Inhibition of tumor-associated fatty acid synthase activity antagonizes estradiol- and tamoxifen-induced agonist transactivation of estrogen receptor (ER) in human endometrial adenocarcinoma cells.Oncogene. 2004 Jun 17; 23(28):4945-58.O
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 (E2)- 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 E2- 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 E2-stimulated ERE-dependent transcription, whereas FAS inhibitors did not significantly increase the levels of ERE transcriptional activity in the absence of E2. 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 E2-inducible gene products were evaluated. FAS blockade induced a dose-dependent decrease in E2-inducible alkaline phosphatase activity. E2-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 E2-stimulated ERalpha expression, and noticeably impaired E2-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 E2- and TAM-related cell cycle proteins revealed an increase of both the expression and the nuclear accumulation of cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1 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 E2 and TAM inducing half-maximal ERE activity (EC50) dramatically increased (>100 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 p21WAF1/CIP1 and p27Kip1 in E2-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.