Association of CLDN6 and CLDN10 With Immune Microenvironment in Ovarian Cancer: A Study of the Claudin Family

Background The claudin family is a group of transmembrane proteins related to tight junctions. While their involvement in cancer has been studied extensively, their relationship with the tumor immune microenvironment remains poorly understood. In this research, we focused on genes related to the prognosis of ovarian cancer and explored their relationship with the tumor immune microenvironment. Methods The cBioPortal for Cancer Genomics database was used to obtain the genetic variation pattern of the claudin family in ovarian cancer. The ONCOMINE and Gene Expression Profiling Interactive Analysis (GEPIA) databases were used to explore the mRNA expression of claudins in cancers. The prognostic potential of these genes was examined via the Kaplan-Meier plotter. The enrichment of immunological signatures was determined by gene set enrichment analysis (GSEA). The correlations between claudins and the tumor immune microenvironment in ovarian cancer were investigated via the Tumor Immune Estimation Resource (TIMER). Results Claudin genes were altered in 363 (62%) of queried patients/samples. Abnormal expression levels of claudins were observed in various cancers. Among them, CLDN3, CLDN4, CLDN6, CLDN10, CLDN15, and CLDN16 were significantly correlated with overall survival in patients with ovarian cancer. GSEA revealed that CLDN6 and CLDN10 were significantly enriched in immunological signatures of B cell, CD4 T cell, and CD8 T cell. Furthermore, CLDN6 and CLDN10 were negatively correlated and positively correlated, respectively, with immune cell infiltration in ovarian cancer. The expression levels of CLDN6 and CLDN10 were also negatively correlated and positively correlated, respectively, with various gene markers of immune cells in ovarian cancer. Thus, CLDN6 and CLDN10 may participate in immune cell infiltration in ovarian cancer, and these mechanisms may be the reason for poor prognosis. Conclusion Our study showed that CLDN6 and CLDN10 were prognostic biomarkers correlated with the immune microenvironment in ovarian cancer. These results reveal new roles for CLDN6 and CLDN10 as potential therapeutic targets in the treatment of ovarian cancer.


INTRODUCTION
Ovarian cancer is the most lethal gynecological cancer among women (Siegel et al., 2020). Although surgical techniques and combined chemotherapy applications have progressed since the 1970s, the 5 year survival rate of advanced ovarian cancer is only 40-45% (Henderson et al., 2018). Therefore, improved treatment of ovarian cancer remains an urgent issue. Immunotherapy is an emerging treatment for several solid tumors, which shows improved outcomes in patients. With the application of various immune-based interventions in ovarian cancer, immunotherapy has been proven useful in advanced disease (Bogani et al., 2020).
The claudin (CLDN) family consists of more than 20 transmembrane proteins, which are major components of tight junctions. They serve as a physical barrier to prevent molecules from passing freely through the paracellular space between epithelial or endothelial cell sheets and also play critical roles in maintaining cell polarity and signal transductions (Weinstein et al., 1976;Wodarz, 2000;Tsukita et al., 2001;Kirschner et al., 2013). Previous research has recognized various claudin gene expression patterns and identified several genes dysregulated in cancers (Hewitt et al., 2006). These genes play roles in the tumorigenesis of solid tumors (Swisshelm et al., 2005;Hagen, 2019) and represent promising targets for cancer detection, prognosis, and therapy (Morin, 2005). However, the relationship between claudins and the tumor immune microenvironment has not yet been elucidated. This study comprehensively analyzed claudin expression in ovarian cancer and further explored the relationship between claudins and the immune microenvironment.

ONCOMINE Database Analysis
Claudin expression levels in various cancers were analyzed via the ONCOMINE database 2 (Rhodes et al., 2007), which includes more than 35 types of cancer and normal samples.

Gene Expression Profiling Interactive Analysis (GEPIA)
GEPIA v2 3 (Tang et al., 2017) is used to analyze the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and GTEx projects using a standard processing pipeline. The expression profile of the claudins in ovarian cancer was explored via GEPIA v2. The p-value cutoff was 0.05 and | log 2 FC| cutoff was 1.5.

Kaplan-Meier Plotter Database Analysis
The Kaplan-Meier plotter 4 (Gyorffy et al., 2012) assesses the effects of 54,000 genes on survival in 21 cancer types. The largest datasets include breast (n = 6234), ovarian (n = 2190), lung (n = 3452), and gastric (n = 1440) cancer. The system includes gene chip and RNA-seq data-sources from the Gene Expression Omnibus (GEO), European Genome-Phenome Archive (EGA), and TCGA databases. The prognostic significance of claudins in ovarian cancer was analyzed via the online database.
Tumor Immune Estimation Resource (TIMER) TIMER 5  allows comprehensive analysis of tumor-infiltrating immune cells. The correlation between claudin expression and immune cell infiltration was analyzed using this database. TIMER v2, an updated and enhanced version of TIMER, was used to analyze immune infiltration across diverse cancer types.

Statistical Analyses
The expression levels of claudins are presented as mean ± standard deviation (SD). Kaplan-Meier survival curves were established based on the log-rank test. The hazard ratio (HR) was determined using the Cox model. Spearman correlation was used for correlation analysis. A p-value of < 0.05 was considered to be significant.

Gene Variation of Claudins in Ovarian Cancer
Twenty-four reviewed proteins of the claudin family were obtained from the UniProt Knowledgebase (UniProtKB) 6 ( Table 1) [an additional file shows this in more detail (see Table 1)]. Firstly, we investigated the genetic variation of the claudin family in ovarian cancer using the cBioPortal for Cancer Genomics. Twenty-four genes were queried in 585 samples of ovarian serous cystadenocarcinoma (TCGA, Pan-Cancer Atlas). Figure 1A shows the alteration frequency of genetic variation in serous ovarian cancer. As shown in Figure 1B, the queried genes were altered in 363 (62%) queried patients/samples. The top three gene variations were CLDN11 (24%), CLDN16 (22%), and CLDN1 (16%). Differences in overall survival (OS) between the altered and unaltered groups were compared using the Kruskal-Wallis test. We found that OS was reduced in the altered group compared to the unaltered group (p = 7.981e-3) ( Figure 1C). Previous studies have shown that the claudin family is dysregulated in a variety of tumors and is involved in diagnosis, tumorigenesis, and prognosis 6 https://www.uniprot.org/ Barros-Filho et al., 2015;Zhou et al., 2018). Thus, the claudin family is worthy of further research in ovarian cancer.

Expression of Claudins Is Dysregulated in Various Cancers
To explore the mRNA expression of the claudin family, we investigated the expression profiles of claudins in various cancers via the ONCOMINE database. The thresholds were: p-value of 0.05, fold change of 1.5, and gene rank of all. Significant analyses are shown in Supplementary Figure 1 (those with < 3 significant analyses were not considered). Results showed that most claudins were dysregulated in various cancers. To verify the expression of claudins in ovarian cancer, GEPIA2 was used to analyze mRNA expression in TCGA and GTEx samples. The | Log 2 FC| cutoff was set to 1.5 and the p-value cutoff was set to 0.05. As shown in Figure 2, eight genes were overexpressed in ovarian cancer samples compared with normal tissue samples and included CLDN1, CLDN3, CLDN4, CLDN6, CLDN7, CLDN9, CLDN10, and CLDN16. Furthermore, three genes showed low expression in the ovarian cancer samples compared with normal tissue samples and included CLDN5, CLDN11, and CLDN15.

Correlation of Claudin Expression With Ovarian Cancer Prognosis
To identify genes with clinical significance, we studied the relationship between differentially expressed genes (DEGs) and ovarian cancer patient prognosis using the Kaplan-Meier plotter. As shown in Figure 3, overexpressed genes CLDN3, CLDN4, CLDN6, and CLDN16 were significantly correlated   Frontiers in Genetics | www.frontiersin.org with poor OS ( Figure 3A) and progression free survival (PFS) ( Figure 3B). In addition, high expression of CLDN10 and CLDN15 were predictive of a good prognosis in ovarian cancer patients (Figures 3C,D). Surprisingly, CLDN10 was overexpressed in cancer, but patients with high expression of CLDN10 showed good OS (HR = 0.73, logrank P = 1.6e-06), PFS (HR = 0.83, logrank P = 0.0067), and post progression survival (PPS, HR = 0.73, logrank P = 0.00029). These results are somewhat counterintuitive, and the underlying mechanism requires further exploration. TCGA projects have identified four molecular subtypes of high-grade serous ovarian carcinoma (HGSOC) (Cancer Genome Atlas Research Network, 2011): (i) the differentiated subtype; (ii) the immunoreactive subtype; (iii) the mesenchymal subtype; and (iv) the proliferative subtype. Among them, T-cell chemokine ligands, CXCL11 and CXCL10, and the receptor, CXCR3, characterized the immunoreactive subtype. Then, Thorsson et al. (2018) developed a global immune classification of solid tumors based on the transcriptomic profiles of 33 cancer types. They identified six distinct immune subtypes: C1 (Wound healing); C2 (IFN-γ dominant); C3 (Inflammatory); C4 (Lymphocyte depleted); C5 (Immunologically quiet); C6 (TGF-β dominant). These six categories represent features of the tumor microenvironment (Charoentong et al., 2017). In this research, we explored the relationships between the expression of differentially expressed genes related to prognosis and molecular subtypes or immune subtypes of ovarian cancer via the TISIDB (Ru et al., 2019). The Kruskal-Wallis test was used. As Supplementary Figure 2 shows, claudins including CLDN3, CLDN6, CLDN10, and CLDN15 are differentially expressed in different immune subtypes. And, claudins including CLDN3, CLDN4, CLDN6, CLDN10, and CLDN16 are differentially expressed in different molecular subtypes (Supplementary Figure 3). Among them, CLDN6 is relatively low expression, and CLDN10 is relatively high expression in the immunoreactive subtype.

GSEA of Immunological Signature Gene Sets
To characterize the potential function of claudins, GSEA was performed using gene expression data from TCGA ovarian cancer patients. Immunological signature gene sets were used. As shown in Figure 4, CLDN6 and CLDN10 were related to the effector differentiation of B cell, CD4 T cell, and CD8 T cell.

Correlation Analyses Between Claudins and Tumor Immune Microenvironment
To understand the role of claudins in immunity, we downloaded 379 RNA-seq FPKM (Fragments per kilobase per million) data of ovarian cancer from TCGA. Subsequently, the FPKM was converted to TPM (transcripts per million) (Li et al., 2010). The ESTIMATE algorithm (Yoshihara et al., 2013) was used to predict tumor purity based on TCGA ovarian cancer samples. Then, the relationship between claudin expression and the immune microenvironment was explored. As shown in Figure 5A, a significant negative correlation between CLDN6 expression and the immune score was observed (Spearman correlation = −0.23, p < 0.001). A significant positive correlation between CLDN10 expression and immune score (spearman correlation = 0.21, p < 0.001) was observed ( Figure 5B). However, the expression levels of CLDN6 and CLDN10 were not correlated with the stromal score.
We next examined the relationship between immune cell infiltration and claudin expression. RNA-seq TPM data (n = 379) from TCGA ovarian cancer were used to assess 22 immune cells subtype concentrations with the CIBERSORT algorithm (Newman et al., 2019). TCGA samples were grouped by the median values of CLDN6 and CLDN10, respectively. Activated dendritic cells differed significantly between the CLDN6_high and CLDN6_low groups. Several cell types were significantly different between the CLDN10_high and CLDN10_low group, including naïve B cells, memory B cells, naïve CD4 T cells, CD4 memory-activated T cells, monocytes, M1 macrophages, and activated dendritic cells ( Figure 5C).
To more accurately describe the relationship between gene expression and immune cell infiltration, we used the TIMER, CIBERSORT, quanTIseq, xCell, MCP-counter, and EPIC algorithms to assess the immune infiltration in tumor tissue (Sturm et al., 2019). TIMER2 provides a platform to analyze immune infiltrates across diverse cancer types based on available TCGA RNA-seq data Li T. et al., 2020). The correlations between claudin expression (CLDN6 and CLDN10) and immune cell infiltration in ovarian cancer are shown in Table 2. As seen in Figure 6C, CLDN6 was negatively correlated with immune cell infiltration, including that of B cells, CD8 + T cells, effector memory CD4 + T cells, M1 macrophages, and myeloid dendritic cells. In contrast, CLDN10 was positively correlated with immune cell infiltration, including that of B cells, CD8 + T cells, effector memory CD4 + T cells, M1 macrophages, and myeloid dendritic cells ( Figure 6D). Relevant evidence suggests that cancer-associated fibroblasts (CAFs) play an important role in the progression of ovarian cancer (Mhawech-Fauceglia et al., 2014;Leung et al., 2018). Interestingly, here, CAFs also showed a positive correlation with CLDN6 expression, but a negative correlation with CLDN10 expression. In ovarian cancer, increased infiltration of tumor-infiltrating lymphocytes (TILs), and more specifically CD8 + T cells, have been proven to be associated with improved clinical outcomes (Sato et al., 2005;Hamanishi et al., 2007;Ovarian Tumor Tissue Analysis et al., 2017). These results suggest that CLDN6 and CLDN10 may participate in immune cell infiltration in ovarian cancer, and these mechanisms may be the reason for poor prognosis.

Relationship Between Claudin Expression and Gene Markers of Immune Cells
To further illustrate the correlation between claudins and the immune microenvironment, we analyzed the relationship between CLDN6 and CLDN10 expression and gene markers of various immune cells in ovarian cancer (TIMER2 database), including B cells, T cells (general), CD8 + T cells, macrophages, dendritic cells, neutrophils, monocytes, natural killer (NK) cells, and regulatory T cells (Tregs) ( Table 3). Purity-adjusted Studies have shown that the tumor-infiltrating immune cells mentioned above are related to the tumor immunotherapy response (Rodriguez et al., 2018). Immune cell-based immunotherapy (Baci et al., 2020), including NK Cells (Nersesian et al., 2019) and dendritic cells (Stiff et al., 2013), play important roles in the treatment of ovarian cancer. Taken together, these analyses and our research indicate that CLDN6 and CLDN10 may play important roles in immunotherapy in the future.

DISCUSSION
CLDN6 and CLDN10 are important components of the claudin family related to tight junctions. Claudins were considered promising targets for diagnosis and therapy since they were involved in uncontrolled cancer growth and metastasis (Martin and Jiang, 2001;Morin, 2005;Bose and Mukhopadhyay, 2010). Moreover, studies have shown that they not only play a vital role in tumorigenesis (Swisshelm et al., 2005;Arabzadeh et al., 2007;Hagen, 2019), but also drug resistance .
CLDN6 had been demonstrated abnormal expression and can be a prognostic marker in cancers including ovarian cancer , endometrial cancer (Kojima et al., 2020), gastric cancer (Kohmoto et al., 2020), breast carcinoma (Liu et al., 2016;Jia et al., 2019), and lung cancer (Micke et al., 2014). Bioinformatic analysis has revealed that CLDN6 is regulated by a diverse set of transcription factors and promotes cancer cell behavior via the ASK1-p38/JNK MAPK secretory signaling pathway (Lin et al., 2017). A study revealed that CLDN6 may be a novel targeted therapy for ovarian cancer as a receptor for clostridium perfringens enterotoxin (Lal-Nag et al., 2012). In addition, 6PHU3, a T-cell-engaging bispecific single chain antibody with anti-CD3/anti-CLDN6 specificities, upregulated the cytotoxicity of T cells and made T cells acquire an effector phenotype (Stadler et al., 2016). Another recent study showed that CLDN6 as a chimeric antigen receptor target in solid tumors can be a strategy to overcome inefficient CAR-T cell stimulation in vivo (Reinhard et al., 2020). These studies suggested that CLDN6 has important research value in the treatment of cancer.
CLDN10, a glandular epithelial marker in epithelial ovarian cancer (Seo et al., 2010), was reported to be a key immune-related    (Yang et al., 2021) and papillary thyroid carcinoma (Xiang et al., 2020). Furthermore, CLDN10 expression has proved to be a prognostic marker for ovarian cancer . The present study combined and analyzed the prognostic potential of CLDN6 and CLDN10 with the tumor immune microenvironment. Consistent with previous reports, both CLDN6 and CLDN10 showed high expression in ovarian cancer. Prognostic analysis showed that the overexpression of CLDN6 was related to a poor prognosis for patients with ovarian cancer. However, CLDN10 overexpression predicted a better prognosis compared to the low CLDN10 expression group. We also found that CLDN6 overexpression was negatively related to immune cell infiltration, whereas CLDN10 overexpression was positively correlated with immune cell infiltration. Moreover, we found that CLDN6 and CLDN10 were related to gene markers of dendritic cells, NK cells, and TAMs. These results may explain why the overexpression of CLDN6 and low expression of CLDN10 predict poor OS in ovarian cancer. This study revealed that the prognostic potential of CLDN6 and CLDN10 is related to the tumor immune microenvironment in ovarian cancer.
Relevant evidence has emerged that immune-related gene expression and TILs are related to the prognosis, recurrence (Ojalvo et al., 2018), and chemotherapeutic response (Choi et al., 2020) of ovarian cancer. Furthermore, the presence of TILs may improve clinical outcomes in ovarian cancer patients (Odunsi, 2017). Immune cell-based immunotherapy (Baci et al., 2020), including NK Cells (Nersesian et al., 2019) and dendritic cells (Stiff et al., 2013), play an important role in the treatment of ovarian cancer. Previous studies and our analyses suggest that CLDN6 may be involved in immune evasion and that they could be an ideal candidate for immunotherapy in ovarian cancer. Future studies on the combined application of claudin-based molecular targeted therapy and immunotherapy are necessary.

CONCLUSION
CLDN6 and CLDN10 were identified as potential prognostic biomarkers and were correlated with immune cell infiltration in ovarian cancer. Our results revealed new roles for CLDN6 and CLDN10 in ovarian cancer and their potential as therapeutic targets in cancer treatment.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/ Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
PeW was responsible for the study conception and design. PG, TP, CC, and SL were involved in data acquisition, data analysis, and interpretation. PG drafted the manuscript and took charge of supervising the manuscript. All authors read and approved the manuscript.