Fatty Acid Inhibition Sensitizes Androgen-Dependent and -Independent Prostate Cancer to Radiotherapy via FASN/NF-κB Pathway

Elevated fatty acid synthase (FASN) has been reported in both androgen-dependent and -independent prostate cancers. Conventional treatment for prostate cancer is radiotherapy (RT); however, the following radiation-induced radioresistance often causes treatment failure. Upstream proteins of FASN such as Akt and NF-κB are found increased in the radioresistant prostate cancer cells. Nevertheless, whether inhibition of FASN could improve RT outcomes and reverse radiosensitivity of prostate cancer cells is still unknown. Here, we hypothesised that orlistat, a FASN inhibitor, could improve RT outcomes in prostate cancer. Orlistat treatment significantly reduced the S phase population in both androgen-dependent and -independent prostate cancer cells. Combination of orlistat and RT significantly decreased NF-κB activity and related downstream proteins in both prostate cancer cells. Combination effect of orlistat and RT was further investigated in both LNCaP and PC3 tumour-bearing mice. Combination treatment showed the best tumour inhibition compared to that of orlistat alone or RT alone. These results suggest that prostate cancer treated by conventional RT could be improved by orlistat via inhibition of FASN.

lines. Significant accumulation in G1 phase and reduction in S phase were found in both cell lines; however, the insignificant sub-G1 population was found in PC3 cells. Radiation survival curves and the D 1 of both cell lines were obtained from the colony formation results. The D 1 s, i.e., the dose required to reduce the fraction of surviving cells to 0.37, were 1.1 Gy and 2 Gy for LNCaP cells and PC3 cells, respectively ( Fig. 2A,B). The D 1 obtained from colony formation assay was used for the following experiments.
orlistat decreases nf-κB activities and changes in protein expressions. The protein expression profiles were altered by orlistat in both cell lines as shown by Western blotting (Fig. 3A,B). Proteins including fatty acid synthase (FASN), pAkt, VEGF and cyclin D1 were inhibited by orlistat in a dose-dependent manner in both cell lines. The protein level of the androgen receptor (AR) was decreased, and p-p53 was increased in LNCaP cell line. Apoptosis-related cleaved caspase-3 was increased, while anti-apoptosis Bcl-2 were decreased in a dose-dependent manner in both cell lines. Since VEGF, cyclin D1 and Bcl-2 were regulated by NF-κB, EMSA was performed to determine the NF-κB activities in both cell lines. The results showed that NF-κB activities were decreased in a dose-dependent manner in both cell lines; however, the inhibition was more profound in PC3 cells (Fig. 3C,D).
Effects of combination treatment on NF-κB activity and protein expressions in LNCaP and PC3 cells.
Here we used EMSA as the endpoint to determine the optimal strategy for combination treatment. Figure 4A,B show the NF-κB activity was decreased in LNCaP cells but increased in PC3 cells 48 hours after X-ray irradiation. The lowest NF-κB activities in LNCaP and PC3 cells were found in the concurrent and pretreatment, respectively. Therefore, these two treatment schedules were used for the following experiments. The radiation and orlistat decreased the expressions of FASN, pAkt, VEGF, and cyclinD1 in LNCaP and PC3 cells. (Fig. 4C). The combination treatment further enhanced the changes in protein expressions induced by orlistat or RT alone in both cell lines.
Orlistat combined with RT enhances the therapeutic efficacy in LNCaP and PC3 tumour-bearing mouse models. Mice with subcutaneous LNCaP or PC3 tumours were randomly divided into four groups (n = 4-5 mice per group) and received different treatments as described in the Materials and methods. Figure 5A shows that the best tumour therapeutic efficacy in LNCaP tumour-bearing mice was found in the combination (COMB) group, and the tumours were almost totally inhibited with a significantly smaller mean size than other groups from Day 21 to Day 32. Both orlistat and RT alone exhibited similar inhibition effects on LNCaP tumours. Figure 5B exhibits the tumour growths in PC3 tumour-bearing mice. Both orlistat and RT showed great tumour suppressions as compared with the CTRL group, and both groups showed significant differences compared with the CTRL group from Day 14 after treatment. RT irradiation had better tumour inhibition than orlistat and kept the tumour sizes close to that of the COMB group in the first twenty days after treatment. However, the tumours received RT began to grow rapidly after Day 45 and had no difference when compared with the ORL group at the following time points.

Discussion
Androgen functions as the fuel for the growth of prostate cancer (PCa), and androgen deprivation therapy (ADT) is applied to patients to slow the tumour growth via blockade of the androgen-related signalling pathway. Although most patients respond to ADT at the beginning, some PCa turns into castration-resistant (CR) form and results in extremely inferior prognosis. Radiotherapy (RT) is delivered to PCa patients with early or late stages for various purposes. Moreover, it has been shown that the androgen receptor (AR) signalling pathway affects the radiosensitivity in bladder cancer 27 and prostate cancer 28 . The clinical results also indicated that ADT could enhance the effects of RT on PCa. These findings suggest that androgen may play a role in modulating the radiosensitivity of PCa to ionising radiation.  www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 4. NF-κB activity change was determined by EMSA assay, and the changes in protein expression profiles were detected by Western blotting. Lower NF-κB activities were found in orlistat and RT-treated (A) LNCaP and (B) PC3 cells. The lowest NF-κB activity among all the combination groups was detected in the concurrent group and pretreated group in LNCaP and PC3, respectively. According to the EMSA findings, LNCaP and PC3 cells were received concurrent and pretreatment for the subsequent Western blotting. (C) RT slightly reduced FASN, AR, and cyclin D1 expressions in LNCaP cells and these reductions were further enhanced by the combination treatment. Also, combination treatment increases p53 and p-p53. (D) Similar effects on protein expression changes were also observed in PC3 cells except for AR, p53, and p-p53, which are not expressed by PC3 cells.
It is known that androgen is primarily made from dehydroepiandrosterone (DHEA), which is derived from cholesterol via four-steps synthesis. Thus, disrupting lipogenesis seems to be a potential way to inhibit the PCa progression since lipogenesis is highly upregulated in PCa. FASN is one of the critical enzymes in lipogenesis. It has been shown that FASN inhibition could arrest the PCa growth both in vitro and in vivo 23,29,30 . Recently, Zadra et al. demonstrated that knockdown of FASN reduced the AR activity in androgen-dependent and CR form of PCa. The authors further proved that FASN inhibition suppressed cell proliferation through reprogramming cancer metabolism and inducing ER stress 31 . Here we used orlistat as a FASN inhibitor and combined with ionising radiation to investigate the combinatorial effects. Figure 1 exhibits that orlistat suppressed cell growth in both androgen-dependent LNCaP and androgen-independent PC3 cells with IC 50 = 120 μM and 80 μM, respectively. Significant accumulation of G1 phase and reduction of S phase were found in both cell lines in a dose-dependent manner (Fig. 1C,D). S phase is known as the most radioresistant phase of the cell cycle 32 , and reduced S phase population may enhance the radiosensitivity of cells. G1 phase blockage caused by FASN inhibition has been reported in several cancer types 24,31,33,34 . The bidirectional regulation of AR and cell cycle has been reported by several groups. Yuan et al. found that shRNA-mediated AR downregulation resulted in G1 arrest through increasing p27 and decreasing p-pRb expressions in androgen-independent CWR22 cells 35 . AR could push the cell cycle forward in LNCaP cells by forming the pre-replication complex, which further cooperates with cyclin A and be used for DNA replication machinery 36 . Moreover, AR not only triggers G1-S transition but also promotes the expression of dihydroceramide desaturase 1 (DEGS1), which could be used as a progression biomarker 37 .
Here the radiation survival curves were built using the results obtained from the colony formation assay on both LNCaP and PC3 cells. The D 1 s obtained from the radiation survival curves are 1.1 and 2 Gy for LNCaP and PC3 cells, respectively ( Fig. 2A,B). These two doses were used for the following experiments to evaluate the combination effects. It is worth noting that LNCaP showed a linear radiation survival curve which was also found in other studies 38,39 . The linear survival curve implies that LNCaP cells might lack double-strand break repair (DSB repair) capability. Collis et al. proved that LNCaP could not correctly repair the DSB with a rapid dual fluorescent assay 40 . Besides, results obtained by Turney et al. also indicated that LNCaP might lack DSB repair by knocking down type 1 insulin-like growth factor receptor (IGF-1R), which regulates DSB repair. The authors found that knockdown of IGF-1R sensitised PC3 and DU145 but not LNCaP cells to ionizing radiation 41 .
We further calculated the mean lethal doses D 0 of LNCaP and PC3 cells, which are 1.2 Gy and 0.7 Gy, respectively. D 0 is known as the mean lethal dose and represents the radiosensitivity of cells. The finding is similar to the www.nature.com/scientificreports www.nature.com/scientificreports/ results recently reported by Ide et al. 27 , suggesting that the androgen receptor (AR) signaling reduces radiosensitivity of cells. RT combined with ADT achieves the better outcomes than RT alone in PCa patients, and the underlying mechanisms have been discovered. Tarish et al. further demonstrated that castration or ADT improves RT responses through impairing DNA DSB repair 42 . Spratt and colleagues found the AR-overexpressed LNCaP-AR cells are more resistant to radiation than the parental LNCaP cells. Higher AR expressions are correlated to more DNA repairs after irradiation 43 .
Moreover, their results also showed that prostate-specific antigen (PSA), TMPRSS2, and KLK2 are upregulated after irradiation. Elevated PSA is usually found in those patients post-ADT due to the failure of the primary therapy 43,44 . Besides, we knocked-down the FASN using shFASN and performed colony formation to prove that FASN could regulate the radiosensitivity in PCa. The shFASN-transduced LNCaP cells showed significantly lower survival rate than parental LNCaP cells when treated with the same X-ray doses (>6 Gy) (Fig. S2), suggesting the correlation between FASN expression and radiosensitivity.
The FASN expression levels between LNCaP and PC3 cells were compared using Western blotting (Fig. S3). LNCaP cells expressed more FASN protein than PC3. It has been found that the FASN expression level does not correlate to the sensitivity to FASN inhibitor 33 , which also shown in Fig. 1A,B. Orlistat slightly increased cleaved caspase-3, decreased Bcl-2, and cyclin D1 expressions in both cell lines (Fig. 3A,B). Reduction of pAkt was also found after orlistat treatment. The PI3k/Akt/FASN pathway in PCa was first demonstrated by Van de Sande and colleagues. In their study, both the activity and expression of FASN were suppressed in LNCaP cells by treating with LY294002, a PI3k inhibitor.
Furthermore, the reduced FASN activity could be rescued by transfecting the construct encoding constitutively active Akt 37,45 . The high-fat diet induces the Akt-FASN activation and promotes tumour progression. A negative correlation of serum FASN reduction and prognosis was observed after ADT in PCa patients 46 . Our results showed that the expression of AR was decreased after FASN inhibition, suggesting that a two-way modulation between AR and FASN (Fig. 3A). The EMSA assay demonstrated NF-κB activity inhibited by the orlistat treatment in a dose-dependent manner in both cell lines (Fig. 3C,D). Increased NF-κB activity results in radioresistant and chemoresistant in various cancer types 47 , and its inhibition could diminish the resistances [48][49][50] . NF-κB is known as a signalling hub and regulates multiple effector proteins, including VEGF, cyclin D1, and Bcl-2. The expressions of these effector proteins were decreased as shown in this study. Here we have shown that the NF-κB activity can be suppressed by FASN inhibitor to enhance the tumour control when combined with radiotherapy. In contrast to our finding, Menendez et al. found that FASN inhibition lowered p53 expression and activated MEK, ERK, and nucleus translocation of NF-κB in breast cancer 51 .
The NF-κB activity strongly correlates to the radioresistance, and its activity was examined by EMSA to determine the optimal strategy for the combination treatment. Since both concurrent and pretreatment showed the lowest NF-κB activity, these two strategies were applied to LNCaP and PC3 cells, respectively (Fig. 4A,B). Combination treatment further enhanced the protein changes after treatments in both cell lines (Fig. 4C,D). Orlistat increased more cleaved caspase-3 expression in LNCaP than PC3, which echoed the results obtained from flow cytometry showing more sub-G1 population in LNCaP cells (Fig. 1C,D). Kao et al. reported that the FASN level is correlated to radioresistance and could be used as a prognostic marker in nasopharyngeal carcinoma 52 . The correlation of radioresistance to FASN activity was also observed in pancreatic cancer 53 . Another FASN inhibitor C75 also was shown to sensitise PCa cells to RT 54 in cell culture. Differing from the orlistat that only targets the thioesterase domain of FASN, C75 mainly acts on the β-ketoacyl synthase domain as other FASN inhibitors. It has also been reported that C75 could target the enoyl reductase and thioesterase domains. C75 is the analogue of natural compound cerulenin 55 . Although C75 shows potent tumor inhibition, it affects the fatty acid synthesis in normal liver and some side effects on normal tissues 56 . In addition, C75 reduces the food intakes in rodents and results in the body weight loss because it also targets carnitine palmitoyltransferase I (CPT1) 57 .
We applied the combination of orlistat and radiation to both LNCaP and PC3 tumour-bearing mouse models and found that the most significant tumour control as compared with that of RT alone or orlistat alone in both mouse models. The combination treatment inhibited LNCaP tumour growth even after orlistat was removed on day 31 post initiation of the treatment (Fig. 5A). Significant differences were found in both RT and COMB groups as compared with the CTRL group in the first 10-day after treatment in PC3 tumour-bearing mice. Better tumour inhibition by RT might be owing to higher radiation dosage given to PC3 tumour-bearing mice compared with that given to LNCaP tumour-bearing mice. Interestingly, PC3 tumours seemed to be more sensitive to orlistat similar to those found in the cell culture.
Moreover, tumours in the COMB group began significantly smaller compared with the ORL group from day 20 after treatment and were significantly smaller compared with the other groups after day 40 (Fig. 5B). These results imply that orlistat-mediated tumour inhibition is not only through FASN inhibition but other signalling regulators such as AR and NF-κB as shown in this study. We further calculated the growth inhibition rates, enhance rates, and combination indices (CIs) to evaluate the efficacy of combination treatment 58 compared with RT or orlistat alone in both LNCaP and PC3 cells as shown in Tables 1 and 2. The CIs for both tumour-bearing mouse models are <1, indicating that the combination treatment is synergistic compared with RT or orlistat treatment alone.
Orlistat is an FDA-approved anti-obesity drug due to its lipase inhibitor capability (IC 50 = 122 ng/ml). The IC 50s of orlistat as a FASN inhibitor in LNCaP and PC3 cells are significantly higher than the IC 50 for orlistat as a lipase inhibitor. Off-target effects due to the differences in IC 50 may be concerned. Hence, body weights were also monitored to assess the general toxicities caused by treatments, and no significant differences or reductions in body weights were found in all groups. Insignificant body weight change may be due to the nutrient compositions in mouse chow, which generally contains 4-4.5% of fat. Orlistat reduces body weights of subjects by blocking fat uptakes. In other words, orlistat would not cause body weight reduction if the fatty acids were relatively low in the food. www.nature.com/scientificreports www.nature.com/scientificreports/ Additionally, orlistat has poor bioavailability and short in vivo half-life. According to the information provided by the FDA drug database, orlistat has poor bioavailability and short in vivo half-life (1-2 hr), and no adverse side-effects were observed in healthy subjects receiving doses above 120 mg three times a day. Recently, orlistat has been shown as a multitargeted agent for cancer therapy. The potential targets include ribosomal proteins 7a, 9,and 14 (RPL 7a,9,and 14), β-tubulin, GAPDH, and Annexin A2 59 . These proteins modulate tumour progression by modulating genomic and chromosomal stability, glycolysis, and membrane trafficking 60 . Even though more experiments have to be carried out to conclude that radiosensitization-mediated by orlistat may also be related to these proteins.
In conclusion, this is the first study, to our knowledge, demonstrates that FASN inhibition could sensitise prostate cancer cells to ionising radiation both in vitro and in vivo. Our results suggest that FASN inhibition improves the outcomes of radiotherapy by redistributing the cell cycle and downregulating the androgen receptor signalling in LNCaP cells. Furthermore, suppression of the NF-κB activity via inhibition of FASN in both LNCaP and PC3 cells may provide a novel strategy for radiotherapy of the prostate cancer.

Materials and Methods
cell culture. Androgen-dependent and -independent human prostate cancer cell lines, LNCaP and PC3, were cultured in RPMI-1640 (Hyclone) and F-12K (Corning) medium containing 10% FBS (Hyclone) and 1% penicillin/streptomycin (Corning), respectively. Both cells were maintained at 37 humidified CO 2 incubator. Drug preparation. For in vitro experiments, the 40 mM orlistat stock solution was obtained by dissolving a Xenical capsule in 6 ml absolute ethanol (Merck) and stored at −20 °C. For in vivo treatment, orlistat was dissolved in 33% absolute ethanol and 66% PEG400 (Sigma). The mice were treated with 240 mg/kg body weight/ day 24 . cytotoxicity assay. 1.5 × 10 4 /well LNCaP and 1 × 10 4 /well PC3 cells were seeded into 96-well plates and treated with various doses of orlistat for 48 hours. The cytotoxicity of orlistat in both cell lines was assessed by AlamarBlue assay (Pierce). Briefly, 20 µl (i.e., 1/10 volume of the culture medium) of AlamarBlue reagent was added into each well. The plate was assayed at 570 and 600 nm with an ELISA reader (TECAN Sunrise) after 4-hour incubation at 37 °C. The cell viability was calculated by the equation provided by the manufacturer. X-ray irradiation. 7.5 × 10 5 LNCaP and 5 × 10 5 PC3 cells were seeded into 6-cm dishes one day before irradiation with cabinet X-ray irradiator (RS-2000, Rad Source Technologies Inc.). Cells exposed to 0-8 Gy IR were