FASN Knockdown Inhibited Anoikis Resistance of Gastric Cancer Cells via P-ERK1/2/Bcl-xL Pathway

Anoikis resistance (AR) is a crucial step in tumor metastasis. The overexpression of fatty acid synthase (FASN) is not only related to the AR of osteosarcoma cells, but also evidenced on gastric cancer (GC). This study investigated the role of FASN in the AR of GC cells. Plates coated with poly-HEMA were used for the culture of cells with AR. Small interfering RNA targeting FASN (siFASN) was transfected into MNK-45 and AGS cells. The number and apoptosis of cells were assessed by a hemacytometer and Annexin-V-FITC/PI assay, respectively. Aggregated cells and colony numbers were manually counted under a microscope. The migration and invasion rates were measured via wound healing and Transwell invasion assays, respectively. The levels of FASN, phosphorylated (p)-ERK1/2, ERK1/2 and Bcl-xL were detected through western blot or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that the cell numbers of MNK-45 and AGS were increased while that of GES-1 cell was decreased during the culture in suspension. A higher apoptosis rate and a smaller number of aggregated cells were observed in GES-1 cells in comparison with MNK-45 and AGS cells. A larger colony number, greater migration and invasion rates, and higher mRNA and protein expressions of FASN were presented in the AR group compared with the control group. Cells transfected with siFASN possessed lower migration and invasion rates, reduced expressions of FASN mRNA and protein, p-ERK1/2 and Bcl-xL, and induced a significantly declined ratio of p-ERK1/2 to ERK1/2. These findings suggest that down-regulation of FASN suppresses the AR of GC cells, which may be related to the inhibition of p-ERK1/2/Bcl-xL pathway.


Introduction
Gastric cancer (GC) is one of the most common malignant tumors in the world and ranks the third leading cause of cancer-related mortality, following lung and liver cancer [1]. Despite the decreased prevalence owing to the progresses made in medical technology, the prognosis of GC still remains poor, with a 5-year survival rate of less than 30% [2]. And metastasis is the main cause of deaths in patients with GC [3]. As a kind of programmed cell death which responds to the detachment of cells from the original extracellular matrix (ECM), anoikis can prevent cells from growth and reattachment in a different site, thus blocking the colonization of cells in distant organs. Unlike the normal cells, cancer cells show a resistance against anoikis, which contribute to the development and metastasis of tumors [4,5]. Therefore, studies on the anoikis resistance (AR) of GC cells are of considerable significance for the prevention and treatment of metastasis in GC and the reduction on GC-induced mortality.
Fatty acid synthase (FASN) is a main enzyme participating in the formation of the newborn fat and is overexpressed in tissues of various tumors, such as prostate cancer and bone tumor [6][7][8][9]. Previous studies have proved an evidence on the interaction between FASN and the process of epithelialmesenchymal transition (EMT) in GC [10], which reveals that FASN may be a promising therapeutic target for the anti-cancer therapy. However, studies on the detailed effect of FASN on GC cells with AR remain inadequate. Moreover, FASN was shown to mediate breast cancer through the regulation of ERK1/2 [11,12]. Extracellular regulated protein kinases (ERK), including ERK1 and ERK2, can be activated into phosphorylated (p)-ERK so as to promote the multiplication and suppress the apoptosis in the cells. Previous studies also revealed the relation on the activation of ERK activation and the cancer and AR [13][14][15]. Bcl-xL, an antiapoptotic member of the B-cell lymphoma-2 (Bcl-2) family, exerts a protective effect on cancer cells, including gastric cancer cells, as reported in previous publications [16][17][18]. Besides, the upregulation of FASN was proved to be associated with the AR of osteosarcoma cells via p-ERK1/2/Bcl-xL [19]. Consequently, we tried to explore whether FASN realizes its effect on AR via ERK1/2/Bcl-xL pathway in GC.
In this study, the anoikis resistant GC cells were cultured in vitro, the purpose of which was to explore the biological characteristics of AR. In addition, the role and molecular mechanism of FASN in GC cells with AR were also investigated in vitro by controlling variables, the results of which demonstrated that FASN mediated AR in GC cells through p-ERK1/2/Bcl-xL pathway, making FASN as a promising therapeutic target for the intervention and remedy of metastasis in GC.

Suspension
Culture of Anoikis Resistant GC Cells. The adherent cells were trypsinized (A610681-0100, Sangon Biotech Co., Ltd., Shanghai, China) so as to obtain the single cell suspension and then seeded at 10 6 per well in Costar ultralow attachment 6-well plates (3471, Corning Incorporation, Corning, NY, USA). Poly-hydroxyethyl methacrylate (poly-HEMA, ST1582, Beyotime Biotechnology, Shanghai, China) was pre-dissolved in 95% ethanol (493511, Sigma-Aldrich, Ontario, Canada), and the solution was added into the culture plates to induce the anoikis in the cell. The cells were passaged every three days. After continuous passages, the anoikis resistant cells were selected and collected as AR group for biological characteristics analysis. Cells under adherent culture were used as the control group and received no treatment.
2.3. Suspension Culture and Detection. The adherent cells of the control and AR groups were trypsinized to the single cell suspension and then seeded at 10 6 per well in costar ultra-low attachment 24-well plates (3473, Corning Incorporation, Corning, NY, USA). A hemacytometer (AMQAF1000, Thermo Fisher Scientific, Waltham, MA, USA) was used to count the numbers of cells on days 0, 2, 4 and 6. The morphology of cells was observed using a phase contrast microscope (magnification: 100x; DYS-810, Shanghai Dianying Optical Instrument Co., Ltd., Shanghai, China).

Cell Apoptosis
Assay. After the culture in suspension for 24 hours (h), the apoptosis of adherent and detached cells was assessed by an Annexin-V-FITC Apoptosis Detection Kit with PI (E606336-0500, Sangon Biotech Co., Ltd., Shanghai, China). The cells were washed 3 times with PBS and then resuspended in binding buffer. Annexin V-FITC and PI were added to the solution containing cells in a dark room at room temperature for 15 minutes (min). The apoptosis rates were analyzed in a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA).

Colony Formation
Assay. Cells in logarithmic phase were trypsinized to single cell suspension and then seeded at 10 5 per well in Costar ultra-low attachment 24-well plates for 7 days of suspension culture. Then the cell suspension was seeded into normal 24-well plates (J00240, Shanghai Jinan Biological Technology Co., Ltd., Shanghai, China) until the formation of colonies. After being fixed with 4% paraformaldehyde (E672002-0500, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 15 min and stained with crystal violet (E607309-0100, Sangon Biotech Co., Ltd., Shanghai, China) for 15 min, the colonies were manually counted.
2.6. Wound Healing Assay. Cells were seeded in 6-well plates (J00660, Shanghai Jinan Biological Technology Co., Ltd., Shanghai, China) at a density of 5 × 10 6 cells per well and cultured until 80-90% confluence. A pipette (Sangon Biotech Co., Ltd., Shanghai, China) tip was used to make a straight scratch which simulated as a wound. Cells were rinsed with PBS and the medium was replaced with serum-free medium at 37°C for 24 h. Images were observed and recorded at 0 and 24 h by a DYS-810 microscope (magnification: 100 x; Shanghai Dianying Optical Instrument Co., Ltd., Shanghai, China) and the distance for the migration was measured by Image J, version 1.48 (National Center for Biotechnology Information, Bethesda, MD, USA). Six visual fields were randomly selected from each room.

Transwell Invasion Assay.
Cell invasion assay was performed in a 24-well Transwell chamber (3379, Corning Incorporation, Corning, NY, USA). The upper filters were pre-coated with Matrigel (354234, Corning Incorporation, Corning, NY, USA), and cells with serum-free medium were seeded into the upper chambers at a density of 4 × 10 5 cell/ml. The medium in the lower chamber contained 10% FBS and functioned as a source of chemoattractants. After the incubation at 37°C for 24 h, cells that passed through the Matrigelcoated membrane were fixed with 4% paraformaldehyde at 4°C for 30 min and then stained with crystal violet at room temperature for 30 min. Cells in six randomly selected visual

Quantitative Reverse Transcription-Polymerase Chain
Reaction (qRT-PCR). Total RNA was extracted from cells using a TRIzol kit (B511321-0100, Sangon Biotech Co., Ltd., Shanghai, China). Reverse transcription was conducted to synthesize cDNA by using a PrimeScript RT Reagent Kit (638314, Takara Bio, Inc., Otsu, Japan). Sequences of the primers (Shanghai GenePharma Co., Ltd., Shanghai, China) were listed in Table 1. Then the cDNA was tracked using SYBR Green qPCR Supermix (1725270, Bio-Rad Laboratories, Inc., CA, USA) through a CFX384 RT-PCR cycler (Bio-Rad Laboratories, Inc., CA, USA) under a thermal cycling program containing 40 cycles of 95°C for 3 min, 95°C for 15 s, and 60°C for 15 s. The expression levels of relative FASN RNA were calculated on the basis of the mean β-actin expression levels in the representative sample. Data were analyzed using the relative quantification method 2 -ΔΔCT [20].

GC Cells Showed Stronger AR Compared with Normal
Gastric Mucosa Epithelial Cells. During the culture in suspension for 2, 4 and 6 days, the number of GES-1 cells decreased while those of MNK-45 and AGS cells increased significantly (Figure 1(a), p<0.001). GES-1, MNK-45 and AGS cells were all attached cells, some of which died during the culture in suspension, and we found the apoptosis rate of the GES-1 cells in suspension was higher than the attached GES-1 cells. And the apoptosis rate of normal gastric mucosa epithelial cells was higher than GC cells in both attached and suspended condition (Figures 1(b), 1(c), p < 0.05, p <0.001). In suspension culture, a multicellular aggregate is more resistant to anoikis than single suspension cells [21]. As suggested by the results of the morphology on the cell, which was observed by microscopy,, MNK-45 and AGS cells formed larger clusters and more aggregated cells when compared with GES-1 cells (Figures 1(d), 1(e), p <0.001).

AR Promoted GC Cell Multiplication, Migration and
Invasion and FASN Was Associated with AR. In the results of colony formation assay, it was presented that the colony numbers of MNK-45 and AGS cells in the AR group were greater than those in the control group, indicating that cells with AR were more capable of proliferation (Figures 1(f), 1(g), p <0.001). During the wound healing assay and Transwell invasion assay, both the migration and invasion rates of MNK-45 and AGS cells were higher in the AR group than in the control group (Figures 2(a)-2(d), p < 0.01, p <0.001). We also found that the relative mRNA and protein levels of FASN were higher in MNK-45 and AGS cells of the AR group as compared with those of the control group, which suggested an association between FASN and AR (Figures 2(e)-2(g), p < 0.01, p <0.001).

SiFASN Promoted Apoptosis Yet Suppressed FASN Expression, Aggregation, Migration and Invasion in GC
Cells. Both the relative mRNA and protein expression levels of FASN in MNK-45 and AGS cells were lower in the AR + siFASN group than in the AR + siNC group (Figures 3(a)-3(c), p <0.001), implying that transfection of siFASN suppressed the expression of FASN. Silence of FASN promoted the apoptosis rates of MNK-45 and AGS cells while decreasing the number of aggregated cells. (Figures 3(d)-3(g), p < 0.01, P <0.001). In addition, the relative migration and invasion rates of MNK-45 and AGS cells in the AR + siFASN group reduced significantly in comparison with the AR + siNC group (Figures 4(a)-4(d), p <0.001).

Discussion
GC is a kind of deadly malignancy with a low early diagnosis rate and unfavorable prognosis. Tumor metastasis is a major reason for the failure in the treatment and mortality in patients in nearly all types of cancer, and was evidenced in approximately 50% of patients during the development of tumor at initial diagnosis [22,23]. Cancer metastasis is a complex process comprising different stages, including local invasion, intravasation, survival in circulation, extravasation, and colonization and metastasis, in which a number of interactions between tumor and stroma have been reported [24,25]. Anoikis, a programmed cell death induced upon the detachment of cells from ECM, is deemed as a crucial mechanism in the preclusion on adherent-independent cell growth and the attachment to an improper matrix, which thereby prevents the metastasis and colonization of cells in distant organs [26]. In addition, Anoikis is one of key procedures to maintain the normal development and homeostasis of organisms [27]. AR, the capability of cells to survive during the detachment from the ECM, is a prerequisite for the development of tumor metastasis [28]. Thus, the competence of cancer cells to resist anoikis has attracted the increasing attention from the scientific community in the field of cancer research, and so far, several mechanisms of AR have been reported and proposed. For instance, integrin-α2β1/-α5β1 interacted with epidermal growth factor receptor (EGFR) in the AR of colon cancer [29]. MiR-141/KLF12/Sp1/survivin signaling pathway enhanced AR in ovarian cancer, and a transcriptomic study addressed the importance of CDKN1A-regulated quiescence for cells to survive from anoikis in head and neck squamous carcinoma [30,31]. In accordance with the discoveries in previous studies, FASN, an enzyme implicated in the process of metabolism, has shown to be positively correlated with the development of various cancers, including GC [32][33][34]. FASN has also been proved to mediate AR during cell growth and metastasis in osteosarcoma. However, the role of FASN in GC cells with AR has not been explored in detail [19].
In the present study, the observations suggested that AR promoted cell multiplication, migration and invasion and led to high expression levels of FASN in cells, which indicated a possible relation between FASN and AR. We also discovered that silence of FASN significantly reduced cell multiplication, migration and invasion as well as p-ERK1/2 and Bcl-xL levels while barely affecting the expression of ERK1/2. P-ERK1/2 may serve as an important hub in the network, as it occurs crosstalk with other pathways, including phosphatidylinositol 3-kinase (PI3K) [35], and Wnt [36]. Moreover, it has also been demonstrated that there exists a regulatory loop between FASN and ERK pathway, in which PI3K/AKT pathway is involved as well [37]. Therefore, the regulation between FASN and ERK pathway may be involved with other molecular pathways. PI3K/AKT pathway also regulates downstream anti-apoptotic proteins, including Bcl-xL [38]. Thus, it was inferred that the functions of FASN on GC cells with AR might be related to p-ERK1/2/Bcl-xL pathway, regardless of other molecular pathways that may be involved. However, there is room for improvement of the current study. Firstly, the length of FASN mRNA is about 8.4 kb, making the clone on the full length a great challenge. Thus, cell lines with overexpressed FASN failed to be established, leaving the results and conclusions with one-sided uncertainty. Also, the specific molecular mechanism of FASN in GC cells with AR was not absolutely settled. Secondly, the experiments for detecting cell apoptosis rates and colony formation could be conducted more scrupulously by the exclusion of the influence of cell proliferation. Thirdly, we directly selected p-ERK1/2/Bcl-xL based on documents and assumption, and this blindness could be avoided by probing into the downstream signaling pathways of FASN. Lastly, researches in vivo should be carried out in the future to further prove the conclusions of experiments in vitro.
In summary, this study demonstrated the stronger AR of GC cells, which may explain the survival of cells during the metastasis and spread of GC. The results revealed that AR facilitated multiplication, migration and invasion while impeding apoptosis of GC cell. Moreover, it was preliminarily verified that the down-regulation of FASN exerted suppressive effects on GC cells with AR, which was associated with the inhibition of p-ERK1/2/Bcl-xL signaling pathway. Taken together, our results and data indicate that FASN  may become a new target for preventing the metastasis of tumor, thus providing a novel reference for the treatment against cancer. Nonetheless, more researches and clinical trials are needed to explore whether FASN is a feasible mean for the prevention and treatment of GC.

Data Availability
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors declare no conflicts of interest.