Tumor Immune Estimation Resource (TIMER) is a public database that uses microarrays for estimating the gene expression. UALCAN is a cancer information analysis platform consisting of a transcriptional expression database, and the corresponding clinical information is based on the TCGA cancer data. In this study, the transcriptional expression data of TP53 family genes were obtained from the TIMER and UALCAN databases.

The correlation between the expression of TP53 family genes and clinicopathological parameters, including cancer stage, lymph node metastasis, tumor grade, and tumor histology was analyzed using the UALCAN database.

Furthermore, the immune infiltration of TP53 family members in ESCA was evaluated using TIMER. The scatterplots of TP53 family members were generated to explore the correlation between the gene expression level and the abundance of immune cell infiltration using Spearman’s correlation analysis.

In this study, we used the Kaplan–Meier plot to analyze the prognostic value of mRNA expression of TP53 family members in ESCA. Patients with ESCC and EAC were categorized into high- and low-expression groups, according to the median values of mRNA expression.

This study was approved by the Medical Ethics Committee of the Affiliated Hospital of North Sichuan Medical College; the hospital is located in Nanchong, Sichuan Province, China. The patients provided their written informed consent to participate in this study. Between July 2015 and March 2016, cancer tissues and paracarcinoma normal tissues samples were obtained from 65 patients with ESCC who had undergone esophagectomy at the Department of Cardiothoracic Surgery, without preoperative chemotherapy or radiation. All patients were pathologically diagnosed with ESCC using surgical specimens and biopsies. All the tissue samples were surgically isolated within 30 min to excise an appropriate amount of tumor and normal tissue, soaked in RNAlater solution (Ambion, Carlsbad, CA, United States), and stored at −80°C until further processing.

Human normal esophageal epithelial cell line (HET-1A) was purchased from American Type Culture Collection (ATCC, Manassas, VA, United States), and human ESCC cell lines (TE1 and Kyse150) were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Medical Sciences, Shanghai, China). All the cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Invitrogen, Waltham, MA, United States) at 37°C with 5% CO₂ in the humidified incubator.

The total RNA was extracted from ESCC tissues and cell lines using the TRIzol reagent (Ambion) according to the manufacturer’s instructions. Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, IN, United States), and subsequently, the cDNA was collected and stored at −80°C.

Quantitative Real-Time PCR was detected by the Lightcycler 480 Real-time PCR system (Roche, Mannheim, Germany) under the following conditions: 95°C for 5 min, followed by 40 cycles of 10 s at 95°C, 15 s at 62°C, 72°C for 10 s, 65°C for 60 s, and 37°C for 30 s. For TP53 mRNA detection, forward primer 5′-CCAGGGCAGCTACGGTTTC-3′ and reverse primer 5′-CTCCGTCATGTGCTGTGACTG-3′. β-Actin was used as an internal control: forward primer 5′-GGACTTCGAGCAAGAGATGG-3′ and reverse primer 5′-AGCACTGTGTTGGCGTACAG-3′. Each experiment was performed in triplicate, and the relative expression of mRNA was normalized to the endogenous expression level of β-actin through the 2^{–Δ Δ Ct} method.

Kyse150 and TE1 cells were selected for further functional research. Plasmids encoding knockdown TP53 (sh-TP53) and corresponding controls (sh-NC) were purchased from Hanbio (Hanbio, Shanghai, China). According to the manufacturer’s protocol, 50–60% of confluent ESCC cells were transfected with plasmids using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, United States). The stably transfected cells were screened in the presence of 2 μg/ml puromycin for 14 days (Sigma, Chicago, IL, United States).

Total protein was extracted from tissues and cell pellet lysis, and the concentration of protein in the lysate was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, United States). Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore; Merck KGaA, Darmstadt, Germany). After blocking with 5% skimmed milk for 1 h, the membranes were probed with primary antibodies at 4°C overnight: anti-TP53 (1:1000) and LC3 (1:500) were purchased from Cell Signaling Technology (CST; Danvers, MA, United States), while anti-p-AKT^Ser473 (1:500), anti-p-mTOR (1:500), anti-p-P70S6K, anti-p-4EBP1, and P62 (1:500) were purchased from Sigma (St. Louis, MO, United States), and anti-GAPDH (1:5000) was purchased from CST, which served as a loading control. Then, the membrane was incubated with a secondary antibody for 2 h. The protein expression was visualized using enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific), and the concentration of proteins was quantified by Image J software.

Cell proliferation was analyzed using the crystal violet assay. Briefly, 5 × 10⁵ stably transfected cells were seeded into each well of a six-well plate in duplicates and cultured for 1–4 days. Then, the cells were fixed with pre-cooled 10% formaldehyde for 20 min and stained with 0.1% crystal violet for 15 min. The reaction was stopped by 10% glacial acetic acid, and the absorbance (450 nm) was measured on a SpectraMax Paradigm microplate reader (Molecular Devices, Sunnyvale, CA, United States).

For the Transwell migration assay, 3 × 10⁵ cells suspended in medium without serum were added to the upper chamber of the Transwell plates (Corning Inc., Corning, NY, United States), while 20% FBS-medium was added to the lower chamber. After incubation for 24 h, the cells in the upper chamber were removed by a cotton swab, while the migrated cells attached to the bottom were fixed with 10% formaldehyde and stained with 0.1% crystal violet. Images were captures using a camera equipped with a BX41 Olympus microscope (Leica, Wetzlar, Germany). Three independent experiments were performed.

Gene set enrichment analysis is an analysis method for genome-wide expression of the microarray data that compares the genes with predefined gene sets. Herein, we used the GSEA approach to analyze the biological functions of TP53 gene with 81 ESCC tissues obtained from TCGA using the ClusterProfiler and ggplot2 package in the R software. Using | NES| > 1 and p-value < 0.05 as the threshold of GSEA, pathways were considered significantly enriched when they fulfilled the sub-conditions.

The two sets of data of cancer tissues and adjacent tissue mRNA expression are skewness distribution, as analyzed by the non-parametric statistical method Wilcoxon test. The difference between TP53 expression and clinicopathologic characteristics was analyzed by Fisher’s exact test. The data in this study were analyzed by GraphPad Prism 7.0 software (GraphPad, San Diego CA, United States) and the R software. p < 0.05 indicates a statistically significant difference.

To explore the transcriptional expression of TP53 family members in ESCA, we analyzed 33 types of cancers tissues and the corresponding normal tissues using the TIMER database. As shown in Figure 1A, the mRNA expression of TP53, TP63, and TP73 was significantly upregulated in ESCA tissues compared to normal tissues. Moreover, the comparison of the expression of TP53 family members based on the TCGA database showed a significantly higher expression of all TP53 members in ESCA tissues than normal specimens (Figures 1B–D).

The investigation of the association between clinicopathological features and mRNA expression of the TP53 family by UALCAN showed that the mRNA expressions of TP53 family members were significantly and positively associated with cancer stages, tumor grade, node metastasis, and tumor histology. Compared to non-cancerous tissues, the upregulated expression of TP53 family members was correlated with stage I, II, III, and IV (Figures 2A–C), while no statistically significant difference was detected in TP63 and TP73 mRNA expression between stages II and IV compared to non-cancerous tissues. However, the expression of TP53 family members only between tumor grade 2 and grade 3 had obvious statistic difference. and TP73 mRNA expression in tumor grade 1 was significantly higher than non-cancerous tissues (Figures 2D–F).

Next, the association of the expression of TP53 family members to the node metastasis of ESCA patients was analyzed. As shown in Figure 2G, the mRNA expression of TP53 was positively associated with the node metastasis of ESCA patients. However, the expression of TP63 and TP73 did not differ significantly with nodal metastasis status in N0, N3 and N2, and N3, respectively (Figures 2H,I).

We also analyzed the association between mRNA expressions of TP53 family members and the tumor histology of ESCA patients. As shown in Figures 2J–L, the mRNA expression of TP53 family members was significantly upregulated in ESCC tissues compared to normal tissues, while that of TP63 and TP73 did not show any remarkable difference in ECA tissues. Taken together, the mRNA expression of TP53 was significantly associated with the clinicopathological parameters of ESCA patients.

The correlation between the mRNA expression of TP53 family members with immune infiltration levels in ESCA was investigated using the TIMER database. The mRNA expression of TP53 was obviously related to tumor purity and neutrophils as shown in Figure 3A. However, B cells, CD8 + T cells, and dendritic cells were weakly not significantly correlated with the mRNA expression of TP63 (Figure 3B). On the other hand, the mRNA expression of TP73 showed a remarkable correlation with infiltrating levels of B cell and CD8 + T cells in ESCA (Figure 3C). The above data illustrated that TP53 family members were remarkably related to the infiltration of immune cells, indicating significant effects of TP53 family members on ESCA.

The prognostic value of TP53 family members in ESCA patients was analyzed by Kaplan–Meier plotter. As shown in Figure 4, TP53 [hazard ratio (HR) = 0.28 and log-rank p = 0.026] showed a significantly good prognosis in ESCC patients when mRNA expression was upregulated, and TP73 (HR = 2.74 and log-rank p = 0.0043) showed a negative correlation between high mRNA expression and significantly positive OS in EAC patients. However, the mRNA expression of TP63 did not show any statistically significant association with the prognosis of both ESCC and EAC patients. These results might provide additional evidence about the prognostic biomarkers for ESCA. Thus, we focused on the role of the TP53 in subsequent experiments.

Furthermore, we verified the mRNA expression of TP53 in 65 ESCC patients by RT-qPCR. The results of expression in ESCC patient tissues and the corresponding adjacent tissues were consistent with the analysis using the TCGA database, wherein the mRNA expression of TP53 was upregulated in ESCC tissues compared to the paired adjacent normal tissues (Figure 5A). Notably, >90% (59/65) of ESCC tissues expressed high levels of TP53, while only 10% (6/65) of matched normal tissues expressed high levels of TP53 (Figure 5B). These findings strongly validated that the mRNA expression of TP53 is upregulated in ESCC.

The association between mRNA expression of TP53 and the clinical factors in 65 ESCC patients was analyzed. As shown in Table 1, high mRNA expression of TP53 in ESCC was positively correlated with TNM stage (p = 0.007) and lymph node metastasis (p = 0.030) in ESCC patients, while no association was observed with other clinicopathological features, including age, gender, and differentiation. Conversely, the correlation between the expression of TP53 and TNM stage and lymph node metastasis was consistent with the analysis using the TCGA database.

To further investigate the potential biological function of TP53, the expression of TP53 in Kyse150 and TE1 ESCC cells was knocked down by stable transfection with sh-TP53 plasmids, while sh-NC plasmid was transfected in ESCC cells as a control. As shown in Figures 6A,B, the mRNA expression and the protein level of TP53 were downregulated by sh-TP53 in ESCC cells, which in turn significantly reduced the growth rate of Kyse150 and TE1 cells, as assessed by cell proliferation assay (Figures 6C,D). The Transwell assay showed that the downregulated expression of TP53 suppressed the migratory ability of ESCC cells (Figure 6E); the wound healing assay also displayed a similar trend (Figure 6F). Together, these findings indicated that TP53 is involved in the regulation of proliferation and migration of ESCC cells.

The above results indicated that TP53 gene plays a vital role in tumorigenesis of ESCC. GSEA was utilized to describe the TP53 gene on the cellular process to investigate the underlying biological function. As shown in Supplementary Table 1, 27 gene sets were significantly enriched; among these, TP53 signaling pathway, mTOR signaling pathway, NOTCH signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and pathway in cancer cell cycle are closely related to tumorigenesis. The enriched gene sets of TP53 and AKT-mTOR were shown by normalized enrichment score value ranking in Figures 7A,B. These findings indicated that TP53 promotes ESCC development by regulating AKT-mTOR signaling pathway. The investigation of AKT-mTOR signaling pathway by Western blotting in stably transfected sh-TP53 ESCC cells elucidated the molecular mechanisms underlying TP53 in tumorigenesis and development of ESCC. As shown in Figure 7C, the expression of TP53 was significantly decreased when transfected with sh-TP53 cells compared to sh-NC cells. The phosphorylation of p-p70S6K and p-4EBP1 was increased in TP53-downregulated ESCC cells, indicating that the downregulation of TP53 significantly enhanced the activation of AKT-mTOR pathway. Also, P62 and LC3-II also showed a declining trend compared to the sh-NC group. Collectively, these results suggested that the downregulation of TP53 activates the transduction pathway of AKT-mTOR and inhibits autophagy, which may be responsible for its tumor-suppressive function.