Renate Wagner

Interaction between fatty acid synthase- and ErbB-systems in ovarian cancer cells.

Fatty acid synthase (FASN) represents a metabolic oncogene. It produces phospholipids for membrane microdomains that accommodate receptor tyrosine kinases including Epidermal Growth Factor-Receptor (EGFR, ErbB1) and ErbB2 (HER2/neu). FASN and ErbBs are overexpressed in ovarian cancer. We examined the effect of FASN and ErbB inhibition on A2780 and SKOV3 ovarian cancer cells. Growth assays reveal that FASN inhibitor C75 sensitizes tumor cells against anti-ErbB drugs (pelitinib [EKB-569], canertinib [CI-1033], erlotinib, cetuximab, matuzumab, trastuzumab) suggesting FASN/ErbB cooperation. qRT-PCR and Western blotting revealed that C75 represses FASN, EGFR, ErbB2, and AKT suggesting that FASN-induced membrane microdomains accommodate/stabilize ErbBs and facilitate AKT recruitment/activation. Our data indicate that AKT is crucial for ErbB/FASN interaction, AKT cross-inhibits ERK and feeds loops that boost FASN and EGFR transcription, and EGFR and ErbB2 must be co-silenced for maximal FASN downregulation. Taken together, interference with FASN and ErbB abrogates their oncogenicity and should be exploited for ovarian cancer treatment.

The PI3 kinase/mTOR blocker NVP-BEZ235 overrides resistance against irreversible ErbB inhibitors in breast cancer cells

Resistance against first and second generation (irreversible) ErbB inhibitors is an unsolved problem in clinical oncology. The purpose of this study was to examine the effects of the irreversible ErbB inhibitors pelitinib and canertinib on growth of breast and ovarian cancer cells. Although in vitro growth-inhibitory effects of both drugs exceeded by far the effects of all reversible ErbB blockers tested (lapatinib, erlotinib, and gefitinib), complete growth inhibition was usually not reached. To define the mechanism of resistance, we examined downstream signaling pathways in drug-exposed cells by Western blot analysis. Although ErbB phosphorylation was reduced by pelitinib and canertinib, activation of the AKT/mTOR pathway remained essentially unaltered in drug-resistant cells. Correspondingly, transfection of tumor cells with constitutively activated AKT was found to promote resistance against all ErbB inhibitors tested, whereas dominant negative AKT reinstalled sensitivity in drug-resistant cells. In a next step, we applied PI3K/AKT/mTOR blockers including the dual PI3K/mTOR kinase inhibitor NVP-BEZ235. These agents were found to cooperate with pelitinib and canertinib in producing in vitro growth inhibition in cancer cells resistant against ErbB-targeting drugs. In conclusion, our data show that ErbB drug-refractory activation of the PI3K/AKT/mTOR pathway plays a crucial role in resistance against classical and second-generation irreversible ErbB inhibitors, and NVP-BEZ235 can override this form of resistance against pelitinib and canertinib.

Blockade of Fatty Acid Synthase Induces Ubiquitination and Degradation of Phosphoinositide-3-Kinase Signaling Proteins in Ovarian Cancer

Aberrations within the phosphoinositide-3-kinase (PI3K) pathway occur in greater than 45% of ovarian carcinomas. The PI3K cascade transmits signals from ErbB receptors downstream to S6 and 4EBP1, which are involved in protein biosynthesis. Many ovarian carcinomas reveal hyperactivation of ErbB1 (epidermal growth factor receptor) or ErbB2 (HER2/neu). Unfortunately, the benefit of anti-ErbB drugs is yet rather limited in ovarian carcinomas. Thus, novel targeting strategies are needed for ovarian carcinomas. The lipogenic enzyme fatty acid synthase (FASN) is overexpressed in approximately 80% of ovarian carcinomas. It stimulates cell growth and signifies poor prognosis. FASN inhibition impedes (ErbB) membrane receptor signaling and sensitizes cells against anti-ErbB drugs. Here, we show that the FASN inhibitor C75 and FASN-targeting siRNAs abrogate growth, induce apoptosis, and downregulate phosphorylation/expression of the PI3K effectors AKT, mTOR, p70S6K, S6, and 4EBP1. In contrast, FASN inhibition impairs expression but only weakly affects phosphorylation of ERK1/2 mitogen-activated protein kinases in ovarian carcinoma cells. Cycloheximide-mediated blockade of protein translation reveals that C75- or FASN siRNA–induced shutdown of FASN accelerates decomposition of signaling proteins. This effect is caused by C75- or FASN siRNA–dependent stimulation of ubiquitination followed by lysosomal-autophagosomal proteolysis. In contrast, PI3K inhibitor LY294002 blocks phosphorylation but does not reduce expression/stability of PI3K effectors. Forced expression of hyperactive (HA) AKT1, unlike HA-MEK1, impairs the growth-inhibitory action of C75. We provide first evidence that the anticancer action of FASN inhibitors is at least partially mediated by drug-dependent proteolysis of PI3K effectors. FASN is a promising cancer target, whose inhibition not only abrogates lipogenesis, which is indispensable for cancer growth, but also downregulates oncogenic PI3K signaling. Mol Cancer Res; 9(12); 1767–79. ©2011 AACR.

Fatty acid synthase is a metabolic marker of cell proliferation rather than malignancy in ovarian cancer and its precursor cells.

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‐inhibitor‐resistant. 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 β‐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 apoptosis, whereas in normal cells they only cause cell cycle deceleration without apoptosis. Thus, normal cells, although growth‐inhibited, may survive and recover from FASN blockade, whereas malignant cells get extinguished. FASN expression and FASN drug sensitivity are directly linked to cell growth and correlate with transformation/differentiation/senescence only indirectly. FASN is therefore a metabolic marker of cell proliferation rather than a marker of malignancy and is a useful target for future drug development.

Upregulation of retinoic acid receptor-β by the epidermal growth factor-receptor inhibitor PD153035 is not mediated by blockade of ErbB pathways

Inhibiting epidermal growth factor‐receptor (ErbB‐1) represents a powerful anticancer strategy. Activation of retinoid pathways is also in development for cancer treatment. Retinoic acid receptor‐β—the tumor suppressor and main retinoid mediator—‐is silenced in many tumors. The ErbB‐1 inhibitor PD153035 cooperates with retinoic acid during growth inhibition and induces retinoic acid receptor‐β suggesting that ErbB‐1 controls retinoic acid receptor‐β. However, here we demonstrate that ErbB pathways are not involved in PD153035‐mediated retinoic acid receptor‐β‐upregulation. PD153035 inhibits ErbB‐1‐phosphorylation, whereas its derivative EBE‐A22 is inactive. Yet both inhibit cell growth and upregulate retinoic acid receptor‐β in ErbB‐1‐overexpressing (MDA‐MB‐468), moderately expressing (OVCAR‐3), ErbB‐1‐negative (MDA‐MB‐453) or ErbB‐negative cells (CEM, Jurkat). Both bind DNA, whereas the closely related ErbB‐1 inhibitors AG1478 and ZD1839, which are inactive on retinoic acid receptor‐β, do not significantly bind DNA. None of the other ErbB‐1/ErbB‐2 inhibitors tested (RG‐14620, LFM‐A12, AG879, AG825) affect retinoic acid receptor‐β. PD153035 decreases methylation of the retinoic acid receptor‐β2 promoter. In OVCAR‐3, it stimulates dislodgement of histone deacetylase 1 from the promoter and acetylation of histones H3 and H4. Consequently, PD153035 facilitates recruitment of RNA polymerase II to the promoter and stimulates transcriptional activity. Moreover, PD153035 increases the retinoic acid receptor‐β mRNA half‐life. No other retinoid receptor, nor estrogen receptor‐α, nor RASSF1A is upregulated by PD153035. Thus PD153035 induces retinoic acid receptor‐β by ErbB‐independent transcriptional and post‐transcriptional mechanisms. This report highlights a triple action for an ErbB‐1 inhibitor (ErbB‐1 inhibition, DNA intercalation, retinoic acid receptor‐β‐induction). Such multitargeting drugs bear great potential for cancer treatment. J. Cell. Physiol. 211: 803–815, 2007.

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells

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.

HER Specific TKIs Exert Their Antineoplastic Effects on Breast Cancer Cell Lines through the Involvement of STAT5 and JNK

Background HER-targeted tyrosine kinase inhibitors (TKIs) have demonstrated pro-apoptotic and antiproliferative effects in vitro and in vivo. The exact pathways through which TKIs exert their antineoplastic effects are, however, still not completely understood. Methods Using Milliplex assays, we have investigated the effects of the three panHER-TKIs lapatinib, canertinib and afatinib on signal transduction cascade activation in SKBR3, T47D and Jurkat neoplastic cell lines. The growth-inhibitory effect of blockade of HER and of JNK and STAT5 signaling was measured by proliferation- and apoptosis-assays using formazan dye labeling of viable cells, Western blotting for cleaved PARP-1 and immunolabeling for active caspase 3, respectively. Results All three HER-TKIs clearly inhibited proliferation and increased apoptosis in HER2 overexpressing SKBR3 cells, while their effect was less pronounced on HER2 moderately expressing T47D cells where they exerted only a weak antiproliferative and essentially no pro-apoptotic effect. Remarkably, phosphorylation/activation of JNK and STAT5A/B were inhibited by HER-TKIs only in the sensitive, but not in the resistant cells. In contrast, phosphorylation/activation of ERK/MAPK, STAT3, CREB, p70 S6 kinase, IkBa, and p38 were equally affected by HER-TKIs in both cell lines. Moreover, we demonstrated that direct pharmacological blockade of JNK and STAT5 abrogates cell growth in both HER-TKI-sensitive as well as -resistant breast cancer cells, respectively. Conclusion We have shown that HER-TKIs exert a HER2 expression-dependent anti-cancer effect in breast cancer cell lines. This involves blockade of JNK and STAT5A/B signaling, which have been found to be required for in vitro growth of these cell lines.

Abstract B01: Multilevel interference with receptor-PI3K-mTORC1 signaling is key mechanism for anticancer activity of fatty acid synthase inhibitors

Abstracts: AACR Special Conference: Metabolism and Cancer; June 7-10, 2015; Bellevue, WA 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. However, forced expression of constitutive active AKT counteracts FASN inhibitor-mediated mTORC1 silencing and abrogates growth arrest. Our data thus suggest that FASN inhibitors by themselves can efficiently eliminate PI3K downstream activity. This mechanism appears crucial for the anticancer effect, which cannot be augmented by co-exposure to drugs that target the very same pathway. 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 these compounds. Supported by Medical Scientific Fund of the Mayor of the City of Vienna & ‘Initiative Krebsforschung’ of the Medical University Vienna, Austria. Citation Format: Renate Wagner, Katharina Prostling, Daniel Veigel, Gerald Stubiger, Michael Grusch, Julian Weghuber, Christian Singer, Heidrun Karlic, Ramon Colomer, Bellinda Benhamu, Maria Luz Lopez-Rodriguez, Fausto Hegardt, Jordi Garcia, Dolors Serra, Peter Valent, Thomas Grunt. Multilevel interference with receptor-PI3K-mTORC1 signaling is key mechanism for anticancer activity of fatty acid synthase inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B01.

269 Molecular interplay between cancer cell fatty acid metabolism and oncogenic signaling as resource for novel treatment strategies against ovarian cancer

1 Medical University Vienna, Comprehensive Cancer Center, Department of Medicine I, Vienna, Austria; 2 Medical University Vienna, Comprehensive Cancer Center, Center of Physiology and Pharmacology, Vienna, Austria; 3 University of Applied Sciences, School of Engineering and Environmental Sciences, Wels, Austria; 4 Medical University Vienna, Comprehensive Cancer Center, Department of Obstetrics/Gynecology, Vienna, Austria; 5 Medical University Vienna, Ludwig Boltzmann Cluster Oncology, Vienna, Austria; 6 Spanish National Cancer Research Center CNIO, Department of Medical Oncology, Madrid, Spain; 7 Universidad Complutense, Departamento de Quimica Organica I, Madrid, Spain; 8 Universitat de Barcelona, Department of Biochemistry and Molecular Biology, Barcelona, Spain; 9 Universitat de Barcelona, Institut de Biomedicina, Barcelona, Spain; 10 Medical University Vienna, Comprehensive Cancer Center, Department of Medicine I & Ludwig Boltzmann Cluster Oncology, Vienna, Austria

Abstract 1229: Fibroblast growth factor signaling in ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epithelial ovarian cancer (OC) is a rapidly progressive, highly lethal disease. It is the fifth leading cause of cancer-related death among women. Over the last decades, there has been only modest improvement in the treatment of OC. Nevertheless, recent clinical trials with inhibitors of transmembrane receptor tyrosine kinases such as fibroblast growth factor (FGF) receptors (FGFRs) yielded some promising results in OC. FGFRs are involved in malignant transformation, angiogenesis and chemoresistance. Inhibition of FGFR-dependent signaling can overcome resistance to standard therapies. Thus, FGFRs represent potential targets for cancer therapy. Unfortunately, however, the biological and therapeutic relevance of the FGF-FGFR system has not been elucidated yet in OC. The aim of this study was to characterize the FGF-FGFR system, its influence on malignancy-related cell properties and its potential role as a druggable signaling pathway in OC cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that expression of FGFs and FGFRs, leading to autocrine signaling loops with a strong mitogenic potential, is a common event in OC. Immunoblotting proved that the FGF signaling system is functionally active in these cells. Conditioned growth medium was able to induce phosphorylation of ERK1/2. Moreover, recombinant FGF-2 significantly elevated the growth rate in approximately 50% of the OC cell lines as demonstrated by formazan dye assay, whereas FGF-1 markedly induced migration in 66% of the cell lines. The dependency of OC cells on FGFR signaling was further evaluated by growth inhibition assays using the two small molecular FGFR inhibitors PD173074 and dovitinib (TKI-258, CHIR-258). A-2780 cells were found to be extremely sensitive to FGFR inhibition, while HEY and SKOV-3 cells showed moderate sensitivity. In contrast, OVCAR-3 cells were not inhibited by these compounds. Together, our data suggest that FGFR inhibitors reveal anticancer activity in a significant proportion of OC cells even as single drugs. Thus, combination of conventional cytotoxic intervention with blockade of the FGF-FGFR system might represent a promising future strategy for the treatment of OC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1229. doi:1538-7445.AM2012-1229