Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-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–S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition 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 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942–53. ©2016 AACR.

Breast cancer is the second leading cause of cancer-related death in women (1). Targeted therapies can significantly reduce recurrence for estrogen receptor (ER)-positive (e.g., selective ER 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, progesterone receptor (PR), or HER2 (5, 6). These triple-negative breast cancers (TNBC) are highly aggressive, have poor prognoses, lack targeted therapies (7–9), and are currently treated with toxic, nonspecific chemotherapy drugs. Consequently, there is an urgent need to identify more effective, less toxic therapeutic strategies.

In addition to ER and HER2 signaling pathways, other growth regulatory pathways have been shown to regulate growth and development of cancers. One of our major goals is to identify signaling pathways and molecules critical for growth of TNBCs. Previously, we identified a set of kinases overexpressed in ER-negative compared with ER-positive breast cancers (10). Further analysis demonstrated that many of these overexpressed kinases are essential for the growth of ER-negative breast cancer cell lines (10). Aberrant phosphorylation has been implicated in many diseases, including cancer (11, 12), and regulate a number of signaling pathways, including those associated with growth, survival, metabolism, and the cell cycle (13–15).

In this study, we hypothesized that phosphatases are differentially expressed in triple-negative as compared with ER-positive tumors. Using transcriptional profiling data to evaluate phosphatase expression in a breast cancer dataset, we identified two sets of phosphatases (those highly expressed or underexpressed) in TNBC as compared with ER-positive tumors. Further analysis of the phosphatases upregulated in triple-negative compared with ER-positive breast cancers, identified several phosphatases critical for anchorage-independent growth in TNBC cells. We selected those phosphatases (PTP4A3, PPAP2B, CDC25B, and TIMM50) that were found critical for the tumorigenicity of TNBC cells for further study. Of these, PTP4A3 (protein tyrosine phosphatase type IVA, member 3, also known as, PRL-3) was required for in vitro and in vivo TNBC cell growth. PTP4A3 has previously been implicated in the regulation of migration, invasion, and metastasis (16), but its role in regulating cancer cell growth is less understood. PTP4A3 inhibition induces G1 arrest in breast cancer cells, reduces growth and Ki67 expression in xenograft tumors, and loss of PTP4A3 in breast cancer cells induces apoptosis specifically in TNBC cells. Our results demonstrate that phosphatases represent promising targets for the treatment and prevention of TNBC, and provide the rationale for further investigations supporting the development of inhibitors of phosphatases or their signaling pathways as viable strategies for women with or at high risk of TNBC.

Study population and design

Tissue samples used for this study were obtained from the Lester and Sue Smith Breast Cancer Tumor bank at Baylor College of Medicine (Houston, TX). We studied 102 breast tumors from this bank (collected from sites in the United States and Europe according to institutionally approved guidelines) for the analysis of gene expression, DNA copy number, and protein change (GEO accession number GSE76275). We used a subset of these tissue samples, which are listed in Supplementary File - Tissue Sample IDs. All studies associated with this research were conducted with the approval of the Baylor College of Medicine and University of Texas MD Anderson Cancer Center Institutional Review Boards. Study demographics define the majority of the population to be postmenopausal women, 97% Caucasian, and a median subject age of 53 years (Supplementary Table S1). After collecting and freezing tumors in liquid nitrogen, DNA, RNA, and protein content was individually isolated. In this cohort, 96% of tumors were invasive ductal carcinomas, 35% presented no lymph node involvement, none were metastasic, and 75% of study participants had tumors ≥2 cm3 at the time of diagnosis. The ER and PR status of each tissue sample was determined through immunohistochemical analysis, and HER2 status was determined by fluorescent in situ hybridization, identifying 49 ER-positive tumors and 53 triple-negative tumors. Of the ER-positive breast cancers, 4 tumors were also HER2-positive (5%), and 42 PR positive (41%; Supplementary Table S1).

Selection of genes for further study

In total, 49 ER-positive and 53 triple-negative breast tumors were profiled using the Affymetrix U133 Plus 2.0 gene expression array. Data analysis was limited to 332 genes identified as phosphatase or phosphatase-interacting proteins. If a gene was represented by more than one probeset on the U133 Plus2 chip, we used the most variable probeset. The list of genes included in the analysis and gene identifiers are presented in Supplementary Table S3. Analysis (expression estimation and group comparison) was performed using BRB-ArrayTools developed by Dr. Richard Simon and the BRB-ArrayTools Development Team (http://linus.nci.nih.gov/BRB-ArrayTools.html). dChip software was used for preparing the clustering picture (http://www.hsph.harvard.edu/cli/complab/dchip). Expression was estimated using Robust Multiarray Average (RMA) procedure. Criterion for group comparison was false discovery rate (FDR) = 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).

Table 1.

Phosphatases differentially expressed ≥1.5-fold in triple-negative compared with ER-positive breast tumors (all FDR ≤ 0.05)

Gene
Type of expressionNameSymbolFold changeFDR
Overexpressed Inositol(myo)-1(or 4)-monophosphatase 2 IMPA2 2.43 <0.001 
 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), mem A PTPLA 2.32 <0.001 
 Protein phosphatase 1, regulatory (inhibitor) subunit 14B PPP1R14B 2.05 <0.001 
 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 PTPRZ1 2.01 0.007 
 Protein tyrosine phosphatase type IVA, member 3 PTP4A3 1.95 <0.001 
 Phosphatidic acid phosphatase type 2B PPAP2B 1.93 <0.001 
 Protein tyrosine phosphatase, receptor type, F PTPRF 1.85 <0.001 
 Cyclin-dependent kinase inhibitor 3 CDKN3 1.83 <0.001 
 CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 1.80 <0.001 
 Discs, large (Drosophila) homolog-associated protein 5 DLGAP5 1.74 0.007 
 Nudix (nucleoside diphosphate linked moiety X)-type motif 11 NUDT11 1.68 <0.001 
 Cell division cycle 25 homolog B (S. pombe) CDC25B 1.66 <0.001 
 Phosphoserine phosphatase PSPH 1.63 0.034 
 Phospholipase D family, member 3 PLD3 1.61 <0.001 
 Cell division cycle 25 homolog A (S. pombe) CDC25A 1.59 <0.001 
 Protein phosphatase 1, regulatory (inhibitor) subunit 11 PPP1R11 1.56 <0.001 
 Nudix (nucleoside diphosphate linked moiety X)-type motif 5 NUDT5 1.55 <0.001 
 Protein tyrosine phosphatase, nonreceptor type 14 PTPN14 1.53 <0.001 
 Translocase 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 <0.001 
 Protein phosphatase 1, regulatory (inhibitor) subunit 3C PPP1R3C 0.37 <0.001 
 Dual specificity phosphatase 4 DUSP4 0.37 <0.001 
 Nudix (nucleoside diphosphate linked moiety X)-type motif 12 NUDT12 0.38 <0.001 
 Fructose-1,6-bisphosphatase 1 FBP1 0.38 <0.001 
 Ectonucleotide pyrophosphatase/phosphodiesterase 1 ENPP1 0.39 <0.001 
 Protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform PPP2R2C 0.41 <0.001 
 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) ENPP4 0.43 <0.001 
 Protein tyrosine phosphatase, receptor type, B PTPRB 0.48 <0.001 
 Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase CILP 0.49 <0.001 
 Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform PPM1A 0.51 <0.001 
 Phosphatase, orphan 2 PHOSPHO2 0.51 <0.001 
 Dual specificity phosphatase 5 DUSP5 0.55 <0.001 
 Protein tyrosine phosphatase, receptor type, T PTPRT 0.55 <0.001 
 Chromosome 12 open reading frame 51 C12orf51 0.58 <0.001 
 Protein phosphatase 1H (PP2C domain containing) PPM1H 0.58 0.001 
 Dual specificity phosphatase 28 DUSP28 0.59 <0.001 
 Protein phosphatase 2, catalytic subunit, alpha isozyme PPP2CA 0.59 <0.001 
 Protein phosphatase 1D magnesium-dependent, delta isoform PPM1D 0.59 <0.001 
 Protein tyrosine phosphatase, receptor type, N polypeptide 2 PTPRN2 0.59 0.002 
 Protein tyrosine phosphatase, nonreceptor type 13 PTPN13 0.60 <0.001 
 Myotubularin related protein 9 MTMR9 0.61 <0.001 
 SAC1 suppressor of actin mutations 1-like (yeast) SACM1L 0.60 <0.001 
 Protein phospahatase 2, regulatory subunit B, alpha PPP2R2A 0.62 <0.001 
 Multiple inositol polyphosphate histidine phosphatase, 1 MINPP1 0.62 <0.001 
 FANCD2/FANCI-associated nuclease 1 FAN1 0.63 <0.001 
 Protein phosphatase 6, catalytic subunit PPP6C 0.63 <0.001 
Gene
Type of expressionNameSymbolFold changeFDR
Overexpressed Inositol(myo)-1(or 4)-monophosphatase 2 IMPA2 2.43 <0.001 
 Protein tyrosine phosphatase-like (proline instead of catalytic arginine), mem A PTPLA 2.32 <0.001 
 Protein phosphatase 1, regulatory (inhibitor) subunit 14B PPP1R14B 2.05 <0.001 
 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 PTPRZ1 2.01 0.007 
 Protein tyrosine phosphatase type IVA, member 3 PTP4A3 1.95 <0.001 
 Phosphatidic acid phosphatase type 2B PPAP2B 1.93 <0.001 
 Protein tyrosine phosphatase, receptor type, F PTPRF 1.85 <0.001 
 Cyclin-dependent kinase inhibitor 3 CDKN3 1.83 <0.001 
 CDC14 cell division cycle 14 homolog B (S. cerevisiae) CDC14B 1.80 <0.001 
 Discs, large (Drosophila) homolog-associated protein 5 DLGAP5 1.74 0.007 
 Nudix (nucleoside diphosphate linked moiety X)-type motif 11 NUDT11 1.68 <0.001 
 Cell division cycle 25 homolog B (S. pombe) CDC25B 1.66 <0.001 
 Phosphoserine phosphatase PSPH 1.63 0.034 
 Phospholipase D family, member 3 PLD3 1.61 <0.001 
 Cell division cycle 25 homolog A (S. pombe) CDC25A 1.59 <0.001 
 Protein phosphatase 1, regulatory (inhibitor) subunit 11 PPP1R11 1.56 <0.001 
 Nudix (nucleoside diphosphate linked moiety X)-type motif 5 NUDT5 1.55 <0.001 
 Protein tyrosine phosphatase, nonreceptor type 14 PTPN14 1.53 <0.001 
 Translocase 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 <0.001 
 Protein phosphatase 1, regulatory (inhibitor) subunit 3C PPP1R3C 0.37 <0.001 
 Dual specificity phosphatase 4 DUSP4 0.37 <0.001 
 Nudix (nucleoside diphosphate linked moiety X)-type motif 12 NUDT12 0.38 <0.001 
 Fructose-1,6-bisphosphatase 1 FBP1 0.38 <0.001 
 Ectonucleotide pyrophosphatase/phosphodiesterase 1 ENPP1 0.39 <0.001 
 Protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform PPP2R2C 0.41 <0.001 
 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) ENPP4 0.43 <0.001 
 Protein tyrosine phosphatase, receptor type, B PTPRB 0.48 <0.001 
 Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase CILP 0.49 <0.001 
 Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform PPM1A 0.51 <0.001 
 Phosphatase, orphan 2 PHOSPHO2 0.51 <0.001 
 Dual specificity phosphatase 5 DUSP5 0.55 <0.001 
 Protein tyrosine phosphatase, receptor type, T PTPRT 0.55 <0.001 
 Chromosome 12 open reading frame 51 C12orf51 0.58 <0.001 
 Protein phosphatase 1H (PP2C domain containing) PPM1H 0.58 0.001 
 Dual specificity phosphatase 28 DUSP28 0.59 <0.001 
 Protein phosphatase 2, catalytic subunit, alpha isozyme PPP2CA 0.59 <0.001 
 Protein phosphatase 1D magnesium-dependent, delta isoform PPM1D 0.59 <0.001 
 Protein tyrosine phosphatase, receptor type, N polypeptide 2 PTPRN2 0.59 0.002 
 Protein tyrosine phosphatase, nonreceptor type 13 PTPN13 0.60 <0.001 
 Myotubularin related protein 9 MTMR9 0.61 <0.001 
 SAC1 suppressor of actin mutations 1-like (yeast) SACM1L 0.60 <0.001 
 Protein phospahatase 2, regulatory subunit B, alpha PPP2R2A 0.62 <0.001 
 Multiple inositol polyphosphate histidine phosphatase, 1 MINPP1 0.62 <0.001 
 FANCD2/FANCI-associated nuclease 1 FAN1 0.63 <0.001 
 Protein phosphatase 6, catalytic subunit PPP6C 0.63 <0.001 

Cell lines, reagents, and plasmids

All cell lines were purchased from ATCC, and were DNA fingerprinted by the Characterized Cell Line Core Facility at MD Anderson Cancer Center in 2012 to insure correct identity. MCF-7, MDA-MB-231, MDA-MB-468, and HS578T cell lines were cultured in DMEM (Cellgro by Mediatech, Inc.), and ZR-75-1, HCC1143, T47D, and BT474 cell lines were cultured in RPMI1640 medium (Cellgro by Mediatech, Inc.). Growth media for all cell lines was supplemented with 10% FBS, penicillin (100 mg/mL), and streptomycin (100 mg/mL). The siRNA oligos were purchased from Sigma-Aldrich. A pool of three siRNA oligos was used at a final concentration of 30 nmol/L using DharmaFECT1 Transfection Reagent (Dharmacon, Inc.), following the manufacturer's instructions. pGIPZ lentiviral short hairpin RNAs (shRNA) for PTP4A3 (Open Biosystems, Inc.) were subcloned into a pTRIPZ lentiviral expression system (Open Biosystems, Inc.). The PTP4A3 overexpression vector and control vector (pCMV6) were commercially obtained and sequence-verified.

RNA preparation and qRT-PCR

RNA was isolated using the RNeasy Kit (Qiagen). cDNA was generated using random primers, and Superscript II Reverse Transcriptase (Invitrogen; Life Technologies). TaqMan assays were designed and qRT-PCR was performed. Cyclophilin was used as an endogenous control, and results were normalized to cyclophilin ± SD.

Generation of stable cell lines

Lentiviral particles were generated by transfecting 293T cells with lentiviral constructs and packaging plasmids encoding VSV-G, Gag, Pol, and Tat using FuGENE 6 Transfection Reagent (Roche Applied Science). Media overlaying the cells was harvested at 24, 48, and 72 hours posttransfection, and filtered through a 0.45-μm MEC filter. Polyethylene glycol (PEG) precipitation was used to concentrate the virus. Stable cell lines expressing inducible shRNAs were generated by lentiviral infection using the pTRIPZ lentiviral expression system in the presence of 4 μg/mL polybrene, followed by puromycin selection. All pTRIPZ stable cell lines were maintained in media with Tet-safe Serum (Clontech Laboratories, Inc.).

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

Growth assays

Cells were transfected with 30 nmol/L final concentration of siRNA. After 24 hours, cells were plated in triplicate in 48-well plates. Cell proliferation was measured using the Countess Automated Cell Counter (Invitrogen; Life Technologies) and Trypan blue staining. Cell count was assessed at days 4 and 6. Each data point was performed in triplicate, with results reported as average percentage ± SD.

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

Anchorage-independent growth assays

Cells were transfected with 30 nmol/L final concentration of siRNA. Anchorage-independent growth assays were performed by 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 were plated per well. Suspended cells were plated on a 0.7% agar base in the same medium. Colonies were counted using the GelCount Colony Counter (Oxford Optronix) 2 and 3 weeks postplating. All experiments were performed in triplicate, and results reported as average colony number ± SD.

Apoptosis assays

Apoptosis assays were performed using Annexin V staining (eBioscience) following the manufacturer's instructions, and replacing propidium iodide (PI) with DAPI staining. Cells were plated on 6-well plates, transfected with siRNA, and harvested after 3 days, then stained and analyzed in triplicate using a Gallios Flow Cytometer (Beckman Coulter, Inc.). Results were reported as average percentage of each population ± SD.

Cell-cycle analysis

Cells were plated on 6-well plates, transfected with siRNA, and harvested after 4 days, at which time they were fixed, stained with PI, and analyzed in triplicate using a Gallios Flow Cytometer (Beckman Coulter, Inc.). Results are reported as the average percentage of each population ± SD.

Immunofluorescence staining and microscopy

Cells were plated on coverslips in 6-well plates. The next day cells were transfected with 30 nmol/L final concentration of siRNA. After 48 hours, cells were washed in PBS, fixed for 20 minutes in 4% paraformaldehyde at room temperature, and blocked for 1 hour at room temperature in 5% goat serum and 1% Triton X-100 in 1× PBS. Coverslips were incubated with primary antibody [phospho-histone H3 (p-H3; 1:500) from Cell Signaling Technology, and Ki67 (Clone SP6) from Thermo 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 then mounted with DAPI-containing mounting media (Vectashield). Fluorescent images were captured with a Nikon microscope. Experiments were performed in triplicate.

IHC and staining analysis

Tumor sections of 5 μm were stained for either Ki67 (Clone SP6 from Thermo Fisher Scientific) or with cleaved caspase-3 (Cell Signaling Technology). Antigen retrieval was performed in citrate buffer, and endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes. Primary antibodies were incubated overnight at 4°C, followed by incubation with biotinylated antirabbit antibody (1:100) for 30 minutes. Peroxidase activity was visualized using the Vector NovaRED Substrate Kit (PK-6101, Vector Laboratories, Inc.).

Western blotting

Cells were transfected with 30 nmol/L final concentration of siRNA. After 48 hours, cells were washed in PBS, lysed in RIPA buffer, run on a 4% to 20% gradient gel, and transferred onto nitrocellulose. Primary antibodies were incubated with the membrane overnight at 4°C and included p-ERK1/2, ERK, cleaved caspase-7, caspase-7, p-p38, and p38 (Cell Signaling Technology; 1:1,000), and vinculin and actin (Sigma-Aldrich; 1:5,000). Blots were washed with PBS-T and incubated with secondary antibodies for 1 hour at room temperature. Bends were detected using enhanced chemiluminescence (ECL) method.

Xenograft experiments

Nude mice were obtained from Charles River Laboratories and experiments were performed in accordance with MD Anderson Institutional 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 into mouse mammary fat pads. For MDA-MB-468 xenograft studies, 5 × 106 pTRIPZ control or pTRIPZ shPTP4A3 cells were mixed with 50% Matrigel and injected into mouse mammary fat pads. For MCF-7 nude mouse xenograft studies, estrogen pellets were given 2 days before injecting 5 × 106 pTRIPZ control or pTRIPZ shPTP4A3 cells mixed with 50% matrigel into mouse mammary fat pads. When tumors reached >30 mm3 in size, mice were randomized to doxycycline treatment or control groups and monitored for tumor growth. Tumors were measured with digital calipers every other day for MDA-MB-231, and every third day for MDA-MB-468 and MCF-7. Volumes (mm3) were calculated as volume = (width2 × length)/2. Statistical differences were calculated by comparison of growth slopes from log transformed tumor volumes plotted versus time.

Assessment of PTP4A3 amplification

PTP4A3 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 percentage of total tumor samples. Coamplification of PTP4A3 and Myc in TNBC samples was determined using three independent datasets: The Cancer Genome Atlas (TCGA), Curtis, and Nikolsky. The amplification status of PTP4A3, Myc, and four genes in between the PTP4A3 and Myc loci (ADCY8, KHDRBS3, FAM135B, and PTK2) was used to determine the independent amplification of PTP4A3.

Survival analysis

The prognostic importance of PTP4A3 expression was evaluated using gene expression profiles and survival data generated by Curtis and colleagues and Kao and colleagues (5, 19). R statistical software was used to analyze data obtained from the Oncomine database to generate Kaplan–Meier survival curves, to determine statistical significance using the log-rank (Mantel–Cox) method, and to perform Cox proportional hazards models analyses. The patients were dichotomized at the mean expression level.

Affymetrics gene expression profiling identifies phosphatases underexpressed and overexpressed in human triple-negative breast tumors

We analyzed the expression levels of phosphatases in 102 breast tumors, 53 triple-negative, and 49 ER-positive, to identify genes differentially expressed in TNBCs. Data analysis was limited to 332 genes identified as phosphatase or phosphatase-interacting proteins. We identified 146 phosphatase genes differently expressed (82 overexpressed, 64 underexpressed) in triple-negative compared with ER-positive breast tumors (FDR = 5%; Supplementary Fig. S1). Further analysis showed 19 of the 82 overexpressed phosphatases had ≥1.5-fold increases in expression, and 27 of the 64 underexpressed phosphatases had ≥1.5-fold reductions in expression and FDR < 0.05 (Table 1). We selected the 19 phosphatases differentially overexpressed by ≥1.5-fold for further study.

Depletion of phosphatases inhibits TNBC growth

We 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 siRNA oligos targeting the 17 phosphatases expressed ≥1.5-fold higher in triple-negative than in ER-positive breast tumors to determine whether they are critical for breast cancer cell growth. Growth rate comparisons of the siRNA oligos- and control-transfected cells identified 12 phosphatases that affected ≥2 breast cancer cell lines. Knockdown of 6 phosphatases (PTP4A3, PPAP2B, DLGAP5, CDC25B, PSPH, TIMM50) showed a growth inhibitory effect in ≥2 of the four TNBC cell lines tested (Table 2,Table 3), and PTP4A3 showed a growth inhibitory effect in ≥3 of the four TNBC cell lines tested. In addition, growth inhibitory effects following phosphatase knockdown were more profound in the triple-negative compared with ER-positive cell lines.

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)
GeneMDA-MB-468HS578THCC1143MDA-MB-231MCF-7T47DBT474ZR-75-1
IMPA2 75a 74a 78a 84a 160a 42a 71a 92 
PTPLA 92 139a 141 88 101 43a 107 89 
PPP1R14B 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 95 
DLGAP5 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 121 
PLD3 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 94 
PPP1R11 85a 90 58a 111 51a 51a 94 94 
PSPH 36a 7a 37a 5a 13a 59a 61a 33a 
TIMM50 45a 34a 86a 52a 32a 80 70a 62a 
Triple-negative cell lines (% of control)ER-positive cell lines (% of control)
GeneMDA-MB-468HS578THCC1143MDA-MB-231MCF-7T47DBT474ZR-75-1
IMPA2 75a 74a 78a 84a 160a 42a 71a 92 
PTPLA 92 139a 141 88 101 43a 107 89 
PPP1R14B 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 95 
DLGAP5 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 121 
PLD3 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 94 
PPP1R11 85a 90 58a 111 51a 51a 94 94 
PSPH 36a 7a 37a 5a 13a 59a 61a 33a 
TIMM50 45a 34a 86a 52a 32a 80 70a 62a 

aP < 0.05.

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)
GeneMDA-MB-468SUM159HCC1143MDA-MB-231MCF-7T47DBT474
PTP4A3 25a 67a 39a 40a 110 55a 68a 
PPAP2B 55a 86 123a 15a 106 39a 94 
DLGAP5 51a 136a 70a 238a 118a 63a 94 
CDC25B 35a 98 55a 70 136a 61a 121 
PSPH 58a 62a 36a 11a 64a 53a 91 
TIMM50 44a 82 82a 48a 156a 71 95 
Triple-negative cell lines (% of control)ER-positive cell lines (% of control)
GeneMDA-MB-468SUM159HCC1143MDA-MB-231MCF-7T47DBT474
PTP4A3 25a 67a 39a 40a 110 55a 68a 
PPAP2B 55a 86 123a 15a 106 39a 94 
DLGAP5 51a 136a 70a 238a 118a 63a 94 
CDC25B 35a 98 55a 70 136a 61a 121 
PSPH 58a 62a 36a 11a 64a 53a 91 
TIMM50 44a 82 82a 48a 156a 71 95 

aP < 0.05.

Depletion of phosphatases reduces TNBC anchorage-independent growth

To determine whether the six phosphatases whose inhibition reduced cell growth were also important for the anchorage-independent growth of TNBC cells, we treated four triple-negative (MDA-MB-468, SUM159, HCC1143, MDA-MB-231), and three ER-positive (MCF-7, T47D, BT474) breast cancer cell lines with siRNAs targeting these phosphatases. Five phosphatases (PTP4A3, PPAP2B, CDC25B, PSPH, TIMM50) showed an inhibitory effect on anchorage-independent growth in ≥2 of the TNBC cell lines (Table 3). PTP4A3 depletion had a larger inhibitory effect on triple-negative as compared with ER-positive breast cancer cell lines, and PTP4A3 depletion inhibited the growth of TNBC cell lines by ≥one-third as compared with control. PPAP2B depletion showed a growth inhibitory effect in MDA-MB-468 and MDA-MB-231 TNBC cells, and in T47D ER-positive cells. Depletion of DLGAP5 had minimal effect on the growth of triple-negative and ER-positive breast cancer cell lines and thus, was not studied further. Depletion of CDC25B and TIMM50 inhibited anchorage-independent growth in two TNBC cell lines, with no or minimal effect on ER-positive breast cancer cell lines. PSPH knockdown affected both triple-negative and ER-positive cell lines equally, and showed no selectivity for TNBC cell lines (Table 3).

PTP4A3 depletion induces G1 arrest

To investigate the effect of depletion of the phosphatases (PTP4A3, PPAP2B, CDC25B, TIMM50) that showed an effect on breast cancer anchorage-independent cell growth selectively in TNBC, 2 triple-negative (MDA-MB-231, HCC1143) and 2 ER-positive breast cancer cell lines (MCF-7, BT474) were treated with siRNA, and assayed for cell-cycle distribution. Both triple-negative and ER-positive BT474 breast cancer cells (and to a lesser extent ER-positive MCF-7 cells) transfected with PTP4A3 siRNA showed an increase of cells in G1 phase accompanied by a significant reduction of cells in S and G2–M (Fig. 1A). PPAP2B depletion 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 cells in the G2–M phase; CDC25B is a critical phosphatase for the progression of cells through mitosis (Fig. 1C). Knockdown of TIMM50 showed little to no effect on the cell cycle (Fig. 1D). We then performed immunofluorescent staining after siRNA transfection of PTP4A3, PPAP2B, CDC25B, and TIMM50. Knockdown of each phosphatase except CDC25B suppressed p-H3 in MDA-MB-231 and HCC1143 cells (Fig. 1E). Knockdown of each of the phosphatases reduced Ki67 in MDA-MB-231 and knockdown of all but PPAP2B reduced Ki67 in HCC2243 cells (Fig. 1F). This indicates that depletion of PTP4A3 has a much greater impact on TNBC cell proliferation than depletion of the other phosphatases tested.

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 siRNAs targeting 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 targeting phosphatases. 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 immunofluorescence techniques. Cells were counted in 10 fields, and the experiment was performed in triplicate. Error bars, SD of the mean.

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 siRNAs targeting 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 targeting phosphatases. 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 immunofluorescence techniques. Cells were counted in 10 fields, and the experiment was performed in triplicate. Error bars, SD of the mean.

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PTP4A3 depletion induces apoptosis in TNBC cell lines

We then studied cell death using the flow cytometry data and found that TNBC cell lines treated with PTP4A3 siRNA had an increase in the proportion of cells in the sub-G1 fraction, suggesting that PTP4A3 and PPAP2B knockdown induces apoptosis in MDA-MB-231 cells (Fig. 2A). Knockdown of PTP4A3 also increased the sub-G1 proportion in HCC1143 cells. To further investigate whether the depletion of PTP4A3, PPAP2B, CDC25B, and TIMM50 induce apoptosis in TNBCs, we performed Annexin V assays of breast cancer cells transfected with phosphatase siRNAs. Knockdown of PTP4A3 increased Annexin V staining in TNBC cell lines, but not in the ER-positive cell lines (Fig. 2B). Conversely, PPAP2 depletion increased Annexin V staining in MDA-MB-231 cells and in the ER-positive MCF-7 cells. These data suggest loss of PTP4A3 induces apoptosis selectively in TNBC cell lines. On the basis of the effect of PTP4A3 knockdown on cell growth (Table 2), cell cycle, and apoptosis, we chose to further investigate the role of PTP4A3 in TNBC.

Figure 2.

PTP4A3 inhibition induces apoptosis selectively in TNBC cell lines. Breast cancer cell lines, MDA-MB-231, HCC1143, MCF-7, and BT474, were transfected with siRNAs targeting five phosphatases or control siRNA targeting luciferase. (*, P < 0.05). A, cells were fixed 72 hours after siRNA transfection in ethanol and were stained with PI. The sub-G1 population is graphed here. Experiment was performed in triplicate, and average with SDEV was graphed. B, cells were stained for Annexin V 72 hours after transfection. Experiment was performed in triplicate, and average with SDEV was graphed.

Figure 2.

PTP4A3 inhibition induces apoptosis selectively in TNBC cell lines. Breast cancer cell lines, MDA-MB-231, HCC1143, MCF-7, and BT474, were transfected with siRNAs targeting five phosphatases or control siRNA targeting luciferase. (*, P < 0.05). A, cells were fixed 72 hours after siRNA transfection in ethanol and were stained with PI. The sub-G1 population is graphed here. Experiment was performed in triplicate, and average with SDEV was graphed. B, cells were stained for Annexin V 72 hours after transfection. Experiment was performed in triplicate, and average with SDEV was graphed.

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Depletion of PTP4A3 reduces TNBC tumor growth in vivo

We next investigated the effect of PTP4A3 knockdown on in vivo tumor growth. MDA-MB-231 cells stably expressing doxycycline-inducible PTP4A3 shRNA constructs or empty vector control constructs were injected into the mammary fat pads of nude mice. Once tumors were established, mice were randomized into two groups and treated with doxycycline-containing or control sucrose water. Tumor growth was significantly inhibited in tumors with PTP4A3 knockdown compared with control (Fig. 3A and Supplementary Fig. S3A and S3B). qRT-PCR confirmed that reduced PTP4A3 expression occurs in tumors treated with doxycycline (Supplementary Fig. S3C). To conclusively determine that PTP4A3 depletion inhibits xenograft growth of TNBC cell lines, we conducted a similar experiment with MDA-MB-468 cells, and found that tumor growth was indeed significantly inhibited in tumors with PTP4A3 knockdown compared with controls (Fig. 3B and Supplementary Fig. S3D). As PTP4A3 knockdown affected the cell cycle and apoptosis in vitro and reduced tumor growth in vivo, we studied the effects of PTP4A3 inhibition in vivo by IHC analysis of Ki67 and cleaved caspase-3 levels of xenograft tumors. PTP4A3 knockdown significantly reduced the number of Ki67-positive and increased the percentage of cleaved caspase-3–positive TNBC cells (Fig. 3C–F). These results confirm our in vitro findings, and show that PTP4A3 inhibition reduces proliferation and induces apoptosis. We also performed an additional xenograft experiment with MCF-7, and found no significant inhibition of tumor growth following depletion of PTP4A3 (Supplementary Fig. S3E and S3F). These results demonstrate that PTP4A3 is necessary for in vivo TNBC tumor growth.

Figure 3.

Inhibition of PTP4A3 reduces tumor growth in TNBC mouse models. A and B, PTP4A3 tumors treated with doxycycline affects tumor growth due to the knockdown of PTP4A3. MDA-MB-231 cells (A) and MDA-MB-468 (B) stably transfected with shRNA-PTP4A3 were injected in the mammary fat pad of nude mice. Mice were randomized when tumor reached 30 mm3. Tumors volumes were measured every other day. Tumor growth rate is significantly reduced after knockdown of PTP4A3 in MDA-MB-231. Tumor growth rates were calculated from the slopes of the growth curves for each tumor. Error bar, SD. C and D, KI67 staining by IHC of xenograft tumors in control and PTP4A3 knockdown mice treated with doxycycline or vehicle. E and F, cleaved caspase-3 staining by IHC of xenograft tumors in control and PTP4A3 knockdown mice treated with doxycycline or vehicle.

Figure 3.

Inhibition of PTP4A3 reduces tumor growth in TNBC mouse models. A and B, PTP4A3 tumors treated with doxycycline affects tumor growth due to the knockdown of PTP4A3. MDA-MB-231 cells (A) and MDA-MB-468 (B) stably transfected with shRNA-PTP4A3 were injected in the mammary fat pad of nude mice. Mice were randomized when tumor reached 30 mm3. Tumors volumes were measured every other day. Tumor growth rate is significantly reduced after knockdown of PTP4A3 in MDA-MB-231. Tumor growth rates were calculated from the slopes of the growth curves for each tumor. Error bar, SD. C and D, KI67 staining by IHC of xenograft tumors in control and PTP4A3 knockdown mice treated with doxycycline or vehicle. E and F, cleaved caspase-3 staining by IHC of xenograft tumors in control and PTP4A3 knockdown mice treated with doxycycline or vehicle.

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PTP4A3 depletion alters critical proliferation and apoptosis signaling pathways

To further explore the mechanism of growth suppression by PTP4A3 knockdown, we analyzed the reverse-phase protein array (RPPA) data from TCGA breast cancer samples. Breast tumors with high (upper tertile) and low (lower tertile) PTP4A3 mRNA expression were selected for further analysis. We compared protein phosphorylation and total protein levels between the two groups, and identified those that significantly differ (P < 0.1) between the high and low PTP4A3-expressing groups. As expected given our in vitro and in vivo findings, cleaved caspase-7 was elevated in tumors with low PTP4A3 expression selectively in basal-like (primarily triple-negative) but not luminal (primarily ER-positive) breast tumors (Supplementary Fig. S2A and S2B). Western blot analysis confirmed increased cleaved caspase-7 expression following PTP4A3 knockdown in triple-negative but not ER-positive breast cancer cell lines (Fig. 4A). These results show that loss of PTP4A3 suppresses TNBC cell growth by inducing apoptosis.

Figure 4.

Signaling proteins involved in proliferation and programmed cell death are altered with PTP4A3 knockdown. Two TNBC cell lines (MDA-MB-231 and HCC1143) and two ER-positive cell lines (MCF-7 and T47D) were transfected with PTP4A3 or control siRNA. After 72 hours, cells were harvested and lysed for Western blot analysis. A, cleaved caspase-7 is increased after knockdown of PTP4A3 in TNBC cell lines only. B, ERK1/2 phosphorylation and protein levels are reduced after knockdown of PTP4A3. C, phosphorylation of p38 is reduced after knockdown of PTP4A3.

Figure 4.

Signaling proteins involved in proliferation and programmed cell death are altered with PTP4A3 knockdown. Two TNBC cell lines (MDA-MB-231 and HCC1143) and two ER-positive cell lines (MCF-7 and T47D) were transfected with PTP4A3 or control siRNA. After 72 hours, cells were harvested and lysed for Western blot analysis. A, cleaved caspase-7 is increased after knockdown of PTP4A3 in TNBC cell lines only. B, ERK1/2 phosphorylation and protein levels are reduced after knockdown of PTP4A3. C, phosphorylation of p38 is reduced after knockdown of PTP4A3.

Close modal

We next investigated the effect of PTP4A3 loss on MAPK signaling. We preformed Western blot analyses to test whether PTP4A3 depletion affects the phosphorylation status of ERK and p38. ERK phosphorylation and total protein levels of 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 of PTP4A3; however, p38 phosphorylation was suppressed in MDA-MB-231, the TNBC cell line with the largest phenotypic effect (Fig. 4C). These changes in ERK and p38 expression and activity may contribute to the selective induction of apoptosis in triple-negative compared with ER-positive breast cancer cell lines.

PTP4A3 is amplified in TNBC and associated with poor overall survival

As loss of PTP4A3 suppresses growth and induces apoptosis in TNBC, we analyzed PTP4A3 expression levels in publicly available human breast cancer datasets (5, 19). PTP4A3 expression is significantly elevated in breast cancer as compared with normal cells, and is increased in triple-negative as compared with ER-positive breast cancers (Supplementary Fig. S4A–S4D).

To determine whether increased PTP4A3 expression increases cancer cell growth, we created stably transfected cell lines (MDA-MB-231, MCF-7) overexpressing PTP4A3. MDA-MB-231 overexpressing PTP4A3 showed increased growth rates, whereas MCF-7 clones overexpressing PTP4A3 exhibited reduced growth compared with vector transfected clones (Fig. 5A and B). Given the high expression of PTP4A3 in TNBC, we investigated the amplification status of PTP4A3 in the TCGA dataset using cBioPortal (17, 18). PTP4A3 is located on 8q.24.3, a region frequently amplified in breast cancer which also includes Myc (20). We discovered that 8q.24.3 is amplified in 21% of all breast cancers and 45% of basal-like breast cancers. In addition, PTP4A3 is amplified in 17% and Myc in 22% of all breast cancers, whereas PTP4A3 is amplified in 29% and Myc in 44% (often concomitantly with PTP4A3) of basal-like breast cancers. The analysis of the TCGA dataset also revealed PTP4A3 amplification without the presence of Myc amplification in 2% of basal-like tumors. This is in contrast to Luminal A/B (primarily ER-positive) breast tumors, in which PTP4A3 is amplified in only 12% (Supplementary Fig. S5A). Similar analysis of the TCGA ovarian cancer dataset gave similar results: PTP4A3 amplification in 34%, Myc amplification in 41%, and PTP4A3 amplification without Myc amplification in 7% of serous ovarian cancers (Supplementary Fig. S5B).

Figure 5.

High levels of PTP4A3 promote TNBC cell growth and associated with poor overall survival in breast cancer patients. A, overexpression of PTP4A3 caused an increased growth in the TNBC cell line MDA-MB-231. Equal amount of cells of MDA-MB-231 cells stably transfected with control or PTP4A3 were plated and assayed for growth for 4 days using Trypan blue staining. B, overexpression of PTP4A3 reduced growth of the ER-positive breast cancer cell lines MCF-7. Equal amount of cells of MCF-7 cells stably transfected with control or PTP4A3 were plated and assayed for growth for 4 days by Trypan blue staining. C, comparative analysis of gene amplification of PTP4A3, Myc, and intermediate genes. The amplification status of PTP4A3, Myc, and genes located in between the PTP4A3 and Myc loci was assessed and graphed in percentage of concurrent of independent amplification D, Kaplan–Meier curves of overall survival of all breast cancer patients in the Kao dataset stratified by PTP4A3 expression. E, proportional hazard ratio analysis demonstrates that high PTP4A3 levels are an independent predictor for worse overall survival in breast cancer patients in the Kao dataset.

Figure 5.

High levels of PTP4A3 promote TNBC cell growth and associated with poor overall survival in breast cancer patients. A, overexpression of PTP4A3 caused an increased growth in the TNBC cell line MDA-MB-231. Equal amount of cells of MDA-MB-231 cells stably transfected with control or PTP4A3 were plated and assayed for growth for 4 days using Trypan blue staining. B, overexpression of PTP4A3 reduced growth of the ER-positive breast cancer cell lines MCF-7. Equal amount of cells of MCF-7 cells stably transfected with control or PTP4A3 were plated and assayed for growth for 4 days by Trypan blue staining. C, comparative analysis of gene amplification of PTP4A3, Myc, and intermediate genes. The amplification status of PTP4A3, Myc, and genes located in between the PTP4A3 and Myc loci was assessed and graphed in percentage of concurrent of independent amplification D, Kaplan–Meier curves of overall survival of all breast cancer patients in the Kao dataset stratified by PTP4A3 expression. E, proportional hazard ratio analysis demonstrates that high PTP4A3 levels are an independent predictor for worse overall survival in breast cancer patients in the Kao dataset.

Close modal

Because of the large size of the 8q24 genomic region, we hypothesized that PTP4A3 and Myc genes might be represented by two different amplicons. To test this hypothesis, we investigated the amplification status of PTP4A3, Myc, and four different genes located between PTP4A3 and Myc in the 8q24 region in the Curtis, TCGA, and Nikolsky datasets (Supplementary Fig. S5C). This analysis revealed that amplification of PTP4A3 but not Myc occurs in 3% and 44% of the Curtis and Nikolsky dataset tumors, respectively (Fig. 5C). We also found that PTP4A3 and Myc coamplification occurs without amplification 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 amplification is not a passenger of Myc amplification, and can be independently involved in breast cancer pathogenesis. Correlation of PTP4A3 copy number and expression levels demonstrated that mRNA expression increases with elevated copy numbers (Supplementary Fig. S5D).

Next, we investigated the importance of PTP4A3 overexpression on survival of breast cancer patients (Kao dataset; ref. 19). Kaplan–Meier analysis of patients dichotomized on median PTP4A3 expression demonstrates poorer overall survival for patients with high versus low PTP4A3-expressing tumors (Fig. 5D). Cox proportional hazard analysis of PTP4A3 above and below median, age, grade, lymph node status, and ER status, indicate PTP4A3 is an independent predictor of decreased overall survival in breast cancer (Kao dataset, Fig. 5E; Curtis dataset, Supplementary Table S2). These results demonstrate that PTP4A3 is an independent predictor of poor overall survival, and suggests that PTP4A3, a critical growth regulatory phosphatase, is a novel potential target for the treatment of breast cancer, particularly TNBC.

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

We also demonstrated that PTP4A3 knockdown reduces phosphorylation of p38 and ERK1/2 in all breast cancer cell lines, consistent with the observed reduced proliferation and growth arrest. p38 inhibition has been shown to induce sensitization to apoptosis and increase sensitivity to cisplatin (21). These results suggest that loss of PTP4A3 causes apoptosis through p38 inactivation. This also suggests that high levels of PTP4A3 might cause resistance to cisplatin or other chemotherapy drugs in TNBCs. Other phosphatases have been shown to induce resistance to cancer treatments, such as mitogen-activated protein kinase phosphatase 3 (MKP3). Increased levels of MKP3 have been associated with tamoxifen resistance in ER-positive breast cancers (22). These findings demonstrate that phosphatases are critical signal transduction regulators in cancer.

PTP4A3 overexpression in TNBC has been confirmed in multiple datasets, and is associated with reduced survival. Using the TCGA dataset and cBioPortal for cancer Genomics, we identified that PTP4A3 gene amplification is enriched in basal-like versus luminal breast cancers, and in some cases is independent of Myc amplification. These results indicate selective importance of PTP4A3 in TNBC. Previous studies have reported PTP4A3 overexpression in several cancers, including breast, gastric, and colon cancer (23, 24), particularly with metastatic colon and breast cancer. Furthermore, PTP4A3 expression is higher in invasive cancer than normal breast tissue, and protein expression is associated with lymph node positivity (25), as well as reduced disease-free survival (23).

PTP4A3 has a well-established role in cell motility and metastasis. Ectopic PTP4A3 overexpression influences migration and invasion of colon cancer cells, whereas PTP4A3 elimination decreases cell motility and metastatic capability in mice (26). PTP4A3 overexpression is primarily observed in metastases (distant or lymph node) versus primary tumors (27). The overexpression of PTP4A3 in triple-negative compared with ER-positive breast cancer supports the concept that high PTP4A3-expressing tumors are more aggressive. We have demonstrated that loss of PTP4A3 reduces cell proliferation and induces apoptosis, and are currently studying PTP4A3 overexpression in cancerous and precancerous cells to test whether PTP4A3 transforms premalignant cells into fully invasive cancer cells.

Although PTP4A3 regulation of molecular pathways is not well studied and has focused primarily on migration and invasion, Ezrin and Integrin-beta 1 are direct targets of PTP4A3 in adhesion and motility (28, 29). Recently, PTP4A3 has 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 understood. Our current studies show that loss of PTP4A3 alters ERK and p38 signaling. In addition, PTP4A3 has been shown to regulate p53 and p21 reporter activity (31). These results provide a potential mechanism for PTP4A3 regulation of proliferation and apoptosis in cancer cells. Other studies have shown that PTP4A3 activates the NF-kB and PI3K pathways, which are important for survival and proliferation of cancer cells (32, 33). PTP4A3 overexpression is associated with increased p65 phosphorylation (32), and reduced PTEN expression (33). Alteration of these multiple pathways may collectively contribute to the reduced proliferation and apoptosis following PTP4A3 knockdown in breast cancer cells. We are currently performing experiments to elucidate direct targets of PTP4A3 in breast cancer cells that regulate cell cycle and cell death.

Here, we have identified phosphatases overexpressed in triple-negative as compared with ER-positive breast cancer, many of which are critical for TNBC cell growth. One of these phosphatases, PTP4A3, is a critical regulator of proliferation and apoptosis, and inhibition of PTP4A3 induces cell-cycle arrest, apoptosis, and inhibition of TNBC tumor growth in vivo. These data strongly suggest that targeting PTP4A3 or its signaling pathway in TNBC is a promising strategy for women with these aggressive breast cancers.

P.H. Brown is a consultant/advisory board member for Susan G. Komen Foundation. No potential conflicts of interest were disclosed by the other authors.

Conception 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. Brown

Acquisition 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. Osborne

Analysis 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. Brown

Writing, 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. Brown

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Mazumdar, G.B. Mills

Study supervision: P.H. Brown

Other (partial funding):C.K. Osborne

The authors thank Svasti Haricharan for the data analysis of the 8q24 region, Michelle Savage for editing the manuscript, and Sam Short for assisting in the submission.

This 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), and the 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 article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data