| Summary: | 博士 === 國立成功大學 === 臨床醫學研究所 === 103 === Background: Patients with esophageal squamous cell carcinomas (ESCCs) have poor survival and frequently developed with synchronous head and neck squamous cell carcinomas (HNSCCs) . Disabled-2 (DAB2) is a crucial tumor suppressor for multiple signaling pathways (ERK, TGF-β etc.) and emerging evidences suggest TGF-β-induced regulatory T cells (Treg) enable tumor cells to escape immunosurveillance, resulting in tumor progression. We aimed to investigate the significance of DAB2 expression and Treg infiltration in ESCC and the field cancerization of esophagus at clinical and cellular levels.
Methods: Tumor tissues of 200 patients (100 ESCC only, 50 HNSCC only, and 50 synchronous SCCs) were evaluated for DAB2 expression and quantitatively accessed for the tumor infiltrating Treg by immunohistochemistry, and then correlated to the clinical features. Expressions of DAB2, ERK, β-catenin, E-cadherin and N-cadherin in ESCC cell lines and cell migration abilities were evaluated by western blot and wound healing assay, respectively. siRNA knockdown and over-expression studies were performed to validate the association between DAB2 and cell behavior. The density of Treg was also correlated to the level of Treg-associated inhibitory cytokines (IL 10, IL-35 and TGF β), and chemokine (CCL22). To explore the upstream regulatory mechanism of DAB2, the methylation status of DAB2 promoter were analyzed by Methylation-specific PCR.
Results: Forty-five ESCC patients had low-DAB2 expression, and significantly had larger tumor size, deeper invasion depth, lymph node metastasis, worse survival, and higher recurrence rate (P〈0.05). The Cox-regression model revealed low-DAB2 expression was an independent factor of poor survival (P〈0.05), and also of tumor recurrence with the predictive performance superior to clinical TNM stage (P〈0.05). Low-DAB2 cancer cells, validated by DAB2 knockdown or over-expression, had higher phosphorylated ERK and migration abilities. Promoter hypermethylation accounted only for a small subset of low-DAB2 cancers. TGF-β-induced epithelial-mesenchymal transition (EMT) only existed in the high-DAB2 cells, and related to worse prognosis of high-DAB2 ESCC (P〈0.05). Tumor infiltrating Treg densities were not associated with DAB2 expression levels (P=0.77), and frequently inversely correlated with the tumor stage. The density of tumor infiltrating Treg in the index tumor (i.e. the first malignancy diagnosed) of synchronous SCC group was higher than those of HNSCC or ESCC only (P〈0.05). Selecting the optimal cut-off value of Treg density as 34.6 cells/mm2 by ROC curve, an increased Treg density of the index tumor can be an independent factor for developing synchronous SCCs (OR: 6.13; 95% CI: 2.84-13.26). The Treg density was positively correlated with serum IL-10 level and the degree of CCL22-positive cells infiltration in tumor. Furthermore, the serum inhibitory cytokine levels were higher in synchronous SCC group. Concomitant elevation of serum IL-35, TGF-β and IL-10 level is an independent risk factors of developing synchronous cancers (OR: 7.86, 95% CI: 2.14-28.90). The finding of a higher prevalence of tuberculosis infection in the patients with synchronous phenotype than those with non-synchronous group (20% vs. 4%, p=0.001), also provided an indirect evidence that the cellular immunosuppression in the patients with synchronous multiple cancers.
Conclusions: DAB2 expression could be a biomarker to identify patients with worse survival and high recurrence. Our data suggest a means to stratify patients who require more aggressive surveillance and may benefit clinically from anti-ERK or anti-TGF-β therapies. Also, a more severe defect in cellular immunity may predispose to multifocal tumor. The Treg cell number in SCC may serve as a novel predictive biomarker for the risk of synchronous cancer development to initiate a proper surveillance strategy.
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