Zip6-attenuation promotes epithelial-to-mesenchymal transition in ductal breast tumor (T47D) cells

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Research Article

Zip6-attenuation promotes epithelial-to-mesenchymal transition in ductal breast tumor (T47D) cells Veronica Lopez, Shannon L. Kelleher⁎ Department of Nutritional Sciences, The Pennsylvania State University, 222 Chandlee, University Park, PA 16802-6110, USA

ARTICLE INFORMATION

AB S TR AC T

Article Chronology:

Breast cancer is associated with zinc (Zn) hyper-accumulation in breast tissue which is postulated

Received 6 May 2009

to be potentiated by the over-expression of Zn importing proteins. Zip6 (LIV-1) over-expression

Revised version received

has been documented in estrogen receptor-positive (ER+) breast tumors. Anti-estrogens, such as

21 September 2009

fulvestrant, are typically prescribed for ER+ breast cancer and thus may play a role in modulating

Accepted 14 October 2009

cellular Zn hyper-accumulation. Herein, we investigated the physiological relevance of Zip6 over-

Available online 21 October 2009

expression and the consequences of Zip6-attenuation in breast tumor cells as a mechanism in the development of anti-estrogen resistance. We documented that over-expression of Zip6 was

Keywords:

associated with significantly higher cellular Zn levels in tumor cells compared with normal breast

Zinc

cells. Fulvestrant significantly reduced Zn accumulation in tumor cells, without robust effects on

Breast cancer

Zip6 protein abundance. Zip6-attenuation significantly reduced cellular Zn pools, which was

T47D

associated with increased mitochondrial membrane potential (ΔΨm) and decreased apoptotic

Zip6

stimuli (cytoplasmic cytochrome C release, caspase- 3 and -9 activities). Importantly, decreased

Apoptosis

apoptosis significantly increased tumor colony formation in soft agar and was associated with

Epithelial-to-mesenchymal transition

reduced E-cadherin expression. Our data suggest that anti-estrogen treatment regulates Zn level and importantly verify that Zip6 over-expression is not an underlying mechanism initiating breast cancer, but in fact may play a “tumor-constraining” role. © 2009 Elsevier Inc. All rights reserved.

Introduction Zinc (Zn) accumulation in breast tissue is one of the most consistent characteristics of breast cancer [1,2]. Recent epidemiological studies indicate that Zn accumulation in breast tissue is correlated with breast cancer development [3]. Moreover, human tumor biopsies [4,5] and N-methyl-N-nitrosourea-induced mammary tumors in rats [6] have been shown to have significantly elevated Zn levels when compared with normal breast tissue. Data such as these implicate Zn hyper-accumulation in the development and progression of breast cancer. The Zn importer Zip6 (LIV1) was first identified in a genetic screen of estrogen-responsive

factors in breast cancer tissue [7]. As a result, Zip6 expression has been suggested as a useful prognostic marker for estrogen receptor-positive (ER+) breast cancer [8,9]. Curiously, Zip6 protein levels have been demonstrated to correspond with better breast cancer prognosis in human patients [8], suggesting that Zip6 may perform a “protective” role. Clearly, the relationship between aberrant Zip6 expression and Zn hyper-accumulation on cellular function in breast tumors requires further investigation. Anti-estrogens are widely administered for the management of ER+ breast cancer. However, acquired resistance to anti-estrogen therapy is a real and unfortunate event as anti-estrogens such as fulvestrant has been shown to promote invasion of cancer cells

⁎ Corresponding author. Fax: +1 814 863 6103. E-mail address: [email protected] (S.L. Kelleher). Abbreviations: Zn, zinc; ER+, estrogen receptor-positive; EMT, epithelial-to-mesenchymal transition; T47D, human tumorigenic cells; HME, human mammary epithelial cells; CHO, Chinese hamster ovary cells; DTPA, diethylene triamine pentaacetic acid; DTT, dithiothreitol 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.10.011

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[10] and may precipitate metastasis. Recently fulvestrant has been shown to significantly reduce Zip6 mRNA expression in MCF-7 cells [11]. This suggests that fulvestrant may modulate Zn hyperaccumulation in malignant breast cells through effects on Zip6 expression. We speculate that fulvestrant may alter cellular Zn levels and represent a mechanism affecting cell phenotype through effects on cell growth, adhesion and apoptosis as studies have shown that aberrant cellular Zn levels are associated with malignancy. For example, Zip4 over-expression has been shown to increase Zn-dependent cell proliferation in pancreatic cancer [12] whereas diminished Zn level in prostate is a consistent feature of malignancy [13]. The maintenance of cell–cell interaction is one mechanism by which tumor invasiveness and malignancy may be constrained as Zn depletion diminishes cell–cell interactions via Ecadherin degradation in lung epithelium [14]. Interestingly, Zip6attenuation in non-tumorigenic MCF-7 breast cancer cells reduced E-cadherin expression and increased cell growth [15]. This finding specifically suggests that Zip6 may modulate a Zn pool responsible for regulating cell adhesion, and implies that Zip6 over-expression in breast cancer tissue may actually serve to keep adhesion and proliferative mechanisms constrained. Additionally, it has been well established that alterations in cellular Zn pools can modulate apoptosis. In prostate cancer cells, Zip1 over-expression [16] and thus Zn accumulation [17] inhibit cell growth, tumor invasiveness and growth [18] and increase cellular sensitivity to apoptotic stimuli through the induction of mitochondria-mediated apoptosis [19,20]. Thus, these observations support our postulate that alterations in cellular Zn pools mediate changes in malignant cell phenotype. In light of the importance of understanding the relationship between dysregulated Zn metabolism and breast cancer and the mechanisms responsible for negative outcomes associated with anti-estrogen therapy, we aimed to explore the relationship between Zip6 expression and tumorigenicity, in vitro. The objective of this study was to determine if Zip6 over-expression in ER+ malignant breast cells potentiates breast cancer progression or “protects” against tumorigenicity. Herein, we demonstrated that gene attenuation of Zip6 expression reduced cellular Zn pools but significantly increased the apoptotic threshold in T47D ductal breast tumor cells, in vitro. Importantly, our data indicated that Zip6attenuation increased tumor formation and diminished expression of E-cadherin reflecting the potential for epithelial-to-mesenchymal transition which would ultimately potentiate tumorigenicity.

Material and methods Cell culture Human tumorigenic (T47D) and normal human mammary epithelial (HME) cells were obtained from the American Type Culture Collection (ATCC). T47D cells were routinely cultured in plastic 75 cm2 flasks, maintained in “growth medium” containing RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with fetal bovine serum (10%), insulin (0.2 units/mL), sodium pyruvate (1.0 mM) and penicillin (100 IU/mL)–streptomycin (100 μg/mL). For fulvestrant studies, cells were plated in 6-well dishes until 60% confluent and then treated with fulvestrant for 7 days in phenolfree RPMI 1640 supplemented with fetal bovine serum (10%), insulin (0.2 units/mL), sodium pyruvate (1.0 mM) and penicillin (100 IU/mL)–streptomycin (100 μg/mL). HME cells were cultured

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in 75 cm2 flasks and grown in 171 medium (Invitrogen, Carlsbad, CA) supplemented with mammary epithelial growth supplement (Invitrogen). Cells were maintained in 5% CO2 at 37 °C in a humidified atmosphere.

Cellular zinc concentration Cells were plated in 15 cm2 polycarbonate dishes and cultured in growth medium until 90–100% confluent. For cells treated with fulvestrant (Sigma-Aldrich), cells were rinsed twice with PBS, then treated with fulvestrant (10 μM) in serum-free Opti-MEM for 24 h. Cells were initially rinsed with 1× PBS, then rinsed with 1× PBS plus EDTA (1 mM) to remove any loosely bound Zn. Samples were collected by scraping with PBS, centrifuged at 2000 × g for 10 min at 4 °C and resuspended in 0.5 mL PBS. Aliquots were removed for the Bradford assay, then samples were centrifuged at 2000 × g for 10 min, resuspended in 0.5 mL Ultrex II Nitric Acid (J T Baker) in mineral-free polypropylene vials and digested overnight at room temperature. Mineral concentration was analyzed using a Tracescan ICP-OES (Thermo Elemental). The standard curve was generated using serial dilutions of QC 21 Spex Instrument Standards (Spex) and NIST 1577b and QC 21 (0.5 ppm dilution) as controls analyzed in triplicate. Cellular protein concentration was determined by Bradford assay and measurements were normalized to total protein concentration.

Immunoblotting Cells were washed in PBS, scraped into lysis buffer containing protease inhibitors as previously described [21] and sonicated for 20 s on ice. Cellular debris and nuclei were pelleted by centrifugation at 500 × g for 5 min. To isolate crude membrane fraction, the post-nuclear supernatant was centrifuged at 100,000 × g for 20 min at 4 °C and protein concentration of the crude membrane was determined by the Bradford assay. Protein (25–100 μg) was diluted in Laemmli sample buffer containing DTT (100 mM) and incubated at 95 °C for 5 min. Proteins were separated by 10% SDS-PAGE (200 V), transferred to nitrocellulose for 60 min at 100 V then immunoblotted with affinity-purified rabbit anti-Zip6 antibody (1:1000, a gift from Dr. Liping Huang, Western Human Nutrition Research Center, Davis, CA) and detected with horseradish peroxidase-conjugated IgG. Membranes were stripped in Restore Western Blot Stripping Buffer (Thermo Fisher) for 15–30 min at room temperature and then reprobed with mouse β-actin (1:5000; Sigma). For EMT studies, membranes were incubated with anti-E-cadherin (1:2000; Abcam, Cambridge, MA), then reprobed with anti-vimentin (2 μg/mL; Abcam) and mouse β-actin. Proteins were visualized by chemiluminescence after exposure to autoradiography film, and relative band density and molecular mass relative to standard molecular mass markers (Amersham Pharmacia) were assessed using the Chemi-doc Gel Quantification System (BioRad).

Real-time semi-quantitative RT-PCR Total RNA was isolated using TriZOL (Invitrogen, per manufacturer's instruction and diluted to 1 μg/μL in RNase-free water) as previously described [21]. cDNA was generated from 1 μg RNA by reverse transcription (Applied Biosystems, Foster City, CA) performed at 48 °C for 30 min followed by 95 °C for 5 min. Real-

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time PCR was performed using the cDNA reaction mixture (1.0 μL) using the DNA Engine Opticon 2 System real-time thermocycler (BioRad, Hercules, CA) coupled with SYBR Green technology (BioRad) using gene-specific primers to either human Zip6 (forward: 5′-AGAGCCCTCCCACTTTGATT-3′, reverse: 5′-GCCGAGTGTATCGTGGAAAT-3′), human E-cadherin (forward: 5′-TGCCCAGAAAATGAAAAAGG-3′, reverse: 5′-GGATGACACAGCGTGAGAGA3′), human vimentin (forward: 5′-CCCTCACCTGTGAAGTGGAT-3′, reverse: 5′-TCCAGCAGCTTCCTGTAGGT-3′) or human β-actin (forward: 5′-AAGATGACCCAGGTGAGTGG-3′, reverse: 5′-CCTGCAGAGTTCCAAAGGAG-3′). Primers were designed by Primer 3 Input v4.0 (frodo.wi.mit.edu.) The PCR cycling parameters were as follows: 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The linearity of the dissociation curve was analyzed by Opticon 2 System software and the mean cycle time of the linear part of the curve was designated Ct. Each sample was analyzed in duplicate and normalized to β-actin using the following equation: ΔCtgene = Ctgene − Ctβ-actin. The fold change in expression was calculated using the following equation: 2(ΔΔCt) where ΔΔCt = mean ΔCt of control − mean ΔCt of Zip6-specific knockdown. Values represent mean fold change± SD, relative to mocktransfected controls (set to 100%).

siRNA-mediated gene attenuation T47D cells were transfected using Amaxa nucleofactor solution (Gaithersburg, MD) following the manufacturer's instructions. Cells (5 × 106/0.100 mL) were transfected without or with 200 pmol of SLC39A6-specific (sense: 5′-GGGUUCAGAAAAUUACUUC-3′, antisense: 5′-GAAGUAAUUUUCUGAACCC-3′) siRNA (Ambion, Applied Biosystems, Austin, TX) in DMEM containing FBS and antibiotic-free medium for 48 h.

Plasmid generation, transient transfection and luciferase reporter assays The 4×-MRE (metal-responsive element) pGL3-luciferase reporter was kindly provided by Dr. Colin Duckett (University of Michigan Medical School, Ann Arbor, MI). Large scale plasmid purification was carried out using the Plasmid Midi Kit (Sigma). For luciferase reporter assay, T47D cells were plated into 24-well plates and transfected using the Amaxa nucleofactor kit. Each well was transfected with either pGL3 empty vector (0.8 μg) or 4×-MREpGL3 luciferase reporter (0.8 μg) and pRL-TK renilla vector (0.05 μg) without or with Zip6 siRNA (200 pmol). After 48 h, cells were treated with ZnSO4 (7 μM) for 24 h to activate the promoter and then analyzed for luciferase activity as previously described [22]. After incubation, cells were rinsed with 1× PBS and harvested in 1× passive lysis buffer (Promega, Madison, WI) following the manufacturer's instructions and measured by luminometry (Turner Biosystems, Sunnyvale CA) using DualLuciferase reporter assay system (Promega) for firefly and renilla luciferase activity. Relative light units (RLU) were determined by the ratio of firefly:renilla luciferase activity.

FluoZin-3 and RhodZin-3 fluorometry assays To verify that Zip6 facilitates the accumulation of Zn into vesicles containing labile Zn, cells were transiently transfected with Zip6 specific siRNA for 48 h prior to experiments. Cells were rinsed with

1× PBS, pH 7.4, and loaded with either FluoZin-3 AM (2 μM, Invitrogen) or RhodZin-3 AM (1 μM, Invitrogen) for 1 h as recommended by the manufacturer in Opti-MEM containing 0.2% pluoronic acid 127. Cells were rinsed two times with PBS, pH 7.4, incubated for 30 min at 25 °C with constant shaking then treated with ZnSO4 (10 μM) for 30 min at 37 °C. Fluorescence of FluoZin-3 (emission 485 nm/excitation 520 nm) and RhodZin-3 (emission 560 nm/excitation 595 nm) was measured at 25 °C using FLUOstar OPTIMA plate reader (BMG Labtech) spectrofluorimeter with FLUOstar OPTIMA software version 1.32R2. Cellular protein concentration was determined by the Bradford assay and fluorescence measurements were normalized to total protein concentration.

Cytosolic and mitochondria-enriched fractions Cells were transfected with Zip6 siRNA as described above for 48 h. Transfected cells were rinsed with 1× PBS, collected in lysis buffer containing protease inhibitors [21]. Samples were prepared as previously described [23]. Briefly, samples were homogenized with a glass dounce homogenizer (10–15 strokes) and centrifuged at 15,000 × g for 15 min at 4 °C. The resulting pellet (mitochondria-enriched fraction) was resuspended lysis buffer, while the supernate (cytoplasmic fraction) was re-centrifuged at 20,000 × g for 15 min at 4 °C. Total protein was diluted in equal volumes of Laemmli sample buffer containing DTT (100 mM) and separated by 10% SDS-PAGE. Cytochrome C (0.5 μg/mL; BD Biosciences, San Jose, CA) was detected by immunoblotting.

Mitochondrial membrane potential (ΔΨ) To determine if Zip6-attentuation affects mitochondrial membrane potential, cells were transfected with Zip6-specific siRNA as described above or treated with ZnSO4 (75 μM) for 4 h, as a positive control. Mitochondrial membrane potential was analyzed using JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide). JC-1 is a cationic carbocyanine dye that accumulates in mitochondria. JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence in healthy cells, whereas in apoptotic or unhealthy cells JC-1 remains in the monomeric form, which elicits only green fluorescence indicating low ΔΨ. Cells were stained as previously described [24]. Briefly, cells were stained with JC-1 dye (2 μM) for 30 min at 37 °C, rinsed with 1× PBS and intensity of fluorescence was immediately measured at the corresponding wavelength for the aggregate (red; emission 560 nm/excitation 595 nm) and monomer (green; emission 485 nm/excitation 520 nm) as described above. De-depolarization was calculated as the ratio of aggregate:monomer (red:green). Fluorescence measurements were normalized to total protein concentration.

Fluorimetric caspase activity To determine the effect of Zip6 gene attenuation on apoptosis, cells were transfected with Zip6-specific siRNA as described above or treated with ZnSO4 (75 μM) for 24 h a control. Caspase activity was assessed using SensoLyte AFC Caspase Profiling Kit (AnaSpec, San Jose, CA). Samples were prepared as recommended by the manufacturer. Briefly, cells were scraped with 1× lysis buffer and incubated for 30 min at 4 °C with constant rotation. Samples were

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subsequently centrifuged at 2500 × g for 10 min at 4 °C. Supernate was loading onto a pre-coated plate according to the manufacturer's recommendation. Quantitation of activity was achieved by measuring fluorimeter (excitation 405 nm; emission 520 nm), as described above. Fluorescence measurements were normalized to total protein concentration.

Soft agar colony formation To verify that Zip6 plays a role in tumor formation, cells were transiently transfected with Zip6 siRNA as described above and plated on soft agar. The agar base was prepared by melting agar (1%) in water at 40 °C which was mixed with an equal volume of RPMI containing 20% FBS then allowed to solidify for 15 min at room temperature. The top agarose was prepared by melting agarose (0.7%) in water which was mixed with an equal volume of RPMI containing 20% FBS then allowed to cool. Cells were added and gently poured onto the culture dish. T47D cells were incubated for 72 h at 37 °C. Colonies (>3 cells) and individual cells were counted using a Zeiss Invertoskop 40C microscope (Thermo Fisher Scientific).

Statistical analysis Results are presented as mean ± standard deviation from triplicate samples from three independent experiments. Statistical comparisons were performed using Student's t-test (Prism Graph Pad, Berkeley, CA) and a significant difference was demonstrated at p < 0.05.

Results Endogenous expression of Zip6 is correlated with cellular Zn levels and fulvestrant reduces cellular Zn levels in T47D cells Previous studies have shown elevated Zip6 expression and Zn concentration in human breast tumor tissue. Herein, we demonstrated that T47D cells had significantly higher (p < 0.05) cellular Zn content than normal HME cells (Fig. 1A). Additionally, immunoblotting indicated that Zip6 protein abundance was significantly higher in T47D cells compared with HME cells (Fig. 1B), validating the human ductal tumor cell line T47D as an appropriate model for breast cancer associated Zn hyper-accumulation. Given the observation that fulvestrant reduced Zip6 mRNA expression in MCF-7 breast cancer cells [11], we examined if fulvestrant diminished Zn levels in T47D cells. Our data illustrated that cells treated with fulvestrant had significantly lower (1.3fold) cellular Zn level compared with non-treated cells (Fig. 2A). This effect was not associated with decreased Zip6 abundance (Fig. 2B). Together, our data demonstrated that Zip6 over-expression is directly associated with high cellular Zn levels and that fulvestrant can reduce intracellular Zn levels through Zip6-independent mechanisms in breast tumor cells.

Zip6-attenuation affects distinct cellular Zn pools in T47D cells Based on observations by Kasper et al. [8] and our data, we next aimed to determine if (1) Zip6 played a significant role in Zn uptake and (2) the elimination of Zip6 expression would result in

Fig. 1 – Zip6 over-expression correlates with cellular Zn levels in human breast cells. (A) T47D cells had significantly higher total cellular Zn content compared with HME cells. Data represent mean ± SD (n = 8–11 samples/group), ⁎p < 0.005. (B) Representative immunoblot of Zip6 in total membrane proteins (50 μg) isolated from T47D and HME cells. The protein abundance of Zip6 was ∼10-fold higher in T47D cells (n = 2 independent samples) compared with HME cells (n = 2 independent samples). Equal loading was verified by immunoblotting with β-actin antibody.

deleterious effects in T47D cells. We attenuated Zip6 expression and determined if Zip6-attenuation affected changes in cytoplasmic, vesicular and mitochondrial Zn pools. Cytoplasmic Zn pools were assessed with a luciferase reporter assay which is a sensitive method of quantifying subtle changes in cytoplasmic Zn pools. Cells were co-transfected with a luciferase reporter construct containing a 4×-metal-response element (4×-MRE) in the absence or presence of Zip6-specific siRNA. This novel transcription-based Zn biosensor is based on the endogenous capacity of cells to activate metal-responsive transcription factor-1 (MTF-1) and bind to metal-responsive elements (MRE) in the promoter region of specific genes. Preliminary studies indicated no difference in luciferase activity between mock-transfected (8.7 ± 1.4 relative light units) and Zip6-attenuated (8.0 ± 0.35 relative light units) cells under basal culture conditions. Thus, we reasoned that preactivation of MTF-1 and subsequent activation of luminescence is required to detect a decrease in luciferase activity in response to Zip6-attenuation. Based on this reasoning, we pre-incubated cells with Zn (7 μM) for 24 h to activate luciferase activity. Following transfection with Zip6-specific siRNA (Fig. 3), we observed a significant decrease in luciferase activity in Zip6-attentuated cells compared with mock-transfected cells (Fig. 4A). These data clearly indicated that Zip6 had a significant effect on Zn uptake and cytoplasmic Zn levels in T47D cells. Within the cell, almost all non-protein bound (labile) Zn is localized within vesicles or organelles, such as mitochondria. Vesicular and mitochondrial Zn pools were assessed with FluoZin3 and RhodZin-3, respectively. FluoZin-3 and Rhodzin-3 are Znspecific fluorophores that upon binding to labile Zn trap the Zn

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a reduction in the amount of cytochrome C released from the mitochondria compared with mock-transfected cells (Fig. 5A). Valinomycin was used as a positive control as it has been shown to induce apoptosis by inducing mitochondria permeability. Our next objective was to determine if the reduction in cytochrome C release observed with Zip6-attenuation was a consequence of decreased mitochondrial membrane permeability, since a reduction in mitochondria membrane potential (ΔΨ) is an early hallmark of apoptosis. Loss of ΔΨm was observed in T47D cells treated with Zn as demonstrated by others [24]. Consistent with our effects on cytochrome C oxidase release, we noted a significant increase in ΔΨm in Zip6-attentuated cells compared with mocktransfected cells (Fig. 5B). These data indicated that mitochondrial membrane permeability was decreased by Zip6-attenuation in T47D cells consistent with reduced cytochrome C release. Next, we

Fig. 2 – Cellular Zn levels are responsive to anti-estrogens. (A) Fulvestrant significantly decreased total cellular Zn content compared with non-treated control cells. Data represent mean ± SD (n = 4 samples/group), ⁎p < 0.005. (B) Representative immunoblot of Zip6 in total membrane proteins (25 μg) isolated from T47D cells treated with fulvestrant (+fulv) compared with untreated cells (−fulv). Equal loading was verified by immunoblotting with β-actin antibody. Fulvestrant treatment minimally increased Zip6 protein abundance. Data represent mean Zip6 abundance as a ratio of Zip6:β-actin ± SD (n = 3) for two independent experiments, ⁎p < 0.05.

within these compartments and fluoresce, permitting quantification of changes in labile Zn pools in vesicles and mitochondria, respectively [25,26]. In fact, FluoZin-3 and RhodZin-3 fluorescence was significantly lower in Zip6-attenuated cells compared with mock-transfected cells (Figs. 4B and C), suggesting that vesicular and mitochondrial Zn pools were significantly reduced by Zip6attenuation. These data indicated that Zip6-attenuation depleted total cellular Zn pools.

Zip6-attenuation inhibits apoptosis in T47D cells Once we determined that Zip6 modulated total cellular Zn pools, we explored the functional consequences of reduced Zn levels on tumor cells. Given the importance of Zn in the regulation of apoptosis and our ability to reduce mitochondrial Zn pools by Zip6-attenuation, we explored whether Zip6-mediated Zn import played a role in mitochondria-mediated cytochrome C release, the initial effector of the mitochondria-mediated apoptotic cascade. Intriguingly, our results indicated that Zip6-attenuation resulted in

Fig. 3 – Verification of Zip6-attentuation in T47D cells. Cells were transfected with Zip6-specific siRNA in antibiotic-free medium. Samples were analyzed for mRNA and protein abundance by real-time PCR and immunoblotting, respectively. (A) Transfection with Zip6-specific siRNA decreased Zip6 mRNA abundance ∼80% compared with mock-transfected cells. Data represent mean ± SD (n = 3) from two independent experiments, ⁎p < 0.05. (B) Representative immunoblots of Zip6 in total membrane proteins (25 μg) isolated from T47D cells from non-transfected (mock) and cells transfected with Zip6-specific RNA (Zip6 KO). Equal loading was verified by immunoblotting with β-actin antibody. Data represent mean Zip6 abundance as a ratio of Zip6:β-actin ± SD (n = 3) for two independent experiments, ⁎p < 0.05.

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Fig. 4 – Zip6-attenuation reduced intracellular Zn pools in T47D cells. (A) Cytoplasmic Zn level was measured using a 4×-metal-response element (MRE) luciferase reporter construct in cells treated with Zip6 siRNA (Zip6 KO) and compared with mock-transfected cells (Mock). Zip6 gene attenuation significantly reduced luminescence suggesting that cytoplasmic Zn pools were lower compared with mock-transfected cells. Data represent mean fluorescence (arbitrary units/μg protein) (n = 3) from two independent experiments, ⁎p < 0.05. Intracellular Zn pools were assessed using (B) FluoZin-3 and (C) Rhodzin-3 fluorescence in Zip6-attentuated cells (Zip6 KO) and compared with mock-transfected cells (Mock). Zip6 gene attenuation significantly reduced vesicular (B) and mitochondrial (C) Zn accumulation compared to mock-transfected cells. Data represent mean % fluorescence relative to mock-transfected control cells ± SD (n = 6–8 samples/group), ⁎p < 0.05.

determined if Zip6-attenuation reduced cleaved-caspase activity downstream of cytochrome C release, directly implicating alterations in Zip6-mediated Zn pools in the regulation of apoptogenic mechanisms. Using an ELISA-based method to measure caspase

Fig. 5 – Zip6-attenuation inhibited apoptotic pathways in T47D cells. Cytoplasmic release of cytochrome C was measured by immunoblotting in cells transfected with Zip6 siRNA (Zip6 KO) and compared to mock-transfected cells (Mock). (A) Representative immunoblot of cytochrome C in cytoplasmic (C. cytochrome C) and mitochondria (M. cytochrome C) fractions in Zip6-expressing (Mock) and Zip6-attenuated (Zip6 KO) T47D cells. Zip6 gene suppression was associated with a reduction in cytochrome C release compared with mock-transfected controls, while mitochondria cytochrome C was increased in Zip6 KO compared with mock-transfected cells. To validate our assay of mitochondria release of cytoplasmic cytochrome C in T47D cells, cells were treated with valinomycin (+Val). (B) Mitochondrial membrane potential in T47D cells transfected with Zip6 siRNA (Zip6 KO) was significantly higher than in Zip6expressing cells. To validate our assay, Zn (75 μM) was used as a positive control as it has been shown to reduce mitochondria membrane potential. Data represent mean fluorescence ratio (red:green) ± SD (n = 4–5 samples/group), ⁎p < 0.05.

activity, we found that Zip6-attenuation significantly reduced cleaved caspase-9 and -3 activities compared with mock-transfected cells (Table 1). To verify that the diminution in caspase activity was specific to the loss of cellular Zn we treated nontransfected cells with Zn. We found that Zn treatment stimulated caspase activity in T47D cells (Table 1) consistent with published data [20]. Collectively, these data suggest that downstream mediators of mitochondria-mediated apoptogenesis were further impaired when cellular Zn pools are reduced.

Table 1 – Effects of Zip6-attenuation on caspase activity. Activity/μg protein

Caspase-3 Caspase-9

Mock

Zip6 KO

+Zn

0.587 ± 0.01 0.178 ± 0.01

0.448 ± 0.02* 0.142 ± 0.02*

0.683 ± 0.02** 0.301 ± 0.02**

*p < 0.01, t-test analysis of Zip6 suppression (Zip6 KO) versus mock. **p < 0.01, t-test analysis of +Zn versus mock.

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Loss of Zip6 expression is physiologically associated with enhanced tumorigenicity and epithelial-to-mesenchymal transition in T47D cells An important consideration as it relates to the diminution of apoptosis via Zip6-attenuation in breast cancer cells is the functional consequence. We reasoned that if apoptotic regulators were reduced and cells experienced an “anti-apoptotic-state,” cell number would be synergistically higher [15] and tumorigenicity enhanced. To explore the consequences of Zip6-attenuation on tumor cell number and tumorigenesis, we assessed effects of Zip6attenuation on tumor cell number and colony formation by quantifying and characterizing tumor colonies in soft agar. We arbitrarily characterized a “tumor” as an aggregate of >3 cells and counted the number of “tumors” and the total number of tumor cells in 6 different microscopic fields (10× magnification) from three different wells in three independent experiments. We found that Zip6-attenuation was associated with increased tumorigenesis compared with mock-transfected cells (Table 2). Interestingly, Zip6-attenuation also resulted in greater tumor cell number compared with mock-transfected cells; however, these differences did not reach statistical significance (Table 2). A devastating consequence of increased cell number and tumor formation may be metastasis. To determine whether the increase in tumor formation associated with reduced cytoplasmic Zn via Zip6-attenuation was related to specific adhesion molecules which are hallmarks of metastasis (epithelial-to-mesenchymal transition, EMT) we quantified changes in the expression of two major EMT markers, E-cadherin and vimentin. Hallmarks of EMT are the loss of E-cadherin expression (epithelial marker) coupled with the gain of vimentin expression (mesenchymal marker), resulting in increased motility and invasivity potentially contributing to a malignant-metastatic phenotype. We observed a significant reduction in E-cadherin mRNA (− 25%, p < 0.05) (Fig. 6A) and protein expression (Fig. 6B) in Zip6-attenuated cells compared with mock-transfected cells. Additionally, we observed a significant increase in vimentin mRNA expression (+34%, p < 0.05) (Fig. 5A). We were unable to detect a corresponding increase in vimentin protein expression in Zip6-attenuated cells compared with mock-transfected cells. We speculate that the increase in mRNA was not robust enough to affect vimentin protein expression in these cells [27]. Collectively, our data support previous reports on the negative correlation between Zip6 expression and tumor size, grade and stage [8]. Moreover our data illustrate the ability of Zip6 over-expression to reduce the apoptotic threshold and minimize tumor formation in T47D cells potentially protecting breast tumor cells from metastatic transformation.

Table 2 – Effects of Zip6-attenutation on T47D cell number and colony formation.

Mean cell number a Mean number of clusters b a

Mock (n = 6)

Zip6 KO (n = 6)

1206 ± 290.1 23 ± 1.0

1619 ± 428.5 35.3 ± 4.1 c

Data represent number of cells/10× magnification field. Data represent mean number of tumors/10× magnification field (tumors represent clusters of > 3 cells/field). c p < 0.001. b

Fig. 6 – The reduction in Zip6 expression modulated expression of EMT markers. Real-time PCR and immunoblotting were used to examine the effects of Zip6-attenuation on E-cadherin and vimentin expression. (A) Zip6 gene suppression resulted in a significant reduction in E-cadherin and an increase in vimentin mRNA expression compared to mock-treated cells. Data represent the mean mRNA expression in Zip6-attenatuated cells relative to Zip6-expressing cells (Mock, set at 100%, n =3), for two independent experiments, ⁎ p<0.05. (B) Representative immunoblots of E-cadherin in total membrane protein (25 μg) from Zip6-attenatuated (Zip6 KO) and Zip6-expressing (Mock) cells. Zip6-attentation was associated with a significant reduction in E-cadherin protein abundance; however, vimentin protein was not detected (data not shown). Equal loading was verified by immunoblotting for β-actin. Data represent mean abundance as a ratio of β-actin ± SD (n = 3) for two independent experiments, ⁎p < 0.05.

Discussion There is compelling evidence implicating Zn hyper-accumulation in breast tissue in the pathophysiology of breast cancer [1,2,28]. In this study we demonstrated that similar to reports in human breast tumor biopsies [3–5], Zn levels are higher in tumorigenic T47D cells

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than in normal breast cells. The over-expression of the Zn importer Zip6 in malignant breast tissue implicates Zip6 as a contributing factor to Zn hyper-accumulation, particularly in ER+ breast cancer. Consistent with observations in human tumor biopsies [8] Zip6 was clearly over-expressed in T47D cells validating these tumor cells as a useful model for exploring the functional consequences of dysregulated Zip6 expression in breast cancer. Zip6 expression is estrogen-responsive [7]; however, our data suggest that factors other than estrogen participate in its over-expression. Unlike observations by Taylor et al. [11] in MCF-7 cells our data illustrated that Zip6 expression was not robustly affected by the anti-estrogen fulvestrant. We speculate that the dissimilarity in these observations may reflect cell-specific regulation of Zn transporters. Alternatively, effects of fulvestrant on Zip6 mRNA expression [11] may not result in parallel changes in Zip6 protein abundance. Importantly our data suggest that the deleterious effects of antiestrogens do not appear to be facilitated through direct effects on Zip6 abundance. In addition to estrogen, our observation that Zip6 is over-expressed in T47D cells may also suggest that the “accumulation” of Zip6 reflects alterations in Zip6 expression in response to changes in cellular Zn pools. In neuronal cells Zn accumulation induced degradation of Zip6 [29,30]; therefore the “over-expression” of Zip6 in tumor cells may be in response to an unknown tumor-related stimuli inhibiting degradation or low cytoplasmic Zn levels due to tightly sequestered intracellular Zn pools. Consistent with a role in Zn uptake, but in contrast to a previously published report utilizing ER− and invasive breast cells (MDA-MB-231) [31] we found that attenuation of Zip6 expression significantly decreased Zn uptake in ER+, non-invasive tumor cells. This may reflect the differential Zip6 localization or dysregulation of Zn transporting mechanisms in malignant breast cell types. While we demonstrated that cellular Zn pools can be modulated by Zip6 we cannot negate the contribution of other Zip proteins in this process. Importantly, we verified that Zip6-attenuation was able to significantly reduce several cellular Zn pools. There is mounting evidence illustrating the importance of tight regulation of cellular Zn pools in cell function although we still lack specific information directly linking dysregulated Zn metabolism with aberrant cellular function and breast cancer etiology. With that said, the notion that Zn transporters can play distinct and unique physiological roles is only now being appreciated. Recently, our group has shown that reduced Zn uptake via Zip3 attenuation in normal mammary cells resulted in increased mammary cell apoptosis in vitro [32] and in vivo [33]. In contrast to these observations, our data indicated that Zip6-attenuation resulted in decreased apoptosis. This dichotomy may reflect malignant versus non-malignant regulation and/or that specific intracellular Zn pools modulate discrete mechanisms. Towards that end, Taylor et al. demonstrated that Zip7 overexpression stimulated cell signaling resulting in increased invasivity [34]. This is very interesting because Zip7 has been exclusively localized to the Golgi apparatus [35] and the Golgi functions as a regulatory axis for various signaling cascades [36]. This is again consistent with the concept that specific intracellular Zn pools regulate discrete cellular processes. These data and our recent findings suggest mechanisms underlying compartment-specific Zn-transport related events and support the notion that Zn transporting mechanisms are not redundant and may in fact play very specific physiological roles. A relationship between cellular Zn metabolism and apoptosis has long been appreciated. Mitochondria-mediated apoptosis

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(intrinsic apoptosis) is initiated by the loss of mitochondrial membrane potential with the release of cytochrome C into the cytosol thus activating caspase-9 and subsequently caspase-3 activity and triggering DNA fragmentation as the final step in the apoptotic cascade. Mitochondria-mediated apoptosis has been shown to be directly affected by Zn, whereby Zn accumulation results in the release of cytochrome C release initiating caspase activity [20]. Mechanistically, it has been shown that Zndependent mitochondria-mediated apoptosis is regulated by the expression and mitochondrial translocation of the pro-apoptotic Bcl-2 family member Bax [37]. Herein, our data illustrated increased mitochondrial membrane potential associated with Zip6-attentuation consistent with a Zn-limiting phenotype, although Zip6 was not localized to the mitochondria (data not shown). Consistent with this observation was the constitutive release of cytoplasmic cytochrome C from mitochondria and caspase-9 and -3 activation in T47D cells, all of which were reduced in Zip6-attenuated cells. Caspases are cysteine proteases which reside as latent precursors in cells [38]. Some studies suggest that Zn inactivates caspase activity by binding to the catalytic site [38], while others suggest Zn directly inhibits caspases-3 and -9 activation, independent of cytochrome C release [39]. It is unclear as to the precise mechanism(s) through which Zn depletion inhibits caspase activity in these cells although our data clearly document that Zip6 over-expression in breast tumor cells modulates a Zn pool which lowers apoptosis sensitivity and illustrates that subtle fluctuations in the cellular Zn microenvironment clearly play a role in regulating cellular function. Thus far, our data indicate that while Zip6 over-expression is associated with Zn hyper-accumulation, this does not suppress apoptogenic mechanisms and thus may not be an initial event in breast cancer transformation. In fact “normalizing” Zip6 expression appears to further halt apoptogenesis in breast tumor cells. A key finding from this study provides compelling evidence that a consequence of Zip6-attenuation and decreased apoptosis in tumor cells is a greater number of cells and significantly greater tumor formation, suggesting that Zip6 over-expression in tumor cells (or more specifically, Zn pools modulated by Zip6) may actually function to “constrain” tumor growth. Importantly, these data provide direct mechanistic information behind the curious observation that Zip6 expression is negatively correlated with tumor size, grade and stage. To identify mechanisms that may play a role in this process, we have shown that consistent with other reports in non-tumor MCF-7 breast cancer cells [15], Zip6attenuation diminished E-cadherin mRNA expression in tumorigenic T47D breast cancer cells, suggesting that Zip6 mediated Zn pools modulate E-cadherin expression [14]. Importantly, we further detected increased expression of vimentin mRNA, a mesenchymal-cell marker indicating a strong potential for cell transition. Vimentin plays a role in cell adhesion by regulating integrin function and gene ablation has been shown to decrease cell adhesion in highly invasive breast cancer cells (MDA-MB-231) [40]. Additionally, over-expression of vimentin is associated with enhanced cellular migration [41]. The significance of our findings is that concomitantly decreased E-cadherin and increased vimentin mRNA is a molecular signature for epithelial-to-mesenchymal transition and suggests that loss of Zip6 expression may be associated with movement of malignant cells from the primary tumor, or metastasis. We speculate that the mechanism(s) by which Zip6 modulates tumorigenicity and potentially metastasis

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may be in part via Zn regulation of NF-κB-activated genes in tumor cells [42,43] and current studies aim to explore this hypothesis. In conclusion, the importance of our exploration of Zip6 function in breast tumor cells reflects the fact that anti-estrogen treatment by therapeutic agents such as tamoxifen and fulvestrant has been associated with acquired hormone therapy resistance [10] and adverse outcomes including enhanced motility and invasive phenotype [10,44,45]. In our study, fulvestrant did not robustly affect Zip6 protein expression in T47D cells. Nevertheless, we have identified distinct physiological functions associated with Zip6 expression, which may shed light to previous observations that Zip6 expression is associated with positive breast cancer prognosis [8]. An adverse outcome of inhibiting Zip6-mediated Zn pools in breast tumor cells is that apoptogenesis is further impaired and cell-to-cell interactions are diminished. Taken together, our results suggest that reducing Zip6 expression may be associated with adverse outcomes such as metastatic transition in vivo and current studies are underway to explore this possibility.

[9]

[10]

[11]

[12]

[13]

[14]

Acknowledgments We thank Dr. Liping Huang (Western Human Nutrition Research Center) for the generous gift of anti-Zip6 antibody, Dr. Colin Duckett (University of Michigan Medical School) for the 4×-MREluciferase plasmid, Farnaz Foolad for excellent technical support and Dr. Andrea Mastro for critical reading of the manuscript. All confocal microscopy was done at the Cytometry Facility, University Park (Huck Institutes of the Life Sciences, Penn State University). This work was supported by DOD#BC062742 to S.L.K.

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