Blockade of Fatty Acid Synthase Triggers Significant
Apoptosis in Mantle Cell Lymphoma
Pascal Gelebart1., Zoulika Zak1., Mona Anand1, Andrew Belch2, Raymond Lai1,2*
1Department of Laboratory Medicine and Pathology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada, 2Department of Oncology, University
of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada
Fatty acid synthase (FASN), a key player in the de novo synthetic pathway of long-chain fatty acids, has been shown to
contribute to the tumorigenesis in various types of solid tumors. We here report that FASN is highly and consistently
expressed in mantle cell lymphoma (MCL), an aggressive form of B-cell lymphoid malignancy. Specifically, the expression of
FASN was detectable in all four MCL cell lines and 15 tumors examined. In contrast, benign lymphoid tissues and peripheral
blood mononuclear cells from normal donors were negative. Treatment of MCL cell lines with orlistat, a FASN inhibitor,
resulted in significant apoptosis. Knockdown of FASN expression using siRNA, which also significantly decreased the growth
of MCL cells, led to a dramatic decrease in the cyclin D1 level. b-catenin, which has been previously reported to be
upregulated in a subset of MCL tumors, contributed to the high level of FASN in MCL cells, Interesting, siRNA knock-down of
FASN in turn down-regulated b-catenin. In conclusion, our data supports the concept that FASN contributes to the
pathogenesis of MCL, by collaborating with b-catenin. In view of its high and consistent expression in MCL, FASN inhibitors
may hold promises for treating MCL.
Citation: Gelebart P, Zak Z, Anand M, Belch A, Lai R (2012) Blockade of Fatty Acid Synthase Triggers Significant Apoptosis in Mantle Cell Lymphoma. PLoS
ONE 7(4): e33738. doi:10.1371/journal.pone.0033738
Editor: Arun Rishi, Wayne State University, United States of America
Received November 2, 2011; Accepted February 16, 2012; Published April 2, 2012
Copyright: ? 2012 Gelebart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study is supported by operating research grants from the Canadian Institute of Health Research, Alberta Cancer Foundation and the Canadian
Cancer Society Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: RL is an employee of DynalifeDx. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Fatty acids play an important role in a variety of cellular
processes. They serve as the building blocks for cell membranes,
target anchor proteins to the cell membranes, function as
precursors in the synthesis of lipid second messengers and act as
important substrates for energy metabolism . Fatty acids are
also implicated in specialized biological functions including the
production of lung surfactants and milk lipids . There are two
sources of fatty acids, namely the dietary source and that
synthesized endogenously. The production of endogenous fatty
acids is catalyzed by the multifunctional homodimeric lipogenic
enzyme called fatty acid synthase (FASN) . In this process,
FASN catalyses the condensation of acetyl-CoA and malonyl-CoA
to generate long-chain fatty acids, and the predominant product of
FASN is palmitate, a 16-carbon fatty acid . The de novo fatty
acid synthesis is extremely active during embryogenesis and in
proliferating fetal cells. In adult human tissues, FASN is mainly
expressed in adipocytes, hepatocytes and hormone-sensitive cells
such as lactating breast and cycling endometrial cells [3,4]. In most
of the other normal human tissues, FASN is expressed at a
relatively low level, as these cells preferentially utilize dietary fatty
It has been recently found that FASN is highly expressed in
many types of human solid tumors [5,6], such as carcinomas of the
breast [7,8], prostate , colon , ovary , thyroid , lung
 and stomach . It has been suggested that a high level of
FASN expression correlates with a shorter survival in patients with
ovarian cancer . These findings led to the hypothesis that the
fatty acid synthetic pathway may contribute to tumorigenesis and
FASN may be a useful anti-cancer target [5,6,9]. In support of
this, an inhibitor of FASN and a FDA-approved anti-obesity drug,
Orlistat, was reported to show antitumor activity . Specifically,
Orlistat has demonstrated potent anti-proliferative and pro-
apoptotic effects in prostate, breast, colon, stomach and ovarian
cancer cells, with no significant effects on normal cells . Orlistat
has also shown significant anti-tumor properties in a prostate
cancer xenograft mouse model, without inducing signs of toxicity
. While the concept that FASN is a useful therapeutic target
for epithelial cell malignancies is relatively supported, the role of
FASN in hematologic cancer has not been extensively examined.
Mantle cell lymphoma (MCL) is a distinct type of B-cell non-
Hodgkin’s lymphoma defined by a constellation of pathologic,
cytogenetic and clinical features . One of the characteristic
features of MCL is the recurrent chromosomal translocation,
t(11;14)(q13;q32), which brings the cyclin D1 gene under the
control of the enhancer of the immunoglobulin heavy chain gene,
leading to over-expression of the cyclin D1 protein. While it is
widely accepted that cyclin D1 plays an important role in the
pathogenesis of MCL, accumulating evidence suggests that MCL
often has defects in many other cellular processes, such as those
involved in cell-cycle regulation, apoptosis and DNA repair
[16,17]. With regard to apoptosis, MCL is well known to be
resistant to apoptosis induced by a variety of conventional
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chemotherapeutic agents . Recent studies have revealed a
number of biochemical defects that may contribute to its relatively
high resistance to apoptosis , including constitutive activation
of the NFkB pathway [19–21], overexpression of several anti-
apoptotic proteins and the absence of Fas receptor . Aberrant
cellular signaling such as the PI3K/Akt pathway also may
contribute to the chemo-resistance of MCL [23,24]. Despite the
advent of several new therapeutic agents , a significant
proportion of MCL patients continues to have a relatively poor
clinical outcome . Thus, there is a need to continue to develop
new therapeutic strategies for this disease. In this study, we found
that FASN is highly and consistently expressed in MCL cell lines
and tumors. Importantly, blockade of FASN can induce significant
apoptosis in MCL. Our findings suggest that FASN may represent
a useful therapeutic target for MCL.
Materials and Methods
1. Cells, tissue culture and FASN inhibitors
Previously described MCL cell lines, including Jeko-1, Mino,
SP53 and Rec-1, were used in this study . Briefly, these cell
lines are positive for cyclin D1, and they carry an mature B-cell
immunophenotype and the t(11;14)(q13;q32) cytogenetic abnor-
mality. All of these cell lines are negative for the Epstein-Barr
virus nuclear antigen and they were grown in RPMI 1640
supplemented with 10% fetal bovine serum (FBS) and glutamine.
To test the sensitivity of MCL cells to FASN inhibitors, MCL
cells were plated at a density of 16105cells/mL and treated with
control diluent (DMSO) or different concentrations of two FASN
inhibitors, namely C75 (Cayman Chemical Company, Ann
Arbor, MI) and Orlistat (Alexis Biochemicals, San Diego, CA).
CellTiter-Blue fluorescence was used to monitor the cell viability
according to the manufacturer’s protocols (Promega, Madison,
2. MCL patient samples and immunohistochemistry
All MCL primary tumors samples were diagnosed at the Cross
Cancer Institute and the diagnostic criteria were based on those
described in the World Health Organization Classification
Scheme . Formalin-fixed, paraffin-embedded tumor samples
from 15 cases of MCL were subjected to immunohistochemistry
based on a protocol previously published . Briefly, tissue
sections of 3–4 mM thickness were deparaffinized and hydrated.
Heat-induced epitope retrieval was obtained by using a pressure
cooker and citrate buffer (pH 6.0). The endogenous peroxidase
activity was blocked using 0.3% H2O2in methanol for 5 minutes.
Tissue sections were then incubated with a mouse monoclonal
anti-FASN antibody (Santa-Cruz Biotechnology, Santa Cruz, CA,
1:200) overnight at 4uC in a humidified chamber. After 2 washes
with phosphate buffered saline (PBS, pH 7.5), tissue sections were
incubated with biotinylated linked universal secondary antibody
and subsequently with streptavidin–HRP complex as per the
manufacturer’s instructions (LSAB+ system, Dako, Burlington,
Ontario, Canada). Tissue sections were incubated with 3,39-
diaminobenzidine/H2O2 (Dako) for color development and
counter-stained with hematoxylin. To assess the sensitivity of
leukemic MCL to a FASN inhibitor, peripheral blood mononu-
clear cells (PBMC) from 3 leukemic patients (all of whom had an
absolute lymphocyte count of .56109/L) were harvested using
Ficoll-Paque density centrifugation, plated at a density of 16105
cells/mL in culture medium, and treated with either DMSO or
different concentrations of Orlistat.
3. Apoptotic assays
The FITC-Annexin V Apoptosis detection kit (BD Biosciences
Pharmingen, San Diego, CA) was used to detect the phosphati-
dylserine translocation from the inner to the outer leaflet of the
plasma membrane. Briefly, 16106cells were diluted in 100 mL of
Annexin V buffer, to which 5 mL of Annexin V-FITC was
subsequently added. After incubation for 15 min at room
temperature in the dark, 400 mL of additional binding buffer
was added. Flow cytometry analysis was conducted within 1 hour.
Caspase 3/7 activities were measured using the Apo-ONE
Homogeneous Caspase-3/7 Assay kit (Promega) according to the
manufacturer’s protocol. Briefly, 100 mL of Caspase 3/7 Apo-One
reagent was added to 100 mL of cells culture treated with or
without FASN inhibitors. After incubation, the fluorescence of
each sample was measured in a fluorescent plate-reading
FLUOstar Optima (BMG Labtechnologies, Offenburg, Germany).
Triplicate experiments were performed and results are presented
as the mean 6 the standard deviation.
4. siRNA and transfection
Cells were transfected with a pool of 4 siRNA targeting FASN
or b-catenin (Dharmacon Inc., Lafayette, CO). Cells treated with
scrambled siRNA served as the negative control. Transient
transfections of MCL cells (56106cells) were performed using
the Electro square electroporator, BTX ECM 800 (225V, 8.5 ms,
3 pulses, Harvard apparatus). A concentration of 200 pmol of
siRNA per 16106cells was used. The efficiency of target gene
inhibition was assessed using Western blot analysis.
5. Western blot analysis
Cells harvested for Western blot analysis were washed with ice-
cold PBS and lysed in buffer containing 1% Triton X-100 and a
complete protease and phosphatase inhibitor cocktail. Protein
samples were electrophoresed through 10% SDS-polyacrylamide
gels and transferred to nitrocellulose. The membrane was stained
with 0.05% Ponceau S (Sigma-Aldrich, Oakville, Ontario,
Canada) to ensure equal protein loading. Primary antibodies
reactive with the following antigens were used: cyclin D1,
poly(ADP-ribose) polymerase-cleaved (PARP), cleaved caspase 3,
cleaved caspase 7 (Cell Signaling Technology, Beverly, MA),
PPARa (Rockland Immunochemicals, Gilbertsville, PA) and b-
actin, (Santa Cruz Biotechnology). Immunoreactivity was detected
using peroxidase-conjugated anti-mouse or anti-rabbit IgG and
visualized by enhanced chemiluminescence (Pierce, Rockford, IL).
6. Reverse transcriptase-polymerase chain reaction (RT-
PCR) and Real-time reverse transcription-PCR
RT-PCR was used to detect FASN mRNA in MCL cell lines.
Total RNA was prepared with the Trizol (Invitrogen, Burlington,
Ontario, Canada) in accordance with the manufacturer’s suggest-
ed protocol. Briefly, cDNA synthesis was carried out for
50 minutes at 42uC using the superscript reverse transcriptase II
(Invitrogen). The PCR was performed for 30 cycles in a thermal
cycler (Applied Biosystems, Streetville, On, Canada), with each
consisting of denaturation (94uC for 1 min), primer annealing
(60uC for 1 min) and DNA extension (72uC for 1 min). Amplified
products were electrophoresed in 2% agarose gel containing
ethidium bromide and visualized using Alpha Imager 3400 (Alpha
Innotech, San Leandro, CA). The following sets of primers were
used: FASN- forward: 59-AAGGTCATCCCTGAGCTGAA-39;
FASN reverse: 59-CCCTGTTGCTGTAGCCAAAT-39 (expected
size, 292 bp); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
used as internal control, GAPDH housekeeping primers forward:
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59-AAGGTCATCCCTGAGCTGAA-39, reverse: 59-CCCTGT-
TGCTGTAGCCAAAT-39 (expected size, 316 bp). Twenty-four
hours after transfection of b-catenin siRNA, total RNA isolated
from cells was reverse transcribed as described above. The cDNA
was then amplified for: b-catenin primers forward: 59-CCCAC-
TAATGTCCAGCGTTT-39, reverse: 59-CTCACGATGATGG-
7. Flow cytometry
MCL cells were fixed in the CytoFix Buffer from Becton
Dickinson Biosciences (Franklin Lakes, NJ) washed in cold PBS,
centrifuged, and re-suspended in the fluorescence-activated cell
scanner (FACS) staining buffer purchased from Becton Dickinson.
Cells were incubated with primary antibodies for 60 minutes at
4uC in the dark, and washed twice using cold buffer between
incubations. The following antibodies were employed: unconju-
gated mouse IgG1 as the isotype control (10 mg/mL, Santa Cruz
biotechnology), unconjugated mouse anti-human CD19 (Becton
Dickinson Biosciences) and anti-human FASN (R and D systems,
Minneapolis, MN). Flow cytometry was performed using the
FACScan (Becton Dickinson Biosciences) and the data was
analyzed using the accompanying CELLQuest software as per
8. Statistical analysis
Statistical analysis was performed using the StatView 4.5
software. Results are expressed as mean 6 standard deviations.
Student’s t-test is used to assess the statistical significance whenever
Figure 1. FASN was highly expressed in MCL patient samples.
A) Immunohistochemical studies revealed strong cytoplasmic FASN
staining in breast carcinoma cells (i.e. positive control). In comparison,
the adjacent benign mammary epithelial cells showed only faint
cytoplasmic staining (white arrow). B) Normal tonsillar lymphoid tissue
showed no definitive staining in the mantle zones (white arrow). The
germinal center cells were weakly positive (black arrow). C and D) Two
MCL tumors showed relatively homogeneous FASN cytoplasmic
Figure 2. FASN was highly expressed in MCL cell lines. A) RT-PCR studies demonstrated the high level of FASN mRNA in four MCL cell lines.
SKBR3 (a breast cancer cell line) and HepG2 (a hepatocarcinoma cancer cell line) were used as positive controls. B) The FASN protein expressed in
MCL cell lines was readily detectable by Western blots. SKBR3 was used as a positive control. In contrast, protein lysates prepared from isolated
peripheral blood mononuclear cells (PBMCs) from a healthy donor were negative for the FASN protein. C) Flow cytometry analysis revealed the
expression of FASN in Jeko-1 cells.
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appropriate. Differences between two sets of experimental data are
considered to be statistically significant when the P value is less
than 0.05. The regression coefficient (R) is determined by linear
1. FASN is highly expressed in MCL but not normal
Using immunohistochemistry, the expression of FASN was
examined in 15 MCL tumors, and the results are illustrated in
figure 1. Breast carcinomas were used as the positive controls. We
found that all 15 cases of MCL showed intense cytoplasmic
staining throughout the tumors. In contrast, with the exception of
the germinal centers, lymphocytes in benign tonsillar tissues
(n=5), including those in the mantle zone, were negative.
We then analyzed FASN expression in MCL cell lines using
RT-PCR. As shown in figure 2A, the FASN transcript was readily
detectable in SKBR3 and HepG2 (a breast cancer cell line and a
hepatocarcinoma cell line, respectively), which served as the
positive controls. The FASN transcript was readily detectable in all
4 MCL cell lines examined. By Western blots, FASN protein was
detectable in all 4 MCL cell lines whereas PBMC from a healthy
donor were negative (figure 2B). By flow cytometry, we were able
to detect a relatively high level of FASN expression in Jeko-1 cells;
in contrast, CD19-positive B-cells in the peripheral blood from a
healthy donor were negative for FASN (not shown).
2. Pharmacologic inhibition of FASN induces significant
inhibition in cell-growth in MCL cell lines
To determine the biological importance of FASN in MCL cells,
we inhibited FASN in MCL cell lines using two previously
published pharmacological inhibitors, namely C75 and Orlistat.
We first determined the concentrations of these agents at which
the viability of the cells can be reduced to 50% (i.e. IC50). As
shown in figure 3, C75 and Orlistat induced a significant reduction
in the number of viable cells in a dose-dependent manner. At
24 hours after the treatment, the IC50 value was in the range of
2.5–5 mM for C75 and 5–10 mM for Orlistat. As illustrated in
figure 3C, Orlistat induced no detectable loss of cell viability in
Figure 3. FASN inhibitors induced cell death of MCL cells. A and B) Four MCL cell lines were treated with two widely-used FASN inhibitors,
namely C75 and Orlistat, in concentrations ranging to 0 to 20 mg/mL for C75 and 0 to 20 mM for Orlistat. At 48 hours, the number of viable cells, as
determined by using the trypan blue exclusion assay, decreased in a dose-dependent manner. Experiments were performed in triplicate and the
means +/2 standard deviations are shown. C) In contrast to MCL cells, PBMC showed no appreciable effects to Orlistat. D and E) To establish a more
direct link between fatty acid production and the observed loss of cell viability, we pre-treated the cells with 30 mM of palmitic acid (labeled as PA).
Palmitic acid significantly blocked the cell-growth inhibitory effects of Orlistat, but it did not completely abrogate the effects of this drug in MCL cells.
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PBMC from a healthy donor. To establish a more direct link
between fatty acid production and the observed loss of cell
viability, we pre-treated the cells with 30 mm of palmitic acid. As
shown in figure 3D and 3E, this pre-treatment significantly
reduced the cell-growth inhibitory effects of Orlistat. The
observation that palmitic acid did not completely abrogate the
effects of Orlistat suggests that Orlistat probably inhibited MCL
cells via mechanisms unrelated to fatty acid production.
We then assessed if the FASN inhibitors induce apoptosis in
MCL cells. As shown in figure 4A, MCL cells treated with 5 or
10 mM of Orlistat for 24 hours contained a subset of Annexin V-
positive cells. Furthermore, we found that increasing concentra-
tions of Orlistat induced a dose-dependent increase in Annexin V-
positive cells, with a corresponding decrease in the cell viability
(p,0.0001; figure 4A). We also employed an in-vitro enzymatic
caspases 3/7 assay, which provides a quantitative assessment of
apoptotic activity. Following 24 hours incubation with 5 or 10 mM
of Orlistat, the caspase 3/7 activities in two MCL cell lines (Rec-1
and Mino) were increased by up to 50 and 8 fold, respectively,
with a corresponding decrease in cell viability (p,0.0001; figure 4B
and 4C). By Western blots, we found that treatment with 10 mM of
Orlistat induced cleavage of caspase-3 and PARP in all four MCL
cell lines (figure 4D).
3. Knockdown of FASN by siRNA induces cell death of
MCL and cyclin D1 downregulation
To further determine the biological significance of FASN in
MCL, we blocked FASN expression using siRNA and assessed the
biological effects. By Western blots, siRNA treatment induced a
substantial reduction in FASN protein expression, ranging from
30–60% within 24 hours of transfection (figure 5A and 5B). As
shown in figure 5C, blockade of FASN expression using siRNA
significantly decreased cell growth, at approximately 20% for
Mino and 40% for SP53 cells at 96 hours post-transfection
(figure 5C). Simultaneously, the number of viable cells, determined
by trypan blue staining, was reduced significantly after treatment
with specific FASN siRNA (P,0.05) in Mino and SP53 cells as
compared with cells transfected with scramble siRNA (figure 5D).
As shown in figure 5E, inhibition of FASN expression by siRNA in
SP53 cells induced cleavage of caspase-3.
Figure 4. FASN inhibitors induced apoptosis of MCL cells. The four MCL cell lines were treated with two different concentrations of Orlistat. A)
The degree of apoptosis was assessed based on the cell surface expression of Annexin V in MCL cell lines detectable by flow cytometry. In addition,
the cell viability in two MCL cells, Rec-1(B) and Mino (C) treated with 5 mM or 10 mM Orlistat for 48 hours, was significantly correlated with the
enzymatic activity of caspase 3/7. D) Using western blot, cleaved capase 3 and PARP were detectable in MCL cells treated with 10 mM Orlistat.
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4. Orlistat induces significant apoptosis in leukemic MCL
To investigate the effect of FASN inhibition on primary MCL
cells, leukemic MCL cells from three patients were treated with
different concentrations of Orlistat, and the results are illustrated
in figure 6. Orlistat induced a significant decrease in the cell
viability, as determined by trypan blue staining (figure 6A). This
also correlated with increased caspase-3/7 activity (figure 6B). In
one of these three patients, we also performed propidium iodide
(PI) staining (figure 6C), and increasing concentrations of Orlistat
induced a higher proportions of PI-positive cells.
5. FASN inhibition decreases b-catenin expression
It has been recently observed that FASN is associated with b-
catenin stabilization in prostate cancer . In addition, we have
recently reported that b-catenin is activated in a subset of MCL
. Thus, we hypothesize that the relatively high level of FASN
expression in MCL cells may be linked to b-catenin in these cells.
To test this possibility, Mino, SP53 and Jeko-1 cells were
transfected with FASN siRNA or scramble siRNA. As shown in
figure 7A, inhibition of FASN using Orlistat induced a dose-
dependent decrease in the protein level of b-catenin (figure 7A).
Similar results were observed when the cells were transfected with
the siRNA to knock-down FASN (figure 7B).
6. b-catenin contributes to FASN expression in MCL
Our analysis of the promoter region of the FASN gene has
revealed the presence of TCF/LEF binding sites. Thus, we
hypothesize that b-catenin may in turn contribute to the
overexpression of FASN. To test this possibility, MCL cell lines
were treated with b-catenin siRNA or scramble siRNA. As shown
on figure 8, inhibition of b-catenin expression using siRNA led to a
Figure 5. Knockdown of FASN decreased the expression of cyclin D1 and induced cell death of MCL cells. A) Two MCL cell lines, Mino
and SP53, were treated with siRNA to knockdown FASN. The expression of FASN and cyclin D1 were assessed by western blot. The expression of
FASN and cyclin D1 was dramatically decreased after FASN siRNA treatment. B) Quantification studies showed that FASN expression was decreased in
MCL after treatment with FASN siRNA as compared to cells treated with scramble siRNA. Triplicate experiments were performed. (C) Two MCL cell
lines were treated with FASN siRNA for up to 96 hours. The cell growth was evaluated using trypan blue exclusion assay. Knockdown of FASN
expression significantly decreased the growth of MCL cells (for Mino cells -#p,0.005 at 72 hours;##p,0.005 at 96 hours; for SP53 cells - * p,0.05
at 72 hours; ** p,0.05 at 96 hours). D) Treatment of two MCL cell lines with FASN siRNA significantly decreased the number of viable cells, as
assessed by using the MTS assay. The differences are statistically significant for Mino (#p,0.05 at 48 hours and##p,0.005 at 72 hours) and for
SP53 (* p,0.05 at 48 hours and ** p,0.005 at 72 hours). Triplicate experiments were performed. E) Treatment of SP53 cell line with siRNA FASN
induced caspase 3 cleavage.
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substantial decrease of FASN protein expression. Similar results
were observed when two individual b-catenin siRNA were used
(figure S1). As shown in figure 8D, b-catenin siRNA did not
induce appreciable downregulation of FASN mRNA, suggesting b-
catenin regulates the FASN protein level primarily via post-
transcriptional mechanisms. Since previous reports have shown
that FASN can be regulated by the mTor signaling pathway and
USP2a-induced protein stabilization in cancer cells [30,31], we
asked if the effect on FASN expression mediated by b-catenin
involves these two pathways. As shown in figure 9, siRNA knock-
down of b-catenin expression led to a substantial down-regulation
of USP2a protein expression, and this finding suggests that b-
catenin may indeed increase the protein expression of FASN by
inhibiting its protein degradation. In contrast, the same treatment
did not result in detectable alterations of mTor activation in MCL
FASN, a protein playing important roles in the endogenous
fatty acid production, has been recognized to contribute to
oncogenesis in solid tumors. It has been well documented that
FASN is over-expressed in many types of epithelial malignancies,
such as those derived from the prostate and breast. However, the
relevance of FASN in hematologic malignancies has not been fully
examined. The expression status and functional significance of
FASN has never been examined in MCL. In the present study, we
report that FASN is consistently expressed at a high level in MCL
cell lines and tumors. These findings are in contrast with the
undetectable FASN level in benign lymphoid tissues, including
normal lymphocytes present in the mantle zone. The lack of FASN
expression in normal peripheral blood mononuclear cells also has
been previously reported . Of note, the high expression level of
FASN is not a universal phenomenon in hematologic malignan-
cies. Based on our literature search, we identified reports
Figure 6. Orlistat induced cell death in primary leukemic MC cells. A) Leukemic cells from three patients were treated with two different
concentrations of Orlistat. The number of viable cells was assessed by trypan blue assay after 24 hours of treatment. Results illustrated represented
the ‘pooled’ data of these three samples. B) In the same experiment, the caspase-3/7 activity was assessed using a commercially available Caspase-3/7
Apo-One kit. Again, results illustrated represented the ‘pooled’ results of three samples. Triplicate experiments were performed. C) Representative
results of propidium iodine staining of leukemic MCL samples treated with Orlistat showed a dose-dependent increase of staining detectable by flow
Figure 7. FASN inhibition decreased b-catenin expression. The
MCL cell line Mino was treated with Orlistat (A) or siRNA (B) to inhibit or
knock-down FASN, respectively. The b-catenin protein expression was
determined by western blot after 48 hours of treatment. FASN
inhibition by either Orlistat or siRNA substantially decreased the
protein expression of b-catenin. Triplicate experiments were performed.
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describing a high level of FASN expression in myelomas  and
in diffuse large B-cell lymphomas . In contrast, the expression
of FASN was not detected in cDNA microarray studies of chronic
lymphocyte leukemia, another CD5-positive B-cell lymphoprolif-
erative disorder .
We found that the high level of FASN expression in MCL is
biologically important. Specifically, our results suggest that FASN
confers anti-apoptotic effects in these cells. In support of this
concept, the use of FASN pharmacologic inhibitors (including C75
and Orlistat) was found to induce apoptosis in a dose-dependent
fashion in all MCL cell lines examined as well as 3 samples of
primary MCL leukemic cells. FASN inhibition using siRNA was
also found to induce apoptosis, associated with a downregulation
of cyclin D1 and b-catenin. We noted that the siRNA-induced
inhibition of FASN did not induce apoptosis as efficient as the two
pharmacological drugs did. This observation can be explained by
the fact that FASN siRNA did not completely abrogate the
expression of FASN; approximately 20 to 30% of FASN protein
remained to be detectable in MCL cell lines after the siRNA
treatment. Alternatively, it is likely that the pharmacologic agents
had off-target effects, as this concept is supported by our
observation that pre-treatment of palmitic acid did not completely
abrogate the cell-growth inhibitory effects of Orlistat (figure 3C
The mechanisms by which FASN is upregulated in cancer cells
have been described in a number of epithelial malignancies. For
instance, the Akt signaling pathway and HER-2 have been
reported to play a role in upregulating FASN expression in breast
cancer  as well as in thyroid papillary carcinoma . While
the Akt pathway may contribute to the high expression level of
FASN in MCL cells, we believe that it is highly unlikely that it is
the sole mechanism, as constitutive activation of the Akt pathway
is largely restricted to blastic MCL cases in one study . To
further examine the mechanism by which FASN expression is
driven in MCL cells, we analyzed the promoter region of the FASN
gene, in an attempt to identify specific transcriptional factors that
may be responsible for the high level of FASN expression in MCL
cells. Our analysis led us to identify the consensus binding
sequence for b-catenin (unpublished finding). In keeping with the
importance of b-catenin in inducing FASN in MCL, we found that
inhibition of b-catenin in MCL cells using siRNA led to a
substantial decrease in the protein level of FASN (Figure 8 and
figure S1). Rather surprisingly, the level of FASN mRNA was not
significantly downregulated. These observations suggest that b-
catenin does not exert transcriptional control over FASN
expression in MCL cells; instead, it is likely that it regulates the
FASN protein level by modulating its stability/protein degrada-
tion. In this regard, two previous reports have observed that the
protein expression of FASN in prostate and breast cancer is
Figure 8. Knock-down of b-catenin substantially decreased FASN protein expression in MCL cells. A–C) Three MCL cell lines were treated
with siRNA b-catenin or scramble siRNA and the expression of FASN was evaluated by Western blot. siRNA treatment induced a substantial decrease
in FASN protein in all the three MCL cell lines studied. Triplicate experiments were performed. D) The mRNA level of FASN was determined in MCL cell
lines after treatment with siRNA b-catenin. Knock-down of b-catenin did not significantly decrease the mRNA level of FASN. Triplicate experiments
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regulated at the post-transcriptional level [30,31]. One of these
two reports highlights the role of USP2a (ubiquitin-specific-protase
2a), an isopeptidase [30,31]. In light of this information, we asked
if b-catenin may contribute to the overexpression of FASN protein
by decreasing USP2a. As shown in figure 9a, it turns out to be the
case. Interestingly, our analysis of the human promoter of USP2a
has revealed the presence of TCF/LEF binding site (unpublished
finding). Taken together, it appears that b-catenin increases the
protein expression of FASN in MCL cells by promoting it
stabilization via modulation of USP2a. We also asked if the mTor
pathway is involved. No modulation of mTor activation was
observed after siRNA b-catenin.
We also have demonstrated, for the first time, the existence of a
positive feedback regulatory loop involving b-catenin and FASN.
Specifically, we have shown that inhibition of FASN by using
siRNA leads to a significant downregulation of b-catenin
expression in MCL cells. In view of the biological significance of
b-catenin in MCL , our findings suggest that one mechanism
by which FASN exerts its oncogenic function in MCL cells is by
upregulating b-catenin. These new information suggests that
inhibiting FASN and b-catenin in combination may be a useful
Despite the fact that FASN overexpression has been observed in
different forms of cancer, there is still no clear mechanism
explaining how FASN mediates its oncogenic effects. It has been
postulated that FASN, by virtue of its normal functions, provide a
source of fatty acids for membrane production as well as energy
supply [9,37]. Moreover, FASN has been associated with post-
translational modifications (such as palmitoylation) of proteins
, a phenomenon that has been described to promote the
activation of the Src-family tyrosine kinases . As mentioned
above, FASN may mediate its oncogenic effects by upregulating b-
catenin, a protein known to carry oncogenic functions.
To conclude, the present study describes the high level of FASN
expression is a consistent finding in MCL cell lines and tumors.
Our data has supported the concept that FASN confers anti-
apoptotic effects in MCL cells. Our results also uncovered a
positive feedback loop involving FASN and b-catenin, a signaling
protein previously reported to be important in the pathobiology of
Figure 9. b-catenin decreased USP2a protein expression in MCL cells. A and B) two MCL cell lines were treated with two different siRNA b-
catenin species or scramble siRNA, and the expression of phospho-mTor (ser2481) and USP2a were evaluated by Western blots. siRNA treatment
induced a substantial decrease in the levels of the USP2a protein in both cell lines. In contrast, no appreciable modulation of phospho-mTor (as a
surrogate marker of mTor activation) was observed. Triplicate experiments were performed and represented results are illustrated.
FASN Inhibition Induces Cell Death of MCL
PLoS ONE | www.plosone.org9 April 2012 | Volume 7 | Issue 4 | e33738
MCL. Thus, inhibition of FASN, possibly in combination with the Download full-text
blockade of b-catenin, may be a useful approach to treat MCL.
two different siRNA sequences (labeled 1 and 2). Both
siRNA species induced a dramatic decrease in FASN protein
detectable by western blots. Two MCL cell lines, Jeko-1 (A) and
Downregulation of b-catenin with the use of
Mino (B), were used for this experiment. Cell lysates were
prepared 48 hours after the siRNA transfection.
Conceived and designed the experiments: PG ZZ RL. Performed the
experiments: PG ZZ MA. Analyzed the data: PG ZZ MA RL. Contributed
reagents/materials/analysis tools: AB. Wrote the paper: PG RL.
1. Maier T, Leibundgut M, Boehringer D, Ban N (2010) Structure and function of
eukaryotic fatty acid synthases. Q Rev Biophys 43: 373–422.
Jayakumar A, Tai MH, Huang WY, al-Feel W, Hsu M, et al. (1995) Human
fatty acid synthase: properties and molecular cloning. Proc Natl Acad Sci U S A
Kusakabe T, Maeda M, Hoshi N, Sugino T, Watanabe K, et al. (2000) Fatty
acid synthase is expressed mainly in adult hormone-sensitive cells or cells with
high lipid metabolism and in proliferating fetal cells. J Histochem Cytochem 48:
Semenkovich CF, Coleman T, Fiedorek FT, Jr. (1995) Human fatty acid
synthase mRNA: tissue distribution, genetic mapping, and kinetics of decay after
glucose deprivation. J Lipid Res 36: 1507–1521.
Menendez JA, Lupu R (2007) Fatty acid synthase and the lipogenic phenotype in
cancer pathogenesis. Nat Rev Cancer 7: 763–777.
Kridel SJ, Lowther WT, Pemble CWt (2007) Fatty acid synthase inhibitors: new
directions for oncology. Expert Opin Investig Drugs 16: 1817–1829.
Jin Q, Yuan LX, Boulbes D, Baek JM, Wang YN, et al. (2010) Fatty acid
synthase phosphorylation: a novel therapeutic target in HER2-overexpressing
breast cancer cells. Breast Cancer Res 12: R96.
Menendez JA, Vellon L, Mehmi I, Oza BP, Ropero S, et al. (2004) Inhibition of
fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpres-
sion in cancer cells. Proc Natl Acad Sci U S A 101: 10715–10720.
Migita T, Ruiz S, Fornari A, Fiorentino M, Priolo C, et al. (2009) Fatty acid
synthase: a metabolic enzyme and candidate oncogene in prostate cancer. J Natl
Cancer Inst 101: 519–532.
10. Kusakabe T, Nashimoto A, Honma K, Suzuki T (2002) Fatty acid synthase is
highly expressed in carcinoma, adenoma and in regenerative epithelium and
intestinal metaplasia of the stomach. Histopathology 40: 71–79.
11. Gansler TS, Hardman W, 3rd, Hunt DA, Schaffel S, Hennigar RA (1997)
Increased expression of fatty acid synthase (OA-519) in ovarian neoplasms
predicts shorter survival. Hum Pathol 28: 686–692.
12. Uddin S, Siraj AK, Al-Rasheed M, Ahmed M, Bu R, et al. (2008) Fatty acid
synthase and AKT pathway signaling in a subset of papillary thyroid cancers.
J Clin Endocrinol Metab 93: 4088–4097.
13. Cerne D, Zitnik IP, Sok M (2010) Increased fatty acid synthase activity in non-
small cell lung cancer tissue is a weaker predictor of shorter patient survival than
increased lipoprotein lipase activity. Arch Med Res 41: 405–409.
14. Kridel SJ, Axelrod F, Rozenkrantz N, Smith JW (2004) Orlistat is a novel
inhibitor of fatty acid synthase with antitumor activity. Cancer Res 64:
15. Swerdlow S, Berger F, Isaacson P, Muller-Hermelink H (2001) Mantle cell
lymphoma. In World Health Organization Classification of Tumours. Pathology
and Genetics of Tumours of Haematopoietic and Lymphoid Tissues
Harris NL, Jaffe ES, Stein H, Vardiman JW, eds. Lyon: IARC Press.
16. Jares P, Colomer D, Campo E (2007) Genetic and molecular pathogenesis of
mantle cell lymphoma: perspectives for new targeted therapeutics. Nat Rev
Cancer 7: 750–762.
17. Smith MR (2008) Mantle cell lymphoma: advances in biology and therapy. Curr
Opin Hematol 15: 415–421.
18. Martinez N, Camacho FI, Algara P, Rodriguez A, Dopazo A, et al. (2003) The
molecular signature of mantle cell lymphoma reveals multiple signals favoring
cell survival. Cancer Res 63: 8226–8232.
19. Pham LV, Tamayo AT, Yoshimura LC, Lo P, Ford RJ (2003) Inhibition of
constitutive NF-kappa B activation in mantle cell lymphoma B cells leads to
induction of cell cycle arrest and apoptosis. J Immunol 171: 88–95.
20. Shishodia S, Amin HM, Lai R, Aggarwal BB (2005) Curcumin (diferuloyl-
methane) inhibits constitutive NF-kappaB activation, induces G1/S arrest,
suppresses proliferation, and induces apoptosis in mantle cell lymphoma.
Biochem Pharmacol 70: 700–713.
21. Fu L, Lin-Lee YC, Pham LV, Tamayo A, Yoshimura L, et al. (2006)
Constitutive NF-kappaB and NFAT activation leads to stimulation of the BLyS
survival pathway in aggressive B-cell lymphomas. Blood 107: 4540–4548.
22. Tucker CA, Kapanen AI, Chikh G, Hoffman BG, Kyle AH, et al. (2008)
Silencing Bcl-2 in models of mantle cell lymphoma is associated with decreases
in cyclin D1, nuclear factor-kappaB, p53, bax, and p27 levels. Mol Cancer Ther
23. Rizzatti EG, Falcao RP, Panepucci RA, Proto-Siqueira R, Anselmo-Lima WT,
et al. (2005) Gene expression profiling of mantle cell lymphoma cells reveals
aberrant expression of genes from the PI3K-AKT, WNT and TGFbeta
signalling pathways. Br J Haematol 130: 516–526.
24. Rizzatti EG, Mora-Jensen H, Weniger MA, Gibellini F, Lee E, et al. (2008)
Noxa mediates bortezomib induced apoptosis in both sensitive and intrinsically
resistant mantle cell lymphoma cells and this effect is independent of constitutive
activity of the AKT and NF-kappaB pathways. Leuk Lymphoma 49: 798–808.
25. Ghielmini M, Zucca E (2009) How I treat mantle cell lymphoma. Blood.
26. Amin HM, McDonnell TJ, Medeiros LJ, Rassidakis GZ, Leventaki V, et al.
(2003) Characterization of 4 mantle cell lymphoma cell lines. Arch Pathol Lab
Med 127: 424–431.
27. Delsol G FB, Muller-Hermelink HK, Campo E, Jaffe ES, Gascoyne RD, et al.
(2008) Anaplastic large cell lymphoma (ALCL), ALK-positive.; In:
Swerdlow SHCE, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J,
Vardiman JW, eds. Lyon: IARC.
28. Gelebart P, Anand M, Armanious H, Peters AC, Dien Bard J, et al. (2008)
Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma.
Blood 112: 5171–5179.
29. Fiorentino M, Zadra G, Palescandolo E, Fedele G, Bailey D, et al. (2008)
Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1
and cytoplasmic stabilization of beta-catenin in prostate cancer. Lab Invest 88:
30. Yoon S, Lee MY, Park SW, Moon JS, Koh YK, et al. (2007) Up-regulation of
acetyl-CoA carboxylase alpha and fatty acid synthase by human epidermal
growth factor receptor 2 at the translational level in breast cancer cells. J Biol
Chem 282: 26122–26131.
31. Graner E, Tang D, Rossi S, Baron A, Migita T, et al. (2004) The isopeptidase
USP2a regulates the stability of fatty acid synthase in prostate cancer. Cancer
Cell 5: 253–261.
32. Wang WQ, Zhao XY, Wang HY, Liang Y (2008) Increased fatty acid synthase
as a potential therapeutic target in multiple myeloma. J Zhejiang Univ Sci B 9:
33. Uddin S, Hussain AR, Ahmed M, Bu R, Ahmed SO, et al. (2010) Inhibition of
fatty acid synthase suppresses c-Met receptor kinase and induces apoptosis in
diffuse large B-cell lymphoma. Mol Cancer Ther 9: 1244–1255.
34. Pallasch CP, Schwamb J, Konigs S, Schulz A, Debey S, et al. (2008) Targeting
lipid metabolism by the lipoprotein lipase inhibitor orlistat results in apoptosis of
B-cell chronic lymphocytic leukemia cells. Leukemia 22: 585–592.
35. Mashima T, Seimiya H, Tsuruo T (2009) De novo fatty-acid synthesis and
related pathways as molecular targets for cancer therapy. Br J Cancer 100:
36. Rudelius M, Pittaluga S, Nishizuka S, Pham TH, Fend F, et al. (2006)
Constitutive activation of Akt contributes to the pathogenesis and survival of
mantle cell lymphoma. Blood 108: 1668–1676.
37. Baron A, Migita T, Tang D, Loda M (2004) Fatty acid synthase: a metabolic
oncogene in prostate cancer? J Cell Biochem 91: 47–53.
38. Sandilands E, Brunton VG, Frame MC (2007) The membrane targeting and
spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct
RhoB/RhoD endosome requirements. J Cell Sci 120: 2555–2564.
FASN Inhibition Induces Cell Death of MCL
PLoS ONE | www.plosone.org10 April 2012 | Volume 7 | Issue 4 | e33738