title: Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.
abstract: The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.
authors.affiliation: Department of Medicine, Breast Cancer Translational Research Laboratory, Evanston Northwestern Healthcare Research Institute (ENHRI), Evanston, IL 60201, USA. jmenendez@enh.org
authors.forename: Javier A
authors.initials: JA
authors.lastname: Menendez
authors.forename: Luciano
authors.initials: L
authors.lastname: Vellon
authors.forename: Ramon
authors.initials: R
authors.lastname: Colomer
authors.forename: Ruth
authors.initials: R
authors.lastname: Lupu
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D001943:Breast Neoplasms / Q000201:enzymology* / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D064429:Fatty Acid Synthases / Q000378:metabolism*; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009707:Nucleosomes / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D006657:Histones; D009707:Nucleosomes; D034741:RNA, Small Interfering; D012694:Serine; D064429:Fatty Acid Synthases; C482128:MAP2K2 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; D017239:Paclitaxel