1. Gut-enriched Krüppel Like Factor (GKLF) Is Associated With Cell Differentiation And Regulates Keratin 13 In Human Esophageal Squamous Cell Carcinoma 2. The Role Of P53 And MDM2 Polymorphisms In The Risk Of Esophageal Squamous Cell Carcinoma

Squamous cell differentiation requires the coordinated activation and repression of genes specific to the differentiation process, disruption of this program accompanies malignant transformation of epithelium.GKLF (Gut-enriched Krüppel like factor) a member of the KLF family of transcription factors, plays key roles in cellular proliferation and differentiation. The aberrant expression of GKLF is observed in many kinds of malignant tumor .Our previous cDNA microarray results show that the transcription factor GKLF is obviously down-regulated in human esophageal squamous cell carcinoma (ESCC). Nonetheless, the roles of GKLF in esophageal tumor progression are not known. Using tissue microarray technology, we investigated the endogenous expression pattern of GKLF in ninety-five human ESCC and matched normal mucosal samples. We find that GKLF is significantly down-regulated in ESCC samples compared with normal counterparts. Furthermore, the expression of GKLF appeares to be correlated with differentiation status of ESCC.Using KYSE150 cells derived from a patient with poorly differentiated esophageal squamous cancer, normally lack GKLF, we demonstrate that the overexpression of GKLF by transiently transfecting the eukaryotic expression vector pcDNA3.1-GKLF could upregulate the expression of Keratin 13 gene at mRNA and protein levels. Luciferase reporter assay, eletrophoretic mobility shift assay(EMSA), chromatin immunoprecipitation (CHIP) assay and site-directed mutagenesis demonstrates a GKLF sequence motif at-399/-411 of Keratin 13 promoter is crucial for GKLF to activate the transcript expression of Keratin 13. The significant association between GKLF and Keratin 13 in both ESCC tissue samples and ESCC cell lines further validates the regulation of Keratin 13 by GKLF and indicates the key role of GKLF in the regulation of the Keratin 13.In summary, GKLF might play important roles in the differentiation process through regulating a number of genes vital for esophageal epithelial differentiation. The decreased expression of GKLF might lead to the abnormal differentiation and malignant transform of esophageal epithelial. The tumor suppressor P53 pathway plays a crucial role in preventing carcinogenesis and genetic variations of this pathway may be associated with cancer susceptibility. We tested this hypothesis by examining the contribution of functional polymorphisms in P53 and MDM2 to risk of esophageal squamous cell carcinoma (ESCC). DNA from 758 ESCC patients and 1,420 controls were genotyped for P53 codon 72Arg＞Pro and MDM2 309T＞G polymorphisms. Odds ratios (OR) and 95% confidence intervals (CI) of ESCC were estimated by logistic regression. We observed an increased risk of ESCC associated with the P53 Pro/Pro (OR, 1.83; 95% CI, 1.43-2.35; P＜0.001) or MDM2 GG (OR, 1.49; 95% CI, 1.16-1.91; P=0.002) genotype, compared with the P53 Arg/Arg or MDM2 TT genotype, respectively. Interaction between these P53 and MDM2 polymorphisms increased risk of ESCC in a multiplicative manner, with the OR being 3.10 (95% CI, 2.07-4.69) for subjects carrying both P53 Pro/Pro and MDM2 GG genotypes. Significant interactions were observed between these polymorphisms and smoking, with risk being the highest (OR, 5.29; 95% CI, 2.91-9.61) in smokers having both P53 Pro/Pro and MDM2 GG genotypes. The MDM2 GG genotype was also associated with risk of developing poorly differentiated and advanced ESCC compared with the GT or TT genotype (OR for high-grade and stagesⅢ-Ⅳversus low-grade and stagesⅠ-Ⅱ=1.60; 95% CI, 1.00-2.64; P=0.049). The P53 and MDM2 polymorphisms may be genetic determinants for the development of ESCC.