Expression of the CLDN18 gene in different cancer types and corresponding normal tissues
In the Oncomine database, 312 unique analyses compared the CLDN18 mRNA expression difference between cancer and normal tissues; among these analyses, 49 showed significance(P < 0.05). The results revealed that CLDN18 expression was downregulated in GC, lung cancer, breast cancer and sarcoma, whereas CLDN18 expression was upregulated significantly in esophageal and pancreatic cancer compared to their normal counterparts (Figure 1A). The results in GEPIA2 showed that in normal tissues, the mRNA expression of CLDN18 was restricted to the lung and stomach, while the expression nearly disappeared in lung cancer tissues and was significantly downregulated in GC tissues. In contrast, obvious ectopic expression of CLDN18 was observed in PC (Figure 1B).
mRNA expression analysis of CLDN18 in pancreatic normal and tumor tissues
In the Oncomine database, 5 of 8 analyses from 7 datasets reported upregulated CLDN18 mRNA expression in PC tissues, while no analysis reported downregulated CLDN18 mRNA expression (Figure 1A). Because the sample sizes of analyses were small in studies included in Oncomine, we further compared the expression difference between PC and normal tissues using series from GEO. Seven series were obtained from GEO based on our predefined criterion, including 381 cancer samples and 209 normal samples. A total of 15 probes reported the expression level of CLDN18. All probes reported an upregulated CLDN18 mRNA expression difference with significance (log2FC > 1, P < 0.05) (Table 1). In addition, the results from GEPIA2 showed that the expression of CLDN18 was clearly higher in PC tissues than in normal tissues (Figure 3A).
Expression of alternatively spliced transcript variants of CLDN18
Analysis using GEPIA2 showed that CLDN18-001, which encodes isoform 2, was mostly expressed in normal gastric and cancer tissues. When compared with CLDN18-001 expression in normal tissues, the expression was downregulated in GC tissues (Figure 2A). The expression of CLDN18-002, which encodes isoform 1, was restricted to pulmonary normal tissues and was obviously downregulated in lung cancer tissues (Figure 2B). The ectopic expression in PC tissues was mainly CLDN18-001 (Figure 2A), and the transcript level of CLDN18-001 was obviously higher than that of CLDN18-002 and CLDN18-003; the latter is an instance of nonsense-mediated decay (Figure 2C).
Correlation between CLDN18 expression and clinicopathological characteristics
Gene expression and currently available clinical data of 177 patients were extracted from the TCGA database based on our search strategies. We compared the expression differences among groups with different clinicopathological characteristics. As shown in Figure 3, there were no relationships between CLDN18 expression and age, sex, race, node metastasis, distant metastasis, alcohol history or tumor location. However, increased expression of CLDN18 correlated significantly with the infiltration depth, tumor stage and histology type, and the expression was higher in tumor tissues with advanced local invasion (T3, T4), later clinical stage (TNM III, IV), infiltrating duct or mucinous carcinoma types (Figure 3B-K).
Protein expression of CLDN18 in normal and various cancer tissues
Three antibodies against claudin 18 were available in the HPA database, of which antibody HPA018446 was used to detect both isoforms of claudin 18, while the detected isoforms of antibodies CAB013010 and CAB013243 were not exactly known. A high staining score was only found in gastric glandular cells among normal tissues, but a high positive rate was found in pancreatic, gastric, lung and ovarian cancer tissues (Figure 4). In PC tissues, claudin 18 was detected in the cytoplasm and on the membrane, and the positive rates of claudin 18 were 58.3% (7/12), 81.8% (9/11), and 30% (3/10) for antibodies HPA018446, CAB013010 and CAB013243, respectively.
Analysis of CLDN18 genetic alterations in PC
The TCGA PanCan Atlas studies were used to analyze genetic alterations of CLDN18 in different cancers. The results showed that CLDN18 alterations mainly occurred in lung squamous, cervical, esophageal, head and neck, and ovarian cancers with the main type being amplification, uterine cancer, with the main type being mutation, and stomach cancer, with the main type being fusion (Figure 5A). cBioPortal has 10 archived datasets of CLDN18 alterations in human PC, and two datasets including PC patients from different cohorts (Queensland Centre for Medical Genomics and TCGA) were used for analysis. Finally, CLDN18 was altered in only 0.4% (2/640) of patients with pancreatic adenocarcinoma. These alterations were truncating mutations with unknown significance in 1 case and amplification in 1 case (Figure 5B).
Coexpression relationship between CLDN18 and the CREB gene family
The expression levels of 9 CREB genes were detected by TCGA-PAAD project. Of these genes, 3 were negatively coexpressed, and 6 were positively coexpressed. In total, 4 genes reported significant coexpression associated with CLDN18. Only the CREBL2 gene reported significant negative coexpression associated with CLDN18 with a Pearson r of 0.2165 (P=0.0037), while CREB3L1, CREB3L3, and CREB3L4 were reported to have significant positive coexpression associated with CLDN18 with Pearson r values of 0.7236 (P < 0.0001), 0.5425 (P < 0.0001), and 0.3506 (P < 0.0001), respectively (Figure 6).
CLDN18 expression is downregulated through methylation in PC
As shown in Figure 7, the methylation of CLDN18 was tested by 21 probes distributed in different regions of the gene (the localization of each probe is represented in the figure, and those localized in the promoter region are highlighted in dark blue). Nineteen regions including all the promoter regions analyzed presented a negative correlation with respect to CLDN18 gene expression (Pearson’s correlation coefficients are indicated on the right), and only one region at the end of the gene analyzed presented a positive correlation with respect to CLDN18 gene expression (Figure 7). The results showed that CLDN18 expression increased as methylation gradually decreased.
Prognostic value of CLDN18
There were 171 patients with eligible survival data in the TCGA-PAAD cohort. Through searching the GEO database, 5 datasets with a total of 359 patients (GSE21501: N=102; GSE57495: N=63; GSE62452: N=66; GSE79229: N=49; GSE85916: N=79) were obtained together for survival analysis. Two and three probes were used to test the mRNA expression of CLDN18 in GSE57495 and GSE85916, respectively. All analyses showed that the expression of CLDN18 is not related to the overall survival of PC patients (Figure 8).