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

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Combined proteasome and histone deacetylase inhibition attenuates epithelial– mesenchymal transition through E-cadherin in esophageal cancer cells Matthew D. Taylor, MD, Yuan Liu, MD, PhD, Alykhan S. Nagji, MD, Nicholas Theodosakis, BS, David R. Jones, MD The Journal of Thoracic and Cardiovascular Surgery Volume 139, Issue 5, Pages 1224-1232.e1 (May 2010) DOI: 10.1016/j.jtcvs.2010.01.039 Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

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. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

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. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

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. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

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. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

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. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

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. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

A, Morphologic analysis of SKGT4 cell line associated with treatment groups. Tumor necrosis factor α (TNF) and transforming growth factor β (TGFβ) treatment demonstrated increased mesenchymal cell morphology associated with induction of epithelial–mesenchymal transition. Treatment with vorinostat, bortezomib (Bortez), or both (V+B) restored epithelial phenotype from mesenchymal phenotype. B, Real-time polymerase chain reaction demonstrated significant upregulation of vimentin and N-cadherin after treatment with tumor necrosis factor α and transforming growth factor β. Asterisk denotes P < .05 for control versus tumor necrosis factor α and transforming growth factor β. Crosshatch denotes P < .03 for control versus tumor necrosis factor α and transforming growth factor β. mRNA, Messenger RNA; GAPDH, gene for glyceraldehyde-3-phosphate dehydrogenase. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 1224-1232.e1DOI: (10.1016/j.jtcvs.2010.01.039) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions