Following the description and introduction of the surgically induced reflux model of esophagitis in rats, it has been extensively used to study esophageal carcinogenesis in order to develop novel preventive and treatment strategies[8, 21–23]. Among various surgical approaches available, EGDA is currently the most effective surgical method to induce animal reflux models[8, 24]. Different from other surgical approaches that encourage acid or alkaline reflux only, EGDA diverts both the gastric contents and some duodenal secretions to the esophagus, which is superior in promoting and simulating the process of human gastroesophageal reflux disease (GERD) from esophagitis to Barret’ esophagus (BE), leading to esophageal dysplasia and consequently adenocarcinoma. In our study, the combination of EGDA with frequent iron injection (not as a carcinogenic agent) promoted tumorigenesis while the application of iron alone had a small influence, indicating that iron dextrin could potentiate further the oxidative stress in the inflamed esophageal epithelium. Higher oxidative stress could in turn aggravate the inflammatory reaction, resulting in abnormal cell proliferation and transformation, with eventual esophageal carcinogenesis[3, 8, 25].
We reported previously DSG2, DSG2 and PKP2 were differently expressed in both esophagitis and esophageal cancer based on bioinformatics. DSC2 and DSG2 are members of the desmosomal cadherin family. Desmosomes not only contribute to an intercellular adhesive which is essential in the normal organization and stabilization of epithelial tissues, but also participate in cellular differentiation, transformation, and tumorigenesis. Moreover, during malignant transformation of epithelial cells, alterations in the expression and function of intercellular junctions might result in tumor invasion and metastasis. There are four types of intercellular junction presenting in vertebrates: desmosomes (DSMs), adherens junctions (AJs), tight junctions (TJs), and gap junctions (GJs),which are crucial for maintaining epithelial homeostasis. All these complexes work as a single unit, interdependently rather than individually. The formation of DSMs is dependent on classic cadherin-mediated adhesion. E-cadherin is the classical cadherins of adherens junctions. It has been reported that E-cadherin can associate with some Dsgs (DSG2), and this interaction may help initiate early stages of DSM assembly. Gap junctions metabolically and electrically connect the cytoplasm of adjacent cardiomyocytes. Meanwhile, some articles summarized the involvement of gap junctions in carcinogenesis . Connexin 43 (Cx43), an important gap junction protein, has been reported associated with esophageal squamous cell carcinoma. DSMs could contribute to Cx assembly and GJ function. The relationship between the DSM and Cxs was first studied in the heart, showing that PKP2 is critical for Cx43 expression and function. The above reports suggested that DSMs might impact gap junction. The connection between them will be explored further in the following study. As outlined above, there were tight connection among these intercellular junctions. The aim of our study is to elucidate the mechanism underlying the effect of DSG2, DSC2 and PKP2 on tumor progression in esophageal adenocarcinoma.
Our findings confirmed the differential expression of DSC2, DSG2, and PKP2 from benign esophagitis to esophageal adenocarcinoma. The upregulation of all these at mRNA expression levels in esophageal adenocarcinoma and the differences when compared to the normal esophagus or benign esophagitis were significant. Besides, the analyses of immunohistochemistry and western blot further validated the above observation, in which the protein expression levels of DSC2, DSG2, and PKP2 were all significantly elevated in EAC. Furthermore, in addition to quantitative changes of these protein expressions in the development of esophageal adenocarcinoma, immunohistochemical staining also demonstrated changes in their cellular localization. Several studies have described alterations of protein expression leading to structural changes of desmosomes, which can induce cell-transformation phenotype and promote carcinogenesis[19, 31, 32]. However, the mechanism involved in the changes of various desmosomal components remains unclear.
Both DSC2 and DSG2 are members of the desmosomal cadherin family. They are transmembrane proteins that maintain intercellular connectivity. The ectodomains of DSC are connected with the DSG at the extracellular region of apposing cells, and the intracellular regions of DSC and DSG directly or indirectly interact with the armadillo proteins including Plakophilins (PKPs), Junction plakoglobin (JUP), Desmoplakin (DSP), Intermediate filaments (IFs), and other desmosomal complexes, which mediate intercellular adhesion in epithelial tissues[12, 19] and may have a direct or indirect role in regulating cell differentiation and tumorigenesis. JUP, which is an homologous protein withβ-catenin, can replace β-catenin in adherens junctions and stimulate the transcription of WNT target genes, including oncogenic targets such as CCND1 (encoding cyclin D1). So, we inferred that desmosomes dysfunction can promote cancer by WNT-β-catenin signalling pathway indirectly. Additionally, PKP2 has been reported functioned as an intracellular inhibitor of the Wnt/β-catenin pathway in colon cance. We will explore the connection between desmosomal cadherin family and Wnt/β-catenin pathway furtherly.
Through modified cell adhesive strength or changes in intracellular and intercellular signaling, the expression patterns of desmosomal cadherin may have been altered, which can affect cell behavior and drive proliferation under some circumstances. In our study, the levels of mRNA and protein expression of DSC2 and DSG2 progressively increased from the epithelial of esophagitis to dysplasia and carcinoma, suggesting an important role of DSC2 and DSG2 overexpression in tumorigenesis of esophageal epithelia. These findings were consistent with previous studies, in which both the DSC2 and DSG2 were overexpressed in squamous cell cancers of the skin and head and neck (HNSCCs). This could enhance internalization, modulate extracellular vesicles (Evs) secretion and paracrine signaling, and increase local and distant mitogenic effects that encourage tumor progression[23, 35]. Besides, the loss of desmoglein-2 (DSG2) has been associated with decreased epithelial cell proliferation and suppressed xenograft tumor growth in mice. In addition, DSC2-positive urothelial carcinomas (UC) have a more advanced stage disease than those tumors without DSC2. Meanwhile, other studies have reported that DSG2 overexpression may promote tumorigenesis in basal cell carcinoma (BCC) by activating Stat3 in the basal layer of the skin of mice, which potentiates the proliferation of CRC through up-regulation of PNN and activating EGFR/ERK signaling pathway. Furthermore, DSG2 has been shown to induce and activate urokinase-type plasminogen activator receptor-related signaling cascade and accelerate cutaneous wound healing, which plays a critical role in the vasculature that is independent of its canonical role as a component of desmosomes in a distinct subpopulation of progenitor cells .
PKP2 is a member of the armadillo family generally localized in the nucleus and cytoplasm of epithelial tissues and cardiomyocytes, which binds the desmosomal cadherins and desmoplakin[42, 43]. Lately, PKP2 has been implicated in tumorigenesis and/or invasion and metastasis of cancer. For instance, in some soft tumors and highly proliferative colonies of cultured mesenchymal stem cells, the upregulation of PKP2 and its integration into adherent junctions (Ajs) have been observed. PKP2 may excite Topflash activity, leading to the translocation of desmosomes. Another study has revealed that PKP2 can promote actin recombination and regulate the assembly of the desmosomal complex by connecting Ras homolog A and protein kinase C-dependent pathways. In our study, the mRNA and protein levels of PKP2 were significantly upregulated in esophageal adenocarcinoma tissues compared with tissues of normal esophagus and esophagitis in rats. Particularly, the expression of PKP2 progressively increased from esophagitis to esophageal adenocarcinoma, and significantly elevated in EAC. These findings were consistent with several previous studies and suggested that PKP2 might be essential for carcinogenesis. Indeed, reduced expression of PKP2 has been associated with inhibition of glioma cell proliferation and migration. Moreover, decreased PKP2 expression reduces Epidermal growth factor (EGF)-dependent and EGF-independent epidermal growth factor receptor (EGFR) dimerization and phosphorylation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development[47, 48]. Besides, PKP2 has been implicated in the invasiveness of bladder cancer as a result of loose adhesion via the epithelial-mesenchymal transition (EMT) and β-catenin–mediated signaling pathways. The redistribution of PKPs could promote adhesion, differentiation, and the localization of PKPs from desmosomes to the nucleus that contribute to tumorigenesis. In a previous study, strong PKP2 immunoreactivity has been observed in 85.7% of metastatic tumors of oropharyngeal carcinoma. Thus, an increase of plakophilin could promote carcinogenesis via the stimulation of translation and proliferation, while the loss of plakophilin may contribute to carcinogenesis and/or metastasis via loss of contact inhibition and increased motility.