Ovarian cancer (OC) is caused by genetic aberrations in networks that control growth and survival. Importantly, aberrant cancer metabolism interacts with oncogenic signaling providing additional drug targets. Tumors overexpress the lipogenic enzyme fatty acid synthase (FASN) and are inhibited by FASN blockers, whereas normal cells are FASN-negative and FASN-inhibitorresistant. Here, we demonstrate that this holds true when ovarian/oviductal cells reside in their autochthonous tissues, whereas in culture they express FASN and are FASN-inhibitor-sensitive. Upon subculture, nonmalignant cells cease growth, express senescence-associated b-galactosidase, lose FASN and become FASN-inhibitor-resistant. Immortalized ovarian/oviductal epithelial cell lines-although resisting senescence-reveal distinct growth activities, which correlate with FASN levels and FASN drug sensitivities. Accordingly, ectopic FASN stimulates growth in these cells. Moreover, FASN levels and lipogenic activities affect cellular lipid composition as demonstrated by thin-layer chromatography. Correlation between proliferation and FASN levels was finally evaluated in cancer cells such as HOC-7, which contain subclones with variable differentiation/senescence and corresponding FASN expression/FASN drug sensitivity. Interestingly, senescent phenotypes can be induced in parental HOC-7 by differentiating agents. In OC cells, FASN drugs induce cell cycle blockade in S and/or G2/M and stimulate

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions–from FASN towards receptor-PI3K-mTORC1–are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2–EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

The metabolic oncogene fatty acid synthase (FASN) is overexpressed in 80% of ovarian cancers (OC) and indicates poor prognosis. Exposure of OC to inhibitors of FASN elicits a complex stress response that interferes with receptor-PI3K-mTORC1 signaling (briefly designated 'PI3K pathway'). Here we demonstrate that FASN inhibitors capitalize on multiple mechanisms to interfere with the PI3K pathway, and that silencing this cascade is crucial for the anticancer action of the drugs. Data were obtained using thin-layer chromatography, ELISA, Western blotting, quantitative micropatterning and growth assays, respectively. Exposure of OC cells (SKOV3, OVCAR-3) to FASN inhibitors (C75, G28UCM) causes lipid redistribution toward storage lipids, whereas membrane lipid rafts and signaling lipids are diminished, which significantly impairs EGF receptor/ErbB/HER function and expression. A severe depletion of phosphatidylinositol (3,4,5) trisphosphate (PIP3), which represents the crucial product of PI3K action, is associated with drug-dependent silencing of AKT. Moreover, FASN blockers rapidly stimulate expression of the stress response gene REDD1 (RTP801/Dig2/DDIT4) followed by slow activation of the energy sensor AMPK. Induction of these mTORC1 upstream repressors has been found to block downstream phosphorylation of ribosomal S6 protein. Moreover, long-term stress imposed by persistent FASN blockade leads to accelerated degradation of signaling proteins. Interestingly, concurrent targeting of the PI3K pathway using the dual PI3K/mTOR blocker NVP-BEZ235 does not aggravate the FASN anticancer drug effects, indicating that PI3K is already maximally silenced due to FASN blockade. In contrast, exogenous expression of constitutive active AKT counteracts FASN inhibitor-mediated mTORC1 silencing and abrogates growth arrest. Elimination of PI3K downstream activity thus appears crucial for the anticancer effect of FASN blockers. On the other hand, silencing PI3K signaling by FASN inhibitors was found to release a negative feedback loop toward MAPK. Thus, FASN drug-mediated PI3K silencing is associated with cross-activation of ERK1/2. Accordingly, co-treatment with the MEK inhibitor selumetinib (AZD6244) significantly improves the anticancer action of FASN inhibitors, whereas introduction of constitutive active MEK does not alter FASN drug-induced growth inhibition. Collectively these data demonstrate that FASN inhibitors utilize a whole panel of different mechanisms to abrogate receptor-PI3K-mTORC1 signaling, which represents at least one of the crucial mechanisms of anticancer action of FASN targeting drugs.