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Cancer Research 68, 1760, March 15, 2008. doi: 10.1158/0008-5472.CAN-07-3177
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

14-3-3{zeta} Down-regulates p53 in Mammary Epithelial Cells and Confers Luminal Filling

Christopher G. Danes1,2, Shannon L. Wyszomierski1, Jing Lu1,2, Christopher L. Neal1, Wentao Yang1 and Dihua Yu1

1 Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center; 2 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas

Requests for reprints: Dihua Yu, Department of Molecular and Cellular Oncology, Unit 108, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3636; E-mail: dyu{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3{zeta} is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3{zeta} overexpression occurs, and we found that increased expression of 14-3-3{zeta} begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3{zeta} overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3{zeta} in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3{zeta} overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3{zeta} overexpression conferred resistance to anoikis. Additionally, 14-3-3{zeta} overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3{zeta} transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3{zeta} induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3{zeta}–overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3{zeta} overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3{zeta} alters MEC acini structure and increases the risk of breast cancer. [Cancer Res 2008;68(6):1760–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tissue homeostasis is achieved through a highly regulated balance of cell proliferation and programmed cell death (1). Tissue integrity depends on this balance, both during development and in tissue maintenance, to sustain proper function. Disruptions of this balance often lead to tissue malfunction and human disease. Disrupted tissue cytoarchitecture is a hallmark of cancer (2).

The human mammary gland exemplifies these principles. It is organized into ducts and lobules surrounding lumens formed by apoptosis during development (3). Breast tumorigenesis is a multistep process featuring distinct morphologic and cytologic changes. These steps, from normal epithelium to adenosis, to ductal hyperplasia (DH), to atypical DH (ADH), to ductal carcinoma in situ (DCIS), and finally to invasive ductal carcinoma (IDC), correspond to increasingly worse patient outcome after diagnosis (4). For example, epidemiologic studies have shown that patients with ADH have a four to five times greater likelihood of developing carcinoma compared with patients with DH (5). Advances in imaging and screening have vastly improved the detection of preneoplastic lesions (6); however, the molecular mechanisms that cause these morphologic and cytologic changes are poorly understood.

One of the key morphologic changes in ADH is luminal filling (7). A proper lumen is formed as a result of apoptosis of centrally localized cells that are no longer attached to the basement membrane (BM; ref. 8). The death of these cells is due to a specialized mode of apoptosis, known as anoikis, induced by detachment from extracellular membrane components and mediated by the mitochondrial cell death machinery (9). Resistance to anoikis is postulated to cause lumen filling and consequently involved in early neoplastic transformation (10).

14-3-3 proteins are vital in maintaining an appropriate balance between cell survival and programmed cell death (11). The 14-3-3 proteins are a family of ubiquitously expressed, dimeric proteins with seven members (β, {gamma}, {varepsilon}, {eta}, {sigma}, {tau}, and {zeta}; ref. 12). These 29-kDa to 31-kDa proteins bind predominantly to phosphorylated serine/threonine motifs, modulating the activities of their binding partners (13, 14). Over 200 of 14-3-3 binding proteins which control an array of processes important for cellular homeostasis, including apoptosis, cell cycle progression, metabolism, and signal transduction, have been identified (13–18). The expression of some 14-3-3 proteins and the activity of pathways modulated by 14-3-3s are often altered during the development of cancer (19). For example, 14-3-3{sigma} is a known tumor suppressor protein, which is lost in some breast cancers (20). However, 14-3-3{sigma} is considered a noncanonical member of the 14-3-3 family. The other 14-3-3s are elevated in certain cancer types and modulate pathways critical for transformation (19). 14-3-3s sequester proapoptotic regulators, such as FOXO transcription factors and BAD, away from their sites of action resulting in increased cellular survival (reviewed in ref. 11).

Accumulation of p53, which typically indicates a loss of p53 function due to p53 mutation, is associated with increased risk of progression of early-stage, benign breast disease to breast cancer (21, 22), and mutations that compromise p53 function are found in ~50% of human cancers (23). p53 is up-regulated during cellular stress and induces expression of genes that control cell cycle progression and programmed cell death. In addition to well-studied role of p53 in regulating cellular response to DNA damage and other genotoxic stresses, p53 also mediates anoikis (24, 25). p53 is a critical regulator of anoikis in fibroblasts, thyroid epithelial cells, and endothelial cells (reviewed in ref. 9). Intriguingly, one study showed that {alpha}6β4 integrin, a cell membrane receptor that binds to extracellular matrix components and increases tumor cell survival, could only promote survival when p53 was nonfunctional (24).

Our laboratory discovered that 14-3-3{zeta} is overexpressed in >40% of advanced breast tumor tissues.3 Here, we investigated 14-3-3{zeta} expression levels in proliferative breast lesions representative of different early stages of breast disease that can progress toward breast cancer. We discovered that 14-3-3{zeta} expression increased significantly at the stage of ADH compared with DH and normal mammary tissue. Furthermore, 14-3-3{zeta} overexpression severely disrupted the tissue architecture of nontransformed mammary epithelial cells (MEC) in three-dimensional cultures, causing irregular shape and luminal filling, features morphologically similar to ADH. The 14-3-3{zeta}–overexpressing cells were resistant to anoikis due, at least partly, to reduced p53. Mechanistically, 14-3-3{zeta} overexpression increased p53 protein degradation via a hyperactivation of the phosphoinositide 3-kinase (PI3K)–Akt signaling pathway that phosphorylates MDM2. Replacement of p53 led to luminal cell apoptosis. Thus, 14-3-3{zeta} overexpression and the consequent down-regulation of p53 may be critical early events causing the morphologic and cytologic changes in early-stage breast disease that promote the development of breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Contructs, cell lines, and antibodies. The 14-3-3{zeta} gene was cloned into the pLNCX2 vector (Clontech) and transfected into HEK293 packaging cell line. Transfected cells were selected with 400 µg/mL of neomycin (G418). Then, conditioned media was collected every 48 hours for 2 weeks. The MCF10A cell line was a kind gift from Dr. Robert Pauley (Karmonos Cancer Institute). Retroviral supernatant was added in a 1:1 ratio with complete media and 8 µg/mL of polybrene. The MCF10A cells were selected with 400 µg/mL neomycin, and four populations of pooled clones were isolated. Cell lines were routinely cultured in DMEM/F12 high glucose supplemented with 5% horse serum (Invitrogen), 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 10 ng/mL epidermal growth factor (Sigma), and 0.1 µg/mL cholera toxin (Calbiochem). Antibodies used are 14-3-3{zeta}, p53 (DO1), MDM2 (SMP14; Santa Cruz), activated caspase-3, pMDM2 166/186, pAkt 473, pAkt 308, Akt (Cell Signaling), β-actin (Sigma), Ki67 (DAKO), hemagglutinin (HA; Covance), and horseradish peroxidase–conjugated secondary antibodies (GE Healthcare).

Immunoblot assay. Cellular proteins were isolated using lysis buffer [2% Triton X-100, 300 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), 2 mmol/L EDTA, 2 mmol/L EGTA, 1% NP40, 1 mmol/L orthovanadate, 0.8 phenylmethylsulfonyl fluoride]. Protein concentration was determined using bicinchoninic acid protein assay reagent (Pierce). Cell fractionation assay performed as previously described (26). SDS-PAGE and Western blotting were performed as previously described (27). Enhanced chemiluminescence reagent (GE Healthcare) was used for detection. LY294002 (Calbiochem) treatment was performed for 2 hours at the indicated concentrations.

Three-dimensional culture assays. Three-dimensional culture assay were performed on MCF10A.vec and MCF10A.14-3-3{zeta} cell lines as previously described (28). Assay medium (DMEM/F12 supplemented with 2% donor horse serum, 10 µg/mL insulin, 1 ng/mL cholera toxin, 100 µg/mL hydrocortisone, 50 units/mL penicillin, and 50 µg/mL streptomycin) containing 5 ng/mL epidermal growth factor and 2% growth factor–reduced Matrigel (BD Biosciences) was replaced every 4 days. Cells were collected using Cell Recovery Solution (BD BioSciences) to remove Matrigel.

Immunofluourescence on three-dimensional cultures. Cultures were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, washed thrice with PBS/glycine buffer (130 mmol/L; 7 mmol/L, Na2HPO4, 3.5 mmol/L NaH2PO4, and 100 mmol/L glycine), and incubated with IF buffer (130 mmol/L; 7 mmol/L Na2HPO4, 3.5 mmol/L NaH2PO4, 7.7 mmol/L NaN3, 0.1% bovine serum albumin, 0.2% Triton X-100, and 0.05% Tween 20) for 1 hour. Antibodies were suspended in IF buffer and incubated overnight. The cultures were incubated with Alexaflour-conjugated rabbit or mouse secondary antibodies (Invitrogen), counterstained with 4',6-diamidino-2-phenylindole (DAPI), and mounted with antifade solution (Molecular Probes). Cultures were visualized using a Nikon Eclipse Fluorescent Microscope (Nikon) and processed using Metamorph 2D deconvolution software (Molecular Devices Co.). Confocal microscopy was done using an Olympus FV300 laser scanning confocal microscope (Olympus America, Inc.).

Immunohistochemistry staining. The samples were staged by a pathologist (W.Y.) according to standardized pathologic criteria. The samples were separated into stages normal tissue, adenosis, DH, ADH, DCIS, and IDC. Immunohistochemistry was done as previously described (29). Slides were incubated with 14-3-3{zeta} antibody at a 1:2,000 dilution. Staining was quantitated using a SAMBA 4000 image analyzer (Samba Technologies). Fields for study were identified under a 20x objective using the area mode of analysis. By using a grid system, nine fields containing tumors were chosen randomly from each slide. Debris, lymphocytes, stromal tissue, and necrotic areas were not included. The final analysis value is the consideration of two variables, the percentage of immunostained surfaces, and the mean absorbance depending on staining intensity.

Reverse transcriptase PCR. To determine p53 mRNA levels, two-step reverse transcriptase PCR was done using Superscript III First Strand Synthesis System according to manufacturer's protocols (Invitrogen). p53 primer sequences were from the MGH-Harvard primer bank and are available upon request.

To quantitate the RNA, we used a quantitative, real-time PCR using the FullVelocity SYBR Green QPCR Master Mix kit (Stratagene) and the Stratagene Mx4000 Quantitative PCR instrument according to the manufacturers' specifications. Using the amplification plots, the threshold cycles for p53 in the 10A.vec and 10A.14-3-3{zeta} cells were normalized to the threshold cycle of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was calculated as a threshold cycle difference.

Small interfering RNA. ON-TARGET plus SMART Pool small interfering RNA (siRNA) for 14-3-3{zeta}, MDM2, Akt1, Akt2, and Akt 3 and ON-TARGET plus control siRNA were purchased from Dharmacon. Each ON-TARGET plus SMART Pool siRNA contains four siRNAs to target mRNA. MDM2 and 14-3-3{zeta} siRNAs (100 nmol/L each) were transfected using Lipofectamine Plus (Invitrogen) in Optimem according to manufacturer's protocol. Akt1, Akt 2, and Akt3 siRNAs were transfected together at a concentration of 35 nmol/L each. Lysates were collected 72 hours after transfection.

Anoikis/apoptosis assays. Plates were coated with 2-hydroxyethyl methacrylate (PolyHEMA, Sigma) to prevent attachment. 10A.vec or 10A.14-3-3{zeta} cells (5 x105) were grown for 48 and 72 hours. Apoptosis assays were performed using the Annexin V–Fluos staining kit (Roche) according to manufacturer's protocol.

Ethidium bromide exclusion assay. Cells were grown in three-dimensional cultures for 10 days, incubated with 1 µmol/L of ethidium bromide, and analyzed by fluorescent microscopy (28). Acini with ethidium bromide–positive cells in lumen were quantified. At least 100 acini were quantified. Statistics were performed in PRISM software (Graphpad Software, Inc.) using an unpaired t test.

Transgenic mice. Transgenic mice were generated with the mouse whey acidic protein (WAP) gene promoter driving expression of HA-tagged 14-3-3{zeta}.4 The inguinal mammary glands were collected from nursing dams. Protein extracts were made by homogenizing these lactating mammary glands in tissue lysis buffer [10 mmol/L sodium phosphate (pH 7.3), 154 mmol/L NaCl, 5% sodium deoxycholate, 1% SDS] using a tissue grinder, followed by centrifugation to remove particulate matter and lipids.

Protein stability assay. MG-132 (10 µmol/L; Calbiochem) was given for 6 hours to inhibit proteasome-mediated protein degradation. Cyclohexamide (75 µg/mL; Calbiochem) was added 16 hours after and was given for annotated time. p53 and actin levels were assessed by immunoblot and quantified by densitometry using Scion Image software. p53 levels were normalized to β-actin levels and calculated as a percentage of p53 protein present in untreated cells.

p53 adenoviruses. Recombinant adenoviruses expressing FLAG-tagged p53 or β-galactosidase reporter gene under the control of the cytomegalovirus promoter (AdFLAGp53 and AdLacZ, respectively) have been previously described (30–32). Two days after plating the three-dimensional culture, adenoviruses (250 viral particles per cell) were applied for 24 hours. The cells were fixed after 10 days in three-dimensional culture and analyzed by confocal microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
14-3-3{zeta} is overexpressed at an early stage of breast disease and causes disruption of the tissue architecture. Our laboratory previously found that 14-3-3{zeta} is overexpressed in >40% of breast cancers from patients with late-stage, advanced, metastatic disease.3 To further investigate 14-3-3{zeta} and its role in early breast disease initiation and cancer progression, we analyzed 14-3-3{zeta} protein levels in tissue samples representative of different stages of early breast disease using immunohistochemistry, including normal mammary epithelium, adenosis, DH, ADH, DCIS, and IDC. 14-3-3{zeta} expression was profoundly increased at the stage of ADH compared with DH, adenosis, and normal mammary tissue (Fig. 1A ). ADH is an early-stage breast disease that confers increased risk for the development of breast carcinoma. These data suggested a role for 14-3-3{zeta} in the early stages of breast disease initiation that could contribute to progression toward breast cancer.


Figure 1
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  		Figure 1. 14-3-3{zeta} is overexpressed at an early stage in breast tumor development and is responsible for disruption of tissue architecture. A, samples from different stages of breast disease were stained with anti-14-3-3{zeta} antibody and quantitated using a SAMBA 4000 image analyzer. The final value is the sum of two variables: percentage of stained surface and mean absorbance of staining intensity. B, 14-3-3{zeta} was stably overexpressed in MCF10A cells using a retroviral expression system. Four separate pools of the transfected clones were isolated. 14-3-3{zeta} protein expression was determined by immunoblot. The exogenous HA tagged 14-3-3{zeta} (*) migrated slower than endogenous 14-3-3{zeta} in SDS PAGE. C, 10A.vec and 10A.14-3-3{zeta} cells were grown in three-dimensional culture, fixed, and stained with HA antibody. The nuclei were counterstained with DAPI. Bar, 20 µm.

 
To examine the relationship between 14-3-3{zeta} expression and the morphologic changes in the characteristics of ADH, we tested the effects of 14-3-3{zeta} overexpression in MCF10A cells, a nontransformed MEC line which, when grown in three-dimensional culture, forms acini structures that simulate the acini structure of the mammary gland in vivo (28). We stably overexpressed 14-3-3{zeta} with an HA tag or an empty vector as control, in the MCF10A cell line, using a retroviral expression system to generate the 10A.14-3-3{zeta} and 10A.vec lines (Fig. 1B). When cultured under three-dimensional conditions, the 10A.vec cells developed into regular acini with hollow lumens (Fig. 1C, top) similar to previously reported MCF10A acini structure (33). In contrast, the 10A.14-3-3{zeta} cells developed into asymmetrical aggregates with filled lumens (Fig. 1C, bottom). Luminal filling is a feature of early-stage, benign breast epithelial lesions (7). Thus, 14-3-3{zeta} overexpression in MCF10A cells recapitulated the disrupted tissue architecture seen in early mammary lesions.

14-3-3{zeta} causes luminal filling by inhibiting anoikis. Luminal clearance in MCF10A cells occurs largely through anoikis (10). The 10A.vec cells underwent apoptotic luminal clearance 8 to 12 days after plating on BM as shown by caspase-3 activation in the luminal cells in the three-dimensional cultures (Fig. 2A, top ). However, caspase-3 was not activated in 10A.14-3-3{zeta} cells under the same culture conditions (Fig. 2A, bottom). We quantified the number of acini with activated caspase-3 in the luminal cells and found a dramatic loss of caspase-3 activation in 10A.14-3-3{zeta} cells in three-dimensional cultures (Fig. 2A, right). Similarly, there is a marked decrease in nonviable cells within the lumen of the 10A.14-3-3{zeta} acini, as assessed by an ethidium bromide exclusion assay (Fig. 2B). To examine whether hyperproliferation may also contribute to luminal filling, we compared growth between 10A.14-3-3{zeta} and 10A.vec cells. We found that 14-3-3{zeta} overexpression did not cause an increase in proliferation; therefore, luminal filling was not due to hyperproliferation (Supplementary Fig. S1). Because the cells within the center of the acini are no longer attached to the BM, we grew the cells in suspension cultures to determine whether 14-3-3{zeta} overexpression results in resistance to anoikis. 10A.vec cells grown in suspension for 72 hours were highly apoptotic (>50%), whereas 10A.14-3-3{zeta} cells were not (<10% apoptosis; Fig. 2C and Supplementary Fig. S2). Bax, a Bcl family proapoptotic regulator, is frequently up-regulated during anoikis (34). Indeed, Bax protein levels were also markedly lower in 10A.14-3-3{zeta} cells, growing in suspension cultures compared with 10A.vec cells cultured under the same conditions (Fig. 2D). Thus, when 14-3-3{zeta} was overexpressed, MCF10A cells became anoikis resistant, failed to up-regulate Bax, a proapoptotic protein, and lost the ability to form a hollow lumen.


Figure 2
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  		Figure 2. 14-3-3{zeta} overexpression induces luminal repopulation in three-dimensional cultures and reduces sensitivity to anoikis. A, 10A.vec and 10A.14-3-3{zeta} cell lines were cultured in three-dimensional conditions for 10 d and fixed. The cells were stained for activated caspase-3 (green) and counterstained with DAPI. Acini with or without activated caspase-3 were counted (>200 acini). Bar, 20 µm. B, cells were grown in three-dimensional culture for 10 d and then treated with a solution containing ethidium bromide. Percentage of total acini with ethidium bromide–positive cells within the lumen (indicating apoptosis) was determined. *, significant difference in EtBR+ cells between the cell lines (P < 0.03). C, cells were plated on polyHEMA-coated plates to prevent attachment and collected after 72 h. Annexin V–positive staining (indicating apoptosis) was determined by flow cytometry and assessed as percentage of the total population. D, cells were plated on PolyHEMA-coated plates and collected after 48 h. Bax, caspase-3, and β-actin levels were assessed by immunoblot.

 
14-3-3{zeta} overexpression disrupts apoptotic signaling by down-regulating p53. p53 is a transcriptional regulator of Bax and modulates anoikis (9, 35). Because 10A.14-3-3{zeta} acinar cultures displayed luminal filling and a failure to up-regulate Bax in response to BM detachment, we examined whether 14-3-3{zeta} may modulate p53 activity. Remarkably, 14-3-3{zeta} overexpression decreased p53 protein levels in three-dimensional cultures of MCF10A cells, as determined by both immunofluorescent and Western blot analyses (Fig. 3A and B ). Conversely, down-regulating 14-3-3{zeta} expression in the 10A.14-3-3{zeta} cells, via siRNAs, led to an increased p53 protein expression (Fig. 3C). These findings were confirmed in the MCF7 cells, a tumorigenic MEC line which contains wild-type p53 (Supplementary Fig. S3). We also examined p53 expression in 14-3-3{zeta} transgenic mice with a WAP gene promoter-driven HA-tagged 14-3-3{zeta} transgene (WAP-HA-14-3-3{zeta}), which is expressed at high levels during lactation.4 No p53 was detected in the mammary glands of lactating WAP-HA-14-3-3{zeta} transgenic mice, although it was present in the lactating mammary tissue of wild-type mice (Fig. 3D). Thus, 14-3-3{zeta} overexpression corresponded with a loss of p53 protein expression in vivo in the mouse mammary gland, as well as in vitro in human MECs. These results illustrate a novel finding, namely that 14-3-3{zeta} overexpression can result in a loss of p53 expression.


Figure 3
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  		Figure 3. 14-3-3{zeta} overexpression results in loss of p53 protein expression. A, after 8 d in three-dimensional culture, 10A.vec and 10A.14-3-3{zeta} cells were stained with p53 antibody (DO1) and counterstained with DAPI. The images were then analyzed as above. Bar, 20 µm. B, protein lysates from four pooled clones (10A.14-3-3{zeta}) were subject to immunoblot assay with 14-3-3{zeta} and p53 antibodies. C, 10A.vec and 10A.14-3-3{zeta} were transfected with siRNAs specific to 14-3-3{zeta} (Dharmacon) or a scrambled control at a concentration of 100 nmol/L. The cells were collected 72 h later and lysates were subjected to immunoblot assay for 14-3-3{zeta}, p53, and β-actin. D, protein extracts from lactating mouse mammary glands were analyzed for 14-3-3{zeta}, p53, and β-actin. Two wild-type mice and 3 WAP-14-3-3{zeta} transgenic mice were examined. 10A.vec and 10A.14-3-3{zeta} lysates were included as controls.

 
14-3-3{zeta} overexpression decreases p53 protein expression by increasing proteasomal degradation of p53. Next, we sought to determine how 14-3-3{zeta} overexpression causes p53 protein loss. We first examined whether reduced p53 protein results from reduced p53 mRNA expression. We found that 14-3-3{zeta} overexpression did not alter p53 mRNA levels suggesting that 14-3-3{zeta} did not regulate p53 at the RNA level but at the protein level (Fig. 4A ). It is well known that p53 protein levels are controlled by the proteasomal degradation pathway (36). We, therefore, investigated whether 14-3-3{zeta} overexpression may reduce p53 protein level by increasing p53 degradation. When proteosome-dependent protein degradation was inhibited with MG132, p53 levels in 10A.14-3-3{zeta} were similar to the levels in 10A.vec cells (Fig. 4B, top). Similarly when p53 was sequestered away from the proteosome via inhibition of its nuclear export by leptomycin, p53 levels in 10A.14-3-3{zeta} cells were not reduced but were similar to those in the 10A.vec cells (Fig. 4B, bottom). To confirm these findings, we tested p53 protein stability after cycloheximide treatment and found that the p53 was degraded faster in 10A.14-3-3{zeta} cells than in control cells (Supplementary Fig. S4). These data indicated that increased p53 proteasomal degradation was largely responsible for decreased p53 levels in 14-3-3{zeta}–overexpressing cells.


Figure 4
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  		Figure 4. 14-3-3{zeta} overexpression decreases p53 protein stability via the proteasome degradation pathway. A, RNA was collected from 10A.vec and 10A.14-3-3{zeta} cells and was reverse transcribed to cDNA. The cDNA was quantified using a FullVelocity SYBR green Q-PCR mix analyzing p53 and GAPDH as a control. The samples were amplified using a Stratagene Mx4000 Quantitative PCR instrument. The p53 levels were adjusted to the GAPDH control 10A.vec and 10A.14.3.3{zeta}, respectively. The fold difference is a ratio difference of 10A.vec (normalized to 1) to 10A.14.3.3{zeta}. B, 10A.vec and 10A.14-3-3{zeta} were treated with 10 µmol/L proteasome inhibitor (MG132) for 6 h. Lysates were collected and immunoblotted for p53. 10A.vec and 10A.14-3-3{zeta} cell lines were treated with 5 ng/mL of nuclear export inhibitor (Leptomycin) for 6 h, and lysates were collected and immunoblotted for p53 and actin. C, cytoplasmic extracts and nuclear extracts were collected from 10A.vec and 10A.14-3-3{zeta} cells. 50 µg of each lysate was immunoblotted for pSerines166/186 MDM2 (Cell Signaling), MDM2 (smp14, Santa Cruz), tubulin (cytoplasmic marker), and Lamin A/C (Nuclear marker). For the MDM2 immunoblot, the arrow indicates nonphosphorylated MDM2 and the arrowhead points to the phosphorylated MDM2. D, 10A.vec and 10A.14-3-3{zeta} cells were transfected with four individual siRNAs specific to MDM2 (Dharmacon) at a concentration of 100 nmol/L. Lysates were collected after 72 h and were immunoblotted (50 µg) for MDM2, p53, and β-actin.

 
MDM2 is the E3 ligase responsible for the ubiquitination and subsequent degradation of p53 (36). Phosphorylation of MDM2 facilitates its nuclear translocation. Increased nuclear translocation of MDM2 leads to increased ubiquitination and degradation of p53 (37). Therefore, we investigated whether MDM2 expression and phosphorylation is altered by 14-3-3{zeta} overexpression. We found higher levels of phosphorylated MDM2 in the 10A.14-3-3{zeta} cells compared with the control 10A.vec cells (Fig. 4C). Additionally, down-regulation of MDM2 with siRNA partially rescued p53 expression in the 10A.14-3-3{zeta} cells (Fig. 4D). Thus, 14-3-3{zeta} decreases p53 protein levels by increasing MDM2-dependent proteasomal degradation of p53.

14-3-3{zeta} overexpression increases MDM2-dependent proteasomal degradation of p53 via activation of PI3K-Akt. The serine/threonine kinase Akt phosphorylates MDM2 on S166 and S186 resulting in nuclear translocation of MDM2 and increased p53 ubiquitination and degradation (37). Our laboratory recently discovered that 14-3-3{zeta} overexpression activates Akt in the MCF7 and MDA-MB-435 breast cancer cell lines.3 To determine if Akt activation also contributes to MDM2-dependent degradation of p53, we first examined Akt phosphorylation (an indicator of Akt activity) in 10A.14-3-3{zeta} and 10A.vec cells. We found that 14-3-3{zeta} overexpression in 10A.14-3-3{zeta} cells led to increased Akt phosphorylation in both two-dimensional and three-dimensional cultures (Fig. 5A ). To determine whether increased Akt phosphorylation contributes to p53 destabilization in the 10A.14-3-3{zeta} cells, we blocked PI3K activation in 10A.14-3-3{zeta} cells using the chemical inhibitor, LY294002. PI3K is an upstream activator of Akt. Increasing concentrations of LY294002 resulted in a corresponding stepwise increase in p53 expression (Fig. 5B). Additionally, transfection of the 10A.14-3-3{zeta} cells with Akt siRNAs to knockdown expression of Akt1, Akt2, and Akt3 caused an increase in p53 expression (Fig. 5C). To ensure that hyperactivation of the Akt pathway, downstream of 14-3-3{zeta}, led to down-regulation of p53, we transiently transfected constitutively active Akt into MCF10A parental cells. Indeed, we detected decreased p53 protein as a result of hyperactivation of the Akt pathway (Supplementary Fig. S5). Therefore, activation of the PI3K/Akt pathway by 14-3-3{zeta} contributes to MDM2-mediated p53 protein degradation.


Figure 5
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  		Figure 5. 14-3-3{zeta} overexpression increases MDM2 E3 ligase activity by hyperactivating the PI3K-Akt pathway. A, 10A.vec and 10A.14-3-3{zeta} were fixed after culture for 10 d in three-dimensional conditions. The acini were stained with a pAkt473 antibody (red) and counterstained with DAPI. The images were processed as above. Bar, 20 µm. Lysates from 10A.vec and 10A.14-3-3{zeta} from both two-dimensional and three-dimensional cultures were collected and immunoblotted for 14-3-3{zeta}, total Akt, pAkt 473, and β-actin. B, 10A.vec and 10A.14-3-3{zeta} cells were treated with increasing concentrations of PI3K inhibitor (LY294002) for 2 h, and lysates were collected and immunoblotted for pAkt 473 and pAkt 308, p53, 14-3-3{zeta}, total Akt, and β-actin. C, 10A.vec and 10A.14-3-3{zeta} cells were transfected with siRNA specific to Akt1, Akt2, and Akt3 (35 nmol/L each). The lysates were collected after 72 h, and immunoblots for p53, 14-3-3{zeta}, total Akt, and β-actin were done.

 
Restoration of p53 protein expression rescues luminal clearance in three-dimensional cultures. Because 14-3-3{zeta} overexpression both induced luminal filling in the 10A.14-3-3{zeta} three-dimensional MEC model and decreased p53 protein levels, we reasoned that anoikis resistance and luminal filling were a direct result of 14-3-3{zeta} modulation of p53 protein levels. To test this, we restored p53 expression in the 10A.14-3-3{zeta} acini by infecting the developing acini with adenovirus expressing p53 (Fig. 6B ). In the acini with increased p53 expression, caspase-3 was activated, particularly in cells that were centrally located and therefore not attached to the BM (Fig. 6A, bottom). In contrast, caspase-3 was not activated in acini infected with LacZ adenovirus as control (Fig. 6A, top). These data show that restoration of p53 protein levels reestablished the balance between apoptotic and survival regulators in 10A.14-3-3{zeta} MECs and allowed for luminal clearance in three-dimensional cultures.


Figure 6
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  		Figure 6. Reconstitution of p53 in three-dimensional cultures results in luminal clearance. A, 10A.vec and 10A.14-3-3{zeta} cell lines were grown in three-dimensional culture conditions. Two days after plating, the cultures were infected with a p53 delivering adenovirus at a concentration of 250 viral particles per cell for 24 h. The adenovirus was removed and cells were grown for seven more days and then fixed. The acini were then stained with active caspase-3, p53 (DO1), and counterstained with DAPI. The cultures were then analyzed with a confocal microscope, and images were collected. B, 10A.vec and 10A.14-3-3{zeta} were infected with 250 viral particles per cell of either a LacZ or p53 adenovirus. The cells cultured for 3 d, and lysates were immunoblotted for p53 (DO1) and actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found a significant increase in the expression of 14-3-3{zeta} in ADH, an early lesion in the multistep process toward breast cancer. We determined that 14-3-3{zeta} overexpression led to luminal filling, a hallmark of early neoplastic transformation, by inhibiting anoikis in cells which were no longer attached to the basement membrane. Furthermore, we discovered that 14-3-3{zeta} down-regulation of p53 was a key mechanism underlying this luminal filling. To our knowledge, this is a previously unreported mechanism of 14-3-3 action. Although the morphologic changes that are hallmarks of preneoplastic breast lesions are well-characterized, the molecular mechanisms underlying these alterations are poorly understood. Our findings are important because they link a molecular change which occurs during the early stages of breast disease (14-3-3{zeta} overexpression and consequent p53 down-regulation) directly to a structural alteration (luminal filling).

Tissue remodeling and homeostasis are dependent on a balance between signaling pathways that control proliferation and apoptosis (1). It has been theorized that luminal filling can occur by hyperproliferation of the basal epithelium; however, studies in MEC three-dimensional cultures have shown that when the epithelium is hyperproliferative, an equal and opposite activation of apoptosis will keep the lumen open (38). Thus, another theory has been proposed: alterations in apoptotic pathways are more important than alterations in proliferative pathways for luminal repopulation (39). Our studies support the latter theory since 14-3-3{zeta} overexpression induces luminal filling in acini of MCF10A cells in three-dimensional cultures by inhibiting luminal cell apoptosis and anoikis.

p53 is activated in response to ECM detachment in a variety of cell types and a role for p53 in anoikis is becoming increasingly well established (9). p53 inactivation is necessary for {alpha}6β4 integrin to promote survival in human carcinoma cell lines (24). Interestingly, suppression of p53 function in early passage human MECs by introducing HPV-16 E6 increased sensitivity to ECM-induced apoptosis, whereas late passage human MECs with reduced p53 became resistant to this apoptosis (40). Our findings in the MCF10A immortalized human MECs are consistent with that of the late passage human MECs. Specifically, we have shown that 14-3-3{zeta} overexpression-mediated p53 loss confers resistance to anoikis leading to luminal filling in three-dimensional cultured MCF10A MECs. These findings suggest that p53 plays a significant role in lumen formation and maintenance by modulating apoptotic response.

The 14-3-3 family of proteins has been linked previously to p53 cellular responses. 14-3-3{sigma} binds directly to p53 and stabilizes p53 by restricting its interaction with MDM2 (41). The 14-3-3{sigma} promoter has a p53 response element and is up-regulated in response to DNA damage (42). Conversely, down-regulation of 14-3-3{zeta} in lung cancer cell lines increased their sensitivity to DNA damage by ionizing radiation (43). These data, together with our data in this report, suggest that 14-3-3{zeta} and 14-3-3{sigma} could have opposite roles in the cellular response to DNA damage. Further investigation is necessary to determine if 14-3-3{sigma} modulates anoikis and if the two isoforms functionally oppose each other in this process as well.

In this study, we discovered that 14-3-3{zeta} modulated the expression of the tumor suppressor p53 by decreasing p53 protein stability. This effect of 14-3-3{zeta} overexpression was PI3K/Akt-dependent and MDM2-dependent, strongly supporting a model in which 14-3-3{zeta} enhances the PI3K/Akt-dependent phosphorylation of MDM2 leading to destabilization of p53. Although 14-3-3{zeta} modulation of Akt activity clearly contributes to destabilization of p53, 14-3-3{zeta} might also play a direct role in increasing nuclear localization of MDM2 (Fig. 4C). 14-3-3 proteins frequently alter the localization of their binding partners, and the Akt phosphorylation sites at S166 and S186 in MDM2 are similar to known 14-3-3 binding motifs. This is an important area for future investigation.

Because our data showed a key role of 14-3-3{zeta} in the formation of early breast lesions, assessment of 14-3-3{zeta} expression levels could provide clinicians with valuable information. Advances in detection and screening have allowed more breast cancers to be diagnosed at early, preinvasive stages. However, the molecular alterations important for the formation and maintenance of these early lesions and the factors which predispose them for progression are largely unknown. Our findings indicate that 14-3-3{zeta} might be a useful molecular marker in assessing the prognosis of and appropriate treatment for newly detected early-stage breast lesions. This notion is supported by a recent study which identified 14-3-3{zeta} as one gene in a two-gene signature that predicted disease recurrence after tamoxifen treatment in women with estrogen receptor–positive breast cancers (44). Additionally, 14-3-3{zeta} is an attractive therapeutic target because it modulates cancer cell survival through interaction with and regulation of many proteins, including p53.


   Acknowledgments
 
Grant support: Rosalie B. Hite Fellowship MDACC (C.G. Danes), NIH grants P30-CA 16672 (MDACC), 1RO1-CA109570, 1RO1-CA119127, PO1-CA0099031 project 4, and P50 CA116199 project 4 (D. Yu).

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.

Note: Dr. Dihua Yu is the Nylene Eckles Distinguished Professor of Breast Cancer Research at MDACC.

We thank the University of Texas M. D. Anderson Cancer Center Support Grant Flow Cytometry and Cell Imaging core facility and Genetically Engineered Mouse core facility, Dr. Richard Giles, Dr. Joan Brugge, Dr. Mina Bissell, Dr. Walter Hittleman, Dr. Jonathan Hannay, Dr. Jeuhui Liu, Dr. Xiaoyan Zhou, and Dr. Timothy McDonnell for scientific advice, experimental assistance, and providing of reagents.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Current address for W. Yang: Shanghai Cancer Hospital, Fudan University, Shanghai, China.

3 Neal et al., unpublished data. Back

4 S.L. Wyszomierski, D.X. Fang, D. Yu, unpublished results. Back

Received 8/16/07. Revised 12/13/07. Accepted 1/17/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. L. Neal, J. Yao, W. Yang, X. Zhou, N. T. Nguyen, J. Lu, C. G. Danes, H. Guo, K.-H. Lan, J. Ensor, et al.
14-3-3{zeta} Overexpression Defines High Risk for Breast Cancer Recurrence and Promotes Cancer Cell Survival
Cancer Res., April 15, 2009; 69(8): 3425 - 3432.
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