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 |
---|
![]() ![]() ![]() ![]() |
---|
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/neucoded 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 surfaceassociated 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).
|
|
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/neupositive 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 nonbreast 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/neuoverexpressing 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/neuoverexpressing 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.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() |
---|
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).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
(1) Horrobin DF. Unsaturated lipids and cancer. In: Horrobin DF, editor. New approaches to cancer treatment. Edinburgh (Scotland): Churchill; 1994. p. 129.
(2) Das UN. Gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid as potential anticancer drugs. Nutrition 1990;6:42934.[ISI][Medline]
(3) Jiang WG, Bryce RP, Horrobin DF. Essential fatty acids: molecular and cellular basis of their anti-cancer action and clinical implications. Crit Rev Oncol Hematol 1998;27:179209.[ISI][Medline]
(4) Begin ME, Ells G, Das UN, Horrobin DF. Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 1986;77:105362.[ISI][Medline]
(5) Begin ME, Ells G, Horrobin DF. Polyunsaturated fatty acid-induced cytotoxicity against tumor cells and its relationship to lipid peroxidation. J Natl Cancer Inst 1988;80:18894.[Abstract]
(6) Menendez JA, del Mar Barbacid M, Montero S, Sevilla E, Escrich E, Solanas M, et al. Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells. Eur J Cancer 2001;37:40213.[ISI][Medline]
(7) Menendez JA, Ropero S, del Barbacid MM, Montero S, Solanas M, Escrich E, et al. Synergistic interaction between vinorelbine and gamma-linolenic acid in breast cancer cells. Breast Cancer Res Treat 2002;72:20319.[ISI][Medline]
(8) Menendez JA, Ropero S, Lupu R, Colomer R. Omega-6 polyunsaturated fatty acid gamma-linolenic acid (18:3n-6) enhances docetaxel (Taxotere) cytotoxicity in human breast carcinoma cells. Oncol Rep 2004;11:124152.[ISI][Medline]
(9) Menendez JA, Colomer R, Lupu R. Omega-6 polyunsaturated fatty acid gamma-linolenic acid (18:3n-6) is a selective estrogen-response modulator in human breast cancer cells: gamma-linolenic acid antagonizes estrogen receptor-dependent transcriptional activity, transcriptionally represses estrogen receptor expression and synergistically enhances tamoxifen and ICI 182,780 (Faslodex) efficacy in human breast cancer cells. Int J Cancer 2004;109:94954.[CrossRef][ISI][Medline]
(10) Kenny FS, Pinder SE, Ellis IO, Gee JM, Nicholson RI, Bryce RP, et al. Gamma linolenic acid with tamoxifen as primary therapy in breast cancer. Int J Cancer 2000;85:64348.[CrossRef][ISI][Medline]
(11) van der Merwe CF, Booyens J, Joubert HF, van der Merwe CA. The effect of gamma-linolenic acid, an in vitro cytostatic substance contained in evening primrose oil, on primary liver cancer: a double-blind placebo controlled trial. Prostaglandins Leukot Essent Fatty Acids 1990;40:199202.[CrossRef][ISI][Medline]
(12) Fearon KC, Falconer JS, Ross JA, Carter DC, Hunter JO, Reynolds PD, et al. An open-label phase I/II dose escalation study of the treatment of pancreatic cancer using lithium gammalinolenate. Anticancer Res 1996;16:86774.[ISI][Medline]
(13) Harris NM, Crook TJ, Dyer JP, Solomon LZ, Bass P, Cooper AJ, Birch BR. Intravesical meglumine gamma-linolenic acid in superficial bladder cancer: an efficacy study. Eur Urol 2002;42:3942.[CrossRef][ISI][Medline]
(14) Harris NM, Anderson WR, Lwaleed BA, Cooper AJ, Birch BR, Solomon LZ. Epirubicin and meglumine gamma-linolenic acid: a logical choice of combination therapy for patients with superficial bladder carcinoma. Cancer 2003;97:718.[CrossRef][ISI][Medline]
(15) Das UN. Occlusion of infusion vessels on gamma-linolenic acid infusion. Prostaglandins Leukot Essent Fatty Acids 2004;70:2332.[CrossRef][ISI][Medline]
(16) Menendez JA, Ropero S, Lupu R, Colomer R. Dietary fatty acids regulate the activation status of Her-2/neu (c-erbB-2) oncogene in breast cancer cells. Ann Oncol 2004;15:171921.
(17) Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:17782.[ISI][Medline]
(18) Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:70712.[ISI][Medline]
(19) Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology 2001;61 Suppl 2:113.
(20) Ross JS, McKenna BJ. The HER-2/neu oncogene in tumors of the gastrointestinal tract. Cancer Invest 2001;19:55468.[CrossRef][ISI][Medline]
(21) Xu R, Perle MA, Inghirami G, Chan W, Delgado Y, Feiner H. Amplification of Her-2/neu gene in Her-2/neu-overexpressing and -nonexpressing breast carcinomas and their synchronous benign, premalignant, and metastatic lesions detected by FISH in archival material. Mod Pathol 2002;15:11624.[CrossRef][ISI][Medline]
(22) Hoque A, Sneige N, Sahin AA, Menter DG, Bacus JW, Hortobagyi GN, et al. Her-2/neu gene amplification in ductal carcinoma in situ of the breast. Cancer Epidemiol Biomarkers Prev 2002;11:58790.
(23) Tan M, Yao J, Yu D. Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities. Cancer Res 1997;57:1199205.[Abstract]
(24) Eccles SA. The role of c-erbB-2/HER2/neu in breast cancer progression and metastasis. J Mammary Gland Biol Neoplasia 2001;6: 393406.[CrossRef][ISI][Medline]
(25) Chen X, Yeung TK, Wang Z. Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3. Biochem Biophys Res Commun 2000;277:75763.[CrossRef][ISI][Medline]
(26) Orr MS, O'Connor PM, Kohn KW. Effects of c-erbB2 overexpression on the drug sensitivities of normal human mammary epithelial cells. J Natl Cancer Inst 2000;92:98794.
(27) Nelson NJ. Can HER2 status predict response to cancer therapy? J Natl Cancer Inst 2000;92:36667.
(28) Valagussa P. HER2 status: a statistician's view. Ann Oncol 2001;12 Suppl 1:S29S34.[Medline]
(29) Yamauchi H, Stearns V, Hayes DF. The role of c-erbB-2 as a predictive factor in breast cancer. Breast Cancer 2001;8:17183.[Medline]
(30) Yu D, Hung MC. Role of erbB2 in breast cancer chemosensitivity. Bioessays 2000;22: 67380.[CrossRef][ISI][Medline]
(31) Yu D, Hung MC. Overexpression of ErbB2 in cancer and ErbB2-targeting strategies. Oncogene 2000;19:611521.[CrossRef][ISI][Medline]
(32) Dowsett M. Overexpression of HER-2 as a resistance mechanism to hormonal therapy for breast cancer. Endocr Relat Cancer 2001;8:19195.
(33) Jarvinen TA, Tanner M, Rantanen V, Barlund M, Borg A, Grenman S, et al. Amplification and deletion of topoisomerase IIalpha associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer. Am J Pathol 2000;156:83947.
(34) Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 2002;62:413241.
(35) Kraus MH, Popescu NC, Amsbaugh SC, King CR. Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J 1987;6:60510.[Abstract]
(36) Xing X, Wang SC, Xia W, Zou Y, Shao R, Kwong KY, et al. The ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis. Nat Med 2000;6:18995.[CrossRef][ISI][Medline]
(37) Wang SC, Hung MC. Transcriptional targeting of the HER-2/neu oncogene. Drugs Today (Barc). 2000;36:83543.[Medline]
(38) Menendez JA, Vellon L, Mehmi I, Oza BP, Ropero S, Colomer R, et al. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc Natl Acad Sci USA 2004;101:1071520.
(39) Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992;89:42859.
(40) Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 2002;20:71926.
(41) Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:78392.
(42) Bosher JM, Williams T, Hurst HC. The developmentally regulated transcription factor AP-2 is involved in c-erbB-2 overexpression in human mammary carcinoma. Proc Natl Acad Sci USA 1995;92:7447.
(43) Bosher JM, Totty NF, Hsuan JJ, Williams T, Hurst HC. A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma. Oncogene 1996;13:17017.[ISI][Medline]
(44) Hurst HC. Update on HER-2 as a target for cancer therapy: the ERBB2 promoter and its exploitation for cancer treatment. Breast Cancer Res 2001;3:3958.[CrossRef][ISI][Medline]
(45) Vernimmen D, Gueders M, Pisvin S, Delvenne P, Winkler R. Different mechanisms are implicated in ERBB2 gene overexpression in breast and in other cancers. Br J Cancer 2003;89:899906.[CrossRef][ISI][Medline]
(46) Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst 2001;93:18527.
(47) Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:11727.[CrossRef][ISI][Medline]
Manuscript received December 21, 2004; revised September 6, 2005; accepted September 8, 2005.
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |