Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer Growth and Predicts Poor Patient Survival

Abstract

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.

Figure 1. PTP4A3 inhibition induces G1 arrest in triple-negative breast cancer cell lines

Breast cancer cell lines, MDA MB231, HCC1143, MCF-7 and BT474, were transfected with siRNA targeting 5 phosphatases or control siRNA targeting Luciferase. A–E) After 72hrs cells were fixed in ethanol, and stained with PI (* indicates p<0.05). The 5 phosphatases were: A) PTP4A3 B) PPAP2B C) CDC25B D) TIMM50 E) MDA MB231 and HCC1143 cells transfected with siRNA targeting phosphatases, were fixed 72hrs after siRNA transfection and stained for phospho-Histone H3 using immunofluorescence techniques. Cells were counted in 10 fields, and the experiment was performed in triplicate. F) Cells were fixed 72hrs after siRNA transfection and stained for Ki-67 using immunofluorescence techniques. Cells were counted in 10 fields, and the experiment was performed in triplicate. Error bars show standard deviation of the mean.

Figure 2. PTP4A3 inhibition induces apoptosis selectively in triple-negative breast cancer cell lines

Breast cancer cell lines, MDA MB231, HCC1143, MCF-7 and BT474, were transfected with siRNA targeting 5 phosphatases or control siRNA targeting Luciferase. (* indicates p<0.05). A) Cells were fixed 72hrs after siRNA transfected in ethanol, and 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 72hrs after transfection. Experiment was performed in triplicate, and average with SDEV was graphed.

Figure 3. Inhibition of PTP4A3 reduces tumor growth in triple-negative breast cancer mouse models

A–B)) PTP4A3 tumors treated with doxycycline affects tumor growth due to the knockdown of PTP4A3. MDA MB231 cells (A) and MDA MB468 (B) stably transfected with shRNA-PTP4A3 were injected in the mammary fat pad of nude mice. Mice were randomized when tumor reached 30mm3. Tumors volumes were measured every other day. Tumor growth rate is significantly reduced after knockdown of PTP4A3 in MDA MB231. Tumor growth rates were calculated from the slopes of the growth curves for each tumor. Error bar shows standard deviation. C–D) KI-67 staining by IHC of xenograft tumors in control and PTP4A3 knockdown mice treated with doxycycline or vehicle. E–F) Cleaved caspase 3 staining by IHC of xenograft tumors in control and PTP4A3 knockdown mice treated with doxycycline or vehicle.

Figure 4. Signaling proteins involved in proliferation and programmed cell death are altered with PTP4A3 knockdown

Two TNBC cell lines (MDA MB231 and HCC1143), and two ER-positive cell lines (MCF-7 and T47D) were transfected with PTP4A3 or control siRNA. After 72hrs cell 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 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 MB231. Equal amount of cells of MDA MB231 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 implication 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 data set stratified by PTP4A3 expression E) Proportional hazard ratio analysis demonstrates that high PTP4A3 levels are an independent predictor for worse over-all survival in breast cancer patients in the Kao data set.