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Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Tumor Growth Through Inhibiting the mTOR Signaling Pathway

Ethyl ferulate is a phenylpropanoid compound isolated from the medicinal herb Ferula. Although ethyl ferulate has anti-inflammatory, antioxidant, and neuroprotective activities with potential use in the nutraceutical and pharmaceutical industry, its anticancer effects and underlying molecular mechanisms against esophageal squamous cell carcinoma (ESCC) have not been investigated. This study investigates the anticancer activity and molecular mechanism of ethyl ferulate in ESCC. MTT, focus formation, soft agar, and cell cycle analysis were used to determine the effect of ethyl ferulate on cell proliferation and cell cycle. Potential candidate proteins were screened and verified via Western blotting, in vitro kinase assay, and in vitro pull-down assay. Mammalian target of rapamycin (mTOR) knockdown cell lines were established by lentiviral infection with shmTOR. The effect of ethyl ferulate on tumor growth was assessed using ESCC patient-derived xenograft models. Ethyl ferulate significantly inhibited cell growth and induced G1 phase cell cycle arrest in ESCC cells. Ethyl ferulate reduced the activity of mTOR in vitro. The inhibition of ESCC cell growth by ethyl ferulate is dependent on mTOR expression. In addition, ethyl ferulate strongly reduced ESCC patient-derived xenograft tumor growth in an in vivo mouse model. Ethyl ferulate is an mTOR inhibitor that can suppress ESCC progression and may be a novel candidate compound for esophageal cancer chemoprevention.


INTRODUCTION
Esophageal cancer is one of the most common malignant digestive tract tumors worldwide, with a poor prognosis and high mortality (1). Esophageal squamous cell carcinoma (ESCC) accounts for approximately 90% of esophageal cancer cases (2). Treatment strategies for managing esophageal cancer have greatly improved; however, the 5-year survival rate of esophageal cancer patients is roughly 10%. Additionally, only 15%-40% of patients survive more than 5 years after post esophagectomy (3). Although epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors have been used to treat ESCC therapeutically, patients initially benefiting from targeted therapies inevitably develop drug resistance (4,5); therefore, it remains a worthwhile endeavor to investigate novel effective therapeutic targets and inhibitors to overcome these challenges.
The mammalian target of rapamycin (mTOR) is a serine/ threonine kinase that drives cell growth and proliferation by linking a variety of extracellular signals and intracellular cues (6). In multicellular organisms, mTOR regulates cell growth and metabolism in response to growth factors, cellular energy conditions, and nutrients (7). The functions of mTOR are exerted via two different multicomponent complexes, mTORC1 and mTORC2, which cooperate with one another and several elements of other signaling pathways (8). The mTORC1 complex regulates protein synthesis and cell growth through the downstream molecules Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa S6 kinase (p70S6K) (9). mTORC2 is responsive to growth factor signaling via phosphorylating downstream V-Akt murine thymoma viral oncogene homolog (AKT), Serum and glucocorticoid-regulated kinase (SGK); it also plays a vital role in the maintenance of normal and cancer cells by associating with ribosomes and participating in cellular metabolic regulation (10). AKT is a serine/threonine kinase and is a crucial Phosphoinositide 3kinase (PI3K) signaling pathway component (11). The p70S6K is a serine/threonine protein kinase that plays an important role in cell growth, cell cycle, and cell differentiation (12). The p70S6K protein is an mTOR pathway effector, and the activation of the mTOR/p70S6K signaling pathway can stimulate protein synthesis and cell growth (13). mTOR is activated in a variety of malignant tumors and is an important regulator of cell proliferation and protein translation (14). mTOR might be a potential target strategy for ESCC treatment in the future because it is highly expressed in many human ESCC tumor tissues and is associated with various clinical characteristics (15)(16)(17). The deregulation of the mTOR signaling pathway in many cancers suggests that mTOR inhibitors may have broad therapeutic use in various tumor types (18). mTOR inhibitors have been used as monotherapy or combination therapy in preclinical and clinical trials to test its efficacy in treating various cancers; however, the effects were marginal, suggesting that the full therapeutic potential of targeting mTOR has not been exploited (19,20). Thus, investigating more potent mTOR inhibitors for cancer treatment remains an ongoing challenge.
Ethyl ferulate, a monomeric phenylpropanoid compound isolated from the medicinal herb Ferula, has attracted extensive interest because of its anti-inflammatory activity, neuroprotective properties, and antioxidant activity (21,22). Ethyl ferulate was found to prevent lipopolysaccharide (LPS)-induced acute lung injury (ALI) through regulating inflammatory responses (22). Ethyl ferulate was observed to ameliorate diabetes-induced oxidative stress and inflammation via activating nuclear factor erythroid 2-related factor 2 (Nrf2) pathway (23). Ethyl ferulate induced the level of heme oxygenase-1 (HO-1) expression and protected oxidative and neurodegenerative condition in brain cells (24). Additionally, ethyl ferulate prevented age-related macular degeneration through regulating the morphological and functional retinal degeneration (25). However, the anticancer activity of ethyl ferulate and its underlying molecular mechanisms against ESCC remain unclear. Herein, we investigated the anticancer effects of ethyl ferulate on the malignant growth of ESCC in vivo and in vitro. MTT Assay KYSE30 (2.5 × 10 3 cells per well), KYSE70, KYSE450, or KYSE510 (2.0 × 10 3 cells per well) cells suspended in complete growth medium were seeded in 96-well plates (100 ml/well). After overnight incubation, different concentrations of ethyl ferulate or isometric DMSO were diluted in complete growth medium and added to each well (100 ml/well). After 72 h, 20 ml of the MTT solution (Solarbio, Beijing, China) were added to each well and the cells were incubated for 2 h at 37°C. Next, the cell culture medium was removed and 150 ml of DMSO were added to each well. The formazan crystals were dissolved by gentle agitation, and cell proliferation was measured at 570 nm absorbance using the Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA, USA).

Anchorage-Independent Cell Growth Assay
KYSE450 or KYSE510 cells (8 × 10 3 cells per well) were suspended in 0.3% agar medium (RPMI 1640 containing various concentrations of ethyl ferulate) and then plated on a 0.6% agar containing the same concentrations of ethyl ferulate in 6-well plates. Cells were maintained at 37°C in a humidified incubator under 5% CO 2 for 2 weeks. Colonies were then photographed with an inverted microscope and counted using the Image-Pro Plus software (v.6) program (Media Cybernetics, Rockville, MD, USA).

Focus Formation Assay
KYSE450 or KYSE510 cells (1 × 10 3 cells per well) were seeded into 6-well plates and incubated overnight before treatment with various concentrations of ethyl ferulate. Cells were maintained at 37°C in a humidified incubator under 5% CO 2 for 10 days. Foci were stained by 0.4% crystal violet staining solution, and the number of foci was counted.

Cell Cycle Analysis
KYSE30, KYSE450, or KYSE510 cells were seeded (9 × 10 4 cells per 60-mm dish) and incubated for 24 h. The cells were then treated with various concentrations of ethyl ferulate or DMSO for 48 h. The cells were then collected by trypsinization, washed with 1× PBS, and fixed in 70% ethanol at -20°C overnight. After rehydration, cells were incubated in RNase (10 mg/ml, Solarbio) for 1 h and then stained with propidium iodide (PI; 20 mg/ml, Solarbio). The cell cycle was analyzed by flow cytometry.

In Vitro Kinase Assay
The kinase assay was performed according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, the active recombinant mTOR (50 ng) protein was mixed with different concentrations of ethyl ferulate in reaction buffer and incubated at room temperature for 15 min. Next, the inactive p70S6K recombinant protein (100 ng) and ATP (Cell Signaling Technology) were added and incubated for 30 min at 30°C. The reaction was stopped by adding 10 ml protein loading buffer. The protein band was detected by Western blot analysis. mTOR activity was assessed using a p70S6K phosphorylation antibody.

In Vitro Pull-Down Assay
Recombinant mTOR (200 ng) protein or ESCC cell lysate from KYSE510 (1 mg) was incubated with ethyl ferulate-Sepharose 4B or Sepharose 4B beads (100 ml, 50% slurry) in reaction buffer. Incubated samples were constantly rotated at 4°C overnight. The following day, the beads were washed 5 times and the protein bands were detected by Western blot analysis.

Establishing mTOR Knockdown Cells
Small hairpin RNA plasmids against mTOR were constructed as previously described (26). Viral vectors were co-transfected with packaging vectors into Lentix-293T cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) to generate lentivirus. At 48 h after transfection, the virus-enriched medium was harvested and passed through a 0.45-mm filter. The virusenriched medium was then supplemented with 10 mg/ml of polybrene and added to KYSE450 and KYSE510 cells grown tõ 60% confluence. The cells were incubated overnight. The following day, the cell culture medium was replaced with fresh complete medium. Infected cells were then selected with puromycin (1 mg/ml) for 48 h.

In Vivo Patient-Derived Esophageal Squamous Cell Carcinoma Xenograft Model and Ethics Statement
The study was conducted according to guidelines established for the care and use of laboratory mice by the Zhengzhou University Institutional Animal Care and Use Committee (Zhengzhou, China). Severe combined immunodeficiency (SCID) female mice (6-9 weeks old) were used to examine the effect of ethyl ferulate on ESCC patient-derived tumor growth. Human ESCC tumor tissues were excised from Affiliated Cancer Hospital in Zhengzhou University patients who received neither chemotherapy nor radiotherapy treatments before surgery. Tissue histology was confirmed by a pathologist. Informed consent was given by each patient. After tumors grew to an average volume of approximately 200 mm 3 , the mice were randomly divided into 2 groups containing 20 animals of the LEG110 tissue and 14 animals of the LEG45 tissue as follows: 1) untreated vehicle group (n = 10 or 7, respectively) and 2) 100 mg ethyl ferulate/kg of body weight (n = 10 or 7, respectively). Ethyl ferulate or vehicle (20% DMSO in 20% Tween 80) was administered by oral gavage once per day Monday through Friday. Tumor volumes were measured by Vernier caliper and calculated according to the following formula: tumor volume (mm 3 ) = (length × width × height × 0.52). Mice were monitored until the tumor volume reached approximately 1.5 cm 3 . The mice were then euthanized, and their tumors, blood, livers, kidneys, and spleens were extracted.

Hematoxylin-Eosin Staining and Immunohistochemistry
The liver, spleen, kidney, and tumor tissues from mice were prepared for hematoxylin-eosin (H&E) staining or immunohistochemistry (IHC). For H&E staining, the tissue sections were deparaffinized, hydrated, stained with H&E, and then dehydrated. For IHC, tumor tissue sections were baked at 65°C and hydrated. Afterward, the tissue sections were boiled in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval and then treated with H 2 O 2 for 5 min. After incubation with a primary antibody (Ki-67, 1:100) at 4°C in a humidified chamber overnight, the sections were incubated with an HRPconjugated goat anti-rabbit or mouse IgG antibody (ZSGB-BIO, Beijing, China) for 30 min. Tissue sections were then stained with 3,3′-diaminobenzidine (DAB; ZSGB-BIO, Beijing, China). The IHC staining was observed by microscope and quantified using the Image-Pro Plus software program.

Statistical Analysis
All statistical results were presented as mean values ± SD. The Student's t test or one-way analysis of variance (ANOVA) was used to compare significant differences. Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) for Windows (IBM, Inc., Armonk, NY, USA). Statistical significance was considered at P value <0.05.

Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Cell Growth
Ethyl ferulate is a 4-hydroxy-3-methoxycinnamate compound ( Figure 1A). SHEE normal esophageal cells were used to assess the toxicity of ethyl ferulate. Cell viability was determined by MTT assay after cells were treated with several concentrations of ethyl ferulate for 72 h. Results showed that SHEE normal esophageal cell proliferation was not affected by ethyl ferulate treatment ( Figure 1B). Therefore, we used ethyl ferulate at concentrations of 20, 40, and 60 mM for the subsequent experiments. To assess the effect of ethyl ferulate on ESCC cell proliferation, we next treated KYSE30, KYSE70, KYSE450, or KYSE510 ESCC cells with ethyl ferulate at different concentrations for 72 h. The MTT assay results revealed that treatment with ethyl ferulate significantly inhibited the growth of ESCC cells in a dose-dependent manner ( Figure 1C). Furthermore, the focus formation and anchorage-independent ESCC cell growth assays showed that ethyl ferulate suppressed focus number and anchorage-independent growth of KYSE450 and KYSE510 ESCC cells in a dose-dependent manner ( Figures 1D, E).

Ethyl Ferulate Increases G1 Phase Cell Cycle Arrest in Esophageal Squamous Cell Carcinoma Cells
Flow cytometry analysis was performed to determine whether treatment with ethyl ferulate affected cell cycle regulation in ESCC cell lines. KYSE30, KYSE450, or KYSE510 ESCC cells were treated with ethyl ferulate at several concentrations for 48 h. The results indicated that ethyl ferulate induced G1 phase cell cycle arrest in a dose-dependent manner (Figures 2A, B). Based on the perturbation of the cell cycle induced by ethyl ferulate, we determined the expression of cell cycle marker proteins in KYSE30, KYSE450, and KYSE510 cells after ethyl ferulate treatment. KYSE30, KYSE450, or KYSE510 ESCC cells were treated with ethyl ferulate for 48 h, and cell cycle marker proteins were analyzed by Western blotting. The results of Western blotting indicated that ethyl ferulate increased the expression of p21 and decreased the expression of cyclin D1 ( Figure 2C).

Ethyl Ferulate Is a Novel mTOR Inhibitor
To determine the underlying molecular mechanism induced upon treatment with ethyl ferulate, we first investigated whether it could affect various EGF-induced signaling molecules in JB6 cells. After 24-h serum starvation, cells were treated with ethyl ferulate for 6 h and then subsequently treated with EGF for 0.5 h. The results indicated that the phosphorylation of mTOR, AKT, GSK3b, and p70S6K was strongly decreased by treatment with ethyl ferulate in a dosedependent manner, whereas the protein level of phosphorylated ERK1/2 was not affected ( Figure 3A). Therefore, to further identify the molecular targets of ethyl ferulate, we evaluated the effect of ethyl ferulate on signaling pathways in KYSE30, KYSE450, and KYSE510 cells after treatment with ethyl ferulate for 24 h. Phosphorylated mTOR, AKT, GSK3b, and p70S6K protein levels were strongly reduced after treatment with ethyl ferulate; however, the level of phosphorylated ERK1/2 was unaffected ( Figure 3B). To determine potential targets of ethyl ferulate, we performed in vitro pull-down assays using ethyl ferulate-conjugated Sepharose 4B beads, Sepharose 4B with a recombinant mTOR protein, or KYSE510 cell lysates. The results showed that ethyl ferulate directly binds with mTOR but not AKT or p70S6K proteins ( Figure 3C). Furthermore, we performed in vitro kinase assays using an active recombinant mTOR protein and an inactive p70S6K protein to confirm the effect of ethyl ferulate on mTOR activity. The result indicated that ethyl ferulate suppressed the phosphorylation of p70S6K by directly targeting mTOR in a dose-dependent manner ( Figure 3D). Additionally, to determine whether the activities of other signaling proteins were affected by ethyl ferulate, the activities of a panel of 36 recombinant active kinases and their respective substrates were screened in the presence or absence of ethyl ferulate. The results indicated that mTOR was the sole kinase that was strongly inhibited by ethyl ferulate (Supplementary Figures S1A, B).  Figures  S2A, B). We next examined the effect of mTOR knockdown on ESCC cell growth using an MTT assay. The results indicated that the anchorage-dependent cell growth of KYSE450 and KYSE510 ESCC cells was decreased upon knockdown of mTOR ( Figure 4A). Furthermore, the focus formation and soft agar assay results showed that focus number and anchorageindependent growth of ESCC cells were significantly reduced upon knockdown of mTOR ( Figures 4B, C).

The Inhibition of Esophageal Squamous Cell Carcinoma Cell Growth by Ethyl Ferulate Is Dependent on mTOR Signaling
We next assessed whether the effect of ethyl ferulate on ESCC cell proliferation is dependent on the expression of mTOR. shmTOR cells were treated with ethyl ferulate for 72 h, and then  anchorage-dependent growth was assessed by MTT assay. Results indicated that shmTOR #1 cells were resistant to the growth inhibitory effect of ethyl ferulate compared with shControl cells ( Figure 5A). Additionally, the focus formation assay and soft agar assay results showed that the inhibitory effect of ethyl ferulate in shmTOR cells is reduced relative to shControl growth ( Figures 5B, C).

Ethyl Ferulate Inhibits Esophageal Squamous Cell Carcinoma Tumor Growth In Vivo
We first assessed the toxicity profile of ethyl ferulate to determine the appropriate concentration for use in our in vivo studies. Mice were orally administered vehicle, ethyl ferulate at 50 mg/kg, or ethyl ferulate at 100 mg/kg once a day Monday through Friday for 2 weeks by the gavage method. Blood samples from mice were collected and used to analyze alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity. The results showed that ALT and AST activities were not significantly altered in mice treated with ethyl ferulate at 50 or 100 mg/kg compared with the vehicle-treated group (Supplementary Figures S3A, B; n = 4). Therefore, we used 100 mg/kg ethyl ferulate for the ESCC patient-derived xenograft (PDX) study. To evaluate the antitumor activity of ethyl ferulate in vivo, we used two PDX models: LEG110 and LEG45. LEG110 or LEG45 human ESCC tumor tissues were implanted into the back of the necks of SCID mice, and ethyl ferulate at 100 mg/kg or vehicle was administered by gavage once a day for 32 (LEG110 tissue; n = 10) or 53 (LEG45 tissue; n = 7) days. The results indicated that the treatment of mice with ethyl ferulate significantly suppressed the tumor volume relative to the vehicle-treated group (Figures 6A, B). Additionally, mice that were treated with ethyl ferulate exhibited no significant loss of body weight compared to the vehicletreated group (Supplementary Figures S4A, B). Next, we measured the AST and ALT activity from serum to determine the potential toxicity of ethyl ferulate on PDX mice. Results showed that the ALT and AST activities were not significantly altered in mice treated with ethyl ferulate compared with the vehicle-treated group (Supplementary Figure S4C). We next prepared the tumor tissues that were isolated from both PDX cases for IHC staining of Ki-67, a cell proliferation marker protein. Results indicated that the expression of Ki-67 was . Furthermore, the liver, kidney, and spleen tissues were stained with H&E to evaluate the potential toxicity of ethyl ferulate. The results of the H&E staining showed that there were no obvious morphological changes between treated and untreated groups (Supplementary Figures S5A, B; n = 5). Next, Western blotting was performed to investigate whether ethyl ferulate could inhibit protein expression of mTOR and its downstream signaling targets in PDX tumor tissues. The results showed that the phosphorylation of mTOR, AKT, and p70S6K was strongly inhibited in the ethyl ferulate-treated group ( Figures 6E, F). We could suggest that inhibition of mTOR activity by ethyl ferulate exerts multiple effects on tumor progression, cancer growth, and cell cycle by modulating the mTOR signaling pathway (Supplementary Figure S6).

DISCUSSION
Despite comprehensive molecular characterization of mTOR, available targeted therapies for ESCC are still lagging behind (27). Ethyl ferulate is a phenylpropanoid compound that has been reported to have potential uses in the nutraceutical and pharmaceutical industry (28). However, the potential therapeutic effects of ethyl ferulate and its molecular targets have not been investigated in cancer. In the present study, we report that ethyl ferulate suppresses ESCC growth by targeting mTOR in vitro and in vivo.
Ribosomal protein S6 kinase (P70S6K) is the downstream effector of mTORC1, and AKT is a key substrate of mTORC2 (29,30). mTORC1 is sensitive to nutrients, and mTORC2 is regulated through PI3K and growth factor signaling. Both complexes influence each other, as AKT regulates PRAS40 (C) Effect on anchorage-independent growth of ESCC cells by mTOR knockdown (shmTOR). After incubation for 2 weeks, colony number was counted. For all, data are shown as means ± SD of values from 3 independent experiments, and the asterisk (*) indicates a significant (P < 0.05) inhibitory effect of mTOR knockdown (shmTOR). One-way analysis of variance (ANOVA) with Dunnett's post-hoc test was used to compare significant differences.
phosphorylation to facilitate mTORC1 activity, while p70S6K regulates Sin1 to modulate mTORC2 activity (31). Through the results of target screening, in vitro pull-down assays, and in vitro kinase assays, we identified that ethyl ferulate is a potent mTORC1/mTORC2 protein kinase inhibitor that can reduce mTOR/AKT/p70S6K signaling in ESCC cells ( Figures 3A, B). Ethyl ferulate directly bound mTOR protein ( Figure 3C) and suppressed the activity of mTOR protein kinase ( Figure 3D). Additionally, shmTOR cells were resistant to anticancer effects of ethyl ferulate (Figures 5A-C). However, high concentrations of ethyl ferulate still reduced cell growth in mTOR knockdown cells ( Figures 5A-C). Therefore, ethyl ferulate may have additional molecular targets. To determine other molecular targets of ethyl ferulate, we conducted a cancer-related kinase screening. However, we did not identify other targets within the kinase panel (Supplementary Figures S1A, B). Therefore, we will investigate additional potential target proteins of ethyl ferulate in the future.
As reported, the disappointing performance of investigational anticancer candidates implies that there are some shortcomings in the translation of preclinical in vitro and in vivo models to humans and that heterogeneity in the patient population presents a significant challenge (32). However, PDX models have become the most credible in vivo human cancer model, as the primary patient tumor characteristics, including gene expression profiles and drug responses, are retained (33). Therefore, the PDX model is useful for drug screening, biomarker development, and the preclinical evaluation of personalized medicine (34). In this study, we first reported the anticancer activity of ethyl ferulate using ESCC PDX The effect of ethyl ferulate on ESCC cancer cell growth and focus formation ability was assessed in cells stably expressing shmTOR or cells stably expressing shControl. Cells were treated with or without ethyl ferulate for 72 h or 10 days, respectively. Cell growth was analyzed by MTT and focus formation assays. (C) The effect of ethyl ferulate on anchorage-independent ESCC cell growth was assessed in cells stably expressing shmTOR or cells stably expressing shControl. Cells were treated with or without ethyl ferulate for 2 weeks, and then colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. All data are shown as means ± SD of values from 3 independent experiments. The asterisk (*) indicates a significant effect (P < 0.05) of ethyl ferulate treatment between mTOR knockdown cells and shControl cells. The Student's t test with one-way ANOVA was used to compare significant differences. models. The results indicated that ethyl ferulate treatment reduced patient-derived esophageal xenograft tumor growth by inhibiting the mTOR/AKT/p70S6K signaling pathway ( Figures 6A, B, E, F). Therefore, targeting mTOR protein with ethyl ferulate may provide antineoplastic effects against esophageal cancer.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. Three slices per PDX tissue were analyzed. Treated or untreated groups of tumor tissues were stained with antibodies to detect Ki-67 (×100; scale bar: 100 mm; ×20; scale bar: 50 mm). (E, F) Ethyl ferulate inhibits phosphorylated mammalian target of rapamycin (mTOR), p70S6K, and AKT protein expression in esophageal tumor tissues. Tumor tissues from each group were immunoblotted with antibodies to detect mTOR, p70S6K, and AKT and phosphorylated mTOR, p70S6K, AKT, and b-actin. b-Actin was used to verify equal protein loading. Band density was measured using the ImageJ (NIH) software program, and the results are shown as a bar graph. One-way ANOVA was used to compare significant differences. The asterisk (*) indicates a significant (*P < 0.05; **P < 0.01) inhibitory effect of ethyl ferulate treatment.

ACKNOWLEDGMENTS
We greatly appreciate the help of Mrs. Ran Yang for preparing immunohistochemistry slides.

SUPPLEMENTARY MATERIAL
The