The roles of tight junction protein cingulin in human endometrioid endometrial cancer

ABSTRACT

The bicellular tight junction molecule cingulin (CGN) binds to microtubules in centrosomes. Furthermore, CGN contributes to the tricellular tight junction (tTJ) proteins lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC). CGN as well as LSR decreased during the malignancy of endometrioid endometrial cancer (EEC). Although tTJ protein LSR is involved in the malignancy of some cancers, including EEC, the role of CGN is unknown. In this study, we investigated the roles of CGN with tTJ proteins in human EEC cells by using the CGN-overexpressing EEC cell line Sawano. In 2D cultures, CGN was colocalized with LSR and TRIC at tTJ or at γ-tubulin-positive centrosomes. In immunoprecipitation with CGN antibodies, CGN directly bound to LSR, TRIC, and β-tubulin. Knockdown of CGN by the siRNA decreased the epithelial barrier and enhanced cell proliferation, migration and invasion, as well as knockdown of LSR. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the permeability of FD-4 to the lumen. In 2D and 2.5D cultures, treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and the epithelial permeability barrier and enhanced cell migration, and pretreatment with EW7197+AG1478, U0126 or an anti-IL-6 antibody prevented this. In conclusion, CGN, with tTJ proteins might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and growth factors derived from stromal cells.

KEYWORDS:

• CGN
• epithelial permeability barrier
• estrogen
• human endometrioid endometrial cancer
• LSR
• malignancy
• TRIC
• tricellular tight junction

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors contributions

AK, Takayuki K, TS, and Takashi K conceived and designed the study. AK, KS, TO, SN, DI, and Takumi K performed the experiments and verified data quality. MM, SN, and DI performed the statistical analysis of obtained data. AK and Takashi K wrote the paper and submitted it to the evaluation of the whole consortium. All authors have read and agreed to the published version of the manuscript.

Ethics statement

The protocol for the human study was reviewed and approved by the ethics committee of the Sapporo Medical University School of Medicine. All experiments were carried out in accordance with the approved guidelines and the Declaration of Helsinki.

supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21688370.2024.2361976.

Additional information

Funding

This work was supported by JSPS KAKENHI Grant Numbers 19K07464 [TK], 19K18674 [HS] and 20K18194 [TO].