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The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.

Cell adhesion is essential for proper tissue architecture and function in multicellular organisms. Cell adhesion molecules not only maintain tissue integrity but also possess signaling properties that contribute to diverse cellular events such as cell growth, survival, differentiation, polarity, and migration; however, the underlying molecular basis remains poorly defined. Here we identify that the cell adhesion signal initiated by the tight-junction protein claudin-6 (CLDN6) regulates nuclear receptor activity. We show that CLDN6 recruits and activates Src-family kinases (SFKs) in second extracellular domain-dependent and Y196/200-dependent manners, and SFKs in turn phosphorylate CLDN6 at Y196/200. We demonstrate that the CLDN6/SFK/PI3K/AKT axis targets the AKT phosphorylation sites in the retinoic acid receptor γ (RARγ) and the estrogen receptor α (ERα) and stimulates their activities. Interestingly, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors. We propose that a similar link between diverse cell adhesion and nuclear receptor signalings coordinates a wide variety of physiological and pathological processes.

Recent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin‐6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins and drug metabolism‐associated proteins (aldo‐keto reductase [AKR] family proteins) were significantly increased in CLDN6‐overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell‐cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell‐cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6‐targeting therapy, against cervical ADC. High claudin‐6 (CLDN6) expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor of cervical adenocarcinoma. Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins and drug metabolism‐associated proteins (aldo‐keto reductase [AKR] family proteins) were significantly increased in CLDN6‐overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell‐cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin.

Malignant mesothelioma is a cancer with a poor survival rate. It is difficult to diagnose mesotheliomas because they show a variety of histological patterns similar to those of various other cancers. However, since currently used positive markers for mesotheliomas may show false positives or false negatives, a novel mesothelial positive marker is required. In the present study, we screened 25 claudins and found that claudin-15 is expressed in the mesothelial cells. We made new rat anti-human claudin-15 (CLDN15) monoclonal antibodies that selectively recognize CLDN15, and investigated whether CLDN15 is a good positive marker for malignant pleural mesotheliomas (MPMs) using MPM tissue samples by immunohistochemistry and semi-quantification of the expression level using an immunoreactive score (IRS) method. Of 42 MPM samples, 83% were positive for CLDN15. The positive ratio was equal to or greater than other positive markers for MPMs including calretinin (81%), WT-1 (50%), and D2-40 (81%). In 50 lung adenocarcinoma sections, four cases were positive for CLDN15 and the specificity (92%) was comparable with other markers (90–100%). Notably, CLDN15 was rarely detected in 24 non-mesothelial tumors in the tissue microarray (12/327 cases). In conclusion, CLDN15 can be used in the clinical setting as a positive marker for MPM diagnosis.

The nuclear receptor liver receptor homolog 1 (LRH1)/NR5A2 is aberrantly expressed in diverse cancer types, including liver and lung cancers. Since we previously showed that excessive phosphorylation of human LRH1 at S510 (hLRH1 pS510 -high) is predictable of hepatocellular carcinoma recurrence, we here clarified the clinicopathological and biological significance of hLRH1 pS510 -high in lung cancer. By immunohistochemistry using an anti-hLRH1 pS510 monoclonal antibody, we evaluated the hLRH1 pS510 signals in 151 and 150 cases of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues, respectively, and performed clinicopathological analysis. hLRH1 pS510 was localized in the nucleus of tumor cells in LUAD and LUSC tissues with different intensity and proportions among the patients. Of note, the strong hLRH1 pS510 signal was occasionally detectable in LUAD and LUSC cells at the expanding tumor edges. A semi-quantitative analysis revealed that 28 (18.4%) and 36 (24.0%) of LUAD and LUSC cases, respectively, exhibited hLRH1 pS510 -high. Kaplan-Meier plots showed significant differences in the disease-free survival (DFS) between the hLRH1 pS510 -high and hLRH1 pS510 -low groups in LUSC, but not in LUAD patients. hLRH1 pS510 -high was also significantly correlated with recurrence in LUSC patients. Additionally, by multivariate analysis, hLRH1 pS510 -high represented an independent biomarker for the DFS of LUSC patients. Furthermore, the impact of hLRH1 pS510 on the viability of LUSC cells was evaluated by comparing phenotypes among two distinct LUSC cell lines expressing wild-type LRH1, LRH1S510A, and LRH1S510E. Consequently, we demonstrated that phosphorylation of hLRH1S510 accelerates the viability of LUSC cells. Thus, hLRH1 pS510 is attractive not only as the predictive biomarker for LUSC but also as the potential therapeutic target.

Background Cell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression. Methods By generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRβ (liver X receptor β) expression in breast cancer tissues from 187 patients. Results We uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRβ. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRβ, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRβ- and LXRβS432-dependence. Furthermore, among triple-negative breast cancer subjects, the \"CLDN4-high/LXRβ-high\" and \"CLDN4-low and/or LXRβ-low\" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively. Conclusions The identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.

The tight-junction protein claudin-9 (CLDN9) is barely distributed in normal adult tissues but is ectopically expressed in various cancer types. Although multiple databases indicated upregulation of CLDN9 in endometrial cancers at the mRNA level, its protein expression and biological roles remain obscure. In the present study, the prognostic significance of CLDN9 expression in endometrial cancer was evaluated by immunohistochemical staining and semi-quantification using formalin-fixed paraffin-embedded specimens obtained from 248 endometrial carcinoma cases. A total of 43 cases (17.3%) had high CLDN9 expression, whereas 205 cases (82.7%) exhibited low CLDN9 expression. The 5-year disease-specific survival rates in the high and low CLDN9 expression groups were 62.8 and 87.8% (P<0.001), respectively. In addition, multivariate analysis revealed that high CLDN9 expression was an independent prognostic factor (hazard ratio, 4.99; 95% CI, 1.96-12.70; P<0.001). Furthermore, CLDN9 expression was significantly correlated with the expression of CLDN6 (P<0.001), which is the closest CLDN member to CLDN9 and a poor prognostic factor for endometrial carcinoma. The 5-year disease-specific survival rate of cases with CLDN6-high/CLDN9-high, CLDN6-high/CLDN9-low and CLDN6-low/CLDN9-high status was 30.0, 37.5 and 72.7%, respectively, whereas that of CLDN6-low/CLDN9-low was 89.8% (P=0.004). In conclusion, aberrant CLDN9 expression is a predictor of poor prognosis for endometrial cancer and may be utilized in combination with CLDN6 to achieve higher sensitivity.

Background Aberrant cell adhesion signaling is known to either accelerate or inhibit cancer progression, but the underlying molecular basis has yet to be established. The immunoglobulin superfamily 11 (IgSF11) functions as a cell adhesion protein and is overexpressed in several types of cancer, including high-grade glioma. However, it remains unknown whether and how IgSF11 stimulates malignant phenotypes. Methods Using The Cancer Genome Atlas (TCGA), we first examined the expression of IgSF11 gene in various types of cancer tissues. Next, we developed an anti-hIgSF11 monoclonal antibody (mAb) and evaluated the clinicopathological significance of high IgSF11 expression in 187 cutaneous melanoma patients via immunohistochemistry using this selective mAb. We also generated human melanoma cell lines A375 and 888mel expressing IgSF11, as well as 888mel: IgSF11 KO and 888mel: IgSF11 KO : IgSF11 cells, and compared their phenotypes with those of control cells both in vitro and in vivo. Immunoprecipitation-mass spectrometry was applied to identify an IgSF11-interacting protein, followed by validation of its association with IgSF11 and of the specific IgSF11 region responsible for the complex formation and promoting melanoma cell migration. Results IgSF11 mRNA was highly expressed in glioblastoma tissues and skin cutaneous melanoma tissues, but not in other malignant tumors. High IgSF11 expression was observed in 57 out of the 187 melanoma cases (30.5%) and was significantly correlated with Clark’s level and high budding, both of which are parameters of melanoma invasion. Using a series of established cell lines, we demonstrated that IgSF11 promotes melanoma cell migration and invasion, as well as the enrichment of a gene set associated with epithelial-mesenchymal transition (EMT). Importantly, we identified that IgSF11 forms a complex with RAS-associated protein 1 (RAP1). Furthermore, the L372–R378 region of IgSF11 was required for recruiting RAP1 and driving melanoma cell migration. Conclusions We found that IgSF11–RAP1 signaling facilitates the migration and invasion of melanoma cells. The identification of IgSF11–RAP1 machinery highlights a novel link between cell adhesion and signaling molecules in promoting the malignant phenotypes of melanoma and may serve as a promising therapeutic target for this malignancy.

Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk−/− and F9:Cldn6:Src−/− cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling.

Background: Among the claudin (CLDN) family, CLDN6 exhibits aberrant expression in various cancers, but its biological relevance has not yet been established. We generated a monoclonal antibody (mAb) against human CLDN6 and verified its specificity. By immunohistochemical staining and semi-quantification, we evaluated the relationship between CLDN6 expression and clinicopathological parameters in tissues from 173 cases of endometrial cancer. Results: The established mAb selectively recognized CLDN6 protein. Ten of the 173 cases (5.8%) showed high CLDN6 expression (score 3+), whereas 19 (11.0%), 18 (10.4%) and 126 (72.4%) cases revealed low CLDN6 expression (score 2+, 1+ and 0, respectively). In addition, intratumor heterogeneity of CLDN6 expression was observed even in the cases with high CLDN6 expression. The 5-year survival rates in the high and low CLDN6 groups was approximately 30% and 90%, respectively. Among the clinicopathological factors, the high CLDN6 expression was significantly associated with surgical stage III/IV, histological type, histological grade 3, lymphovascular space involvement, lymph node metastasis and distant metastasis. Furthermore, the high CLDN6 expression was an independent prognostic marker for overall survival of endometrial cancer patients (hazard ratio 3.50, p = 0.014). Conclusions: It can be concluded that aberrant CLDN6 expression is useful to predict poor outcome for endometrial cancer and might be a promising therapeutic target.