Research Article
RNA Interference–Mediated Silencing of the Acetyl-CoACarboxylase-a Gene Induces Growth Inhibition and
Apoptosis of Prostate Cancer Cells
Koen Brusselmans, Ellen De Schrijver, Guido Verhoeven, and Johannes V. Swinnen
Laboratory for Experimental Medicine and Endocrinology, University of Leuven, Leuven, Belgium
Abstract
Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the
exploration of lipogenic enzymes as targets for cancer
intervention have focused on fatty acid synthase (FAS), the
enzyme catalyzing the terminal steps in fatty acid synthesis.
Chemical inhibition or RNA interference (RNAi)–mediated
knockdown of FAS consistently inhibits the growth and
induces death of cancer cells. Accumulation of the FAS
substrate malonyl-CoA has been implicated in the mechanism
of cytotoxicity of FAS inhibition. Here, using RNAi technology,
we have knocked down the expression of acetyl-CoA
carboxylase-A (ACC-A), the enzyme providing the malonylCoA substrate. Silencing of the ACC-a gene resulted in a
similar inhibition of cell proliferation and induction of
caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant
cells with low lipogenic activity, no cytotoxic effects of
knockdown of ACC-A or FAS were observed. These findings
indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumorassociated lipogenesis and suggest that in addition to FAS,
ACC-A is a potential target for cancer intervention. (Cancer
Res 2005; 65(15): 6719-25)
Introduction
Enhanced expression of lipogenic enzymes is increasingly
recognized as a common characteristic of a wide variety of tumors
(1–3). Overexpression of fatty acid synthase (FAS), a key lipogenic
enzyme that catalyzes the terminal steps in the de novo
biosynthesis of long-chain fatty acids (4), is found already in the
earliest stages of tumor development (5, 6). In many tumor types,
FAS overexpression is further pronounced as the tumor progresses
towards a more advanced stage (2, 5, 7). In breast and prostate
cancers, similar changes have been found in the expression of
acetyl-CoA-carboxylase-a (ACC-a), the rate-limiting enzyme of
fatty acid synthesis that catalyzes the condensation of malonyl-CoA
using acetyl-CoA and CO2 as precursors (1, 8).
The finding that lipogenesis is low in nearly all nonmalignant
adult tissues (9, 10), whereas it is up-regulated in many tumors, has
led to the exploration of endogenous lipogenesis as a novel target
for prevention and/or treatment of cancer. Up to now, nearly all
Note: K. Brusselmans and E. De Schrijver contributed equally to this work.
Requests for reprints: Johannes V. Swinnen, Laboratory for Experimental
Medicine and Endocrinology, Gasthuisberg, O&N niv 9, Herestraat 49, bus 902,
B-3000, Leuven, Belgium. Phone: 32-16-345974; Fax: 32-16-345934; E-mail:
[email protected].
I2005 American Association for Cancer Research.
www.aacrjournals.org
attempts to examine this possibility have focused on FAS. Both
chemical inhibitors of FAS (cerulenin, c75, Orlistat, EGCG) and the
use of more selective approaches such as gene silencing with small
interfering RNA (siRNA) targeting FAS have resulted in growth
arrest and cell death in tumor cells (2, 11–21). Although it has been
suggested that cytotoxicity induced by FAS inhibition is the result
of accumulation of the toxic intermediate malonyl-CoA (22, 23), the
exact mechanism by which inhibition of FAS induces tumor cell
death remains a matter of debate.
In the present work, we examined the effect of siRNA-mediated
knockdown of ACC-a on prostate cancer cells and compared the
effects with those of knockdown of FAS. It is shown that inhibition
of ACC-a induces a similar growth arrest and tumor cell death as
observed after blockage of FAS, despite the fact that there is no
accumulation of malonyl-CoA.
Materials and Methods
Cell culture. The human LNCaP prostate cancer cell line was obtained
from the American Type Culture Collection (Manassas, VA). Nonmalignant
skin fibroblasts were provided by Prof. Dr. J.J. Cassiman (K.U. Leuven,
Belgium). Cells were cultured at 37jC in a humidified incubator with a 5%
CO2/95% air atmosphere in RPMI 1640 supplemented with 3 mmol/L Lglutamine and 10% FCS (Invitrogen, Carlsbad, CA). For experiments
examining biological effects in the absence of androgens, charcoal-treated
FCS was used to reduce background steroid levels. For analyzing cellular
responses to androgens, R1881 (methyltrienolone; DuPont/NEN, Boston,
MA) was dissolved in ethanol and added to the cultures. For culturing cells
in the presence of palmitate-bovine serum albumin (BSA) complex,
palmitate (Sigma, St. Louis, MO) was first complexed to fatty acid–free
BSA (Invitrogen) as described (24, 25). Briefly, 4 volumes of a 4% BSA
solution in 0.9% NaCl were added to 1 volume of 5 mmol/L palmitate in
ethanol and incubated at 37jC for 1 hour, to obtain a 1 mmol/L stock
solution of BSA-complexed palmitate.
RNA interference. Transfection procedures with siRNA using Oligofectamine (Invitrogen) have been described previously (17); siRNA oligonucleotides were purchased from Dharmacon (Lafayette, CO). Sequences of
siRNA oligonucleotides targeting FAS and luciferase (Luc) have been
described (17). The siRNA oligonucleotides targeting ACC-a were sense,
CAAUGGCAUUGCAGCAGUGdTdT and antisense, CACUGCUGCAAUGCCAUUGdTdT.
RNA analysis. An 840-bp cDNA probe for ACC was synthesized by PCR
on human cDNA (generated by reverse transcription as described; ref. 26)
using 5V-TTCTCAGAGCTTCCGAACTTGCT and 5V-CTAACCTGGCTCTACCAACCAC as forward and reverse primer, respectively. PCR products were
cloned into the pGEM-T vector (Promega, Madison, WI). Probes for FAS and
18S rRNA, RNA preparation, and Northern blot procedures have been
described previously (26).
2-14C-acetate incorporation assay and TLC analysis. At 24, 48, 72, 96,
or 120 hours after transfection with siRNA, 2-14C-labeled acetate (57 mCi/
mmol; 2 ACi/dish; Amersham International, Aylesbury, United Kingdom)
was added to the cell culture medium. After 4 hours incubation, cells
were collected by centrifugation and resuspended in 0.8 mL PBS. Lipids
were extracted using the Bligh Dyer method as previously described (17);
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Figure 1. Effect of ACC-a RNAi and FAS
RNAi on ACC-a and FAS expression and
on lipid biosynthesis in LNCaP cells.
Expression of ACC-a and FAS was
analyzed by Northern blot (A) and Western
blot analysis (B ) at 24, 48, 72, 96, and 120
hours after transfection of LNCaP cells with
siRNA targeting ACC-a, FAS, or Luc.
18S RNA and cytokeratin-18 (CK18 )
expression were used as internal control
for Northern blot and Western blot analysis,
respectively. C, measurement of
2-14C-labeled acetate incorporation into
cellular lipids of LNCaP cells at 24, 48, 72,
96, and 120 hours after transfection with
siRNA targeting ACC-a, FAS, or Luc, as
revealed by scintillation counting. D,
measurement of 2-14C-labeled acetate
incorporation into different lipid classes of
LNCaP cells at 72 hours after transfection
with siRNA targeting ACC-a, FAS, or Luc,
as quantitated by TLC analysis and
Phosphor Imaging. PL, phospholipids; TG,
triglycerides; Chol, cholesterol. Columns,
means (n = 6-18); bars, FSD. *,
significantly different from control
(Luc siRNA-transfected cells) by
Tukey test.
2-14C-acetate incorporation into cellular lipids was quantitated by
scintillation counting. Obtained results were normalized for sample
protein content. To analyze 2-14C-acetate incorporation into different lipid
classes, TLC analysis was done and lipids were quantitated using a
PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) as previously
described (17).
Proliferation/cytotoxicity assays and fluorescence-activated cell
sorting analysis. At the indicated time after transfection with siRNA, cells
were collected and cell number and viability were determined using a
trypan blue dye exclusion assay as described previously (17). Cell
proliferation was quantitated using a bromodeoxyuridine (BrdUrd) labeling
Cancer Res 2005; 65: (15). August 1, 2005
and detection kit (Roche, Basel, Switzerland). For fluorescence-activated cell
sorting (FACS) analysis, cells were trypsinized; fixed in ice-cold 70% ethanol
for 1 hour; washed twice with PBS containing 0.05% Tween 20; and
resuspended in PBS containing 0.05% Tween 20, 0.5 mg/mL propidium
iodide, and 1 mg/mL RNase A (Sigma). Samples were analyzed on a FACSort
cytometer using the CellQuest and Modfit programs (Becton Dickinson,
Franklin Lakes, NJ).
Hoechst staining. LNCaP cells were plated in Lab-Tek II chamber
slides (Nalge Nunc International, Naperville, IL) at a density of 1.2 105
cells per chamber, incubated overnight, and transfected with siRNA as
described (17). At 96 hours after transfection, Hoechst 33342 (Sigma) was
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ACC-a RNAi Induces Apoptosis in Cancer Cells
added to the culture medium of living cells; changes in nuclear
morphology were detected by fluorescence microscopy using a filter for
Hoechst 33342 (365 nm). For quantification of Hoechst 33342 stainings,
the percentages of Hoechst-positive nuclei per optical field were counted.
To investigate the involvement of caspases, the irreversible caspase
inhibitor z-VAD-fmk (N-benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethylketone) was added 24 hours after siRNA transfection at a final concentration
of 50 Amol/L.
Immunoblot analysis. At the indicated time after transfection with
siRNA, cells were washed with PBS and lysed in a reducing buffer
containing 62.5 mmol/L Tris (pH 6.8), 2% SDS, 0.715 mol/L 2-mercaptoethanol, and 8.7% glycerol. Protein concentrations were measured using the
bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL). Generation of
antibodies against FAS and Western Blot procedures using antibodies
against FAS, cytokeratin-18 (Santa Cruz Biotechnology, Santa Cruz, CA),
ACC-a, or cleaved poly(ADP-ribose) polymerase (PARP; both from Cell
Signaling Technology, Inc., Beverly, MA) have been previously described
(17, 25).
Malonyl-CoA measurement. Sample preparation and malonyl-CoA
quantification in LNCaP cells was done as described (27, 28). Cells were
collected in PBS and after centrifugation resuspended in 0.6 mL ice-cold 0.6
N trichloroacetic acid. Cell suspensions were sonicated and 100 pmol of
isobutyryl-CoA was added to each sample as internal standard. After
centrifugation at 12,000 g, supernatants were washed six times with 0.7
mL diethyl ether and were evaporated to dryness by vacuum centrifugation.
Acyl-CoA esters were separated and quantitated using a reverse-phase
high-performance liquid chromatography (HPLC) with a 3.5-Am Zorbax SBC8 column (Rockland Technologies, Newport, DE), an LKB 2150 HPLC
pump, an LKB 2152 LC controller, a UV detector (at 254 nm) with UV-M
monitor, and an LKB 2210 recorder (all from Amersham Pharmacia
Biotech, LKB, Uppsala, Sweden). The buffers used for the mobile phase
were 0.1 mol/L potassium phosphate (pH 5.0; buffer A) and 0.1 mol/L
potassium phosphate (pH 5.0) with 40% acetonitrile (buffer B). Using a
constant flow rate of 1 mL/min, runs started with a 12-minute period with
100% buffer A; at 12 minutes, the percentage of buffer B gradually increased
from 0% to 10% (over 28 minutes); at 40 minutes, buffer B was kept at 10%
for 4 minutes; at 44 minutes, the percentage of buffer B was gradually
increased to 80% (over 36 minutes). For identification, sample peaks and
retention times were compared with those of pure malonyl-CoA and
isobutyryl-CoA standards.
Statistical analysis. Obtained results were analyzed by one-way ANOVA
using a Tukey test as post-test. Ps < 0.05 were considered statistically
significant. All data are means F SD.
cells, as a control. Northern blot and Western blot analysis showed
a marked suppression of ACC-a and FAS expression in LNCaP
cells after transfection with ACC-a siRNA and FAS siRNA,
respectively, when compared with cells transfected with Luc
siRNA (Fig. 1A-B).
Results
RNA interference–mediated silencing of ACC-A and fatty
acid synthase decreases the synthesis of fatty acids in prostate
cancer cells. To specifically silence ACC-a and FAS gene
expression in LNCaP prostate cancer cells, cells were transiently
transfected with appropriate siRNA oligos or with siRNAs
targeting Luc, which is not endogenously expressed in LNCaP
Figure 2. Impact of ACC-a RNAi and FAS RNAi on LNCaP cell proliferation
and viability. LNCaP cells were transfected with siRNA targeting ACC-a, FAS, or
Luc. A, at the indicated time points, cells were collected and stained with trypan
blue; viable cells were counted. B, at the indicated time points, cells were
exposed to BrdUrd for 2 hours. Thereafter, BrdUrd incorporation in LNCaP cells
was measured colorimetrically and normalized for the number of viable cells at
the start of the BrdUrd exposure [expressed as percentage BrdUrd incorporation
of the control (Luc siRNA-transfected cells)]. C, the percentage of dead cells was
counted after staining the cells with trypan blue at the indicated time points.
Columns, means (n = 6-7); bars, FSD. *, significantly different from control
(Luc siRNA-transfected cells) by Tukey test. D, cell cycle analysis on LNCaP
cells at 72 hours after transfection with FAS siRNA, ACC-a siRNA, or Luc siRNA.
Data are expressed as percentage of the total population of cells. The data from
the FACS-based cell cycle analysis are the means F SD of three independent
experiments.
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Figure 3. ACC-a RNAi and FAS RNAi
induce apoptosis of LNCaP cells.
A, Western blot analysis showing the
cleavage of PARP in LNCaP cells,
transfected with siRNA targeting ACC-a,
FAS, or Luc, at 24, 48, 72, 96, and 120
hours after transfection. Cytokeratin-18
(CK18 ) expression was used as an internal
control. At 96 hours after transfection with
Luc siRNA (B), ACC-a siRNA (C ), or FAS
siRNA (D ), LNCaP cells were stained
with Hoechst 33342 and apoptosis was
analyzed by fluorescence microscopy.
Addition of z-VAD-fmk, 24 hours after
transfection, suppressed LNCaP cell
apoptosis induced by inhibition of ACC-a
and FAS as revealed at 96 hours after
transfection with ACC-a siRNA (E ) or FAS
siRNA (F ). Bar, 100 Am.
To evaluate the effect of reduced ACC-a and FAS expression on
endogenous lipid synthesis, LNCaP cells were exposed to 2-14Clabeled acetate for 4 hours at different time points after
transfection with siRNA targeting ACC-a, FAS or Luc. Thereafter,
cellular lipids were extracted and the incorporation of 2-14Clabeled acetate into cellular lipids was quantitated by scintillation
counting. Starting from 72 hours after transfection, ACC-a RNA
interference (RNAi) markedly reduced the synthesis of new lipids
compared with the control levels, and this effect lasted for at least
till 120 hours after transfection (Fig. 1C). A similar inhibition was
observed for FAS siRNA (Fig. 1C).
To investigate the effect of ACC-a RNAi and FAS RNAi on
different lipid species, lipid extracts of LNCaP cells were analyzed
by TLC. The majority of 14C-label in control cells was incorporated
into phospholipids and smaller but substantial amounts of label
were found in triglycerides and in free cholesterol. Both ACC-a and
FAS RNAi caused a significant and comparable decrease in the
synthesis of phospholipids and triglycerides (Fig. 1D). The
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synthesis of cholesterol was not affected by FAS RNAi but was
reduced by ACC-a RNAi.
Both ACC-A and fatty acid synthase RNA interference
inhibit cell proliferation and induce apoptosis of prostate
cancer cells. To investigate the effect of ACC-a RNAi and FAS
RNAi on cell proliferation and cell survival, LNCaP cells were
stained with trypan blue at different time points after transfection
with siRNA targeting ACC-a, FAS or Luc. Cell counting showed that
silencing of the ACC-a gene caused a stagnation of the number of
viable cells, whereas control cells continued proliferating normally
(Fig. 2A). BrdUrd incorporation experiments revealed that ACC-a
RNAi significantly decreased proliferation of LNCaP cells (Fig. 2B).
Counting the percentage of dead cells after trypan blue staining
showed a significant increase in cell death starting at 72 hours after
transfection (Fig. 2C). Similar effects on cell proliferation and
survival were observed in LNCaP cells transfected with siRNA
targeting FAS. In contrast, no effects on cell proliferation or
viability were observed after transfection with siRNA targeting Luc.
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ACC-a RNAi Induces Apoptosis in Cancer Cells
To investigate the effects on cell proliferation more in detail, we
analyzed the cell cycle distribution of LNCaP cells using a FACS. In
agreement with the cell death observed after trypan blue staining,
ACC-a and FAS RNAi increased the percentage of sub-G1 cells
(which are assumed nonviable) at 72 hours after transfection
(Fig. 2D). Both inhibition of ACC-a and of FAS also induced a 2-fold
decrease of the fraction of cells in the S phase (Fig. 2D).
To study the mechanism of cell death induced by inhibition of
fatty acid synthesis, we analyzed cleavage of PARP, a nuclear
polymerase which is one of the main targets for cleavage by
caspases during the apoptotic process. As revealed by Western
blot analysis, LNCaP cells transfected with ACC-a or FAS siRNA
showed positive staining for cleaved PARP, confirming apoptosis
(Fig. 3A). No cleaved PARP was observed in control cells. In
addition, Hoechst 33342 staining of LNCaP cells at 96 hours after
transfection with siRNA targeting ACC-a or FAS (Fig. 3C-D)
showed the presence of nuclear fragmentation and chromatin
condensation, typical hallmarks of apoptosis, which were not
observed in Luc siRNA–transfected LNCaP cells (Fig. 3B). To
confirm the involvement of caspases, we examined the effects of
z-VAD-fmk, a general caspase inhibitor, on the internucleosomal
degradation of DNA. To this end, LNCaP cells were treated with
z-VAD-fmk 24 hours after transfection with siRNA targeting
ACC-a or FAS. Hoechst 33342 staining at 96 hours after
transfection revealed that apoptosis induced by inhibition of
ACC-a as well as by inhibition of FAS were both significantly
suppressed by z-VAD-fmk (Fig. 3E-F ). Using fluorescence
microscopy, quantitative analysis of Hoechst 33342 stainings at
96 hours after transfection was done by counting the
percentages of Hoechst-positive cells per optical field: 3.4 F
1.9% for Luc siRNA, 21.2 F 7.1% for ACC-a siRNA, 21.3 F 4.9%
for FAS siRNA, 6.3 F 3.1% for ACC-a siRNA supplemented with
z-VAD-fmk, and 5.8 F 2.2% for FAS siRNA supplemented with zVAD-fmk [ACC-a siRNA and FAS siRNA conditions in the
absence of z-VAD-fmk were significantly different (P < 0.05) from
the control (Luc siRNA), n = 7-12].
In an attempt to find out whether tumor cell death, observed
after inhibition of fatty acid synthesis, is due to an accumulation of
the toxic intermediate malonyl-CoA or to starvation of fatty acids,
we quantitated the levels of malonyl-CoA in acid-soluble extracts
from LNCaP cells transfected with siRNA targeting ACC-a, FAS, or
Figure 4. Effect of ACC-a RNAi and FAS RNAi on malonyl-CoA accumulation.
LNCaP cells were transfected with siRNA targeting ACC-a, FAS, or Luc and after
72 hours intracellular malonyl-CoA levels were quantitated using
reversed-phase HPLC. Columns, means (n = 8); bars, FSD. *, significantly
different from control (Luc siRNA-transfected cells) by Tukey test.
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Luc using reversed-phase HPLC. FAS inhibition markedly increased
the levels of malonyl-CoA. Although inhibition of ACC-a induced
tumor cell death, ACC-a RNAi had no effect on malonyl-CoA levels
(Fig. 4).
In a second step, we investigated the effects of exogenous
palmitate, the predominant product of the fatty acid synthesis
pathway, on the viability of LNCaP cells transfected with siRNA
targeting FAS, ACC-a, or Luc. The cytotoxicity induced by FAS
inhibition was decreased f2-fold after addition of palmitate
(Fig. 5). To a lesser extent, palmitate also rescued the cytotoxic
effects induced by ACC-a RNAi.
Effect of RNA interference–mediated inhibition of ACC-A
and fatty acid synthase on nonmalignant human fibroblasts.
To investigate whether the cytotoxic effects induced by silencing of
ACC-a are specific for cancer cells, we analyzed the effect of ACC-a
RNAi on nonmalignant human fibroblasts, which show a low
lipogenic activity (17, 25). 2-14C-labeled-acetate incorporation
assays revealed that ACC-a and FAS RNAi significantly inhibited
the already low de novo synthesis of lipids in these fibroblasts
(Fig. 6A). However, in contrast with LNCaP cells, silencing of the
ACC-a gene or of the FAS gene did not affect proliferation nor
viability of fibroblasts (Fig. 6B-C).
Discussion
To gain more insight into the mechanisms underlying cytotoxicity induced by FAS inhibition and to explore whether also other
lipogenic enzymes are potential targets for cancer intervention, we
have selectively knocked down ACC-a expression in prostate
cancer cells using RNAi and have compared the effects with those
of FAS RNAi. Our data show that down-regulation of ACC-a and
FAS evoked a comparable inhibition of LNCaP cell proliferation
and resulted in both cases in caspase-mediated apoptosis. These
similar effects were observed despite the fact that FAS siRNA
increased intracellular malonyl-CoA levels 9-fold, whereas no
accumulation of this metabolite was observed after transfection
with ACC-a siRNA. Moreover, administration of exogenous
palmitate, the end product of FAS, rescued the cytotoxic effects
of FAS RNAi and to a lesser extent also those of ACC-a RNAi,
suggesting that depletion of end products ( fatty acids) may be the
major component in the observed cytotoxic effects. The observation that the rescue effect of palmitate was more outspoken in
LNCaP cells transfected with FAS siRNA than in those transfected
with ACC-a siRNA, may be explained by at least two facts: (a) In
contrast to FAS, ACC-a is also involved in fatty acid elongation. (b)
Whereas FAS RNAi did not affect the synthesis of cholesterol, ACCa RNAi slightly reduced the synthesis of cholesterol, which fits in
with previous studies in human liver reporting that malonyl-CoA
rather than acetyl-CoA is incorporated in mevalonate, a precursor
of cholesterol (29). Both fatty acid elongation and synthesis of
cholesterol can not be rescued by administration of palmitate.
Additional involvement of changes in the NADPH levels in the
induction of cell death can not be excluded (30). It is worth
mentioning that ACC-a and FAS RNAi also cause inhibition of
lipogenesis, growth arrest, and cell death in LNCaP cells cultured in
the absence of androgens (charcoal-treated FCS) or in the presence
of 0.1 nmol/L R1881 (data not shown), thereby indicating that ACCa or FAS RNAi-mediated growth inhibition/cytotoxicity of prostate
cancer cells does not depend on the presence or absence of
androgens. Interestingly, nonmalignant fibroblasts with a low basal
rate of lipogenesis show no growth retardation or cell death after
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lipogenesis in cancer cells may also affect key processes such as
signal transduction, intracellular trafficking, and tumor cell
migration. Interestingly, in yeast, inactivation of ACC-a completely
inhibits vegetative growth and causes cell death even after
supplementation of fatty acids (38). To investigate the specific
function of ACC-a in Saccharomyces cerevisiae, a temperaturesensitive mutant of ACC-a has recently been constructed. When
Figure 5. Exogenous palmitate partially rescues the cytotoxicity induced by
inhibition of fatty acid synthesis. LNCaP cells were transfected with
siRNA targeting ACC-a, FAS, or Luc; 4 hours after transfection palmitate-BSA
was added to the culture medium to a final concentration of 80 Amol/L. After
72 hours of incubation, cells were collected and stained with trypan blue and the
percentage of dead cells was counted. Columns, means (n = 3); bars, FSD.
Representative of two independent experiments.
treatment with RNAi targeting ACC-a and this despite a marked
further decrease in lipogenesis, suggesting that cytotoxic effects
may have some selectivity for tumor cells overexpressing lipogenic
enzymes. Why similar data where not obtained with TOFA [5(tetradecyloxy)-2-furoic acid], an allosteric inhibitor of ACC,
remains to be explored (23).
The significance of increased lipogenesis in tumor cells and the
mechanisms by which interference with this increased lipogenesis
may result in tumor cell death certainly merit further investigation.
It is obvious that one of the basic characteristics of malignant cells
is uncontrolled proliferation and that proliferation requires
membrane synthesis. Both RNAi-mediated silencing of ACC-a and
of FAS induced growth arrest of LNCaP cells in the G1 phase and
membrane synthesis has been linked to the late G1 phase of the cell
cycle (31, 32). It is less obvious why tumor cells would derive the
required lipids for membrane synthesis preferentially from endogenous lipid synthesis. Moreover, lipogenesis is also increased in
nonproliferating tumor cells (1). Enhanced endogenous lipogenesis
may not only have quantitative effects on lipid and membrane
synthesis but also qualitative effects. In fact, in view of the inability
of human cells to synthesize polyunsaturated fatty acids from fully
saturated precursors produced by FAS, the newly synthesized
phospholipids in tumor cells are mainly saturated and monounsaturated. Recent reports indicate that saturated and monounsaturated
phospholipids together with cholesterol and sphingolipids tend to
partition into detergent-resistant membrane microdomains (33).
Membrane microdomains or ‘‘lipid rafts’’ have been shown to be
involved in several key cellular processes including signal transduction, intracellular trafficking, cell polarization, and migration
(33–36). We observed that both inhibition of ACC-a and of FAS
mainly affected the synthesis of raft-associated lipids in prostate
cancer cells, whereas non–raft-associated lipids were less dependent on ACC-a and FAS activity (37).1 The finding that fatty acid
synthesis in cancer cells preferentially affects the composition of
membrane microdomains opens the possibility that the increased
1
Unpublished results.
Cancer Res 2005; 65: (15). August 1, 2005
Figure 6. Effect of ACC-a RNAi and FAS RNAi on nonmalignant human
fibroblasts. A, fibroblasts were transfected with siRNA targeting ACC-a, FAS or
Luc. After 72 hours, cells were treated with 2-14C-labeled acetate for 4 hours.
Lipid extracts were prepared and 14C-incorporation was quantitated. Proliferation
(B ) and viability (C) of fibroblasts transfected with ACC-a, FAS, or Luc siRNA,
as revealed by counting the number of viable cells and the percentage of dead
cells respectively, using the trypan blue exclusion assay. Columns, means
(n = 3-8); bars, FSD. *, significantly different from control (Luc siRNA-transfected
cells) by Tukey test.
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ACC-a RNAi Induces Apoptosis in Cancer Cells
grown at the restrictive temperature, these yeast cells developed an
altered nuclear envelope, showed severe abnormalities in spindle
formation, became arrested in the G2-M phase of the cell cycle, and
finally lost viability (39). In this system, inactivation of ACC-a
caused a sharp decline in the production of inositol-ceramides and
very long chain fatty acids. It would certainly be worthwhile to study
whether similar alterations in lipid synthesis are also observed in
human cancer cells treated with RNAi-targeting ACC-a.
In conclusion, the present data suggest that not only FAS but
also ACC-a may be an interesting target for the development of
novel forms of cancer prevention and therapy. Selective inhibition
of ACC-a indicates that at least in the prostate tumor cell line
LNCaP cell death is not related to accumulation of malonyl-CoA.
The ability to selectively inhibit different steps in the lipogenic
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Acknowledgments
Received 2/18/2005; revised 5/10/2005; accepted 5/18/2005.
Grant support: Fortis Bank Insurance and VIVA Cancer Research grants;
Concerted Research Action (Research Fund, Katholieke Universiteit Leuven); Fund
for Scientific Research-Flanders (F.W.O., Belgium) research grants and postdoctoral
fellowship (K. Brusselmans); Interuniversity Poles of Attraction Programme-Belgian
State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural
Affairs; and Stichting Emmanuel Van Der Schueren, Belgium (E. De Schrijver).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank S. Organe and F. Vanderhoydonc for technical assistance.
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Cancer Res 2005; 65: (15). August 1, 2005
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2005 American Association for Cancer
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RNA Interference−Mediated Silencing of the
Acetyl-CoA-Carboxylase-α Gene Induces Growth Inhibition
and Apoptosis of Prostate Cancer Cells
Koen Brusselmans, Ellen De Schrijver, Guido Verhoeven, et al.
Cancer Res 2005;65:6719-6725.
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