Abstract LB-279: Proteasome inhibitor bortezomib induces E-cadherin reexpression through inhibiting NF- B mediated DNA methylation in esophageal cancer

Abstract

Abstract Background: Esophageal cancer represents a significant challenge as most patients present with advanced stage disease. This is due to the development of early nodal or distant metastasis. Metastatic progression is initiated by a process termed epithelial-mesenchymal transition (EMT). Recently, bortezomib, as an anti-tumor reagent, has been used as first-line regimen for patients with metastatic esophageal cancer in a Phase II clinical trial. However, the molecular mechanisms through which bortezomib inhibits tumor progression have not been well described. E-cadherin is a hallmark of EMT in tumor metastatic progression. Its aberrant hypermethylation is significantly associated with a poor prognosis in esophageal cancer. Our prior work indicated that bortezomib increased E-cadherin at transcriptional level in esophageal cancer. We hypothesized that NF-κB inhibition with bortezomib would reduce DNA methylation of E-cadherin and result in increased E-cadherin expression in esophageal cancer. Methods: Esophageal squamous and adenocarcinoma cancer cells were treated with TNF to simulate the pro-inflammatory tumor milieu and TGF-ß, a cytokine critical for the induction of EMT, followed by treatment with bortezomib (50nM) for 24 hrs. Cellular morphological changes were evaluated. E-cadherin promoter methylation status and mRNA was assessed using Methylation Specific PCR (MSP) and real-time PCR, respectively. To evaluate the effect of NF-κB on the methylation of the E-cadherin promoter in esophageal cancer, cells were either stimulated with TNF or transfected with an expression vector encoding the NF-κB subunit RelA/p65 or a super-suppressor IκB (SR- IκB), then treated with or without bortezomib. Results: We examined the effects of bortezomib on cellular morphology and found that bortezomib completely reverses TNF/TGF -induced EMT of esophageal cancer cells, correlating with an increase of E-cadherin transcript (5∼6 fold). Simultaneously, bortezomib dramatically inhibits methylation of the E-cadherin promoter in esophageal cancer cells. Furthermore, treatment with TNF or overexpression of the RelA/p65 induced hypermethylation of E-cadherin promoter. While inactivating NF-κB using SR- IκB expression vector significantly inhibits methylation of E-cadherin promoter in esophageal cancer cell lines. Interestingly, bortezomib failed to induce either hypomethylation or transactivation of E-cadherin in the absence of RelA/p65 when using SR-IκB in esophageal cancer cell lines. Conclusion: These findings indicate that bortezomib inhibits EMT of esophageal cancer cells by demethylation of the E-cadherin promoter, and that this function is NF- B dependent. In addition to functioning as a transcription activator, RelA/p65 appears to function as a transcription repressor by participating in methylation and silencing of E-cadherin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-279.