Inhibition of tumor-associated fatty acid synthase activity antagonizes estradiol- and tamoxifen-induced agonist transactivation of estrogen receptor (ER) in human endometrial adenocarcinoma cells
Inhibition of tumor-associated fatty acid synthase activity antagonizes estradiol- and tamoxifen-induced agonist transactivation of estrogen receptor (ER) in human endometrial adenocarcinoma cells
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DOI：
10.1038/sj.onc.1207476
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发表时间：
2004-06-17
期刊：
影响因子：
8
通讯作者：
Lupu, R
中科院分区：
文献类型：
--
作者：
Menendez, JA;Oza, BP;Lupu, R
脂肪生成酶脂肪酸合酶（FAS）的过度表达是性类固醇相关肿瘤亚群的一个常见分子特征，包括子宫内膜癌和乳腺癌，这些肿瘤与不良预后相关。肿瘤相关 FAS 过度活跃的药理学抑制作用正在作为化疗靶点进行研究。我们研究了霉菌毒素蓝藻素（一种共价 FAS 灭活剂）和新型小化合物 C75（一种缓慢结合的 FAS 抑制剂）对石川细胞（一种分化良好的人类子宫内膜癌的体外模型）中雌二醇 (E-2) 和他莫昔芬 (TAM) 刺激的 ER 驱动分子反应的影响。我们通过使用雌激素反应元件报告基因构建体（ERE-荧光素酶）进行瞬时共转染测定，评估了 FAS 抑制对 E-2 和 TAM 诱导的雌激素受体 (ER) 转录活性的影响。通过对 E-2 刺激的 ERE 依赖性转录的剂量依赖性抑制，观察到浅蓝素和 C75 的抗雌激素作用，而在 E-2 不存在的情况下，FAS 抑制剂不会显着增加 ERE 转录活性的水平。此外，FAS 活性的药理学阻断完全消除了 TAM 刺激的 ERE 活性。为了解决瞬时转染测定的可靠性问题，评估了 FAS 抑制剂对 E-2 诱导基因产物的影响。 FAS 阻断诱导 E-2-诱导型碱性磷酸酶活性呈剂量依赖性下降。 FAS 阻滞剂存在时，E-2 刺激的孕酮受体 (PR) 和 HER-2/neu 癌基因的积累被消除。 FAS 抑制还导致 E-2 刺激的 ERα 表达显着下调，并显着损害 E-2 诱导的 ERα 核积累。 FAS 阻断后观察到细胞增殖和细胞活力呈剂量依赖性下降。细胞死亡 ELISA 检测 DNA 片段，证明 FAS 抑制剂刺激 Ishikawa 细胞凋亡。对关键 E-2 和 TAM 相关细胞周期蛋白的分析表明，FAS 抑制后，细胞周期蛋白依赖性激酶抑制剂 p21(WAF1/CIP1) 和 p27(Kip1) 的表达和核积累均增加。为了排除非 FAS 浅蓝蛋白和 C75 相关的影响，我们最终使用高度序列特异性的 RNA 干扰 (RNAi) 机制在 FAS 基因表达沉默后监测 ER 信号传导。在 FAS RNAi 转染的 Ishikawa 细胞中，诱导半最大 ERE 活性 (EC50) 的 E-2 和 TAM 浓度显着增加（4100 倍）。此外，RNAi对FAS的消耗还导致E-2刺激的Ishikawa细胞中ERα表达的丧失、PR的下调以及p21(WAF1/CIP1)和p27(Kip1)的积累。如果化学稳定的 FAS 抑制剂或能够递送靶向 FAS 基因的 RNAi 的细胞选择性载体系统在体内表现出全身抗癌作用，我们的结果将使 FAS 成为预防和治疗子宫内膜癌的新靶点。
Overexpression of the lipogenic enzyme fatty acid synthase (FAS) is a common molecular feature in subsets of sex-steroid-related tumors including endometrium and breast carcinomas that are associated with poor prognosis. Pharmacological inhibition of tumor-associated FAS hyperactivity is under investigation as a chemotherapeutic target. We examined the effects of the mycotoxin cerulenin (a covalent FAS inactivator), and the novel small compound C75 ( a slow-binding FAS inhibitor) on estradiol (E-2)- and tamoxifen (TAM)-stimulated ER-driven molecular responses in Ishikawa cells, an in vitro model of well-differentiated human endometrial carcinoma. We evaluated the effects of FAS inhibition on E-2- and TAM-induced estrogen receptor ( ER) transcriptional activity by using transient cotransfection assays with an estrogen-response element reporter construct (ERE-Luciferase). Antiestrogenic effects of cerulenin and C75 were observed by dose-dependent inhibition of E-2- stimulated ERE-dependent transcription, whereas FAS inhibitors did not significantly increase the levels of ERE transcriptional activity in the absence of E-2. Moreover, pharmacological blockade of FAS activity completely abolished TAM-stimulated ERE activity. To address the reliability of transient transfection assays, the effects of FAS inhibitors on E-2- inducible gene products were evaluated. FAS blockade induced a dose-dependent decrease in E-2- inducible alkaline phosphatase activity. E-2- stimulated accumulation of progesterone receptor (PR) and HER-2/neu oncogene was abolished in the presence of FAS blockers. FAS inhibition also resulted in a marked downregulation of E-2-stimulated ERalpha expression, and noticeably impaired E-2- induced ERalpha nuclear accumulation. A dose-dependent decrease in cell proliferation and cell viability was observed after FAS blockade. A Cell Death ELISA, detecting DNA fragmentation, demonstrated that FAS inhibitors stimulated apoptosis of Ishikawa cells. The analysis of critical E-2- and TAM-related cell cycle proteins revealed an increase of both the expression and the nuclear accumulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(Kip1) following FAS inhibition. To rule out non-FAS cerulenin- and C75-related effects, we finally monitored ER signaling after silencing of FAS gene expression using the highly sequence-specific mechanism of RNA interference (RNAi). The concentrations of E-2 and TAM inducing half-maximal ERE activity (EC50) dramatically increased (4100 times) in FAS RNAi-transfected Ishikawa cells. Moreover, depletion of FAS by RNAi also caused loss of ERalpha expression, downregulation of PR, and accumulation of p21(WAF1/CIP1) and p27(Kip1) in E-2- stimulated Ishikawa cells. If chemically stable FAS inhibitors or cell-selective vector systems able to deliver RNAi targeting FAS gene demonstrate systemic anticancer effects in vivo, our results render FAS as a novel target for the prevention and treatment of endometrial carcinoma.