Antitumoral actions of the anti-obesity drug orlistat (Xenical™) in breast cancer cells: blockade of cell cycle progression, promotion of apoptotic cell death and PEA3-mediated transcriptional repression of Her2/neu (erbB-2) oncogene

Background: Orlistat (Xenicale), a US Food and Drug Administration (FDA)-approved drug for bodyweight loss, has recently been demonstrated to exhibit antitumor properties towards prostate cancer cells by virtue of its ability to block the lipogenic activity of fatty acid synthase (FAS). FAS (oncogenic antigen-519) is up-regulated in about 50% of breast cancers, is an indicator of poor prognosis, and has recently been functionally associated with the Her2/neu (erbB-2) oncogene. Materials and methods: We assessed the antitumoral effects of orlistat against the human breast cancer cell line SK-Br3, an in vitro paradigm of FAS and Her2/neu overexpression in breast cancer. Results: Cell cycle analyses revealed that micromolar concentrations of orlistat induced, in a timeand dose-dependent manner, significant changes in the distribution of cell populations including a complete loss of G2-M phase, S-phase accumulation and a concomitant increase in the emerging sub-G1 (apoptotic) cells. Poly (ADP-ribose) polymerase (PARP) cleavage, an early event required for cells committed to apoptosis, was more predominant in orlistat-treated G1 phase cells. When we characterized signaling molecules participating in the cellular events following orlistat-induced inhibition of FAS activity and preceded inhibition of breast cancer cell proliferation, a dramatic downregulation of Her2/neu-coded p185 Her2/neu oncoprotein was found in orlistat-treated SK-Br3 cells (>90% reduction). Interestingly, a significant accumulation of the DNA-binding protein PEA3, a member of the Ets transcription factor family that specifically targets a PEA3-binding motif present on the Her2/neu gene promoter and down-regulates its activity, was observed in orlistat-treated SKBr3 cells. When a Luciferase reporter gene driven by the Her2/neu promoter was transiently transfected in SK-Br3 cells, orlistat exposure was found to dramatically repress the promoter activity of Her2/neu gene, whereas a Her2/neu promoter bearing a mutated binding DNA sequence was not subject to negative regulation by orlistat, thus demonstrating that the intact PEA3 binding site on the Her2/neu promoter is required for the orlistat-induced transcriptional repression of Her2/neu overexpression. RNA interference (RNAi)-mediated silencing of FAS gene expression similarly repressed Her2/neu gene expression in a PEA3-dependent manner, thus ruling out a role for non-FAS orlistat-mediated effects. When the combination of orlistat and the anti-Her2/neu antibody trastuzumab (Herceptine) in either concurrent (orlistat +trastuzumab) or sequential (orlistat ! trastuzumab; trastuzumab ! orlistat) schedules was tested for synergism, addition or antagonism using the combination index (CI) method of Chou‐Talalay, co-exposure of orlistat and trastuzumab demonstrated strong synergistic effects (CI10‐90=0.110‐0.847), whereas sequential exposure to orlistat followed by trastuzumab (CI10‐90=0.380‐1.210) and trastuzumab followed by orlistat (CI10‐90=0.605‐1.278) mainly showed additive or antagonistic interactions. Indeed, orlistatinduced FAS inhibition synergistically promoted apoptotic cell death when concurrently combined with trastuzumab as determined by an ELISA for histone-associated DNA fragments. Importantly, the degree of FAS expression in a panel of human breast cancer cell lines was predictive of sensitivity to orlistat-induced anti-proliferative effects as determined by a MTT-based characterization of