Overexpression of the DEC1 protein induces senescence in vitro and is related to better survival in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related death in China and has limited effective therapeutic options except for early surgery, since the underlying molecular mechanism driving its precursor lesions towards invasive ESCC is not fully understood. Cellular senescence is the state of the permanent growth arrest of a cell, and is considered as the initial barrier of tumor development. Human differentiated embryo chondrocyte expressed gene 1 (Dec1) is an important transcription factor that related to senescence. In this study, DEC1 immunohistochemical analysis was performed on tissue microarray blocks constructed from ESCC combined with adjacent precursor tissues of 241 patients. Compared with normal epithelia, DEC1 expression was significantly increased in intraepithelial neoplasia and DEC1 expression was significantly decreased in ESCC in comparison with intraepithelial neoplasia. In vitro, DEC1 overexpression induced cellular senescence, and it inhibited cell growth and colony formation in ESCC cell line EC9706. Fresh esophagectomy tissue sections from five ESCC patients were detected by immunohistochemistry of DEC1 and senescence-associated β-galactosidase (SA-β-Gal) activity, and strongly positive expression of DEC1 was correlated to more senescent cells in these fresh tissue sections. Kaplan-Meier method analysis of the 241 patients revealed that DEC1 expression levels were significantly correlated with the survival of ESCC patients after surgery. The expression levels of DEC1 were also correlated with age, tumor embolus, depth of invasion of ESCC, lymph metastasis status and pTNMs. These results suggest that DEC1 overexpression in precursor lesions of ESCC is a protective mechanism by inducing cellular senescence in ESCC initiation, and DEC1 may be a potential prognostic marker of ESCC.

Figure 1. Representative photographs of DEC1 expression in different lesions by immunohistochemistry.

Red arrows indicate the enlarged parts. (A) Normal esophageal epithelia. (B) Low grade intraepithelial neoplasia. (C) High grade intraepithelial neoplasia. (D) ESCC.

Figure 2. DEC1 induces cellular senescence and inhibits cell growth in vitro.

(A) SA-β-Gal staining assay was performed and senescent cell percentage was counted 48 h after transfection in EC9706 cells. (B) Protein expressions were analyzed by western blotting 48 h after transfection in EC9706 and HEK293 cells. (C) Growth curves of EC9706 and HEK293 cells after transfection with pCMV6-XL5-DEC1 and control plasmids. (D) Cells were seeded 24 h after transfection and colony number was counted two weeks G418 selection. Cotransfection efficiency was analyzed by western blotting. (*, P<0.05 as compared with ctrl.).

Figure 3. Detection of DEC1 and SA-β-Gal activity in fresh tissue sections.

DEC1 expression and SA-β-Gal activity in ESCC and adjacent normal epithelia were detected by immunohistochemistry and SA-β-Gal staining assay in consecutive frozen sections.

Figure 4. Survival curves of DEC1 expression in ESCC patients analyzed by Kaplan-Meier method.