Induction of Thioredoxin-Interacting Protein by a Histone Deacetylase Inhibitor, Entinostat, Is Associated with DNA Damage and Apoptosis in Esophageal Adenocarcinoma - PubMed (original) (raw)

. 2018 Sep;17(9):2013-2023.

doi: 10.1158/1535-7163.MCT-17-1240. Epub 2018 Jun 22.

Deborah R Surman 1, Kate Brown 1, Yuan Xu 1, Lucas A McDuffie 1, Vivek Shukla 1, Emily S Reardon 1, Daniel R Crooks 2, Jane B Trepel 3, Sunmin Lee 3, Min-Jung Lee 3, Shaojian Gao 1, Sichuan Xi 1, Kaitlin C McLoughlin 1, Laurence P Diggs 1, David G Beer 4, Derek J Nancarrow 4, Leonard M Neckers 2, Jeremy L Davis 1, Chuong D Hoang 1, Jonathan M Hernandez 1, David S Schrump 1, R Taylor Ripley 5

Affiliations

Induction of Thioredoxin-Interacting Protein by a Histone Deacetylase Inhibitor, Entinostat, Is Associated with DNA Damage and Apoptosis in Esophageal Adenocarcinoma

Paul L Feingold et al. Mol Cancer Ther. 2018 Sep.

Abstract

In 2017, an estimated 17,000 individuals were diagnosed with esophageal adenocarcinoma (EAC), and less than 20% will survive 5 years. Positron emission tomography avidity is indicative of high glucose utilization and is nearly universal in EAC. TXNIP blocks glucose uptake and exhibits proapoptotic functions. Higher expression in EAC has been associated with improved disease-specific survival, lack of lymph node involvement, reduced perineural invasion, and increased tumor differentiation. We hypothesized that TXNIP may act as a tumor suppressor that sensitizes EAC cells to standard chemotherapeutics. EAC cell lines and a Barrett epithelial cell line were used. qRT-PCR, immunoblot, and immunofluorescence techniques evaluated gene expression. TXNIP was stably overexpressed or knocked down using lentiviral RNA transduction techniques. Murine xenograft methods examined growth following overexpression of TXNIP. Apoptosis and DNA damage were measured by annexin V and γH2AX assays. Activation of the intrinsic apoptosis was quantitated with green fluorescence protein-caspase 3 reporter assay. In cultured cells and an esophageal tissue array, TXNIP expression was higher in Barrett epithelia and normal tissue compared with EAC. Constitutive overexpression of TXNIP decreased proliferation, clonogenicity, and tumor xenograft growth. TXNIP overexpression increased, whereas knockdown abrogated, DNA damage and apoptosis following cisplatin treatment. An HDAC inhibitor, entinostat (currently in clinical trials), upregulated TXNIP and synergistically increased cisplatin-mediated DNA damage and apoptosis. TXNIP is a tumor suppressor that is downregulated in EACC. Its reexpression dramatically sensitizes these cells to cisplatin. Our findings support phase I/II evaluation of "priming" strategies to enhance the efficacy of conventional chemotherapeutics in EAC. Mol Cancer Ther; 17(9); 2013-23. ©2018 AACR.

©2018 American Association for Cancer Research.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1.

Figure 1.. Glutamine is Required for Proliferation EACC. TXNIP is higher in esophageal adenocarcinoma samples and is repressed by glutamine.

A. Flo-1, Esc2, OE33, and CP-C were cultured in DMEM with 1mM pyruvate. 10 mM glucose (G) and / or 2 mM glutamine (Q) are added as indicated. Cells were grown in 96-well plates; proliferation was measured using Cyquant DNA quantification kits. B. Flo-1, Esc2, OE33, and CP-C were treated for 72 hours with L-Glutamic acid γ-(p-nitroanilide) hydrochloride (GPNA). Cells were grown in 96-well plates and proliferation was measured using Cyquant DNA quantification kits at day 5. C. Samples were categorized into 4 levels of expression patterns. 22 normal esophageal tissue were compared to 20 EAC samples from a US Biomax microarray. The brown staining indicates TXNIP expression in a representative sample. The top row is magnified by 4x and the bottom row by 10x. D. Immunoblot of TXNIP in EACC lines and Barrett’s Esophageal cell lines. E. qRT-PCR and immunoblot normalized to b-actin measured TXNIP mRNA and protein expression after proliferation with glucose only, glutamine only, or both.

Figure 2.

Figure 2.. TXNIP Overexpression Was Associated with Modest Glycolytic Inhibition.

A. Realtime Seahorse extracellular flux analysis of extracellular acidification rate (ECAR) measures glycolytic activity based on sequential glucose, oligomycin (oligo), and 2-DG injections at the time points indicated. ECAR is measured in Flo-1 and Esc2 with TXNIP overexpressing cells versus controls. B. YSI analyzer measured lactate secretion over a 5-day period in Flo-1 and Esc2 with overexpression of TXNIP. C. Reactive Oxygen Species (ROS) production was measured using DCFDA kit with a Calibur flow cytometer after glutamine withdrawal.

Figure 3.

Figure 3.. TXNIP Acts As a Tumor Suppressor in EACC.

A. After selection, EACC overexpressing TXNIP were grown in non-puromycin growth media for 5 days and proliferation was quantified using Cyquant DNA measurement kits. DNA measurements were translated into cell numbers by a standard curve. Flo-1and Esc2 cells were seeded at 1,500 and 1,000 cells per well, respectively. B. Colonies of EACC overexpressing TXNIP in soft agar were stained with crystal violet, photographed, and counted (mean ± SD). C. Esc2 overexpressing TXNIP versus control cells were injected subcutaneously into flanks of nude mice (10 mice / 20 flanks per experiment). Tumor growth curve in nude mice were measured by caliper (mean ± SEM). D. Mice were sacrificed and tumors harvested 30 days after injection. Photographs of each harvested tumors derived from both groups are displayed. The upper row is tumors from control cells and the bottom row is tumors from cells overexpressing TXNIP. E. Tumor weight was measured (mean ± SEM). F. RNA was extracted from emulsified fragments of each tumor and TXNIP mRNA expression was quantified using qRT-PCR normalized to β-actin.

Figure 4.

Figure 4.. Platinum-Based Chemotherapy Increased TXNIP Expression and Overexpression of TXNIP Enhanced the Effectiveness of this Therapy.

A. Cells were treated with cisplatin for 72 h at the concentrations indicated. qRT-PCR and immunoblots of TXNIP expression were normalized with β-actin. B. Flo-1 overexpressing TXNIP were treated with 4 ug/mL of cisplatin and Esc2 overexpressing TXNIP were treated with 1.0 ug/mL of cisplatin for 24 h. qRT-PCR and immunoblots of TXNIP expression were normalized with β-actin. C. Esc2 cells with lentiviral shRNA knockdown of TXNIP were treated with 1.5 ug/mL of cisplatin for 24 h. qRT-PCR and immunoblots of TXNIP expression were normalized with β-actin. D. Cell lines were treated with the same time and concentrations as in (B.), after which, γH2AX immunofluorescent staining was performed. In Flo-1, puncts were counted in 50 cells from each group. Esc2 had very high γH2AX uptake and so levels were categorized based on percent staining intensity. E. - F. Annexin V apoptosis assay was performed after cisplatin treatment as described in (B.).

Figure 5.

Figure 5.. Combined Entinostat and Cisplatin Treatment Is Associated with TXNIP Induction and Increased DNA Damage and Apoptosis.

A. Flo-1 cells were treated with 1 uM entinostat and 1 ug/mL cisplatin for 48 hours. Esc2 cells were treated with 1 uM entinostat and 0.35 ug/mL cisplatin for 48 hours. qRT-PCR and immunoblots of TXNIP expression were normalized with β-actin. B. After treatment conditions as described in (A.), γH2AX immunofluorescent staining was performed. γH2AX levels were categorized based on percent staining intensity. C. After treatment conditions as described in (A.), cellular apoptosis was analyzed with annexin V assays. D. After treatment conditions as described in (A.), cellular apoptosis was analyzed with annexin V assays for Esc2 cells with lentiviral shRNA knockdown of TXNIP versus controls. E. After treatment conditions as described in (A.), caspase 3 and cleaved caspase 3 levels were quantified based by immunoblot. F. Flo-1 cells were treated with the proteasome inhibitor, MG132, and the anti-bcl-2 drug, ABT-137, as positive controls. A cleaved caspase 3 GFP reporter assay was generated with cells transfected with a GFP-labeled reporter attached to a ubiquitin domain by a caspase 3 site. Cells were treated in conditions as described in (A.). Pictures were obtained with a fluorescent microscope at 4x magnification.

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