Pharmacological and small interference RNA‐mediated inhibition of breast cancer‐associated fatty acid synthase (oncogenic antigen‐519) synergistically enhances Taxol (paclitaxel)‐induced cytotoxicity

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. © 2004 Wiley‐Liss, Inc.