Download Dietinduced obesity increases melanoma progression: Involvement

IJC
International Journal of Cancer
Diet-induced obesity increases melanoma progression:
involvement of Cav-1 and FASN
Vimal Pandey, Maleppillil Vavachan Vijayakumar, Amrendra Kumar Ajay, Parmanand Malvi and Manoj Kumar Bhat
Recent population-based epidemiological studies strongly hint towards a link between obesity and its occurrence as well as
progression of several cancers including melanoma. Although effects of obesity on breast, colon and liver cancers have been
extensively investigated, the links between obesity and melanoma remain largely unexplored. Present study aimed to
understand the effect of high fat diet-induced weight gain on susceptibility of C57BL/6J mice to melanoma. For this, mice
routinely were fed on high fat diet for 6 months (HFD mice). Subsequently, mouse melanoma cells were injected
subcutaneously in control as well as HFD mice and followed for tumor initiation and progression. We provide strong evidence
that diet-induced obesity leads to increased melanoma progression in male C57BL/6J mice. We observed that increased
melanoma progression is associated with enhanced Cav-1 and FASN expression in tumors from HFD mice. Cav-1 and FASN are
co-ordinately regulated and Cav-1 interacts with FASN in melanoma cells. Enhanced levels of Cav-1, FASN and pAkt control
melanoma cell proliferation. Our study establishes a causative relationship between diet-induced obesity and melanoma
progression as well as demonstrates that obesity affects important tumorigenic pathways in melanoma.
Many studies have lately emerged providing plausible evidence for the role of obesity, an indispensable component of
metabolic syndrome and a severe metabolic disorder, in
pathogenesis and progression of cancer. Study by American
Cancer Society states that 14% of all cancer deaths in men
and 20% of all cancer deaths in women from range of cancer
types can be ascribed to excess body weight.1 Data from the
National Health and Nutrition Examination Survey
(NHANES) shows increased prevalence of overweight and
obese adults in US population2–4 with a similar trend in children.2,4 Traditionally, cancers that are associated with obesity
are breast, colon, pancreas, liver, cervix, stomach and kidney.2,4 Among postmenopausal women in UK, 5% of all cancers are attributable to being overweight or obese,5 and obese
Swedish men are at significantly increased risk of occurrence
of various cancers.6
In the recent past, several reports have emerged highlighting a possible link between obesity and melanoma cancers.7–12
Key words: obesity, melanoma, Cav-1, FASN
Additional Supporting Information may be found in the online
version of this article.
DOI: 10.1002/ijc.26048
History: Received 4 Aug 2010; Accepted 7 Feb 2011; Online 8 Mar
2011
Amrendra Kumar Ajay’s current address is: Renal Division,
Brigham and Women’s Hospital, Harvard Medical School, Boston,
MA
Correspondence to: Manoj Kumar Bhat, National Centre for Cell
Science, Pune University Campus, Ganeshkhind, Pune-411 007,
India, Tel.: þ91-20-25708066, Fax: þ91-20-25692259,
E-mail: [email protected] or [email protected]
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Solar radiation has been identified as a principal causal factor
for melanoma. However, the role of changing lifestyle patterns
associated with obesity may also contribute to the development and progression of melanoma. In a study by Dennis
et al., occurrence of melanoma had significant association with
highest category of body surface area and body mass index
[weight (kg)/height (m2)].7 In another study, it has been
clearly demonstrated that obesity increases the risk of melanoma11 and body mass index also relates with the risk of melanoma occurence.13 All these studies provide a firm basis for an
association between obesity and increased risk of melanoma
occurrence thereby suggesting that strategies to control obesity
may be beneficial in reducing risk for melanoma development.
Though the mechanisms by which obesity facilitates carcinogenesis have been elucidated for several cancer types, epidemiological studies suggest that they may not be similar for all
cancer types. Surprisingly, the effects of diet-induced obesity
on melanoma occurrence and progression are yet to be
detailed. Moreover, the mechanisms or factors that contribute
towards increased melanoma progression in obese condition
remain inconclusive and poorly understood. Recently, using
genetic mouse models a study detailed the causative effects of
obesity on melanoma.14 All these observations thus emphasize
the urgency to understand biological mechanisms linking obesity with melanoma.
We explored the effects of diet-induced obesity on occurrence and progression of melanoma in male C57BL/6J mice
and probed into underlying mechanisms. In the present
study, we demonstrate that in HFD mice, melanoma progression was significantly increased in comparison to their counterparts fed on regular diet. Factors contributing to this phenomenon must involve increased expression and activation of
survival molecules or oncogenes. Therefore, we investigated
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Cav-1 and FASN mediate obesity-induced increase in melanoma progression
the status of several important signalling intermediates
involved in tumorigenesis. Very interestingly, we observed
that in tumors from HFD mice Cav-1 and FASN expression
and pAkt levels were increased significantly which are associated with rapid progression of melanoma.
Material and Methods
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Animal experiments
Mice with normal and obese phenotype were generated by
manipulating their caloric intake as described previously.15
Briefly, 30 male C57BL/6J mice 4–5 weeks of age were divided into two groups. Group one referred to as control
group was fed on normal diet (Amrut Laboratory Animal
Feed, Pune, India) whereas group two mice fed on high fat
diet (Provimi Animal Nutrition, Bangalore, India) supplemented with 400 g groundnut and 200 g dried coconut per
kg bodyweight of mice were termed as HFD mice. Both
the groups of mice were fed for 6 months till they showed
8–10 g difference in body weight. Body weight, blood glucose,
triglycerides, total serum cholesterol was measured once every
month to monitor changes during the experiment. All animal
experiments have been performed following the requirement
of the Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), Government of India,
and after obtaining permission of the Institute’s Animal Care
and Use Committee (IACUC).
Glucose, cholesterol and triglycerides estimation
Blood (random and fed state) was collected by an approved
tail cap method to estimate blood glucose using rapid glucose
analyser (Accu-Chek Sensor Comfort, Roche Diagnostics,
Germany). Triglyceride and cholesterol levels were estimated
as reported earlier.15
Estimation of serum insulin, leptin and adiponectin
Serum collected from mice was used for estimation of insulin, leptin and adiponectin by mouse specific respective
ELISA kits. Insulin levels were estimated using ultra sensitive
mouse insulin ELISA kit, Mercodia, Sweden. Leptin was
measured using leptin mouse insulin EIA kit from Assay
Designs. Adiponectin was measured using Quantikine mouse
Adiponectin/Acrp30 immunoassay kit from R&D Systems.
All assays were done according to manufacturer’s protocol.
For leptin and adiponectin estimation, serum from respective
groups of animals was pooled and estimation was carried out
in triplicate.
Cells and culture conditions
Murine melanoma cell line B16F10 and human melanoma
cell line A375 were obtained from American Type Culture
Collection (ATCC; Manassas, VA) and maintained in our inhouse cell repository. Cells were routinely cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with
10% heat inactivated fetal bovine serum (Hyclone, UT), peni-
cillin (100 U/ml) and streptomycin (100 lg/ml) (Invitrogen
Corporation, CA) at 37 C with 5% CO2.
Tumor challenge
Mice were injected subcutaneously (sc) with 2 105
B16F10 cells and monitored daily for the presence of palpable tumors. Once the tumors became palpable, measurements
were taken every alternate day. Tumor volume was calculated
using the formula: Tumor volume ¼ 0.52 a b2 (a—longest diameter and b—shortest diameter). Tumor growth was
followed up to 10 days after its initiation as tumors in obese
mice attained a size of 2 cm by this time. After 10 days,
mice were killed by cervical dislocation. Excised tumors volume and weight measurements were taken and samples were
immediately preserved in Trizol for RNA preparation and at
80 C for lysate preparation and immunoblotting.
Immunoblotting
Approximately 2 mm tumor sections were taken in eppendorf tubes. These were washed five times with ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold lysis buffer
(RIPA, 50 mM Tris-HCl, pH 7.5, with 120 mM NaCl, 10
mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM
EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40 and protease inhibitor cocktail).
Samples were homogenized in RIPA using a Dounce Homogeniser, vortexed for 5 min and kept on ice for 3 hr. Samples
were again vortexed for 5 min. Lysates were repeatedly
passed through a 27 gauge syringe and centrifuged at 12,000
rpm for 40 min. Alternatively, melanoma cells were treated
with inhibitors as per the experimental conditions and lysed
in RIPA as described above. Clear supernatant was stored at
80 C. Equal amounts of protein samples were resolved on
8–10% SDS-polyacrylamide gel and then transferred onto
PVDF membrane. The membranes were probed with antibodies against pSTAT-3, STAT-3, PPAR-c, b-Catenin, pERK,
ERK, pCav-1 (Tyr-14), Cav-1, FASN, pAkt (Ser-473), Akt,
p53, p21, Bax and b-Tubulin (Santa Cruz Biotechnology,
CA). Whenever required, blots were stripped and reprobed
with required antibodies. Otherwise, gels run in duplicates
were probed for desired proteins.
MTT cytotoxicity assay
Cells were seeded at a density of 5,000 cells per well into 96
well plates and allowed to adhere for 24 hr at 37 C. Next
day, cells were treated with inhibitors as per the experimental
requirements. Control cells were treated with vehicle (DMSO
or ethanol). After treatment, medium was removed and 50 ll
of MTT (methylthiazole tetrazolium, 1 mg/ml in DMEM
without phenol red) was added in each well and further incubated for 4 hr at 37 C. Formazan crystals thus formed were
solubilized in 50 ll iso-propanol and absorbance was measured at 570 nm using 630 nm as reference filter.
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RNA extraction, cDNA synthesis and RT-PCR
Long-term survival assay
Tumor sections were preserved at 80 C in TRIzol reagent
(Invitrogen, Carlsbad) after excision until processing for RTPCR. Total RNA from tumors of control and HFD mice was
extracted using TRIzol reagent according to the manufacturer’s instructions. cDNA synthesis and RT-PCR were performed as described earlier.15 The primer pairs used were as
follows: Cav-1 50 -AGA CTC GGA GGG ACA TCT CTA
CAC-30 (F), 50 -ACT GTG TGT CCC TTC TGG TTC TG-30
(R) and b-Actin 50 -ATC TGG CAC CAC ACC TTC TAC
AAT GAG CTG CG-30 (F), 50 -CGT CAT ACT CCT GCT
TGC TGA TCC ACA TCT GC-30 (R). The annealing temperature used for Cav-1 and b-actin was 58 C.
Approximately 1 103 B16F10 cells and 2 103 A375 cells/
well were plated in 12 well plates. Next day, these were
treated with inhibitors as per the experimental requirements.
After 48 hr, medium was removed and fresh medium was
added. Cells were allowed to grow for 7 days with intermittent medium change every third day. Thereafter, these were
fixed with 3% paraformaldehyde for 10 min and stained with
0.05% crystal violet for 2 hr at room temperature. Plates
were then photographed with Gel Doc (Biorad, Hercules).
Almost 80% confluent cells in 96 well plates or 35 mm plates
were transfected with 100 nM or 250 nM of non-specific
control SiRNA (Ctrl SiRNA) or Cav-1 and FASN specific
SiRNA reconstituted in SiRNA dilution buffer using Lipofectamine 2000. Six hour post-transfection, medium was
removed and fresh medium was added. For Cav-1 siRNA,
single as well as double transfection was performed. In case
of double transfection, 6 hr post-first-transfection, the second
transfection mixture was again added for additional 6 hr.
Cells were further grown for 48 hr. MTT assay was performed or whole cell lysates were prepared for
immunoblotting.
Immunofluorescence confocal microscopy
B16F10 cells were plated in Labtek chambered slides and
allowed to grow for 24 hr. Next day, medium was changed to
DMEM supplemented with 5% serum from control or HFD
mouse and cells were cultured in 5% mouse serum for 12
days. These were then washed with PBS and immunofluorescence staining was done as described previously.15
Co-immunoprecipitation
Approximately 3 106 cells were plated in 100 mm petri
plates and allowed to grow for 48 hr. Cells were collected by
scraping and lysed in RIPA buffer without DTT. Equal
amount of protein (600 lg) was taken and lysates were precleared with 50 ll protein A/G-plus agarose for 30 min. Fifty
microgram lysates were run as input. Agarose beads were
pelleted and supernatant was incubated with Cav-1 specific
antibody overnight at 4 C. Fifty microliter protein A/G-plus
agarose was added in antibody-antigen complex with gentle
shaking for 4 hr at 4 C. Ag-Ab complexes were centrifuged
at 3,000 rpm for 5 min. Pellet was washed twice with low
salt buffer (50 mM Tris Cl pH 7.5, 25 mM NaCl and 1% Triton X-100) and high salt buffer (50 mM Tris Cl pH 7.5, 500
mM NaCl and 1% Triton X-100) each. Target and its associated proteins were disrupted and resolved on 10% SDSPAGE. The expression levels of FASN and Cav-1 were
detected by immunoblotting.
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Data are expressed as the mean and standard deviation. In
most cases, bars represent variations within the wells of an
experiment. Statistical comparisons were made using Student’s two-tailed unpaired t test and p value < 0.05 was considered significant.
Results
High fat diet causes significant weight gain in wild-type
C57BL/6J mice and alters serum lipids and
adipokine profiles
Mice were maintained on control or high fat diet and monitored at regular intervals for weight gain as per the experimental plan given in Figure 1a. After 25 weeks, mice from
control and HFD group showed a significant difference in
the body weight (Fig. 1b). Average weight of mice from control group was 18.9 þ/ 2.8 as compared to 26.4 þ/ 3.9 in
HFD mice (p < 0.05, Fig. 1c). Increased body weight was
also accompanied by increase in serum triglyceride and a significant increase in total cholesterol (p < 0.05). Serum triglycerides and cholesterol increased by 1.8-fold and also,
blood glucose levels increased 1.3-fold as compared to the
control mice (Fig. 1c). Moreover, HFD mice showed a noticeable increase in serum insulin as well as leptin levels and
decrease in serum adiponectin levels. Though there was 2.4fold increase in serum insulin levels in HFD mice, it was insignificant (p ¼ 0.098) (Fig. 1d). Interestingly, in HFD mice,
leptin levels increased significantly (p < 0.01) by 6-fold and
adiponectin levels decreased by 2.3-fold as compared to control mice (p < 0.05) (Figs. 1e and 1f).
Melanoma progresses rapidly in mice fed on high fat diet
To test whether diet-induced obesity has any impact on the
growth and progression of melanoma, tumor growth rates
were compared between control and HFD mice. When a
weight difference of 8–10 g was attained between the control
and HFD group, tumors were induced by sc injection of 2 105 B16F10 cells and mice were observed for the initiation
and progression of melanoma tumors. Tumors in both control as well as HFD mice were detected after 11 days of
injecting cells. Although, there was no noticeable difference
in the time of initiation of tumor formation between the two
groups, tumors in HFD mice progressed rapidly as compared
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Figure 1. High fat diet induces significant weight gain in C57BL/6J mice and alters serum lipids and adipokine profile. (a) Experimental
layout for studying the effects of diet-induced obesity on initiation and progression of melanoma. (b) Pictures of control and HFD male
C57BL/6J mice showing increase in body size and deposition of fat in the peritoneal cavity. Black arrows indicate the adipose tissue
deposits. No fat deposition in control mice was detected. (c) Relative fold levels of weight (g), blood glucose (mg/dl), triglycerides (mg/dl)
and cholesterol (mg/dl) in control and HFD mice. Levels of (d) serum insulin (lg/l), (e) serum leptin (pg/ll) and (f) serum adiponectin (ng/
ml) in control and HFD fed mice. *p < 0.05.
to control mice (Fig. 2A). The average tumor weight and tumor volume in HFD mice was 3.52 g and 1,920 mm3 as
compared to 0.92 g and 924 mm3, respectively, in control
mice, [Fig. 2B(a,b)]. This clearly indicates that diet-induced
obesity favors progression of melanoma in male C57BL/6J
mice [Fig. 2B(c)].
Increased caveolin-1 expression is involved in the
proliferation of melanoma cells
It has been reported that changes in the serum lipid profile
and adipokines such as leptin and adiponectin have severe consequences on the progression of tumors.16–18 However, no
study till date has highlighted the impact of diet-induced obesity and altered serum profile on the changes in the cellular signalling that may have effect on melanoma cell growth. Very
recently, it has been indicated that changes in the angiogenic
effector molecules such as VEGF may be involved.14
Though in several tumor types the involvement of JAKSTAT pathways in obesity enhanced tumorigenesis has been
reported,19–21 however, the mechanism still remains obscure
for melanoma. We checked for the activation and expression
of several molecules that have been implicated in increased
tumor progression under obese conditions. No changes in
activation status of STAT-3 and ERK were detected. Also, no
changes in the expression levels of b-catenin and PPAR-c
were detected whereas ObR expression was slightly enhanced
(Supplementary Fig. 1). We detected strikingly increased
expression of caveolin-1 (Cav-1) as well as its Tyr-14 phosphorylated form in tumors from HFD mice (Fig. 3A). Also,
we detected an increase in the expression level of Cav-1
mRNA in tumors from HFD mice (Fig. 3A). Cav-1 belongs
to a family of scaffolding proteins necessary for the formation
of 50–100 nm plasma membrane invaginations, named caveolae. Cav-1 can either act as a tumor promoter or a tumor
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Figure 2. Melanoma progresses rapidly in mice fed on high fat diet. (A) Tumor initiation and progression in control and HFD mice. (B) Final
weight (a) and volume (b) of tumors in control and HFD mice at termination of the experiment. (C) Representative pictures of tumors from
control and HFD mice in situ and after being excised. For control and HFD groups n ¼ 8. *p < 0.05. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
suppressor depending on the tumor type. In melanoma, Cav1 is reported to be involved in tumor promotion.
To confirm the significance of Cav-1 in melanoma,
B16F10 and A375 cells were treated with increasing concentrations of MCD, an inhibitor of Cav-1. In both cell lines
MCD in a dose dependent manner decreased cell survival
and at 10 mM MCD concentration <20% cells survived [Fig.
3B(a,b)]. This concentration of MCD did not reduce survival
of other cell types as reported previously.22 To ascertain that
the increased Cav-1/pCav-1 levels are a direct consequence of
altered serum composition in HFD mice, we cultured B16F10
cells in 5% serum obtained from control and HFD mice and
performed immunofluorescence analysis for Cav-1 as well as
other molecules. Increased levels of Cav-1 as well as its Tyr14 phosphorylated form were detected in B16F10 cells cultured in serum from HFD mice (Fig. 3C). Significance of
Cav-1 was re-verified by utilizing its specific SiRNA. Silencing
of Cav-1 by SiRNA inhibited growth of A375 cells (Fig. 3D).
Put together, these results clearly indicate that over expression and activation of Cav-1 is likely to be involved in the
growth of melanoma tumors in HFD mice.
p53 is a known transcription factor of Cav-1 in several
cellular models. Its expression increases with increasing
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1 and p53 is not very clear in melanomas. Though p53 levels
in melanoma tumors from HFD mice were increased (Supplementary Figs. 2a and 2b), it did not result in its activation
as evident by unaltered levels of p21 or Bax (Supplementary
Fig. 2a). Also, over expression of p53 in A375 did not induce
either cell death or alter Cav-1 expression (Supplementary
Fig. 2c).
Melanoma from HFD mice have increased expression of
FASN which interacts with Cav-1
Fatty acid synthase (FASN) is an enzyme required for de
novo synthesis of fatty acids in animals. It has been implicated in survival and proliferation of melanoma cells24,25 and
we observed that its expression in melanoma tumors from
HFD mice is enhanced in comparison to mice on normal
diet (Fig. 4A). Also, FASN expression increased in B16F10
cells cultured in serum from HFD mice (Fig. 4B). To confirm
the role of FASN in proliferation of melanoma, B16F10 and
A375 cells were treated with inhibitors cerulenin and orlistat.
In the presence of inhibitors growth of cells was retarded in
a dose dependent manner [Fig. 4C(a–d)]. Cerulenin is a specific and more potent inhibitor of FASN as compared to orlistat.24 In B16F10 cells cultured in 2% FBS in presence of orlistat cell growth was reduced by 60% [Fig. 4C(c)].
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Figure 3. Increased Cav-1 expression is involved in proliferation of melanoma cells. (A) Protein level and phosphorylation at tyrosine 14
residue of Cav-1 as well as Cav-1 mRNA level in representative tumor samples from control and HFD mice. (B) Dose-dependent reduction in
cell survival in B16F10 (a) mouse melanoma cells and A375 (b) human melanoma cells in the presence of MCD, an agent known to cause
Cav-1 depletion as assessed by MTT assay. (C) Effect of serum from control and HFD mice on Cav-1 (a) and pCav-1(Tyr-14) (b) expression in
B16F10 melanoma cells. Cells were cultured in 5% mouse serum for 12 days and then immuno-stained for the expression of pCav-1 and
Cav-1. Cells show an increased expression of pCav-1 and Cav-1 when cultured in serum from HFD mice. (D) Inhibition of A375 melanoma
cell growth in the presence of Cav-1 specific SiRNA as assessed by MTT assay.
Interestingly, in the presence of 10% FBS, orlistat did not
cause reduction in growth (data not shown) because the basal
expression of FASN in 10% serum in B16F10 cells is very
high.24 On the other hand, cerulenin in a dose dependent
manner reduced the survival of B16F10 cells cultured in 10%
FBS [Fig. 4C(a)]. In A375 cells, both cerulenin and orlistat
reduced the survival by 50% [Fig. 4C(b,d)]. Silencing of
FASN gene with the corresponding SiRNA decreased A375
cells survival by 50% (Fig. 4D). Thus, both Cav-1 and
FASN are overexpressed in melanoma tumors from HFD
mice (Figs. 3A and 4A). Recently, it has been demonstrated
that Cav-1 interacts with FASN in the membrane of prostate
cancer cells.26 To explore this in B16F10 and A375 melanoma cells, immunoprecipitation was carried out using Cav-1
specific antibody. Cav-1 and FASN were detected in the
immune complex by immunoblotting. IgG heavy chain
served as loading control. As shown in Figure 4E, we
detected FASN in the immunocomplex suggesting a functional interaction between Cav-1 and FASN. To the best of
our knowledge, this is the first report demonstrating direct
interaction between Cav-1 and FASN in melanoma cells.
Akt is activated in melanoma tumor from HFD mice
Cav-1 is upregulated and activated in rapidly growing melanoma tumors in mice fed on HFD. We checked the expression as well as activation status of Akt, an important downstream effector of Cav-1 signalling. Increased activation of
Akt (pAkt) was detected in the tumors from HFD mice (Fig.
5A). To verify the significance of Akt in melanoma cells
growth, B16F10 and A375 cells were treated with increasing
concentrations of LY294002. Treatment with the inhibitor in
a dose dependent manner inhibited proliferation of these
cells. At 100 lM, concentrations of LY294002 survival was
reduced by 70% and 50% in B16F10 and A375 cells,
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Figure 4. Melanoma from HFD mice have increased expression of FASN, which interacts with Cav-1. (A) Expression levels of FASN in
representative tumor samples from control and HFD mice. (B) Effect of serum from control and HFD mice on FASN expression in B16F10
melanoma cells. (C) Dose-dependent reduction in cell growth in B16F10 mouse melanoma cells and A375, human melanoma cells in the
presence of cerulenin and orlistat, known inhibitors of FASN, as assessed by MTT assay. (a) Dose-dependent inhibition of growth in B16F10
cells in the presence of cerulenin, (b) Dose-dependent inhibition of growth in A375 cells in the presence of cerulenin, (c) Dose-dependent
inhibition of growth in B16F10 cells cultured in 2% fetal bovine serum in the presence of orlistat and (d) Dose-dependent inhibition of
growth in A375 cells cultured in normal condition of 10% fetal bovine serum in the presence of orlistat. (D) Inhibition of growth in the
presence of FASN specific SiRNA as assessed by MTT assay. (E) Co-immunoprecipitation of Cav-1 and FASN in B16F10 mouse melanoma
cells and A375, human melanoma cells. DU145, human prostate cancer cells were used as a positive control. Immunoprecipitation was
carried out using Cav-1 specific antibody. Cav-1 and FASN were detected in the immune complex by immunoblotting. IgG heavy chain
served as loading control.
respectively (Fig. 5B). To ascertain the involvement of Cav-1,
FASN and Akt in proliferation of cells, B16F10 and A375
cells were treated with inhibitors MCD, orlistat, cerulenin
and expression levels of pAkt, Akt, Cav-1 and FASN were
checked. Treatment with MCD reduced Cav-1 levels in both
B16F10 and A375 cells and decreased the levels of pAkt
without affecting basal expression of Akt. MCD had no visual
effect on the expression of FASN [Fig. 5C(a)]. Treatment
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with FASN inhibitors not only reduced FASN levels but also
diminished Cav-1 and pAkt levels without affecting basal Akt
levels. Cerulenin being more potent FASN inhibitor, it causes
significant decrease in pAkt levels. No decrease in the levels
of Cav-1 mRNA was detected in cells treated with cerulenin
and orlistat [Fig. 5C(b)]. Silencing of Cav-1 and FASN by
their specific SiRNAs also reduced pAkt levels. Interestingly,
silencing of Cav-1 by its specific SiRNA diminishes FASN
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Figure 5. Involvement of Akt in mediating the proliferative functions of Cav-1 and FASN. (A) Expression levels of FASN in representative
tumor samples from control and HFD mice. (B) Dose-dependent reduction in cell growth in (a) B16F10 mouse melanoma cells and (b)
A375, human melanoma cells in the presence of LY294002, an Akt inhibitor, as assessed by MTT assay. (C) (a) B16F10 and A375 cells
were treated with MCD, cerulenin and orlistat and whole cell lysates were probed for pAkt, Akt, Cav-1 and FASN. Figure shows that Cav-1
and FASN inhibition abrogate Akt activation and FASN inhibition also diminishes Cav-1 levels showing probable modulation of Cav-1
stabilization by FASN, (b) FASN inhibitors do not alter Cav-1 mRNA levels. (D) A375 cells were transfected with Cav-1 and FASN specific
SiRNA and whole cell lysates were immunoblotted for pAkt, Akt, Cav-1 and FASN. Figure shows that silencing of Cav-1 and FASN abrogate
Akt activation. FASN silencing also diminishes Cav-1 levels. (E) Increased sensitivity of B16F10 cells cultured in 5% HFD mice serum to
MCD and Cerulenin as compared to cells cultured in 5% sera from control mice. dIndicates double transfection. **Indicates p ¼ 0.001.
levels also (Fig. 5D). To check dependence of obesity promoted melanoma growth on Cav-1 or FASN, we cultured
B16F10 cells in 5% mouse serum from control and HFD
mice, for 12 days. These cells were then treated with MCD
and cerulenin and interestingly, cells cultured in HFD mice
serum were more sensitive to growth inhibition by MCD
and cerulenin (Fig. 5E). We also performed long-term survival assays in the presence of inhibitors of Cav-1, FASN
and Akt to investigate whether the reduction in survival by
these inhibitors is reversed or persisted even after removal
of the inhibitors. Almost all A375 cells were eliminated at
both the doses of MCD, cerulenin and orlistat, whereas
in B16F10 cells growth inhibition was dose-dependent
(Fig. 6a). Though Akt inhibitor caused dose-dependent inhibition of cell growth, it did not eliminate cells completely
(Fig. 6a).
Discussion
Both obesity and melanoma incidences have increased in the
recent past. Higher body weight has been associated with
increased melanoma occurrence in several epidemiological
studies.7–12 Despite sufficient basis for this interrelationship,
not many studies have probed into effects of obesity on progression of melanoma and investigated the underlying mechanisms. In particular, a direct effect of diet-induced obesity
on the occurrence and progression of melanoma has not
been documented so far. Though, genetic predisposition to
obesity has been highlighted, the contribution of diet cannot
be under estimated. Diet-induced obesity is prevalent in a
large population of people and therefore it is important to
study its impact on the progression of cancer. This study for
the first time demonstrates that diet-induced obesity causes
increased melanoma progression in male C57BL/6J mice.
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Figure 6. (a) Long-term survival assays confirm involvement of Cav-1, FASN and partial involvement of Akt in melanoma cell proliferation. Cells
were treated with indicated concentrations of inhibitors for 48 hr. After that medium was replaced with fresh DMEM supplemented with 10%
FBS and cells were allowed to grow for 7 days with medium change every third day. (b) Graphical representation of the molecular changes
caused by diet-induced obesity in the increased melanoma progression. When intake of energy outweighs its consumption, obesity develops.
This causes secretion of adipokines and changes in the inflammatory status thereby inducing oxidative stress. Mechanistically, these obesity
related alterations bring about increased expression of Cav-1, FASN and increased activation of Akt which together contribute to significantly
enhanced progression of melanoma. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
High fat diet regime utilized in this study causes increase
in fat deposition, body weight, serum triglycerides and cholesterol levels. Also, serum glucose and insulin levels
increased but they were insignificant. In accordance with
what is known, diet-induced obesity also caused significant
increase in serum leptin level and decrease in adiponectin
level. No difference in tumor initiation was observed and
interestingly diet-induced obesity did accelerate melanoma
progression in C57BL/6J mice.
To probe into the changes at cellular level involved in mediating these effects, we checked expression level of Cav-1 along
with many other signalling intermediates associated with melanoma cell proliferation. Caveolins are a family of scaffolding
proteins necessary for the formation of 50–100 nm plasma
membrane invaginations, named caveolae.27 The prototype of
caveolin protein family is Cav-1, which has a very broad tissue
expression pattern, with highest level being in terminally differentiated cells such as adipocytes, endothelial cells, Type I pneuC 2011 UICC
Int. J. Cancer: 130, 497–508 (2012) V
mocytes and epithelial cells.28 In lipid homeostasis and obesity
Cav-1 role has been implicated and the deficiency of Cav-1
causes resistance to diet-induced obesity.28 In cancers, Cav-1 is
associated with tumor promoter or tumor suppressor activity
depending on the tumor type.29 Because of its low expression
in transformed mammary epithelial cell lines tumor suppressor
role of Cav-1 has been proposed in breast cancers.30,31 On the
other hand, Cav-1 expression is elevated in prostate, bladder
and oesophagus tumors.32–36 In melanoma, the role of Cav-1 is
debatable though it is also reported to be involved in tumor
promotion.37–39 Cav-1 has been reported to be present in the
secretory cellular components of pancreas and salivary
glands,40 differentiating osteoblasts,41 as well as in adipocytes42
and these secreted micro vesicles may participate in tumorigenicity in vitro and in vivo as described earlier.37
One of the novel findings of this study is that Cav-1 as
well as pCav-1 levels are significantly elevated in tumors
developed in HFD mice. Disruption of Cav-1 function in
Carcinogenesis
506
Cav-1 and FASN mediate obesity-induced increase in melanoma progression
melanoma cells by MCD treatment or with Cav-1 specific
SiRNA diminishes cell survival. Interestingly, obesity has a positive impact on Cav-1 protein and its mRNA levels. Regulation
of Cav-1 at mRNA level in tumors from obese mice is an interesting research finding which warrants further investigation.
Another molecule whose levels are elevated in tumors from
HFD mice is FASN. It is an enzyme essential in lipid biosynthesis that controls obesity through regulation of feeding behaviour.43,44 Treatment of mice with FASN inhibitor cerulenin
results in reduced food intake and substantial weight loss.43
FASN also functions as a tumor promoter in melanoma.24 In
addition, p53 levels in melanoma tumors from HFD mice were
enhanced and it did not result in its activation as evident by
detection of unaltered levels of p21 or Bax. p53 does not modulate Cav-1 transcription in melanoma cells as over expression
of p53 did not cause any change in Cav-1 expression level.
Treatment of B16F10 and A375 cells with FASN inhibitors or
FASN specific SiRNA not only reduces its protein level but also
inhibits proliferation significantly in addition to diminishing
Cav-1 as well as pAkt levels. These results are in agreement with
the findings that FASN inhibitors reduce melanoma progression
and metastasis in animal models.26 Also, we observed that cells
cultured in sera from HFD mice were more sensitive to the
growth inhibition by MCD and cerulenin. Taken together, it can
be implied that the increased expression of Cav-1 and FASN in
tumors is likely to be involved in rapid proliferation of melanoma cells under obese conditions since these are elevated in
tumors of HFD mice. Recently, in prostate cancer cells it has
been reported that Cav-1 and FASN interact together and FASN
controls Cav-1 signalling by adding a palmitic acid residue to
it.24 Our finding that Cav-1 and FASN interact with each other
in melanoma cells is indicative of similarities in the modulation
of Cav-1 by FASN and vice versa between melanoma cells and
prostate cancer cells, in which it has been previously reported.26
Alterations in the functional molecules are communicated
to the downstream effector molecules through modulation of
signalling network. One of the main pathways crucial to cell
growth and survival is Akt/PI3K pathway.45 Activation of
Akt contributes to malignant phenotypes in various human
cancers.46 In the present study, we observed an increased
activation of Akt and enhancement in Cav-1 and FASN levels. Therefore, it is likely that activation of Akt and Cav-1 as
well as upregulation of Cav-1 and FASN protein levels are
involved in the increased proliferation of melanoma cells in
HFD mice, which is in agreement with other reports.47,48
Treatment with MCD reduces Cav-1 level in B16F10 and
A375 cells. It also decreases pAkt level without affecting basal
expression of Akt. Silencing of Cav-1 using specific SiRNA
reduced FASN as well as pAkt levels in A 375 cells. Also, FASN
SiRNA reduces Cav-1 protein and pAkt levels. Though, MCD
treatment decreases Cav-1 level, no change in FASN level was
detected whereas downregulation of Cav-1 by its specific SiRNA
diminishes FASN. This discrepancy may be because MCD is an
indirect inhibitor of Cav-1 via depletion of lipids rafts and may
not directly target Cav-1. Interestingly, Cav-1 was recently
shown to be required for the upregulation of FASN.26
It is reported that FASN may be involved in regulation of
Cav-1 protein by adding palmitic acid residue, which is crucial to its functioning.24 Moreover, palmitoylation is known
to promote protein stabilization.49,50 Therefore, it is likely
that palmitoylation of Cav-1 protein may be one of the
mechanism responsible for its stabililization at protein level
in melanoma cells. FASN inhibitors also decreased the activation of Akt without affecting basal levels.
To conclude, in the present study, we provide firm evidence that diet-induced obesity causes increased melanoma
progression in C57BL/6J mice (Fig. 6b). We demonstrate that
obesity induced metabolic alterations directly affect the transcriptional regulation of Cav-1, protein expression of Cav-1
and FASN, both being important mediators of proliferation
in melanoma cells. We, for the first time, provide evidence
that Cav-1 and FASN interact in melanoma cells. Also,
FASN and Cav-1 co-ordinately regulate each other which has
an impact on activation status of Akt and growth of melanoma cells. Thus, further investigation of molecular interplay
between Cav-1, FASN and Akt activation as well as therapeutic intervention of these pathways may be helpful in developing newer approaches for preventing rapid progression of
melanoma in obese individuals.
Acknowledgements
The authors thank Dr. G. C. Mishra, Director, NCCS, for being very supportive and giving all the encouragement to carry out this work. They also
thank Department of Biotechnology (DBT), Government of India, for providing financial support. V.P. thanks Council for Scientific and Industrial
Research (CSIR), A.K.A. thanks Indian Council for Medical Research
(ICMR) and P.M. thanks University Grants Commission (UGC), New
Delhi, India, for providing research fellowships. They also thank Animal
house, Confocal and FACS facility for assistance. This work was done in partial fulfillment of a Ph.D. thesis (of V.P.) submitted to the University of
Pune, Pune, India.
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