FGFC1 Exhibits Anti-Cancer Activity via Inhibiting NF-κB Signaling Pathway in EGFR-Mutant NSCLC Cells

FGFC1, an active compound isolated from the culture of marine fungi Stachybotrys longispora FG216, elicits fibrinolytic, anti-oxidative, and anti-inflammatory activity. We have previously reported that FGFC1 inhibited the proliferation, migration, and invasion of the non-small cell lung cancer (NSCLC) cells in vitro. However, the precise mechanisms of FGFC1 on NSCLC and its anti-cancer activity in vivo remains unclear. Hence, this study was focused to investigate the effects and regulatory mechanisms of FGFC1 on two NSCLC cell lines, EGFR-mutant PC9 (ex19del) and EGFR wild-type H1299. Results suggested that FGFC1 significantly inhibited proliferation, colony formation, as well as triggered G0/G1 arrest and apoptosis of PC9 cells in a dose- and time-dependent manner, but no obvious inhibitory effects were observed in H1299 cells. Subsequently, transcriptome analysis revealed that FGFC1 significantly down-regulated 28 genes related to the NF-κB pathway, including IL-6, TNF-α, and ICAM-1 in the PC9 cells. We further confirmed that FGFC1 decreased the expression of protein p-IKKα/β, p-p65, p-IκB, IL-6, and TNF-α. Moreover, NF-κB inhibitor PDTC could strengthen the effects of FGFC1 on the expression of CDK4, Cyclin D1, cleaved-PARP-1, and cleaved-caspase-3 proteins, suggesting that the NF-κB pathway plays a major role in FGFC1-induced cell cycle arrest and apoptosis. Correspondingly, the nuclear translocation of p-p65 was also suppressed by FGFC1 in PC9 cells. Finally, the intraperitoneal injection of FGFC1 remarkably inhibited PC9 xenograft growth and decreased the expression of Ki-67, p-p65, IL-6, and TNF-α in tumors. Our results indicated that FGFC1 exerted anti-cancer activity in PC9 cells via inhibiting the NF-κB signaling pathway, providing a possibility for FGFC1 to be used as a lead compound for the treatment of NSCLC in the future.


Introduction
Lung carcinoma is one of the most prevalent types of cancer and exhibits the highest mortality rate among all cancer types globally [1]. Non-small-cell lung cancer (NSCLC) accounts for over 80% of all lung cancers, with the characteristics of a low cure rate and high mortality [2]. Epidermal growth factor receptor (EGFR) gene mutation is an important driving factor in non-small cell lung cancer (NSCLC) [3]. Common sensitizing mutations were detected in 79.8% of patients: 57.1% had exon 19 deletions [4]. In recent years, selective targeted EGFR tyrosine kinase inhibitors (TKIs) have been widely developed and used for the treatment of NSCLC in those who have an EGFR mutation [5]. However, most of them are still far from being used clinically owing to their poor selectivity or potential toxicity [6]. The post-operative recurrence rate of NSCLC is still very high, and the overall survival rate of NSCLC patients remains low [7]. The effectiveness of the available treatment options remains limited. Therefore, it is of great importance to develop new chemotherapeutic agents and treatment strategies. In recent years, investigators have focused on searching for new bioactive compounds from natural products and exploring their anti-cancer effect and underlying mechanism, which would undoubtedly provide valuable information on NSCLC treatment [8].
The cell cycle is a complex and strictly controlled process, consisting of different phases, which plays a key role in cell growth and proliferation [9]. The cell cycle consists of the G0/G1 phases, S phases, and G2/M phases [10]. The G0/G1 phase is characterized by the synthesis of mRNA and protein required for DNA replication [11]. Cell cycle disturbance is one of the important markers of cancer. Numerous proteins play a critical role in cell cycle progression from recent studies, such as cyclins and cyclin-dependent kinase (CDKs) complex [12]. There was increasing laboratory evidence showing that many anti-cancer drugs can inhibit tumor growth by inducing cell cycle arrest [13]. Therefore, arresting the tumor cell cycle can suppress cell proliferation and induce cell apoptosis [14]. Cell apoptosis is a kind of programmed cell death, which is related to tumor formation and development. Many kinds of mechanisms such as gene regulation and signal transduction were involved. Inducing cell apoptosis is an effective way of cancer treatment [15]. Caspase-3 is known as an important mediator of programmed cell death and cell apoptosis [16]. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly ADP-ribose polymerase (PARP), leading to cell apoptosis [17,18]. Therefore, inducing cell apoptosis is the therapeutic mechanism for many anti-cancer drugs.
Nuclear factor-κB (NF-κB) is a family of critical transcription factors of the inflammatory pathway and plays a significant role in the progression of various cancers, such as lung, breast, liver, pancreatic, multiple types of lymphoma, etc [19,20]. NF-κB normally exists in the cytoplasm in an inactive form as it is bound to IκB. NF-κB activation is regulated by the IκB kinase (IKK) complex, which phosphorylates IκB, resulting in subsequent ubiquitination and proteasome-dependent degradation [21]. Next, released NF-κB translocates to the nucleus and facilitates the transcription of its target genes, e.g., interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and intercellular cell adhesion molecule-1 (ICAM-1) [22,23]. NF-κB is a crucial factor that controls the expression of pro-inflammatory cytokines, prosurvival, anti-apoptotic, and cell cycle regulators [24]. In consideration of the significant role of NF-κB signaling in carcinogenesis and tumor progression, targeting NF-κB has gradually become a hotspot of cancer therapy research [25]. Tingting Long et al. reported that Polygonatum sibiricum polysaccharides play an anti-cancer effect through NF-κB signaling pathways in lung cancer [22]. Zengyang Pei et al. also found that low doses of selenium may protect the prostate from prostatitis-induced cancer by inhibiting nuclear translocation of the NF-κB [26]. Given the key role of NF-κB in the development of cancers, it has become an excellent target for cancer therapy.
In the past few decades, natural products have been considered as a potential and valuable source of anti-cancer drug candidates because of their multi-targeting potential and overcoming the disadvantages of monotherapy, such as side effects [27] and drug resistance. Fungi fibrinolytic compound 1 (FGFC1, 2,5-bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7dihydroxy-8-methyl-3-keto-1,2,7,8-tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid, the chemical structure is shown in Figure 1A), is a rare compound with a molecular weight of 869 Da isolated from the culture of marine fungi Stachybotrys longispora FG216 (CCTCCM 2012272) [28]. FGFC1 has been proven to exhibit various biological activities such as thrombolytic, anti-inflammatory, and antioxidant activities [29]. Wu and others discovered that FGFC1 could enhance fibrinolysis, with no risk of bleeding through degrading fibrin protein in vitro and in vivo [30,31]. Keita Shibata et al. reported that FGFC1 effectively inhibited cerebrovascular inflammation by suppressing inflammatory cytokine mRNA expression [32]. Shike Yan et al. found that FGFC1 exerted an inhibitory effect on NSCLC PC9 cells in vitro [33]. However, the underlying mechanisms of FGFC1 on NSCLC and the anti-cancer effect in vivo deserve further investigation.
In this study, we evaluated the effects of FGFC1 on NSCLC cells both in vitro and in vivo and determined the inhibitory property of FGFC1 on the NF-κB pathway in NSCLC cells for the first time. Our results showed that FGFC1 possessed a significant anti-proliferative effect on EGFR-mutant PC9 cells via inducing cell cycle arrest and cell apoptosis through inhibition of the NF-κB signaling pathway. These results provide available information for understanding the anti-cancer effects and regulatory mechanism of FGFC1 on NSCLC, which will provide a sufficient base for FGFC1 to be promoted as a lead compound in NSCLC therapy.

FGFC1 Significantly Inhibited PC9 Cells Viability In Vitro
To investigate the anti-cancer effects of FGFC1 on the NSCLC and the non-cancerous  In this study, we evaluated the effects of FGFC1 on NSCLC cells both in vitro and in vivo and determined the inhibitory property of FGFC1 on the NF-κB pathway in NSCLC cells for the first time. Our results showed that FGFC1 possessed a significant anti-proliferative effect on EGFR-mutant PC9 cells via inducing cell cycle arrest and cell apoptosis through inhibition of the NF-κB signaling pathway. These results provide available information for understanding the anti-cancer effects and regulatory mechanism of FGFC1 on NSCLC, which will provide a sufficient base for FGFC1 to be promoted as a lead compound in NSCLC therapy.

FGFC1 Significantly Inhibited PC9 Cells Viability In Vitro
To investigate the anti-cancer effects of FGFC1 on the NSCLC and the non-cancerous cells, dose-and time-dependent changes in PC9, H1299, and normal renal epithelial 293T cells viability were determined using the CCK8 assay after incubation periods of 24, 48, and 72 h. PC9 cells harbor EGFR mutation with exon 19 deletion, whereas H1299 cells carry wild-type EGFR. Here, H1299 cells were used as a control cell line to study the selective effect of FGFC1 on EGFR-mutant NSCLC [34,35]. Strikingly, treatment with FGFC1 caused a significant anti-proliferative effect on PC9 cells as indicated by dose-and timedependent changes in cell viability, whereas a slight inhibitory effect only was observed in EGFR wild-type H1299 cells and 293T cells ( Figure 1B). Figure 1C summarized the IC 50 values of FGFC1 in these three cell lines. In PC9 cells, which were the most sensitive to FGFC1, the IC 50 of FGFC1 after 24, 48, and 72 h of exposure were 41.73 ± 5.85, 22.29 ± 2.43, and 8.97 ± 0.94 µM, respectively. In H1299 and 293T cells, the IC 50 was >100 µM after 72 h of exposure, suggesting that these cells were relatively less sensitive to FGFC1. As shown in Figure 1D, the observation under optical microscope confirmed that after a low concentration FGFC1 treatment, the pseudopodia of PC9 cells extended, part of cellular morphology turned into a spindle shape, resulting in significant changes in cell morphology and dose-dependent decrease in cell number. However, barely morphological changes were observed in H1299 cells and 293T cells, indicating that FGFC1 indeed exerted certain selectivity to PC9 cells with EGFR mutations. In addition, we performed a colony assay which is one of the indicators to evaluate the inhibitory effect of drugs on tumor cell proliferation. Compared with H1299 cells and 293T cells, FGFC1-treated PC9 cells reduced the number and size of the colonies in a dose-dependent manner. As shown in Figure 1F, after FGFC1 treatment, the number of cell colonies decreased from 1580 to 231, showing significant reductions in colony formation, further confirming the cell growth inhibition effect of FGFC1 on EGFR-mutant PC9 cells ( Figure 1E,F).

FGFC1 Induced G0/G1-Phase Arrest and Apoptosis in PC9 Cells
To further determine whether or not the cytotoxic effect on NSCLC cells of FGFC1 is mediated by cell cycle arrest and/or apoptosis, PC9 and H1299 cells were treated with different doses of FGFC1 for 48 h. Subsequently, PI staining was used to analyze the distribution of cells at different stages of cell cycle by flow cytometry. The result demonstrated that following 0, 5, 10, and 20 µM FGFC1 treatment, the percentage of cells in the G0/G1 phase increased from 44.83% to 50.79%, 62.38, and 76.45% in PC9 cells, respectively. Nevertheless, limited effects of FGFC1 on the G0/G1 phase were observed in EGFR wildtype H1299 cells (Figure 2A,B). Given the effect of FGFC1 on the cell cycle, we further examined the expression of cell cycle-related proteins via Western blotting analysis. As expected, PC9 cells showed a dose-dependent decrease in CDK4 (0.95-fold, 0.56-fold, and 0.35-fold, respectively), and Cyclin D1 (0.61-fold, 0.50-fold, and 0.36-fold, respectively), after treatment with FGFC1 for 24 h compared with the DMSO-treated cells, but not in H1299 cells ( Figure 2C,D). To further confirm the effects of FGFC1 on apoptosis in NSCLC cells, PC9 and H1299 cells were treated with different concentrations of FGFC1, and cell apoptosis was analyzed by flow cytometry. As presented in Figure 2E,F, a significant increase was observed in the percentage of PC9 apoptotic cells (5.42%, 17.48%, and 28.07% Mar. Drugs 2022, 20, 76 6 of 23 following 48 h treatment with FGFC1, respectively). Concomitantly, this was further evidenced by the increased level of cleaved-caspase-3/pro-caspase-3 (1.96-fold, 3.22-fold, and 5.65-fold, respectively), and cleaved-PARP-1/PARP-1 (1.76-fold, 3.30-fold, and 5.80-fold, respectively), in FGFC1-treated PC9 cells ( Figure 2G,H). In contrast, limited effects were observed in EGFR wild-type H1299 cells, which confirmed the selective inhibitory effects of FGFC1 on PC9 cells. These results suggested that FGFC1 exerted in vitro anti-proliferative effects on EGFR-mutant PC9 NSCLC cells through inducing G0/G1 cell cycle arrest and cell apoptosis.  cleaved-PARP-1/PARP-1 and cleaved-caspase-3/pro-caspase-3 ratios in Western blotting. All data were represented as the means ± SD for at least three independent experiments. (* p < 0.05 and ** p < 0.01 vs the DMSO control).

FGFC1 Affected NF-κB Signaling Activation in PC9 Cells
FGFC1 was proven to exert anti-proliferative effects on NSCLC PC9 cells. To further explore its anti-cancer mechanism in NSCLC, a high-throughput RNA sequencing (RNAseq) was employed for the analysis of gene expressions in PC9 cells after DMSO and 10 μM FGFC1 treatment, respectively. The genes with p-value ≤ 0.05 and log2FC fold change cleaved-PARP-1/PARP-1 and cleaved-caspase-3/pro-caspase-3 ratios in Western blotting. All data were represented as the means ± SD for at least three independent experiments. (* p < 0.05 and ** p < 0.01 vs. the DMSO control).

FGFC1 Inhibited NF-κB p-p65 Nuclear Translocation
Nuclear translocation of activated transcription factor NF-κB commonly occurs in cancer and the inhibition of NF-κB has been proven to be an efficient therapeutic pathway for various cancers [37]. NF-κB activity is triggered with EGFR signaling in EGFR-driven NSCLC progression and NF-κB must translocate to the nucleus to regulate the target genes [38,39]. To investigate the effect of FGFC1 on p-p65 translocation in EGFR-mutant PC9 cells, we assessed the nuclear translocation of p-p65 using the immunofluorescence assay. The mean fluorescence intensity was 71 ± 4.26 and 52 ± 6.23, respectively, in different FGFC1-treated groups ( Figure 4A,B).

FGFC1 Inhibited Tumor Growth In Vivo
Finally, we examined whether or not FGFC1 could prevent PC9 tumor xenograft progression in BALB/c athymic nude mice. The data showed that intraperitoneal injection of FGFC1 at 10 mg/kg efficiently suppressed tumor growth compared with vehicle treatment ( Figure 5). The mean tumor volume increased from 98 ± 1.4 to 1166 ± 116.29 mm 3 between days 1 and 21 in the vehicle control group, whereas the mean tumor volume increased from 98.5 ± 1.87 to 411.9 ± 36.33 mm 3 in the FGFC1 group ( Figure 5A). However, the body weight of mice did not decrease significantly ( Figure 5B), suggesting that the toxicity of these therapies was tolerable. The average tumor weight in the FGFC1-treated group was lower than that in the control group ( Figure 5C,D). In addition, the immunohistochemical analysis indicated that FGFC1 treatment remarkably reduced the expression of Ki-67

FGFC1 Inhibited Tumor Growth In Vivo
Finally, we examined whether or not FGFC1 could prevent PC9 tumor xenograft progression in BALB/c athymic nude mice. The data showed that intraperitoneal injection of FGFC1 at 10 mg/kg efficiently suppressed tumor growth compared with vehicle treatment ( Figure 5). The mean tumor volume increased from 98 ± 1.4 to 1166 ± 116.29 mm 3 between days 1 and 21 in the vehicle control group, whereas the mean tumor volume increased from 98.5 ± 1.87 to 411.9 ± 36.33 mm 3 in the FGFC1 group ( Figure 5A). However, the body weight of mice did not decrease significantly ( Figure 5B), suggesting that the toxicity of these therapies was tolerable. The average tumor weight in the FGFC1-treated group was lower than that in the control group ( Figure 5C,D). In addition, the immunohistochemical analysis indicated that FGFC1 treatment remarkably reduced the expression of Ki-67 (0.56-fold), a marker for tumor cell proliferation [40], and significantly reduced the expression of p-p65, IL-6, and TNF-α (0.49-fold, 0.47-fold, and 0.50-fold, respectively), compared with the control group in tumor cells, which were consistent with the in vitro studies ( Figure 5E,F). In conclusion, the results demonstrated that FGFC1 treatment significantly suppresses the growth of PC9 cells via regulating NF-κB signaling pathways, without a significant effect on the total body weight of the mice.
(0.56-fold), a marker for tumor cell proliferation [40], and significantly reduced the expression of p-p65, IL-6, and TNF-α (0.49-fold, 0.47-fold, and 0.50-fold, respectively), compared with the control group in tumor cells, which were consistent with the in vitro studies (Figure 5E,F). In conclusion, the results demonstrated that FGFC1 treatment significantly suppresses the growth of PC9 cells via regulating NF-κB signaling pathways, without a significant effect on the total body weight of the mice. (F) The IHC score of Ki67, p-p65, IL-6, and TNF-α was quantified by the IRS system (n = 10 fields of view). All data were represented as the means ± SD for at least three independent experiments. and ** p < 0.01 vs. the DMSO control).

Discussion
As a marine-derived compound, FGFC1 consists of several bioactivities, including thrombolytic, anti-inflammatory, anti-oxidant, and anti-cancer activities [31][32][33]. The present study further investigated the anti-cancer effect of FGFC1 on NSCLC and its inhibitory effect on the NF-κB signaling pathway. The results showed that the semi-inhibitory concentration and colony formation abilities of the EGFR-mutant PC9 cells treated with FGFC1 were much lower than that of H1299. As known, cell cycle arrest and apoptosis are two effective mechanisms in the induction of cell death [41,42]. The cell cycle distribution diagram showed that FGFC1 arrested EGFR-mutated PC9 cells at the G0/G1 phase. Nevertheless, no remarkable effect was acquired in the EGFR wild-type H1299 cells. These results suggested that cell cycle arrest was one of the mechanisms in the growth inhibitory effect of FGFC1 against NSCLC. Cell cycle progression and division are regulated by checkpoint controls and sequential activation of cyclin-dependent kinases (CDKs), which are major targets for down-regulation in cancer [43]. To further explore the molecular mechanisms of the G0/G1 phase arrest induced by FGFC1, we examined the expression levels of cell cycle checkpoint proteins, Cyclin D1 and CDK4, which are essential for cell progression from G1 to the S phase for the start or G1/S checkpoint [44,45]. Our results showed that compared with H1299 cells, FGFC1 treatment significantly down-regulated the protein levels of CDK4 and Cyclin D1 in PC9 cells. In addition to cell cycle arrest, Annexin V and PI staining revealed that FGFC1 also induced cell apoptosis of PC9 cells. Consistent with flow cytometry results, FGFC1 treatment increased the protein levels of apoptosisassociated proteins cleaved-PARP-1 and cleaved-caspase-3 in PC9 cells. However, the same concentration of FGFC1 treatment could not induce cell cycle arrest and apoptosis in EGFR wild-type H1299 cells. As known, PC9 cells are defective in the EGFR gene with the exon19 deletion, but there is a wild-type EGFR in H1299 cells. These results indicated that FGFC1-induced cell death might be dependent on the EGFR status of NSCLC. However, the detailed mechanism needs to be further explored in the future.
NF-κB plays a well-known role in regulating immune response and inflammation, and growing evidence supports a major role in tumorigenesis [46]. Abnormal or constitutive activation of NF-κB p65 has been detected in many human malignancies [47]. Mingyue Lun et al. reported that the proteasome inhibitor Velcade is the first anti-cancer drug targeting NF-κB activation on the market. Velcade could inhibit the proliferation of breast cancer cells by suppressing the NF-κB pathway [48]. Nifedipine, known as a calcium channel blocker, had also been proved to inhibit the NF-κB pathway in lung cancer cells, as to explain the immunosuppressive effect of lung cancer [49]. Thus, NF-κB p65 is considered to be an interesting therapeutic target for cancer treatment [27]. To date, FGFC1-treatment has significantly decreased the expressions of activated NF-κB in transient middle cerebral artery occlusion (tMCAO) mice [50]. In addition, we have found that FGFC1 significantly down-regulated the expression of EGFR protein in PC9 cells [33]. However, the effect of FGFC1 on the NF-κB pathway in cancer cells is not clear. In the present study, RNAseq results showed that FGFC1 down-regulated the expression of NF-κB target genes, including IL-6, TNF-α, and ICAM-1 in PC9 cells and qRT-PCR confirmed that FGFC1 indeed reduces the transcription levels of IL-6, TNF-α, and ICAM-1. Moreover, Western blot results revealed that FGFC1 can significantly decrease the phosphorylation of IKKα, IKKβ, p65, and IκBα and thus suppressed protein levels of NF-κB targets, IL-6 and TNF-α. Correspondingly, FGFC1 also inhibited the nuclear translocation of NF-κB p65 in PC9 cells. As expected, the activities of the NF-κB signaling pathway and the nuclear translocation of NF-κB p65 had not been affected by FGFC1 in EGFR wild-type H1299 cells. Furthermore, the PDTC and FGFC1 combined treatment group was found to have aggravated cell cycle arrest and apoptosis by enhancing the inhibitory effects of FGFC1 on the NF-κB pathway, finally inhibiting the colony formation of PC9 cells. The above results clarified that FGFC1 inhibited cell viability and induced cell arrest and apoptosis of EGFR-mutant NSCLC PC9 cells via negative regulation of the NF-κB signaling pathway. Additionally, as a crucial regulator of survival, cell cycle arrest, and apoptosis of cancer cells, the NF-κB signaling pathway has not only been linked to the oncogenic potential of the EGFR but is also involved in the acquisition of EGFR-TKIs resistance [39,51]. EGFR mutations are detected in approximately 30-65% of NSCLC patients in Asia [52]. Many studies illustrated that the EGFR pathway was crossed with the NF-κB-dependent pathway [53]. Mutations of EGFR triggers NF-κB activation via the proteasome-mediated degradation of IκBα. Meanwhile, transcriptional induction of NF-κB target genes exerted a positive feedback effect on EGFR signaling [54]. Activation of NF-κB and EGFR in a constitutive manner could be observed in multiple solid tumors and combined to provide oncogenic signals to cancer cells [53]. Recently, activation of NF-κB signaling has been proven to be vital for promoting resistance to EGFR-TKIs in cancers [55]. Masashi Fukuoka et al. revealed that the activation of NF-κB immediately after EGFR-TKIs treatment in PC9 cells acquired EGFR-TKI resistance [56]. Bivona et al. found that knocking down several components of the NF-κB pathway specifically enhanced EGFR-TKIs-induced cell death [57]. Galvani E et al. showed that a new type of irreversible EGFR-TKI CNX-2006 inhibited the cell proliferation via the NF-κB pathway, and was active against EGFR-mutated NSCLC models, both in vitro and in vivo [58]. In the subsequent study, whether FGFC1 can reverse the resistance-related mechanism of EGFR-TKI through the NF-κB signaling pathway, deserves further investigation. Our study would provide a sound rationale for future investigation in the clinical setting.
In vivo studies showed that FGFC1 was effective in reducing the tumor volume, size, and weight in a PC9 cell-xenograft mouse model. Compared with the control group, FGFC1 significantly inhibited tumor growth. However, mice body weight did not change following FGFC1 treatment. Additionally, immunohistochemical results showed that FGFC1 decreased the expression of Ki67, an indicator of cell proliferation in tumors [41], suggesting that FGFC1 effectively inhibited PC9 cells proliferation in vivo. To confirm whether FGFC1 exerts anti-cancer effects via the NF-κB signaling pathway in vivo, we analyzed the expression of p-p65, IL6, and TNF-α in PC9 xenograft tumors. Consistent with the in vitro results, the expression levels of these NF-κB pathway-related proteins expressions were also significantly decreased by FGFC1 ( Figure 6).

Cell Lines and Cell Culture
The PC9 cell line (ATCC, CRL-32727, Manassas, VA, USA) and the H1299 cell line (ATCC, CRL-5803, Manassas, VA, USA) were from the American Type Culture Collection. PC9 cells contain EGFR exon 19 deletion mutation (E746-A750) [59], whereas H1299 cells possess the wild-type EGFR gene that can be used as control cells. These 2 cell lines were cultured in RPMI-1640. 293T normal renal epithelial cell lines were purchased from the Institute of American Type Culture Collection (ATCC, CRL-3216, Manassas, VA, USA) and cultured in DMEM. All culture media were supplemented with 10% FBS with 100 U/mL of penicillin and 100 µg/mL of streptomycin, and cells were cultured at 37 • C in 5% CO 2 . The cell lines were routinely tested to confirm that they were free of Mycoplasma.

Cell Proliferation Assay
PC9, H1299, and 293T cells were seeded into 96-well plates with 3 × 10 3 cells/well in a culture medium and allowed to adhere overnight. The cells were then treated with various concentrations of FGFC1 (0, 0.5, 5, 10, 20, and 40 µM) for 24 h, 48 h, and 72 h, respectively, and 0.1% DMSO served as vehicle control. Each dosage was repeated in triplicate, and 3 independent experiments were performed. After treatment, 10 µL of CCK8 reagent was added to each well and incubated at 37 • C for 2 h. The medium was removed, followed by the DMSO dissolution. Finally, the absorbance of each well was measured at 450 nm by a Microplate Reader (BIO-TEK, Inc., Winooski, VT, USA). The concentration of FGFC1 required to inhibit cell proliferation by 50% (IC 50 ) was calculated using GraphPad Prism 8.0 (San Diego, CA, USA) software for semi-log curve fitting with regression analysis.

Colony Formation Assay
PC9, H1299, and 293T cells were seeded at approximately 400 cells per well in 6-well plates and were cultured with 4 mL of medium for 24 h. Then, the cells were treated with different concentrations (0, 5, 10, and 20 µM) of FGFC1 for 10 days, and 0.1% DMSO served as vehicle control. Next, cells were washed with PBS 2 times, fixed with 5 mL 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 15 min at room temperature. Finally, cell colonies were photographed under a microscope and the number of colonies in each well was counted for analysis.

Cell Cycle Analysis
The NSCLC cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and were treated with FGFC1 (0, 5, 10, and 20 µM) for 48 h; DMSO served as vehicle control. Subsequently, cells were collected, washed with PBS, and fixed with 70% ice-cold ethanol at 4 • C overnight. After fixation, cells were washed with cold PBS and stained with 500 µL of propidium iodide (PI)/RNase staining buffer, followed by incubation on ice for 30 min. Cell cycle distribution was performed using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). All experiments were performed in triplicate and yielded similar results. Obtained data were analyzed with Modfit LT 4.1 software (Verify Software House, Topsham, ME, USA).

Apoptosis Assay
Cell apoptosis was detected by flow cytometry using an Annexin V-FITC apoptosis detection kit. Briefly, NSCLC cells were seeded at 1 × 10 5 cells/well into a 6-well plate overnight and were treated with FGFC1 at different concentrations (0, 5, and 10 µM) for 48 h. Subsequently, apoptotic cells were collected and then stained with Annexin V and PI in 1 × binding buffer at room temperature for 15 min. Finally, stained cells were analyzed using the FACSCelesta flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with FlowJo V10 software (Flowjo, OH, USA).

Transcriptome Analysis
PC9 cells were selected for RNA-seq analysis using Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Briefly, after treatment with FGFC1, total RNA was extracted using Trizol reagent and was treated with mRNA enrichment method after it was purified by Oligo(dT)-attached magnetic beads. Purified mRNA was fragmented into small pieces, and the fragments were reverse transcribed and validated for quality control to get the final sequencing library. The library was sequenced on the BGISEQ-500 platform (BGI, Shenzhen, China). The sequencing data were provided by The Beijing Genomics Institute (BGI). The differentially expressed genes analysis was performed using the DESeq2 (v1.4.5) with p-value ≤ 0.05.

Quantitative RT-PCR (qRT-PCR)
After being treated with 0, 5, 10, and 20 µM FGFC1 for 24 h, NSCLC cells were collected for RNA extraction. RNA samples were then reverse transcribed into cDNA using the RT Master Mix from the Reverse Transcription kit. Quantitative analysis of target genes including IL-6, TNF-α, and ICAM-1 was conducted in triplicate using SYBR ® Green Real-time PCR Master Mix with 7500 Real-Time PCR System (Thermo Fisher Scientific, Invitrogen, Waltham, MA, USA). The relative expression of each targeted gene was calculated and normalized using the 2 −∆∆Ct method relative to reduced glyceraldehydephosphate dehydrogenase (GAPDH). The sequences of the primers used for qRT-PCR were as follows: IL-6 forward and reverse primers: 5 -GATGGATGCTTCCAATCTGGAT-3 and 5 -AGTTCTCCATAGAGAACAACATA-3 ; TNF-α forward and reverse primers: 5 -GCCAACGCCCTCCTGGCCAATG-3 and 5 -CCCTTCTCCAGCTGGAAGAC-3 ; ICAM-1 forward and reverse primers: 5 -ACCATGGAGCCAATTTCTC-3 and 5 -ACAATCCCTC TCGTCCAG-3 , and the primers for the GADPH forward and reverse primers were 5 -CATATGGGGAAGGTGAAGGTCGGAGTC-3 and 5 -GAATTCTTACTCCTTGGAGGCCA TGTGG-3 . The GADPH primer was used as the negative control. Each assay was performed in triplicate.

Western Blotting Analysis
Cells were treated with different doses of FGFC1 (0, 5, 10, and 20 µM) or 10 µM PDTC for 24 h before being lysed with RIPA buffer on ice and quantified with a BCA assay kit. Equivalent amounts of proteins were resuspended in a loading buffer, boiled at 100 • C for 5 min, and separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a 0.45 µM PVDF membrane. The membranes were blocked for 1 h at room temperature using 5% non-fat dry milk and subsequently incubated overnight at 4 • C with specific primary antibodies (1:1000). Then, the membranes were washed with TBST 3 times, followed by incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP) (1:10,000) for 1 h. Protein bands were visualized with highly sensitive chemiluminescence (Bio-Rad, Hercules, CA, USA) and quantified using Image Lab software. β-actin were used as loading controls.

Immunofluorescence Detection
The expression of p-p65 in PC9 was determined by immunofluorescence staining. Cells were plated at 1.2 × 10 5 cells/mL on glass coverslips in 6-well plates and treated with FGFC1 at different concentrations (0, 5, and 10 µM) for 24 h. After treatment, cells were washed with 1× PBS, fixed using 4% paraformaldehyde for 20 min, and then were permeabilized with 0.1% Triton X-100 in PBS containing 0.5% BSA (PBS-BSA) for 30 min. Subsequently, cells were blocked for 1 h using 1% BSA, incubated overnight at 4 • C with primary antibodies, p-p65. The slides were washed 3 times with PBS and stained for 30 min at room temperature with Goat anti-Rabbit IgG (1:500, Invitrogen, Waltham, MA, USA). Nuclei were counterstained with DAPI (4 ,6-diamidino-2-phenylindole) (Invitrogen, Waltham, MA, USA) and fluorescence of the samples was captured using a confocal laser scanning microscope (A1þR, Nikon, Tokyo, Japan).

In Vivo Antitumor Experiment
BALB/c nude mice (male, 5-6 weeks old, 10~14 g) were obtained from the Jiesijie Experimental Animal Co., Ltd. (Shanghai, China), and were raised in a pathogen-free and temperature-controlled environment. PC9 cells (5 × 10 6 ) were subcutaneously implanted into the right flank of the nude mice. Xenograft growth was monitored every other day using a digital caliber, and the tumor volumes (V) were measured using the following formula V = (L × W 2 ) × 0.5, where L is the length and W is the width of the xenograft [53]. When tumors reached a volume of approximately 100 mm 3 , the mice were randomly divided into 2 groups of 6 mice each: vehicle control (5% DMSO, 3 times weekly, intraperitoneal (ip) injection) and FGFC1(10 mg/kg, 3 times weekly, ip injection). During the whole experimental period, the feed intake and motor activity of the mice were carefully observed, their body weights and tumor measurements were performed 3 times per week using a sterile Vernier caliper. At the end of the study (day 21), the mice were sacrificed, the tumors were excised carefully, and the tumor weights were measured. All animal experiments were by institutional animal care guidelines and protocols were approved by the institutional animal care and use committee (Permit Number: SHOU-DW-2018-054) of Shanghai Ocean University (Shanghai, China).

Statistical Analysis
All experiments were repeated at least 3 times and the quantified data are expressed as the means ± SD unless otherwise indicated. Comparisons were made using one-way ANOVA analysis. p-value < 0.05 indicates statistical significance. Statistical analyses were performed with GraphPad Prism 8.0 software.

Conclusions
In conclusion, our results demonstrated that FGFC1 exhibited anti-cancer effects in EGFR-mutant NSCLC PC9 cells in vitro and in vivo via induction of G0/G1 cell cycle arrest and cell apoptosis mediated by the NF-κB signaling pathway. This study suggests that FGFC1 might be a promising lead compound that can be used as NSCLC treatment, therefore it merits further exploration.