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. 2010 May;139(5):1224-32, 1232.e1.
doi: 10.1016/j.jtcvs.2010.01.039.

Combined proteasome and histone deacetylase inhibition attenuates epithelial-mesenchymal transition through E-cadherin in esophageal cancer cells

Matthew D Taylor  1 , Yuan Liu, Alykhan S Nagji, Nicholas Theodosakis, David R Jones
Affiliations

Combined proteasome and histone deacetylase inhibition attenuates epithelial-mesenchymal transition through E-cadherin in esophageal cancer cells

Matthew D Taylor et al. J Thorac Cardiovasc Surg. 2010 May.

Abstract

Objective: Metastasis is thought to be governed partially by induction of epithelial-mesenchymal transition. Combination of proteasome and histone deacetylase inhibitors has shown significant promise, but no studies have investigated this in esophageal cancer. This study investigated effects of vorinostat (histone deacetylase inhibitor) and bortezomib (proteasome inhibitor) on esophageal cancer epithelial-mesenchymal transition.

Methods: Three-dimensional tumor spheroids mimicking tumor architecture were created with esophageal squamous and adenocarcinoma cancer cells. Cells were treated with tumor necrosis factor alpha (to simulate proinflammatory tumor milieu) and transforming growth factor beta (cytokine critical for induction of epithelial-mesenchymal transition). Tumor models were then treated with vorinostat, bortezomib, or both. Cytotoxic assays assessed cell death. Messenger RNA and protein expressions of metastasis suppressor genes were assessed. After treatment, Boyden chamber invasion assays were performed.

Results: Combined therapy resulted in 3.7-fold decrease in adenocarcinoma cell invasion (P = .002) and 2.8-fold decrease in squamous cell invasion (P = .003). Three-dimensional invasion assays demonstrated significant decrease in epithelial-mesenchymal transition after combined therapy. Quantitative reverse transcriptase polymerase chain reaction and Western blot analyses revealed robust rescue of E-cadherin transcription and protein expression after combined therapy. Importantly, inhibition of the E-cadherin gene resulted in abolition of the salutary benefits of combined therapy, highlighting the importance of this metastasis suppressor gene in the epithelial-mesenchymal transition process.

Conclusions: Combined vorinostat and bortezomib therapy significantly decreased esophageal cancer epithelial-mesenchymal transition. This combined therapeutic effect on esophageal cancer epithelial-mesenchymal transition was associated with upregulation of E-cadherin protein expression.

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Figures

FIGURE 1
FIGURE 1
Two-dimensional esophageal cancer invasion decreased with vorinostat and bortezomib (Bortez) treatment after induction of epithelial–mesenchymal transition. A, Treatment with vorinostat and bortezomib in esophageal squamous carcinoma (TE2) resulted in significantly decreased cell invasion. B, Treatment with vorinostat and bortezomib in esophageal adenocarcinoma (SKGT4) resulted in significantly decreased cell invasion. Crosshatch denotes P<.05 for control versus for tumor necrosis factor (TNF) and transforming growth factor (TGF). Asterisk denotes P<.01 for treatment group versus tumor necrosis factor and transforming growth factor. Plus sign denotes P<.05 for bortezomib treatment group versus combined vorinostat and bortezomib treatment group.
FIGURE 2
FIGURE 2
Combined vorinostat and bortezomib (Bortez) treatment induced modest esophageal cancer cell death in both squamous carcinoma (TE2) and adenocarcinoma (SKGT4) histologic subtypes after 24 hours of treatment.
FIGURE 3
FIGURE 3
Metastasis suppressor gene transcription (CDH1, gene for E-cadherin; BRMS1, breast cancer metastasis suppressor 1 gene; NME1, gene for NM23A; ARHGDIB, gene for Rho guanosine diphosphate dissociation inhibitor [RhoGDI2], CD44; KISS1, gene for KiSS-1) after induction of epithelial-mesenchymal transition and with treatment with vorinostat, bortezomib (Bortez), or both. E-cadherin transcription was significantly upregulated in both esophageal squamous (TE2) and adenocarcinoma cell lines (SKGT4) after combined treatment of vorinostat and bortezomib. In squamous esophageal cancer, combined vorinostat and bortezomib resulted in increased nm23 and RhoGDI2 transcription. Asterisk denotes P <.01 for treatment group versus tumor necrosis factor and transforming growth factor. Plus sign denotes P<.05 for bortezomib treatment versus combined vorinostat and bortezomib treatment. Crosshatch denotes P<.05 for vorinostat treatment versus combined vorinostat and bortezomib treatment. Caret denotes P<.05 for control versus tumor necrosis factor (TNF) and transforming growth factor (TGF). mRNA- Messenger RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIGURE 4
FIGURE 4
E-cadherin gene promoter activity was increased with combined treatment with vorinostat and bortezomib in both esophageal squamous carcinoma (TE2) and adenocarcinoma (SKGT4). Plus sign denotes P<.05 for vorinostat treatment versus bortezomib (Bort) treatment. Crosshatch denotes P <.05 for bortezomib treatment versus combined vorinostat and bortezomib treatment. Asterisk denotes P <.02 for given treatment group versus tumor necrosis factor and transforming growth factor. Caret denotes P <.05 for control versus tumor necrosis factor (TNF) and transforming growth factor (TGF). RLU, Relative luminescence units; Bgal, β-galactosidase.
FIGURE 5
FIGURE 5
Combined treatment with vorinostat (Vorino) and bortezomib (Bort) induces protein expression of E-cadherin in esophageal cancer (TE2) cells. TNF, Tumor necrosis factor; TGF, transforming growth factor.
FIGURE 6
FIGURE 6
Three-dimensional esophageal cancer metastasis decreased with combined vorinostat (V) and bortezomib (B) treatment. Addition of E-cadherin–blocking antibody (E-cad anti) to tumor cells treated with vorinostat and bortezomib induced increased metastasis in both squamous carcinoma (TE2, A) and adenocarcinoma (SKGT4, B) cell lines.
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