In 1998, claudins were identified as the major integral membrane proteins essential for tight junction (TJ) assembly (1). Since then, 27 members of this gene family have been found in mammals. Our contemporary understanding of claudins is based on their canonical barrier and fence functions. Recently, this view has expanded considerably following increasing evidence suggesting that claudins may be involved in signal transduction and that they may be causally important in tumorigenesis (2). Obvious dysregulation of claudin expression has been found in a number of cancer tissues. Owing to the specific expression profile and differences between normal and tumor cells, claudins are attractive targets that can theoretically enable selective drug delivery with minimal adverse events (3). To effectively target claudins for clinical applications, it is essential to understand their roles and fully elucidate the complicated mechanisms by which the expression of claudins is dysregulated in cancer. The present study reviews the current knowledge describing the dysregulation of claudin expression in cancer and discusses the intrinsic and extrinsic determinants of the context-specific expression patterns of claudins.

Epithelial and endothelial cells in most living systems form a sheet-like structural interface between the external environment and internal compartments that not only protects the internal organs, but also acts as a selective barrier between the body and the external environment that restricts free exchange across the paracellular space (4). The paracellular space between neighboring cells in the epithelial sheet is connected by several types of cell-cell junctions, which can be categorized as TJs, adherens junctions (AJs) and desmosomes (Fig. 1) (5). AJs bind with the cytoskeleton to impart support and serve as signaling hubs, playing important roles in regulating gene expression (6). Desmosomes create a strong structural network that binds cells together, but is not continuous and cannot prevent solute transport (7). Therefore, TJs are the chief intercellular junctions that act as permeability barriers and confer polarity to epithelial cells in close proximity to adjacent plasma membranes containing TJ strands formed by TJ proteins (8). TJ proteins are diverse and can be divided into three groups: Transmembrane proteins, adaptor proteins and signaling proteins associated with TJs (9). These proteins interact with each other and other proteins to form a multimeric protein architecture. As key proteins, claudins are a family of integral membrane proteins that are essential for TJ strand assembly.

Claudin (which means ‘to close’, named after the Latin word ‘claudere’) was identified as the major integral membrane protein forming TJ strands by Tsukita in 1998 (1). A total of 27 homologous claudin genes have been identified in mammals, and numerous more claudin proteins are selectively expressed by alternative splicing in various tissues. According to homology, claudins 1–10, 14, 15, 17 and 19 are homologous classic claudins, while claudins with less homology (claudins 11–13, 16, 18 and 20–27) are non-classical claudins (10). Based on their functions, claudins can also be classified as paracellular barrier-forming claudins function in intracellular space sealing, and paracellular channel-forming claudins function in selective permeability. The paracellular barrier-forming group includes claudins 1, 3–5, 7, 11, 14 and 18, which are responsible for restricting transport and enhancing the barrier function of the cell. The claudins involved in paracellular channel-forming ensure proper availability of water and ions for cellular functions; for example, claudins 10a and 17 allow transport of anions, claudins 10b and 15 allow passage of cations, and claudin-2 allows transport of both anions and water (11). The structure of claudins includes four transmembrane domains, the intracellular N and C termini, and two extracellular loops (ECLs) (ECL1 and ECL2) (Fig. 1). ECLs are involved in the formation of interactions between claudin strands and determine the characteristics of claudin-based TJs, as their homology is low among claudins (12). ECLs are also specific binding sites for Clostridium perfringens enterotoxin and monoclonal antibodies, which have been utilized for imaging and therapy in pathological conditions (9).

Our contemporary understanding of claudins is based on their canonical barrier and fence functions, which work on the basis of forming TJ complexes. Based on the crystallographic structure of claudin-15 and cysteine cross-linking experiments with mutated claudin-15 and claudin-2, as well as freeze-fracture electron microscopy images, Suzuki et al (13) proposed a model of paracellular pores in which β-barrel-like channels are formed by the apposition of two anti-parallel double rows of claudins in the membrane of two adjacent cells. Given the results of the crystallization of some other claudins (claudins 3, 4 and 19), this structure seems to be common to other claudins, with differences in residues of their extracellular regions accounting for their different permeabilities and selectivities (14–16). According to physiological studies, two mechanisms have been proposed for paracellular permeability: The ‘pore’ mechanism, in which solutes or ions pass through a paracellular channel formed by the TJ strands (enabling transport of molecules with an estimated diameter of ~4 Angstrom), and the ‘leak’ mechanism, in which solutes supposedly pass through breaks in the TJ strands (permitting permeation of molecules up to a size limit of ~60 Angstrom) (17). Claudins are also essential for the asymmetric localization of membrane proteins and lipids in the exoplasmic leaflet, acting as a membrane fence in epithelial cells (18).

Our understanding of claudins has expanded considerably over decades of research, following increasing evidence suggesting that claudins may be involved in signal transduction. As the PDZ domain-binding motif located in the cytoplasmic C-terminal tail of claudins can directly interact with zonula occluden (ZO) family proteins, which play important roles in a number of cellular processes, it is not unexpected that claudins participate in signal transduction (9). Claudins mainly function as inhibitory factors through interaction with numerous other signaling pathway elements (such as yes-associated protein/transcriptional co-activator with PDZ-binding motif and β-catenin), retaining them in the submembrane compartment (19,20). On the other hand, a number of signaling pathways can modify claudins to regulate their protein localization, protein interactions and overall turnover (2). Moreover, mounting evidence suggests that claudins are localized to sites outside the TJ complex, such as the basolateral membrane and nucleus (21). Little is known about the functions of these claudins, and available data support a cell-extracellular matrix interaction and signaling function that integrates, codifies and transports information into the cell (21). These functions are mainly achieved through the formation of a complex between claudins and other molecules, such as integrins, epithelial cell adhesion molecules and matrix metalloproteinases, which leads to the activation of signal transduction (Fig. 2) (22–24).

In line with their aforementioned important functions, dysregulation of claudin-mediated barrier function and signaling is a precursor for the pathogenesis of numerous human diseases, including a number of cancer types (9). For example, mutations of CLDN1, CLDN5, CLDN14, CLDN16, and CLDN19 have been reported to cause human neonatal ichthyosis, sclerosing cholangitis, non-syndromic deafness, familial hypomagnesaemia and other symptoms (9). Knockout of CLDN18 leads to increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), and tumorigenesis in mice (20). The dysregulated expression of claudins in cancer has attracted attention, given the increased burden of cancer worldwide.

The claudin expression profile of numerous different tissues and cells, including normal and tumor tissues, has been assessed at the mRNA and protein levels. Studies have revealed a range of outcomes that reflect the complexity of claudins in terms of spatial localization, tumor type and stage of disease. Through a comprehensive review of the transcription data in The Cancer Genome Atlas (TCGA), using the online tool GEPIA (http://gepia.cancer-pku.cn) (Fig. 3), the protein expression data in the Human Protein Atlas (https://www.proteinatlas.org) and individual studies, most of the claudins, if not all, have been reported to show the following expression features.

First, the protein level of claudins is not necessarily correlated with the mRNA level. Although 26 claudin genes have been found in humans (CLDN13 is absent), not each one has been validated at the protein level. Currently, only claudins 1–7, 10–12, 15, 16, 18, 19 and 23 have been identified at the mass spectrometry level, although claudins 8 and 9 have been identified at the protein level by immunoblotting, and some claudins may be expressed in tissues that have not been probed (25). Of them, claudins 1, 3, 4, 6, 7 and 18 are the most extensively studied in tumors.

Second, claudin transcripts can be alternatively spliced to produce isoforms, and some of these isoforms are only found in cancer. Alternatively spliced transcript variants have been found for the majority of CLDN family members, although the protein forms of some of these isoforms have not been detected. Currently, credible immunological evidence exists for the presence of the claudin 10 and 18 protein isoforms claudins 10a and 10b and claudins 18.1 and 18.2, respectively (26–28). In addition to splice variants that result from differing physiological conditions, many more transcript variants that do not exist in normal tissues may result from alterations that affect RNA splicing, which dysregulate the expression of CLDN genes, contributing to cancer progression (29).

Third, the expression of claudins changes dynamically during development and differentiation. Claudin-6 expression was reported to be 90-fold higher in undifferentiated human pluripotent stem cells than in differentiated cells, while in adult tissues, it was nearly completely absent (30,31). The expression of claudin-1 declines with differentiation, whereas the expression of claudin-4 increases and is highest in the late stage, in which human embryonic stem cells transition into hepatocyte-like cells (32). In the colon, claudin-7 expression gradually increases with the differentiation of epithelial cells into luminal cells, which results in an expression gradient from the basal side to the surface of the intestinal lumen (33). Age-related changes in expression have also been reported, including changes in claudin-3 expression, which is downregulated with aging (34).

Fourth, although various claudins are expressed in the same tissue simultaneously, distinct claudin members are expressed in a tissue-specific manner. Claudins 18 and 19 are two typical examples. Claudin-18 expression is restricted to the lung, where it is present as the isoform claudin-18.1, and to the stomach, where it is present as the isoform claudin-18.2 (27). Claudin-19 is detected mainly in the thick ascending limb of the loop of Henle, the retinal epithelium and peripheral neurons in normal tissues (35). The expression of claudins 1, 4 and 7 is relatively widespread and closely related to cancer (36,37). Single-site specificity within one organ was also discovered for claudin-1 expression, as claudin-1 mRNA expression was found to be higher in the distal site than in the proximal site of the colon (38).

Fifth, the expression of claudins appears to be reversed with carcinogenesis; that is, a claudin that is downregulated in a specific tissue will be upregulated in tumors arising from that tissue, while a claudin that is upregulated in a specific tissue will be expressed at low levels in tumors derived from that tissue. This is especially true for claudin-18 and −6. In one study, the expression of claudin-18 nearly disappeared in lung cancer tissues and was significantly downregulated in gastric cancer tissues, while it was ectopically upregulated in pancreatic cancer tissues (27). Claudin-6 is a strictly oncofetal cell surface antigen whose expression is tightly regulated and completely absent in normal human tissues, but reactivated in germline tumors such as testicular, ovarian and uterine cancer (31).

Sixth, the expression levels of some claudins are heterogeneous within tumors and may differ in different cancer subtypes. For example, in estrogen receptor (ER)-positive luminal A and luminal B breast cancer, the expression of claudin-1 was found to be downregulated, while in the ER-negative basal-like breast cancer subtype, increased expression and cytoplasmic delocalization were observed (39). Consensus molecular subtype 2 and the transit-amplifying and C5 subtypes of metastatic colorectal cancer exhibit higher expression of claudin-1 than other subtypes (40). The expression levels of claudins are also not homogeneous intratumorally. Claudin-1 expression was shown to be retained in the central regions, but lost in the invasive edges in colorectal cancer (CRC) tissues (41). Although the majority of studies have agreed with these findings, some studies have reported the opposite results, especially for claudins that are widely expressed in a number of cancer types (claudins 1, 4 and 7) (36,37).

Seventh, changes in the expression of claudins are a very early event, although these changes are always exacerbated with the progression of the cancer. Claudin-1 was shown to be upregulated in the course of colitis, while claudin-7 mRNA levels were decreased in adenomas, suggesting that changes in claudin-7 mRNA levels are an early event in the development of CRC (42,43). Claudin-18 mRNA levels were also found to be decreased in intestinal metaplasia and intraepithelial neoplasia tissues before gastric cancer formation, but were commonly increased in precursor lesions of pancreatic cancer (27,44).

Since the expression patterns of claudins are diverse and dynamic, the transcription and function of claudins must be tightly controlled via a wide range of regulatory mechanisms, including genetic background, epigenetic alterations, transcriptional changes and post-translational modifications (PTMs), which are commonly modulated by oncogenic signaling pathways. Extrinsic factors, including the cancer cell of origin, differentiation status, pre-existing or acquired tumor microenvironment and environmental factors, also determine the expression profile of claudins (Table I; Fig. 4).

Consistent with the diversity of claudin expression in cancer, numerous studies on the roles of claudins in carcinogenesis have reported contradictory results. However, the data suggest that even if it does not function as a cancer driver, claudin dysregulation assists in the initiation and progression of cancer and is involved in nearly all aspects of tumor biology and all steps of tumor development. In addition to their various roles in cancer, from a translational perspective, as classic cell surface molecules, claudins may be ideal targets for therapy. As aforementioned, claudin expression has tissue specificity and is dysregulated in cancer. Furthermore, it has been observed that most claudins are buried in the TJ complex in normal tissues, while higher accessibility is caused by extrajunctional mislocalization in malignant tissues (96). Owing to this specific expression profile and difference between normal and tumor cells, claudin-targeting strategies seem to hold substantial promise, and this idea has been validated by clinical applications using specific claudin-targeting therapies (targeting claudin-6 and −18.2) (97). For the majority of claudins, with the exception of claudin-6 and −18.2, although the antitumor effect has been verified in proof-of-concept experiments, they remain at the laboratory stage, and their translation into clinical practice is eagerly awaited.

Overall, the dysregulated expression of claudins is a common phenotype associated with a number of different cancer types. The tissue-, cancer- and stage-specific expression patterns of claudins are the result of the regulation of various factors at different levels, from genetic background to tumor microenvironment alterations. Along with the understanding of the dysregulation of claudin expression in cancer, the exploration of their role in tumorigenesis and thus in cancer treatment and prevention will be advanced.