Claudins (CLDNs) are the most important tight junction proteins, which are mainly expressed in endothelial cells or epithelial cells in a tissue-specific manner. As a member of the CLDNs family, CLDN6 is highly expressed in fetal tissues such as the stomach, pancreas, lung, and kidney, but is not expressed in corresponding adult tissues. The expression of CLDN6 is regulated by a variety of factors, including but not limited to stimuli and transcription factors, DNA methylation, and post-translational modifications. CLDN6 has been found to have a key role in the formation of barriers, especially the lung epithelial barrier and the epidermal permeability barrier (EPB). Importantly, the role of CLDN6 in cancer has attracted attention. Strong evidence indicates that the altered expression of CLDN6 is linked to the development of various cancers. Malignant phenotypes of tumors affected by CLDN6 include proliferation and apoptosis, migration and invasion, and drug resistance, which are regulated by CLDN6-mediated key signaling pathways. Given the important role in tumors and its low or no expression in normal tissues, CLDN6 is an ideal target for tumor therapy.

Structure of CLDN6

CLDN6 is a four-transmembrane protein with a short cytoplasmic N terminus and a C-terminal cytoplasmic domain, two extracellular domains (ECL1, which is larger and a smaller ECL2), and one short intracellular loop, which is consistent with other CLDNs. ECL1 and COOH-terminal PDZ-binding motif (PBM) are the typical domains of CLDNs. The ECL1 interdigitates between cells to form ion-selective pores. CLDN6 decreased chloride permeability when transfected into MDCK II cells. Furthermore, the ECL1 of CLDN6 helps the Hepatitis C virus (HCV) enter into cells. The PBM of CLDN6 can bind to proteins with PDZ domains such as ZO-1 to mediate cell signaling transduction. In addition, the ECL2 of CLDN6 contributes to the binding for clostridium perfringens enterotoxin (CPE).

Fig. 1 Schematic presentation and molecular diagram of CLDN6.

Regulation of CLDN6

• Stimuli and transcription factors

The expression of CLDN6 is regulated by the degree of cell fusion and junctional maturation, as evidenced by the increase in expression with time and a high dependence upon plating density. CLDN6 expression is also regulated by oxygen and estrogen, which are mediated by transcription factors such as HIF-1αand Estrogen receptor α/β (Erα/β).

• DNA Methylation

DNA methylation is a major epigenetic mechanism that leads to gene silencing by various mechanisms including, for example, methyl-DNA-binding proteins, changes in histone acetylation or inhibition of DNA-binding factors, as well as repositioning of nucleosomes thus preventing RNA polymerase II (Pol II) initiation. Methylation of CLDN6 is associated with a decrease in mRNA expression in esophageal squamous cell carcinoma and breast cancer. The methylation mechanism of CLDN6 has been studied mainly in breast cancer cells. DNA methylation of the CLDN6 promoter inhibits its expression through binding to MeCP2, deacetylating H3 and H4. At the transcriptional start site of CLDN6, Pol II is stalled and phosphorylated at ser2, which is relieved after treatment with demethylating agents such as Azacytidine, and the mRNA expression of CLDN6 is upregulated. Additionally, lymphoid-specific helicase (LSH), a member of the chromatin remodeling family, increases the DNA methylation and Pol II stalling of CLDN6 by enhancing the binding of DNMT3b to CLDN6.

• Posttranslational Modifications

Posttranslational modifications such as phosphorylation, ubiquitination, glycosylation, and palmitoylation are processes affecting the conformation, stability, trafficking, and function of proteins. The intracellular segment of CLDN6 contains several potential phosphorylation sites. Tyr-196/200 are phosphorylated by Src-family kinases (SFKs), and strong binding of SFKs to CLDN6-p-Tyr-196/200 fully activates SFKs, resulting in stimulating the downstream PI3K/AKT pathway. Tyr-213 is another site shown to be phosphorylated, which is mediated by EphA7 (one of the Eph receptor tyrosine kinases family), reducing the distribution of CLDN6 at the cell surface.

Emerging Roles in Cancers

The loss of cell-to-cell junctions is one of the important factors in cellular transformation and tumorigenesis. More than simply being static components that establish tight junctions, CLDNs are also cellular signaling components to receive environmental cues and transmit signals within the cell, which explains their involvement in cancer growth and progression. As a member of CLDNs, CLDN6 is also associated with tumor initiation and progression in a range of cancers.

• The expression of CLDN6 in cancers

The expression pattern of CLDNs varies among cancer types and the expression of CLDN6 has been explored from the perspective of pan-cancer analysis, in which CLDN6 was found significantly upregulated in 20 types of cancers while it is downregulated in glioblastoma multiforme (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), acute myeloid leukemia (LAML), and brain lower-grade glioma (LGG).

• The malignant phenotypes of CLDN6 affected in cancers

Studies have shown that CLDN6 inhibits the development and progression in some cancers such as breast cancer, cervical cancer, and meningioma. In hepatocellular cancer, gastric cancer, endometrial cancer, and ovarian cancer, CLDN6 has the opposite roles.

• Proliferation and apoptosis

The relationship between CLDN6 and tumor cell proliferation and apoptosis is confirmed by gain- and loss-of-function experiments in a variety of tumor cells. In MCF-7 breast cancer cells, CLDN6 overexpression inhibits proliferation and anchorage-independent growth and induces apoptosis via ASK1-P38/JNK signaling in MCF-7 cells. Besides, suppression of CLDN6 in MCF-7 or HBL-100 cells results in increased cell proliferation, and CLDN6 exerts this role in HBL-100 cells in association with P38/MAPK signaling activation. Similarly, CLDN6 overexpression in cervical carcinoma cells inhibits tumor growth in vitro and in vivo. However, CLDN6 promotes the proliferation of human hepatocellular carcinoma cells. CLDN6 also acts as a tumor promoter to increase cell proliferation in gastric cancer. Knockdown of CLDN6 significantly inhibited HEC-1B endometrial cancer cell proliferation via PI3K/Akt/mTOR signaling pathway. In summary, CLDN6 is involved in the regulation of cancer cell proliferation and apoptosis through different pathways , which results in pro- or anti-cancer effects of CLDN6 in different cancers.

• Migration and invasion

CLDN6 plays an important role in the migration and invasion of tumor cells in vitro. In breast cancers with low CLDN6 expression, CLDN6 overexpression inhibits tumor cell migration and invasion, while in cancers such as hepatocellular cancer with high CLDN6 expression, CLDN6 acts as a promoter of migration and invasion. Further mechanistic studies are needed to determine the context-dependent mechanisms. It is worth noting that several studies have used nude mice to establish tumor metastasis models, such as the liver metastasis model by injecting gastric cancer cells into the spleen and lung metastasis model by tail vein injection of breast cancer cells to observe the effect of CLDN6 on tumor metastasis, and obtained results consistent with the in vitro experiments, which profoundly demonstrated the effect of CLDN6 on tumor metastasis.

• Drug resistance

CLDNs such as CLDN1, 2, 3, 7, and 11 are known to modulate drug resistance in cancers. In addition, CLDN6 is highly expressed in MCF-7 multidrug-resistant (MDR) cells, and CLDN6 up-regulates the expression of glutathione S-transferase-p1 (GSTP1) and promotes chemoresistance in MCF-7 cells. However, in triple-negative breast cancer cells MDA-MB-231, GSTP1 is lost, CLDN6 enhances chemoresistance to ADM via activating the AF-6/ERK signaling pathway and up-regulating cancer stem cell characters. Cancer cells with high plasticity can undergo phenotypic switching to a drug-resistant state to avoid drug toxicity, which made tumor plasticity an important mechanism of drug resistance and tumor relapse. CLDN6 activates the Hippo signaling and induces a phenotypic shift from hepatic lineage to biliary lineage, which makes HCC cells more resistant to sorafenib. These findings highlight the significance of CLDN6 as a novel target in improving the efficacy of cancer therapy.

Signaling Pathways Involved in CLDN6

The mechanism of how CLDN6 transmits signaling is not very clear, but there are two theories currently. On the one hand, CLDN6 binds to PDZ-containing proteins through its PBM to mediate signaling transduction. For example, CLDN6 combines with PDZ-containing protein ZO-1, and then ZO-1, UVRAG, and BECLIN1 form complexes to regulate autophagosome formation in breast cancer cells. In MDA-MB-231 breast cancer cells, CLDN6 binds to another PDZ-containing protein AF-6 to inhibit its downstream ERK activation. CLDN6 is found upregulated and interacted with ZO-2/YAP1, which activates the Hippo signaling in HCC cells. In gastric cancer cells, CLDN6 interacted with LATS1/2 and decreased the conversion of LATS into p-LATS, thereby inhibiting the Hippo signaling pathway. But the mechanisms of how CLDN6 binds to LATS1/2 are not very clear, may be associated with ZO-1.

On the other hand, CLDN6 activates a series of kinases depending on ECL2 and Tyr-196/200 to transmit cell adhesion signals to the nucleus and regulate gene expression. The retinoic acid receptors (RARs), one member of the nuclear receptor superfamily, transcriptionally regulate the expression of many target genes. RARγ heterodimers can trigger epithelial differentiation, which is similar to CLDN6, so researchers tried to find the link between CLDN6 and RARγ. It was concluded that ECL2-dependent, CLDN6-mediated cell–cell adhesion recruits and activates the SFK/PI3K/AKT signaling pathway and stimulates the RARγ and ERα activities in F9 stem cells. Also, CLDN6 regulates the PI3K/AKT/mTOR signaling pathway in HEC-1B endometrial carcinoma cells and EGFR/AKT/mTOR signaling pathway in HepG2 cells, but the mechanisms of how CLDN6 affects the signaling pathway in both HEC-1B and HepG2 cells remain to be verified.

CLDN6 Related Products & Applications

As a member of the tight junction protein CLDNs family, CLDN6 has two extracellular loops, one amino-terminal and one carboxy-terminal. CLDN6 is regulated by stimuli and transcription factors, DNA methylation, phosphorylation, and palmitoylation. CLDN6 has a traditional barrier function in regulating the lung epithelial barrier and the EPB. Importantly, CLDN6 has emerging roles in cancers. The PBM at the carboxyl terminus and the second extracellular loop mediate the signaling effects of CLDN6, which is an important mechanism by which CLDN6 affects tumor cell proliferation and apoptosis, migration and invasion, and drug resistance. Since CLDN6 is not or lowly expressed in most adult tissues, vaccines and drugs targeting CLDN6 are ideal for tumor prevention and treatment in solid tumors with high CLDN6 expression.

Creative Biogene provides customers with a range of cutting-edge tools and solutions designed to facilitate CLDN6 research. We offer a range of ready-to-use transfected stable cell lines, preformed viral particles and virus-like particles, providing researchers with powerful tools to support their CLDN6 functional studies and related drug development efforts.

Transfected stable cell lines

Our stable cell lines are specifically engineered to express CLDN6 or its variants, providing a reliable and consistent model for studying CLDN6-related functions, signaling pathways, and drug responses. These cell lines serve as valuable tools to investigate the role of CLDN6 in various biological processes, including cell adhesion, barrier function, and cellular behavior.

Preformed viral particles

Our preformed viral particles, such as lentiviral or adenoviral particles, carry CLDN6 genes or shRNA constructs designed to modulate CLDN6 expression. These particles offer an efficient and targeted approach to deliver CLDN6-related genetic material into specific cell types. Researchers can utilize these particles to study CLDN6 function, assess its role in disease progression, or explore potential therapeutic interventions.

Virus-like particles (VLPs)

Our CLDN6-specific VLPs are synthetic structures that mimic the outer shell of a virus. These VLPs are engineered to display CLDN6 on their surface, allowing researchers to study CLDN6 interactions, cellular adhesion, and potential therapeutic applications. CLDN6 VLPs serve as valuable tools for immunological studies, vaccine development, drug discovery, and high-throughput screening.