation with biotinylated anti-rabbit antibody (1:100) for 30 minutes. Peroxidase activity wasvisualized using the Vector NovaRED Substrate Kit (PK-6101,Vector Laboratories, Inc.).

Western blottingCells were transfected with 30 nmol/L final concentration of

siRNA. After 48 hours, cells were washed in PBS, lysed in RIPAbuffer, run on a 4% to 20% gradient gel, and transferred ontonitrocellulose. Primary antibodies were incubated with the mem-brane overnight at 4�C and included p-ERK1/2, ERK, cleavedcaspase-7, caspase-7, p-p38, and p38 (Cell Signaling Technology;1:1,000), and vinculin and actin (Sigma-Aldrich; 1:5,000). BlotswerewashedwithPBS-T and incubatedwith secondary antibodiesfor 1 hour at room temperature. Bends were detected usingenhanced chemiluminescence (ECL) method.

Xenograft experimentsNude mice were obtained from Charles River Laboratories and

experiments were performed in accordance with MD AndersonInstitutional Animal Care and Use Committee (IACUC)-approved protocols. For MDA-MB-231 xenograft studies, 1.5 �106 pTRIPZ control or pTRIPZ shPTP4A3 cells were injected intomouse mammary fat pads. For MDA-MB-468 xenograft studies, 5� 106 pTRIPZ control or pTRIPZ shPTP4A3 cells were mixed with50% Matrigel and injected into mouse mammary fat pads. ForMCF-7 nude mouse xenograft studies, estrogen pellets were given2 days before injecting 5 � 106 pTRIPZ control or pTRIPZshPTP4A3 cells mixed with 50% matrigel into mouse mammaryfat pads. When tumors reached >30 mm3 in size, mice wererandomized to doxycycline treatment or control groups andmonitored for tumor growth. Tumors were measured with digitalcalipers every other day for MDA-MB-231, and every third day forMDA-MB-468 and MCF-7. Volumes (mm3) were calculated asvolume ¼ (width2 � length)/2. Statistical differences were calcu-lated by comparison of growth slopes from log transformedtumor volumes plotted versus time.

Assessment of PTP4A3 amplificationPTP4A3 and Myc gene amplification status was obtained from

the cBioPortal database (17, 18). Breast cancer samples were

assessed for PTP4A3 gene amplification, and represented in per-centage of total tumor samples. Coamplification of PTP4A3 andMyc in TNBC samples was determined using three independentdatasets: The Cancer GenomeAtlas (TCGA), Curtis, andNikolsky.The amplification status of PTP4A3, Myc, and four genes inbetween the PTP4A3 andMyc loci (ADCY8, KHDRBS3, FAM135B,and PTK2) was used to determine the independent amplificationof PTP4A3.

Survival analysisThe prognostic importance of PTP4A3 expression was eval-

uated using gene expression profiles and survival data gener-ated by Curtis and colleagues and Kao and colleagues (5, 19).R statistical software was used to analyze data obtained fromthe Oncomine database to generate Kaplan–Meier survivalcurves, to determine statistical significance using the log-rank(Mantel–Cox) method, and to perform Cox proportionalhazards models analyses. The patients were dichotomized atthe mean expression level.

ResultsAffymetrics gene expression profiling identifies phosphatasesunderexpressed and overexpressed in human triple-negativebreast tumors

We analyzed the expression levels of phosphatases in 102breast tumors, 53 triple-negative, and 49 ER-positive, to identifygenes differentially expressed in TNBCs. Data analysis was limitedto332 genes identified as phosphatase or phosphatase-interactingproteins. We identified 146 phosphatase genes differentlyexpressed (82 overexpressed, 64 underexpressed) in triple-nega-tive compared with ER-positive breast tumors (FDR ¼ 5%; Sup-plementary Fig. S1). Further analysis showed 19 of the 82 over-expressed phosphatases had �1.5-fold increases in expression,and 27 of the 64 underexpressed phosphatases had �1.5-foldreductions in expression and FDR < 0.05 (Table 1). We selectedthe 19 phosphatases differentially overexpressed by �1.5-foldfor further study.

Depletion of phosphatases inhibits TNBC growthWe transfected four triple-negative (MDA-MB-468, MDA-MB-

231, HS578T, HCC1143) and four ER-positive (MCF-7, T47D,ZR75-1, BT474) breast cancer cell lines with a pool of three siRNAoligos targeting the 17 phosphatases expressed �1.5-fold higherin triple-negative than in ER-positive breast tumors to determinewhether they are critical for breast cancer cell growth. Growth ratecomparisons of the siRNA oligos- and control-transfected cellsidentified 12 phosphatases that affected �2 breast cancer celllines. Knockdownof 6phosphatases (PTP4A3, PPAP2B,DLGAP5,CDC25B, PSPH, TIMM50) showed a growth inhibitory effect in�2 of the four TNBC cell lines tested (Table 2), and PTP4A3showed a growth inhibitory effect in �3 of the four TNBC celllines tested. In addition, growth inhibitory effects followingphosphatase knockdown were more profound in the triple-neg-ative compared with ER-positive cell lines.

Depletion of phosphatases reduces TNBC anchorage-independent growth

To determine whether the six phosphatases whose inhibitionreduced cell growth were also important for the anchorage-inde-pendent growth of TNBC cells, we treated four triple-negative






(MDA-MB-468, SUM159, HCC1143, MDA-MB-231), and threeER-positive (MCF-7, T47D, BT474) breast cancer cell lineswith siRNAs targeting these phosphatases. Five phosphatases(PTP4A3, PPAP2B, CDC25B, PSPH, TIMM50) showed aninhibitory effect on anchorage-independent growth in �2 ofthe TNBC cell lines (Table 3). PTP4A3 depletion had a largerinhibitory effect on triple-negative as compared with ER-pos-itive breast cancer cell lines, and PTP4A3 depletion inhibitedthe growth of TNBC cell lines by �one-third as compared withcontrol. PPAP2B depletion showed a growth inhibitory effectin MDA-MB-468 and MDA-MB-231 TNBC cells, and in T47DER-positive cells. Depletion of DLGAP5 had minimal effect onthe growth of triple-negative and ER-positive breast cancer celllines and thus, was not studied further. Depletion of CDC25Band TIMM50 inhibited anchorage-independent growth in twoTNBC cell lines, with no or minimal effect on ER-positive breastcancer cell lines. PSPH knockdown affected both triple-negativeand ER-positive cell lines equally, and showed no selectivity forTNBC cell lines (Table 3).

PTP4A3 depletion induces G1 arrestTo investigate the effect of depletion of the phosphatases

(PTP4A3, PPAP2B, CDC25B, TIMM50) that showed an effecton breast cancer anchorage-independent cell growth selectivelyin TNBC, 2 triple-negative (MDA-MB-231, HCC1143) and 2ER-positive breast cancer cell lines (MCF-7, BT474) were treatedwith siRNA, and assayed for cell-cycle distribution. Both triple-negative and ER-positive BT474 breast cancer cells (and to alesser extent ER-positive MCF-7 cells) transfected with PTP4A3siRNA showed an increase of cells in G1 phase accompanied by

a significant reduction of cells in S and G2–M (Fig. 1A). PPAP2Bdepletion caused an increase in the proportion of cells in the G2

–M phase in MDA-MB-231 cells (Fig. 1B). Not surprisingly,siRNA knockdown of CDC25B increased the proportion of cellsin the G2–M phase; CDC25B is a critical phosphatase for theprogression of cells through mitosis (Fig. 1C). Knockdown ofTIMM50 showed little to no effect on the cell cycle (Fig. 1D).We then performed immunofluorescent staining after siRNAtransfection of PTP4A3, PPAP2B, CDC25B, and TIMM50.Knockdown of each phosphatase except CDC25B suppressedp-H3 in MDA-MB-231 and HCC1143 cells (Fig. 1E). Knock-down of each of the phosphatases reduced Ki67 in MDA-MB-231 and knockdown of all but PPAP2B reduced Ki67 inHCC2243 cells (Fig. 1F). This indicates that depletion ofPTP4A3 has a much greater impact on TNBC cell proliferationthan depletion of the other phosphatases tested.

PTP4A3 depletion induces apoptosis in TNBC cell linesWe then studied cell death using the flow cytometry data and

found that TNBC cell lines treated with PTP4A3 siRNA had anincrease in the proportion of cells in the sub-G1 fraction,suggesting that PTP4A3 and PPAP2B knockdown induces apo-ptosis in MDA-MB-231 cells (Fig. 2A). Knockdown of PTP4A3also increased the sub-G1 proportion in HCC1143 cells. Tofurther investigate whether the depletion of PTP4A3, PPAP2B,CDC25B, and TIMM50 induce apoptosis in TNBCs, we per-formed Annexin V assays of breast cancer cells transfected withphosphatase siRNAs. Knockdown of PTP4A3 increased AnnexinV staining in TNBC cell lines, but not in the ER-positive cell lines(Fig. 2B). Conversely, PPAP2 depletion increased Annexin V

Table 3. Anchorage-independent growth of triple-negative and ER-positive cell lines treated with individual phosphatase siRNAs or control (siLuc)

Triple-negative cell lines (% of control) ER-positive cell lines (% of control)Gene MDA-MB-468 SUM159 HCC1143 MDA-MB-231 MCF-7 T47D BT474

PTP4A3 25a 67a 39a 40a 110 55a 68a

PPAP2B 55a 86 123a 15a 106 39a 94DLGAP5 51a 136a 70a 238a 118a 63a 94CDC25B 35a 98 55a 70 136a 61a 121PSPH 58a 62a 36a 11a 64a 53a 91TIMM50 44a 82 82a 48a 156a 71 95aP < 0.05.

Table 2. Growth of triple-negative and ER-positive cell lines treated with individual phosphatase siRNAs or control (siLuc)

Triple-negative cell lines (% of control) ER-positive cell lines (% of control)Gene MDA-MB-468 HS578T HCC1143 MDA-MB-231 MCF-7 T47D BT474 ZR-75-1

IMPA2 75a 74a 78a 84a 160a 42a 71a 92PTPLA 92 139a 141 88 101 43a 107 89PPP1R14B 70a 73a 95 62a 80 78a 126a 169a

PTP4A3 43a 55a 48a 47a 107 73a 83 121a

PTPRZ1 81a 108 156a 118a 130a 46a 76 76a

PPAP2B 70a 22a 144a 27a 49a 29a 124 174a

CDKN3 76a 88 90 67a 84a 72a 90 95DLGAP5 52a 36a 170a 14a 89 61a 86 40a

PTPRF 72a 78 95 100 107 77 40a 61a

CDC14B 90 112 161a 198a 130a 70 63a 160a

CDC25B 48a 34a 34a 58a 47a 34a 50a 121PLD3 67a 88 141 58a 82a 51a 56a 66a

NUDT11 55a 115 80 68a 117a 69a 92 73a

CDC25A 88 149a 258a 117a 142a 25a 70 94PPP1R11 85a 90 58a 111 51a 51a 94 94PSPH 36a 7a 37a 5a 13a 59a 61a 33a

TIMM50 45a 34a 86a 52a 32a 80 70a 62a

aP < 0.05.






staining in MDA-MB-231 cells and in the ER-positive MCF-7cells. These data suggest loss of PTP4A3 induces apoptosisselectively in TNBC cell lines. On the basis of the effect ofPTP4A3 knockdown on cell growth (Table 2), cell cycle, andapoptosis, we chose to further investigate the role of PTP4A3 inTNBC.

Depletion of PTP4A3 reduces TNBC tumor growth in vivoWe next investigated the effect of PTP4A3 knockdown on

in vivo tumor growth. MDA-MB-231 cells stably expressing

doxycycline-inducible PTP4A3 shRNA constructs or emptyvector control constructs were injected into the mammary fatpads of nude mice. Once tumors were established, mice wererandomized into two groups and treated with doxycycline-containing or control sucrose water. Tumor growth was signif-icantly inhibited in tumors with PTP4A3 knockdown com-pared with control (Fig. 3A and Supplementary Fig. S3A andS3B). qRT-PCR confirmed that reduced PTP4A3 expressionoccurs in tumors treated with doxycycline (SupplementaryFig. S3C). To conclusively determine that PTP4A3 depletion

Figure 1.PTP4A3 inhibition induces G1 arrest in TNBC cell lines. Breast cancer cell lines, MDA-MB-231, HCC1143, MCF-7, and BT474, were transfected with siRNAstargeting five phosphatases or control siRNA targeting luciferase. A–E, after 72 hours, cells were fixed in ethanol and stained with PI (� , P < 0.05).The five phosphatases were: PTP4A3 (A) PPAP2B (B), CDC25B (C), TIMM50 (D), MDA-MB-231 and HCC1143 (E) cells transfected with siRNA targetingphosphatases. They were fixed 72 hours after siRNA transfection and stained for p-H3 using immunofluorescence techniques. Cells were counted in 10 fields,and the experiment was performed in triplicate. F, cells were fixed 72 hours after siRNA transfection and stained for Ki67 using immunofluorescencetechniques. Cells were counted in 10 fields, and the experiment was performed in triplicate. Error bars, SD of the mean.






inhibits xenograft growth of TNBC cell lines, we conducted asimilar experiment with MDA-MB-468 cells, and found thattumor growth was indeed significantly inhibited in tumors withPTP4A3 knockdown compared with controls (Fig. 3B andSupplementary Fig. S3D). As PTP4A3 knockdown affected thecell cycle and apoptosis in vitro and reduced tumor growth in vivo,we studied the effects of PTP4A3 inhibition in vivo by IHC analysisof Ki67 and cleaved caspase-3 levels of xenograft tumors. PTP4A3knockdown significantly reduced the number of Ki67-positiveand increased the percentage of cleaved caspase-3–positive TNBCcells (Fig. 3C–F). These results confirm our in vitro findings, andshow that PTP4A3 inhibition reduces proliferation and inducesapoptosis.We also performed an additional xenograft experimentwithMCF-7, and found no significant inhibition of tumor growthfollowing depletion of PTP4A3 (Supplementary Fig. S3E and

S3F). These results demonstrate that PTP4A3 is necessary for invivo TNBC tumor growth.

PTP4A3 depletion alters critical proliferation and apoptosissignaling pathways

To further explore the mechanism of growth suppression byPTP4A3 knockdown, we analyzed the reverse-phase proteinarray (RPPA) data from TCGA breast cancer samples. Breasttumors with high (upper tertile) and low (lower tertile) PTP4A3mRNA expression were selected for further analysis. We com-pared protein phosphorylation and total protein levels betweenthe two groups, and identified those that significantly differ (P <0.1) between the high and low PTP4A3-expressing groups. Asexpected given our in vitro and in vivo findings, cleaved caspase-7 was elevated in tumors with low PTP4A3 expression

Figure 2.PTP4A3 inhibition induces apoptosisselectively in TNBC cell lines. Breast cancer celllines, MDA-MB-231, HCC1143, MCF-7, andBT474, were transfected with siRNAs targetingfive phosphatases or control siRNA targetingluciferase. (� , P < 0.05). A, cells were fixed72 hours after siRNA transfection in ethanoland were stained with PI. The sub-G1

population is graphed here. Experiment wasperformed in triplicate, and average withSDEV was graphed. B, cells were stained forAnnexin V 72 hours after transfection.Experiment was performed in triplicate, andaverage with SDEV was graphed.






selectively in basal-like (primarily triple-negative) but notluminal (primarily ER-positive) breast tumors (SupplementaryFig. S2A and S2B). Western blot analysis confirmed increasedcleaved caspase-7 expression following PTP4A3 knockdown intriple-negative but not ER-positive breast cancer cell lines (Fig.4A). These results show that loss of PTP4A3 suppresses TNBCcell growth by inducing apoptosis.

We next investigated the effect of PTP4A3 loss on MAPKsignaling. We preformed Western blot analyses to test wheth-er PTP4A3 depletion affects the phosphorylation status ofERK and p38. ERK phosphorylation and total protein levelsof ERK were reduced after PTP4A3 depletion in both ER-positive and ER-negative breast cancer cell lines (Fig. 4B).Conversely, p38 total protein was not affected by loss ofPTP4A3; however, p38 phosphorylation was suppressed inMDA-MB-231, the TNBC cell line with the largest phenotypiceffect (Fig. 4C). These changes in ERK and p38 expression andactivity may contribute to the selective induction of apoptosisin triple-negative compared with ER-positive breast cancercell lines.

PTP4A3 is amplified in TNBC and associated with poor overallsurvival

As loss of PTP4A3 suppresses growth and induces apoptosisin TNBC, we analyzed PTP4A3 expression levels in publiclyavailable human breast cancer datasets (5, 19). PTP4A3expression is significantly elevated in breast cancer as com-pared with normal cells, and is increased in triple-negativeas compared with ER-positive breast cancers (SupplementaryFig. S4A–S4D).

To determine whether increased PTP4A3 expression in-creases cancer cell growth, we created stably transfected celllines (MDA-MB-231, MCF-7) overexpressing PTP4A3. MDA-MB-231 overexpressing PTP4A3 showed increased growthrates, whereas MCF-7 clones overexpressing PTP4A3 exhibitedreduced growth compared with vector transfected clones(Fig. 5A and B). Given the high expression of PTP4A3 inTNBC, we investigated the amplification status of PTP4A3 inthe TCGA dataset using cBioPortal (17, 18). PTP4A3 is locatedon 8q.24.3, a region frequently amplified in breast cancerwhich also includes Myc (20). We discovered that 8q.24.3 isamplified in 21% of all breast cancers and 45% of basal-likebreast cancers. In addition, PTP4A3 is amplified in 17% andMyc in 22% of all breast cancers, whereas PTP4A3 is amplifiedin 29% and Myc in 44% (often concomitantly with PTP4A3) ofbasal-like breast cancers. The analysis of the TCGA dataset alsorevealed PTP4A3 amplification without the presence of Mycamplification in 2% of basal-like tumors. This is in contrast toLuminal A/B (primarily ER-positive) breast tumors, in whichPTP4A3 is amplified in only 12% (Supplementary Fig. S5A).Similar analysis of the TCGA ovarian cancer dataset gavesimilar results: PTP4A3 amplification in 34%, Myc amplifica-tion in 41%, and PTP4A3 amplification without Myc amp-lification in 7% of serous ovarian cancers (SupplementaryFig. S5B).

Because of the large size of the 8q24 genomic region, wehypothesized that PTP4A3 and Myc genes might be repre-sented by two different amplicons. To test this hypothesis, weinvestigated the amplification status of PTP4A3, Myc, and fourdifferent genes located between PTP4A3 and Myc in the 8q24region in the Curtis, TCGA, and Nikolsky datasets (Supple-

mentary Fig. S5C). This analysis revealed that amplification ofPTP4A3 but not Myc occurs in 3% and 44% of the Curtis andNikolsky dataset tumors, respectively (Fig. 5C). We alsofound that PTP4A3 and Myc coamplification occurs withoutamplification of the region between PTP4A3 and Myc in 10%and 11% in the Curtis and Nikolsky datasets, respectively(Fig. 5C, purple bars). These data suggest that PTP4A3 ampli-fication is not a passenger of Myc amplification, and can beindependently involved in breast cancer pathogenesis. Cor-relation of PTP4A3 copy number and expression levels dem-onstrated that mRNA expression increases with elevated copynumbers (Supplementary Fig. S5D).

Next, we investigated the importance of PTP4A3 over-expression on survival of breast cancer patients (Kao data-set; ref. 19). Kaplan–Meier analysis of patients dichoto-mized on median PTP4A3 expression demonstrates pooreroverall survival for patients with high versus low PTP4A3-expressing tumors (Fig. 5D). Cox proportional hazard anal-ysis of PTP4A3 above and below median, age, grade, lymphnode status, and ER status, indicate PTP4A3 is an indepen-dent predictor of decreased overall survival in breast cancer(Kao dataset, Fig. 5E; Curtis dataset, Supplementary TableS2). These results demonstrate that PTP4A3 is an indepen-dent predictor of poor overall survival, and suggests thatPTP4A3, a critical growth regulatory phosphatase, is a novelpotential target for the treatment of breast cancer, particu-larly TNBC.

DiscussionIn this study, we identified phosphatases differentially over-

expressed or underexpressed in TNBC as compared with ER-positive breast cancer, and showed that several highly expressedphosphatases are essential for growth of TNBCs. This is the firstreport using an unbiased global approach demonstrating thegrowth-promoting effect of phosphatases overexpressed inTNBC. We identified four phosphatases (PTP4A3, PPAP2B,CDC25B, and TIMM50) that are critical for TNBC anchorage-independent growth. We further demonstrated that PTP4A3 isrequired for proliferation of TNBC cell lines, and that PTP4A3knockdown induces a G1 cell-cycle block in both TNBC and ER-positive breast cancer cell lines, and apoptosis specifically inTNBC cell lines.

We also demonstrated that PTP4A3 knockdown reducesphosphorylation of p38 and ERK1/2 in all breast cancer celllines, consistent with the observed reduced proliferation andgrowth arrest. p38 inhibition has been shown to induce sen-sitization to apoptosis and increase sensitivity to cisplatin (21).These results suggest that loss of PTP4A3 causes apoptosisthrough p38 inactivation. This also suggests that high levelsof PTP4A3 might cause resistance to cisplatin or other chemo-therapy drugs in TNBCs. Other phosphatases have been shownto induce resistance to cancer treatments, such as mitogen-activated protein kinase phosphatase 3 (MKP3). Increasedlevels of MKP3 have been associated with tamoxifen resistancein ER-positive breast cancers (22). These findings demonstratethat phosphatases are critical signal transduction regulators incancer.

PTP4A3 overexpression in TNBC has been confirmed in mul-tiple datasets, and is associated with reduced survival. Using theTCGA dataset and cBioPortal for cancer Genomics, we identified






that PTP4A3 gene amplification is enriched in basal-like versusluminal breast cancers, and in some cases is independent of Mycamplification. These results indicate selective importance ofPTP4A3 in TNBC. Previous studies have reported PTP4A3 over-expression in several cancers, including breast, gastric, and coloncancer (23, 24), particularly with metastatic colon and breastcancer. Furthermore, PTP4A3 expression is higher in invasivecancer than normal breast tissue, and protein expression is asso-ciatedwith lymphnode positivity (25), aswell as reduced disease-free survival (23).

PTP4A3 has a well-established role in cell motility andmetastasis. Ectopic PTP4A3 overexpression influences migra-

tion and invasion of colon cancer cells, whereas PTP4A3elimination decreases cell motility and metastatic capabilityin mice (26). PTP4A3 overexpression is primarily observed inmetastases (distant or lymph node) versus primary tumors(27). The overexpression of PTP4A3 in triple-negative com-pared with ER-positive breast cancer supports the concept thathigh PTP4A3-expressing tumors are more aggressive. We havedemonstrated that loss of PTP4A3 reduces cell proliferationand induces apoptosis, and are currently studying PTP4A3overexpression in cancerous and precancerous cells to testwhether PTP4A3 transforms premalignant cells into fully inva-sive cancer cells.

Figure 3.Inhibition of PTP4A3 reducestumor growth in TNBC mousemodels. A and B, PTP4A3 tumorstreated with doxycyclineaffects tumor growth due tothe knockdown of PTP4A3.MDA-MB-231 cells (A) and MDA-MB-468 (B) stably transfectedwith shRNA-PTP4A3 wereinjected in the mammary fatpad of nude mice. Mice wererandomized when tumor reached30 mm3. Tumors volumes weremeasured every other day.Tumor growth rate issignificantly reduced afterknockdown of PTP4A3 inMDA-MB-231. Tumor growthrates were calculated from theslopes of the growth curvesfor each tumor. Error bar, SD.C and D, KI67 staining by IHC ofxenograft tumors in controland PTP4A3 knockdown micetreated with doxycycline orvehicle. E and F, cleavedcaspase-3 staining by IHC ofxenograft tumors in control andPTP4A3 knockdown micetreated with doxycycline orvehicle.






Although PTP4A3 regulation of molecular pathways is notwell studied and has focused primarily on migration andinvasion, Ezrin and Integrin-beta 1 are direct targets ofPTP4A3 in adhesion and motility (28, 29). Recently, PTP4A3has been shown to participate in src-mediated Rho signaling,thereby inducing cell motility and invasion (30). Similarly,PTP4A3 regulation of cancer cell growth is poorly under-stood. Our current studies show that loss of PTP4A3 altersERK and p38 signaling. In addition, PTP4A3 has been shownto regulate p53 and p21 reporter activity (31). These resultsprovide a potential mechanism for PTP4A3 regulation ofproliferation and apoptosis in cancer cells. Other studies

have shown that PTP4A3 activates the NF-kB and PI3K path-ways, which are important for survival and proliferation ofcancer cells (32, 33). PTP4A3 overexpression is associatedwith increased p65 phosphorylation (32), and reduced PTENexpression (33). Alteration of these multiple pathways maycollectively contribute to the reduced proliferation and apo-ptosis following PTP4A3 knockdown in breast cancer cells.We are currently performing experiments to elucidate directtargets of PTP4A3 in breast cancer cells that regulate cell cycleand cell death.

Here, we have identified phosphatases overexpressed intriple-negative as compared with ER-positive breast cancer,

Figure 4.Signaling proteins involved in proliferation and programmedcell death are altered with PTP4A3 knockdown. Two TNBC celllines (MDA-MB-231 and HCC1143) and two ER-positive celllines (MCF-7 and T47D) were transfected with PTP4A3 orcontrol siRNA. After 72 hours, cells were harvested and lysedfor Western blot analysis. A, cleaved caspase-7 is increasedafter knockdown of PTP4A3 in TNBC cell lines only. B, ERK1/2phosphorylation and protein levels are reduced afterknockdown of PTP4A3. C, phosphorylation of p38 is reducedafter knockdown of PTP4A3.






many of which are critical for TNBC cell growth. One of thesephosphatases, PTP4A3, is a critical regulator of proliferationand apoptosis, and inhibition of PTP4A3 induces cell-cyclearrest, apoptosis, and inhibition of TNBC tumor growth in vivo.These data strongly suggest that targeting PTP4A3 or its signal-ing pathway in TNBC is a promising strategy for women withthese aggressive breast cancers.

Disclosure of Potential Conflicts of InterestP.H. Brown is a consultant/advisory board member for Susan G. Komen

Foundation. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: P. den Hollander, J.C. Chang, C.K. Osborne,P.H. Brown

Development of methodology: P. den Hollander, J.C. Chang, C.K. Osborne,G.B. Mills, P.H. BrownAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. den Hollander, K. Rawls, J. Hill, S.A.W. Fuqua,J.C. Chang, C.K. OsborneAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. den Hollander, A. Tsimelzon, J. Shepherd,S.G. Hilsenbeck, G.B. Mills, P.H. BrownWriting, review, and/or revision of the manuscript: P. den Hollander, S.A.W.Fuqua, J.C. Chang, C.K. Osborne, S.G. Hilsenbeck, G.B. Mills, P.H. BrownAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Mazumdar, G.B. MillsStudy supervision: P.H. BrownOther (partial funding): C.K. Osborne

AcknowledgmentsThe authors thank Svasti Haricharan for the data analysis of the 8q24

region, Michelle Savage for editing the manuscript, and Sam Short forassisting in the submission.

Figure 5.High levels of PTP4A3 promote TNBCcell growth and associated with pooroverall survival in breast cancerpatients. A, overexpression of PTP4A3caused an increased growth in theTNBC cell line MDA-MB-231. Equalamount of cells of MDA-MB-231 cellsstably transfected with control orPTP4A3 were plated and assayed forgrowth for 4 days using Trypan bluestaining. B, overexpression of PTP4A3reduced growth of the ER-positivebreast cancer cell lines MCF-7. Equalamount of cells of MCF-7 cells stablytransfected with control or PTP4A3were plated and assayed for growth for4 days by Trypan blue staining.C, comparative analysis of geneamplification of PTP4A3, Myc, andintermediate genes. The amplificationstatus of PTP4A3, Myc, and geneslocated in between the PTP4A3 andMyc loci was assessed and graphedin percentage of concurrent ofindependent amplification D, Kaplan–Meier curves of overall survival of allbreast cancer patients in the Kaodataset stratified by PTP4A3expression. E, proportional hazard ratioanalysis demonstrates that highPTP4A3 levels are an independentpredictor for worse overall survival inbreast cancer patients in the Kaodataset.






Grant SupportThis work was funded by an NCI Cancer Center Support Grant

(P30CA016672 to P.H. Brown, G.B. Mills), a Susan G. Komen Promise Grant(KG081694 to P.H. Brown, G.B. Mills), a Komen SAB Grant (P.H. Brown), andthe Norman Brinker Award for Research Excellence (P.H. Brown). In addition,this research utilized the shared resources of the Cancer Center Support Grant(CCSG)-funded Characterized Cell Line Core (P30CA016672 to G.B. Mills).

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

Received March 10, 2014; revised September 30, 2015; accepted October 15,2015; published OnlineFirst February 26, 2016.

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2016;76:1942-1953. Published OnlineFirst February 26, 2016.Cancer Res Petra den Hollander, Kathryn Rawls, Anna Tsimelzon, et al. Growth and Predicts Poor Patient SurvivalPhosphatase PTP4A3 Promotes Triple-Negative Breast Cancer

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Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer … · Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer Growth and Predicts Poor Patient Survival Petra den Hollander1,