cerulenin and Breast-Neoplasms

Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cerulenin; Fatty Acid Synthases; Glycolysis; Humans; Receptor, ErbB-2; Signal Transduction; Thyroid Hormone-Binding Proteins

Both fatty acid synthase (FASN) and ErbB2 have been shown to promote breast cancer cell migration. However, the underlying molecular mechanism remains poorly understood and there is no reported evidence that directly links glycolysis to breast cancer cell migration. In this study, we investigated the role of FASN, ErbB2-mediated glycolysis in breast cancer cell migration. First, we compared lactate dehydrogenase A (LDHA) protein levels, glycolysis and cell migration between FASN, ErbB2-overexpressing SK-BR-3 cells and FASN, ErbB2-low-expressing MCF7 cells. Then, SK-BR-3 cells were treated with cerulenin (Cer), an inhibitor of FASN, and ErbB2, LDHA protein levels, glycolysis, and cell migration were detected. Next, we transiently transfected ErbB2 plasmid into MCF7 cells and detected FASN, LDHA protein levels, glycolysis and cell migration. Heregulin-β1 (HRG-β1) is an activator of ErbB2 and 2-deoxyglucose (2-DG) and oxamate (OX) are inhibitors of glycolysis. MCF7 cells were treated with HRG-β1 alone, HRG-β1 plus 2-DG, OX or cerulenin and glycolysis, and cell migration were measured. We found that FASN, ErbB2-high-expressing SK-BR-3 cells displayed higher levels of glycolysis and migration than FASN, ErbB2-low-expressing MCF7 cells. Inhibition of FASN by cerulenin impaired glycolysis and migration in SK-BR-3 cells. Transient overexpression of ErbB2 in MCF7 cells promotes glycolysis and migration. Moreover, 2-deoxyglucose (2-DG), oxamate (OX), or cerulenin partially reverses heregulin-β1 (HRG-β1)-induced glycolysis and migration in MCF7 cells. In conclusion, this study demonstrates that FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration. These novel findings indicate that targeting FASN, ErbB2-mediated glycolysis may be a new approach to reverse breast cancer cell migration.

Topics: Breast Neoplasms; Cell Movement; Cerulenin; Fatty Acid Synthase, Type I; Fatty Acid Synthesis Inhibitors; Female; Glycolysis; Humans; MCF-7 Cells; Receptor, ErbB-2

This study aimed to investigate the role of fatty acid synthase (FASN) in the epithelial-mesenchymal transition (EMT) of breast cancer cells. MCF-7 cells and MCF-7 cells overexpressing mitogen-activated protein kinase 5 (MCF-7-MEK5) were used in this study. MCF-7-MEK5 cells showed stable EMT characterized by increased vimentin and decreased E-cadherin expression. An In vivo animal model was established using the orthotopic injection of MCF-7 or MCF-7-MEK5 cells. Real-time quantitative PCR and western blotting were used to detect the expression levels of FASN and its downstream proteins liver fatty acid-binding protein (L-FABP) and VEGF/VEGFR-2 in both in vitro and in vivo models (nude mouse tumor tissues). In MCF-7-MEK5 cells, significantly increased expression of FASN was associated with increased levels of L-FABP and VEGF/VEGFR-2. Cerulenin inhibited MCF-7-MEK5 cell migration and EMT, and reduced FASN expression and down-stream proteins L-FABP, VEGF, and VEGFR-2. MCF-7-MEK5 cells showed higher sensitivity to Cerulenin than MCF-7 cells. Immunofluorescence revealed an increase of co-localization of FASN with VEGF on the cell membrane and with L-FABP within MCF-7-MEK5 cells. Immunohistochemistry further showed that increased percentage of FASN-positive cells in the tumor tissue was associated with increased percentages of L-FABP- and VEGF-positive cells and the Cerulenin treatment could reverse the effect. Altogether, our results suggest that FASN is essential to EMT possibly through regulating L-FABP, VEGF and VEGFR-2. This study provides a theoretical basis and potential strategy for effective suppression of malignant cells with EMT.

Topics: Animals; Breast Neoplasms; Cell Movement; Cerulenin; Epithelial-Mesenchymal Transition; Fatty Acid Synthases; Fatty Acid Synthesis Inhibitors; Fatty Acid-Binding Proteins; Female; Gene Expression; Humans; MAP Kinase Kinase 5; MCF-7 Cells; Mice; Mice, Nude; Vascular Endothelial Growth Factor A

Patients with ER/HER2-positive breast cancer have a poor prognosis and are less responsive to selective estrogen receptor modulators; this is presumably due to the crosstalk between ER and HER2. Fatty acid synthase (FASN) is essential for the survival and maintenance of the malignant phenotype of breast cancer cells. An intimate relationship exists between FASN, ER and HER2. We hypothesized that FASN may be the downstream effector underlying ER/HER2 crosstalk through the PI3K/AKT/mTOR pathway in ER/HER2-positive breast cancer. The present study implicated the PI3K/AKT/mTOR pathway in the regulation of FASN expression in ER/HER2-positive breast cancer cells and demonstrated that rapamycin, an mTOR inhibitor, inhibited FASN expression. Cerulenin, a FASN inhibitor, synergized with rapamycin to induce apoptosis and inhibit cell migration and tumorigenesis in ER/HER2-positive breast cancer cells. Our findings suggest that inhibiting the mTOR-FASN axis is a promising new strategy for treating ER/HER2-positive breast cancer.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Cerulenin; Drug Synergism; Estrogen Receptor alpha; Fatty Acid Synthase, Type I; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Protein Kinase Inhibitors; Real-Time Polymerase Chain Reaction; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Fatty acid synthase (FAS) has been proposed to be a new drug target for the development of anticancer agents because of the significant difference in expression of FAS between normal and tumour cells. Since a n-hexane-soluble extract from Ginkgo biloba was demonstrated to inhibit FAS activity in our preliminary test, we isolated active compounds from the n-hexane-soluble extract and evaluated their cytotoxic activity in human cancer cells. Three ginkgolic acids 1-3 isolated from the n-hexane-soluble extract inhibited the enzyme with IC(50) values 17.1, 9.2 and 10.5 µM, respectively, and they showed cytotoxic activity against MCF-7 (human breast adenocarcinoma), A549 (human lung adenocarcinoma) and HL-60 (human leukaemia) cells. Our findings suggest that alkylphenol derivatives might be a new type of FAS inhibitor with cytotoxic activity.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fatty Acid Synthases; Female; Ginkgo biloba; Hexanes; HL-60 Cells; Humans; Inhibitory Concentration 50; Lung Neoplasms; Molecular Structure; Plant Extracts; Plant Leaves; Salicylates

To investigate the endogenous signaling pathways associated with high proliferation potential of breast cancer cells.. Breast cancer cell lines LM-MCF-7 and MCF-7 with high and low proliferation capability were used. The promoter activity of fatty acid synthase (FASN) was examined using luciferase reporter gene assay. The expression level of FASN mRNA was measured using RT-PCR and real time PCR, respectively. The level of leukotriene B4 (LTB4) was determined with ELISA. The expression levels of 5-lipoxygenase (5-LOX) was analyzed using RT-PCR and Western blot, respectively. 5-Bromo-20-deoxyuridine (BrdU) incorporation assay was used to study the proliferation of LM-MCF-7 and MCF-7 cells.. The promoter activity of FASN was significantly higher in LM-MCF-7 cells than MCF-7 cells. Treatment of LM-MCF-7 cells with ERK1/2 inhibitor PD98059 (30-50 μmol/L) or LOX inhibitor NDGA (25 μmol/L) abolished the activation of FASN. Moreover, treatment of LM-MCF-7 cells with the specific 5-LOX inhibitor MK-886 (20-40 μmol/L) or 5-LOX siRNA (50-100 nmol/L) decreased the promoter activity of FASN. The level of LTB4, the final metabolite produced by 5-LOX, was significantly higher in LM-MCF-7 cells than MCF-7 cells. Administration of exogenous LTB4 (1-10 nmol/L) was able to stimulate the promoter activity of FASN in MCF-7 cells. Treatment of LM-MCF-7 cells with the FASN inhibitor cerulenin (10 μmol/L) reduced all the levels of p-ERK1/2, 5-LOX, and LTB4. Treatment of LM-MCF-7 cells with cerulenin, PD98059, or MK-886 abolished the proliferation. Administration of exogenous LTB4 (10 nmol/L) significantly increased BrdU incorporation in MCF-7 cells.. THESE results suggest a novel positive feedback loop involving FASN/p-ERK1/2/5-LOX/LTB4/FASN contributes to the sustaining growth of breast cancer LM-MCF-7 cells.

Topics: Arachidonate 5-Lipoxygenase; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cerulenin; Fatty Acid Synthases; Fatty Acid Synthesis Inhibitors; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; Leukotriene B4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction

Certain fatty acids in canola oil (CAN) have been associated with a reduced risk of breast cancer. This study assessed the effects of CAN on proliferation and death of human breast cancer cells in vitro and in vivo in chemically induced mammary carcinogenesis. We hypothesize that CAN reduces breast cancer cell growth by inducing cell death. In a series of in vitro experiments, human breast cancer T47D and MCF-7 cells were cultured and treated with CAN and two chemotherapeutic drugs, tamoxifen and cerulenin. Cell proliferation and caspase-3 and p53 activities were measured. Reduced cancer cell growth and increased expression of caspase-3 and p53 were seen in T47D and MCF-7 cells treated with CAN. Moreover, CAN showed synergistic cancer cell growth inhibition effects with tamoxifen and cerulenin. In a subsequent live animal experiment, 42 female Sprague-Dawley rats were randomly assigned to corn oil (CORN) or CAN diets, and mammary tumors were chemically induced by N-nitroso-N-methylurea. CAN-dieted rats had reduced tumor volumes and showed an increased survival rate as compared to CORN-dieted rats. We demonstrated that CAN has suppressive effects on cancer growth, and reduces tumor volumes. The results suggest that CAN may have inhibitory effects on breast cancer cell growth, and warrants further investigation of the synergistic effects of CAN with anti-cancer drugs.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cerulenin; Drug Synergism; Fatty Acids, Monounsaturated; Female; Humans; Rapeseed Oil; Rats; Rats, Sprague-Dawley; Tamoxifen; Tumor Suppressor Protein p53

The lipogenic enzyme fatty acid synthase (FASN) is differentially overexpressed and hyperactivated in a biologically aggressive subset of breast carcinomas and minimally in most normal adult tissues, rendering it an interesting target for anti-neoplastic therapy development. We previously reported that the FASN blockade can induce a synergistic chemosensitization of breast cancer cells to microtubule interfering agents (MIAs) such as docetaxel, paclitaxel and vinorelbine. Upon pharmacological inhibition of FASN activity using the natural antibiotic cerulenin [(2S,3R)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide], we evaluated the role of FASN-catalyzed endogenous fatty acid biogenesis on the sensitivity of SK-Br3, MCF-7 and MDA-MB-231 breast cancer cell lines to the anti-metabolite 5-fluorouracil (5-FU). Cells were exposed simultaneously to cerulenin and 5-FU, sequentially to 5-FU followed by cerulenin or cerulenin followed by 5-FU. Cell viability was determined by MTT assays and the increase in 5-FU-induced cell growth inhibition was measured by dividing 5-FU IC30 and IC50 values (i.e., 30% and 50% inhibitory concentrations, respectively) that were obtained in the absence of cerulenin by those in its presence. Co-exposure to cerulenin enhanced 5-FU efficacy up to 20-, 81-, and 58-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. Pre-treatment with cerulenin followed by the addition of 5-FU increased 5-FU efficacy up to 31-, 87-, and 126-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. Pre-treatment with 5-FU followed by the addition of cerulenin augmented 5-FU efficacy up to 107-, 20-, and 18-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. When isobologram transformations of multiple dose-response analyses were performed to detect in vitro synergy, we concluded that the nature of the interaction between cerulenin and 5-FU in individual breast cancer cells lines generally exhibited sequence-dependency. Thus, while synergism was mainly observed when breast cancer cells were exposed to 5-FU prior to cerulenin, moderate synergism or additive interactions was obtained either when the chemical FASN blocker preceded 5-FU or when both drugs were concurrently administered. Of note, no antagonist interactions occurred upon any schedule of combined treatment with cerulenin and 5-FU. Our current findings revealing a schedule-dependent synergistic interaction between 5-FU and cerulenin represents, to the best of our knowledge, the first eviden

Topics: Breast Neoplasms; Cell Survival; Cerulenin; Drug Interactions; Drug Resistance, Neoplasm; Drug Synergism; Fatty Acid Synthase, Type I; Fluorouracil; Humans; Tumor Cells, Cultured

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cerulenin; Chromones; Cytochromes c; Cytosol; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Mitochondria; Morpholines; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2

A biologically aggressive subset of human breast cancers has been demonstrated to overexpress fatty acid synthase (FAS), the key enzyme of endogenous FA biosynthesis. This breast cancer-specific activation of FAS-dependent lipogenesis, an anabolic-energy-storage pathway of minor importance in normal cells, would render breast cancer cells more vulnerable to anti-metabolite interventions with FAS as therapeutic target. Not surprisingly, pharmacological inhibitors of FAS have been reported to produce both cytostatic and cytotoxic effects in human breast cancer cells, as well as to suppress DNA replication. However, the signal transduction pathway(s) that link FAS hyperactivity and breast cancer cell growth has been unresolved. Here, we have attempted to provide a systematic approach to assess the role of FAS signaling on the survival and proliferation of human breast cancer cells. First, we assessed the level of FAS protein in a panel of human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-435, ZR-75B, T47-D, BT-474, and SK-Br3). FAS expression was graded from ++++ (overexpression) in SK-Br3 cells to + (very low expression) in MDA-MB-231 cells. No correlation was noted between FAS overexpression and estrogen receptor (ER) or progesterone receptor (PR) status, whereas a positive correlation was found between high levels of FAS expression and the amplification and/or overexpression of HER-2/neu oncogene. Because metabolic adaptation of breast cancer cells to the ambient fatty acid concentration may be relevant to the goal of utilizing FAS inhibition as a chemotherapeutic target, we evaluated the effect of exogenous dietary fatty acids on the cytotoxicity resulting from the inhibition of FAS activity. Pharmacological inhibition of FAS activity by the natural antibiotic cerulenin [(2S,3R)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide] resulted in a dose-dependent cytotoxicity which positively paralleled the endogenous level of FAS. Supraphysiological levels of exogenous oleic acid (OA), a omega-9 monounsaturated fatty acid synthesized from a primary-end product of FAS palmitate, significantly diminished cell toxicity caused by cerulenin. Indeed, OA exposure significantly reduced FAS activity and expression by 55% in FAS-overexpressing SK-Br3 cells. omega-3 (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid) and omega-6 (linoleic acid and arachidonic acid) polyunsaturated fatty acids (PUFAs), however, were unable to rescue breast can

Topics: Active Transport, Cell Nucleus; BRCA1 Protein; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cerulenin; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; DNA Fragmentation; Dose-Response Relationship, Drug; Fatty Acid Synthases; Fatty Acids; Humans; Immunoblotting; In Situ Nick-End Labeling; Inhibitory Concentration 50; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; NF-kappa B; Signal Transduction; Time Factors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The lipogenic enzyme fatty acid synthase (FAS) is differentially overexpressed and hyperactivated in a biologically aggressive subset of breast carcinomas and minimally in most normal adult tissues, rendering it an interesting target for antineoplastic therapy development. Recently, a molecular connection between the HER -2/ neu (c- erb B-2) oncogene and FAS has been described in human breast cancer cells. Here, we examined the relationship between breast cancer-associated FAS hyperactivity and HER -2/ neu -induced breast cancer chemoresistance to taxanes. Co-administration of docetaxel (Taxotere) and the mycotoxin cerulenin, a potent and non-competitive inhibitor of FAS activity, demonstrated strong synergism in HER -2/ neu -overexpressing and docetaxel-resistant SK-Br3 cells, modest synergism in moderately HER -2/ neu -expressing MCF-7 cells, and it showed additive effects in low HER -2/ neu -expressing and docetaxel-sensitive MDA-MB-231 cells. Sequential exposure to cerulenin followed by docetaxel again yielded strong synergism in SK-Br3 cells, whereas antagonistic and moderate synergistic interactions were observed in MCF-7 and MDA-MB-231 cells, respectively. Importantly, inhibition of FAS activity dramatically decreased the expression of HER -2/ neu oncogene in SK-Br3 breast cancer cells. To the best of our knowledge this is the first study demonstrating that FAS is playing an active role in HER -2/ neu -induced breast cancer chemotherapy resistance.

Topics: Antifungal Agents; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cerulenin; Docetaxel; Drug Resistance, Neoplasm; Fatty Acid Synthases; Female; Genes, erbB-2; Humans; Taxoids; Transfection

Activity and expression of fatty acid synthase (FAS), a critical enzyme in the de novo biosynthesis of fatty acids in mammals, is exquisitely sensitive to nutritional regulation of lipogenesis in liver or adipose tissue. Surprisingly, a number of studies have demonstrated hyperactivity and overexpression of FAS (oncogenic antigen-519) in a biologically aggressive subset of human breast carcinomas, suggesting that FAS-dependent neoplastic lipogenesis is unresponsive to nutritional regulation. We have assessed the role of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) on the enzymatic activity and protein expression of tumor-associated FAS in SK-Br3 human breast cancer cells, an experimental paradigm of FAS-overexpressing tumor cells in which FAS enzyme constitutes up to 28%, by weight, of the cytosolic proteins. Of the omega-3 PUFAs tested, alpha-linolenic acid (ALA) dramatically reduced FAS activity in a dose-dependent manner (up to 61%). omega-3 PUFA docosahexaenoic acid (DHA) demonstrated less marked but still significant inhibitory effects on FAS activity (up to 37%), whereas eicosapentaenoic acid (EPA) was not effective. Of the omega-6 fatty acids tested, gamma-linolenic acid (GLA) was the most effective dose-dependent inhibitor of FAS activity, with a greater than 75% FAS activity reduction. Remarkably, omega-6 PUFAs linoleic acid (LA) and arachidonic acid (ARA), suppressors of both hepatic and adipocytic FAS-dependent lipogenesis, had no significant inhibitory effects on the activity of tumor-associated FAS in SK-Br3 breast cancer cells. Western blotting studies showed that down-regulation of FAS protein expression tightly correlated with previously observed inhibition of FAS activity, suggesting that ALA-, DHA-, and GLA-induced changes in FAS activity resulted from effects at the protein level. We investigated whether the FAS inhibitory effect of GLA and omega-3 PUFAs correlated with a cytotoxic effect related to a peroxidative mechanism. Measurement of cell viability by MTT assay indicated a significant cellular toxicity after ALA and GLA exposures. Furthermore, we observed a significant correlation between the ability of PUFAs to repress FAS and cause cell toxicity. In the presence of anti-oxidants (vitamin E), ALA and GLA dramatically lost their ability to inhibit FAS activity. Interestingly, a combination of ALA and GLA was FAS inhibitory in an additive manner, and this FAS repression was only partially reversible by vitamin E. In exam

Topics: Adipocytes; alpha-Linolenic Acid; Arachidonic Acids; Blotting, Western; Breast Neoplasms; Cell Division; Cerulenin; Dietary Fats; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Fatty Acid Synthases; Female; Fluorescent Antibody Technique; gamma-Linolenic Acid; Humans; Mitogen-Activated Protein Kinases; Tumor Cells, Cultured

Fatty acid synthase (FAS) activity is a potential therapeutic target to treat cancer and obesity. Here, we have identified a molecular link between FAS and HER2 (erbB-2) oncogene, a marker for poor prognosis that is overexpressed in 30% of breast and ovarian cancers. Pharmacological FAS inhibitors cerulenin and C75 were found to suppress p185(HER2) oncoprotein expression and tyrosine-kinase activity in breast and ovarian HER2 overexpressors. Similarly, p185(HER2) expression was dramatically down-regulated when FAS gene expression was silenced by using the highly sequence-specific mechanism of RNA interference (RNAi). Pharmacological and RNAi-mediated silencing of FAS specifically down-regulated HER2 mRNA and, concomitantly, caused a prominent up-regulation of PEA3, a transcriptional repressor of HER2. A cytoplasmic redistribution of p185(HER2) was associated with marked morphological changes of FAS RNAi-transfected cells, whereas chemical inhibitors of FAS promoted a striking nuclear accumulation of p185(HER2). The simultaneous targeting of FAS and HER2 by chemical FAS inhibitors and the humanized antibody directed against p185(HER2) trastuzumab, respectively, was synergistically cytotoxic toward HER2 overexpressors. Similarly, concurrent RNAi-mediated silencing of FAS and HER2 genes synergistically stimulated apoptotic cell death in HER2 overexpressors. p185(HER2) was synergistically down-regulated after simultaneous inhibition of FAS and HER2 by either pharmacological inhibitors or small interfering RNA. These findings provide evidence of an active role of FAS in cancer evolution by specifically regulating oncogenic proteins closely related to malignant transformation, strongly suggesting that HER2 oncogene may act as the key molecular sensor of energy imbalance after the perturbation of tumor-associated FAS hyperactivity in cancer cells.

Topics: 4-Butyrolactone; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Line; Cell Size; Cell Survival; Cerulenin; Fatty Acid Synthases; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Humans; Ovarian Neoplasms; Receptor, ErbB-2; RNA, Small Interfering; Signal Transduction; Transcription Factors; Trastuzumab

We designed our experiments to evaluate whether fatty acid synthase (FAS), a lipogenic enzyme linked to tumor virulence in population studies of human cancer, is necessary for the malignant transformation induced by Her-2/neu (erbB-2) oncogene, which is overexpressed not only in invasive breast cancer but also in premalignant atypical duct proliferations and in ductal carcinoma in situ of the breast. To avoid the genetic complexities associated with established breast cancer cell lines, we employed NIH-3T3 mouse fibroblasts engineered to overexpress human Her-2/neu coding sequence. NIH-3T3/Her-2 cells demonstrated a significant upregulation of FAS protein expression, which was dependent on the upstream activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT pathways. Remarkably, pharmacological FAS blockade using the mycotoxin cerulenin or the novel small compound C75 completely suppressed the state of Her-2/neu-induced malignant transformation by inhibiting the ability of NIH-3T3/Her-2 cells to grow under either anchorage-independent (i.e., to form colonies in soft agar) or low-serum monolayer conditions. Moreover, NIH-3T3/Her-2 fibroblasts were up to three times more sensitive to chemical FAS inhibitors relative to untransformed controls as determined by MTT-based cell viability assays. In addition, pharmacological FAS blockade preferentially induced apoptotic cell death of NIH-3T3/Her-2 fibroblasts, as determined by an ELISA for histone-associated DNA fragments and by the terminal deoxynucleotidyltransferase (TdT)-mediated nick end labeling assay (TUNEL). Interestingly, the degree of Her-2/neu oncogene expression in a panel of breast cancer cell lines was predictive of sensitivity to chemical FAS inhibitors-induced cytotoxicity, while low-FAS expressing and chemical FAS inhibitors-resistant MDA-MB-231 breast cancer cells became hypersensitive to FAS blockade when they were engineered to overexpress Her-2/neu. Our observations strongly suggest that inhibition of FAS activity may provide a new molecular avenue for chemotherapeutic prevention and/or treatment of Her-2/neu-related breast carcinomas.

Topics: 4-Butyrolactone; Animals; Antifungal Agents; Apoptosis; Breast Neoplasms; Cell Adhesion; Cell Transformation, Neoplastic; Cells, Cultured; Cerulenin; Chemoprevention; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; fas Receptor; Fatty Acid Synthases; Fatty Acids; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Mice; Mitogen-Activated Protein Kinases; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Tumor Necrosis Factor

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy.

Topics: Acetyl-CoA Carboxylase; Apoptosis; Breast Neoplasms; Carnitine O-Palmitoyltransferase; Cell Survival; Cerulenin; Enzyme Inhibitors; Epoxy Compounds; Fatty Acid Synthases; Fatty Acids; Female; Furans; Humans; Hypolipidemic Agents; Kinetics; Malonyl Coenzyme A; Models, Biological; Oxidation-Reduction; Tumor Cells, Cultured

A biologically aggressive subset of human breast cancers and other malignancies is characterized by elevated fatty-acid synthase (FAS) enzyme expression, elevated fatty acid (FA) synthesis, and selective sensitivity to pharmacological inhibition of FAS activity by cerulenin or the novel compound C75. In this study, inhibition of FA synthesis at the physiologically regulated step of carboxylation of acetyl-CoA to malonyl-CoA by 5-(tetradecyloxy)-2-furoic acid (TOFA) was not cytotoxic to breast cancer cells in clonogenic assays. FAS inhibitors induced a rapid increase in intracellular malonyl-CoA to several fold above control levels, whereas TOFA reduced intracellular malonyl-CoA by 60%. Simultaneous exposure of breast cancer cells to TOFA and an FAS inhibitor resulted in significantly reduced cytotoxicity and apoptosis. Subcutaneous xenografts of MCF7 breast cancer cells in nude mice treated with C75 showed FA synthesis inhibition, apoptosis, and inhibition of tumor growth to less than 1/8 of control volumes, without comparable toxicity in normal tissues. The data suggest that differences in intermediary metabolism render tumor cells susceptible to toxic fluxes in malonyl-CoA, both in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Survival; Cerulenin; Enzyme Inhibitors; Fatty Acid Synthases; Female; Furans; Humans; Hypolipidemic Agents; Malonyl Coenzyme A; Mice; Mice, Nude; Tumor Cells, Cultured; Tumor Stem Cell Assay

One of the key limiting factors in the treatment of advanced stage human epithelial malignancies is the lack of new, selective molecular targets for antineoplastic therapy. A substantial subset of human breast, ovarian, endometrial, colorectal, and prostatic cancers express elevated levels of fatty acid synthase, the major enzyme required for endogenous fatty acid biosynthesis, and carcinoma lines are growth inhibited by cerulenin, a noncompetitive inhibitor of fatty acid synthase. We have shown previously that the difference in fatty acid biosynthesis between cancer and normal cells is an exploitable target for metabolic inhibitors in the in vitro setting and in vivo in a human ovarian carcinoma xenograft in nude mice. Here, we report that cerulenin treatment of human breast cancer cells inhibits fatty acid synthesis within 6 h after exposure, that loss of clonogenic capacity occurs within the same interval, and that DNA fragmentation and morphological changes characteristic of apoptosis ensue.

Topics: Antifungal Agents; Apoptosis; Breast Neoplasms; Cerulenin; DNA, Neoplasm; Fatty Acid Synthases; Fatty Acids; Female; Humans; Tumor Cells, Cultured

OA-519 is a prognostic molecule found in tumor cells from breast cancer patients with markedly worsened prognosis. We purified OA-519 from human breast carcinoma cells, obtained its peptide sequence, and unambiguously identified it as fatty acid synthase through sequence homology and enzymology. Tumor fatty acid synthase is an approximately 270-kDa polypeptide which specifically abolished immunostaining of human breast cancers by anti-OA-519 antibodies. Tumor fatty acid synthase oxidized NADPH in a malonyl-CoA-dependent fashion and synthesized fatty acids composed of 80% palmitate, 10% myristate, and 10% stearate from acetyl-CoA, malonyl-CoA, and NADPH with a specific activity of 624 nmol of NADPH oxidized per min per mg. Tumor cell lines with elevated fatty acid synthase showed commensurate increases in incorporation of [U-14C]acetate into acylglycerols demonstrating that fatty acid synthase increases occur in the context of overall increases in endogenous fatty acid synthesis. Cerulenin inhibited acylglycerol synthesis in tumor cells and fibroblast controls in a dose-dependent fashion and also caused a growth inhibition which generally paralleled the level of endogenous fatty acid synthesis. Supraphysiologic levels of palmitate, 14 microM in dimethyl sulfoxide, significantly reversed the growth inhibition caused by cerulenin at concentrations of up to 5 micrograms/ml, indicating that cerulenin-mediated growth inhibition was due to fatty acid synthase inhibition.

Topics: Acetates; Antibodies; Antigens, Neoplasm; Biomarkers, Tumor; Blood Proteins; Breast Neoplasms; Carbon Radioisotopes; Cell Division; Cell Line; Cerulenin; Drug Design; Fatty Acid Synthases; Fatty Acids; Female; Haptoglobins; Humans; Immunohistochemistry; Kinetics; Malonyl Coenzyme A; Palmitic Acid; Palmitic Acids; Prognosis; Tumor Cells, Cultured