Abstract 188: Interferon stimulated gene expression as a potential resistance mechanism in DNA damage from epigenetic inhibitors
Authors: Luszczek Wioleta , Cheriyath Venugopalan , Mekhail Tarek , Borden Ernest C. 
Published: February 2011

Since epigenetic inhibitors, such as inhibitors of histone deacetylases (HDAC) and DNA methyltransferases (DNMT) can induce inhibition of cancer cell proliferation we tested the hypothesis that concurrent inhibition of DNA methylation by 5-AZA-dC and histone acetylation by LBH589 or MGCD0103 could synergistically reduce the viability of human small cell lung carcinoma (SCLC) cell lines.

SCLC cells were treated with 5-AZA-dC in combination with HDAC inhibitors. Growth inhibition of cells was assessed after 72 h with Alamar blue assay. Synergistic effect was determined by Chou and Talalay analysis. Inhibition of DNMT1 expression by 5-AZA-dC was verified by Western blot. TUNEL and Caspase 3 cleavage assays were used to detect apoptosis in SCLC. DNA damage was assessed by Comet assay and analyzed using CometScoreTM software. Illumina microarray analysis was performed to compare basal gene expression in sensitive H526 and resistant H196 cells. Real-time RT-PCR was used to confirm gene array data.

Although sub-IC50 concentrations of 5-AZA-dC and the HDAC inhibitors (LBH589 or MGCD0103) synergistically reduced proliferation of 4/6 SCLC cell lines, loss of viability of SCLC cells did not correlate with the inhibition of either DNMT1 or HDACs suggesting non-epigenetic mechanisms for the synergy. Since combinations of 5-AZA-dC and HDAC inhibitors had marginal effects on cell cycle profiles or apoptosis index, comet assay was undertaken to assess DNA damage. MGCD0103 and 5AZA-dC co-treatment augmented DNA damage in SCLC cells resulting in increased tail length or moment in comet assays by 24 hrs in sensitive cell lines. Comparison of ratio of basal gene expression profiles of resistant H196 to sensitive H526 cells identified expressed (≥2x) mRNA differences of 2197 genes in H196 cells. Of these, &gt;x10x expression of 273 genes was detected in resistant H196 cells compared to sensitive H526. More than 17% of genes from highly expressed genes (&gt;60 fold) belonged to interferon stimulated gene (ISG) pathways. IFI27 was the most highly expressed gene (379 fold difference) in H196 cells compared to H526 cells; other constitutively expressed ISGs included IFIT1, IFI44, IFI35, MX1 and GBP2. In confirmation of this unexpected microarray result, qRT-PCR identified marked upregulation of 15 selected genes in H196 cells. To further confirm correlation between upregulation ISGs with DNA damage resistance, basal expression of ISGs in sensitive (H526, H82, H146 and DMS114) and resistant (H196, SW1271) cells were compared. Box and Whiskers analysis identified markedly higher median mRNA levels for most of ISGs in the resistant cells.

Constitutive expression of IFN-stimulated genes may predict SCLC cells resistance to epigenetic modulators or other DNA damage agents; these unexpected findings may contribute to enhance mechanistic understanding of cell growth inhibition by both IFNs and epigenetic inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 188.