Acetyl-CoA Carboxylase A Is Essential to Breast Cancer Cell Survival

Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase A (ACC A ), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACC A in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACC A or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)–resistant form of ACC A mRNA prevented the effect of ACC A -RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACC A and FAS siRNA–induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACC A or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy. (Cancer Res 2006; 66(10): 5287-94) France). Helium was used as carrier gas at a flow rate of 1.5 mL/min, with N 2 as make-up gas for the flame ionization detector. The temperature was programmed to increase from 65 j C to 135 j C at a rate of 3 j C/min and from 135 to 220 j C at a rate of 2 j C/min, then kept constant for 5 minutes. Identification of fatty acid methyl esters was obtained by comparison with the retention times of pure standard mixtures (Supelco, St. Quentin Fallavier, France). Deuterated palmitate and palmitic acid contents in tumor cells were quantified using the internal standard L -A-phosphatidylcholine, dimyristoyl-d54 and normalized for cell number. Statistical analysis. Data are expressed as mean F SD (three independent experiments). Comparison of values was done using a non- parametric Mann-Whitney U test. P < 0.05 was considered as statistically significant.


Introduction
Numerous lines of evidence suggest that activation of fatty acid synthesis is required for carcinogenesis. Tumor cells synthesize 95% of saturated and monosaturated fatty acids de novo despite adequate nutritional lipid supply ( for review, see ref. 1). Hence, fatty acid accumulation due to induction of de novo lipogenesis might lead to specific alterations that we previously reported in membrane lipid composition of breast tumor tissues (2) and consequently might affect fundamental cellular processes such as signal transduction pathways and gene expression.
The fatty acid synthesis pathway involves two key enzymatic reactions. The first is the carboxylation of acetyl-CoA in the cytosol to form malonyl CoA. This step, catalyzed by acetyl-CoA carboxylase a (ACCa), is a key regulatory step in the fatty acid synthesis pathway, which is preferentially controlled by the equilibrium between active and less active forms of ACCa involving reversible phosphorylation. Malonyl-CoA is sequentially used by the subsequent enzyme in the pathway, fatty acid synthase (FAS), as a donor of acetate moieties to the growing acyl chain, resulting in the synthesis of palmitic acid (16:0).
Although both ACCa and FAS govern de novo lipogenesis, most attention in the past few years on the role of this pathway in tumor biology has been directed on the consequences of FAS expression, which is markedly increased in many human epithelial cancers, including breast and prostate cancers (3)(4)(5)(6)(7). Induction of FAS probably occurs via transcriptional mechanisms (8,9) and more specifically via posttranslational events in prostate cancer cells (10). Previous studies showed that an inhibition of FAS by chemical inhibitors (cerulenin and C75) or by RNA interference (RNAi)selective gene silencing leads to a rapid decline in fatty acid synthesis, associated with growth inhibition and cell apoptosis (11)(12)(13)(14)(15)(16)(17)(18). The growth arrest response triggered by FAS inhibition has been attributed to altered lipid production (11,15). The apoptosis induced by FAS inhibition has been attributed to cytoplasmic malonyl-CoA accumulation (14,16,17) that was recently contradicted for prostate cancer cells (19).
We recently described an interaction between ACCa and BRCA1, product of the breast cancer susceptibility gene, through its BRCT domain (20). We hypothesize that BRCA1 mutation may lead to a disruption of BRCA1-ACCa complex, which in turn increases ACCa release and consequently lipogenesis in breast/ovarian tumor cells. This suggests that ACCa activity might be essential for breast cancer cells survival. This last hypothesis has been taken as the start point for the present investigation, aimed at examining the role of ACCa in mammary cell survival and consequently in mammary carcinogenesis. For this purpose, we used a strategy based on specific gene silencing by RNAi. We showed that exogenously added synthetic 21-nucleotide small interfering RNA (siRNA) duplexes act as specific agents to silence ACCa gene expression. In all human breast cancer cell lines tested, this selective ACCa gene silencing, as well as selective FAS gene silencing, is correlated to a decline in fatty acid synthesis, growth arrest, and induction of apoptosis associated with alteration of mitochondrial function. On the contrary, in human mammary epithelial cells, selective FAS or ACCa inhibition leads to a decline in fatty acid synthesis without affecting both cell growth and cell survival. Our data provide evidence that fatty acids newly synthesized through de novo lipogenesis are essential to breast cancer cell survival and, thus, that ACCa might be considered as a key target for antineoplastic therapy. Our data also increase the potent interference between therapy based on lipogenesis inhibition and dietary lipids intake.

Materials and Methods
Cell culture. Human breast carcinoma cell lines MCF-7, MDA-MB-231, breast transformed cell line HBL100, and prostate cancer cell line LNCaP were used and tested for mycoplasm-free cell lines. Stock cultures were grown in RPMI 1640 containing 10% FCS and 1% antibiotics. The cells were grown in growth medium at 37jC in a 95% air, 5% CO 2 -humidified incubator. Human mammary epithelial cells (Cambrex) were grown in MEGM Bulletkit (Cambrex, Emerainville, France) containing growth factors, cytokines, and supplements according to the recommendation of the manufacturer.
RNAi. To design siRNA oligonucleotides targeting ACCa, DNA sequences of the type AA (N19; 5 ¶-GATGAGACTAGCCAAACAA-3 ¶) were selected according to the protocol of the manufacturer (Proligo Primers & Probes) and submitted to a BLAST search against human genome sequence to ensure reliable specificity. This sequence corresponded to exon 13, nucleotides 1,043 to 1,062 located 3 ¶ to the first nucleotide of the start codon of the human ACCa cDNA (NM198836). The siRNA oligonucleotides targeting FAS gene were derived from previous report (18). As a nonspecific siRNA control, the luciferase siRNA duplex was used as control. Transfections of human breast cancer cells with siRNA-targeting ACCa, FAS, or luciferase were done in six-well culture plates at a density of 1 Â 10 4 per well using Lipofectamine 2000 (Invitrogen, Cergy-Pontoise, France) and 0.2 nmol of siRNA duplex, resulting in a final siRNA concentration of 100 nmol/ L. SiRNA-transfected cells were collected at 0, 24, 48, 72, and 96 hours after transfection for real-time PCR analysis. Cells were used 72 hours after transfection for fatty acid analysis and 96 hours after transfection for Western blot analysis. Cells were harvested at different time points after transfection according to the cell type for apoptosis analysis (96 hours for HBL-100 and MDA-MB-231, 144 hours for MCF-7 and LNCaP).
Fluorescence-activated cytometry sorter analysis. Both attached and detached cells, transfected or untransfected by siRNA, were harvested at different time points, depending on cell lines, and resuspended in 500 AL medium containing 40 nmol/L DiOC 6 , 0.1 mg/mL Hoechst 33342, and 5 Amol/L hydroethidium (Molecular Probes, Inc., Eugene, OR; Synthetic) for 15 minutes at 37jC. Cells were immediately analyzed in a flow cytometer equipped with argon and krypton lasers. Hoechst was excited with 350-to 356-nm light from a krypton laser and emitted light was collected using a 467-nm bandpass filter. DiOC 6 was excited at 488 nm and detected at 525 nm. Hydroethidium was excited at 490 nm and detected at 620 nm.
Fatty acid analysis. Seventy-two hours after transfection, cells were fed with serum-free fresh medium for 4 hours. Then, they were supplemented with deuterated acetate (Cambridge Isotope Laboratories, Inc., Andover, MA; 5 mg/mL) for 4 hours à 37jC. One million of cells were then collected and total lipids were extracted from tumor cells with 4 mL of chloroform/ methanol (2:1) containing 100 AL of antioxidant butylated hydroxytoluene (1 mg/mL in methanol) and 40 AL of L-A-phosphatidylcholine, dimyristoyl-d54 (Cambridge Isotope Laboratories; 0.5 mg/mL in methanol) as internal standard. After centrifugation, the extract was washed with 1.0 mL NaCl. The separated chloroform layer was evaporated to dryness. Dichloromethane (80 AL) was added to dissolve the lipids, followed by 25 AL Methyl-Prep II (Alltech, Deerfield, IL) to convert the fatty acids to their methyl esters. The mixture was incubated for 10 minutes at room temperature. Fatty acid methyl esters were extracted twice into hexane, which was evaporated and dissolved in 200 AL hexane. Fatty acid methyl ester composition was determined by capillary gas chromatography (Agilent Technologies, Massy, France). One microliter was injected through an on-column injector at 65jC into a gas chromatograph with the aid of an automatic injector (Agilent Technologies) operated at column and detector temperatures of 220jC and 250jC, respectively. A 30 m Â 0.32 mm internal diameter fused silica capillary column with 0.25-Am film thickness was used for the separation of fatty acid methyl esters (SGE, Courtaboeuf, France). Helium was used as carrier gas at a flow rate of 1.5 mL/min, with N 2 as make-up gas for the flame ionization detector. The temperature was programmed to increase from 65jC to 135jC at a rate of 3jC/min and from 135 to 220jC at a rate of 2jC/min, then kept constant for 5 minutes. Identification of fatty acid methyl esters was obtained by comparison with the retention times of pure standard mixtures (Supelco, St. Quentin Fallavier, France). Deuterated palmitate and palmitic acid contents in tumor cells were quantified using the internal standard L-Aphosphatidylcholine, dimyristoyl-d54 and normalized for cell number.
Statistical analysis. Data are expressed as mean F SD (three independent experiments). Comparison of values was done using a nonparametric Mann-Whitney U test. P < 0.05 was considered as statistically significant.

Results
Effect of siRNA-targeting ACCA on mRNA and protein levels in human breast cancer cells. To specifically silence the ACCa gene, breast tumor cell lines (MDA-MB-231 and MCF-7) and mammary epithelial transformed cell line (HBL100) were transfected with siRNA-targeting ACCa mRNA. To further compare ACCa and FAS inhibition effect, silencing of FAS gene by RNAi was also done according to previous report (18). As shown in Fig. 1, siRNA-targeting ACCa severely suppressed expression of ACCa when compared with control cells. For the HBL100 cell line, basal ACCa expression level was too low to determine ACCa protein level after siRNA treatment (see basal ACCa expression in Fig. 1A, top). In the same way, siRNA-targeting FAS suppressed expression of FAS in tumor cell lines when compared with control. Interestingly, although treatment of the three cell lines with siRNA-targeting ACCa had no effect on the levels of FAS, depletion of FAS by siRNA in both MCF-7 and MDA-MB-231 cells also slightly reduced the levels of ACCa (Fig. 1B).
Effect of siRNA-targeting ACCA on cell growth and cell apoptosis. To examine the effect of ACCa siRNA on cell phenotype, we examined several hallmarks of cell apoptosis and cell proliferation. ACCa siRNA-transfected cells were analyzed by three-color flow cytometry after staining with hydroethidium that stains cells producing reactive oxygen species (ROS) and with DiOC 6 that stains cells with normal mitochondrial function. Indeed, some apoptotic pathways are linked to mitochondrial perturbations, such as the loss of the mitochondrial membrane integrity, resulting in the collapse of the mitochondrial membrane potential reflected by decreased binding of DiOC 6 ( for review, see ref. 22). Cells were also stained with Hoechst 33342, allowing quantification of DNA content. In parallel, the cell numbers were determined with a cell counter. Before applying this method, we had done trypan blue exclusion and Annexin V staining to establish the apoptotic pathway. The prostate cancer cell line LNCaP previously described as sensitive to ACCa and FAS siRNA-induced apoptosis was used as a positive control (18,19). In both MDA-MB-231 and HBL100 tumor cell lines, transfection with siRNA-targeting ACCa or FAS for 96 hours resulted in an increase in DiOC 6 -negative cells, from 1% to 3% in the control cells to up to 80% to 90% and 70% to 80% in the ACCa and FAS siRNA-treated cells, respectively ( Fig. 2A, black columns). This effect is already significant at 72 hours posttransfection, with an increase in DiOC 6 -negative cells to up to 55% and 40% in the ACCa and FAS siRNA-treated cells, respectively (data not shown). Moreover, whatever the cell line and in both ACCa and FAS siRNA, ROS formation is restricted to DiOC 6 -negative cells ( Fig.  2A, gray columns). This effect is less marked in MCF-7 and LNCaP cell lines, with apoptotic cells increasing from 5% in the control cells to up to 25% to 40% in cells treated by ACCa or FAS siRNA (data not shown).
Finally, percentage of Hoechst low-intensity cells matched with the percentage of DiOC 6 -negative/ROS-positive cells ( Fig. 2A, white  columns). Counting the number of cells revealed that ACCa and FAS siRNA results in a stagnation of the number of cells (Table 1) whereas control cells continue to proliferate. Apparently, for both cell lines, ACCa or FAS silencing-induced apoptosis did not result in the accumulation of cells in a particular phase of the cell cycle (Table 1).
To address whether ROS production is the cause of HBL-100 and MDA-MB-231 cell death induced by ACCa and FAS siRNA, we next used the free radical-scavenging antioxidant a-tocopherol, the most biologically active form of vitamin E. For both siRNA transfections, we showed that a-tocopherol is able to block the ROS formation, as well as the loss of mitochondrial membrane potential and the occurrence of hypoploidy ( Fig. 2A), without affecting cell cycle phase distribution (Table 1), except for the HBL-100 cell line in which a-tocopherol slightly increased the ratio of S-phase cells.
Our present data provide evidence that RNAi-mediated ACCa or FAS gene silencing inhibits cell growth and leads to apoptosis induced by ROS production, which is suppressed by the lipidsoluble radical-scavenging antioxidant a-tocopherol.
Effect of siRNA-targeting ACCA on de novo fatty acid synthesis and fatty acid composition in human breast cancer cells. To determine the metabolic consequences of RNAi treatment, de novo fatty acid synthesis was quantified by measuring deuterated acetate incorporation into palmitic acid using gas chromatography. As expected, deuterated acetate is incorporated into palmitic acid in control cells (Fig. 2B, histogram). In ACCa and FAS siRNA-transfected cells, incorporation of deuterated acetate into 16:0 was drastically reduced. The magnitude of this decrease ranged from f91% with FAS siRNA to f97% with ACCa siRNA Lipogenesis and Breast Cancer Cell Survival www.aacrjournals.org relative to nontransfected cells or to control cells transfected with luciferase siRNA, showing that de novo fatty acid synthesis is inhibited by ACCa or FAS siRNA treatment. The effect of reduced ACCa and FAS activities was evaluated by quantifying the absolute amount of cellular palmitic acid. Consistent with the determination of lipogenic fluxes above, ACCa and FAS RNAi reduced the pool of palmitic acid (Fig. 2B, graph).
Effect of siRNA-targeting ACCA and FAS on normal human mammary epithelial cells. To investigate whether the cytotoxic effects of ACCa and FAS inhibition are specific for cancer cells, we transfected nonmalignant human mammary epithelial cells with ACCa or FAS siRNA and analyzed their effects. In contrast with carcinoma cell lines and transformed cell line, even de novo fatty acid synthesis declines in human mammary epithelial cells     (Fig. 2C, right); this decrease did not affect proliferation nor viability of these normal epithelial cells (Fig. 2C, left).
Rescue of ACCA knockdown-induced apoptosis by overexpressing a siRNA-resistant ACCA mutant. To confirm the specificity of ACCa-RNAi, synthetic genes expressing either wildtype ACCa (ACCa-wt) or siRNA-resistant ACCa mutant (ACCa-m) were constructed and transfected into cells 24 hours before ACCa siRNA transfection. The ectopically expressed proteins were detected with an anti-myc antibody. As a negative control, both vectors expressing wild-type ACCa-wt and ACCa-m were cotransfected with FAS siRNA. The overexpression of the ACCa-m in the ACCa siRNA-treated cells completely reversed the siRNA phenotype, preventing cell death (Fig. 3A) and restoring cell viability (compared the final number of cells in Tables 1 and 2). As predicted, overexpression of the ACCa-wt fails to prevent apoptosis induced by either ACCa or FAS knockdown (Fig. 3A). Palmitic acid content and de novo fatty acid synthesis were assessed in the siRNA-treated cells cotransfected with the ACCa-wt and ACCa-m synthetic genes. As shown in Fig. 3B (histogram), the drastic decrease of deuterated 16:0 induced by ACCa siRNA is blocked by cotransfection with the ACCa-m (95% versus 34%). Moreover, the pool of palmitic acid is fully restored by ACCa-m overexpression in MDA-MB-231 (Fig. 3B, right graph). In contrast, ACCa-m is incapable of restoring the levels of deuterated 16:0 decreased by FAS siRNA (90% versus 86%). Similarly, overexpression of ACCa-wt fails to restore deuterated 16:0 level in both ACCa and FAS siRNA-treated cells (95% versus 70.5% and 90% versus 85%, respectively).
Rescue of ACCA and FAS knockdown-induced apoptosis by supplementation with palmitic acid 16:0. To determine whether ACCa silencing-induced apoptosis is triggered by palmitic acid depletion, we tested the effect of supplementation with 100 Amol/L 16:0 on siRNA-transfected cell lines by three-color flow cytometry. As shown in Fig. 4A (histogram), 16:0 prevents ROS production, loss of mitochondrial membrane potential, as well as hypoploidy caused by ACCa knockdown, showing that 16:0 is sufficient to block the occurrence of siRNA-induced apoptosis. A dose-response assay reveals that the lower dose used (100 nmol/L 16:0), which is 10-fold higher than the background levels of palmitic acid in the medium, is also sufficient to prevent apoptosis (data not shown). Palmitic acid supplementation also rescues cells from apoptosis induced by FAS RNAi (Fig. 4A). De novo lipogenesis is not restored by palmitic acid supplementation of either ACCa-or FAS-silenced cells, as expected (Fig. 4B). Cell cycle progression and final cell numbers of siRNA-transfected cells as well as nontransfected cells are not affected by 16:0 supplementation (data not shown).
Taken together, a critical concentration of palmitic acid or metabolic derivatives of palmitic acid, resulting from either de novo lipogenesis or from circulating lipids, is essential for the survival of breast cancer cells.

Discussion
The present study was undertaken to examine the role of ACCa activity in breast cancer cell survival. Previous studies showed that FAS inhibitors, cerulenin and C75, and RNAi-silencing FAS are proapoptotic for breast cancer cells (11-13, 15-18, 23). On the other hand, the specific ACCa inhibitor 5-(tetradecyloxy)-2-furoic acid had no effect on cell proliferation and cell apoptosis, and preincubation of cells with 5-(tetradecyloxy)-2-furoic acid protected them against FAS inhibitor cytotoxicity (14,17).
Here, we show that, in contrast to the chemical inhibition by 5-(tetradecyloxy)-2-furoic acid, the inhibition of ACCa activity by RNAi results in a marked decrease of lipogenesis, leading to induction of cell apoptosis. This observation on breast cancer cells corroborates recent data on the prostate cancer cell line LNCaP that showed that cytotoxic mechanism of FAS inhibition is unrelated to malonyl-CoA accumulation (19). The ACCa silencing by RNAi is specific as the restoration of cellular palmitic acid content by palmitic acid supplementation or ACCa overexpression Figure 3. Rescue of ACCa knockdowninduced apoptosis and restoration of ACCa enzymatic function by overexpressing a siRNA-resistant ACCa mutant. Synthetic genes expressing either wild-type ACCa (ACCa-wt) or siRNA-resistant ACCa mutant (ACCa-m) were transfected to cells before ACCa and FAS siRNA transfection. A, the effect of cotransfection on apoptosis and ROS was determined by flow cytometry after staining with DiOC 6 (black columns ) and hydroethidium (gray columns ). Columns, mean of three experiments done in duplicate; bars, SD. Moreover, in HBL-100, Western blot showed the level of expressed proteins detected with an anti-myc antibody. B, the effect of cotransfection on ACCa enzymatic function was followed by analyzing total palmitic acid content (graph ) and incorporation of deuterated acetate into palmitic acid (histogram ). Columns and points, mean of three independent experiments; bars, SD. *, significantly different from control (luciferase siRNAtransfected cells) by Mann-Whitney test.
fully restores cell viability. The specific suppression of ACCa as well as the activity of FAS results in oxidative stress associated with mitochondrial impairment; these are prevented by the lipid-soluble antioxidant vitamin E. The discrepancy between the effect of ACCa gene silencing by specific RNAi and the effect of 5-(tetradecyloxy)-2-furoic acid evokes a nonspecific pleiotropic effect of 5-(tetradecyloxy)-2-furoic acid, affecting pathways other than fatty acid synthesis, which, in turn, might counteract the induction of oxidative stress and apoptosis. Thus, our data showed that, together with FAS, ACCa could be considered as a target for breast cancer therapy.
Exogenous palmitic acid intake counteracts the beneficial effect of ACCa and FAS inhibition against cancer cell survival. Thus, the potential of using the lipogenesis pathway as a target for antineoplastic therapy based on dietary intervention with palmitic acid depends on the daily intake and uptake of this fatty acid. Quantification of palmitate in healthy women sera reveals an average content of 1,200 Amol/L 3 , 12-fold higher than the dose of 100 Amol/L used in our study. Even if equivalence between human serum and cell-culture medium is unknown, the potential of using ACCa as a target for antineoplastic therapy based on dietary intervention with palmitic acid seemed to be unlikely. In this respect, additional investigations, such as dietary interventions (i.e., N-3 fatty acids or conjugated linoleic acid; ref. 24), are required to improve and to confirm the anticancer potential of inhibition of lipogenesis.
Interestingly, dietary palmitic acid has been described as an allosteric inhibitor of fatty acid synthesis in lactating mammary gland (25), likely through the allosteric inhibition of ACCa. It is obvious that, in this study, breast tumor cells lack the feedback regulation of fatty acid synthesis by dietary long-chain saturated palmitic acid independently to ACCa level expression. We hypothesize that this feedback inhibition deficiency of fatty acid synthesis is one of the features of premalignant cell survival strategy, maybe to adjust the insufficient supply of dietary fatty acids. The loss of feedback inhibition of ACCa may occur at numerous levels of transcriptional or posttranslational regulation, as ACCa is subject to global and local regulation (i.e., dietary and hormonal transcriptional regulation, adenosine monophosphateactivated protein kinase-dependent phosphorylation, and intracellular abundance of citrate or palmitoyl-CoA).
Although there is strong evidence that cancer cell proliferation and survival are dependent on de novo fatty acid synthesis, the physiologic significance of this pathway is largely unknown. It is reasonable to propose that, in dividing tumor cells, synthesis of fatty acids might provide a large pool of available lipids required for the newly synthesized cellular membrane. Thus, as we have shown in this study, lipogenesis may be not essential for high cell proliferation when a sufficient amount of exogenous fatty acids is present in the microenvironment. Fatty acid is likely essential to the control of membrane potential to maintain mitochondria integrity as lipid depletion per se induces oxidative stress by impairing mitochondrial function.
On the other hand, according to the extremely attractive theory recently proposed by Hochachka et al. (26), it is also conceivable that enhanced lipogenesis in tumor cells represents an adaptive response to control redox imbalance caused by glycolytic metabolism. It is well known that cancer cells have an unusual tolerance to limiting O 2 availability with an unusual carbohydrate metabolism exhibiting excessive lactate production by aerobic glycolysis. In both normal and malignant prostate cells, three major pathways-lactate dehydrogenase, the respiratory chain, and lipid synthesis-are involved in the regulation of the redox balance. Interestingly, in prostate cancer cells, because intracellular O 2 supplies decline (i.e., hypoxic condition), the respiratory chain cannot fully oxidize the reducing equivalents being formed and, in this metabolic context, increased lipogenesis has been proposed to be the main physiologic function to maintain redox balance through oxidation of NADPH during fatty acid synthesis. This hypothesis envisaged fatty acid synthesis as a means to mop up excess reducing potential in cancer cells. Finally, the three above speculations-roles of lipogenesis in proliferation, mitochondria function, and hypoxia defense-are not exclusive. A recent publication has just strengthened the link between glycolysis, lipogenesis, and tumor cell proliferation (27). Hatzivassiliou and colleagues have shown that down-regulation of ATP citrate lyase, a key enzyme linking glucose metabolism to lipid synthesis via the conversion of citrate to acetyl-CoA, affects glucose-dependent de novo lipid synthesis in tumor cells, which leads to an inhibition of tumor cell proliferation. Cancer cells displaying high rates of glucose metabolism are strongly affected whereas those displaying a low rate of aerobic glycolysis are unaffected. Those data reinforce our analysis of the importance of lipogenesis for tumor cell proliferation.
According to experimental studies, a-tocopherol seems to exhibit a dual and opposite role on mammary carcinogenesis considering its preventive or curative effect. If tocopherol may be chemopreventive to breast cancer (28), it could enhance tumor growth and metastasis in animal model developing breast tumor (29)(30)(31). The fact that a-tocopherol blocks apoptosis induced by ACCa or FAS deficiency is in agreement with a stimulatory effect of antioxidants in tumor growth.
Our previous epidemiologic findings involving biological measurements of dietary fat intake support a metabolic role of lipids in mammary carcinogenesis. In humans, dietary saturated palmitic acid seems to be associated with an increased risk of breast cancer (32,33), an observation which fits well with our experimental results on effects of palmitic acid supplementation in breast cancer cells. Moreover, the fatty acid composition in serum seems to influence the fatty acid composition of cell membranes, showing that dietary fatty acids influence cellular fatty acid composition (34). In already established breast cancer, membrane-saturated fatty acid composition is profoundly altered compared with the surrounding healthy tissue (2). Such an alteration, from both metabolic and dietary origins, can have profound effects on many cellular processes, such as hormonal response. Although there is good evidence that cellular lipid content is essential in mammary carcinogenesis, the question of how fatty acids could enhance mammary carcinogenesis still needs to be clarified.
Taken together, RNAi-mediated silencing of ACCa gene establishes ACCa as a potential target for anticancer therapy and would allow to define the role of ACCa, and consequently saturated palmitic acid, in cancer cell biology. However, dietary intake of lipids, such as palmitate and vitamin E, might be taken into account to fully estimate efficacy of anticancer therapy based on inhibition of lipogenesis. Figure 4. Rescue of ACCa and FAS knockdown-induced apoptosis by supplementation with palmitic acid. ACCa and FAS siRNA-transfected cells were supplemented with 100 Amol/L palmitic acid (16:0). A, tumor cells were analyzed by flow cytometry for apoptosis and ROS after staining with DiOC 6 (black columns ) and hydroethidium (gray columns ). Columns, mean of three independent experiments done in duplicate; bars, SD. B, de novo fatty acid synthesis and total palmitic acid content were determined by measuring incorporation of deuterated acetate into palmitic acid (histogram ) and absolute amount of palmitic acid (graph ) in cells by gas chromatography. Columns and points, mean of three independent experiments; bars, SD. *, significantly different from control (luciferase siRNA-transfected cells) by Mann-Whitney test.