Table7_Evaluation of the Prognostic Relevance of Differential Claudin Gene Expression Highlights Claudin-4 as Being Suppressed by TGFβ1 Inhibitor in Colorectal Cancer.XLSX

Background: Claudins (CLDNs) are a family of closely related transmembrane proteins that have been linked to oncogenic transformation and metastasis across a range of cancers, suggesting that they may be valuable diagnostic and/or prognostic biomarkers that can be used to evaluate patient outcomes. However, CLDN expression patterns associated with colorectal cancer (CRC) remain to be defined.
Methods: The mRNA levels of 21 different CLDN family genes were assessed across 20 tumor types using the Oncomine database. Correlations between these genes and patient clinical outcomes, immune cell infiltration, clinicopathological staging, lymph node metastasis, and mutational status were analyzed using the GEPIA, UALCAN, Human Protein Atlas, Tumor Immune Estimation Resource, STRING, Genenetwork, cBioportal, and DAVID databases in an effort to clarify the potential functional roles of different CLDN protein in CRC. Molecular docking analyses were used to probe potential interactions between CLDN4 and TGFβ1. Levels of CLDN4 and CLDN11 mRNA expression in clinical CRC patient samples and in the HT29 and HCT116 cell lines were assessed via qPCR. CLDN4 expression levels in these 2 cell lines were additionally assessed following TGFβ1 inhibitor treatment.
Results: These analyses revealed that COAD and READ tissues exhibited the upregulation of CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN12 as well as the downregulation of CLDN5 and CLDN11 relative to control tissues. Higher CLDN11 and CLDN14 expression as well as lower CLDN23 mRNA levels were associated with poorer overall survival (OS) outcomes. Moreover, CLDN2 and CLDN3 or CLDN11 mRNA levels were significantly associated with lymph node metastatic progression in COAD or READ lower in COAD and READ tissues. A positive correlation between the expression of CLDN11 and predicted macrophage, dendritic cell, and CD4+ T cell infiltration was identified in CRC, with CLDN12 expression further being positively correlated with CD4+ T cell infiltration whereas a negative correlation was observed between such infiltration and the expression of CLDN3 and CLDN15. A positive correlation between CLDN1, CLDN16, and neutrophil infiltration was additionally detected, whereas neutrophil levels were negatively correlated with the expression of CLDN3 and CLDN15. Molecular docking suggested that CLDN4 was able to directly bind via hydrogen bond with TGFβ1. Relative to paracancerous tissues, clinical CRC tumor tissue samples exhibited CLDN4 and CLDN11 upregulation and downregulation, respectively. LY364947 was able to suppress the expression of CLDN4 in both the HT29 and HCT116 cell lines.
Conclusion: Together, these results suggest that the expression of different CLDN family genes is closely associated with CRC tumor clinicopathological staging and immune cell infiltration. Moreover, CLDN4 expression is closely associated with TGFβ1 in CRC, suggesting that it and other CLDN family members may represent viable targets for antitumor therapeutic intervention.

背景：紧密连接蛋白（Claudins, CLDNs）是一类亲缘关系紧密的跨膜蛋白家族，已被证实与多种癌症的致癌转化及转移相关，提示其可作为极具价值的诊断或预后生物标志物，用于评估患者预后结局。然而，与结直肠癌（colorectal cancer, CRC）相关的CLDN表达模式仍有待明确。
方法：本研究通过Oncomine数据库分析了20种肿瘤类型中21个不同CLDN家族基因的mRNA表达水平。为阐明不同CLDN蛋白在结直肠癌中的潜在功能作用，本研究借助GEPIA、UALCAN、人类蛋白质图谱（Human Protein Atlas）、肿瘤免疫估计资源（Tumor Immune Estimation Resource）、STRING、Genenetwork、cBioportal及DAVID数据库，分析了这些基因与患者临床结局、免疫细胞浸润、临床病理分期、淋巴结转移及突变状态的相关性。采用分子对接分析探究CLDN4与转化生长因子β1（transforming growth factor β1, TGFβ1）的潜在相互作用。通过定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）检测临床结直肠癌患者样本及HT29、HCT116细胞系中CLDN4与CLDN11的mRNA表达水平，并在经TGFβ1抑制剂处理后，额外检测了这两种细胞系中的CLDN4表达水平。
结果：分析结果显示，相较于对照组织，结肠腺癌（colon adenocarcinoma, COAD）与直肠腺癌（rectal adenocarcinoma, READ）组织中CLDN1、CLDN2、CLDN3、CLDN4、CLDN7及CLDN12的表达上调，而CLDN5与CLDN11的表达下调。更高的CLDN11、CLDN14表达水平及更低的CLDN23 mRNA水平与较差的总生存期（overall survival, OS）结局相关。此外，在结肠腺癌或直肠腺癌中，CLDN2与CLDN3、或CLDN11的mRNA水平分别与淋巴结转移进展显著相关，且其在结肠腺癌与直肠腺癌组织中的表达水平更低。在结直肠癌组织中，CLDN11的表达与预测的巨噬细胞、树突状细胞及CD4+ T细胞浸润呈正相关；CLDN12的表达与CD4+ T细胞浸润进一步呈正相关，而此类浸润与CLDN3、CLDN15的表达呈负相关。本研究还检测到CLDN1、CLDN16的表达与中性粒细胞浸润呈正相关，而中性粒细胞水平与CLDN3、CLDN15的表达呈负相关。分子对接分析表明，CLDN4可通过氢键与TGFβ1直接结合。相较于癌旁组织，临床结直肠癌肿瘤组织样本中CLDN4表达上调、CLDN11表达下调。LY364947可抑制HT29与HCT116细胞系中CLDN4的表达。
结论：综上，本研究结果表明，不同CLDN家族基因的表达与结直肠癌肿瘤的临床病理分期及免疫细胞浸润密切相关。此外，CLDN4的表达与结直肠癌中的TGFβ1存在紧密关联，提示其及其他CLDN家族成员或可成为抗肿瘤治疗干预的潜在可行靶点。