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Human Cancer Genomic Research (S.U., A.K.S., M.A.-R., M.A., R.B., P.B., A.R.H., K.S.A.-K.), King Fahad National Center for Children’s Cancer and Research, Research Center, and Departments of Endocrinology (A.A.-N.), Surgery (S.A.-S.), and Pathology (F.A.-D.), King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia; and Department of Head and Neck Surgery (J.N.M.), M. D. Anderson Cancer Center, Texas 77030
Address all correspondence and requests for reprints to: Khawla S. Al-Kuraya, M.D., FCAP, Department of Human Cancer Genomic Research, King Fahad National Center for Children’s Cancer and Research, King Faisal Specialist Hospital and Research Cancer, MBC#98-16, P.O. Box 3354, Riyadh 11211, Saudi Arabia. E-mail: Kkuraya{at}kfshrc.edu.sa.
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Abstract |
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Objective: Our objective was to investigate the role of FASN and its relationship with phosphatidylinositol 3-kinase/AKT activation in a large series of PTC in a tissue microarray format followed by studies using PTC cell lines and Nude mice.
Design: Analysis of apoptosis and cell cycle were evaluated by flow cytometry and DNA fragmentation assays. FASN and phospho-AKT protein expression was determined by immunohistochemistry and Western blotting.
Results: Our data show that expression of FASN is associated with activated AKT (phospho-AKT) in a subset of PTC. Treatment of PTC cell lines (NPA-187, ONCO-DG-1, and B-CPAP) with C-75, an inhibitor of FASN, suppresses growth and induces apoptosis in all cell lines. Treatment of PTC cells with C-75 or expression of FASN small interfering RNA causes down-regulation of FASN and inactivation of AKT activity. Furthermore, treatment of PTC cell lines with C-75 results in apoptosis via the mitochondrial pathway involving the proapoptotic factor Bad, activation of Bax, activation of caspases, and down-regulation of antiapoptotic proteins. Finally, treatment of NPA-187 xenografts with C-75 results in growth inhibition of tumors in Nude mice via down-regulation of FASN expression and inactivation of AKT.
Conclusions: Our results suggest that FASN and activated AKT pathway may be a potential target for therapeutic intervention for the treatment of PTC.
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Introduction |
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Fatty acid synthase (FASN) is a key enzyme that plays a critical role in a number of metabolic functions by catalyzing the terminal steps in the synthesis of long-chain saturated fatty acids (4, 5). In normal cells, FASN expression level is found to be low due to the presence of abundant amounts of dietary lipids (5). However, there is renewed interest in the ultimate role of FASN in cancer pathogenesis. Tumor-associated FASN, by conferring growth and survival advantages rather than functioning as an anabolic energy-storage pathway, has been reported in many types of cancers including breast, ovarian, prostate, and stomach (6, 7, 8, 9, 10, 11, 12). Inhibition of FASN activity preferentially inhibits tumor cell growth and induces apoptosis in a number of tumor cells (13, 14). Considerable evidence demonstrates that phosphatidylinositol 3-kinase (PI3K)/AKT signaling plays an important role in oncogenic transformation and cancer progression and has been linked with FASN expression in tumor cells (8, 15).
In this study, we examined the expression of FASN in PTC and determined its relationship with PI3K/AKT, clinicopathological features, and prognosis in large series of human PTC. We then used a panel of PTC cell lines to examine the effect of C-75, a synthetic inhibitor of FASN, on PTC cell proliferation and apoptosis. In addition, we also evaluated the effect of C-75 on PTC cell line xenografts for tumor regression using a Nude mouse model. Our present findings strongly suggest that a tight functional association between FASN and AKT is taking place in a subset of PTC cells and also highlights that inhibition of FASN activity may provide a new molecular avenue for chemotherapeutic treatment of PTC.
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Materials and Methods |
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C-75 was purchased from Calbiochem (San Diego, CA). Antibodies against phospho-AKT (p-AKT), cleaved caspase-3, and BH3 interacting domain death agonist were purchased from Cell Signaling Technologies (Beverly, MA). FASN, cytochrome c, β-actin, caspase-9 and -3, and poly ADP ribose polymerase antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). X-linked inhibitor of apoptosis protein and caspase-8 antibodies were purchased from R&D (Minneapolis, MN). Annexin V was purchased from Molecular Probes (Eugene, OR). Apoptotic DNA-ladder kit was obtained from Roche (Penzberg, Germany). JC1 was purchased from Aexis (San Diego, CA).
Cell lines and culture conditions
PTC cell lines, ONCO-DG-1 and B-CPAP, were obtained from DSMZ (Braunschh, Germany) and cultured in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin at 37 C in a humidified atmosphere containing 5% CO2. All experiments were performed in RPMI 1640 containing 5% serum. NPA-187 cells were cultured as described previously (16).
Animals and xenograft study
Six-week-old Nude mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in a pathogen-free animal facility at least 1 wk before use. All animal studies were done in accordance with institutional guidelines. For xenograft study, mice were inoculated sc in the right abdominal quadrant with 5 million NPA-187 cells in 200 µl PBS. After 1 wk, mice were randomly assigned into three groups, two groups receiving two doses of C-75 and the remaining group receiving 0.9% saline. Treatment with two doses of C-75, 10 mg/kg and 20 mg/kg (animal), was given ip twice weekly. The control group received the vehicle alone at the same schedule. The body weight and tumor volume of each mouse were monitored weekly. The tumor volume was measured as described previously (17).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays
A total of 104 cells was incubated in triplicate in a 96-well plate in the presence or absence of indicated doses of C-75 for 48 h. The ability of C-75 to suppress cell growth was determined by MTT cell proliferation assays, as previously described (18).
DNA laddering
DNA laddering assay was performed as described earlier (19). Briefly, cells were treated with and without C-75 for 48 h, DNA was extracted, and 2 µg DNA was electrophoresed on a 1.5% agarose gel containing ethidium bromide at 75 V for 2 h.
Annexin V/propidium iodide dual staining
PTC cell lines were treated with the indicated concentrations of C-75. The cells were harvested, and the percentage of cells undergoing apoptosis was measured by flow cytometry after staining with fluorescein-conjugated annexin V and propidium iodide as previously described (20).
Gene silencing using small interfering RNA (siRNA)
FASN siRNA (catalog nos. S100059752 and SS100059759 pooled) and Scrambled control (catalog no.102781) siRNA were purchased from QIAGEN (Valencia, CA). For transient expression, cell lines were transfected by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. After incubating the cells for 6 h, the lipid and siRNA complex was removed, and fresh growth medium was added. Cells were lysed 48 h after transfection, and specific protein levels were determined by Western blot analysis with specific antibodies against the targeted proteins and actin as a loading control.
Cell lysis and immunoblotting
Cells were treated with C-75 as described in the legends and lysed as previously described (21). Proteins (15–20 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Billerica, MA). Immunoblotting was done with different antibodies and visualized by the enhanced chemiluminescence (Amersham, Piscataway, NJ) method.
Measurement of mitochondrial membrane potential by JC1 assay
After treatment of PTC cell lines with C-75 for 48 h, cells were incubated with 10 µM JC1 at 37 C in dark for 15 min, and mitochondrial membrane potential (percentage of green and red aggregates) was determined by flow cytometry as described previously (22).
Assays for cytochrome c release
Release of cytochrome c from mitochondria was assayed as described earlier (23). Briefly, cells were treated with and without C-75 as described in the figure legend, and proteins were extracted. Twenty micrograms of protein from cytosolic and mitochondrial fraction of each sample were analyzed by immunoblotting using anti-cytochrome c antibody.
Patient selection and tissue microarray (TMA) construction
A total of 536 patients with papillary carcinoma of the thyroid, diagnosed between 1988 and 2004, were selected from the files of the King Faisal Specialist Hospital and Research Centre. All samples were analyzed in a TMA format. TMA construction was performed as described earlier (23, 24). Briefly, tissue cylinders with a diameter of 0.6 mm were punched from representative tumor regions of each donor tissue block and brought into a recipient paraffin block using a modified semiautomatic robotic precision instrument (Beecher Instruments, Sun Prairie, WI). Two cores of papillary carcinoma of the thyroid were arrayed from each case. Patients were reclassified into three histology subtypes of papillary carcinoma, classical papillary carcinoma, follicular variant of papillary carcinoma, and tall-cell variant, according to well-established histopathological criteria. The Institutional Review Board of the King Faisal Specialist Hospital and Research Centre approved the study.
Mice tumors and TMA construction and immunohistochemistry
Paraffin blocks were made from the tumor tissue of all mice, and muscle/soft tissue was used from control mice where the NPA-187 cell line was not injected. Paraffin blocks were made from liver and spleen tissue, which also served as controls. After mapping the hematoxylin and eosin slides, a TMA was constructed comprising two cores from tumor tissue, one core from liver, and one from spleen of each mouse.
Immunohistochemical studies on formalin-fixed, paraffin-embedded tissue sections were performed as described in earlier studies (25, 26). TMA from human tumors and mice NPA-187 cell line xenograft tumors were processed and stained manually. Each TMA spot was assigned an intensity score from 0–3 (I0, I1–3), and the proportion of the tumor staining for that intensity was recorded as 5% increments from a range of 0–100 (P0, P1–3). A final H score (range 0–300) was obtained by adding the sum of scores obtained for each intensity and proportion of area stained (H score = I1X P1 + I2XP2 + I3XP3). Adipose tissue served as positive control for FASN expression as has been reported previously (27). PTC tumors were grouped into two groups: one with complete absence of staining (H score = 0) and the other group showing some degree of staining (H score > 0) depending on the H score (Fig. 1
). p-AKT scoring was done as described earlier (25, 26). Briefly, p-AKT was scored as levels on an intensity scale ranging from 0–3. For purposes of statistical analysis, all cases staining at level 0 and 1 were grouped as p-AKT negative and all cases staining at level 2 and level 3 were grouped as p-AKT positive.
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The software used for statistical analysis was Statview 5.0 (SAS Institute Inc., NC). P values < 0.05 were considered significant. The correlation coefficients were calculated between pairs of variables using Pearson’s correlations. Two-sided tests were used throughout the analyses.
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Results |
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Levels of FASN were examined by immunohistochemistry in a series of 536 PTC (Fig. 1
). High levels of FASN expression were seen in 6.2% (30 of 487) of the PTC. p-AKT overexpression was seen in 55.1% (255 of 463) of the PTC. Representative information for FASN was available in 487 spots, and immunohistochemical analysis failure of the remaining cases was due to missing spots or fixation artifacts. As shown in Table 1, FASN overexpression was not associated with age, gender, histology type, extrathyroidal extension, and American Joint Cancer Committee stage. However, FASN overexpression was significantly associated with overexpression of p-AKT (P = 0.0261). Although patients with overexpression of FASN had a poor disease-free survival of 67.8% at 5 yr as compared with 83.9% for patients with negative FASN, this difference did not reach statistical significance (P = 0.2884).
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We initially sought to determine whether C-75 treatment led to inhibition of PTC cell proliferation. PTC cells were treated with various doses of C-75 for 48 h, and proliferation was assayed using MTT assays. Figure 2A showed that as the dose of C-75 increased from 10–200 µM, cell growth inhibition increased in a dose-dependent fashion in all the PTC cell lines. C-75-induced growth inhibition was found to be statistically significant (P < 0.05) (Student’s t test) at most of the doses tested in all cell lines.
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, the sub-G1 population of cells increased from 1.29% in control to 61.36% with 50 µM C-75 in NPA-187 cells. Similar results were obtained with ONCO-DG1 and in B-CPAP cells. This increase in the sub-G1 population was accompanied by loss in G0/G1, S, and G2/M phase in C-75-treated thyroid cancer cells, suggesting that PTC cells were dying due to apoptosis (23, 27). C-75-induced apoptosis in PTC cells was further confirmed by annexin V/PI dual staining (Fig. 2C) and DNA laddering (Fig. 2D) methods. These results suggest that suppression of growth by C-75 treatment in PTC cells is via inducing apoptosis. Constitutive expression of FASN and activation of protein kinase B/AKT signaling pathways in PTC cells
The overexpression of FASN has been shown to cooperate with survival pathways including the PI3K/AKT pathway (28). Using PTC cell lines, we sought to determine whether treatment of these cells with C-75 caused any effects on FASN expression and AKT activity. PTC cell lines were treated with 25 and 50 µM C-75 for 24 h, cells were lysed, and proteins were analyzed by Western blot. As shown in Fig. 3A, all three cell lines expressed constitutive FASN and p-AKT, and treatment of PTC cells with C-75 suppressed FASN expression and dephosphorylated constitutive activated AKT. C-75 treatment dephosphorylated AKT and p-Bad as early as 1 h in B-CPAP (supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org), suggesting that this was an early event. To independently verify that the effects of C-75 could be attributed to its ability to block FASN, similar experiments were conducted with siRNA targeting FASN. Like C-75, the siRNA targeting FASN down-regulated the expression of FASN protein and decreased the phosphorylation of AKT (Fig. 3B). On the other hand, AKT inhibitor did not show any effect on FASN expression (supplemental Fig. 1B). Expression of constitutively activated AKT in PTC cells partially protected FASN siRNA-induced activation of caspase-3 and caspase-9 (supplemental Fig. 1C), suggesting that activity of AKT is regulated by FASN expression.
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, constitutive phosphorylation of FKHR and GSK3 were seen in all PTC cell lines, and C-75 treatment caused dephosphorylation of these proteins, suggesting that FASN down-regulation induces apoptosis via inactivation of AKT and its downstream effectors in PTC cell lines. C-75 treatment of PTC cells induced apoptosis via mitochondrial pathway-mediated caspase activation in PTC cell lines
In the next series of experiments, we sought to determine whether down-regulation of FASN and p-AKT signaling involves the mitochondria in PTC cell lines. We first examined the activation of Bax in response to C-75 treatment. As shown in Fig. 4A, inhibition of FASN led to conformational changes and activation of Bax protein starting within 2 h of C-75 treatment. Interestingly, overexpression of Bcl-xL protected C-75-mediated activation of caspase-9 in B-CPAP cells (supplement Fig. 2A). We then tested the effect of C-75 on the mitochondrial membrane potential and release of cytochrome c in these cells. Cells were treated with C-75 for 24 h and labeled with JC1 dye and mitochondrial membrane potential was measured by flow cytometry. As shown in Fig. 4B, treatment of cells with C-75 resulted in loss of mitochondrial membrane potential in PTC cells and caused release of cytochrome c to the cytosol. We then sought to determine whether C-75-induced release of cytochrome c was capable of activation of caspase-9 and -3 and PARP. Figure 4C shows that C-75 treatment resulted in the activation of caspase-9 and -3 and cleavage of PARP in PTC cells. It has been shown that caspase-3 is able to activate caspase-8 and cause BID cleavage downstream of the mitochondria to potential the apoptotic stimuli (19). Our data showed that there was activation of caspase-8 and BID after C-75 treatment (data not shown). Furthermore, pretreatment of PTC cell lines with zVAD-fmk, a universal caspase inhibitor, prevented C-75-mediated activation of caspase-3, cleavage of PARP, and apoptosis (supplement Fig. 2, B and C), further indicating that C-75-induced apoptosis occurs via caspase activation.
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, caspase-9 was found to be activated after 9 h and caspase-8 after 12 h of C-75 treatment. In addition, B-CPAP pretreated with caspase-8 or -9 or a universal caspase inhibitors (zVAD-fmk) followed by treatment with C-75 did not show any effect on C-75-mediated Bax conformational changes (Fig. 4E), indicating that Bax conformation does not depend on caspase activation. These data confirm the finding that FASN-induced apoptosis causes activation of the intrinsic mitochondrial apoptotic pathway. Inhibitors of apoptosis proteins (IAP) have been shown to be physiological substrates of AKT that are stabilized to inhibit programmed cell death and have direct effects on caspase-9 and -3 (32). We therefore also examined whether C-75 induced cell death by modulating the expression of IAP family members that ultimately determine the cell’s response to apoptotic stimuli. PTC cell lines were treated with 25 and 50 µM C-75 for 24 h, and expression of cIAP1, XIAP, and survivin were determined using Western blotting. As shown in supplemental Fig. 2D, C-75 treatment caused down-regulation of cIAP1, XIAP, and survivin. These results implicate that these survival proteins may be modulated by FASN and AKT for the survival of PTC cells.
In vivo activity of FASN inhibitor C-75 against PTC cells xenograft
Our observation that thyroid cancer cells exhibit enhanced sensitivity to FASN inhibitor-induced apoptosis in vitro suggests the potential for therapeutic responses to treatment of thyroid cancer with FASN inhibitor in vivo. C-75 has previously been shown to inhibit tumor progression, including formation of ascites in an ovarian cancer xenograft model (8). Therefore, the ability of C-75 to inhibit thyroid tumor growth was examined in a mouse xenograft model of PTC cancer. Tumor development in Nude mice and treatment with C-75 were performed as described in Materials and Methods. After 6 wk treatment, mice were killed and tumors were collected. As shown in Fig. 5A, C-75 treatment caused a time-dependent regression of NPA-187 xenograft tumor in mice compared with vehicle-treated mice. The regression reached significance (P < 0.05) at the end of fourth week of treatment by C-75. A significant reduction in tumor weight (Fig. 5B) was also observed in mice treated with C-75 vs. the control group (P < 0.05). Additionally, images of tumor before and after necropsy showed that C-75 treatment resulted in shrinkage of tumor size (Fig. 5C). We analyzed the FASN and p-AKT level in primary tumors derived from vehicle-treated mice and tumors treated with C-75 mice by Western blot analysis. As shown in Fig. 5D, the levels of FASN and p-AKT were markedly decreased in mice treated with C-75 compared with vehicle-treated mice. Immunohistochemistry of FASN protein on these tumors showed a reduced level of FASN staining after C-75 treatment as well as activation of caspase-3 and cleavage of PARP, suggesting induction of apoptosis (supplemental Fig. 3). In the mice xenografts, FASN H scores ranged from 220–250 in the untreated group (mean score 230; SD 17.3), from 150–190 (mean score 167; SD 20.8) in the 10 mg/kg-dose C-75-treated group, and from 110–130 in the 20 mg/kg-dose C-75-treated group (mean score 130; SD 17.3). There was a reduction in FASN levels after treatment, and this difference was statistically significant in both groups: untreated vs. 10 mg/kg-dose C-75-treated group (P = 0.015) and untreated group vs. 20 mg/kg-dose C-75-treated group (P = 0.002). Animals treated with C-75 did not exhibit weight loss (data not shown).
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Discussion |
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Our in vitro experiments with PTC cell lines indicate that constitutive levels of FASN and activated AKT (p-AKT) are present in these cell lines. Furthermore, inhibition of FASN activity either by C-75 treatment or by gene silencing using siRNA of FASN causes down-regulation of FASN protein leading to dephosphorylation of AKT in PTC cell lines. In addition, treatment of PTC cell lines with C-75 also causes dephosphorylation of downstream targets of AKT including FKHRL1 and GSK3, further suggesting that FASN overexpression may modulate AKT activity. The mechanism by which FASN inhibitor C-75 decreases AKT activity is still not fully elucidated, but one possible mechanism is that fatty acids synthesized by FASN are incorporated into membrane phospholipids, which are known modulators of AKT activation (33). Therefore, when FASN expression is decreased by the treatment of C-75, there will be less fatty acid synthesis and lower phospholipid levels available. This decreased level of phospholipids may result in inhibition of AKT activity in PTC cells lines after C-75 treatment. Numerous studies demonstrate a critical role of PI3K/AKT signaling in oncogenic transformation and cancer progression (15, 34). AKT prevents apoptosis by generating antiapoptotic signals through regulation of a number of apoptotic and survival molecules such as Bad, GSK3, and caspase-9 and activation of transcriptional factors such as Forkhead (FOXO1) and nuclear factor-
B (31, 35, 36).
FASN inhibition has been shown to induce apoptosis in human cancer cells (37). Our data show that C-75 treatment of PTC cells induces apoptosis and growth inhibition. Apoptosis is a multistep process, and an increasing number of genes have been identified that are involved in the control or execution of apoptosis (38). FASN inhibition by C-75 in PTC cells causes apoptosis via the mitochondrial pathway and activation of caspase cascade. Furthermore, pretreatment of PTC cells with a broad-spectrum caspase inhibitor abrogates C-75-induced apoptosis. These data suggest that inhibition of FASN induces apoptosis via inactivation of p-AKT and dephosphorylation of Bad, resulting in disruption of mitochondrial membrane potential due to conformational changes and activation of Bax leading to release of cytochrome c into cytosol. Release of cytochrome c into cytosol results in activation of downstream caspases, eventually resulting in apoptosis. Recently, a similar study showed that inhibition of PI3K/AKT by cerulenin, another specific inhibitor of FASN, induces apoptosis in breast cancer cell lines via release of cytochrome and activation of caspases (39), further supporting our findings. Because our in vitro studies using PTC cell lines show that C-75 suppresses growth and induces apoptosis via down-regulation of FASN and inactivation of AKT activity, we confirm these cellular finding in vivo using NPA-187 xenograft tumors in Nude mice. Treatment of xenograft tumors with C-75 results in regression of tumor volume in a time- and dose-dependent manner and loss in xenograft tumor weight. Tumor regression is accompanied by down-regulation of FASN expression and decrease in the level of phosphorylated AKT. The body weight of controls and experimental animals did not differ throughout the experiments, suggesting that C-75 treatment significantly inhibits NPA-187 xenograft growth without causing any visible side effects to mice. Thus, C-75 may have a therapeutic potential for the treatment of a subset of PTC.
In summary, our data indicate the presence of a cross talk between FASN and PI3K/AKT signaling pathway that modulates AKT activation and may play a role in the pathogenesis of PTC. This may have significant clinical implications. Elucidating the molecular link between FASN overexpression and p-AKT may be important for the purpose of developing molecularly targeted treatment against this biologically separate subset of PTC.
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Acknowledgments |
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Footnotes |
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First Published Online August 5, 2008
Abbreviations: FASN, Fatty acid synthase; GSK3, glycogen synthase kinase-3; IAP, inhibitor of apoptosis protein; p-AKT, phospho-AKT; PI3K, phosphatidylinositol 3-kinase; PTC, papillary thyroid carcinoma; PVDF, polyvinylidene difluoride; siRNA, small interfering RNA; TMA, tissue microarray; XIAP, X-linked IAP.
Received March 4, 2008.
Accepted July 29, 2008.
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References |
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B is a target of AKT in anti-apoptotic PDGF signaling. Nature 401:86–90[CrossRef][Medline]
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