Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors

Abstract

The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10⁻⁴ M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10⁻⁸, 10⁻⁶ M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.