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* Department of Medicine, Breast Cancer Translational Research Program, Evanston Northwestern Healthcare Research Institute, Evanston, Illinois; and Department of Medicine, Northwestern University Feinberg School of Medicine, The Lurie Comprehensive Cancer Center, Chicago, Illinois
1 To whom requests for reprints should be addressed at Department of Medicine, Northwestern University Feinberg School of Medicine, The Lurie Comprehensive Cancer Center, Chicago, Illinois; or Breast Cancer Translational Research Program, Evanston Northwestern Healthcare Research Institute, 1001 University Place, Evanston, Illinois 60201. E-mail: jmenendez{at}enh.org; r-lupu{at}northwestern.edu
The U.S. Food and Drug Administration (FDA)-approved antiobesity drug Orlistat (marketed by Roche [Basel, Switzerland] as Xenical) is a semisyn-thetic derivative of lipstatin that irreversibly inhibits pancreatic and gastric lipases within the gastrointestinal (GI) tract (1). Interestingly, an intense public interest has been generated by the characterization of Orlistat as a potent inhibitor of prostate and breast tumor growth (2). Mechanistically, it has been demonstrated that Orlistat exhibits antiproliferative and antitumor properties toward prostate and breast cancer cells by virtue of its ability to block the lipogenic activity of fatty acid synthase (FAS) (2, 3). Fatty acid synthase is the key metabolic muti-enzyme that is responsible for the terminal catalytic step in the de novo fatty acid biosynthesis (4). Upregulation of FAS expression and activity, an anabolic-energy-storage pathway largely considered of minor importance in humans, is a very early and nearly universal neoplastic marker that positively correlates with aggressive behaviors and poorer clinical outcome prognoses in many human cancers (5). Interestingly, we recently demonstrated that cancer-associated FAS, which to date appeared to be part of a growth factor-driven pleiotropic change in the genetic program controlling lipogenesis, actively contributes to the cancer phenotype by regulating the expression, activity, and cellular localization of the Her-2/neu (erbB-2) oncogene (6), a master player in the etiology and aggressive behavior of several cancers (7).
Because Orlistat possesses extremely low oral bioavailability, a novel formulation will be required for treating tumors such as prostate or breast carcinomas. However, we hypothesized that Orlistat-induced inhibition of FAS activity may represent a valuable therapeutic strategy to test in GI tumors overexpressing Her-2/neu oncogene, that is, associated with approximately one fourth of all GI tract malignancies (8). To test this hypothesis, we employed the highly metastatic and Her-2/neu-overexpressing NCI-N87 stomach carcinoma cell line. Micromolar concentrations of Orlistat (up to 40 µM) induced dose-dependent antiproliferative effects against NCI-N87 cells when relative cell numbers were determined using a 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT)-based characterization of metabolically viable cells (Fig. 1a
). Cell cycle analyses revealed that Orlistat exposure induced a complete loss of G2-M cell population with a concomitant increase of cells in G1 by 48 hrs. This G1 blockade of Orlistat-treated NCI-N87 cells was accompanied with significant increases (~3-fold) of cells in the emerging sub-G1 (apoptotic) cell population (Fig. 1b). Moreover, flow cytometric analyses established the ability of Orlistat (10 µM, 48-hr exposure) to dramatically decrease (>90% reduction) the expression levels of Her-2/neu-coded p185Her-2/neu oncoprotein in NCI-N87 cells (Fig. 1c). To gain additional insight into the molecular mechanisms underlying the downregulation of Her-2/neu induced by Orlistat, we examined the expression of Her-2/neu regulator PEA3 following Orlistat-induced blockade of FAS activity in NCI-N87 cells. The DNA-binding protein PEA3, a member of the Ets transcription factor family, specifically targets a DNA sequence on the Her-2/neu promoter and downregulates its promoter activity, thus suppressing Her-2/neu overexpression and inhibiting tumorigenesis (9). Interestingly, a significant accumulation of PEA3 protein was observed in immunoblotting analyses of Orlistat-treated NCI-N87 cells (Fig. 1d). We next tested whether Orlistat-induced accumulation of PEA3 correlated with a transcriptional response of Her-2/ neu gene. When a luciferase reporter gene driven by the Her-2/neu promoter (pNulit) was transiently transfected into the NCI-N87 stomach carcinoma cell line, Orlistat exposure for 48 hrs was found to significantly repress the promoter activity of Her-2/neu by ~60% (Fig. 2a, left panel). When we characterized Orlistat’s effects on the activity of a Her-2/ neu promoter bearing a mutated PEA3 binding sequence (5'-AGGAAG-3' to 5'-AGCTCG-3'), we concluded that the intact PEA3 binding site on the Her-2/neu promoter is required for the Orlistat-mediated transcriptional repression of Her-2/neu gene expression in NCI-N87 cells. Thus, the luciferase reporter gene driving the PEA3 site-mutated sequence was not subject to negative regulation by Orlistat (Fig. 2a, right panel). Importantly, RNA interference (RNAi)-mediated silencing of the FAS expression following transfection with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene (6) similarly repressed Her-2/neu promoter activity in a PEA3-dependent manner (Fig. 2a), thus ruling out a role for non-FAS Orlistat-mediated effects in Her-2/neu downregulation. These findings, altogether, constitute an independent strategy to confirm our hypothesis that FAS inhibition is accompanied by the specific suppression of Her-2/neu oncogene expression in cancer cells (6).
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). Because the cyclin-dependent kinase inhibitor (CDKi) p27Kip1 plays a key role in the onset and progression of Her-2/neu-related carcinomas (10), low p27Kip1 expression is regarded as an important adverse prognostic factor in GI cancers (11), and trastuzumab resistance may be associated with decreased p27Kip1 levels and may be susceptible to treatments that induce p27Kip1 expression (12), we finally examined the expression levels of p27kip1 following Orlistat treatment. NCI-N87 cells exposed to 10 µM Orlistat over a 48-hr time course demonstrated a dramatic accumulation of the p27Kip1 protein concomitantly with Orlistat-induced downregulation of p185Her-2/neu expression (Fig. 2c). Although caution must be applied when extrapolating in vitro results into clinical practice, the significant cytotoxicity, the blockade of cell cycle progression, and the dramatic repression of Her-2/neu expression that occur following Orlistat treatment strongly suggest that this selective lipase inhibitor used for treating obesity may represent a suitable drug candidate for treating Her-2/neu-overexpressing GI carcinomas through its ability to block tumor-associated FAS hyperactivity. Moreover, Orlistat supra-additively interacts with trastuzumab-based anti-Her-2/neu immunotherapy, which is associated with a PEA3-dependent transcriptional repression of Her-2/neu gene and an increase in p27Kip1 protein levels, thus providing appropriate surrogate markers to follow treatment efficacy in vivo. Although the efficacy of Orlistat in a wider panel of GI carcinoma cell lines expressing different amounts of Her-2/neu remains to be elucidated, we recently observed that MCF-7 breast cancer cells, which express physiological amounts of Her-2/neu oncogene, became hypersensitive to Orlistat when they were engineered to overexpress Her-2/ neu (data not shown). These findings, altogether, support the concept that inhibition of FAS activity may provide a new molecular avenue for chemotherapeutic prevention and/or treatment of Her-2/neu-dependent carcinomas (6, 13). Considering that studies evaluating Orlistat’s long-term effects have begun to emerge (14), future analyses of the incidence and clinical outcome of GI carcinomas in a representative obese population earlier receiving Orlistat treatment may illustrate further the chemopreventive effects of this statin to GI tumors.
Acknowledgments
We thank Dr. Mien-Chie Hung (The University of Texas M.D. Anderson Cancer Center, Department of Cancer Biology, Houston, TX) for kindly providing Her-2/neu promoter constructs. Javier A. Menendez is the recipient of a Translational Research Pilot Project (PP2) and of a Career Development Award from the Specialized Program of Research Excellence—SPORE—in Breast Cancer (Robert H. Lurie Comprehensive Cancer Center, Chicago, U.S.A.), of a Basic, Clinical and Translational Award (BRCTR0403141) from the Susan G. Komen Breast Cancer Foundation (U.S.A.), and of a Breast Cancer Concept Award (BC033538) from the Department of Defense (U.S.A).
References
This article has been cited by other articles:
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  M. Prentki and S. R. M. Madiraju Glycerolipid Metabolism and Signaling in Health and Disease Endocr. Rev., October 1, 2008; 29(6): 647 - 676. [Abstract] [Full Text] [PDF]
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 J. A. Menendez, L. Vellon, and R. Lupu Antitumoral actions of the anti-obesity drug orlistat (XenicalTM) 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 Ann. Onc., August 1, 2005; 16(8): 1253 - 1267. [Abstract] [Full Text] [PDF]
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