Dietary fatty acids regulate the activation status of Her-2/neu (c-erbB-2) oncogene in breast cancer cells.

Research from several sources provides strong evidence that dietary or exogenously derived fatty acids (FAs) may play an important role in the etiology, evolution and/or progression of breast cancer [1]. However, the type of individual FAs in a diet, rather than the amount of total dietary fat, may be more important in breast cancer disease. In this regard, epidemiological, experimental and mechanistic data implicate v-9 oleic acid (OA; 18:1n-9), the main FA of olive oil, a-linolenic acid (ALA; 18:3n-3), the v-3 FA of vegetable oils, and eicosapentaneoic (EPA; 22:5n-3) and docosahexaenoic (DHA; 22:6n-3) FAs, the two main v-3 FAs of fish oils, as inhibitors of the development and progression of human breast cancer, and v-6 FAs such as linoleic acid (LA; 18:2n-6), the main FA of corn and sunflower oils, as stimulators of the disease. Moreover, one of the most interesting yet controversial dietary approaches has been the possible role of dietary FAs in treating breast cancer. However, little is know about the ultimate mechanisms underlying FA-regulated breast cancer risk and progression. Interestingly, we previously reported that exogenous supplementation with FAs such as g-linolenic acid (GLA; 18:3n-6), an essential FA found in the plant-seed oils of evening primrose, blackcurrant and borage oils, synergically sensitizes cultured breast cancer cells to anti-mitotic drugs such as paclitaxel (Taxole) [2], vinorelbine (Navelbinee) [3] and docetaxel (Taxoteree) [4]. Moreover, this GLA-induced sensitization was also synergic with the anti-estrogens tamoxifen and ICI 182,780 (Faslodexe) [5]. Since the human Her-2/neu (c-erb B-2) oncogene and its p185 orphan receptor oncoprotein, which are overexpressed in 25–30% of invasive breast cancers, have been associated with cytotoxic and endocrine therapy resistance [6], we recently envisioned that FA-induced breast cancer chemosensitization may involve regulation of Her-2/neu-dependent signaling. In the last few years it has become clearer that the activation status of Her-2/neu, and not just its overexpression, is a crucial event that determines both the aggressive biological behavior of breast carcinomas and the breast cancer response to chemotherapy, anti-estrogens and the anti-Her-2/neu antibody trastuzumab (Herceptine) [7, 8]. Although the mechanism of activation of the Her-2/neu oncoprotein is not completely understood, in vitro and in vivo Her-2/neu activation has been demonstrated to occur as a consequence of proteolytic cleavage of its extracellular domain (ECD), thereby resulting in the production of truncated membranebound fragment with kinase activity, a key event for downstream signaling. In this regard, we recently reported that Her-2/neu ECD expression correlates with a diminished efficacy of the combination chemotherapy of biweekly gemcitabine plus paclitaxel [9]. This study validated our previous results, showing that both the probability of obtaining a complete response and the duration of clinical response to a paclitaxel–doxorubicin chemotherapy regimen were significantly lower and shorter, respectively, in metastatic breast cancer patients with increased Her-2/neu ECD concentrations compared with the cases with non-increased concentrations [10]. The present investigation was performed to evaluate the effects of exogenous supplementation with dietary FAs on Her-2/neu ECD concentrations in cultured breast cancer cells. First, the Oncogene Science Her-2/neu Microtiter ELISA (Cambridge, MA, USA) was used to compare the baseline expression of Her-2/neu ECD in BT-474, SK-Br3, MCF-7 and MDA-MB-231 human breast cancer cell lines. This sandwichtype enzyme immunoassay utilizes two monoclonal antibodies, NB-3 and TA-1, that recognize the extracellular, ligand-binding domain of the Her-2/neu oncoprotein, and quantifies the Her-2/neu ECD in cell cultures. As expected, very high levels of Her-2/neu ECD [expressed as human Neu units (HNU) per mg of protein] were found in BT-474 and SK-Br3 breast cancer cells (105±12 and 95±11 HNU per mg protein, respectively), whereas MCF-7 (1.6 HNU/mg protein) and MDA-MB-231 (0.5 HNU/mg protein) cells contained physiological and low levels of Her-2/neu ECD, respectively. Thus, Her-2/neu ECD levels in Her-2/neu-overexpressing SKBr3 and BT-474 breast cancer cells were 200-fold higher than in Her-2/neu-negative MDA-MB-231 cells. To assess the effects of FAs on Her-2/neu ECD concentration, SK-Br3 and BT-474 cells, after a 24 h starvation period in media without serum but with 0.5% FA-free bovine serum albumin (BSA), were incubated for 48 h with 0.5% BSA FA-free BSA (controls) or various concentrations (1.25, 2.5, 5 and 10mM) of BSA-bound FAs (Table 1). Her-2/neu ECD levels in SK-Br3 cells were slightly decreased by 24% at 10mM ALA, while Her-2/neu ECD expression dramatically decreased by 53% with 10mM ALA in BT-474 cells. Culture of SK-Br3 cells with EPA significantly decreased Her-2/neu ECD expression by 47% at 10mM EPA. A less marked, but significant, decrease in Her-2/neu ECD expression was observed in BT-474 cultured in the presence of 10mM EPA (28% reduction). SK-Br3 cells treated with 10mM DHA showed a 38% decrease in Her-2/neu ECD expression. Annals of Oncology 15: 1719–1723, 2004