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Effect of {gamma}-Linolenic Acid on the Transcriptional Activity of the Her-2/neu (erbB-2) Oncogene

Javier A. Menendez, Luciano Vellon, Ramon Colomer, Ruth Lupu

Affiliations of authors: Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston, IL (JAM, LV, RL); Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL (JAM, LV, RL); Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL (JAM, RL); Medical Oncology, Institut Catala d'Oncologia, Hospital Universitari Dr. Josep Trueta, Girona, Spain (RC)

Correspondence to: Ruth Lupu, PhD, Evanston Northwestern Healthcare Research Institute, 1001 University Place, Evanston, IL 60201 (e-mail: r-lupu{at}northwestern.edu) or Javier A. Menendez, PhD, Evanston Northwestern Healthcare Research Institute, 1001 University Place, Evanston, IL 60201 (e-mail: jmenendez{at}enh.org).


    ABSTRACT
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The {omega}-6 polyunsaturated fatty acid {gamma}-linolenic acid (GLA; 18:3n-6), which is found in several plant oils and is used as an herbal medicine, has antitumor activity in vitro. We examined the effect of GLA on the expression of the Her-2/neu (erbB-2) oncogene, which is involved in development of numerous types of human cancer. Flow cytometric and immunoblotting analyses demonstrated that GLA treatment substantially reduced Her-2/neu protein levels in the Her-2/neu–overexpressing cell lines BT-474, SK-Br3, and MDA-MB-453 (breast cancer), SK-OV3 (ovarian cancer), and NCI-N87 (gastrointestinal tumor derived). GLA exposure led to a dramatic decrease in Her-2/neu promoter activity and a concomitant increase in the levels of polyomavirus enhancer activator 3 (PEA3), a transcriptional repressor of Her-2/neu, in these cell lines. In transient transfection experiments, a Her-2/neu promoter bearing a PEA3 site–mutated sequence was not subject to negative regulation by GLA in Her-2/neu–overexpressing cell lines. Concurrent treatments of Her-2/neu–overexpressing cancer cells with GLA and the anti–Her-2/neu antibody trastuzumab led to synergistic increases in apoptosis and reduced growth and colony formation.


The oil from seeds of the evening primrose is used in traditional medicine as a treatment for a variety of chronic diseases. This oil (and that from seeds of borage and black currant) contains {gamma}-linolenic acid (GLA), a member of the {omega}-6 family of polyunsaturated fatty acids. GLA exerts selective cytotoxic effects on cancer cells without affecting normal cells (15). In addition, exogenous supplementation with GLA sensitizes breast cancer cells to antimitotic drugs and endocrine therapies (69). A recent phase II study suggested that GLA may have activity against endocrine-sensitive breast cancer with low systemic toxicity (10), and GLA treatments have led to some tumor responses in other advanced solid malignancies (1115). Although enhanced lipid peroxidation has been proposed as the main mechanism of action of GLA (15), the ultimate molecular pathways underlying GLA's anticancer actions remain largely unknown.

A novel molecular explanation concerning the anticancer actions of GLA may relate to its ability to specifically regulate oncoproteins. We recently reported that exogenous supplementation of cultured breast cancer cells with GLA significantly diminished proteolytic cleavage of the extracellular domain of the Her-2/neu–coded p185Her-2/neu tyrosine kinase oncoprotein and, consequently, its activation (16). Considering that activation and overexpression of Her-2/neu oncogene are crucial for the etiology, progression, and cell sensitivity to various treatments in ~30% of breast carcinomas (1732), these findings showed a previously unrecognized mechanism by which GLA might regulate breast cancer cell growth, metastasis formation, and response to chemotherapy and endocrine therapy. However, two main questions remained to be addressed: (1) Does GLA-induced deactivation of p185Her-2/neu relate to GLA-induced changes in Her-2/neu gene expression? (2) Is the ability of GLA to regulate Her-2/neu oncogene a common mechanism of GLA's action against other types of cancer or is it restricted to breast cancer?

To characterize the effects of GLA on the expression of Her-2/neu oncogene, we first treated BT-474 and SK-Br3 breast cancer cells, which naturally contain Her-2/neu oncogene amplification (33,34), with GLA (10 µg/mL for 48 hours). In flow cytometry analyses, levels of cell surface–associated Her-2/neu protein were substantially lower in GLA-treated cells than in vehicle-treated cells (Fig. 1, A). Similarly, immunoblot analysis indicated that GLA treatment led to a substantial reduction in Her-2/neu protein levels in both cell lines (Fig. 1, B).



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Fig. 1. {gamma}-Linolenic acid (GLA)–induced PEA3-dependent inhibition of Her-2/neu promoter activity and synergism with trastuzumab. BT-474 (breast cancer), SK-Br3 (breast cancer), MDA-MB-453 (breast cancer), SK-OV3 (ovarian cancer), NCI-N87 (gastrointestinal cancer), and MDA-MB-231 (breast cancer) cell lines were obtained from the American Type Culture Collection and routinely grown in phenol red–containing improved minimal essential medium (IMEM; Biosource International, Camarillo, CA) containing 5% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. Cells were maintained at 37 °C in a humidified atmosphere of 95% air–5% CO2. Before experiments, cells were serum starved overnight and then cultured in IMEM containing 0.1% FBS in the absence or presence of GLA (Sigma Chemical Co., St. Louis, MO). GLA was prepared at a concentration of 1 mg/mL in ethanol and was added to a final concentration of 0, 5, 10, or 20 µg/mL; an equal volume of ethanol was added to control cells. Trastuzumab was solubilized in water containing 1.1% benzyl alcohol (stock solution 21 mg/mL), stored at 4 °C, and used within 1 month. A) Cell surface expression of Her-2/neu protein in BT-474 and SK-Br3 cells was determined by flow cytometry using a mouse anti–Her-2/neu antibody directed against the extracellular domain of the p185Her-2/neu oncoprotein (clone Ab-5; Oncogene Research Products; San Diego, CA). Briefly, after GLA or control treatment, cells were washed, harvested, resuspended in phosphate-buffered saline (PBS) containing 1% FBS, and incubated with 5 µg/mL Ab-5 antibody for 1 hour at 4 °C. The cells were washed again and then incubated with a fluorescein isothiocyanate (FITC)–conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:200 in cold PBS containing 1% FBS for 45 minutes at 4 °C. The cells were washed once more in cold PBS, and flow cytometric analysis was performed with a FACScalibur flow cytometer (Becton Dickinson, San Diego, CA) equipped with Cell Quest Software (Becton Dickinson). Representative flow cytometry immunofluorescence profiles in untreated control cells (blue line) and in GLA-treated cells (orange line) are shown. As a control for nonspecific immunofluorescence, cells were stained with the secondary antibody alone (solid black line). The mean fluorescence signal associated with cells for labeled p185Her-2/neu was quantified using the Geo Mean (GM) fluorescence parameter provided with the software. Data are the means and 95% confidence intervals (CIs) of three independent experiments. B) Immunoblot analysis of Her-2/neu and PEA3 proteins in control- and GLA-treated BT-474 and SK-Br3 cells. Cells were washed twice with PBS and then lysed in buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) for 30 minutes on ice. Equal amounts of protein (10 µg) were heated in sodium dodecyl sulfate (SDS) sample buffer for 10 minutes at 70 °C, subjected to electrophoresis on either 3%-8% NuPAGE Tris-acetate (p185Her-2/neu) or 10% SDS-polyacrylamide gel electrophoresis (PEA3) gels, and transferred to nitrocellulose membranes. Nonspecific binding was blocked by incubation for 1 hour with TBS-T (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.05% Tween-20) containing 5% nonfat dry milk. The treated filters were washed in TBS-T and then incubated with primary antibodies (anti–p185Her-2/neu mouse monoclonal antibody–Clone Ab-3 [Oncogene Research Products, San Diego, CA] and anti-PEA3 mouse monoclonal antibody–sc-113 [Santa Cruz Biotechnology, Santa Cruz, CA] for 2 hours in TBS-T containing 5% (w/v) nonfat dry milk. The membranes were washed in TBS-T, horseradish peroxidase–conjugated secondary antibodies in TBS-T were added for 45 min, and immunoreactive bands were detected by enhanced chemiluminescence reagent (Pierce, Rockford, IL). Blots were reprobed with an anti-{beta}-actin goat polyclonal antibody (Santa Cruz Biotechnology) to control for protein loading and transfer. Densitometric values of protein bands were quantified using Scion Imaging Software (Scion Corp., Frederick, MD). Representative blots are shown; similar results were obtained in three independent experiments. C) Reverse transcription (RT)–polymerase chain reaction (PCR) analysis of Her-2/neu expression in BT-474 and SK-Br3 breast cancer cells. Total RNA from control-treated BT-474 and SK-Br3 cells or cells treated with varying amounts of GLA (0, 5, 10, or 20 µg/mL) was extracted with the TriPure Isolation Reagent (Boehringer-Mannheim). One microgram of total RNA was reverse-transcribed and amplified with the Access RT-PCR System (Promega Inc.) using 1 mM of specific primers for Her-2/neu (sense: 5'-GGGCTGGCCCGATGTATTTGAT-3'; antisense: 5'-ATAGAGGTTGTCGAAGGCTGGGC-3'). As an internal control, {beta}-actin primers were used. The RT reaction was carried out for 45 minutes at 48 °C. Her-2/neu and {beta}-actin complementary DNAs were amplified for 20 cycles of the following conditions: 96 °C for 30 seconds, 60 °C for 1 minute, and 68 °C for 2 minutes. The PCR products were separated on 2% agarose gels and detected by ethidium bromide staining. Results are representative of three independent experiments. D) MDA-MB-453, SK-OV3, NCI-N87, and MDA-MB-231 cells were treated for 48 hours with either ethanol or GLA (10 µg/mL) and then subjected to immunoblot analysis of Her-2/neu, PEA3, and {beta}-actin (as a control). Representative blots from three independent experiments are shown. E) Apoptosis was analyzed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick and labeling (TUNEL) analysis using the DeadEnd Fluorometric TUNEL System (Promega Inc.). Briefly, BT-474 cells were split at a density of 2 x 104 cells/well in eight-well Lab-Tek chamber slides. After 24 hours, the cells were treated with trastuzumab (10 µg/mL) and/or GLA (10 µg/mL) for 48 hours. Cells were then washed twice with PBS, fixed in 4% methanol-free paraformaldehyde for 10 minutes, washed twice with PBS, and permeabilized with 0.2% Triton X-100 for 5 minutes. After two more washes, each slide was covered with equilibration buffer for 10 minutes more. The buffer was then aspirated, and the slides were incubated with terminal deoxynucleotidyl transferase buffer at 37 °C for 1 h. The reaction was stopped with 2x standard saline citrate, washed with PBS and mounted with Vectashield + 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Representative immunofluorescence photographs of cells undergoing apoptosis (green TUNEL staining) and the corresponding DAPI-counterstained photomicrographs are shown in the top panel. Apoptosis was quantified by determining the percentage of BT-474 and SK-Br3 cells containing nuclei with complete TUNEL-associated staining per total cells, as determined by DAPI staining of four random fields (bottom panel). For each pair of columns, the height of the columns on the left represents the sum of the effects of each agent alone, and the total height of the columns on the right indicates the observed apoptosis when the agents are used in combination. Data are the means and 95% confidence intervals of four independent experiments. One-factor analysis of variance was used to analyze differences in the percentage of apoptosis between the various treatment groups and the control group. *Two-sided P<.001 for the GLA + trastuzumab group versus all other groups. All statistical tests were two-sided.

 
Although Her-2/neu overexpression was originally attributed solely to erbB-2 gene amplification, an elevation in Her-2/neu mRNA levels per gene copy is also observed in all cell lines that exhibit gene amplification (35). We used reporter gene expression and reverse transcription–polymerase chain reaction (RT-PCR) analyses to characterize the effects of GLA on the transcription of the Her-2/neu gene. Treatment of BT-474 and SK-Br3 cells that had been transfected with a construct containing a luciferase reporter gene driven by a wild-type Her-2/neu promoter fragment with GLA (10 µg/mL for 48 hours) led to a strong reduction in reporter gene expression in both lines (Table 1).


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Table 1.  Luciferase activity in transiently transfected cells*

 
For semiquantitative RT-PCR analyses, we treated BT-474 and SK-Br3 cells with varying concentrations of GLA (5, 10, or 20 µg/ml for 48 hours) and then extracted total RNA from the cells. One microgram of total RNA was then reversed transcribed and amplified with specific primers for Her-2/neu, and the products were separated on agarose gels (Fig. 1, C). We observed strong, dose-dependent decreases in transcription of the Her-2/neu gene in both cells lines with GLA treatment.

We next investigated the possibility that GLA-induced repression of Her-2/neu transcription is mediated by the DNA binding protein PEA3. This protein, a member of the Ets transcription factor family, specifically targets a DNA sequence in the Her-2/neu promoter, thus suppressing Her-2/neu overexpression in cancer cells (3638). Immunoblot analysis of BT-474 and SK-Br3 cells that were treated for 48 hours with 10 µg/mL GLA showed an increase in the levels of PEA3 protein in both cell lines relative to levels in control-treated cells (Fig. 1, B).

To examine whether the increased PEA3 levels might mediate the inhibition of Her-2/neu transcription in GLA-treated cells, we examined the effect of GLA on transcription from a Her-2/neu promoter bearing a mutated PEA3 binding sequence, at –33 to –28, that is known to abolish PEA3 binding (36). For this analysis we used the same luciferase reporter gene construct described above but containing the HER-2/neu promoter mutation at the PEA3 binding site (5'-GAGGAA-3' to 5'-GAGCTC-3'). The mutant promoter was much less active than the wild-type promoter in untreated control cells (Table 1), supporting the notion that a PEA3 binding site on the Her-2/neu promoter acts as a positive regulatory element necessary for elevated expression of Her-2/neu oncogene in cancer cells (3537). In addition, transcription from the mutant promoter was not reduced in GLA-treated cells.

To gain insight into the possible role of PEA3 in GLA-mediated repression of Her-2/neu transcription, we evaluated Her-2/neu and PEA3 protein levels by immunoblot analysis in a panel of cancer cells with high or low levels of endogenous Her-2/neu (Fig. 1, D). In MDA-MB-453 breast cancer, SK-OV3 ovarian cancer, and NCI-N87 gastrointestinal carcinoma cells, all of which overexpress Her-2/neu, levels of PEA3 protein were low to undetectable in control cells. In cells treated with GLA (10 µg/mL for 48 hours), by contrast, Her-2/neu protein levels were markedly decreased and PEA3 protein expression was increased. In transient transfection experiments with the luciferase reporter gene construct, the activity of the Her-2/neu promoter bearing the intact PEA3 binding site was strongly reduced in all three cell lines after GLA treatment relative to control treatment (Table 1). In addition, mutation of the PEA3 binding site greatly reduced reporter gene activity in untreated cells, and GLA exposure did not further reduce reporter gene activity (Table 1). The effects of GLA were different in MDA-MB-231 breast cancer cells, which naturally express low to undetectable levels of Her-2/neu (Fig. 1, D). First, GLA exposure did not affect Her-2/neu protein levels in these cells. Second, MDA-MB-231 cells constitutively exhibited high levels of PEA3 protein, and these levels were not changed substantially following GLA exposure. Finally, the transcriptional activity of the Her-2/neu promoter was reduced only marginally by either GLA treatment or mutation of the PEA3 binding sequence (Table 1).

GLA has been shown to sensitize cells to the effects of other anticancer therapies, including antimitotic drugs such as paclitaxel, docetaxel, and vinorelbine, and antiestrogens such as tamoxifen and ICI 182,780 (69). We therefore investigated whether GLA-induced transcriptional repression of Her-2/neu affects the growth-inhibitory effects of trastuzumab, a humanized monoclonal antibody that binds with high affinity to Her-2/neu and has therapeutic effects in patients with Her-2/neu–positive breast cancer (3941). For these analyses, we determined apoptosis in BT-474 cells treated with GLA and/or trastuzumab by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick and labeling (TUNEL) using the DeadEnd Fluorometric TUNEL System (Promega Inc., Madison, WI). Immunofluorescence microscopy revealed many strongly positive nuclei in BT-474 cells treated with both drugs, whereas such nuclei were rare in untreated, GLA-treated, and trastuzumab-treated cells (Fig. 1, E, top panel). Counts of apoptotic nuclei from four random fields indicated that 5% (95% confidence interval [CI] = 4% to 6%) of the cells underwent apoptosis in the presence of 10 µg/mL GLA, 13% (95% CI = 11% to 15%) of cells treated with trastuzumab underwent apoptosis, and 38% (95% CI = 32% to 44%) of cells treated with trastuzumab plus GLA underwent apoptosis (Fig. 1, E). In SK-Br3 cells, those figures were 3% (95% CI= 2 to 4%), 11% (95% CI = 9% to 13%), and 43% (95% CI = 39% to 47%), respectively. A two-way analysis of variance (ANOVA) showed that concurrent exposure to GLA and trastuzumab synergistically increased the apoptotic effects achieved with GLA and trastuzumab as single agents (Fig. 1, E, bottom panel).

We also analyzed synergism with 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT)–based cell viability assays and the isobologram analysis (see Supplemental Figure 1 available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue21). Concurrent administration of 10 µg/ml GLA increased the sensitivity of BT-474 and SK-Br3 cells to trastuzumab by approximately 30- and 40-fold, respectively, and two-way ANOVAs showed that these increases were statistically significant. This combination yielded combination index50 (CI50) values of 0.697 and 0.615 in BT-474 and SK-Br3 cells, respectively, thus suggesting that the interaction was truly synergistic (CI50 = 1, additive; CI50 <1, synergism). When we used soft-agar assays to investigate the actions of GLA on Her-2/neu-induced anchorage-independent cancer cell growth (23,24), a two-way ANOVA showed that GLA cotreatment enhanced the growth-inhibitory effects of suboptimal doses of trastuzumab in a statistically significant manner in both BT-474 and SK-Br3 breast cancer cell lines (see Supplemental Figure).

Together, our results suggest that GLA-promoted accumulation of PEA3, a potent repressor of the Her-2/neu promoter (3638), is a key mechanism underlying GLA-induced suppression of Her-2/neu overexpression in cancer cells. It is possible that GLA activation of other factors that interact with the Her-2/neu promoter, such as AP-2, may account for the reduced Her-2/neu promoter activity in GLA-treated cells; however, recent studies suggest that AP-2-regulated Her-2/neu promoter regions have different roles in breast and non–breast cancer cells (4245) and we found that GLA does not appear to regulate AP-2 expression (data not shown). Whether GLA-induced transcriptional repression of Her-2/neu oncogene may represent a potential molecular approach to treating Her-2/neu–overexpressing carcinomas, i.e., in combination with trastuzumab, is a possibility that can be tested only after extensive preclinical and clinical studies of GLA. Considering that GLA mitigates Her-2/neu overexpression via PEA3 binding to the Her-2/neu promoter, GLA's anti-Her-2/neu actions should not be affected by the mechanisms of resistance described for trastuzumab (46,47). Therefore, our results raise the possibility that GLA-induced transcriptional repression of the Her-2/neu oncogene may represent a molecular approach to treat Her-2/neu–overexpressing carcinomas, i.e., in combination with trastuzumab. Although extensive preclinical and clinical studies are necessary before GLA can enter clinical trials, our findings suggest that future studies assessing the clinical relevance of GLA-regulated Her-2/neu promoter activity are warranted.


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This work was supported by the Basic, Clinical and Translational Award (BRCTR0403141) from the Susan G. Komen Breast Cancer Foundation (United States), the Breast Cancer Concept Award (BC033538) from the Department of Defense (United States), and by the Special Program For Research Excellence in Breast Cancer Career Development award P50CA89018-03 (to J. A. Menendez) and the RO1 Project of the Special Program For Research Excellence in Breast Cancer Career Development awards P50CA89018–03 (to R. Lupu).

The authors wish to thank Prof. Mien-Chie Hung (The University of Texas M. D. Anderson Cancer Center) for kindly providing Her-2/neu promoter constructs. Trastuzumab was kindly provided by the Evanston Northwestern Healthcare Hospital Pharmacy (Evanston, IL).

Funding to pay the Open Access publication charges for this article was provided by the Northwestern University Breast Cancer Special Program for Research Excellence (SPORE).


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Manuscript received December 21, 2004; revised September 6, 2005; accepted September 8, 2005.



             
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