Esophageal cancer is the sixth leading cause of cancer-related deaths in the world and the eighth most common cancer globally; esophageal squamous cell carcinoma (ESCC) is the major histological type of esophageal cancers worldwide (Arnal et al., 2015). Despite the improved management and treatment of ESCC patients, the overall 5-year survival rate (10%) and 5-year survival rate following resection (15–40%) have remained low (Huang and Yu, 2018). The poor prognosis and rising morbidity of ESCC highlights the importance of improving detection methods, which is essential before treatment. As early clinical symptoms are not obvious, many ESCC patients are not diagnosed until the clinical symptoms appear in the late stage. In order to improve the prognosis of patients after surgery, patients were often treated with surgery combined with radiotherapy and chemotherapy, but this has not significantly increased the survival rate of ESCC patients. Since we have failed to fully understand the mechanism of the occurrence and development of ESCC, it is necessary and meaningful to explore its potential molecular mechanism to identify new prognostic biomarkers and potential ESCC therapeutic targets.

In recent years, studies have shown that transcription factors might play an important role in the occurrence and development of tumors, which has prompted us to study the clinical value and mechanism of transcription factor PTTG1 in ESCC. PTTG1 was the first proto-oncogene detected from rat pituitary tumor cells, encoding the securin protein—a protein that prevented separins from promoting the separation of sister chromatids (Pei and Melmed, 1997; Arenas et al., 2018). PTTG1 was seen highly expressed in a variety of tumors and was related to the degree of tumor differentiation, invasion, and metastasis (Vlotides et al., 2007). It was shown that PTTG1 played an essential role in the biological function of tumor cells. Its high expression in colorectal cancer promoted proliferation and metastasis (Ren and Jin, 2017). Its high expression in hepatocellular carcinoma, small cell lung carcinoma, and prostate cancer had value for clinical diagnosis and prognosis (Kong et al., 2019; Yang et al., 2019; Ersvær et al., 2020). Although studies found that PTTG1 was likely to exhibit high expression in ESCC, the number of cases has been small and the research was based on a single center study, using a single detection method (Shibata et al., 2002; Ito et al., 2008; Feng et al., 2017). There have been only two reports on the mechanism of PTTG1 in ESCC. It was found that PTTG1 promoted the movement and migration of ESCC cells by activating related genes (Ito et al., 2008; Feng et al., 2017). The possible target genes of PTTG1 as a transcription factor in ESCC had not been reported yet, and the practice of using integrated computational biology to mine ChIP-seq data to study the underlying molecular mechanism of PTTG1 had not been reported.

Based on the existing research on PTTG1 in ESCC, this study used clinical samples to make tissue chips, performed immunohistochemistry (IHC) to detect the expression level of PTTG1 protein in ESCC tissues, and used our clinical sample tissues to conduct RNA sequencing (RNA-seq) to verify the expression of PTTG1 mRNA. The comprehensive analysis and evaluation of PTTG1 expression in ESCC were completed by synthesizing in-house IHC, clinical sample tissue RNA-seq (in-house RNA-seq), public high-throughput data, and literature data. We also explored the possible signaling pathways and target genes of PTTG1 in ESCC by combining the target genes of PTTG1 (displayed by ChIP-seq), differentially expressed genes (DEGs) of ESCC, and PTTG1-related genes (Supplementary Figure 1), revealing the potential molecular mechanism of PTTG1 in ESCC and providing a new target for ESCC research.

Although some studies reported on the expression level of PTTG1 in ESCC, its clinical significance had yet to be verified, and the mechanism of abnormal PTTG1 expression in ESCC was not clear. To address these issues, this study used a variety of detection methods to investigate the expression level of PTTG1 in ESCC, including in-house IHC, in-house RNA-seq, gene chips, public RNA-seq, and a published study. We conducted a comprehensive integrated analysis of the expression data of 2,721 samples from independent studies to calculate SMD and SROC. This study on PTTG1 expression in ESCC had the largest sample size of any related study by far. The analysis results showed that the expression of PTTG1 in ESCC was upregulated. As a transcription factor, PTTG1 could target and regulate specific genes to perform biological functions. Combining the analysis results of DEGs, PTTG1-related genes of ESCC, and ChIP-seq of PTTG1, we found for the first time that SLC25A17 and ERH were likely to be the target genes of PTTG1 in ESCC. These findings could help to study the key role of PTTG1 in the regulation mechanism in ESCC.

So far, there have been only three reports on the clinical significance of PTTG1 expression in ESCC. The sample size of these was relatively small, and they were based on a single center study. The use of a single detection method failed to fully represent the role of PTTG1 in ESCC. At the time of these reports, the widely used high-throughput technology had not been adopted. Differences existed among the results of several studies. One study using RT-PCR of 48 cases of ESCC and its matched samples revealed that PTTG1 expression was upregulated in ESCC, and the average expression level in tumor tissue was 2.19 times than that of normal tissue (0.105 vs. 0.048) (Shibata et al., 2002). In another study, the immunohistochemistry of 50 ESCC samples and normal esophageal tissues suggested that PTTG1 was highly expressed in ESCC. Of 50 ESCC tissues, 31 (62%) were positive, while only 17 of 50 normal tissues (34%) were positive (Feng et al., 2017). Another study, using 113 cases of ESCC immunohistochemistry and immunoblotting, revealed the overexpression of PTTG1. It was found that 14 tumors in 113 cases were negatively expressed; 31 cases expressed 0–10% of tumor cells (1+), 52 cases showed 10–30% tumor cell expression (2+), and 16 cases showed 30% tumor cell expression (3+). PTTG1 expression of 2+ or 3+ was determined as PTTG1 positive (68/113, 60.2%), while PTTG1 staining of normal esophageal epithelium showed no intracytoplasmic staining and occasionally intranuclear staining (about 10%) (Ito et al., 2008). In order to expand the sample size and integrate various detection methods and multi-center data, in this study, we employed various detection methods (immunohistochemistry of tissue chips, RNA-seq, microarrays, and RT-PCR) to study the clinical significance of PTTG1 expression in ESCC tissues. The single detection method and the comprehensive results indicated that the PTTG1 protein and mRNA were highly expressed in ESCC, which was consistent with the reported results (Shibata et al., 2002; Ito et al., 2008; Feng et al., 2017). In the study, SMD = 1.17 (95% CI: 0.72–1.62, P < 0.01), and this result was strongly supported by the SROC combined with AUC 0.86 (95% CI: 0.83–0.89). This study used a large sample to verify the upregulation of PTTG1 in ESCC, suggesting that high expression of PTTG1 might promote the occurrence of ESCC.

In addition to its role in promoting ESCC, we also discussed the possibility of PTTG1 advancing tumor progression. It was reported in the literature that the high expression of PTTG1 was closely related to the pathological stage and lymph node metastasis (Shibata et al., 2002). In vitro, knocking down PTTG1 in EC-1 and Eca-109 cell lines could inhibit EMT, migration, and metastasis (Ito et al., 2008; Feng et al., 2017). This study also examined the relationship between the progress of PTTG1 and ESCC on a variety of data sets. In our study, we have confirmed that PTTG1 was significantly overexpressed in ESCC tissues, and in the prognosis analysis, the survival condition of patients with high PTTG1 expression was worse than that of patients with low PTTG1 expression (HR = 1.29), but no significant statistical difference was found. The reason for this situation may be that overexpression of PTTG1 plays an important role in the occurrence of ESCC, but it may have little effect on the development and progression of ESCC. Of course, this hypothesis needs to be further verified by our future experiments. We did not establish a relationship between the high expression of PTTG1 and other pathological parameters that represented the clinical process; however, the expression of PTTG1 in patients less than or equal to 60 years old was higher than that of patients older than 60, and a higher expression of PTTG1 was correlated to a lower age. The mechanism of the relationship between PTTG1 and the patient’s age was unclear and requires further study.

We then investigated the reasons for the high expression of PTTG1. The mechanism of PTTG1 in ESCC has been reported in previous studies. PTTG1 induces EMT and metastasis in ESCC by activating GLI1 (Feng et al., 2017). PTTG1 might also regulate the expression of multiple genes related to cell movement, including members of the Ras–Rho oncogene superfamily (Ito et al., 2008). As a transcription factor, PTTG1 may achieve its biological function through targeted regulation of a certain set of genes. However, there was no report on the potential target genes of PTTG1 in ESCC. By combining the target genes displayed by ChIP-seq of PTTG1, DEGs of ESCC, PTTG1-related genes, and the possible signaling pathways of PTTG1, it was observed that PTTG1 may interact with these genes through chemokines and cytokine signaling pathways. The inflammatory response, integrin signaling pathway, ubiquitin proteasome pathway, and apoptosis signaling pathway played a role in ESCC. By constructing a PPI network and combining ChIP-seq data, we obtained four potential target genes, SPTAN1, SLC25A17, IKBKB, and ERH. These hub genes all had binding sites with PTTG1. Interestingly, these genes had been reported as tumor related, and SPTAN1 was a family of filamentous cytoskeletal proteins that acted as an essential scaffold protein for stabilizing the plasma membrane and organizing intracellular organelles. The encoded protein had functions of DNA repair and cell cycle regulation. The expression of SPTAN1 in metastatic colon cancer was lower than that in non-metastatic colon cancer. The low expression of SPTAN1 reduced the ability of cell-to-cell contact, which facilitated the metastasis of colon cancer cells (Hinrichsen et al., 2014; Ackermann et al., 2019). It was found that SPTAN1 expression increased in ovarian cancer after chemotherapy (L’Espérance et al., 2006). The SLC25A17 gene was the only member of the mitochondrial carrier family that had previously been confirmed to be located in the peroxisomal membrane. It encoded a peroxisomal membrane protein of the mitochondrial solute carrier family and display varied expression levels in 21 human tissues (including brain, esophagus, lung, liver, and kidney tissues) (Agrimi et al., 2012). However, only one relevant study reported its role in tumors, showing that genetic changes in SLC25A17 (CNV deletion) were closely associated with the overall survival rate and relapse-free survival rate of neuroblastoma patients (Khan et al., 2015). Because SLC25A17 is positively correlated with TF PTTG1 expression, it was worth further study. IKBKB, also known as IKKβ, is a nuclear factor κB kinase subunit β inhibitor, affecting cell physiology in many ways and phosphorylating proteins to regulate cellular processes from the cell cycle to metabolism and differentiation (Page et al., 2017). Excessive activity of IKBKB in oral and dental epithelial cells leads to changes in the composition of epithelial cells and the expression of several tumor suppressor proteins and miRNAs. This results in cells in a pre-tumor state, and the lack of p53 or p16 tumor suppressor proteins and the loss of p19 would cause odontogenic tumors and metastases to local lymph nodes and lungs. IKBKB was observed in the areas of tumor ameloblasts that express keratin K5 at high levels (Page et al., 2020). IKBKB could be activated by α-KG produced by GDH1 and then upregulate GLUT1 to promote glucose uptake and tumor cell survival, thus accelerating the occurrence of glioma (Wang et al., 2019). ERH was an mRNA splicing and mitotic factor, and its function was related to pyrimidine biosynthesis and cell cycles (Gelsthorpe et al., 1997). The ERH gene might affect the apoptosis of bladder cancer T24 cells through TLR, NF-κB, TNF, or TGF-β signaling pathways, activating the growth of malignant tumors (Pang et al., 2020). The research team also proved that ERH knockdown could inhibit the transfer of BUC T24 cells in vitro and in vivo through nude mice tail vein metastasis experiments. They found that ERH gene knockdown suppressed MYC-mediated migration and invasion in bladder cancer T24 cells (Pang et al., 2019). In primary human breast cancer and breast cancer cell models, quantitative RT-PCR found that ERH expression was significantly more upregulated in tumorigenic breast cancer cell lines than in non-tumorigenic cell lines (Zafrakas et al., 2008). This study also found a significant positive correlation between ERH and PTTG1 expression; the molecular relationship between these is also worthy of further study. These reports, to some extent, supported our hypothesis that PTTG1 may play an important role in the occurrence and development of ESCC by upregulating SLC25A17 and ERH.

Future research could continue this work and improve on the current deficiencies. First, extra research should be conducted on the relationship between PTTG1 and ESCC progress and prognosis to explore whether PTTG1 could act as a factor to evaluate the prognosis of ESCC. Second, the research should investigate whether PTTG1 can be detected in body fluids and whether it contributes to the early diagnosis of ESCC. Third, a ChIP-seq of PTTG1 using the ESCC cell line needs to be performed. Although the target genes of PTTG1 in different tissue cells might partially overlap, they also have tumor specificity. Fourth, in vivo and in vitro experiments need to be carried out to study the biological functions and related molecular mechanisms of PTTG1. Fifth, related experiments need to demonstrate the function of the highlighted potential markers in our study.

This study combined multiple detection methods—IHC, RNA-seq, and gene chip data—to comprehensively demonstrate for the first time that PTTG1 was highly expressed in ESCC tissues. This was true from the protein level to the mRNA level and from individual research to integrated analysis. The results were confirmed by a study based on 2,721 cases. It was found that the high expression of PTTG1 may play an important role in the formation of ESCC. These roles may be completed by PTTG1 regulating the downstream target genes SLC25A17 and ERH; however, this hypothesis requires extra experiments to verify.

The data presented in the study are deposited in the Gene Expression Omnibus (GEO) (https://www.ncbi.nim.nih.gov/geo/) repository, accession number (GSE164158).

The studies involving human participants were reviewed and approved by the First Affiliated Hospital of Guangxi Medical University. The patients/participants provided their written informed consent to participate in this study.

All authors contributed to the study conception and design. H-FZ performed the data extraction and statistical analysis and drafted the manuscript. S-WC and H-JZ conceived and designed the study and edited the manuscript. L-JY, R-QH, and GC contributed to the design of the study, supervised all the experiments, and corrected the manuscript. Z-GH, XY, Y-WD, JL, and Z-WF collected, extracted, and analyzed the data. J-XM, Z-QT, C-BL, RL, L-HY, and JM critically revised the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.