Overexpressed Fatty Acid Synthase in Gastrointestinal Stromal Tumors: Targeting a Progression-Associated Metabolic Driver Enhances the Antitumor Effect of Imatinib

The majority of gastrointestinal stromal tumors (GIST) harbor mutually exclusive KIT or PDGFRA mutations, which result in constitutive activation of the encoded receptor tyrosine kinases (RTK) to drive tumorigenesis and dictate treatment response to imatinib (1, 2). In adults, 10% to 15% of GISTs exhibit wild-type KIT and PDGFA genes but may bear mutated NF1, BRAF, or SDH genes (3, 4). Both the NIH (Bethesda, MD) and National Comprehensive Cancer Network (NCCN) schemes are deemed effective in risk stratification for GISTs, whereas their prognostic utility is not uniformly validated, and the NCCN scheme even lacks sufficient evidence-based data for some uncommon settings (5, 6). Another challenge in the current care of GIST patients is the inevitable resistance to imatinib that emerges following initial responses due to acquired secondary mutations (1, 7). Therefore, it is desirable to characterize aberrations in nonkinase biochemical pathways that may crosstalk with or regulate RTK-driven signaling to overcome the current limitations in prognosis and drug resistance, particularly for cases refractory to imatinib.

Lipid biosynthesis drastically increases to meet the needs of rapidly proliferating tumor cells (8). Being a multifunctional homodimeric enzyme in de novo lipogenesis, fatty acid synthase (FASN) is frequently overexpressed to catalyze palmitate synthesis to maintain the homeostasis of energy substrates and lipid rafts (9, 10). In most carcinomas, overexpressed FASN promotes aggressive phenotypes, predicts decreased patient survival, and has emerged as an attractive therapeutic target because normal tissues primarily import extracellular lipids without a strict requirement for FASN activity (10). However, the relevance of overexpressed FASN in GISTs remains to be clearly established; to date, only one pertinent study documented that FASN overexpression predominates in high-risk and metastatic GISTs (11).

In this study, mRNA and protein expression levels of FASN significantly increased in higher risk tumors. Furthermore, protein overexpression was strongly associated with unfavorable KIT/PDGFR/BRAF mutation types and independently predicted poor disease-free survival (DFS) in primary imatinib-naïve GISTs. In imatinib-resistant GIST cells, stable silencing against endogenous FASN decreased cell growth and migration and enabled resensitization to imatinib, concomitant with significant suppression of the activity and/or expression of kinases in the PI3K/AKT/mTOR/RPS6/4E-BP1 signaling cascade. Apart from inducing apoptosis and autophagy, FASN inhibition using C75 apparently inhibited total and phosphorylated KIT levels through transcriptional repression and inactivated downstream kinases. Compared with either drug alone, the therapeutic additivism of combined therapy of C75 and imatinib was substantiated in vitro and in vivo, with concomitant downregulation of KIT and the phosphorylated levels of PI3K/AKT/mTOR pathway. Taken together, we robustly characterized FASN as a progression-associated metabolic driver for accurate prognostication in GISTs and provided a rationale for dual blockade of KIT and FASN for nonkinase, metabolic derangement-targeting therapies.

GIST48 and GIST430 cells primarily harbor a homozygous V560D mutation and a heterozygous in-frame deletion in KIT exon 11, respectively. Following imatinib therapy, the GIST48 tumors acquired a heterozygous D820A mutation in KIT exon 17, and the GIST430 tumors developed a heterozygous missense mutation in exon 13 (12). Both cell lines were authenticated by short tandem repeat genotyping, periodically confirmed to be mycoplasma free using PlasmoTest (Invivogen) and maintained in IMDM (Invitrogen) supplemented with 15% FBS, 100 U/mL penicillin/streptomycin, and 4 mmol/L l-glutamine (Invitrogen) at 37°C in 5% CO₂ (12, 13).

The Institutional Review Boards approved this study (104-8432C). For IHC, we employed previously constructed tissue microarrays (TMA) of 370 primary GISTs resected before 2009 with triplicate cores in each case (13). Recut TMA sections yielded 350 cases informative for FASN immunoexpression status (Table 1), including 213 successfully genotyped for KIT/PDGFRA/BRAF. Independent of cases in the TMAs, 40 primary GISTs with available frozen samples (Supplementary Table S1) were used to determine FASN mRNA abundance by qRT-PCR. All cases evaluated for immunoexpression and mRNA abundance were imatinib naïve before disease relapse and unrelated to neurofibromatosis type I.

GIST cell lines stably transduced with shFASN or shLacZ or treated with C75, orlistat, or vehicle were extracted for total RNA using an RNeasy Mini kit (Qiagen). To isolate pure tumor cells from frozen tissues, laser capture microdissection (LCM) was applied on cryomold sections of 40 samples, from which total RNA was extracted as described previously (14). The RT, real-time PCR procedures using an ABI StepOnePlus System (Invitrogen), the reagents of a predesigned TaqMan assay (FASN, KIT, and POLR2A as a housekeeping gene for normalization), and the formulae for calculating FASN and KIT mRNA levels were described in Supplementary Methods S1.

The mutational statuses of KIT exons 11, 9, 13, and 17, as well as of PDGFRA exons 12 and 18, were previously determined for 213 primary GISTs (13, 15). Of these, 22 cases with wild-type KIT and PDGFRA genes were screened for possible mutation in the BRAF hotspot exon 15 (16). The prognostically dichotomized grouping of genotypes is detailed in Supplementary Methods S2 and has been reported previously (13–16).

TMA sections were microwave heated for antigen retrieval, incubated with a primary antibody against FASN (clone 23, 1:100; BD Transduction), and detected for protein expression using a ChemMate EnVision Kit (Dako). FASN immunoexpression was recorded as the mean percentage of labeled cells in the tumoral cytoplasm, and overexpression was defined by 50% or more tumor cells with moderate or strong cytoplasmic staining. To screen for defects in the mitochondrial succinate dehydrogenase (SDH) complex, 22 GISTs without KIT or PDGFRA mutations in the TMA cohort were evaluated for immunoexpression of SDHA (1:750; Abcam) and SDHB (1:200; Abcam) on full sections (16).

The methods used to establish stable clones of FASN-silenced GIST48 and GIST430 cells and myristoylated-AKT–transfected GIST430 cells are detailed in Supplementary Methods S3. With pLKO.1-shLacZ (TRCN0000072223) being the control, two lentiviral vectors of pLKO.1-shFASN (Taiwan National RNAi Core Facility), including shFASN#25 (TRCN0000003125) and shFASN#28 (TRCN0000003128), were used to silence FASN expression. The lentiviral vectors harboring constitutively active, myristoylated-AKT and the corresponding EGFP control were purchased from Addgene. The luciferase-tagged pKM2L-phKIT promoter construct was purchased from RIKEN Bioresource Center and transfected into GIST cell lines using Lipofectamine as detailed in Supplementary Methods S4.

In two GIST cell lines, Western blots were performed to evaluate endogenous FASN expression, total and phosphorylated KIT, the efficiency of FASN knockdown and myristoylated AKT transfection, and the expression and activity of ERK, PI3K, AKT, and signaling molecules downstream of mTOR following genetic and pharmacologic manipulations, as described in Supplementary Methods S5.

The FASN inhibitors C75 (Sigma Aldrich) and orlistat (Roche) were obtained and suspended in DMSO. GIST48 and GIST430 cells were seeded in 96-well plates at a density of 5 × 10³ cells per well the day before treatment at the indicated time points with vehicle control (0.9% saline), increasing concentrations of imatinib (Novartis) or C75, or varying combinations of imatinib and C75 at the indicated doses.

To evaluate effects associated with FASN knockdown, pharmacologic interventions, or myristoylated-AKT, we analyzed cancer phenotypic alterations using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), wound healing, and colorimetric caspase activity assays as appropriate and detailed in Supplementary Methods S6–S8.

The animal welfare committee approved the experimental protocol (2013092401). GIST430 cells treated with C75 (50 and 100 μmol/L), imatinib (100 mg/kg), a combination of C75 (50 μmol/L) and imatinib (100 mg/kg), or vehicle controls were inoculated into the flanks of 8-week-old SCID mice to analyze the in vivo tumor growth-inhibiting effects of various therapies. Tumor volume was calculated using the following formula: V = π/6 × length (mm) × width (mm)². Whole sections from formalin-fixed xenograft specimens were analyzed by TUNEL assay, and pertinent antibodies are described in Supplementary Methods S9.

For frozen samples, the Kruskal–Wallis and Mann–Whitney U tests were performed to examine differences in FASN mRNA levels between GISTs of various risk categories. In the TMA cohort, we evaluated associations between FASN immunoexpression and clinicopathologic factors using χ² and Mann–Whitney U tests for categorical and continuous variables, respectively. As of April 2009 (median, 49.9 months; range, 1–247), follow-up data were available for 350 cases informative for FASN immunoexpression. The endpoint was DFS, which would not be confounded by imatinib therapy for relapsed or disseminated diseases. We used the log-rank test to compare univariate prognostic analyses. In multivariate analyses, significant prognosticators with univariate values P < 0.05 were generally introduced, including either the NIH scheme or NCCN guidelines. Size and mitosis, component factors of risk stratification, were not incorporated in the multivariate comparisons. Student t or one-way ANOVA test was used to analyze quantitative RT-PCR and functional or pharmacologic assays for cell and xenograft samples.

From 40 primary GISTs, their endogenous FASN mRNA levels in LCM-isolated tumor cells were quantified, revealing significant upregulation in higher risk categories defined by both NCCN guidelines (P < 0.0001, Fig. 1A) and the NIH scheme (P < 0.001, Supplementary Fig. S1). In regard to FASN mRNA levels, NCCN guidelines more effectively distinguished GISTs of various risk categories (high risk vs. moderate risk, P = 0.023; moderate risk vs. very low/low risk, P = 0.006). In contrast, no significant difference was observed between low-risk and intermediate-risk cases using the NIH scheme. Consistent with the frequent FASN transcriptional upregulation in carcinomas, FASN overexpression in the progression of GISTs likely occurs in part through transcriptional machinery following altered signaling stimuli (9, 10).

To examine the translatability of upregulated mRNA, we analyzed the clinical relevance of overexpressed protein in a large set of primary imatinib-naïve GISTs. There were 350 GISTs informative for FASN immunoexpression status (Table 1) with clinical follow-up data, which comprised 88 no- or very low-risk cases, 100 low-risk cases, 65 moderate-risk cases, and 97 high-risk cases based on NCCN guidelines and corresponded to 127 very low/low-risk cases, 110 intermediate-risk cases, and 113 high-risk cases according to the NIH risk scheme. Of these, 22 cases with wild-type KIT and PDGFRA genes exhibited neither a BRAF hotspot mutation nor aberrant loss of SDHA or SDHB (16). FASN-overexpressing GISTs were frequently nongastric (P = 0.050) and were strongly associated with the presence of epithelioid cells, unfavorable KIT/PDGFR/BRAF genotypes, and increasing tumor size, mitotic rate, and risk levels by both NCCN and NIH criteria (all P ≤ 0.005). Univariately, poor DFS (Table 2; Fig. 1C) was associated with nongastric locations (P = 0.0023), unfavorable genotypes (P = 0.0005), epithelioid histology, increases in tumor size, mitosis, NCCN risk levels, and FASN overexpression (all P < 0.0001). In multivariate analysis, FASN overexpression (P < 0.001; HR, 2.448) independently predicted poor outcomes, along with higher NCCN risk levels (P < 0.001) and epithelioid histology (P = 0.007). However, unfavorable genotypes lost prognostic independence. Analogous to the NCCN guidelines, the alternative incorporation of NIH criteria in multivariate analysis demonstrated very similar influence and statistical significance (Supplementary Table S2). Specifically, FASN overexpression (P = 0.002; HR, 2.253) remained independent as an adverse prognosticator, together with higher NIH risk levels (P < 0.001) and epithelioid histology (P = 0.010).

Given the challenge of imatinib resistance in GISTs (1), we focused on imatinib-resistant GIST48 and GIST430 cell lines to characterize the biological implications of FASN. As both cell lines overexpressed FASN, RNAi was applied, and two shFASN clones achieved stable silencing with different efficiency (Fig. 2A). Compared with shLacZ, shFASN significantly decreased viable cells in both GIST430 and GIST48 cells (Supplementary Fig. S2), consistent with the known growth-promoting function of FASN (9–11). Moreover, a wound healing assay exhibited significantly less migrating cells when FASN was silenced, indicating that FASN has a promigratory attribute (Fig. 2B; Supplementary Fig. S3). Depending on the efficiency of shFASN clones, FASN silencing resulted in a varying but significantly increased percentage of nonviable GIST48 and GIST430 cells treated with imatinib (5–1,000 nmol/L; Fig. 2C), implying that suppression of FASN expression may reduce imatinib resistance. Accumulating evidence has revealed that the PI3K/AKT/mTOR pathway plays important roles in relaying RTK signaling in GISTs and neoplastic lipogenesis of other cancers (12, 17). Therefore, we explored the potential regulatory basis of these kinases linked to overexpression of FASN in GIST cell models, demonstrating that phosphorylated forms of PI3K, AKT, and mTOR were all prominently downregulated by FASN knockdown (Fig. 2D). However, the expression and phosphorylation of KIT protein did not exhibit significantly altered levels in FASN-knockdown GIST48 and GIST430 cells (Fig. 2D), nor did fold changes of KIT mRNA expression and promoter activity as measured by qRT-PCR and luciferase-constructed reporter assays, respectively (Supplementary Fig. S4).

A synthetic analogue of cerulenin, C75, has been shown to potently inhibit FASN and suppress tumor growth of various human cancers in vitro and in vivo (9). However, the therapeutic potential and molecular underpinning of C75 remains unexplored in GISTS, especially in regard to crosstalk with KIT-elicited signal transduction. As measured by XTT assay, C75 had a deleterious effect on the survival of both cell lines, and the IC₅₀ values, close to that reported in lung adenocarcinoma cells (18), were between 60 and 70 μmol/L (Fig. 3A, top). Another FASN inhibitor, orlistat, also dose dependently suppressed the cell viability of both GIST cell lines, although its potency was inferior to C75 (Supplementary Fig. S5). Accordingly, we selected C75 for further assessment of its potential efficacy in combination with imatinib. All four conditions of imatinib (100 and 200 nmol/L) and C75 (25 and 50 μmol/L) combination significantly improved the viability-suppressing capability in imatinib-resistant cell lines in a generally dose-dependent manner (Fig. 3A, bottom). This finding indicated an additive effect between C75 and imatinib and the need for validating the potential of a dual blockade of KIT and FASN in vivo (see below). To decipher the basis of C75 in growth inhibition of imatinib-resistant GIST cells, we examined whether C75 could induce cellular apoptosis, which was validated by significantly increased caspases 3 and 7 activity from 48 hours onward or by induced cleaved caspase-3 in GIST48 and GIST430 cells treated with C75 at both 50 and 100 μmol/L (Fig. 3B; Supplementary Fig. S6). In both GIST cell models, C75 at 50 μmol/L, similar to imatinib, also increased expression of the LC3-II isoform, indicating the role of C75 in triggering autophagy, and C75 at 100 μmol/L induced LC3-II more obviously than imatinib. Compared with monotherapy, GIST48 cells exhibited further increased levels of cleaved caspase-3 and LC3-II when treated with imatinib and 100 μmol/L C75. In contrast, GIST430 cells more readily showed additional increases of LC3-II when combing imatinib with C75 at either 50 or 100 μmol/L, albeit no apparent additive effect on induction of apoptosis. These findings implied an existent but variable additive effect of C75/imatinib combination on the induction of cellular autophagy and apoptosis in imatinib-resistant GIST cells (Supplementary Fig. S6).

Unlike the observed RNAi results, total and phosphorylated KIT levels apparently diminished in C75-treated imatinib-resistant GIST cells (Fig. 3C), which were in line with significantly decreased KIT mRNA levels (Supplementary Fig. S7) and promoter activity (Supplementary Fig. S8). Similarly, orlistat induced significant repression in the KIT promoter activity in both cell lines, along with resultant decreases at the levels of mRNA, protein expression, and phosphorylation (Supplementary Figs. S7–S9). These findings indicate that transcriptional repression, at least partly, implicates the KIT-repressing effect of pharmacologic inhibition by C75, which became more evident when combined with imatinib (Fig. 3D; Supplementary Fig. S10). Furthermore, treatment with C75 or imatinib individually or in combination was examined to determine the modulatory effect on KIT phosphorylation and PI3K/AKT/mTOR signaling activity. Consistent with FASN silencing, FASN-targeting C75 abrogated the activation of PI3K, AKT, mTOR and, conceivably, their downstream kinases RPS6 and 4E-BP1 (Fig. 3C), with relatively slight or no decreases in the expression of the total forms of individual kinases. Generally, similar to C75, orlistat treatment inhibited the expression and activation of KIT as well as on downstream kinases of the AKT/mTOR signaling cascade in both GIST48 and GIST 430 cells (Supplementary Figs. S9 and S10). Paradoxically, in orlistat-treated GIST48 cells, the level of phosphorylated AKT increased between 24 and 48 hours but abated to a barely detectable level at 72 hours.

Compared with monotherapies or vehicle control, the C75/imatinib combination more effectively inactivated the kinases of the AKT/mTOR signaling cascade, except for phosphorylated RPS6 and 4E-BP1 in GIST430 cells. Instead, the activation status of ERK was accentuated by C75 and even more clearly by the C75/imatinib combination in both cell lines (Fig. 3D). Given the known antiapoptotic role of AKT (17), we transfected cells with myristoylated-AKT to elucidate its effect on FASN inhibition–induced apoptosis in GIST430 cells, which had more obvious C75-inactivated AKT than the GIST48 cells (Fig. 3C). The GIST430 cells with myristoylated-AKT exhibited significantly reduced activity of caspases 3 and 7 triggered by C75 (Supplementary Fig. S11A), implying a potential link between AKT inactivation and the C75-induced cytotoxicity. Moreover, myristoylated-AKT transfection increased the phosphorylated mTOR, RPS6, and 4E-BP1 and partially rescued the activities of upstream KIT and PI3K reduced by C75 (Supplementary Fig. S11B).

Given the additivism between C75 and imatinib, a GIST430-derived xenograft model was established to analyze the in vivo therapeutic efficacy and antitumor mechanism of C75 and C75/imatinib combination therapy. The in vivo tumor-inhibiting effect of C75 was significant at both 50 and 100 μmol/L compared with the control group, although no dose-dependent difference was seen between the two doses tested on gross examination (Supplementary Fig. S12A). However, the group receiving 100 μmol/L C75 exhibited broader hyalinized stromal changes with fewer viable cells in the corresponding xenografts (Supplementary Fig. S12B). Next, we evaluated the therapeutic efficacy of the C75/imatinib combination, which from day 21 onward exhibited more prominent growth-suppressing capability than either C75 or imatinib alone (Fig. 4A), corroborating the additivism of the combined therapy in vitro. Notably, the gross fibrotic regression was only present in the xenograft specimens receiving combined therapy, and neither significant body weight loss nor impaired function of vital organs was observed in the treated mice. Microscopically, the PBS-treated xenografts displayed cellular proliferation of epithelioid to spindle cells without necrosis or fibrosis. Tumor cells of the control group exhibited diffuse and strong expression of p-cKIT, p-AKT, p-mTOR, pRPS6, and p-4E-BP1, high Ki-67 proliferative index, and few TUNEL-positive apoptotic cells. In contrast, the xenografts receiving combined therapy displayed remarkably lower cellularity and mitosis, prominent stromal fibrosis, and overt decreases in the levels of p-cKIT and phosphorylated kinases downstream of the AKT/mTOR pathway (all P < 0.001; Fig. 4B). However, these alterations in histomorphology, kinase expression and activity, and proliferative and apoptotic markers were mild to moderate in the monotherapy groups receiving imatinib or C75 alone.

FASN, an enzyme critical for endogenous lipogenesis, catalyzes the condensation of malonyl-CoA and acetyl-CoA to synthesize palmitate de novo and converts excess carbon intake into long-chain fatty acids for storage (8, 10). In various carcinomas and their preinvasive precursors, the expression level of FASN is selectively increased and frequently associated with shorter DFS (10). Specifically, for GISTs, only one prior study on FASN reported its preferential overexpression in high-risk and metastatic tumors and confers a proproliferative function by upregulating cyclin A (11). In this study, GIST tissues exhibited a positive correlation between FASN mRNA abundance and risk levels, being statistically more robust when defined by NCCN guidelines than by NIH criteria. Although this observation suggested possible implications of different stratification criteria in the correlation with FASN mRNA expression, it indicated that FASN overexpression in GISTs is, at least in part, transcriptionally regulated. In prostate and breast cancers, FASN mRNA is known to be transcriptionally upregulated by activated AKT, which enhances the binding of sterol regulatory element–binding protein (SREBP)-1c to the FASN promoter (10, 19). The strong association between FASN overexpression and adverse clinicopathologic variables, such as higher risk levels, was also validated in our large cohort of GISTs. Moreover, we confirmed that FASN overexpression not only correlated with unfavorable genotypes but also conferred an independent negative prognostic impact, with a risk of shorter DFS that was two times higher.

Despite being complex, the molecular underpinning of FASN oncogenicity has been linked to its versatile roles in evading apoptosis, maintaining lipid rafts, and activating central oncogenic pathways, such as the PIK3/AKT/mTOR signaling cascade (9, 10). The biological implications of overexpressed FASN in GISTs were supported by FASN-silenced cell lines that demonstrated decreased cell growth and migration. Although overexpressed FASN has emerged as a potential therapeutic target in various cancer types (9, 20), knowledge about the biology of FASN inhibition in GIST cell and xenograft models is scant. In RTK-targeted therapies, such as imatinib targeting of mutant KIT in GISTs, acquired resistance frequently follows a short-lived treatment response (1, 7). This predicament urges a fundamental elucidation of interdependent KIT-eliciting downstream and collateral pathways essential for sustaining resistance and survival of tumor cells to develop novel therapeutic strategies. In GIST cells harboring refractory mutations, FASN knockdown indeed inhibited the activity of the PI3K/AKT/mTOR axis and downstream RPS6 and 4E-BP1 kinases and notably increased the susceptibility to imatinib. These results highlighted the therapeutic relevance of overexpressed FASN in imatinib-resistant GISTs and the potential of drug combinations for dual blockade of FASN and KIT.

Consequently inducing cellular apoptosis, the chemical anticancer mechanisms of C75 involve abrogation of de novo fatty acid synthesis, toxicity of accumulated malonyl-CoA, and increased reactive oxygen species associated with dissipated mitochondrial membrane potential (21–23). Similar to previously reported tumor types, the apoptosis-inducing effect of pharmacologic FASN inhibition was reproduced in GISTs (9, 24, 25). In imatinib-resistant GIST cells and xenografts, we observed significant additive efficacy with C75/imatinib combination therapy. Exposure to C75 alone or the C75/imatinib combination led to abrogation of AKT activity concomitant with increased caspase activity in cell lines and an increased TUNEL-labeling index in xenografts. In this context, C75-induced apoptosis in GIST cells is potentially linked to AKT inactivation, given that FASN inhibition may perturb membranous lipid rafts needed for the proper subcellular localization of proteins involved in signal transduction, including the antiapoptotic PI3K/AKT/mTOR pathway (17, 20). Moreover, transfection of C75-treated GIST cells with myristoylated-AKT counteracted the activity of caspases 3 and 7 and reactivated not only downstream mTOR, RPS6, and 4E-BP1 but also upstream KIT and PI3K. However, it should be added that the adverse weight-reducing side effect of C75 compromises its clinical utility as a cancer therapeutic (10).

Imatinib, which binds directly to the KIT receptor, was shown to have no appreciable effect on the KIT promoter (26). In contrast, C75 repressed KIT transactivation in imatinib-resistant GIST cells and eventually caused a prominent loss of total KIT protein that further exhausted available receptors for sustaining phosphorylated activation. These findings resonated with the well-characterized example of FASN inhibitors in alleviating the resistance to HER2-targeted therapy in breast carcinomas, given that C75 can inhibit the HER2 gene promoter through upregulation of the PEA3 transcriptional repressor (27, 28). Unlike C75 and orlistat, FASN knockdown did not significantly repress KIT at all levels of transcription, expression, and activity in imatinib-resistant GIST cell models. This discrepancy may pose a query whether C75 exerted a KIT promoter–repressing effect through an FASN-independent mechanism or it indirectly impeded the KIT signaling output through inhibiting docking proteins that interact with activated KIT. Considering the association of FASN overexpression with unfavorable RTK genotypes in GIST samples and the improved sensitivity toward imatinib by FASN knockdown, an interdependent positive feedback loop appears conceivable, which coordinately regulates FASN, KIT-elicited signaling output, and the PI3K/AKT/mTOR signaling pathway. Relevantly, FASN is required to sustain the level of diacylglycerol in the lipid rafts involving the recruitment of KIT-interacting docking proteins, such as protein kinase C (PKC) and Grb2 (29, 30). More intriguingly, PKC-θ isoform has been shown to mediate sustained mutant KIT activation in the Golgi complex in GISTs (31). In this context, both generalized inhibition by C75 and the resultant attenuation of KIT signaling relay might contribute to the inactivation of PI3K/AKT/mTOR and downstream kinases, with or without decreased expression of their total proteins. Collectively, FASN inhibition resulting from combined therapy minimized KIT dependency and improved the sensitivity to imatinib in resistant GISTs in vitro and in vivo.

Accumulating evidence indicates that apoptosis and autophagy share common regulatory elements within oncogenic pathways activated by RTKs, including the PI3K/AKT/mTOR pathway (32, 33). In GIST cells, the endogenous turnover of KIT protein is known to be involved in the autophagic process, and the novel HSP90AA1 inhibitor, NVP-AUY92, has been revealed to enhance the magnitude of autophagy for promoting KIT degradation (34). To date, few studies have described the autophagy-modulating role of FASN inhibition in tumor cells. One such study reported that glioma cells treated with C75 or orlistat revealed increased autophagic activity (24). In imatinib-resistant GIST cells, increased expression of LC3-II, a widely accepted autophagic marker (31–34), was inducible by C75 (also orlistat), and its level could be even higher upon combined treatment with C75 and imatinib. Although AKT inactivation–induced apoptosis directs cells to programmed death (17, 33), the significance of FASN inhibition–induced autophagy in GISTs requires further elucidation. Specifically, the relevant issues regarding C75-induced autophagy include its potential dependency on AKT, engagement in KIT expression and activation, and resultant cytoprotection to maintain energy homeostasis versus cytotoxicity to promote cellular death under nutrient deprivation. A better understanding of these aspects will help decipher the possible benefits or detriments of autophagic inhibition in GIST cell growth in combined therapy against FASN and KIT.

In short, we have characterized the clinical relevance and biological implications of FASN, as well as the potential of dual blockade of FASN and KIT in GISTs. With FASN mRNA being preferentially upregulated in aggressive GISTs, overexpressed FASN confers growth-promoting and promigratory oncometabolic phenotypes that are associated with adverse clinicopathologic factors and unfavorable RTK genotypes, and independently portends decreased DFS. Biologically, an interdependent positive feedback loop may exist to coordinately regulate FASN, KIT-elicited signaling output, and the PI3K/AKT/mTOR pathway in GISTs. Through disrupting this loop, FASN-targeting C75 represses KIT transactivation to exhaust KIT protein, induces apoptosis and autophagy, and inactivates signaling kinases downstream of PI3K/AKT. Therefore, FASN inhibition improves sensitivity to imatinib in resistant cell models and underpins the in vitro and in vivo additivism of C75/imatinib combination therapy. On the basis of the suppressive effects that C75 demonstrated, our study provides a rationale for future investigation of novel FASN inhibitors with a better therapeutic index in combination with imatinib in GISTs that acquire refractory mutations.

No potential conflicts of interest were disclosed.

Conception and design: C.-F. Li, L.-T. Chen, H.-Y. Huang

Development of methodology: C.-F. Li, T.-C. Chan, S.-C. Yu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-T. Li, T.-C. Chan, H.-Y. Huang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F.-M. Fang, Y.-Y. Chen, L.-T. Chen

Writing, review, and/or revision of the manuscript: C.-F. Li, S.-C. Yu, H.-Y. Huang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.-F. Li, F.-M. Fang, Y.-Y. Chen, T.-C. Chan, S.-C. Yu, L.-T. Chen

Study supervision: L.-T. Chen and H.-Y. Huang supervised this work equally

Other (carried out the experiments): T.-T. Liu

The authors thank Dr. Jonathan Fletcher for providing GIST cell lines and Kaohsiung Chang Gung genomic (CLRPG8D0113) and tissue bank (CLRPG8B0033, CLRPG8E0161) core laboratories for technical assistance. The authors also thank Dr. Shiaw-Min Huang at Bioresource Collection and Research Center, Food Industry Research and Development Institute for the authentication of GIST48 and GIST430 cells.

H.-Y. Huang was funded by Taiwan National Ministry of Science and Technology (NSC99-2628-B-182A-064-MY3) and Chang Gung Hospital (CMRPG8C0983). C.-F. Li was funded by Taiwan National Ministry of Science and Technology (NSC99-2320-B-384-001-MY2 and MOST104-2314-B-384 -009-MY3), Ministry of Health and Welfare (MOHW103-TDU-M-221-123017 and MOHW104-TDU-M-212-133004), and Taiwan National Health Research Institutes (CA-106-PP-36 and CA-106-SP-03).

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