If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
[S] The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of eight figures. 1 H. Liu and X. Wu contributed equally to this work. 2 Present address of Z. Luo: Department of Molecular Biology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China.
Fatty acid synthase (FASN) is a key enzyme in the synthesis of palmitate, the precursor of major nutritional, energetic, and signaling lipids. FASN expression is upregulated in many human cancers and appears to be important for cancer cell survival. Overexpression of FASN has also been found to associate with poor prognosis and higher risk of recurrence of human cancers. Indeed, elevated FASN expression has been shown to contribute to drug resistance. However, the mechanism of FASN-mediated drug resistance is currently unknown. In this study, we show that FASN overexpression causes resistance to multiple anticancer drugs via inhibiting drug-induced ceramide production, caspase 8 activation, and apoptosis. We also show that FASN overexpression suppresses tumor necrosis factor-α production and nuclear factor-κB activation as well as drug-induced activation of neutral sphingomyelinase. Thus, TNF-α may play an important role in mediating FASN function in drug resistance.
). Mammalian FASN is a homo-dimer of a multifunctional protein of ∼270 kDa containing seven catalytic domains: β-ketoacyl synthase, malonyl/acetyltransferase, dehydrogenase, enoyl reductase, β-ketoacyl reductase, acyl carrier protein, and thioesterase. Under normal physiological conditions, circulating free fatty acids (FAs) from diet are the preferred source, and de novo FA synthesis is minimally needed. Thus, the FASN expression level is low in normal nonadipose tissues/cells. Cancer cells, however, synthesize more than 90% of their triacylglycerol de novo, requiring increased FASN expression (
), suggesting that increased FASN expression may also contribute to disease progression and treatment failure. In particular, FASN overexpression has been found in a drug-selected breast cancer cell line, and this elevation contributes to cellular resistance to Adriamycin (doxorubicin) and mitoxantrone in breast (
). However, the mechanism by which FASN contributes to drug resistance is currently unknown.
In this study, we investigated the molecular mechanism of FASN-mediated drug resistance using FASN overexpression stable clones of MCF7 cells and found that FASN overexpression causes resistance to multiple anticancer drugs as well as to γ-irradiation via inhibiting treatment-induced ceramide production, caspase 8 activation, and apoptosis. Further studies showed that FASN overexpression suppresses tumor necrosis factor (TNF)-α production, nuclear factor (NF)-κB activation, and drug-induced activation of neutral sphingomyelinase (nSMase). Hence, FASN overexpression may cause drug resistance by inhibiting TNF-α expression, which in turn suppresses activation of NF-κB and nSMase and, thus, reduced ceramide production, caspase 8 activation, and apoptosis.
MATERIALS AND METHODS
All electrophoresis reagents and polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad (Hercules, CA). Doxorubicin, mitoxantrone, cisplatin, etopside, paclitaxel, vinblastine, verapamil, and DTT were from Sigma (St. Louis, MO). Cell culture mediums DMEM and typsin-versene mixture were purchased from Media Tech (Herndon, CA) or Lonza (Walkersville, MD). Monoclonal antibody against human FASN and ceramide were purchased from BD Biosciences (San Jose, CA) and Enzo Life Sciences (Farmingdale, NY), respectively. Monoclonal antibody against cleaved poly(ADP-ribose) polymerase (PARP), caspase-8, cleaved caspase-8, and cleaved caspase-9 were obtained from Cell Signaling (Danver, MA). G418 was from Invitrogen (Carlsbad, CA). Specific siRNA targeting human FASN mRNA (sc-43758) was purchased from Santa Cruz Biotechnology. Specific siRNAs targeting human nSMase-2 and FASN were purchased from Qiagen and Santa Cruz Biotechnology, respectively. The NF-κB reporter expression construct and its negative control plasmid of the PathDetect NF-κB Cis-Reporting System were from Agilent Technologies (Santa Clara, CA). All other chemicals were of molecular biology grade from Sigma (St. Louis, MO) or Fisher Scientific (Chicago, IL).
FASN-overexpressing MCF7 clones (FASN1 and FASN2) and vector-transfected control clone (Vec) were generated previously (
) and maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 400 µg/ml G418. MCF7/AdVp3000 and its stable clone with FASN knockdown were also generated in the previous study (
Cells were washed with phosphate-buffered saline (PBS) and then lysed in a buffer [1% Triton X-100, l50 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, and 0.1% SDS) for 30 min at 4°C with constant agitation. The cell lysates were sonicated briefly followed by centrifugation (16,000 g, 4°C) for 15 min to remove insoluble materials. The protein concentration of the soluble fraction (cell lysates) was determined using a Bio-Rad protein assay kit.
Western blot analysis was done as described previously (
). Briefly, cell lysates were separated by SDS-polyacrylamide gel electrophoresis followed by transfer to a PVDF membrane. The blot was then probed with a monoclonal antibody to FASN, cleaved PARP, AKT, NF-κB p65, c-FLIP, or caspases, followed by reaction with horseradish peroxidase-conjugated secondary antibodies. The signal was captured by X-ray film using enhanced chemiluminescence.
FASN activity assay
FASN activity was determined using a protocol previously described (
). Briefly, 120 μg of particle-free supernatant of cell lysate was mixed with a buffer containing 200 mM potassium phosphate (pH 6.6), 1 mM DTT, 1 mM EDTA, 0.24 mM NADPH, and 30 μM acetyl-CoA in a final volume of 0.3 ml, and the reaction was monitored at 340 nm for 3 min to measure background NADPH oxidation. After addition of 50 μM malonyl-CoA, the reaction was monitored for an additional 15 min to determine FASN-dependent oxidation of NADPH using a Synergy H1 hybrid reader with Gen5 2.0 software (BioTek Instruments). The rate change of OD340nm was corrected for the background rate of NADPH oxidation. The change in concentration of NADPH during oxidation was calculated using the following equation:
where ΔC is change in the concentration of NADPH, ΔA is change in absorbance, and E is extinction coefficient of NADPH (E340nm= 6.22 mM−1∙cm−1). FASN activity was expressed as nmol NADPH oxidized/min/mg protein.
Real-time RT-PCR was performed as described previously (
). Briefly, cells were harvested, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) followed by real-time RT-PCR using the Power SYBR Green RNA-to-CT 1-Step kit (Applied Biosystem, Carlsbad, CA). Data were normalized to an internal control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer pairs used were 5′-GCTGACCCCAGGCTGTGA-3′ (forward) and 5′-TGCTCCATGTCCGTGAACTG-3′ (reverse) for FASN; 5′-CCCAGGCAGTCAGATCATCTTC-3′ (forward) and 5′-GGTTTGCTACAACATGGGCTACA-3′ (reverse) for TNF-α and 5′-AGGACTGGCTGGCTGATTTC-3′ (forward) and 5′-TGTCGTCAGAGGAGCAGTTATC-3′ (reverse) for nSMase-2. Analysis of a single PCR product was confirmed by melt-curve analysis.
TNF-α level in the medium was measured using Human TNF-α ELISA kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's instructions. Briefly, vector-transfected or FASN1 and FASN2 clones of MCF7 cells were seeded at 1 × 105 cells/well in 48-well plates and on the next day treated with 1 µM doxorubicin or control vehicle for 6 h followed by collection of medium and determination of TNF-α protein concentration.
The cytotoxicity of the cells to anticancer drugs or γ-irradiation was determined using the sulforhodamine B (SRB) colorimetric assay as previously described (
). Briefly, cells in 96-well plates were cultured for 24 h and treated with different concentrations of anticancer drugs for three days. The culture medium was then removed, and the cells were fixed and stained by 0.4% (w/v) sulforhodamine B (Sigma) in 1% acetic acid followed by washing with 1% acetic acid to remove the unbound SRB. The bound SRB was then solubilized with 10 mM Tris base, and the OD570 nm was determined using a 96-well plate reader (MRX; Dynex Technologies, Chantilly, VA).
TNF-α promoter reporter construct engineering
TNF-α promoter was amplified using genomic DNA from MCF7 cells as template and primers 5′-ACTCGAGGCCGCCAGACTGCTGCAGGG-3′ (forward) and 5′-ACCATGGAGAGGGTGGAGCCGTGGGTCA-3′ (reverse) derived from the sequence of human TNF-α promoter in GenBank (AY274896). The PCR product was first cloned into pGEM-T easy vector (Promega, Madison, WI) and reengineered into the promoterless luciferase reporter vector pGL4.10 (Promega). The final luciferase report construct was verified by double-strand DNA sequencing.
Luciferase reporter assay
For the assay of TNF-α promoter activity, MCF7 cells with stable FASN overexpression or MCF7/AdVp3000 cells with stable FASN knockdown and their respective control cells were seeded at 5 × 104 cells/well in 24-well plates and transfected with the firefly luciferase reporter construct driven by the TNF-α promoter. Renilla luciferase reporter with a TK promoter was used as a control for transfection efficiency. Forty-eight hours after transfection, cells were washed three times with PBS and lysed in passive lysis buffer (Promega, Madison, WI). Luciferase activity was measured using the dual luciferase reporter assay system according to the manufacturer's instructions (Promega).
For the assay of NF-κB activity, the same procedures were followed as described above using the PathDetect NF-κB Cis-Reporting System along with the renilla luciferase reporter driven by a TK promoter as a control for transfection efficiency. Basal NF-κB activity was detected 48 h after transfection. For doxorubicin-induced NF-κB activity, MCF7/AdVp3000 cells were treated with 0.1 µM doxorubicin for 24 h in the presence of 10 µM FTC after reporter transfection, followed by determination of luciferase activity.
Transient knockdown of target genes
For transient knockdown of FASN and nSMase-2, ∼30 nM siRNA duplexes along with control scrambled siRNAs were transiently transfected into cells using Metafectene Pro transfection reagent (Biontex, CA) as previously described (
The cell death detection ELISA (Roche, Mannheim, Germany) assay was conducted to determine apoptosis in control and FASN-overexpression MCF7 cells after treatment with or without 1 μM doxorubicin for 48 h. PARP cleavage, another indicator of apoptosis, was analyzed using Western blot probed with an antibody that detects only the large fragment of the cleaved PARP to avoid potential confusion from detection of the uncleaved PARP.
Determination of ceramide level
Ceramides were extracted as previously described (
). Briefly, MCF7 cells with stable FASN overexpression and vector-transfected control cells were resuspended in 0.5 ml PBS, mixed with 10 µl of 2.5 ng/l 17:0 ceramide as internal standards and 3 ml of methanol/chloroform (2:1), followed by incubation on ice for 10 min. Chloroform (1 ml) and water (1.3 ml) were then added to the mixture and vortexed for 1 min prior to centrifugation at 1,750 g for 10 min at 10°C. The organic phase was transferred to a new glass tube for evaporation of the solvent under nitrogen at room temperature. The dried lipids were dissolved in 1 ml of methanol for ESI-MS analyses using API-4000 (Applied Biosystems/MDS SCIEX, Forster City, CA) with an Analyst data acquisition system. Samples (10 µl each) were delivered into the electrospray ionization (ESI) source through a LC system (Agilent 1100) with an auto sampler. The mobile phase was methanol/water/ammonium hydroxide (90:10:0.1, v/v/v). Standard curves for all ceramides were established quantitative analyses.
The effect of GW4869 on total ceramide production was determined using immunofluorescence staining as previously described (
). Briefly, MCF7 cells were treated with 10 μM GW4869 or 5% methane sulfonic acid vehicle for 30 min, and then treated with 1 μM ADR or vehicle DMSO for 24 h. Cells were then harvested, fixed, and permeabilized using the BD Cytofix/Cytoperm Plus kit (BD, Franklin Lakes, NJ), followed by staining with monoclonal anti-ceramide antibody and FITC-conjugated secondary antibody. DAPI was used to stain total DNA as a control. Fluorescence was measured using a Synergy H1 plate reader with FITC at excitation/emission of 485/535 nm and DAPI at 350/470 nm. FITC fluorescence intensity was adjusted by that of the DAPI.
nSMase activity assay
nSMase activity was determined using the sphingomyelinase assay kit from Echelon Biosciences (Salt Lake City, UT) following manufacturer's instructions. Briefly, vector-transfected and FASN1 or FASN2 stable clones of MCF7 cells were treated with or without 1 μM doxorubicin for 48 h. Cells were then harvested, lysed, and centrifuged at 20,000 g for 10 min to remove insoluble and noncytosolic proteins. About 100 µg of cytosolic proteins were used for determination of nSMase activity.
FASN overexpression causes resistance to multiple chemotherapeutic agents and γ-irradiation
We have shown previously that ectopic overexpression of FASN increases cellular resistance to doxorubicin and mitoxantrone (
). To determine whether FASN overexpression contributes to multidrug resistance, we tested the response of two previously established MCF7 clones with ectopic overexpression of FASN (FASN1 and FASN2) to multiple anticancer drugs in comparison with control cells transfected with vector (Vec). Fig. 1A–C shows that both FASN1 and FASN2 clones have increased FASN mRNA and protein levels as well as FASN activity compared with the Vec control cells. However, the two clones do not appear to differ significantly in FASN expression and activity. Fig. 1D shows that both FASN1 and FASN2 cells are significantly more resistant than the control Vec clone to doxorubicin, mitoxantrone, etopside, camptothecin, and cisplatin with 1.5- to 3-fold increases in relative resistance factor. However, the relative resistance factor between FASN1 and FASN2 cells is not significantly different, consistent with FASN expression level and activity between the two cells. Interestingly, FASN overexpression had no significant effect on cellular response to vinblastine and paclitaxel. These observations suggest that FASN overexpression may cause cellular resistance to DNA-damaging drugs but not to microtubule modulators, such as vinca alkaloids, in MCF7 cells. To further test this notion, these cells were treated with γ-irradiation followed by survival analysis. Fig. 1D shows that both FASN1 and FASN2 cells are also significantly more resistant to γ-irradiation than the Vec control cells.
FASN overexpression protects cancer cells from drug-induced apoptosis via inhibiting caspase 8 activation
To determine whether FASN overexpression protects cells from drug-induced apoptosis, we first tested the drug-induced apoptosis rate of FASN1 and FASN2 stable clones compared with Vec control cells using the cell death detection ELISA kit that quantifies the level of DNA fragmentation. As shown in Fig. 2A, both FASN1 and FASN2 clones had significantly lower levels of doxorubicin-induced DNA fragmentation compared with the Vec control cells. We next analyzed cleaved PARP, another indicator of apoptosis (
), using Western blot and found that the production of the 89 kDa cleaved PARP was significantly less in response to doxorubicin treatment in FASN1 and FASN2 cells than in the Vec control cells (Fig. 2B), consistent with the findings using ELISA assay. Together, these results show that FASN overexpression likely inhibits drug-induced apoptosis and thus contributes to doxorubicin resistance in MCF7 cells.
We next determined whether FASN affects drug-induced activation of caspases 8 or 9 using Western blot analysis. As shown in Fig. 2C, caspase 8 in the Vec control cells is activated following doxorubicin treatment, whereas this activation in FASN1 and FASN2 clones is inhibited. Caspase 9, on the other hand, failed to be activated by doxorubicin treatment in the cells tested, and there is no difference in caspase 9 activation between the Vec control cells and the FASN1 and FASN2 cells (data not shown). Thus, FASN may protect cells from drug-induced apoptosis by suppressing drug-induced caspase 8 activation.
To determine whether the above observation is cell-line specific and whether knocking down FASN in a parental cell line would have the opposite effect on apoptosis and caspase 8 activation, we transiently knocked down FASN expression in human breast cancer cell line MDA-MB-468 as we previously described (
), followed by doxorubicin treatment and determination of PARP cleavage and caspase 8 activation. As shown in Fig. 2D, FASN knockdown sensitized/enhanced the parental MDA-MB-468 cells to doxorubicin-induced apoptosis and caspase 8 activation. This observation is consistent with our previous finding that FASN knockdown in MDA-MB-468 cells causes sensitization to anticancer drugs (
), we next determined whether FASN overexpression affects drug-induced degradation of c-FLIP using Western blot analysis. As shown in supplementary Fig. I-A, both c-FLIPL and c-FLIPS appeared to be at similar level between the Vec control cells and stable clones with FASN overexpression (FASN1 and FASN2). Following doxorubicin treatment, both c-FLIPL and c-FLIPS are completely degraded in all three cell lines and FASN overexpression does not inhibit drug-induced c-FLIP degradation. Thus, c-FLIP unlikely mediates FASN inhibition of drug-induced caspase 8 activation.
FASN has been shown to have a positive feedback relationship with AKT in multiple studies (for a review, see Ref.
). Consequently, we tested whether FASN overexpression affects doxorubicin-induced AKT activation and phosphorylation at 24 h following doxorubicin treatment using Western blot analysis. Supplementary Fig. I-B shows that the basal level of phosphorylated AKT is not detectable in all three (Vec, FASN1, and FASN2) cell lines. Following a 24 h doxorubicin treatment, the level of phosphorylated AKT increased dramatically in all three cell lines. However, the level of phosphorylated AKT in FASN1 and FASN2 cells is not different from that in the Vec control cells. Thus, FASN overexpression in MCF7 cells does not appear to affect doxorubicin-induced AKT activation. Although AKT has been mainly involved in preventing apoptosis, it is also known to activate c-FLIP (
). In addition, the metabolism of different classes of lipids is highly intertwined. Thus, we tested the potential effects of FASN on ceramide production. As shown in Fig. 3, the basal total ceramide level in FASN1 and FASN2 stable clones is slightly higher than in the Vec control cells (Fig. 3A), and doxorubicin treatment dramatically induced ceramide level in all cell lines (Fig. 3B). However, the doxorubicin-induced increase in total ceramide production is compromised in the FASN stable clone (more significantly in FASN2 clone) compared with that in the Vec control cells (Fig. 3B, C). The basal and doxorubicin-induced production of individual ceramide is shown in supplementary Fig. II. It is clear that the C16:0 ceramide is the major constituent of the total ceramide and that doxorubicin-induced C16:0 ceramide production is compromised in both FASN1 and FASN2 cells compared with the Vec control cells. Doxorubicin-induced production of other ceramides, such as C18:1 and C24:0, also appears to be reduced in FASN-overexpression cells. It is noteworthy that FASN1 cells may be more tolerant than FASN2 cells to ceramide because its basal level of ceramide is significantly higher. Although doxorubicin-induced absolute level of total ceramide is not significantly lower in the FASN1 clone, the fold increase in doxorubicin-induced ceramide production is significantly less in FASN1, similar to FASN2, than in the Vec control cells. The individual ceramides also have the similar trend of decrease.
Ceramide can be generated either by de novo synthesis from palmitoyl CoA and serine or by breaking down sphingomyelin by acid or neutral sphingomyelinase (aSMase or nSMase). To determine which pathway is used in doxorubicin-induced ceramide generation and subsequent cell death in MCF7 cells, several selective inhibitors were used in combination with doxorubicin for survival assay. As shown in Fig. 4, fumonisin B1 and myriosin (inhibitors of de novo ceramide synthesis; Fig. 4A, B) and desipramine (aSMase inhibitor; Fig. 4C) had no effect on doxorubicin inhibition of cell survival. However, GW4869, an nSMase inhibitor that can effectively inhibit doxorubicin-induced ceramide production in MCF7 cells (supplementary Fig. III), blocked the inhibitory effect of doxorubicin on cell survival (Fig. 4D). Because nSMase-2 is the major nSMase in MCF7 cells (
), we next tested whether knocking down nSMase-2 expression using siRNA could block the inhibitory effect of doxorubicin on cell survival. As shown in supplementary Fig. IV, knocking down nSMase-2 expression significantly attenuated the doxorubicin effect on MCF7 cell survival. Thus, the nSMase→ceramide→caspase 8→apoptosis pathway may contribute to doxorubicin-induced apoptosis of MCF7 cells, and this pathway may be inhibited by FASN overexpression.
To test this possibility, we compared the nSMase activity among FASN1, FASN2, and the Vec control cells. Fig. 5A shows that the basal nSMase activity had no significant difference among all three cell lines. Upon doxorubicin treatment, the nSMase activity in the Vec control cells is dramatically increased (Fig. 5B). However, the doxorubicin-induced increase in nSMase activity is significantly suppressed by FASN overexpression in the FASN1 and FASN2 cells. Thus, nSMase may be a major target and mediator of FASN in doxorubicin resistance.
TNF-α expression is suppressed by FASN overexpression
To elucidate the signaling pathways further, a nonbiased comparative real-time RT-PCR SuperArray analysis of FASN1, FASN2, and the Vec control cells was conducted. Of the 89 genes analyzed, we found only 2 genes [interleukin (IL)-8 and TNF-α] that had greater than 1.5-fold change consistently in duplicated experiments between the Vec control and the FASN-overexpressing FASN1 and FASN2 cells (see supplementary Tables I and II). This array-based finding was verified in another independent FASN-overexpressing clone in duplicated experiments (supplementary Table III). The average reduction in IL-8 and TNF-α expression in all three stable clones with FASN overexpression was about 16- and 13-fold, respectively.
To verify the above findings, we performed a real-time RT-PCR analysis of TNF-α expression in the control Vec and FASN-overexpressing FASN1 and FASN2 cells. As shown in Fig. 6A, B, both the basal and doxorubicin-induced TNF-α mRNA levels are significantly reduced in FASN1 and FASN2 cells compared with the Vec control cells. To determine whether the production of TNF-α protein is also inhibited, we performed an ELISA assay. As shown in Fig. 6C, doxorubicin-induced production of TNF-α protein was significantly reduced in the FASN1 and FASN2 cells compared with the Vec control cells. We next performed analysis of TNF-α expression using the stable FASN knockdown MCF7/AdVp3000 cells (
), and we found that FASN knockdown drastically increased both basal and doxorubicin-induced TNF-α expression (Fig. 6D, E), consistent with the findings of FASN-overexpression cells. We also tested a different drug, mitoxantrone, in inducing TNF-α production and found that FASN knockdown similarly inhibited mitoxantrone-induced TNF-α expression (Fig. 6F).
To determine whether the effect of FASN on TNF-α expression is possibly via inhibiting its transcription, FASN-overexpressing MCF7 cells and FASN-knockdown MCF7/AdVp3000 cells along with their vector or scrambled shRNA-harboring control cells were transfected with a firefly luciferase reporter construct driven by TNF-α promoter. A renilla luciferase reporter construct driven by thymidine kinase promoter was cotransfected into these cells as controls for transfection efficiency. As shown in Fig. 7A, both FASN1 and FASN2 cells show significantly lower relative TNF-α promoter activity than do the Vec control cells. MCF7/AdVp3000 cells with FASN knockdown (Si) have significantly higher relative TNF-α promoter activity than the control cells (Scr) (Fig. 7B).
Because TNF-α is a known upstream regulator of and activates NF-κB, we hypothesize that the effect of FASN on TNF-α expression likely will consequently affect NF-κB activity. To test this possibility, we performed a reporter assay of NF-κB activity in MCF7/AdVp3000 cells with stable FASN knockdown and its scramble shRNA transfected control cell by transiently transfecting luciferase reporter construct driven by a promoter containing NF-κB response element followed by treatment with or without doxorubicin and determination of luciferase expression. As shown in Fig. 7C, D, both the basal and doxorubicin-induced NF-κB activity are dramatically increased in the cells with FASN knockdown compared with the control cells. Furthermore, we found that the expression level of NF-κB in the FASN overexpression cell lines is dramatically reduced compared with that of the Vec control cells (Fig. 7E). These observations are consistent with the negative relationship between FASN expression and TNF-α expression. Together, these findings suggest that FASN may downregulate the transcription and production of TNF-α, which in turn downregulates NF-κB level and activity and suppresses doxorubicin-induced ceramide production and apoptosis.
Altered and increased metabolism is one of the hallmarks of cancer (
). Despite the fact that the use of anticancer agents in appropriate combinations has led to major improvements in the treatment of malignant tumors, resistance to chemotherapy frequently occurs and is a major obstacle in cancer treatment. Hence, understanding metabolic-related drug resistance becomes critical. In this study, we show that FASN overexpression in breast cancer cell line MCF7 causes resistance to DNA damage-induced cell death possibly via inhibition of TNF-α production, which in turn increases NF-κB activity and reduces drug-induced production of ceramide possibly by nSMase and caspase 8-mediated apoptosis.
Previously, FASN overexpression was found in Adriamycin (doxorubicin)-selected and multidrug-resistant MCF7 cells, and it contributes to the observed drug resistance of these cells (
). Interestingly, we did not find that FASN overexpression in MCF7 cells causes resistance to non-DNA-damaging drugs, such as paclitaxel and vinblastine. This finding is contradictory to the observations by Menendez et al. (
) who found that inhibiting FASN activity enhanced the cytotoxicity of docetaxel in the Her2-overexpressing breast cancer cell lines. The same group has also reported that inhibiting FASN expression or activity could sensitize breast cancer cells with a high Her2 level to vinorelbine (
). The reason for this discrepancy is currently unknown; however, it is possible that the high level of Her2 may affect the role of FASN in cellular response to docetaxel.
Although the detailed mechanism is not yet known, FASN has been shown to play a role in regulating gene expression. A previous genomic profiling analysis of a breast cancer cell line MDA-MB-435 showed that FASN knockdown upregulated the expression of IL-8 (
), the observed reduction of IL-8 in FASN-overexpressing clones may be the result of TNF-α reduction. Indeed, we also found that NF-κB activity in FASN knockdown cells is significantly upregulated, and its expression in FASN-overexpression cells is downregulated. Thus, the TNF-α signaling pathway may be inversely regulated by FASN, and it may mediate FASN-induced drug resistance.
It is noteworthy that the effect of FASN on TNF-α promoter activity (Fig. 7) is lesser compared with the effect on the expression of endogenous TNF-α (Fig. 6). The reason for this difference is currently unknown. However, it is likely that transfection efficiency of the reporter construct contributed to the lower effect of FASN on the promoter activity assay. It is also possible that the TNF-α promoter in the reporter construct does not constitute the proper structure as does the endogenous promoter for a more efficient effect. Further, it cannot be ruled out that the endogenous TNF-α mRNA is less stable in FASN-overexpression cells, whereas the stability of reporter mRNA is not affected by FASN expression. All these possibilities may contribute to the lower effect of FASN on the reporter assay than on the endogenous TNF-α, and further investigations are needed to delineate the detailed mechanism of FASN effect on TNF-α expression.
GW4869, among several other inhibitors, was found to be the only inhibitor that improved survival against doxorubicin treatment. GW4869 is a cell-permeable, noncompetitive, specific inhibitor of nSMase-2 (
). Furthermore, it has been shown that anthracycline drugs, such as doxorubicin, induce ceramide production in cancer cells by activating nSMase and that inhibiting nSMase attenuates doxorubicin-induced ceramide production (
). Thus, we conclude that the FASN function in drug resistance and in suppression of drug-induced ceramide production is likely mediated by nSMase. This conclusion is corroborated by our observation that specifically knocking down nSMase-2 significantly attenuates the doxorubicin effect on MCF7 cell survival and that inhibiting nSMase reduces doxorubicin-induced ceramide production.
It is noteworthy that FASN appears to inhibit doxorubicin-induced fold increase in ceramide level in two independent clones, although the absolute level of increase in one of the two clones is also significantly reduced by FASN overexpression (Fig. 3). Currently, it is not clear whether the inhibition in fold increase or absolute level of ceramide is the key to FASN function in doxorubicin resistance. Future studies will be needed to clarify this issue.
It has also been shown that TNF-α activates nSMase and initiates a rapid increase in ceramide production (
Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2.
). On the basis of our findings and previous observations, we propose that FASN overexpression suppresses production of TNF-α, which in turn reduces doxorubicin-induced production of ceramide possibly by reducing nSMase activity, resulting in lowered caspase 8 activation and apoptosis (Fig. 8). Because mitoxantrone, camptothecin, and etopside, similar to doxorubicin, are all topoisomerase II inhibitors, FASN may also use the same mechanism of resistance to these drugs as to doxorubicin (see Fig. 8). Indeed, we found that mitoxantrone-induced TNF-α expression is significantly increased by knocking down FASN expression. However, how FASN overexpression inhibits TNF-α production is currently unknown. Previously, it has been shown that supplementation of palimitate causes cellular resistance to doxorubicin and mitoxantrone (
). Thus, it is possible that palmitate, the final product of FASN, plays some role in inhibiting TNF-α production; this possibility is awaiting further investigation.
It is also noteworthy that the anticancer drugs (doxorubicin, mitoxantrone, camptothecin, and etopside) that FASN causes resistance to are topoisomerase II inhibitors and that they as well as γ-irradiation induce double-strand DNA breaks. Cisplatin also causes DNA damage. Thus, it is tempting to speculate that FASN overexpression may protect MCF7 cells against DNA damage or facilitate the repair of damage. In support of this argument, it has been observed that FASN suppresses the expression of DNA damage-inducible transcript 4, a stress-response gene, which may also mediate the function of FASN in resistance to DNA-damaging treatments. We are currently investigating this possibility.
The authors thank Joleyn Khoo for her technical support in PCR analysis.
Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker.
Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2.
This work was supported in part by National Institutes of Health Grant CA-113384. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. Hailan Liu and Xi Wu are recipients of US Department of Defense Predoctoral Fellowships W81XWH-06-1-0490 and W81XWH-10-1-0057.