Activity and expression of
fatty acid synthase (FAS), a critical
enzyme in the de novo biosynthesis of
fatty acids in mammals, is exquisitely sensitive to nutritional regulation of lipogenesis in liver or adipose tissue. Surprisingly, a number of studies have demonstrated hyperactivity and overexpression of FAS (oncogenic antigen-519) in a biologically aggressive subset of human
breast carcinomas, suggesting that FAS-dependent neoplastic lipogenesis is unresponsive to nutritional regulation. We have assessed the role of omega-3 and omega-6
polyunsaturated fatty acids (PUFAs) on the enzymatic activity and
protein expression of
tumor-associated FAS in SK-Br3 human
breast cancer cells, an experimental paradigm of FAS-overexpressing
tumor cells in which FAS
enzyme constitutes up to 28%, by weight, of the cytosolic
proteins. Of the omega-3 PUFAs tested,
alpha-linolenic acid (ALA) dramatically reduced FAS activity in a dose-dependent manner (up to 61%). omega-3 PUFA
docosahexaenoic acid (DHA) demonstrated less marked but still significant inhibitory effects on FAS activity (up to 37%), whereas
eicosapentaenoic acid (EPA) was not effective. Of the
omega-6 fatty acids tested,
gamma-linolenic acid (GLA) was the most effective dose-dependent inhibitor of FAS activity, with a greater than 75% FAS activity reduction. Remarkably, omega-6 PUFAs
linoleic acid (LA) and
arachidonic acid (ARA), suppressors of both hepatic and adipocytic FAS-dependent lipogenesis, had no significant inhibitory effects on the activity of
tumor-associated FAS in SK-Br3
breast cancer cells. Western blotting studies showed that down-regulation of FAS
protein expression tightly correlated with previously observed inhibition of FAS activity, suggesting that ALA-, DHA-, and GLA-induced changes in FAS activity resulted from effects at the
protein level. We investigated whether the FAS inhibitory effect of GLA and omega-3 PUFAs correlated with a cytotoxic effect related to a peroxidative mechanism. Measurement of cell viability by MTT assay indicated a significant cellular toxicity after ALA and GLA exposures. Furthermore, we observed a significant correlation between the ability of PUFAs to repress FAS and cause cell toxicity. In the presence of
anti-oxidants (
vitamin E), ALA and GLA dramatically lost their ability to inhibit FAS activity. Interestingly, a combination of ALA and GLA was FAS inhibitory in an additive manner, and this FAS repression was only partially reversible by
vitamin E. In examining the molecular mechanisms underlying resistance of
breast cancer-associated FAS to normal dietary
fatty acid-induced suppression, a dramatic decrease of FAS accumulation was found after exposure of SK-Br3 cells to
mitogen-activated protein kinase (MAPK)
extracellular signal-regulated kinase (MAPK ERK1/2) inhibitor
U0126, phosphatidylinositol-3'-kinase (PI-3'K) blocker
LY294002, and/or anti-HER-2/neu antibody
trastuzumab. Interestingly, a long-term exposure to pharmacological inhibitors of FAS activity
cerulenin [(2S,3R) 2,3-epoxy-4-oxo-7E,10E-dodecadienamide] or C75 also resulted in a significant reduction of FAS accumulation. These data indicate that: a) GLA- and omega-3 PUFA-induced repression of
tumor-associated FAS may result, at least in part, from a non-specific cytotoxic effect due to peroxidative mechanisms; b) alternatively, GLA and omega-3 PUFAs have a suppressive effect on FAS expression and activity that can result in the accumulation of toxic fluxes of the FAS substrate
malonyl-CoA; c) GLA- and/or omega-3 PUFA-induced repression of
tumor-associated FAS may represent a novel mechanism of PUFA-induced cytotoxicity clinically useful against
breast carcinomas carrying overexpression of FAS
enzyme; d) fundamental differences in the ability of FAS gene to respond to normal
fatty acid's regulatory actions in lipogenic tissues may account for the observed extremely high levels of FAS in
breast carcinoma; and e) FAS overexpression in SK-Br3
breast cancer cells is driven by increases in HER-2/neu signaling, acting in major part through a constitutive downstream art through a constitutive downstream activation of the MAPK ERK1/2 and PI-3'K/AKT transduction cascades.