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[Cancer Research 66, 3248-3255, March 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Identification of 14-3-3{sigma} as a Contributor to Drug Resistance in Human Breast Cancer Cells Using Functional Proteomic Analysis

Yang Liu, Hailan Liu, Baoguang Han and Jian-Ting Zhang

Department of Pharmacology and Toxicology, Walther Oncology Center/Walther Cancer Institute and IU Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana

Requests for reprints: Jian-Ting Zhang, IU Cancer Center, Indiana University School of Medicine, 1044 West Walnut Street, R4-166, Indianapolis, IN 46202. Phone: 317-278-4503; Fax: 317-274-8046; E-mail: jianzhan{at}iupui.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Multidrug resistance (MDR) is a major obstacle to successful cancer treatment. To understand the mechanism of MDR, many cancer cell lines have been established, and various mechanisms of resistance, such as ATP-binding cassette (ABC) transporter–mediated drug efflux, have been discovered. Previously, a MDR cell line MCF7/AdVp3000 was selected from breast cancer cell line MCF7 against Adriamycin, and overexpression of ABCG2 was thought to cause MDR in this derivative cell line. However, ectopic overexpression of ABCG2 in MCF7 cells could not explain the extremely high drug resistance level of the selected MCF7/AdVp3000 cells. We hypothesized that MCF7/AdVp3000 cells must have other resistance mechanisms selected by Adriamycin. To test this hypothesis, we compared the global protein profiles between MCF7 and MCF7/AdVp3000 cells. Following two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, 17 protein spots with differential levels between the two cell lines were identified. Although 14-3-3{sigma}, keratin 18, keratin 19, ATP synthase ß, protein disulfide isomerase, heat shock protein 27, cathepsin D, triose-phosphate isomerase, peroxiredoxin 6, and electron transfer flavoprotein were increased, nm23/H1, peroxiredoxin 2, nucleophosmin 1/B23, and inorganic pyrophosphatase were decreased in MCF7/AdVp3000 cells. The differential levels of these proteins were validated using Western blot. Furthermore, functional validation showed that the elevated 14-3-3{sigma} expression contributes considerably to the observed drug resistance in MCF7/AdVp3000 cells. We, thus, conclude that these proteins likely contribute to the resistance selected in the MCF7/AdVp3000 cells, and their altered expression in tumors may cause clinical resistance to chemotherapy. (Cancer Res 2006; 66(6): 3248-55)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Multidrug resistance (MDR) to chemotherapy frequently occurs and represents a major obstacle to successful breast cancer treatment. Studies with tumor cell lines have revealed that MDR can develop and thus cause chemotherapy failure. Many drug-resistant cell lines have been established for studying MDR by selecting tumor cell lines with a stepwise increase in concentration of various anticancer agents. Using this approach of stepwise selection, ATP-binding cassette (ABC) membrane transporters, such as ABCB1 (MDR1) and ABCC1 (MRP1), have been discovered to be responsible for multidrug resistance in many cell lines selected with various anticancer drugs. Previously, a series of drug-resistant breast cancer cell lines were generated with different levels of resistance to anticancer drugs by subjecting MCF7 to stepwise selections using increasing concentrations of Adriamycin in the presence of verapamil, an ABCB1/ABCC1 inhibitor (1, 2). The final derivative cell line MCF7/AdVp3000 with the highest level of resistance was further examined, and, subsequently, it was found that this derivative cell line overexpresses ABCG2 (BCRP/MXR; ref. 3). Overexpression of ABCG2 was, thus, thought to be the main mechanism of drug resistance selected by Adriamycin in MCF7/AdVp3000 cells.

Indeed, transfection of ABCG2 cDNA into the parental MCF7 cells caused drug resistance. However, it has also been found that the drug resistance level of the transfected cells was much lower than that of the drug-selected MCF7/AdVp3000 cells (3). The relative resistance factor of ABCG2-transfected MCF7 cells to mitoxantrone is 32 times, whereas that of the drug-selected MCF7/AdVp3000 cells is 3,902 times compared with parental MCF7 cells (4). There is a 122-fold difference in mitoxantrone resistance between the drug-selected and ABCG2-transfected MCF7 cells, although both express similar levels of ABCG2 and do not differentially express ABCB1 or ABCC1. Thus, other drug resistance mechanisms must have been selected, which are absent in the ABCG2-transfected cells. Indeed, using an AmpArray analysis, we found previously that the expression of other ABC transporters, such as ABCC3, has been selected to contribute to drug resistance in the selected MCF7/AdVp3000 cell line (5).

To further examine whether drug resistance mechanisms other than overexpression of drug efflux pump ABC transporters have been selected by Adriamycin in MCF7/AdVp3000 cells, we applied proteomic approach that combines two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to compare and identify proteins with differential profile between the parental drug-sensitive MCF7 and the drug-selected MCF7/AdVp3000 cells. Seventeen such protein spots were identified, and one of the proteins, 14-3-3{sigma}, was shown functionally to contribute to resistance to both mitoxantrone and Adriamycin in MCF7/AdVp3000 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. All electrophoresis reagents, precast SDS-PAGE slab gels, immobilized pH gradient strips, iodoacetamide, and polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad (Hercules, CA). Adriamycin, mitoxantrone, verapamil, DTT, acetonitrile, and {alpha}-cyano-4-hydroxycinnamic acid were from Sigma (St. Louis, MO). Modified trypsin was purchased from Promega (Madison, WI). Monoclonal antibody BXP-21 against ABCG2 was from ID Labs (London, Ontario, Canada). Antibodies against keratin 18, heat shock protein (hsp27), and 14-3-3{sigma} were from Lab Vision (Fremont, CA). Antibodies against PDI and cathepsin D were from Santa Cruz Biotechnology (Santa Cruz, CA). SYBR Green PCR Master Mix for real-time PCR was purchased from Applied Biosystems (Foster City, CA). LipofectAMINE Plus and G418 were purchased from Invitrogen (Carlsbad, CA). Cell culture medium IMEM and DMEM were purchased from BioSource International (Camarillo, CA) and Media Tech. (Herndon, CA), respectively. All other chemicals were molecular biology grade from Sigma or Fisher Scientific (Pittsburgh, PA).

Cell cultures and transfection. Human breast cancer cell line MCF7 and its derivative lines MCF7/AdVp10, MCF7/AdVp100, MCF7/AdVp3000, and MCF-7/AdVpRev were gifts from Dr. Susan E. Bates (National Cancer Institute). The drug-resistant derivative lines were generated by stepwise selection using 10, 100, and 3,000 ng/mL Adriamycin in the presence of verapamil for MCF7/AdVp10, MCF7/AdVp100, and MCF7/AdVp3000, respectively (1, 2, 6). The revertant cell line MCF-7/AdVpRev that lost most of its drug resistance was produced by culturing the drug-resistant MCF7/AdVp3000 cells in the absence of drug selection. These cell lines were cultured as previously described (1, 2, 6). To maintain the drug resistance phenotype of MCF7/AdVp10, MCF7/AdVp100, and MCF7/AdVp3000 cells, 10, 100, and 3,000 ng/mL Adriamycin were included, respectively, together with 5 µg/mL verapamil. HEK293 and its derivative stable cell clones were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin.

To establish MCF7/AdVp3000 stable cell clones with reduced 14-3-3{sigma} expression, a plasmid pSilencer-{sigma} expressing short hairpin RNAs (shRNA) specifically targeting 14-3-3{sigma} were generated elsewhere (7) and was used to transfect MCF7/AdVp3000 cells using FuGene 6 Transfection Reagents (Roche, Indianapolis, IN) according to the vendor's manual. A plasmid pSilencer-Scr (Ambion, Austin, TX) expressing a scrambled siRNA was also transfected into MCF7/AdVp3000 cells as a control. At 24 hours following transfection, the cells were split at an appropriate dilution into medium supplemented with 800 µg/mL G418 for 2 weeks. Individual stable clones were selected and expanded for further analysis.

To establish MCF7 and HEK293 stable clones overexpressing ectopic 14-3-3{sigma}, the cDNA encoding hemagglutinin-tagged 14-3-3{sigma} at NH2 terminus was engineered into the pcDNA3 vector (7) and transfected into those cells using LipofectAMINE Plus according to the manufacturer's instruction. Two days following transfection, 10% of the transfected cells were replated and selected with 800 µg/mL G418 for 2 weeks, and stable clones were propagated for further analysis.

Cell lysate preparation. The cultured cells were harvested, washed with PBS, and lysed in a lysis buffer [1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 1 mmol/L EGTA (pH 8), 0.2 mmol/L sodium orthovanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride, 0.5% NP40, 0.1% SDS] for 30 minutes at 4°C with constant agitation. The cell lysates were then sonicated briefly and followed by centrifugation (16,000 x g at 4°C) for 15 minutes to remove insoluble materials. The protein concentrations of supernatants were measured by Bradford assay (8).

Two-dimensional gel electrophoresis. Two hundred micrograms each of the cell lysates were first diluted to 2 µg/µL with lysis buffer and precipitated by acetone, and the proteins were collected by centrifugation. The protein pellets were washed once with ice-cold acetone followed by solubilization in 180 µL Bio-Rad rehydration buffer for separation by isoelectrical focusing following by SDS-PAGE (4-15% gradient gels) exactly the same way as we previously described (9). The gels were then stained with Coomassie blue staining solution for overnight followed by destaining with 18% methanol and 5% acetic acid.

Image analysis, MALDI-TOF mass spectrometry, and database search. The image analysis, MALDI-TOF mass spectrometry, and database search were done the same way as previously described (9). Briefly, the images of the two-dimensional gel electrophoresis gels were scanned and analyzed using Fluor-S MAX MultiImager system and PD Quest software (Bio-Rad), respectively. The quantity of each spot on the two-dimensional gel electrophoresis gels was defined as parts per million of the total integrated absorbance. The abundance of the individual protein was calculated using a quantitative analysis set within the PD Quest software. Protein spots of interest were then excised from the gels that have high levels of the protein, and processed robotically using the PROTEAN 2D Spot Cutter (Bio-Rad) and the MassPREP Workstation (Perkin-Elmer, Wellesley, MA), respectively. The excised gel spots in 96-well plates were then processed for destaining, in gel trypsin digestion, and mass determination using MALDI-TOF mass spectrometer (Micromass, Toronto, Ontario, Canada). The peptide matching and protein searching of the National Center for Biotechnology Information database were done using ProFound search engine.

Western blot analysis. Western blot analysis was done as previously described (9, 10). Briefly, cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. The blot was then probed with primary antibody followed by reaction with horseradish peroxidase–conjugated secondary antibody. The signal was detected using enhanced chemiluminescence and recorded on an X-ray film.

Real-time quantitative reverse transcription-PCR. Total RNAs were isolated from cultured cells using a RNeasy mini kit according to the manufacturer's instruction (Qiagen, Valencia, CA) and treated with RQ1 RNase-free DNase I. Four micrograms total RNAs each were reverse transcribed using avian myeloblastosis virus Reverse Transcriptase and Oligo(dT)12-18 primer (Invitrogen). Primers for real-time PCR were designed using Primer Express software version 2.0 (Applied Biosystems) and synthesized by Invitrogen. The primer sequences for 14-3-3{sigma} are 5'-GGCCATGGACATCAGCAAGAA-3' (forward) and 5'-CGAAAGTGGTCTTGGCCAGAG-3' (reverse). The primer sequences for ABCG2 are 5'-GGCTTTCTACCTGCACGAAAACCAGTTGAG-3' (forward) and 5'-ATGGCGTTGAGACCAG-3' (reverse). The primer sequences for internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 5'-AAGGACTCATGACCACAGTCCAT-3' (forward) and 5'-CCATCACGCCACAGTTTCC-3' (reverse). Real-time quantitative PCR was done in a ABI Prism@7000 Sequence Detection System (Applied Biosystems) using SYBR Green diction according to the manufacturer's instruction. The threshold cycle (Ct) was defined as the PCR cycle number at which the reporter fluorescence crosses the threshold reflecting a statistically significant point above the calculated baseline. The Ct of 14-3-3{sigma} and ABCG2 was determined and normalized against that of the internal control housekeeping gene GAPDH. The relative level calculated against that in MCF7 cells = 2{Delta}{Delta}Ct.

Cytotoxicity assay. The cytotoxicity was determined using the sulforhodamine B colorimetric assay (11). Briefly, the cells were seeded in 96-well plate at 3,000 per well and cultured for 24 hours. Adriamycin and mitoxantrone were then added to the cells, and the cells were cultured continuously for 3 to 7 days before the sulforhodamine B assay. For sulforhodamine B assay, the culture medium was aspirated, and the cells were fixed and stained by adding 70 µL 0.4% (w/v) sulforhodamine B in 1% acetic acid solution to each well and incubation at room temperature for 20 minutes. The plates were then washed three to five times with 150 µL of 1% acetic acid to remove the unbound sulforhodamine B and air-dried. The bound sulforhodamine B was then solubilized with 200 µL of 10 mmol/L unbuffered Tris-base, and the A570 nm was determined using a 96-well plate reader (MRX, Dynex Technologies, Chantilly, VA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein profiling and identification of differentially expressed proteins. To compare global protein profiles between the parental drug-sensitive MCF7 and its drug-resistant derivative MCF7/AdVp3000 cells, we first did two-dimensional gel electrophoresis analysis of total lysates prepared from these cell lines. Following Coomassie blue staining (Fig. 1 ), the gels were subjected to PD Quest imaging analysis. Protein spots that have differences (≥1.5-fold) between the two cell lines in triplicate experiments were chosen for further analysis. Thirty-six such spots were found (Fig. 2A ), and they were subjected to MALDI-TOF mass spectrometry analysis. Seventeen of these spots were identified with good peptide coverage, significant Z scores, and similar observed and calculated molecular weight and isoelectric point (pI; Table 1 ; see also Fig. 1).


Figure 1
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  		Figure 1. Two-dimensional gel electrophoresis profile of MCF7 and MCF7/AdVp3000 cell lysates. Proteins (120 µg) of MCF7 (A) and MCF7/AdVp3000 (B) cell lysates were first separated by IEF (pH 3-10 Strip, Bio-Rad) followed by precast SDS-PAGE (10-20% gradient gel, Bio-Rad) and stained with Coomassie blue. The protein profiles were analyzed using PD Quest software (Bio-Rad). The numbered spots are identified proteins with differential expression.

 

Figure 2
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  		Figure 2. Proteins differentially expressed between MCF7 and MCF7/AdVp3000 cells. A, histograms showing 36 protein spots with differential levels between MCF7 and MCF7/AdVp3000 cells. Columns, protein quantity in each gel of three gels. Left three columns, intensity of the protein spots on three separate gels from the parental MCF7 cells; right three columns, intensity of the protein spots on three separate gels from the drug-resistant MCF7/AdVp3000 cells. The ssp numbers are arbitrarily assigned to represent each protein spot. Quantitative changes (y axis) are depicted in parts per million (ppm) determined by PD Quest gel analysis software. B, validation of differentially expressed proteins with Western blot analysis. Cell lysates from MCF7 (lanes 1 and 3) and MCF7/AdVp3000 (lanes 2 and 4) cells were separated by SDS-PAGE followed by Western blot analysis using antibodies specific to ABCG2, 14-3-3{sigma}, cathepsin D, keratin 18, PDI, hsp27, nucleophosmin 1, and nm23 (lanes 1-2) or by Coomassie blue staining as a loading control (lanes 3-4).

 

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  		Table 1. Proteins with different levels between MCF7 and MCF7/AdVp3000 identified by peptide mass fingerprinting

 
Of the 17 protein spots identified, keratin 18, keratin 19, 14-3-3{sigma}, protein disulfide isomerase (PDI), cathepsin D, ATP synthase ß, hsp27, peroxiredoxin 6, and electron transfer flavoprotein (ETF) were increased in expression, whereas nucleophosmin 1/B23, peroxiredoxin 2, and nm23 were decreased in the drug-selected MCF7/AdVp3000 cells. Interestingly, three proteins, triose-phosphate isomerase (TPI), PDI, and peroxiredoxin 2, were each identified twice in two separate spots. Although the TPI in ssp6305 was increased, the protein in ssp7303 was decreased in the drug-resistant MCF7/AdVp3000 cells. Although both spots have similar molecular weight, they have different pIs (Fig. 1), suggesting that the posttranslational modifications of TPI may have been selected in the drug-resistant MCF7/AdVp3000 cells.

Validation of identified proteins. To validate the identified proteins, we did Western blot analysis to determine the level of these proteins in MCF7 and MCF7/AdVp3000 cells. Only the proteins with antibodies commercially available were tested. As shown in Fig. 2B, 14-3-3{sigma}, cathepsin D, keratin 18, hsp27, and PDI are up-regulated, whereas nucleophosmin and nm23 are down-regulated in the drug-resistant MCF7/AdVp3000 cells, consistent with the data shown in Table 1 and Fig. 2A obtained using proteomic approach. As a control, ABCG2 was also detected and was increased in the drug-resistant MCF7/AdVp3000 cells as expected.

Correlation between 14-3-3{sigma} expression and drug resistance. Of the proteins that have altered expression in the drug-resistant MCF7/AdVp3000 cells, 14-3-3{sigma}, which is increased in MCF7/AdVp3000 cells, is thought to be interesting because of its role in cell cycle control and apoptosis (for a review, see ref. 12). To determine if the increased expression of 14-3-3{sigma} potentially contributes to drug resistance selected in the MCF7/AdVp3000 cells, we first did a correlation analysis of its expression level with drug resistance. As stated above, during the stepwise selection to generate MCF7/AdVp3000 cells, other MCF7 derivatives with intermediate drug resistance levels, MCF7/AdVp10 and MCF7/AdVp100, and a revertant cell line MCF7/AdVpRev were also generated (1, 2, 6). The protein and mRNA levels of 14-3-3{sigma} in these cell lines were determined using Western blot and real-time reverse transcription-PCR analyses, respectively. As shown in Fig. 3A and B , the 14-3-3{sigma} protein level increased in the MCF7/AdVp10 cells compared with the parental sensitive MCF7 cells and peaked in the MCF7/AdVp100 cells despite the fact that its mRNA level continued to increase in MCF7/AdVp3000 cells (Fig. 3C). On the other hand, ABCG2, which has been thought to be the major cause of drug resistance in MCF7/AdVp3000 cells, was expressed at very low levels in MCF7/AdVp10 and MCF7/AdVp100 cells compared with MCF7/AdVp3000 cells (Fig. 3A and D). The expression of both 14-3-3{sigma} and ABCG2 was fully reversed in the revertant cell line (Fig. 3A-D). These results suggest that the elevated expression of 14-3-3{sigma} likely contributes to the drug resistance phenotype observed with the selected MCF7 derivative cell lines, and that 14-3-3{sigma} may be one of the early respondent genes to drug assault, whereas ABCG2 is a later one.


Figure 3
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  		Figure 3. Expression level of 14-3-3{sigma} and ABCG2 in MCF7 and its drug-resistant derivative cell lines. A, Western blot analysis. Cell lysates were prepared from the drug-sensitive parental MCF7; its drug-resistant derivative cell lines MCF7/AdVp10 (AdVp10), MCF7/AdVp100 (AdVp100), MCF7/AdVp3000 (AdVp3000); and the revertant cell line MCF7/AdVpRev (AdVpRev) and used for Western blot analysis of 14-3-3{sigma} and ABCG2. B, quantification of 14-3-3{sigma} level. The 14-3-3{sigma} level in MCF7 and its derivative cell lines as determine by Western blot in (A) were quantified using an online software Scion Image. C and D, real-time quantitative reverse transcription-PCR analysis of 14-3-3{sigma} (C) and ABCG2 (D) expression. Real-time quantitative reverse transcription-PCR was done by measuring the mRNA levels using SYBR Green and calculated in the fold change (2{Delta}{Delta}Ct) relative to MCF7 cells after normalization by the internal control, GAPDH.

 
Role of 14-3-3{sigma} in drug resistance. To determine whether the increased 14-3-3{sigma} expression in MCF7/AdVp3000 cells contributes to the drug resistance phenotype, we did an experiment to knock down 14-3-3{sigma} expression in MCF7/AdVp3000 cells followed by determining the possible changes in drug resistance. For this purpose, a plasmid pSilencer-{sigma} expressing siRNA targeting 14-3-3{sigma} was transfected into MCF7/AdVp3000 cells. Stable cell clones with reduced 14-3-3{sigma} expression were generated. As shown in Fig. 4A , the expression of 14-3-3{sigma} was drastically decreased in two independent clones Si40 and Si90 compared with the control clone Scr16 transfected with scrambled shRNA and the untransfected MCF7/AdVp3000 cells. We next did sulforhodamine B assay to determine the effect of reducing 14-3-3{sigma} expression on the resistance of MCF7/AdVp3000 cells to mitoxantrone and Adriamycin. As shown in Fig. 4B and C, reducing 14-3-3{sigma} expression significantly decreased the resistance level of MCF7/AdVp3000 cells compared with the control cells harboring a scrambled shRNA. The IC50 to mitoxantrone and Adriamycin in these clones were decreased by 49% (Si90) to 52% (Si40) and 50% (Si40) to 68% (Si90), respectively, compared with the control cells.


Figure 4
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  		Figure 4. 14-3-3{sigma} silencing and its effect on drug resistance. A, Western blot analysis of 14-3-3{sigma} expression. MCF7/AdVp3000 cells were stably transfected with shRNAs targeting 14-3-3{sigma} as described in Materials and Methods. Cell lysates from two stable clones harboring 14-3-3{sigma} shRNA (Si40 and Si90) and a control stable clone harboring a shRNA with scrambled sequence (Scr16) were analyzed for expression of 14-3-3{sigma} and ABCG2. GAPDH was used as a loading control. B and C, effect of 14-3-3{sigma} on resistance to mitoxantrone (B) and Adriamycin (C). The stable clones with reduced 14-3-3{sigma} expression (Si40 and Si90) were tested for their response to mitoxantrone (B) and Adriamycin (C) along with the control stable clone (Scr16) using sulforhodamine B assay as described in Materials and Methods.

 
To determine if enforced ectopic expression of 14-3-3{sigma} in the parental drug-sensitive MCF7 cells would cause drug resistance, we cloned 14-3-3{sigma} cDNA into pcDNA3 and transfected it into the parental drug-sensitive MCF7 cells followed by analysis of its effect on drug resistance. As shown in Fig. 5A , two stable clones (M{sigma}12 and M{sigma}16) with overexpression of ectopic 14-3-3{sigma} were generated. These clones with overexpression of ectopic 14-3-3{sigma} were subjected to drug resistance analysis using sulforhodamine B assay. As shown in Fig. 5B and C, overexpression of ectopic 14-3-3{sigma} caused resistance to both mitoxantrone and Adriamycin. The IC50 to mitoxantrone and Adriamycin in these clones were increased 3-fold (M{sigma}12) to 5-fold (M{sigma}16) and 2-fold (M{sigma}12) to 5-fold (M{sigma}16), respectively, compared with the control cells. Taken together, the above results showed that the increased expression of 14-3-3{sigma} in the drug-selected MCF7/AdVp3000 cells contributes significantly to the observed drug resistance phenotype in these cells.


Figure 5
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  		Figure 5. Effect of enforced ectopic expression of 14-3-3{sigma} on drug resistance in MCF7 cells. A, Western blot analysis of 14-3-3{sigma} expression. The parental MCF7 cells were transfected with 14-3-3{sigma} cDNA and stable clones with ectopic overexpression of 14-3-3{sigma} were selected as described in Materials and Methods. Cell lysates from two stable clones over-expressing 14-3-3{sigma} (M{sigma}12 and M{sigma}16) and a control stable clone harboring vector (Vec10) were prepared for Western blot analysis of 14-3-3{sigma}. GAPDH was used as a loading control. B and C, effect of 14-3-3{sigma} overexpression on resistance to mitoxantrone (B) and Adriamycin (C). The stable clones with enforced 14-3-3{sigma} expression (M{sigma}12 and M{sigma}16) were tested for their response to mitoxantrone (B) and Adriamycin (C) along with the control stable clone (Vec10) using sulforhodamine B assay as described in Materials and Methods.

 
To determine whether the 14-3-3{sigma}–mediated drug resistance is not specific to MCF7 cells, we conducted similar experiments to ectopically overexpress 14-3-3{sigma} in HEK293 cells. As shown in Fig. 6A , stable HEK293 cell clones (H{sigma}1 and H{sigma}7) overexpressing ectopic human 14-3-3{sigma} were established together with a clone transfected with vector control (Vec2). These clones were then subjected to mitoxantrone resistance analysis using sulforhodamine B assay. As shown in Fig. 6B, overexpression of ectopic 14-3-3{sigma} caused resistance to mitoxantrone in both clones. The IC50 to mitoxantrone were increased 8.6-fold for H{sigma}1 and 8.1-fold for H{sigma}7 clone, respectively, compared with the control cells. Thus, the 14-3-3{sigma}–mediated drug resistance is not specific to MCF7 cells, and its elevated expression causes drug resistance.


Figure 6
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  		Figure 6. Effect of enforced ectopic expression of 14-3-3{sigma} on drug resistance in HEK293 cells. A, Western blot analysis of 14-3-3{sigma} expression. HEK293 cells were transfected with 14-3-3{sigma} cDNA and stable clones with ectopic overexpression of 14-3-3{sigma} were selected as described in Materials and Methods. Cell lysates from two stable clones overexpressing 14-3-3{sigma} (H{sigma}1 and H{sigma}7) and a control stable clone harboring vector (Vec2) were prepared for Western blot analysis of 14-3-3{sigma}. GAPDH was used as a loading control. B, effect of 14-3-3{sigma} overexpression on resistance to mitoxantrone. The stable clones with enforced 14-3-3{sigma} expression (H{sigma}1 and H{sigma}7) were tested for their response to mitoxantrone along with the control stable clone (Vec2) using sulforhodamine B assay as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we used proteomic approach to identify proteins with differential expression between the parental drug-sensitive MCF7 and its drug-resistant derivative MCF7/AdVp3000 cells. We found 36 protein spots with significant alterations in expression between the two cell lines. Seventeen of these protein spots were identified by MALDI-TOF mass spectrometry, and seven proteins with commercially available antibodies were validated using Western blot analysis. We further validated the functional role in drug resistance of one protein, 14-3-3{sigma}, and found that its up-regulated expression in the drug-resistant MCF7/AdVp3000 cells contributes considerably to its drug resistance phenotype. These findings illustrate that multiple mechanisms can be selected to cause drug resistance in breast cancer cells, and they may all contribute partially to chemotherapeutic resistance in breast cancer treatment.

14-3-3{sigma} (also called stratifin) was originally characterized as a human mammary epithelium-specific marker 1 (HME1), primarily expressed in epithelial cells, and its expression increases during epithelial differentiation (13). 14-3-3{sigma} is a member of the 14-3-3 family proteins that include seven isotypes ({alpha}/ß, {gamma}, {varepsilon}, {sigma}, {zeta}, {tau}/{theta}, and {eta}) in mammal and play various roles in intracellular signaling (14, 15). 14-3-3 proteins can interact with >100 cellular proteins at their phosphorylation sites, and the target proteins include various protein kinases, receptor proteins, enzymes, structural and cytoskeletal proteins, and proteins involved in cell cycle control and apoptosis (15–17). Among all seven isoforms, 14-3-3{sigma} has been found to be directly related to human cancers (12, 18) and thought to be a tumor suppressor because its expression has been found decreased in several types of human tumors (9, 19–24), possibly due to gene expression silencing by hypermethylation (19, 25). However, most recently, the decreased 14-3-3{sigma} expression in tumors was found to be sporadic events, and its expression in some tumors, such as pancreatic cancers, is increased (26–28).

Recently, it was also found that 14-3-3{sigma} is an independent prognosis factor for poor survival of patients with colorectal, pancreatic, and breast cancers (29–31). Interestingly, Cheng et al. also found an increase in 14-3-3{sigma} expression as prostate tumor progresses (24). Adenocarcinomas with high Gleason scores had significantly higher staining intensities and higher percentages of 14-3-3{sigma} immunoreactive cells than adenocarcinomas with low Gleason scores. These observations suggest that the advanced prostate adenocarcinomas are likely drug resistant. Indeed, our recent studies showed that the androgen-independent prostate cancer cell lines express more 14-3-3{sigma} than the androgen-dependent ones, and they are more resistant to anticancer drugs (7). Furthermore, elevated expression of 14-3-3{sigma} has also been observed with drug-resistant melanoma and pancreatic adenocarcinoma cell lines (32, 33). These findings, together with the results of this study, suggest that 14-3-3{sigma} is likely a prognosis factor for poor survival by causing drug resistance in chemotherapy of multiple human cancers.

14-3-3{sigma} has been suggested to be a critical G2-M regulator in epithelial cells, and knocking out 14-3-3{sigma} expression caused mitotic catastrophe and cell death following DNA damage due to lack of proper G2-M arrest (34, 35). It seems that 14-3-3{sigma} is critical for sequestration of Cdc2/cyclin B1 complex in the cytoplasm, thereby preventing cells from entering mitosis (34, 35). Many anticancer drugs, such as Adriamycin and mitoxantrone, are DNA-damaging agents, and cancer cells with elevated expression of 14-3-3{sigma}, thus, have the advantage to survive drug treatment by efficiently causing G2-M arrest for DNA repair rather than committing to mitotic catastrophe and cell death.

Given its potential role in drug resistance, novel therapeutics targeting 14-3-3{sigma} may be developed for better treatment of cancers. Recently, the atomic structure of 14-3-3{sigma} has been solved (36), which showed that the dimeric interface of 14-3-3{sigma} is different from the other 14-3-3 isoforms. Small molecular inhibitors that can specifically disrupt the homodimerization of 14-3-3{sigma} may be developed to inhibit dimerization and thus function of 14-3-3{sigma} to enhance chemosensitivity of cancers to currently available anticancer drugs, such as Adriamycin and mitoxantrone.

Of the other 16 protein spots with altered expression in the drug-resistant MCF7/AdVp3000 cells, 6 (nucleophosmin 1/B23, keratin 19, keratin 18, PDI, nm23/H1, hsp27, and cathepsin D) have been associated with cell responses to DNA damage–induced cell death and/or cause drug resistance and poor prognosis (Table 2 ). For example, overexpression of hsp27 has been found to cause resistance to Adriamycin in breast cancer cells (37) by increasing efficiency in repair of Adriamycin-induced DNA damages (38). Overexpression of hsp27 has been thought to cause drug resistance and poor prognosis in breast (39) and ovarian (40, 41) cancers.


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  		Table 2. Possible relationship between the expression of identified proteins and drug resistance

 
Most of the remaining protein spots (peroxiredoxin 2, ATP synthase, inorganic pyrophosphatase, TPI, peroxiredoxin 6, and ETF) are metabolic enzymes that do not have known direct roles in drug resistance (Table 2). However, some of them are involved in generating energy, which may be important for MCF7/AdVp3000 cells to survive chronic environmental stresses, such as drug attack. For example, ATP synthase ß is one of the subunits in the F1-ATPase responsible for ATP regeneration in mitochondria (42). It is possible that the increased needs of ATP for constantly effluxing anticancer drugs by ABC transporters, such as ABCG2 and ABCC3, in MCF7/AdVp3000 cells (5) require elevated ATP synthesis provided by the increased expression of ATP synthase.

It is noteworthy that three proteins, PDI, TPI, and peroxiredoxin 2, were all identified in two separate spots each (see Table 1). Although both spots representing PDI were increased and that representing peroxiredoxin 2 were decreased in the drug-resistant MCF7/AdVp3000 cells, the two spots representing TPI changed in opposite directions. Currently, the cause for existence of two different spots for these proteins is unknown. It is possible that they represent the same protein with differential modifications, considering that they have similar molecular weight but different pIs. It is also noteworthy that none of the ABC transporters found earlier using AmpArray (5) were identified in this study. Because ABC transporter membrane proteins are highly hydrophobic and heterogeneous in sugar moiety, they are unlikely to be easily solublized, focused, separated, and identified by two-dimensional gel electrophoresis and MALDI-TOF. Clearly, the proteomic approach used in this study has limitations, and different approaches and methods may be needed to identify all proteins that may contribute to the drug resistance phenotype of MCF7/AdVp3000 cells. Furthermore, it is unknown if the multiple mechanisms of resistance (e.g., elevated expression of 14-3-3{sigma} and ABC transporters, such as ABCG2) have additive or synergistic effect on drug resistance in MCF7/AdVp3000 cells. We are currently working toward answering this question.

In summary, using proteomic approach, we identified 17 protein spots that have altered expression in the drug-resistant breast cancer cell line MCF7/AdVp3000 compared with the parental sensitive MCF7 cells. We further showed that the elevated 14-3-3{sigma} expression contributes significantly to the drug resistance phenotype of MCF7/AdrVp3000 cells using both siRNA technology and ectopic overexpression. The findings may have important clinical implications. Future studies on the other identified proteins using similar strategies may yield equally important information regarding their role in drug resistance. All these proteins, in addition to 14-3-3{sigma}, may be developed as new target for better treatment of cancers using combination chemotherapy.


   Acknowledgments
 
Grant support: Department of Defense grant DAMD17-03-1-0566 and NIH grant CA94961.

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.

We thank Dr. Susan Bates at National Cancer Institute for providing drug-resistant and revertant MCF7 derivative cell lines and Dr. Mu Wang at Indiana University for technical assistance in the mass spectrometry analysis.

Received 10/21/05. Revised 12/22/05. Accepted 1/18/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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