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Keywords:

  • obesity;
  • melanoma;
  • Cav-1;
  • FASN

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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 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 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

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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 in-house cell repository. Cells were routinely cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (Hyclone, UT), penicillin (100 U/ml) and streptomycin (100 μg/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 = 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-γ, β-Catenin, pERK, ERK, pCav-1 (Tyr-14), Cav-1, FASN, pAkt (Ser-473), Akt, p53, p21, Bax and β-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 μl 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 μl iso-propanol and absorbance was measured at 570 nm using 630 nm as reference filter.

RNA extraction, cDNA synthesis and RT-PCR

Tumor sections were preserved at −80°C in TRIzol reagent (Invitrogen, Carlsbad) after excision until processing for RT-PCR. 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 5′-AGA CTC GGA GGG ACA TCT CTA CAC-3′ (F), 5′-ACT GTG TGT CCC TTC TGG TTC TG-3′ (R) and β-Actin 5′-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3′(F), 5′-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3′(R). The annealing temperature used for Cav-1 and β-actin was 58°C.

SiRNA transfections

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 μg) was taken and lysates were pre-cleared with 50 μl 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% SDS-PAGE. The expression levels of FASN and Cav-1 were detected by immunoblotting.

Long-term survival assay

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).

Statistics

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

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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 +/− 2.8 as compared to 26.4 +/− 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 2.4-fold increase in serum insulin levels in HFD mice, it was in-significant (p = 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).

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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 (μg/l), (e) serum leptin (pg/μl) and (f) serum adiponectin (ng/ml) in control and HFD fed mice. *p < 0.05.

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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 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)].

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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 = 8. *p < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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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 JAK-STAT 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 β-catenin and PPAR-γ 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 suppressor depending on the tumor type. In melanoma, Cav-1 is reported to be involved in tumor promotion.

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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.

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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 Tyr-14 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 grades of melanoma23 and the interrelationship between Cav-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(ad)]. 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)]. 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.

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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.

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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 μM, concentrations of LY294002 survival was reduced by 70% and 50% in B16F10 and A375 cells, 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 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 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).

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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 = 0.001.

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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.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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.

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 pneumocytes 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 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

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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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  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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IJC_26048_sm_SuppFig1.tif192KSupporting Information Figure 1.
IJC_26048_sm_SuppFig2.tif467KSupporting Information Figure 2.
IJC_26048_sm_SuppMaterials.doc27KSupporting Information Materials.

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