A comprehensive analysis of different types of databases reveals that CDH1 mRNA and E-cadherin protein are not downregulated in most carcinoma tissues and carcinoma cell lines

CDH1 is a gene that codes for the epithelial-cadherin (E-cad) protein, which is embedded in the plasma membrane of epithelial cells forming the tissues that cover the body surfaces and line the walls of cavities, channels, and glands [1, 2]. E-cad is a calcium-dependent cell–cell adhesion protein that forms homophilic interactions in adjacent epithelial cells establishing adherens junctions [3, 4]. This protein plays a major role in embryonic development and morphogenesis [5, 6]. In its inactive form, E-cad contains a short signal sequence for import to the endoplasmic reticulum (ER), a 130 amino acid pro-peptide, a single transmembrane domain, a 150 amino acid cytoplasmic domain, and a 550 amino acid ectodomain [7, 8]. E-cad is activated after cleavage of the pro-peptide in the presence of calcium ions [9]. In the extracellular matrix (ECM), the ectodomains of E-cad on adjacent cells bind each other to form adherens junctions, while the cytoplasmic domain of E-cad interacts with β-catenin, which in turn binds α-catenin connecting to the actin cytoskeleton of the cell leading to the stabilization and integrity of the epithelial tissues [10, 11].

The loss of E-cad expression has been considered a hallmark of cancer progression and metastasis [12, 13] via loss of heterozygosity of the chromosomal region 16q22.1 containing the CDH1 locus, nonsense mutations [14], or promoter methylation [15]. E-cad activity as a tumor suppressor manifests via its loss during epithelial-mesenchymal transition (EMT) and/or regulation during metastatic progression, where its loss leads to increased tumor cell migration and invasion [16]. E-cad also plays a role in primary tumor development, progression [17, 18], and metastatic colonization [19]. The loss of E-cad expression is thought to disrupt adherens junctions leading to the acquisition of motility/invasiveness of metastasizing tumor cells. Although some carcinoma cells undergo EMT, many carcinoma cells neither fully lose the ability to produce E-cad nor undergo a mesenchymal‐to‐epithelial transition (MET) during metastasis [20‐22]. Assertions as to the necessity of EMT and its reverse MET in metastasis have been controversial [23‐25] as many metastatic tumor cells still express E-cad [26‐29].

In this study, we analyzed multiple large transcriptomics, proteomics, and immunohistochemistry datasets on clinical cancer samples and cancer cell lines to determine the levels of CDH1 mRNA and E-cad protein in different carcinomas during tumor progression. Strikingly, the levels of CDH1 mRNA and E-cad protein were not reduced in most of the examined tumors, even in the later stages of cancer compared to respective healthy tissues. The only exception to this trend was kidney cancer, which exhibited significantly lower levels of CDH1 mRNA and E-cad protein, the pattern normally described in textbooks. The observations presented in this study demonstrate that the changes in E-cad expression during tumor progression and metastasis are more complex than widely believed.

The loss of E-cad has been widely considered a hallmark of metastatic cancers and critical for metastasizing tumor cells to break away from the epithelial tissues to invade the tumor stroma [12‐14]. This observation was established primarily with the help of invasive lobular breast cancer (ILC) tissues, in which the loss of E-cad has been shown to play a key role [14, 50‐52]. Our analysis of clinical cancer tissues revealed that CDH1/E-cad expression was downregulated only in a few types or subtypes of tumors among the large group of tumor types or subtypes examined; ILC happens to be a subtype of breast tumors in which E-cad expression was downregulated (Fig. 4F). Another major exception is kidney carcinoma, which exhibited the well-described loss of E-cad expression (Figs. 1C and 7). In agreement with recent debates on the role of E-cad in tumor progression and metastasis [63, 64], our analysis demonstrates that CDH1 mRNA and E-cad protein are not downregulated in the majority of carcinomas (Figs. 1, 4, and 5) or during tumor progression in most carcinomas (Figs. 2 and 4E, and Supplementary Figs. 1 and 2—Additional file 1). For a more detailed analysis of the role of E-cad in EMT in tumor progression, tumor samples exhibiting hybrid EMT or partial EMT [65‐68] may be required. Because it is difficult to obtain data on the hybrid EMT or partial EMT samples from cancer patients, at least in large quantities, our studies cannot provide insight into the role of E-cad in complex phenomena like hybrid EMT or partial EMT in metastasis, which shows a limitation of this type of study.

It is interesting to note that CDH1 mRNA and/or E-Cad were upregulated in most cancers in the early stages of tumor development and the levels remained elevated as tumors progressed to later stages across most carcinoma types (Figs. 2 and 4E, and Supplementary Figs. 1 and 2—Additional file 1). These results suggest that most carcinomas may require higher levels of E-cad expression for tumor formation and tumor progression in earlier stages of tumor development, and this requirement needs to be maintained even after metastasis has occurred. One possibility is that the upregulation of CDH1/E-cad expression in carcinoma cells is an adaptive response to the abnormal signaling inside tumor cells, which is known to result in increasingly altered cell–cell adhesion and actin cytoskeleton rearrangement during tumor formation, progression, and invasion [69‐71]. For example, it has been shown that tumor cells can upregulate proteins that are directly related to the rearrangement of the actin cytoskeleton [72, 73] and that there is rearrangement (but not loss) of E-cad-based adherens junctions during neoplastic transformation [69]. Tumor cells may respond to these types of changes in cell–cell adhesion and actin cytoskeleton rearrangement by expressing more E-cad to restore the altered cell–cell adhesion and epithelial tissue integrity during tumor formation, progression, and invasion. It has also been shown that E-cad plays an important role in preventing anoikis, the induction of apoptosis after the loss of attachment to the ECM and neighboring cells [71, 74]. To prevent anoikis induced by truncation of the cytoplasmic domain of E-cad which results in disruption of the binding of the domain to β-catenin, a linker protein that connects the actin cytoskeleton to the cytoplasmic domain of E-cad [74, 75], tumor cells may be required to upregulate E-cad [76, 77].

Since CDH1/E-cad upregulation is widespread in carcinomas (Figs. 1, 4, and 5) and the levels remain elevated as tumors progressed to later stages across most carcinoma types (Figs. 2 and 4E, and Supplementary Figs. 1 and 2—Additional file 1), the effect of higher levels of CDH1 mRNA on carcinoma patient’s survival (Fig. 8) suggest that the role of E-cad on carcinoma development and progression is more complex than previously thought and warrants further investigation. Although the Log-rank tests allowed us to establish a positive correlation between CDH1 mRNA levels and cancer patient’s survival (Fig. 8), the survival tests did not allow us to conclude whether CDH1 expression is functionally linked to cancer patient’s survival, showing another limitation of this study. To gain further insight into the potential value of CDH1 mRNA levels in cancer prognosis and the role of CDH1/E-cad in carcinoma development and progression, future studies should consider additional clinical data, such as median survival time, age, and tumor stages. In addition, since CDH1 mRNA is markedly upregulated in some types of tumors, such as colon and endometrial carcinoma (Fig. 1A), from the early stages of tumor development (Fig. 4E, and Supplementary Fig. 2A-C—Additional file 1), it is worth further investigation to determine whether CDH1 mRNA levels can serve as a reliable biomarker for early diagnosis of these carcinomas.

It is well established that metastatic carcinoma cells invade the stroma and migrate in single cells or collectively in groups [78]. In single-cell invasion/migration, single cells acquire the ability to break away from the primary tumor tissues through the loss of E-cad [79, 80]. In contrast, in collective cell invasion/migration, most of the tumor cells localized in the interior of a cell cluster maintain elevated levels of E-cad expression and only the tumor cells on the edge of the cluster express low levels of E-cad, which allow the cluster of cells to break away from the primary carcinoma tissues [23, 27, 81, 82]. Most previous studies designed to investigate tumor EMT and metastasis normally used in-vitro 2-dimensional cell culture (2D) or 3D scaffold cell culture with a focus on single-cell invasion/migration [53, 59, 83, 84]. Results from these types of studies may not reflect the situation in the collective cell invasion/migration [63, 64, 85, 86]. Our findings that CDH1/E-cad expression is not significantly downregulated when primary tumors progress into metastatic tumors (Fig. 6), which are consistent with the observations from other groups [13, 20, 28, 29], suggest that single-cell invasion/migration may not be the preferred mode of invasion/migration, and collective invasion/migration might be the predominant form of invasion/migration for most carcinomas, a notion that is supported by several studies monitoring metastatic tumors in circulation [81, 87, 88]. Furthermore, after metastatic carcinoma cells settle down in a new place, the metastatic carcinoma cells re-acquire epithelial cell phenotypes via MET [89, 90]. It is also possible that MET can contribute to the elevated or unchanged levels of E-cad in metastatic cancer cells.

CDH1 mRNA and E-cadherin protein are not downregulated in most carcinoma tissues and carcinoma cell lines tested in this study. Thus, the role of E-cad in tumor progression and metastasis may have previously been oversimplified. Because CDH1 mRNA is markedly upregulated in the early stages of tumor development of some types of tumors, such as colon and endometrial carcinomas, CDH1 mRNA levels may serve as a reliable biomarker for the early diagnosis of these carcinomas.