Abstract B10: Vorinostat exposure results in decreased lamin B1 expression and nuclear structure normalization in an in vitro model of esophageal adenocarcinoma progression
Authors: Nandakumar Vivek , Hansen Nanna , Hernandez Kathryn , Senechal Patti , Slaydon Miranda , Glenn Honor , Davies Paul , Johnson Roger H. , Bussey Kimberly J. , Meldrum Deirdre R. 
Published: August 2014

Esophageal adenocarcinoma (EA) has a poor prognosis with five-year survival rates ranging between 5% and 30%, depending on stage at diagnosis. Barrett's esophagus (BE) contributes to a 15-fold increased risk for EA and is thought to be caused by chronic acid reflux. BE is a multi-stage condition believed to progress through metaplasia to dysplasia and then to EA. The progression from normal squamous epithelium through BE to EA is characterized by genetic and epigenetic alterations, but little is known about their association with the gross morphological alterations, including nuclear structure, used for clinical diagnosis. Recent advances in epigenomics increasingly acknowledge the interplay between chromatin remodeling and the role of three-dimensional (3D) nuclear architecture in regulating gene expression. To unravel the epigenetic mediated links between the cytological and molecular scale alterations in BE progression, we systematically investigated the effects of the clinically available histone deacetylase (HDAC) inhibitor vorinostat on gene expression and 3D nuclear architecture using human cell lines representative of normal squamous (EPC2), metaplasia (CPA) and adenocarcinoma (FLO-1) of the esophageal epithelium.

We assessed alterations in viability, gene expression, and three-dimensional (3D) nuclear morphology upon drug treatment. A 12-point, three-fold, serial dilution drug-dose-response (DDR) curve at 24, 48 and 72 hour time points indicated that FLO-1 cancer cells were most sensitive to the drug, with an IC50 value at 48 hours of 0.7928 μM, versus 7.536 μM for CPA and 21.49 μM for EPC2. FLO-1, but not CPA or EPC2, underwent apoptosis after 24 hours of vorinostat exposure as assessed by the Caspase 3/7 Glo. RT-qPCR to assess gene expression demonstrated that p21 (CDKN1A) was induced as expected in all cell lines, with FLO-1 showing the greatest induction. MGMT expression was also increased 12-fold in FLO-1 after vorinostat treatment. To understand the gross morphological correlates of vorinostat treatment in all cell lines, we used optical computed tomography to image untreated, DMSO-treated and vorinostat treated cells individually in 3D with sub-micron, isotropic spatial resolution. Quantitative 3D image analysis revealed a preferential normalization of nuclear structure of cancer and metaplastic cells after 48 hour treatment at the FLO-1 IC50 dose. FLO-1 cells showed markedly reduced cell and nuclear volumes and decreased chromatin clumpiness after vorinostat treatment. Similar morphological trends were present but less pronounced in CPA cells. EPC2 cells showed minimal changes in response to treatment. These morphological trends were accompanied by the expected increase in H3K9 acetylation, which was significant in FLO-1. However, the anticipated corresponding drop in H3K9me3 was not observed. FLO-1 expressed significantly more lamin B1, but not lamin B2 or lamin A/C, compared to the other cell lines. Lamin B1 was significantly reduced in FLO-1 after vorinostat treatment.

These results support further exploration of vorinostat for the treatment of patients with BE and EA. Our data indicate that vorinostat sensitivity increases, with a corresponding increase in apoptosis, as cells progress from normal to esophageal adenocarcinoma. This increased sensitivity to the drug is accompanied by a normalization of nuclear architecture, including a reduction in lamin B1, in surviving abnormal cells after treatment.

Citation Format: Vivek Nandakumar, Nanna Hansen, Kathryn Hernandez, Patti Senechal, Miranda Slaydon, Honor Glenn, Paul Davies, Roger H. Johnson, Kimberly J. Bussey, Deirdre R. Meldrum. Vorinostat exposure results in decreased lamin B1 expression and nuclear structure normalization in an in vitro model of esophageal adenocarcinoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B10.