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PEA-15 Inhibits Tumorigenesis in an MDA-MB-468 Triple-Negative

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PEA-15 Inhibits Tumorigenesis in an MDA-MB-468 Triple-Negative Published OnlineFirst March 9, 2010; DOI: 10.1158/1078-0432.CCR-09-1456 Published Online First on March 9, 2010 as 10.1158/1078-0432.CCR-09-1456 Cancer Therapy: Preclinical Clinical Cancer Research PEA-15 Inhibits Tumorigenesis in an MDA-MB-468 Triple-Negative Breast Cancer Xenograft Model through Increased Cytoplasmic Localization of Activated Extracellular Signal-Regulated Kinase Chandra Bartholomeusz1,2, Ana M. Gonzalez-Angulo2,3, Anna Kazansky1,2, Savitri Krishnamurthy4, Ping Liu5, Linda X. H. Yuan1,2, Fumiyuki Yamasaki1,2, Shuying Liu3, Naoki Hayashi1,2, Dongwei Zhang1,2, Francisco J. Esteva1,2, Gabriel N. Hortobagyi1,2, and Naoto T. Ueno1,2,6 Abstract Purpose: To determine the role of PEA-15 in breast cancer. Experimental Design: A reverse-phase protein array was used to measure PEA-15 expression levels in 320 human breast cancers; these levels were correlated with clinical and tumor characteristics. PEA-15 was overexpressed by an adenovirus vector or by stably expressing PEA-15 in different breast cancer cell lines. The effects on breast cancer cell survival and on the downstream apoptotic signaling pathway were mea- sured in terms of cell proliferation (trypan blue for cell viability, bromodeoxyuridine incorporation for DNA synthesis), anchorage-independent growth (soft agar colony formation), and apoptosis (fluores- cence-activated cell sorter analysis). The preclinical efficacy of Ad.PEA-15 given intratumorally was eval- uated in nude mice bearing tumors from s.c. implanted human MDA-MB-468 triple-negative breast cancer cells. Results: In human breast cancers, low levels of PEA-15 expression correlated with high nuclear grade (P < 0.0001) and with negative hormone receptor status (P = 0.0004). Overexpression of PEA-15 in breast cancer cells resulted in growth inhibition, reduction in DNA synthesis, and onset of caspase-8–dependent apoptosis. In athymic nude mice bearing MDA-MB-468 xenografts, tumor volumes were significantly smaller in mice treated intratumorally with Ad.PEA-15 than in control mice (P < 0.0001). Tumors from mice treated with Ad.PEA-15 had increased levels of activated (phosphorylated) extracellular signal-reg- ulated kinase and reduced levels of Ki-67 compared with tumors from nontreated or control-adenovirus– treated mice. Conclusion: PEA-15 has therapeutic potential in breast cancer. Further preclinical and clinical explo- ration of PEA-15 as a druggable target is warranted. Clin Cancer Res; 16(6); 1802–11. ©2010 AACR. Breast cancer is the most common cancer in women in gesterone receptor, and HER-2 is known as triple recep- the United States and most of the Western world (1). In tor–negative breast cancer. It is the only major type of 2008, 182,460 cases are expected to be diagnosed (26% breast cancer for which no specific targeted therapy is of all cancers in women), and 40,480 women are ex- available to improve patient outcomes (2). A deeper un- pected to die of the disease (accounting for 15% of all derstanding of the molecular mechanisms responsible for cancer deaths in women, making this disease the second breast cancer is imperative if more effective treatments are most common cause of cancer death among women). to be discovered. Breast cancer that is negative for estrogen receptor, pro- PEA-15 (also called PED) is an acidic, serine-phosphor- ylated, 15-kDa phosphoprotein that contains a death effector domain and is associated with microtubules (3). Authors' Affiliations: 1Breast Cancer Translational Research Laboratory, In primary astrocytes (the first cells in which this protein Departments of 2Breast Medical Oncology, 3Systems Biology, 4Pathology, 5Biostatistics, and 6Stem Cell Transplantation and Cellular was identified), PEA-15 was shown to regulate the nuclear Therapy, The University of Texas M.D. Anderson Cancer Center, localization of extracellular signal-regulated kinase (ERK) Houston, Texas and consequently affect the transcription of ERK-dependent Corresponding Author: Naoto T. Ueno, Department of Breast Medical targets such as transcription factor Elk. This function was Oncology, Unit 1354, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713- shown to be important in slowing astrocyte proliferation 745-6168; Fax: 713-794-4385; E-mail: [email protected]. (4). We previously showed that the antitumor activity of doi: 10.1158/1078-0432.CCR-09-1456 the adenovirus protein E1A was dependent on PEA-15 (5) ©2010 American Association for Cancer Research. and that women with ovarian tumors that expressed high 1802 Clin Cancer Res; 16(6) March 15, 2010 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 9, 2010; DOI: 10.1158/1078-0432.CCR-09-1456 In vivo Activity of PEA-15 in Breast Cancer tumor growth compared with that in mice treated with Translational Relevance control virus or nontreated mice. New targeted therapies for breast cancer are needed Materials and Methods because many current treatments become ineffective in advanced disease. We provide evidence that PEA-15, a Cell lines and culture conditions. The human breast ade- phosphoprotein that slows cell proliferation by bind- nocarcinoma cell lines SKBR-3, MDA-MB-231, MCF-7, ing to and sequestering extracellular signal-regulated MDA-MB-468, and MDA-MB-453 were obtained from kinase in the cytoplasm, may be a candidate therapeu- the American Type Culture Collection. The cells were tic gene for breast cancer. Our results from a reverse- grown in DMEM/nutrient mixture F12 (DMEM/F12; Life phase protein lysate microarray showed that low Technologies) supplemented with 10% fetal bovine serum PEA-15 expression directly correlated with negativity and penicillin/streptomycin, and maintained in a humid- for hormone receptors and high nuclear grade. Overex- ified incubator at 37°C containing 5% CO2. pression of PEA-15 in the MDA-MB-468 triple-negative Western blot analysis. Cells were washed thrice with PBS breast cancer cell line inhibited growth and induced (pH 7.4; 1×) and then lysed in lysis buffer [20 mmol/L apoptosis. Correspondingly, in athymic nude mice Na2PO4 (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, bearing human MDA-MB-468 xenografts, tumor vo- 1% aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, lumes were significantly smaller in mice treated intra- 100 mmol/L NaF, and 2 mmol/L Na3VO4] as previously tumorally with Ad.PEA-15 adenovirus than in control described (11). PEA-15 was extracted with NP40 lysis buff- mice. Combined, these results contribute to the ratio- er as previously described (12). The antibodies used were nale for evaluating PEA-15 as a therapeutic gene in rabbit anti–PEA-15 polyclonal antibody (Cell Signaling breast cancer. Technology; used in a 1:1,000 dilution), rabbit anti– pPEA-15 (S116) (Invitrogen; 1:1,000), rabbit anti–pPEA- 15 (S104) (1:1,000; Cell Signaling Technology), ERK levels of PEA-15 survived longer than did women with tu- (1:500; Santa Cruz Biotechnology), pERK (1:500; Cell Sig- mors that expressed low levels of PEA-15 (6). naling Technology), actin (1:5,000; Sigma-Aldrich Chem- A study of PEA-15 suggested that overexpression of PEA- ical Co.), poly (ADP-ribose) polymerase (PARP; 1:1,000; 15 led to the inhibition of both Fas-mediated and tumor BD Biosciences), α-tubulin (1:5,000; Sigma-Aldrich), and necrosis factor receptor-1 (TNFR1)–mediated apoptosis in caspase-8 (1:500; EMD Chemicals]. Secondary rabbit MCF-7 breast cancer cells; this function was thought to in- (1:5,000) and mouse (1:5,000) fluorescent antibodies volve the displacement of FADD-FLICE binding through were from Molecular Probes and were detected with an the death effector domain of PEA-15 (7). However, in an- Odyssey imaging system (Li-Cor Biosciences). other study, PEA-15 was found to have two different Colony formation assay. Three human breast cancer cell effects in a single cell type (NIH3T3)—inhibition of Fas- lines (MDA-MB-468, MCF-7, and MDA-MB-453) were mediated apoptosis but enhancement of TNFR1-induced transfected in 100-mm plates with 10 μg of either a vector apoptosis (8). In breast cancer, PEA-15 expression inhibits containing HA-tagged PEA-15 (4)oracontrolvector invasion by binding to ERK, and reduced PEA-15 expres- (pcDNA3; Stratagene) by means of FuGENE transfection sion is seen in metastatic breast cancer cells, suggesting reagent (Roche Diagnostics) according to the manufac- that PEA-15 is a suppressor of metastasis (9). In breast turer's instructions. Both plasmids contained a neomycin cancer, our comparison of PEA-15 expression levels in a resistance gene. At 48 h after transfection, cells were tryp- small tissue microarray revealed that PEA-15 expression sinized, diluted at a ratio of 1:5 or 1:10, and then plated was lower in high-grade tumors than in low-grade tumors on 100-mm plates and grown in DMEM/F12 containing (10). However, there has not been a large comprehensive 500 μg/mL of the neomycin analogue G418. Three weeks study of the role of PEA-15 in breast cancer. later, colonies were stained with 0.5% crystal violet in 20% The objectives of this project were to look at the cor- ethanol and counted. relations of PEA-15 with clinical and tumor characteris- Transfection conditions. The PEA-15 transfectants were tics in human breast cancers and to study the in vitro established as follows. MDA-MB-453 human breast cancer and in vivo antitumor effects of PEA-15 in breast cancer cells were seeded on 100-mm plates and transfected with cell lines. In vitro, we used adenovirus Ad.PEA-15 and 10 μg of either a HA-PEA-15 vector (4) or a control vector breast cancer cells that stably express PEA-15 to study (pcDNA3; Stratagene) by means of FuGENE transfection its biological function in breast cancer.
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