Analysis of phosphatases in ER-negative breast cancers identifies DUSP4 as a critical regulator of growth and invasion

Abstract

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple-negative" breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells.

Keywords: MAPK pathways; Mouse xenograft; Phosphatase; TNBC.

Fig. 1

Phosphatases differentially expressed in ER-negative and ER-positive breast cancers. a Supervised hierarchical clustering of breast cancers based on phosphatase expression that distinguishes ER-positive from ER-negative breast tumors. Clinical parameters (HER2, ER, Pam50) are annotated for the samples (x-axis), and gene signature is annotated for the genes (y-axis). b Hierarchical clustering analysis of only ER-negative breast tumors using the phosphatases genes (as shown) selected in a

Fig. 2

Genomic alteration and expression of phosphatases. a Percent of all tumors with any deletion among the top five underexpressed genes in the TCGA dataset. b Homozygous deletion of selected phosphatases in TCGA. c All somatic alterations of DUSP4 gene represented by the cBio Oncoprint. d Differential expression of DUSP4 in two breast cancer datasets

Fig. 3

Induced expression of DUSP4 inhibits ER-negative but not ER-positive growth in vitro. a Western blot analysis of doxycycline-induced expression of DUSP4 in SUM159, MDA-MB-231, and MCF7 breast cancer cells. b–d Proliferation analysis of breast cancer cell lines upon expression of DUSP4. e–g Anchorage-independent colony formation assay of breast cancer cell lines upon expression of DUSP4

Fig. 4

Induction of DUSP4 expression inhibits the growth of ER-negative breast cancer cells in vivo. a Induced expression of DUSP4 does not inhibit in vivo xenograft growth of MDA-MB-231 vector clone ±Dox; b calculated slope from MDA-MB-231 vector clone xenograft. c MDA-MB-231-DUSP4 clone ±Dox growth curves; d calculated slope from MDA-MB-231-DUSP4 xenograft. e Induced expression of DUSP4 does not inhibit in vivo xenograft growth of SUM 159-Vector clone ±Dox growth curves; f calculated slope from SUM 159-vector ±Dox, g SUM 159-DUSP4 clone ±Dox growth curves; h calculated slope from SUM 159-DUSP4 clone xenograft. t-test p values are indicated

Fig. 5

Increased DUSP4 expression causes reduced proliferation, invasion, and a G1 cell cycle block. a Representative Ki67 staining of MDA-MB-231 xenograft sections. b Quantitation of Ki67; c representative pictures of Boyden chamber assay for migration for the cells before and after DUSP4 induction. d Relative migration in MDA-MB-231; e representative pictures of Boyden chamber assay for invasion for the cells before and after DUSP4 induction. f Relative invasion in MDA-MB-231. g Cell cycle changes after DUSP4 induction. t-test p values are indicated

Fig. 6

RPPA analysis identifies signaling pathways altered upon induced expression of DUSP4. a RPPA analysis was performed on SUM159 cells with and without induction of DUSP4 (–Dox = No DUSP4 over expression and +Dox = DUSP4 overexpression). Selective markedly altered proteins are indicated after DUSP4 expression. The median expression values for each protein in cells treated with Dox was subtracted from the protein expression value in cells treated with vehicle to obtain a difference value for each protein studied. Those proteins that showed the greatest change (increased or decreased) are shown in Supplementary Table 2. b Changes in MAP Kinase group: p-ERK1/2, p-p38, and p-JNK1/2. c Changes in the other signaling pathways: p-Rb, p-p65 (S536), and p-AMPK (T172)