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Combined Proteasome And Histone Deacetylase Inhibition Upregulates E-Cadherin Metastasis Suppressor Gene Expression And Attenuates Epithelial-Mesenchymal Transition In Esophageal Cancer
Matthew D Taylor, Yuan Liu, Alykhan S Nagji, Nicholas Theodosakis, David R Jones
University of Virginia, Charlottesville, VA
BACKGROUND:
Metastasis is governed in large part by perturbations in the epithelial-mesenchymal transition (EMT) and alterations in metastasis suppressor gene profiles. The combined use of proteasome and histone deacetylase (HDAC) inhibitors has shown significant promise in the treatment of selected solid tumors, including lung cancer. No studies have investigated this molecularly-targeted drug combination in the treatment of esophageal cancer. The purpose of this study is to investigate the effect of Vorinostat, an HDAC inhibitor, and Bortezomib, a proteasome inhibitor, on esophageal cancer metastasis.
METHODS:
Three-dimensional tumor spheroids that mimic tumor architecture were created using esophageal squamous (TE2) and adenocarcinoma (SKGT4) cancer cells. Cells were treated with TNF-α to simulate the pro-inflammatory tumor milieu and TGF-β, a cytokine critical for the induction of the EMT. 3-D tumors were then treated with Vorinostat and/or Bortezomib. Cell proliferation assays were used to assess cell death. Boyden chamber invasion assays were performed to quantify cell invasion following treatment. mRNA expression of the metastasis suppressor genes BRMS-1, E-cadherin, CD44, and KISS-1 was performed using quantitative RT-PCR (QRT-PCR). Statistical analysis was performed using Student’s t-test with p < 0.05 considered significant.
RESULTS:
Combined Vorinostat and Bortezomib resulted in maximal, yet modest, cancer cell death at 24 hours (40% cell death in squamous cell; 45% cell death in adenocarcinoma). TNF/TGF treatment was associated with a 1.7 fold increase in adenocarcinoma invasion (p=0.01) and a 1.5 fold increase in squamous cell cancer invasion (p=0.02) compared to controls. Combined therapy resulted in a 3.7 fold decrease in adenocarcinoma cell invasion (p=0.002) and a 2.8 fold decrease in squamous cell invasion (p=0.003) using 2-D invasion assays. Three dimensional invasion assays shown in Figure 1 demonstrate a significant decrease in metastatic movement following combined therapy of Vorinostat and Bortezomib. QRT-PCR revealed a robust rescue of E-cadherin transcripts following combined therapy in both histologies with no difference in BRMS-1, CD44, and KISS-1 mRNA levels (Table 1).
CONCLUSIONS:
Combined Vorinostat and Bortezomib therapy significantly decreases esophageal cancer EMT and metastatic movement, suggesting a robust tumoristatic function. This combined therapy effect on esophageal cancer metastasis was associated with upregulation of the metastasis suppressor gene, e-cadherin.  
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