Breast Cancer Res Treat DOI 10.1007/s10549-016-3892-y PRECLINICAL STUDY Analysis of phosphatases in ER-negative breast cancers identifies DUSP4 as a critical regulator of growth and invasion 1 1 1 2 Abhijit Mazumdar • Graham M. Poage • Jonathan Shepherd • Anna Tsimelzon • 1 1 1 1 Zachary C. Hartman • Petra Den Hollander • Jamal Hill • Yun Zhang • 3 2 2 2 Jenny Chang • Susan G. Hilsenbeck • Suzanne Fuqua • C. Kent Osborne • 4 1 Gordon B. Mills • Powel H. Brown Received: 28 January 2016 / Accepted: 27 June 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Estrogen receptor (ER)-negative cancers have a phosphoproteins. We identified 48 phosphatase genes are poor prognosis, and few targeted therapies are available for significantly differentially expressed in ER-negative com- their treatment. Our previous analyses have identified pared to those in ER-positive breast tumors. We discovered potential kinase targets critical for the growth of ER-neg- that 31 phosphatases were more highly expressed, while 11 ative, progesterone receptor (PR)-negative and HER2- were underexpressed specifically in ER-negative breast negative, or ‘‘triple-negative’’ breast cancer (TNBC). cancers. The DUSP4 gene is underexpressed in ER-nega- Because phosphatases regulate the function of kinase sig- tive breast cancer and is deleted in approximately 50 % of naling pathways, in this study, we investigated whether breast cancers. Induced DUSP4 expression suppresses both phosphatases are also differentially expressed in ER-neg- in vitro and in vivo growths of breast cancer cells. Our ative compared to those in ER-positive breast cancers. We studies show that induced DUSP4 expression blocks the compared RNA expression in 98 human breast cancers (56 cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, ER-positive and 42 ER-negative) to identify phosphatases JNK1, RB, and NFkB p65 phosphorylation; and inhibits differentially expressed in ER-negative compared to those invasiveness of TNBC cells. These results suggest that that in ER-positive breast cancers. We then examined the DUSP4 is a critical regulator of the growth and invasion of effects of one selected phosphatase, dual specificity phos- triple-negative breast cancer cells. phatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein Keywords TNBC Á Phosphatase Á Mouse xenograft Á microarray analyses of 172 proteins, including MAPK pathways Electronic supplementary material The online version of this Introduction article (doi:10.1007/s10549-016-3892-y) contains supplementary material, which is available to authorized users. Breast cancer is the most frequently diagnosed cancer in & Powel H. Brown women and the second highest cause of cancer-related [email protected] death in United States women in 2016 [1]. Selective 1 estrogen receptor modulators (SERMs) and aromatase Department of Clinical Cancer Prevention, The University of inhibitors are currently used to treat ER-positive breast Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA cancers [2–6], while anti-HER2 drugs are used to treat ER- negative HER2-positive breast cancers [7–9]. However, 2 Department of Medicine and the Dan L. Duncan Cancer Center, Lester and Sue Smith Breast Center, Baylor College there are few treatment options available for breast cancers of Medicine, Houston, USA that do not express ER, progesterone receptor (PR), or the 3 Methodist Cancer Center, The Methodist Hospital Research HER2 protein, otherwise known as ‘‘triple-negative’’ breast Institute, Houston, USA cancers (TNBCs). As no targeted therapy is available for 4 Department of Systems Biology, The University of Texas most TNBC tumors, cytotoxic chemotherapy is the current M.D. Anderson Cancer Center, Houston, USA treatment strategy. 123 Breast Cancer Res Treat We previously conducted cDNA microarray studies to genes. Statistical analysis was done using Partek software identify potential therapeutic targets of ER-negative breast (http://partek.com). The following criteria were used to cancers [10]. These studies identified several kinases crit- find genes differentially expressed between ER-negative ical for the growth of TNBC. In this study, we sought to and ER-positive tumors: Benjamini–Hochberg false dis- identify phosphatases differentially expressed in ER-neg- covery rate (FDR) p value B 0.1, fold change C1.2 ative tumors that also regulate breast cancer growth. We or B0.8. identified 11 underexpressed and 31 overexpressed phos- phatases in ER-negative compared to those in ER-positive Cell lines and cell culture breast cancers. We initially focused on those 11 phos- phatases underexpressed exhibiting decreased expression The MCF-7 (HTB-22), MDA-MB-231 (HTB-26), and in ER-negative tumors and found that of these 11 under- MDA-MB-468 (HTB-132) cell lines (American Type expressed phosphatases, DUSP4 is the most frequently Culture Collection (ATCC), Manassas, VA, USA) were deleted phosphatase in ER-negative breast cancer. cultured in DMEM (Cellgro by Mediatech, Inc., Manassas, DUSP4 specifically regulates extracellular regulated VA, USA) supplemented with 10 % fetal bovine serum, kinase (ERK), and the phosphatase activity of DUSP4 is 100 U/ml penicillin, and 100 mg/ml streptomycin. These enhanced upon interaction with ERK and p38 [11–13]. were grown and maintained as described in Supplementary DUSP4 expression has been shown to play an important Methods. STR profiles were compared to (1) known ATCC role in senescence [14] and emerging evidence suggests fingerprints (ATCC.org); (2) the Cell Line Integrated DUSP4 is involved in the growth and progression of cancer Molecular Authentication database (CLIMA) version [15, 16]. Our results demonstrate that DUSP4 is frequently 0.1.200808 (http://bioinformatics.istge.it/clima/)[17]; and deleted in breast cancer and underexpressed in TNBCs, and (3) the MD Anderson fingerprint database. that DUSP4 overexpression halts TNBC tumor growth and invasion. Our results also demonstrate that all three MAPK Reagents and antibodies proteins (ERK1/2, p38, and JNK1) are negatively regulated by DUSP4. Moreover, re-expression of DUSP4 also inhi- The DUSP4 antibody was purchased from BD Transduc- bits other growth inducing pathways, including NFjB and tion Laboratories (Lexington, KY, USA). Phospho-ERK1/2 Rb. Overall, this study demonstrates that DUSP4 is fre- (#4370) and total ERK1/2 (#4372) were purchased from quently deleted in breast cancers and is a critical regulator Cell Signaling Technology, Inc. (Danvers, MA, USA). of growth and invasion of TNBCs, suggesting DUSP4 is an Antiactin antibody was purchased from Sigma-Aldrich important tumor suppressor gene in these aggressive breast Corp. (St. Louis, MO, USA). Antimouse (#NA931 V) and cancers. antirabbit (NA934 V) secondary antibodies were obtained from GE Healthcare Bio-sciences Corp. (Piscataway, NJ, USA). Materials and methods Viral vectors and modification of cell lines Breast tumor and microarray datasets The DUSP4 ORF clone was obtained from Open Biosystems All tumors were collected by Dr. Jenny Chang and (Huntsville, AL, USA) and cloned into tetracycline (Tet)- approved by the institutional review board of Baylor Col- inducible vector (pTIPZ) through a gateway LR clonase lege of Medicine. This tumor set, including tumor stage, reaction. Final constructs were verified through restriction size, and patient race and menopausal status, has been digestion and sequencing. Lentiviral vectors were prepared previously described [10]. In this study, four samples were as described previously [18]. Stable cell lines expressing removed from the analysis. These four samples had an inducible cDNAs were generated by lentiviral infection unconfirmed ER status or appeared as outliers on the using a pTIPZ lentiviral expression system in the presence of principal component analysis (PCA) plot and thus removed 4 lg/ml polybrene, followed by puromycin selection at 48 h from the dataset. Therefore, 98 samples, 56 ER-positive post infection. All pTIPZ stable cell lines were maintained in and 42 ER-negative invasive breast cancers, were used for media with Tet-safe serum (Clontech Laboratories Inc., this analysis. Gene expression was estimated using the Mountain View, CA, USA). Robust Multi-array Average (RMA) procedure. We limited our data and clustering analysis to 262 genes (454 probe- Quantitative RT-PCR (qRT-PCR) assessments sets on Affymetrix U133A chip), which encode known phosphatases and proteins with phosphatase in their name. Quantitative PCR assays of reverse-transcribed transcripts We used the most variable probeset for each of the 262 (4 replicates per transcript) were carried out using an ABI 123 Breast Cancer Res Treat PRISM 7900 Sequence Detection System (Life Technolo- Reverse phase protein array (RPPA) gies (formerly Applied Biosystems Inc., Foster City, CA, USA), as previously described [10]. RPPA assays were performed in the MD Anderson Core facility. Experimental details are provided in Supplemen- Cell growth assays tary Materials. Cellular proliferation and anchorage-independent growth Mouse experiments were measured as described previously [19]. Experiments using nude mice (The Jackson Laboratory, Cell cycle assays Bar Harbor, ME, USA) were performed in accordance with M.D. Anderson Institutional Animal Care and Use Com- To measure cell cycle distribution, Tet-inducible cells were mittee (IACUC)-approved protocols. Experimental details treated for 4 days with or without doxycycline