Inhibition of Fatty-acid Synthase Suppresses P-AKT and Induces Apoptosis in Bladder Cancer

Basic and Translational Science Inhibition of Fatty-acid Synthase Suppresses P-AKT and Induces Apoptosis in Bladder Cancer Bo Jiang, En-Hui Li, You-Yi Lu, Qi Jiang, Di Cui, Yi-Feng Jing, and Shu-Jie Xia OBJECTIVE METHODS

RESULTS

CONCLUSION

To investigate the role of fatty acid synthase (FASN) in bladder transitional cell carcinoma (BTCC). FASN expression was investigated in non–muscle-invasive BTCC tissue specimens by immunohistochemistry and BTCC cell lines by Western blot. After treatment with FASN-siRNA or FASN inhibitor cerulenin (Cer), the proliferation and apoptosis of BTCC cell lines 5637 and 253 J were determined by cell counting Kit-8 (CCK8) assay and flow cytometry respectively. The expression of p-AKT, cyclin D1 (CCND1), and apoptosis-related proteins were detected by Western blot. High levels of FASN expression were observed in 59% (32/54) of non–muscle-invasive BTCC tissue specimens, and FASN expression was associated with histologic grade (P ⬍ .05) and recurrence (P ⬍ .05). FASN expression was high in 6 BTCC cell lines. FASN inhibitor Cer and FASN-siRNA produced the increased apoptosis and decreased proliferation of bladder cancer cells, and caused inactivity of AKT and downregulation of CCND1. Furthermore, treatment of BTCC cell lines with Cer resulted in apoptosis via the caspase-dependent pathway involving inactivation of antiapoptotic bcl-2 protein. Our data suggest that FASN plays an important role in BTCC development. Targeting FASN may be a new therapeutic strategy for BTCC. UROLOGY 80: 484.e9 – 484.e15, 2012. © 2012 Elsevier Inc.

B

ladder cancer is the fifth most common malignancy throughout Europe, with an estimated 127,000 new cases and 46,000 deaths in 1995.1 Bladder transitional cell carcinoma (BTCC) accounts for 90-95% of bladder cancer. Although the prognosis for BTCC is often favorable, it has a high rate of tumor recurrence, progression, and metastasis. Therefore, a better understanding of the molecular and cellular heterogeneity of BTCC will lead to the discovery of potential therapeutic targets. Fatty-acid synthase (FASN) is a key biosynthetic enzyme involved in lipogenesis and the production of longchain fatty acids from acetylcoenzyme A (CoA) and malonyl-CoA, which play an important role in energy homeostasis by converting excess carbon intake into fatty acids for storage.2 In most normal cells (except liver and adipose tissue), FASN expression level is difficult to find because of the presence of abundant amounts of dietary Financial Disclosure: The authors declare that they have no relevant financial interests. From the Department of Urology, The Affiliated First People’s Hospital of Shanghai Jiao Tong University, School of Medicine, Shanghai, China Reprint requests: Shu-Jie Xia, Ph.D., The Affiliated First People’s Hospital of Shanghai Jiao Tong University, Department of Urology, School of Medicine, Shanghai, China. E-mail: [email protected] Submitted: November 1, 2011, accepted (with revisions): February 27, 2012

© 2012 Elsevier Inc. All Rights Reserved

lipids.3 However, FASN is preferentially overexpressed in many cancers and has been strongly linked to tumor cell proliferation and apoptosis. Therefore, it is considered a potential therapeutic target in prostate and thyroid cancers and multiple myeloma.4-6 Furthermore, FASN germ line polymorphisms have also been reported to be significantly associated with risk of lethal prostate cancer.7 This tumor-associated FASN was often associated with poor prognosis and reduced disease-free survival rather than function as an anabolic energy-storage pathway in these cancer types. In bladder cancer, correlation of FASN expression with tumor recurrence and advanced stage has been indicated.8 However, the role of FASN in BTCC has not been described. In this study, we examined the expression of FASN in 54 non–muscle-invasive BTCC tissue specimens and the effect of FASN-siRNA and FASN inhibitor cerulenin (Cer) on proliferation and apoptosis of BTCC cell lines. These data suggest a possible potential role of FASN as a novel therapeutic target for BTCC.

MATERIAL AND METHODS Tissue Specimens BTCC tissue specimens were collected from 54 patients by transurethral resection of bladder tumor (TURBT) in the Urol0090-4295/12/$36.00 484.e9 doi:10.1016/j.urology.2012.02.046

ogy Department of the First People’s Hospital Affiliated to Shanghai Jiao Tong University from March 2005 to April 2006. Tumor grade was based on pathologic findings following the World Health Organization Classification (1973 WHO grading).9 Clinical staging was based on a combination of cystoscopy, computed tomography, ultrasound, and histopathological data. The follow-up data were obtained from medical records, registry, and patient interviews. The follow-up was based on regular cystoscopies and also included an appropriate patient history, urinalysis, and urine cytology. Patients were assessed every 3 months in the first 2 years after initial diagnosis, followed by every 6 months for the next 3 years, and then annually thereafter. The primary endpoint was a duration to the first recurrence, and the final endpoint was to March 2011 (the follow-up periods: median, 35.5 months; range, 2-72).

Cell Culture and Reagent Human prostate cancer cell line LNCaP and human bladder cancer cell lines RT4, T24, 5637, 253 J, BIU87, and EJ were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured at 37°C with 5% CO2 in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU mL–1 penicillin, and 100 ␮g mL–1 streptomycin. Normal urothelial cells were primarily cultured from fresh bladder tissue of 2 young donors (30 and 32 years old), and cultured at 37°C with 5% CO2 in KSFM media supplemented with 1% fetal bovine serum, 100 IU mL–1 penicillin, and 100 ␮g mL–1 streptomycin. All experimental protocols were approved by the Medical Ethics Committee of the Shanghai First People’s Hospital (Shanghai, China). Cer was obtained from Biomol (Enzo Life Sciences, Farmingdale, NY), dissolved in dimethyl sulfoxide, and stocked in 10 mg/mL concentration.

Immunohistochemistry Archived paraffin-embedded formalin-fixed tissues were used for immunohistochemistry. Antibody against FASN (Cell Signaling Technology, Beverly, MA) was used at 1:50 dilution. Secondary antibody (Long Island Biotech, Shanghai, China) was applied at a dilution of 1:400. A standard avidin-biotin step was applied and diaminobenzidine for detection. FASN immunoreactivity was performed using a combined scoring system based on the fraction of positive tumor cells and the predominant staining intensity in the tumor according to Visca et al’s description.8 The fraction of positive tumor cells was estimated using a 4-tiered scale (10% ⫽ 1%, 11-50% ⫽ 2%, 51-80% ⫽ 3, ⬎80% ⫽ 4). Staining intensity was scored on a 3-tiered scale: 0 ⫽ negative (no staining visible), 1 ⫽ weak (few intracytoplasmic granules visible), and 2 ⫽ strong (many intracytoplasmic granules visible). The overall score in each case was added and scores of ⬍2 were considered negative. Evaluation of the immunostaining was carried out by 2 independent pathologists.

SiRNA Transfection The sequence of the FASN-targeted siRNA (5=- CCC AGG CUG AAG UUU ACA ATT ⫺3=) was chosen out of 3 individual siRNAs because of its most efficiency in reducing FASN mRNA and protein level. We used a scrambled-sequence siRNA duplex as a negative siRNA control. All siRNAs were designed and synthesized by Shanghai Genepharma Company (China). 5637 And 253 J cells were transiently transfected with 20 nM siRNA-targeted FASN or scrambled siRNA in a 6-well plate (2.5 ⫻ 105 cells per well) or a 96-well plate (1 ⫻ 104 cells per well) 484.e10

using Lipofectamine RANIMAX reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Western Blot Analysis After treatment with Cer for 24 hours or transfection for 48 hours in 6-well plates, cells were harvested and resuspended in 60-␮L lysis buffer. Protein samples (30 ␮g) were loaded on 8% SDS–polyacrylamide gel for electrophoresis. Protein samples with molecular weights ⬎70 kD were transferred to a polyvinylidene fluoride membrane at 100 V for 3 hours or ⬍70 kD at 100 V for 1 hour. Western blot analysis was done after blocking the membrane with 5% nonfat milk in Tris-buffered saline solution ⫹ 0.1% Tween 20 (TBST) for 2 hours at room temperature. The membrane was incubated with the specific antibody at 4°C overnight. The following antibodies were used: antibodies against human FASN, cyclin D1 (CCND1), totalAKT, phosphor-AKT, caspase-3, cleaved caspase-3, caspase-7, caspase-8, bcl-2, and ␤-actin at 1:1000 dilution (Cell Signaling Technology). After washing in TBST for 3 ⫻ 10 minutes, the blots were incubated with anti-rabbit IgG horseradish peroxidase conjugate at 1:2000 dilution (Cell Signaling Technology) for 2 hours at room temperature. Antigen was detected using standard chemical luminescence methodology.

Cell Proliferation Assay Cell proliferation was determined with the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan). Cells were suspended at a final concentration of 3 ⫻ 103 cells/well and cultured in 96-well plates. After overnight culture, Cer was added to the specific concentration (0-40 ␮g/mL) for 24 or 72 hours. Then CCK-8 reagent (10 ␮L) was added to each well of a 96-well plate containing 100 ␮L culture medium and the plate was incubated for 2 hours at 37°C. Viable cells were evaluated by absorbance measurements at 450 nm. After 48 hours of incubation, cells transiently transfected with siRNAs were stained in the same way.

Flow Cytometry Assay Apoptotic cells were measured triplicately by flow cytometry according to instructions of the annexin (V) fluorescein isothiocyanite (FITC) Apoptosis Detection Kit (BD Biosciences, Sparks, MD). After 24 hours exposure of 5 ␮g/mL Cer or 48 hours transfection, cells were harvested and washed twice with phosphate-buffered saline and resuspended in binding buffer containing FITC conjugated annexin V antibody (5 ␮L) and propidium iodide (5 ␮L). After incubation in the dark for 30 minutes, cells were analyzed by flow cytometry.

Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction Total RNA from cells were prepared using Trizol reagent (Invitrogen). RNA was reverse-transcribed by an AMV Reverse Transcription System (Promega, Madison, WI) and was amplified by quantitative real-time reverse transcriptase polymerase chain reaction (Q-PCR). The sequences of the primers were as follows: FASN (sense, 5=-TAT GCT TCT TCG TGC AGC AGT T-3=; antisense, 5=-GCT GCC ACA CGC TCC TCT AG-3=). The Ct values were normalized to the house-keeping gene ␤-actin (sense, 5=-CGG GAA ATC GTG CGT GA-3=; antisense, 5=-TGC CCA GGA AGG AAG GCT-3=) and amplification was performed using SYBR Green as fluorescent dye (Takara, China). The specificity of the PCR products was controlled by melting-curve analysis. The relative expression of UROLOGY 80 (2), 2012

Figure 1. FASN expression in bladder cancer. (A) FASN expression in BTCC tissue specimens were detected by immunohistochemistry (magnification x400). Positive expression of FASN in poorly differentiated BTCC (a); negative expression of FASN in well-differentiated BTCC (b). (B) FASN expression in various BTCC cells and primary normal urothelial cells were examined by Western blot; LNCaP cells were the positive control.

mRNA was calculated using the comparative delta ct. (threshold cycle number) to compare the expression levels among different samples.

Table 1. FASN expression and its association with clinicopathologic parameters in BTCC

Total

Statistical Analysis Results were analyzed using chi-square test and Student’s t-test to assess statistical significance, respectively, with values of P ⬍ .05 considered statistically significant. Two-sided tests were used throughout the analyses. All statistical evaluations were performed with SPSS 11.5 (SPSS, Inc., Chicago, IL).

RESULTS FASN Expression and Its Association with Clinicopathological Parameters in BTCC Levels of FASN were examined by immunohistochemistry in 54 non–muscle-invasive BTCC specimens (Fig. 1A). Granular cytoplasmic staining showed the positive expression (Fig. 1A), and high levels of FASN expression were seen in 59% (32/54) of BTCC. As shown in Table 1, FASN overexpression was not associated with age, gender, clinical stage, and disease-free survival at 5 years (P ⫽ .63, .9, .37, and .22, respectively); however, it was significantly associated with poor cell differentiation (P ⬍ .05). Patients with overexpression of FASN had a poor recurrence-free survival of 47% (15/32) at 5 years compared with 86% (19/22) of patients with negative FASN (P ⬍ .05). FASN was Overexpressed in BTCC Cell Lines To further determine different expressions between BTCC cell lines and normal urothelial cells, we measured FASN protein level in 6 different human BTCC cell lines (5637, T24, RT4, 253 J, BIU87, and EJ), and primary human UROLOGY 80 (2), 2012

No. of patients Sex Female Male Age (y) ⬍70 ⱖ70 PT stage PTa PT1 Histologic grade G1 G2 G3 Disease-free survival, 5 y Recurrence-free survival, 5 y

Negative n %

Positive n %

P Value

54

22

41

32

59

24 30

10 12

42 40

14 18

58 60

.90

20 34

9 13

45 38

11 21

55 62

.63

16 38

8 14

50 37

8 24

50 63

.37

20 27 7 45

11 11 0 20

55 41 0 44

9 16 7 25

45 59 100 56

⬍.05 .22

34

19

56

15

44

⬍.05

urothelial cells by Western blot. The prostate cancer cell line LNCaP was used as positive control. As shown in Figure 1B, FASN expression was higher in all bladder cancer cell lines than in normal urothelial cells. FASN Inhibition by Cer and FASN-siRNA–Suppressed BTCC Cell Proliferation To explore the effect of FASN on human bladder cancer, we treated 253 J and 5637 cells with Cer, a known FASN inhibitor, and measured the proliferation effects of Cer on these 2 cell lines by CCK8 assay. Incubating 253 J and 484.e11

Figure 2. FASN inhibition suppressed the proliferation of BTCC cells. FASN inhibitor Cer suppressed the growth of 5637 cells (A) and 253 J cells (B) in a time- and dose-dependent way. Cells mock-transfected or with FASN-siRNA or negativesiRNA, and FASN knockdown efficiency was detected by Q-PCR (C) and Western blot (D). FASN knockdown inhibited the proliferation of 5637 cells (E) and 253 J cells (F). The data represent mean ⫾ SD of triplicate experiments. *P ⬍ .05 statistical significance (Student’s t-test).

5637 cells with Cer led to time- and dose-dependent decreases in cell viability (Fig. 2A,B). To independently verify whether the effect of Cer could be owed to its ability to inhibit FASN, similar experiments were done with siRNA targeting FASN simultaneously. After siRNA transfection, it showed that FASN-siRNA treatment resulted in a significant reduction of FASN mRNA and protein expression in 5637 and 253 J cells (Fig. 2C,D). Like Cer, the siRNA targeting FASN reduced 5637 and 253 J cells’ viability significantly (Fig. 2E,F). FASN Inhibition Downregulated the Expression of p-AKT and CCND1 Furthermore, to identify how FASN inhibition reduced the proliferation of BTCC cells, we examined 2 key factors (CCND1 and AKT) in cell proliferation. CCND1, as an important positive regulator for the G1 to S phase transition, has been shown to be involved in bladder cancer cell proliferation.10 The PI3K/AKT pathway is one of the most important survival pathways in bladder cancer and contributes to G1 cell cycle progression via CCND1 upregulation.11,12 In 5637 and 253 J cells, after treatment with 5 and 484.e12

10 ␮g/mL Cer for 24 hours or transiently transfected with siRNAs for 48 hours, the expression of CCND1 and p-AKT were found to be reduced in both Cer and FASN-siRNA treatment groups (Fig. 3A,B). By contrast, we did not find that Cer had any effect on FASN expression in these cell lines (Fig. 3B). FASN Inhibition Induced BTCC Cells Apoptosis Meanwhile, we measured the effect of FASN inhibition on BTCC cells’ apoptosis. Apoptosis was detected by analyzing the percentage of early apoptotic cells using annexin V/PI double-staining. With 5 ␮g/mL Cer treatment for 24 hours or FASN-siRNA transfection for 48 hours, the percentage of early apoptotic cells (FITC⫹/ PI⫺) increased significantly in 5637 and 253 J cells (Fig. 4A–D). To further investigate the cellular effects of FASN inhibition on 253 J and 5637 cells, we examined apoptosis-related protein after the administration of Cer. Western blot studies after treatment with 5 and 10 ␮g/mL Cer for 24 hours showed significant reductions in the expression of total caspase-3, total caspase-7, total caspase-8, and the antiapoptotic bcl-2 protein in 5637 and 253 J cells, UROLOGY 80 (2), 2012

Figure 3. FASN inhibition downregulated CCND1 and AKT activity in BTCC cell lines. (A) FASN knockdown by FASN-siRNA downregulated CCND1 and p-AKT. (B) FASN inhibitor Cer downregulated CCND1 and p-AKT and had no effect on FASN expression. Cells were treated with 5 or 10 ␮g/mL Cer ⫻ 24 hrs. ␤-actin as the internal control.

as well as increases in the expressions of cleaved caspase-3 in 253-J cells (Fig. 4E,F), which indicated that Cer induced apoptosis via the caspase dependent pathway.

COMMENT Recently there has been renewed interest in the role of lipid metabolism in human cancer. Many reports have shown that FASN, a major lipidic enzyme, was involved in tumor occurrence,13 evolution,14 metastasis,15 and chemotherapeutic resistance16 in various types of cancers. Serum FASN was overexpressed in metastatic breast cancer17 and can be used as a novel marker in pancreatic neoplasia.18 Previously, FASN has been shown to be overexpressed in bladder cancer.8 Here we detected 54 human BTCC tissue specimens and found that FASN expression was examined in 59% of non–muscle-invasive BTCC, and FASN overexpression was associated with histologic grade and could serve as a predictor of recurrence, which was similar to previous reports. In addition, we also showed that FASN was overexpressed in the 6 BTCC cell lines, but not expressed in normal urothelial cells. These data indicated that FASN might play an important role in BTCC development. In some studies, FASN has been shown to be strongly correlated with several kinds of tumor cells’ proliferation and apoptosis.4-6 To explore the role of FASN in BTCC development, we investigated the effect of FASN on the proliferation and apoptosis of BTCC cells. We found that FASN inhibition led to decreased proliferation and increased apoptosis in 5637 and 253 J cells. It indicated that FASN may be a new target for BTCC therapy. It is currently unclear how FASN regulates BTCC development. A previous report has shown that FASN had a significant effect on cell viability in breast cancer and liver cancer via cyclins.19 CCND1 is an important positive regulator for the G1 to S phase transition. Therefore, we tested whether FASN inhibition regulates UROLOGY 80 (2), 2012

this common target of cell proliferation in bladder cancer and found CCND1 protein level was significantly reduced in 5637 and 253 J cells after FASN inhibition. In addition, the PI3K-AKT signaling pathway was believed to be involved in the genesis, cell survival, and chemotherapeutic resistance in various tumor types, including bladder cancer.20,21 P-AKT, as the activated AKT, contributes to the process of cell survival by phosphorylating different substrates that directly or indirectly regulate G1 cell cycle progression and apoptotic program, such as upregulation of CCND111,12 and inhibition of the proapoptotic Bcl-2 family member.22 Inhibition of PI3K/ AKT pathway is regarded as a potential strategy for cancer treatment.23 Recently, it has been reported that FASN took part in regulating AKT phosphorylation. We sought to investigate the effect of p-AKT when the activity or expression of FASN is prevented in BTCC cells. Our results suggested that Cer and FASN-siRNA could lead to a decrease in p-AKT protein levels. The mechanism by which FASN inhibition decreased AKT activity was still not fully elucidated, but one possible explanation was that FASN could affect the production of membrane phospholipids, which were known modulators of AKT activation.24-26 FASN was the key enzyme for synthesis of long-chain fatty acids, which were incorporated into membrane phospholipids. Therefore, once the activity or expression of FASN was inhibited, there would not be enough fatty acids and phospholipids available to maintain the activity of membrane microdomain as platforms for cell signaling. This decreased level of membrane phospholipids might lead to the decreased levels of p-AKT after FASN inhibition. Cer, as a natural inhibitor of FASN activity, was a cytotoxic agent for several tumor cells through induction of apoptosis in some reports.6,27 Consistent with these results, FASN inhibition by Cer was also shown to induce apoptosis in both 5637 and 253 J cells. In view of 484.e13

Figure 4. FASN inhibition induced apoptosis of BTCC cells in a caspase-dependent way. Apoptosis was detected by analyzing the percentage of early apoptotic cells using annexin V/PI double-staining. FCM analysis revealed that FASN knockdown and FASN inhibitor Cer both can induce apoptosis dramatically in 5637cells (A, C) and 253 J cells (B, D) compared with controls. After 24 hours of treatment with 5 or 10 ␮g/mL Cer, cell lysates (30 ␮g) were immunoblotted with antibody against caspase-7, caspase-8, caspase-3, cleaved caspase-3, and bcl-2. ␤-actin as the internal control. Cer activated caspase-7, caspase-8, and caspase-3 expression, and downregulated bcl-2 protein levels in 5637 cells (E) and 253 J cells (F), respectively.

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UROLOGY 80 (2), 2012

the established roles of caspase family proteases in mammalian cell apoptosis, we tried to identify whether Cer induced apoptosis involved in these caspase family proteases’ activation in BTCC cells, and our finding showed that caspase-3, 7, and 8 were activated, which indicated that FASN inhibition led to BTCC cells’ apoptosis through a caspase-dependent apoptotic pathway. Bcl-2, which is a negative apoptosis regulator of cell death, plays an important role in cell survival28 and protects cells from apoptosis by preventing mitochondrial outer membrane permeabilization.29 Further study here demonstrated that bcl-2 protein expression could also be regulated by FASN, and bcl-2 protein expression significantly decreased after inhibition of FASN activity by Cer. This release of many apoptogenic mediators from mitochondria would lead to a cascade of reaction with the assistance of caspases that cleaved key cellular proteins.30 The present study indicated that FASN inhibition–induced apoptosis was related to downregulation of the antiapoptotic bcl-2 and activation of caspases.

CONCLUSIONS Our study suggested that FASN might play an important role in BTCC. FASN inhibition could suppress proliferation significantly and induce caspase-dependent apoptosis in BTCC cells. FASN inhibition could decrease the expression of p-AKT, CCND1, and bcl-2 in bladder cancer cells, which might have been mediated by the FASN roles in bladder cancer. Targeting FASN may be a new therapeutic strategy for BTCC. References 1. Bray F, Sankila R, Ferlay J, et al. Estimates of cancer incidence and mortality in Europe in 1995. Eur J Cancer. 2002;38:99-166. 2. Kuhajda FP, Jenner K, Wood FD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci U S A. 1994;91:6379-6383. 3. Kuhajda FP. Fatty acid synthase and cancer: new application of an old pathway. Cancer Res. 2006;66:5977-5980. 4. Mansour M, Schwartz D, Judd R, et al. Thiazolidinediones/PPAR␥ agonists and fatty acid synthase inhibitors as an experimental combination therapy for prostate cancer. Int J Oncol. 2011;38:537-546. 5. Uddin S, Siraj AK, Al-Rasheed M, et al. Fatty acid synthase and AKT pathway signaling in a subset of papillary thyroid cancers. J Clin Endocrinol Metab. 2008;93:4088-4097. 6. Okawa Y, Hideshima T, Ikeda H, et al. Fatty acid synthase is a novel therapeutic target in multiple myeloma. Br J Haematol. 2008; 141:659-671. 7. Nguyen PL, Ma J, Chavarro JE, et al. Fatty acid synthase polymorphisms, tumor expression, body mass index, prostate cancer risk, and survival. J Clin Oncol. 2010;28:3958-3964. 8. Visca P, Sebastiani V, Pizer ES, et al. Immunohistochemical expression and prognostic significance of FAS and GLUT1 in bladder carcinoma. Anticancer Res. 2003;23:335-339. 9. Epstein JI, Amin MB, Reuter VR, et al. The World Health Organization/international society of Urological pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference committee. Am J Surg Pathol. 1998;22:1435-1448.

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10. Cheng G, Xie L. Parthenolide induces apoptosis and cell cycle arrest of human 5637 bladder cancer cells in vitro. Molecules. 2011;16:6758-6768. 11. Gille H, Downward J. Multiple ras effector pathways contribute to G(1) cell cycle progression. J Biol Chem. 1999;274:22033-22040. 12. Vartanian R, Masri J, Martin J, et al. AP-1 regulates cyclin D1 and c-MYC transcription in an AKT-dependent manner in response to mTOR inhibition: role of AIP4/itch-mediated JUNB degradation. Mol Cancer Res. 2011;9:115-130. 13. Migita T, Ruiz S, Fornari A, et al. Fatty acid synthase: a metabolic enzyme and candidate oncogene in prostate cancer. J Natl Cancer Inst. 2009;101:519-532. 14. Menendez JA, Vellon L, Mehmi I, et al. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc Natl Acad Sci U S A. 2004;101:10715-10720. 15. Murata S, Yanagisawa K, Fukunaga K, et al. Fatty acid synthase inhibitor cerulenin suppresses liver metastasis of colon cancer in mice. Cancer Sci. 2010;101:1861-1865. 16. Zeng L, Biernacka KM, Holly JM, et al. Hyperglycaemia confers resistance to chemotherapy on breast cancer cells: the role of fatty acid synthase. Endocr Relat Cancer. 2010;17:539-551. 17. Vazquez-Martin A, Fernandez-Real JM, Oliveras-Ferraros C, et al. Fatty acid synthase activity regulates HER2 extracellular domain shedding into the circulation of HER2-positive metastatic breast cancer patients. Int J Oncol. 2009;35:1369-1376. 18. Walter K, Hong SM, Nyhan S, et al. Serum fatty acid synthase as a marker of pancreatic neoplasia. Cancer Epidemiol Biomarkers Prev. 2009;18:2380-2385. 19. Kim K, Kim HY, Cho HK, et al. The SDF-1alpha/CXCR4 axis induces the expression of fatty acid synthase via sterol regulatory element-binding protein-1 activation in cancer cells. Carcinogenesis. 2010;31:679-686. 20. Wu D, Tao J, Xu B, et al. Phosphatidylinositol 3-kinase inhibitor LY294002 suppresses proliferation and sensitizes doxorubicin chemotherapy in bladder cancer cells. Urol Int. 2011;87:105-113. 21. Szanto A, Bognar Z, Szigeti A, et al. Critical role of bad phosphorylation by Akt in cytostatic resistance of human bladder cancer cells. Anticancer Res. 2009;29:159-164. 22. Belkhiri A, Dar AA, Zaika A, et al. t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism. Cancer Res. 2008;68:395-403. 23. Kuroda K, Horiguchi A, Sumitomo M, et al. Activated Akt prevents antitumor activity of gefitinib in renal cancer cells. Urology. 2009;74:209-215. 24. Menendez JA, Vellon L, Lupu R. Targeting fatty acid synthasedriven lipid rafts: a novel strategy to overcome trastuzumab resistance in breast cancer cells. Med Hypotheses. 2005;64:997-1001. 25. Swinnen JV, Van Veldhoven PP, Timmermans L, et al. Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains. Biochem Biophys Res Commun. 2003;302:898-903. 26. De Schrijver E, Brusselmans K, Heyns W, et al. RNA interferencemediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells. Cancer Res. 2003;63:3799-3804. 27. Zecchin KG, Rossato FA, Raposo HF, et al. Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis. Lab Invest. 2011;91:232-240. 28. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988;335:440-442. 29. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9:47-59. 30. Ola MS, Nawaz M, Ahsan H. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol Cell Biochem. 2011;351: 41-58.

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