FAM3B promotes progression of oesophageal carcinoma via regulating the AKT–MDM2–p53 signalling axis and the epithelial‐mesenchymal transition

Abstract FAM3B has been suggested to play important roles in the progression of many cancers, such as gastric, oral, colon and prostate cancer. However, little is known about the role of FAM3B in human esophageal squamous cell carcinoma (ESCC). In the present study, we found that FAM3B expression was higher in ESCC tissues than in adjacent normal tissues. Using quantitative real‐time polymerase chain reaction, we found similar results in cell lines. FAM3B expression was significantly related to T/TNM stage. Importantly, Kaplan–Meier analysis revealed that a high expression level of FAM3B predicted a poor outcome for ESCC patients. Overexpression of FAM3B inhibits ESCC cell death, increases oesophageal tumour growth in xenografted nude mice, and promotes ESCC cell migration and invasion. Further studies confirmed that FAM3B regulates the AKT–MDM2–p53 pathway and two core epithelial‐to‐mesenchymal transition process markers, Snail and E‐cadherin. Our results provide new insights into the role of FAM3B in the progression of ESCC and suggest that FAM3B may be a promising molecular target and diagnostic marker for ESCC.

family includes four genes: FAM3A, FAM3B, FAM3C and FAM3D. 11 Over the past decade, intensive studies have suggested that members of the FAM3 gene family may play an important role in the development of a variety of major diseases, including diabetes and cancer. [12][13][14][15] Of the four family members, FAM3B has been studied the most. 16 FAM3B (also called PANDER) was originally identified in the endocrine pancreas and is also expressed in the small intestine, stomach and colon. In addition to identifying its role in glycolipid metabolism, research on FAM3B focuses on its biological role in tumour progression. In 2008, Huang et al 17 first reported that the expression level of FAM3B was lower in human gastric cancer tissues than in adjacent normal tissues and that its lower mRNA level was associated with deeper tumour invasion of gastric cancer tissues. These findings suggested that abnormal expression of FAM3B might be involved in cancer initiation and progression. Since then, the regulatory role of FAM3B in the progression of other tumours, including oral squamous cell carcinoma and colon and prostate cancer, have been reported. [18][19][20] In addition, another FAM3 family member, FAM3C, was reported to be closely involved in the development of oesophageal cancer. 21 These studies have increased interest in exploring whether FAM3B also plays an important role in the progression of oesophageal cancer.
In this study, we demonstrate for the first time that expression of FAM3B is significantly up-regulated in ESCC. Its biological function in ESCC appears to be to inhibit cell death and promote cell migration and invasion rather than regulate the cell cycle. We Control, which is non-governmental organization that exists to help the global health community accelerate the fight against cancer. The clinicopathological information for the samples included age, gender, pathological grade, tumour stage, lymph node metastatic status, and TNM classification. Patients were closely followed up every 3 months during the first 2 postoperative years, and every 6 months thereafter. OS time was calculated from the date of the initial surgery to death. The study was performed with the approval of the hospital ethics committee, and written informed consent was obtained from all patients.

| Tissue microarray and immunohistochemistry
Constructed tissue microarrays (TMAs) contained 40 pairs of ESCC tissue samples and their corresponding adjacent nontumour tissues, as described previously. The TMA sections were first dewaxed and then washed with phosphate buffer saline (PBS) and incubated with 3% H 2 O 2 for 10 minutes. Antigen retrieval was performed by heating the sections from 65°C to 95°C for 20 minutes in a pressure cooker. The temperature was then decreased to room temperature, and the sections were blocked with 5% normal rabbit serum for 1 hour and incubated with rabbit anti-human FAM3B polyclonal antibody (Abcam, Cambridge, UK) overnight at 4°C. The slides were washed and incubated with biotinylated secondary antibody conjugated with haptoglobin related protein (HRP, 1:500) for 1 hour at room temperature. The expression of FAM3B was scored as high or low independently by two experienced pathologists.

| Cell culture
Human ESCC cell lines (ECA109, KYSE150, KYSE9706 and TE-1) and oesophageal epithelial cells (HET-1A) were kindly provided by the State Key Laboratory of Biotherapy of Sichuan University. The cells were cultured in RMPI 1640 medium (Hyclone, USA) with 10% fetal bovine serum (FBS; Gibco, USA) and penicillin, and were maintained in a humidified incubator at 37°C with 5% CO 2 . The cells were passaged when the cell density reached 80%-90%, and the medium was replaced every 2-3 days.

| Real-time PCR
Total RNA was isolated from tissue specimens and cells using the Axygen Mini Kit (Corning, Union City, CA, USA) according to the manufacturer's protocol. The extracted RNA was then reverse transcribed to cDNA using a PrimeScript ™ RT Reagent Kit with gDNA Eraser (Takara, Tokyo, Japan) following the manufacturer's instruction. The cDNA template was mixed with the gene primers of interest and SYBR Green 2X mixture (Bio-Rad, Hercules, CA, USA).
Quantitative real-time polymerase chain reaction analysis (q-RT-PCR) of the reaction mixtures was performed using a Bio-Rad CFX96 Touch (Bio-Rad). All primer sequences used are listed in Table 1. The expression level of the target mRNA was calculated by the 2 -ΔΔCT method. Relative quantification was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript as an endogenous housekeeping control.

| Colony-formation assay
A colony-formation assay was performed to detect cell tumorigenic capability. The cells were seeded in six-well plates (500 cells/well), and the medium was changed every 5 days. The plates were maintained under standard breeding conditions for 14 days, fixed with absolute methanol for 15 minutes, and stained with crystal violet for 20 minutes. Colony counts were based on macroscopic biocenosis.

| Cell cycle assays
In cell cycle assay, cells were first transfected for 48 hours and then fixed with 75% ethanol and stained with 500 μL of PBS containing 50 μg/mL propidium iodide and 0.1% TritonX-100 in the dark at 4°C.

| Protein extraction and Western blot analysis
Cells were lysed in ice-cold lysis buffer containing fresh protease inhibitor. Cellular lysate extracts were scraped from the wells, collected in Eppendorf tubes, and centrifuged at 12 000 g for 10 minutes at 4°C. The total cell proteins were obtained and quantified using a NanoDrop TM 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separate proteins were transferred to a PVDF membrane.
The membrane was blocked with 5% fat-free milk at 37°C for 1 hour and then incubated with the homologous primary antibodies (Abcam) overnight at 4°C. The membranes were rinsed, incubated in peroxidase-conjugated secondary antibody at 37°C for 30 minutes, and analysed using a chemiluminescence system (Amersham Biosciences, UK). The stained bands were scanned and filed, and the pixel intensity was analysed using Alpha processing software (Alpha Innotech, Chengdu, china).

| Lentivirus packaging and transduction
The recombinant vectors were first constructed by cloning full-length human FAM3B cDNA into the pEB-GFP (T2A) PURO vector using a Clon Express II One Step cloning kit (Vazyme, Nanjing, China) according to the manufacturer's protocol and then transformed into  After 4 weeks, the mice were sacrificed and tumour tissues were removed. The protein was extracted from the tumour tissues and used for Western blot analysis.   immunohistochemical results were consistent with the transcription data. That is, FAM3B protein was located primarily in the nucleus of ESCC cells and was expressed at a higher level in tumour tissue than in adjacent normal tissue (Figure 2A,B).

| FAM3B expression is associated with ESCC clinicopathological parameters and prognosis
We explored further whether FAM3B protein expression was significantly related to the clinicopathological parameters of ESCC patients.
FAM3B expression was not significantly related to gender, age, differentiation, or lymph node metastasis. By contrast, increased expression of FAM3B in ESCC was significantly associated with invasion and TNM stage (P = 0.011 and 0.003, respectively; Table 3).
The FAM3B expression measured by immunohistochemistry was used to classify ESCC patients into two groups: a high-expression group (20 patients) and low-expression group (20 patients). Their prognosis was measured as OS. The respective average survival and 5-year survival rates were 58 months and 45% for the low-expression group and 42 months and 15% for the high-expression group.
Kaplan-Meier survival analysis revealed that patients in the high-expression group had poorer OS than those in the low-expression group (P < 0.01; Figure 2C).

| FAM3B promotes ESCC progression in vitro and in vivo
To assess the biological role of FAM3B in ESCC progression, we per-  The colony-formation assay showed that FAM3B silencing signif- To explore further the effect of FAM3B in vivo, we established stably expressed FAM3B using ECA109 cell line, and FAM3B transfection efficiency was measured by Western blot ( Figure 7A).
ECA109 cells that stably overexpressed FAM3B were implanted into the left armpit and control cells into the right armpit of nude mice.
Four weeks after injection, tumours were visible in both armpits of all five mice. As seen in Figure 7B and C, tumours removed from the left armpit were markedly larger in volume than those taken from the right side (P < 0.01), which suggested that FAM3B increased tumour growth in vivo mouse model.

| FAM3B promotes ESCC progression through regulation of the AKT-MDM2-p53 pathway and epithelial-mesenchymal transition
To examine whether the AKT-MDM2-p53 pathway and epithelial-

| DISCUSSION
Although several molecular markers closely associated with tumour progression have been recognized, more sensitive markers are needed because of the poor prognosis of ESCC patients. FAM3B is expressed in many normal human tissues, including the pancreas, small intestine, stomach and oesophagus. Initially, FAM3B was confirmed to be a cytokine that triggers cell apoptosis and was therefore assumed to be a potential tumour marker. 11 In this study, we found that the FAM3B expression level was higher in ESCC tissues than in adjacent normal tissues. Using qRT-PCR, we obtained similar results in cell lines. We also found a significant association between FAM3B expression level and T/TNM stage.  Cell cycle dysregulation is also a major factor affecting cancer cell growth. Of previous FAM3B-related studies, we found only one paper reporting that FAM3B inhibits the cell cycle progression of gastric cancer cells by up-regulation of p21. 42 To provide a better understanding of the molecular functions of FAM3B, we used flow cytometry to analyze the changes in the cell cycle in ECA109/TE-1 cells transfected with the FAM3B siRNA sequence. We found no significant difference (data not shown), which indicates that the mechanism through which FAM3B promotes ESCC progression is independent of cell cycle control. F I G U R E 9 The proposed model for the mechanism of FAM3B-induced ESCC progression. FAM3B overexpression may up-regulate Snail to decrease E-cadherin and to increase N-cadherin, which in turn activates EMT process and consequently promotes ESCC cell migration and invasion. In the meanwhile, FAM3B may inhibit ESCC cell death through the AKT-MDM2-p53 signalling axis HE ET AL.

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In summary, our study is the first to demonstrate that FAM3B functions as a tumour promoter in the progression of oesophageal carcinoma. Our expression data indicated that FAM3B expression is elevated in ESCC tumour cells and cell lines, and showed a positive correlation between FAM3B expression level and T/TNM stage. Our functional experiments confirmed that overexpression of FAM3B inhibits ESCC cell death, increases tumour growth in vivo and promotes ESCC cell migration and invasion. Analysis of the mechanism revealed that FAM3B acts as a regulator of the AKT-MDM2-p53 signalling pathway and the EMT, as proposed in Figure 9. FAM3B may be a potential prognostic marker and molecular target for therapy in clinical treatment. These findings provide a basis for exploring further the molecular mechanisms underlying FAM3B-induced ESCC progression.