HER2 (erbB-2)-targeted effects of the omega-3 polyunsaturated fatty acid, alpha-linolenic acid (ALA; 18:3n-3), in breast cancer cells: the "fat features" of the "Mediterranean diet" as an "anti-HER2 cocktail".
BACKGROUND
Data derived from epidemiological and experimental studies suggest that alphalinolenic acid (ALA; 18:3n-3), the main omega-3 polyunsaturated fatty acid (PUFA) present in the Western diet, may have protective effects in breast cancer risk and metastatic progression. A recent pilot clinical trial assessing the effects of ALA-rich dietary flaxseed on tumor biological markers in postmenopausal patients with primary breast cancer demonstrated significant reductions in tumor growth and in HER2 (erbB-2) oncogene expression.


OBJECTIVE
The molecular mechanism by which ALA inhibits breast cancer cell growth and metastasis formation may involve a direct regulation of HER2, a well-characterized oncogene playing a key role in the etiology, progression and response to some chemo- and endocrine therapies in approximately 20% of breast carcinomas.


METHODS
Using HER2-specific ELISA, flow cytometry, immunofluorescence microscopy, Western blotting, RT-PCR and HER2 promoter-reporter analyses, we characterized the effects of exogenous supplementation with ALA on the expression of HER2 oncogene, a master key player in the onset and metastasis formation of breast cancer disease. Metabolic status (MTT) assays were performed to evaluate the nature of the cytotoxic interaction between ALA and the humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin). To study these issues we used BT-474 and SKBr-3 breast cancer cells, which naturally exhibit amplification of the HER2 oncogene.


RESULTS
ALA treatment dramatically suppressed the expression of HER2-coded p185Her-2/neu oncoprotein as determined by ELISA, flow cytometry, immunofluorescence microscopy and immunoblotting techniques. Interestingly, ALA-induced down-regulation of p185Her-2/neu correlated with a transcriptional response as no HER2 mRNA signal could be detected by RT-PCR upon treatment with optimal concentrations of ALA (up to 20 microM). Consistent with these findings, ALA exposure was found to dramatically repress the activity of a Luciferase reporter gene driven by the HER2 promoter. Moreover, the nature of the cytotoxic interaction between ALA and trastuzumab (Herceptin) revealed a significant synergism as assessed by MTT-based cell viability assays.


CONCLUSIONS
i) These findings reveal that the omega-3 PUFA ALA suppresses overexpression of HER2 oncogene at the transcriptional level, which, in turn, interacts synergistically with anti-HER2 trastuzumab- based immunotherapy. ii) Our results molecularly support a recent randomized double-blind placebo-controlled clinical trial suggesting that ALA may be a potential dietary alternative or adjunct to currently used drugs in the management of HER2-positive breast carcinomas. iii) Considering our previous findings demonstrating the <<HER2 upregulatory actions>> of the omega-6 PUFA linolenic acid (LA; 18:2n-6) and the <<HER2 down-regulatory actions >> of the omega-3 PUFA docosahexaenoic acid (DHA; 22:6n-3) and of the omega-9 monounsaturated fatty acid oleic acid (OA; 18:1n-9), it is reasonable to suggest that a low omega-6/omega-3 PUFA ratio and elevated MUFA levels, the two prominent <<fat features>> of the <<Mediterranean diet>>, should be extremely efficient at blocking HER2 expression in breast cancer cells.
Menéndez
Javier A
JA
Fundació d'Investigació Biomédica de Girona Dr. Josep Trueta (IdIBGi), Girona, Catalonia. Spain. javiermenendez72@yahoo.com
Vázquez-Martín
Alejandro
A
Ropero
Santiago
S
Colomer
Ramón
R
Lupu
Ruth
R
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000517:prevention & control; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000517:prevention & control; D045744:Cell Line, Tumor / Q000187:drug effects* / Q000378:metabolism; D038441:Diet, Mediterranean; D004042:Dietary Fats, Unsaturated / Q000032:analysis; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D004790:Enzyme Induction / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D015525:Fatty Acids, Omega-3 / Q000032:analysis; D043371:Fatty Acids, Omega-6 / Q000032:analysis; D005260:Female; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D005784:Gene Amplification / Q000187:drug effects; D018734:Genes, erbB-2 / Q000187:drug effects*; D006801:Humans; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000068878:Trastuzumab; D014810:Vitamin E / Q000494:pharmacology; D017962:alpha-Linolenic Acid / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D004042:Dietary Fats, Unsaturated; D015525:Fatty Acids, Omega-3; D043371:Fatty Acids, Omega-6; D009363:Neoplasm Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; D014810:Vitamin E; D017962:alpha-Linolenic Acid