CEBPB regulates the bile acid receptor FXR to accelerate colon cancer progression by modulating aerobic glycolysis

Abstract Background Aerobic glycolysis is a main characteristic of tumors, and inhibited glycolysis impedes the tumor development. Farnesoid X Receptor (FXR) mainly regulates bile acid metabolism. In this research, we mainly investigated whether FXR was involved in the regulation of glycolysis in colon cancer. Methods The differential expression analysis was performed on FXR and Enhancer Binding Protein Beta (CEBPB) data in colon cancer downloaded from The Cancer Genome Atlas (TCGA) database. Western blot and qRT‐PCR were used to detect the expression levels of CEBPB and FXR. The upstream gene of FXR was predicted through bioinformatic analysis. ChIP and dual luciferease assays were performed to confirm the targeted relationship between CEBPB and FXR. Gene Set Enrichment Analysis (GSEA) was performed on FXR. Finally, the glycolysis capabilities of cells in each treatment group were detected. CCK‐8, colony formation assay and flow cytometry were performed to test proliferation and apoptosis of colon cancer cells. Results FXR was lowly expressed at the cell level in colon cancer. In vitro assays verified the antitumor effect of FXR on colon cancer. ChIP and dual luciferase assays verified that transcription factor CEBPB bound with the promotor region of FXR, and negatively regulated the expression of FXR. Cell function assays proved that silenced expression of FXR promoted glycolysis, which promoted the development of colon cancer cells. Conclusion The study on FXR‐regulated glycolysis of colon cancer cells helps us to further understand the molecular mechanism by which FXR regulated the development of colon cancer cells.

microenvironment (TME) features (hypoxia, acidosis and immunosuppression). Tumor glycolysis exacerbates hypoxia, acidosis and immunosuppression, and leads to tumor proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), invasion and metastasis. 5 Metabolic reprogramming is a vital feature for tumors. 6,7 Tumor treatment by inhibiting glycolysis is drawing more and more attention.
For instance, Bian et al. 7 found that nuclear receptor Nur77 (nerve growth factor-induced gene B) was a suppressor for hepatocellular carcinoma. The interaction between Nur77 and PEPCK1 (phosphoenolpyruvate carboxykinase) represses the glycolysis, which exhausts ATP and hinders cell growth. Ma et al. 8 discovered that E2F transcription factor 1 manipulated SEC61 Translocon Subunit Gamma to accelerate the development and metastasis of breast cancer by modulating glycolysis. However, few reports focus on colon cancer and glycolysis.
Accordingly, this study investigated their relationship.
Farnesoid X receptor (FXR) is a nuclear receptor that transcribes and mediates the signal transcription viability of bile acid.
FXR belongs to the nuclear receptor (NR) superfamily, and is vital in mammal reproduction, development and metabolism. 9 FXR locates in human chromosome 12q23.1, and contains 11 exons and 10 introns. 10 It mainly regulates bile acid metabolism to keep the concentration of bile acid in a biologically proper range, thereby preventing bile acid-induced cytotoxicity. [11][12][13] Andrea et al. 14 investigated loss of FXR expression in the late stage of colon cancer. At the same time, Sigurd et al. 15 reported that the low expression of FXR in human colon cancer was related to high grading of tumors and poor clinicopathological reaction. These studies indicate that FXR may be a tumor suppressor, but its potential molecular mechanism in the glycolysis process of colon cancer is still unclear.
In the early stage of the study, it was found through bioinformatics analyses that there was an upstream transcription factor (TF) Enhancer Binding Protein Beta (CEBPB) for FXR. CEBPB was significantly upregulated in colon cancer, which was negatively related to FXR. A series of research unveiled that CEBP was an important TF in tumors. 16 Li et al. 17 reported that isoforms of CEBPB participated in aerobic glycolysis, and accelerated growth of breast cancer. Hu et al. 18 found that MIR503 Host Gene repressed the proliferation and invasion of cervical cancer cells, and promoted cell apoptosis through miR-191/CEBPB axis. So far, the role of CEBPB in colon cancer has been rarely reported. Hence, this study aims to discuss the influence of CEBPB targeting FXR on colon cancer, as well as whether CEBPB targeting FXR affects cancer progression through aerobic glycolysis.

| Bioinformatics analyses
The expression data of FXR mRNA (Normal: 41, Tumor: 480) were downloaded from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/). R package "edgeR" was used to perform differential analysis on the normal and the tumor groups (|log FC| > 2, padj < 0.05). Thereafter, hTFtarget and PROMO databases were adopted to predict the upstream regulatory genes of FXR. UALCAN (http://ualcan.path.uab.edu/index.html) was taken to predict the expression of CEBPB protein. ChIPBase (http://rna.sysu.edu.cn/chipb ase/) was used to test the correlation between the expression of FXR and CEBPB. JASPAR database was adopted to predict the putative binding site on FXR and CEBPB. Finally, Gene Set Enrichment Analysis (GSEA) was performed on FXR mRNAs to investigate the enriched pathways in colon cancer.
To control the expression of FXR and CEBPB in cells, oe-FXR and si-FXR, oe-CEBPB and si-CEBPB were established. To acquire oe-FXR and oe-CEBPB, the complete CDS sequence of FXR and CEBPB (from GenBank database) were linked to pcDNA3.1 plasmid. The blank pcDNA3.1 plasmid was taken as a negative control (oe-NC). To silence the expression of FXR and CEBPB, siFXR and siCEBPB were established. All the primer sequences were synthesized by Sangon Biotech.
The Lipofectamine 2000 kit (Thermo Fisher) was used to transfect 2 μg plasmid vectors to colon cancer cells SW620 and HCT116. The transfected cells were cultured in an incubator containing 5% CO 2 at 37°C.

| qRT-PCR
Colon cancer cells in each treatment group were collected. Total RNA extraction kit (Invitrogen, Thermo Fisher Scientific, Inc.) was adopted to extract RNA complying with the protocol. β-actin was taken as an internal reference. The reverse transcription kit (Takara, RR037A) was adopted to reversely transcribe the RNA, while qRT-PCR kit (Takara, RR820A) was applied for quantitative analysis. Each sample has to be repeated at least three times. In the end, the relative expression was calculated using 2 −ΔΔC t . Table 1 lists the involved primer sequences.

| Dual-luciferase assay
The promoter region of FXR containing CEBPB binding site was cloned to Pgl3-basic (Panomics, Fremont), and the sequence was named as FXR WT. The promoter region of FXR containing all the possibly mutated CEBPB sites was cloned to Pgl3-basic (Panomics), and the sequence was named as FXR MUT, with pRLSV40 (Promega) as a control. To study whether silenced or overexpressed CEBPB would impact FXR viability, different concentrations of si-CEBPB and oe-CEBPB were co-transfected to SW620 and HCT116 cells, respectively. Dual-luciferase reporter detection system was used to detect the luciferase viability.

| The measurement of glycolysis pathway viability
This study determined the activity of the glycolysis pathway by quantitatively measuring the levels of different products (including pyruvate, lactic acid, citric acid and malic acid) produced during glycolysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using Seahorse Biosciences XF96 analyzer. Cells were cultured in the petri dish for 24 h, and then cultured in XF medium at 37°C for 2 h. OCR was measured complying with the instruction of XF Cell Mito Stress Test Profile. Oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and rotenone were added in sequence, and then the OCR value was determined.

ECAR was measured complying with XF Glycolysis Stress Test
Profile. Glucose, oligomycin and 2-deoxyglucose were sequentially added to each well until the concentration reached a specified level.  (4)).

| Cell proliferation capability
CCK-8 was utilized to detect the cell proliferation viability of colon cancer cells in each treatment group. A microplate reader was adopted to test the absorbance (450 nm) at 0, 24, 48 and 72 h respectively. Meanwhile, the colony formation assay was applied to determine the cell proliferation capability of colon cancer cells in each treatment group. The transfected colon cancer cells were inoculated into 6-well plates with 5 × 10 2 cells per well. They were cultured in an incubator containing 5% CO 2 at 37°C for 12 days. The visible colonies were manually counted after cells were stained.

| Cell apoptosis test
Firstly, colon cancer cells in each treatment group were collected.
Then cells were washed three times using precooled phosphate buffer saline (PBS). The cells were resuspended in the binding buffer, where fluorescein isothiocyanate (FITC) and propidium iodide (PI) were then added in the dark using double staining method. Finally, a flow cytometer was adopted to detect the cell apoptosis. Each test was repeated at least three times.

| Chromatin immunoprecipitation (ChIP) assay
Colon cancer cells were cultured in 100 mm petri dish to 80% concentration. ChIP assay was undertaken following the protocol of ChIP kit (P2078, Beyotime Biotechnology). Cell lysates were cultured with IgG as a control. PCR was applied to amplify FXR promoter region to Region 2 (located −1488 ~ −1272) and Region 3 (located −838 ~ −630) that contained CEBPB binding sites, and Region 1 (located −553 ~ −175) that excluded CEBPB binding sites. The latter TA B L E 1 Primer sequences used in qRT-PCR was taken as a negative control. Antibodies used in ChIP assay were anti-CEBPB (1:1000, Rabbit monoclonal, ab32358), anti-IgG (1:1000, Rabbit monoclonal, ab109489). Table 2 shows the primers sequences in ChIP assay.

| Statistical analyses
All the data in the study were analyzed using GraphPad Prism 8.0 software and in the form of mean ± standard deviation. The data were subjected to Student-t test or Bonferroni-corrected one-way analysis of variance. * p < 0.05 means a statistically significant difference.

| FXR is downregulated in colon cancer tissue and cells
To investigate the expression of FXR in colon cancer, FXR expression data were first downloaded from TCGA database. FXR expression in colon cancer and adjacent tissue was analyzed.
The result implied that FXR was significantly downregulated in colon cancer ( Figure 1A). To further verify the result, qRT-PCR and Western blot were utilized to test the FXR expression in colon cancer cell lines. The results suggested that compared with normal human colon cell line CCD-18Co, FXR expression was prominently lowered in colon cancer cell lines HCT116, SW620, HT-29, and SW480 ( Figure 1B-D). FXR was highly expressed in colon cancer cell line HCT116, while lowly expressed in SW620 ( Figure 1B-D). Therefore, HCT116 and SW620 were chosen for further studies.

| The aberrant expression of FXR affects the proliferation and apoptosis of colon cancer cells
To

| The aberrant expression of FXR affects the glycolysis of colon cancer
In recent years, more and more studies have been focusing on inhibiting glycolysis to block the malignant progression of tumors. In this study, the low expression of FXR was significantly related to the Glycolysis/Gluconeogenesis metabolic pathway subjecting to GSEA ( Figure 3A). In order to study whether aberrant expression of FXR To further determine the effect of aberrant expression of FXR on cell glycolysis and metabolism, a real-time quantitative glycolysis assay was performed. Seahorse method was adopted to analyze ECAR and OCR. ECAR detected the glycolysis capacity of SW620 and HCT116 cells, while OCR detected the oxidative phosphorylation (OXPHOS) viability of mitochondria ( Figure 3D,E). Their results demonstrated that overexpressed FXR prominently repressed the glycolysis level and capacity of colon cancer cells ( Figure 3D).
Overexpressed FXR resulted in a significant increase in the basic oxygen consumption rate and maximum oxygen consumption rate ( Figure 3E). It was indicated that compared with OXPHOS, the glycolysis level was lowered. In contrast, the silenced expression of FXR prominently impelled the glycolysis level and capacity of colon cancer cells ( Figure 3D). The silenced FXR resulted in a significant decrease in the basic oxygen consumption rate and maximum oxygen consumption rate ( Figure 3E). It was indicated that glycolysis level was increased over OXPHOS. In sum, overexpressed FXR repressed glycolysis in colon cancer cells, while silenced FXR promoted glycolysis. Aberrant FXR expression affected the progression of colon cancer by regulating cellular glycolysis.

| CEBPB directly targets FXR
To dig up the upstream gene of FXR, hTFtarget and PROMO databases were utilized. CEBPB was found to be the only one TF that regulated the expression of FXR ( Figure 4A). CEBPB was significantly highly expressed in colon cancer patients ( Figure 4B). The Pearson correlation analysis revealed that the expression levels of CEBPB and FXR were negatively correlated ( Figure 4C). To further verify the results of bioinformatics analyses, qRT-PCR was adopted to test the expression level of CEBPB in colon cancer cells. It was revealed that CEBPB was remarkably upregulated in cells ( Figure 4D), indicating that CEBPB promoted the progression of colon cancer.
To identify the targeted relationship between CEBPB and FXR, ChIPBase and JASPER databases were first adopted. CEBPB was revealed as the key TF that regulated FXR ( Figure 4E). Through bioinformatics analysis, it was predicted that there were at least two possible binding sites for CEBPB on FXR promoter: Site1 (677-687) and Site2(1340-1350) ( Figure 4E). Then the ChIP assay verified the binding relationship between CEBPB and FXR promoter region.
Hereby, two primers containing CEBPB core binding sequences were designed. The sequences were located in the transcription start sites (Region 2 and Region 3). Region 1 (excluding binding elements) was taken as a negative control ( Figure 4F). The binding of CEBPB and FXR promoter was observed in regions 2 and 3, while there were no bands in the negative control (Region 1) ( Figure 4G), implying that CEBPB targeted Region 2 and Region 3 of FXR promoter.
Thereafter, the dual-luciferase assay determined the molecular regulation mechanism of CEBPB targeting FXR. The reported luciferase gene driven by FXR promoter was utilized to test the transcription viability of FXR, thereby measuring the luciferase viability when CEBPB was overexpressed or silenced. When CEBPB was silenced in SW620 cells, the luciferase viability was significantly increased ( Figure 4H).
In contrast, overexpressed CEBPB in HCT116 led to a significant decrease of luciferase viability ( Figure 4I). Comparing the viability of wild and mutant FXR promoters (FXR WT and FXR MUT, respectively), the experimental results indicated that CEBPB knockdown did not affect the luciferase viability of FXR MUT ( Figure 4I). Similarly, overexpressed CEBPB had no effect on FXR MUT activity ( Figure 4I)  co-transfected oe-CEBPB and oe-FXR groups ( Figure 5A). What is more, the CEBPB protein level was prominently reduced in si-CEBPB treatment group, while the FXR protein level was remarkably risen.
CEBPB protein level was remarkably decreased in co-transfected si-CEBPB and si-FXR groups ( Figure 5A). Thereafter, ECAR was taken to analyze the glycolysis process in each treatment group.

| DISCUSS ION
This study analyzed the regulation between CEBPB and FXR, which was further proven by ChIP and dual luciferase assays. The suppression of FXR on tumor cells was evaluated. Percentages of pyruvic acid, lactic acid, citric acid and malic acid, as well as ECAR and OCR of cells in petri dish were measured. A series of cell assays and molecular assays suggested that CEBPB negatively regulated the expression of FXR and accelerated aerobic glycolysis, which facilitated colon cancer cell growth ( Figure 6).  22 For example, the TF CEBPB represses the proliferation of osteosarcoma by regulating its downstream target gene CLEC5A. 23 Here, we clarified that overexpressed CEBPB could accelerate glycolysis and malignant progression in colon cancer cells. In addition, we also confirmed that CEBPB played as a promoter in colon cancer by suppressing the expression of FXR.
Herein, it was found through bioinformatics approach that low-level FXR was remarkably relevant to glycolysis signaling. In the 1920s, Otto Warburg discovered that glycolysis was the main source of energy for cancer cells even with sufficient oxygen. 24 Oxidative phosphorylation is the main mechanism for normal cells to produce energy. However, during cell growth and proliferation, tumor cells require a large amount of energy in a short time.
OXPHOS cannot effectively provide energy for tumors, while glycolysis pathways do. Therefore, cancer cells mainly produce energy through glycolysis, and the lactic acid produced by glycolysis provides an acidic environment for tumor growth. 25