Fructose-1,6-Bisphosphatase 2 Inhibits Oral Squamous Cell Carcinoma Tumorigenesis and Glucose Metabolism via Downregulation of c-Myc

Background Fructose-1,6-bisphosphatase 2 (FBP2), known as a rate-limiting enzyme in gluconeogenesis, is a tumor suppressor downregulated in various cancers. However, the role of FBP2 in oral squamous cell carcinoma (OSCC) remains largely unclear. Methods The level of FBP2 in OSCC tissues and matched adjacent normal tissues was determined by western blot and RT-qPCR assays. In addition, analysis of FBP2 function in OSCC cells was assessed using both gain-of-function and loss-of-function studies. Results In this study, we found that the expression of FBP2 was remarkably downregulated in OSCC tissues and OSCC cells. Overexpression of FBP2 suppressed the viability, proliferation, migration, and glycolysis of OSCC cells, whereas FBP2 knockdown exhibited the opposite results. Moreover, downregulation of FBP2 promoted the growth and glycolysis of OSCC cells in nude mice in a xenograft model. Specifically, FBP2 colocalizes with the c-Myc transcription factor in the nucleus. Significantly, inhibitory effects of FBP2 overexpression on the viability, proliferation, migration, and glycolysis of OSCC cells were reversed by c-Myc overexpression. Conclusion Collectively, FBP2 could suppress the proliferation, migration and glycolysis in OSCC cells through downregulation of c-Myc. Our study revealed a FBP2-c-Myc signaling axis that regulates OSCC glycolysis and may provide a potential intervention strategy for OSCC treatment.


Introduction
Oral squamous cell carcinoma (OSCC) has been considered to be the most prevalent malignancy in the head and neck region [1,2]. In addition, OSCC has a tendency for local invasion and a potential for cervical lymph node metastasis [3][4][5]. Moreover, OSCC often leads to oral dysfunction including chewing and swallowing disorders, which profoundly worsens a patient's quality of life [6]. Recently, surgery, chemotherapy, radiotherapy, and adjuvant chemoradiotherapy are the main treatment strategies for OSCC treatment [7][8][9]. Despite substantial progress in the diagnosis and treatment of OSCC, the 5-year overall survival rates of OSCC remain no more than 50% [6,10]. Therefore, discovery effective biomarkers and therapeutic targets may help to understand the prognosis and treatment of OSCC.
One remarkable feature of cancer is the reprogramming energy metabolism [11]. Glucose metabolism is a major type of energy metabolism [12]. Under normal conditions, cells generate energy on the process of aerobic respiration [12]. Meanwhile, cells use glycolysis to generate energy when the oxygen is not enough [12]. As early as the 1950s, Otto Warburg performed a study of cancer metabolism and discovered that cancer cells preferentially metabolize glucose through aerobic glycolysis whether there is sufficient oxygen present [13,14]. Glycolysis plays a key role in the growth and metastasis of human cancers [15,16]. Malignant tumors, including OSCC, rely on aerobic glycolysis to maintain rapid cell growth [17].
Fructose-1,6-bisphosphatase 2 (FBP2) is a rate-limiting enzyme in gluconeogenesis [18]. Gluconeogenesis, a reverse process of glycolysis, is important for the maintenance blood glucose levels during starvation [19]. Li et al. found that FBP1 loss could promote glycolysis and cell growth of gastric cancer cells [20]. Huangyang et al. showed that FBP2 restoration could suppress glycolysis activity in sarcoma [18]. However, the role of FBP2 in OSCC remains largely unclear. On the other hand, c-Myc was known to be a crucial mediator in tumor progression [21,22]. In addition, c-Myc could act as a key modulator in the regulation of glucose metabolism through mediation of GLUT1, HK2, PKM2, and LDHA [23]. However, the relation between FBP2 and c-Myc in OSCC remains unexplored.
Thus, this study aimed to investigate the function of FBP2 in glycolysis, proliferation, and migration in OSCC.

Clinical Samples.
A total of 10 paired OSCC tissues and matched adjacent normal tissues were obtained from the Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine between April 2019 and September 2020. This study was approved by the ethics committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The written consent was obtained from each patient.
2.2. RNA-Sequencing (RNA-seq) Analysis. Trizol reagent (Thermo Fisher Scientific) was used to extract total RNA from tissues. Sequencing libraries were prepared from all samples using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Beverly, MA). After that, the libraries were quantified by Agilent Bioanalyzer 2100 and sequenced by Illumina Hiseq sequencer (Illumina, San Diego, CA, USA).

Microarray Data
Analysis. GSE35261 dataset which contain the gene expression data of OSCC tissues and normal tissues was downloaded from GEO database. The differentially expressed genes (DEGs) between OSCC tissues and normal tissues were identified using the limma package.|log2 (fold change)|>1.0 with adjusted P value <0.05 was set as the screening threshold.

Intersected
Analysis. The intersection of DEGs from inhouse RNA-seq analysis (validation dataset) and GSE35261 dataset (testing dataset) was performed using the Venn diagram package.
2.5. KEGG Pathway Analyses. DEGs were evaluated by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Adjusted P < 0:05 was set as the threshold values.
2.6. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). The Trizol reagent (Invitrogen, USA) was used to extract total RNA from tissues and cells. Complementary DNA (cDNA) was synthesized using the ReverTra Ace qPCR RT Kit (TOYOBO, Japan). After that, the qPCR was performed on an Applied Biosystems™ 7500 Real-Time PCR System (Applied Biosystems, USA) with the Power SYBR Green PCR Master kit (Applied Biosystems, USA).
2.12. Wound-Healing Assay. HSC-3 and CAL-27 cells were plated into 6-well culture plates overnight at 37°C up tõ 80% confluence. After that, cells were wounded with a sterile 20-μL pipette tip. The wound closure was photographed with a microscope (ECLIPSE TS2, Nikon, Japan) at 0 h and 48 h.
2.14. Transwell Migration Assay. Cell migration was determined by transwell migration assay using transwell chambers (Corning, NY, USA). Briefly, HSC-3 and CAL-27 cells (1 × 10 4 cells) suspended in serum-free medium were seeded on the upper transwell chamber. 600 μL of DMEM containing 10% FBS was added into the bottom chamber. After 24 h of incubation, the migrated cells were stained with 0.1% crystal violet. Later on, migrating cells were observed and counted under a microscope (ECLIPSE TS2, Nikon, Japan). 2.16. Glucose Uptake Assay. Glucose uptake was measured in HSC-3 and CAL-27 cells using a glucose update assay kit (cat.no. ab136955, Abcam) following the manufacturer's procedure.
2.17. Measurement of Lactate and ATP. The D-lactate levels were measured in HSC-3 and CAL-27 cells using a D-lactate assay kit (cat.no. ab83429, Abcam) according to the manufacturer's procedure. The cellular ATP content was detected in HSC-3 and CAL-27 cells using an ATP colorimetric assay kit (cat.no. ab282930, Abcam) according to the manufacturer's procedure.
2.18. Animal Study. BALB/c nude mice (6-8 weeks old) were obtained from the SiPeiFu (Beijing) Biotechnology Co., Ltd. (Beijing, China). This study was approved by the Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, and conducted according to the institutional guidelines. HSC-3 cells (2.5 × 10 6 ) were injected subcutaneously into the left flank of each mouse. When the mean tumor volume reached 40 mm 3 , mice were divided randomly into four groups: control, shRNA NC, FBP2 shRNA1, and DDP groups. After that, 2.5 × 10 6 HSC-3 or HSC-3 cells stably expressing shRNA NC, FBP2, and shRNA1 in 100 μL PBS were injected into the left flank of nude mice. Mice in the DDP group were treated intraperitoneally (i.p.) with 2 mg/kg cisplatin (MedChemExpress, Shanghai, China) once every two days for 3 weeks. The tumor volume = ðlength x width 2 Þ/2. After 3 weeks, mice were sacrificed, and the tumors were isolated.

2.19
. Coimmunoprecipitation (Co-IP) Assay. Co-IP was carried out using a Pierce™ Classic Magnetic IP/Co-IP Kit (Thermo Scientific, USA). Briefly, HSC-3 and CAL-27 cells were incubated with anti-FBP2 and anti-c-Myc antibodies overnight at 4°C. Later on, the samples were incubated with pierce protein A/G magnetic beads. Subsequently, the protein binding complex was subjected to western blot assay.
2.20. Immunofluorescence (IF) Analysis. HSC-3 and CAL-27 cells were seeded onto 24-well plates and fixed by 4% paraformaldehyde for 15 min and then permeabilized in 0.1% Triton X-100 for 5 min at room temperature. After that, cells were blocked with 1% BSA in PBS, and stained with FBP2 and c-Myc antibodies (Abcam) at 4°C overnight, and then incubated with the corresponding secondary antibody at room temperature for 1 h. The stained cells were observed using a fluorescence microscope (Leica, German).

Statistical Analysis.
All data were repeated in triplicate. One-way analysis of variance (ANOVA) and Tukey's tests were carried out for multiple group comparisons. Values are shown as the mean ± SD. Differences were considered to be statistically significant at * P < 0:05.

Identification of DEGs in the OSCC Tissues and Normal
Tissues. The DEGs between OSCC tissues and normal tissues were screened by GEO database (GSE35261, testing dataset) and validated by our in-house dataset (validation dataset). Our analysis found that a total of 777 DEGs were identified in the testing dataset and 193 DEGs were identi-  Figure 2C). For a more in-depth understanding of these overlapping DEGs, KEGG pathway enrichment analyses were performed. As revealed in Figure 1(b), these overlapping DEGs are mainly enriched in metabolic pathways. 10 overlapping genes (downregulated genes: BHMT, GGT6, GMDS, FBP1, FBP2, CBR3, FUT3; upregulated genes: PFKP, PLCB1, CYP3A5) participated in metabolic pathways (Figures 1(a) and 1(b)).

The
Collectively, FBP2 could suppress the proliferation and migration of OSCC cells.

Overexpression of FBP2 Inhibited the Glycolysis in OSCC
Cells. We further investigated whether FBP2 could change glucose metabolism in OSCC cells. As indicated in Figure 4(a), FBP2 knockdown notably stimulates the glucose uptake in HSC-3 cells, whereas upregulation of FBP2 markedly inhibits the glucose uptake in CAL-27 cells. In addition, the intracellular lactate concentrations were significantly increased in HSC-3 cells transfection with FBP2 shRNA1 (Figure 4(b)). In contrast, CAL-27 cells transfected with FBP2 OE exhibited the opposite results (Figure 4(b)). Next, to determine whether the increased glucose uptake into OSCC cells could result in elevated energy production, we investigated the effect of FBP2 on cellular ATP levels in OSCC cells. As shown in Figure 4(c), downregulation of FBP2 leads to a 2.54-fold increase in ATP content in HSC-3 cells compared with the control group. In contrast, FBP2 overexpression remarkedly decreased the cellular ATP levels in CAL-27 cells (Figure 4(c)). Furthermore, downregulation of FBP2 significantly increased the expressions of the glucose transporter GLUT-1 and glycolytic enzymes HK2, PKM2, and LDHA in HSC-3 cells (Figure 4(d)). Conversely, upregulation of FBP2 in CAL-27 cells resulted in marked decrease in their expressions (Figure 4(e)). To sum up, overexpression of FBP2 could inhibit the glycolysis in OSCC cells.            (Figures 6(e) and 6(f)). These data showed that FBP2 could bind with c-Myc in the cell nucleus.
Meanwhile, the expression of FBP2 in OSCC cells was significantly downregulated by FBP2 shRNA but upregulated by FBP2 OE (Supplementary Figure 6A). Consistently, the level of c-Myc in OSCC cells was notably decreased by c-Myc shRNA but increased by c-Myc OE (Supplementary Figure 6B). In addition, FBP2 negatively regulated the level of c-Myc (Supplementary Figure 6B), and the effect of FBP2 shRNA/OE on c-Myc level was partially reversed by c-Myc shRNA/OE (Supplementary Figure 6B). Furthermore, FBP2 knockdown markedly promoted the viability, proliferation, and migration and inhibited the apoptosis of HSC-3 cells; however, c-Myc inhibition largely abolished the promoting effect of FBP2 knockdown on the growth of HSC-3 cells (Figures 7(a)     Significantly, downregulation of FBP2 stimulated the glucose uptake in HSC-3 cells, followed by elevation of intracellular lactate concentrations and cellular ATP levels, whereas these phenomena were largely reversed by c-Myc knockdown (Figures 8(a)-8(c)). In contrast, overexpression of FBP2 inhibited the glucose uptake in CAL-27 cells, followed by reduced intracellular lactate concentrations and cellular ATP levels, whereas these phenomenon were largely reversed by c-Myc overexpression (Figures 8(d)-8(f )). Collectively, FBP2 could inhibit glycolysis in OSCC cells through downregulating c-Myc.

Discussion
In this study, we found that FBP2 is downregulated in OSCC tissues. In addition, overexpression of FBP2 significantly inhibited the proliferation, migration and glycolysis of OSCC cells via downregulation of c-Myc. Mechanistically, the ability of FBP2 to inactivate c-Myc signaling contributes to decreased glycolysis, leading to the inhibition of OSCC progression.
Deregulated glucose metabolism is considered as a cancer hallmark that contributes to tumor progression [27,28]. In contrast to healthy cells, most cancer cells rely on aerobic glycolysis as the main energy to sustain uncontrolled tumor cell proliferation, i.e., the Warburg effect [29]. In addition, gluconeogenesis is a reverse process of glycolysis and can antagonize aerobic glycolysis in cancer [30]. Glucose metabolism is balanced by the anabolic (e.g., gluconeogenesis) and catabolic (e.g., glycolysis) processes [31]. FBP2 is a key enzyme for gluconeo-genesis, which plays important roles in energy and glucose metabolism [24]. Thus, re-expression of FBP2 might antagonize glycolysis and then decrease glucose uptake by cancer cells. In the present study, we found that overexpression of FBP2 significantly suppressed the glucose uptake in OSCC cells, followed by reduced intracellular lactate concentrations and cellular ATP levels. In addition, overexpression of FBP2 decreased the expressions of glycolytic genes of GLUT1, LDHA, HK2, and PKM2 in OSCC cells, which resulted in reduced glycolytic activity. Collectively, overexpression of FBP2 could inhibit glycolysis in OSCC.
It has been shown that gluconeogenic enzymes such as FBP localize in the nucleus and could affect gene transcription [18,32]. Li et al. found that nuclear FBP1 could inhibit transcription factor HIF activity and then decrease its downstream targets (PDK1, LDHA, GLUT1), therefore inhibiting glycolytic activity [33]. In addition, the transcription factor c-Myc plays important roles in the regulation of cellular growth and metabolism [34]. Ectopic c-MYC expression could drive aerobic glycolysis in cancers via the direct upregulation of GLUT1, LDHA, HK2, and PKM2 [35,36]. In this study, we found that FBP2 directly binds to c-Myc in the nucleus. In addition, overexpression of FBP2 significantly downregulated the expression of c-Myc in OSCC cells, which led to decreased glycolytic activity. In contrast, overexpression of c-Myc reversed FBP2 overexpression-mediated glycolytic inhibition. These data suggested that FBP2 might act as a nuclear c-Myc transcriptional corepressor, which was consistent with the previous study [18]. Collectively, FBP2 could inhibit glycolysis in OSCC cells through downregulating c-Myc.

Conclusion
In this study, we provided the evidence that FBP2 overexpression could inhibit the migration and invasion and suppressed the glycolysis in OSCC cells by inhibiting c-Mycmediated glycolysis. Our results may provide a new treatment strategy for OSCC.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
All experiments were approved by the Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, which adheres to the National Institutes of Health Guide for the Care and Use of laboratory animals.

Conflicts of Interest
The authors have declared that no competing interest exists.