Characterization of tumor suppressive role of DESC1 in esophageal squamous cell carcinoma

Abstract

Esophageal cancer (EC) is ranked as the eighth most common cancer and the sixth leading cause of cancer deaths worldwide. In Hong Kong, esophageal squamous cell carcinoma (ESCC) is the prominent histological type of EC. Previously, a cDNA microarray analysis using paired tissue samples was performed in our lab to identify candidate tumor suppressor genes related to ESCC development. Differentially Expressed in Squamous cell Carcinoma 1 (DESC1) was one of the top hits identified in the study, suggesting a putative tumor suppressive role of DESC1 in ESCC. In the present study, the in vitro functions of DESC1 were investigated. DESC1-overexpressing cells showed a significant reduction in cell viability under serum deprivation, as evaluated by the MTT assay. The increase in apoptotic events caused by the overexpression of DESC1 was further verified by sub-G1 analysis, Annexin V-PI staining, and caspase cleavage. Since serum deprivation is known to trigger apoptosis, it was hypothesized that DESC1 may sensitize cells to apoptosis under apoptotic stress. To further verify this hypothesis, cells were treated with 5-fluorouracil (5-FU), an anti-cancer drug that induces apoptosis. Consistent with the previous result, DESC1-overexpresssion caused more cells to undergo apoptosis, when cells were treated with 5-FU. Western blotting showed that DESC1-overexpression down-regulated the phosphorylation level of a pro-survival protein, AKT1, when cells were under serum deprivation or 5-FU treatment. By introducing a constitutively active AKT, myr-AKT, into DESC1-overexpressing cells, the reduction of cell viability caused by DESC1-overexpression was abolished, validating the role of AKT1 in DESC1 function. Further analysis showed that the total level, as well as the phosphorylation level, of Epidermal Growth Factor Receptor (EGFR) was also reduced notably in DESC1-overexpressing cells. Since EGFR is an upstream regulator of AKT, this result suggests that DESC1 may regulate AKT1 phosphorylation by down-regulating the EGFR pathway. The importance of the proteolytic activity of DESC1 in its functions was investigated by introducing a point mutation to abolish its catalytic activity. The MTT assay demonstrated that the sensitizing effect of DESC1 to apoptosis was abrogated in the mutant. Also, the molecular changes, including the downregulation of EGFR protein level and AKT1 activation, and the increase in caspase cleavage, were abolished in the mutant, indicating that the protease activity is critical to its function. As a transmembrane protein, the extracellular environment may be critical to the function of DESC1. This idea was supported by the observation that DESC1-overexpressing cells showed a reduction in colony formation ability, when cells were cultured with Matrigel. Western blotting analysis showed that DESC1-overexpression caused a down-regulation in the EGFR protein level and AKT activation level, which coincided with the previous experiment that shows DESC1 regulated the EGFR/AKT1 pathway. In summary, these results suggest that DESC1 sensitizes cells to apoptosis, when they experience apoptotic stress, such as serum deprivation or 5-FU treatment. The present study provides valuable insight for how this recently discovered gene participates in tumor development and suggests the usefulness of DESC1 as a biomarker or prognostic indicator of ESCC.