Abstract POSTER-BIOL-1319: Temporal shifts in the epigenetic regulation of tight junctions from primary to recurrent ovarian cancer
Authors: Huang Zhiqing , Visco Zachery , Sfakianos Gregory , Whitaker Regina , Berchuck Andrew , Murphy Susan 
Published: August 2015

Purpose: Despite a complete clinical response following treatment with surgery and first-line chemotherapy, about 70-90% of patients with advanced stage ovarian cancer (OC) will inevitably experience disease recurrence. Our objective was to identify potential new therapeutic targets for prevention of recurrence by identifying changes in gene regulation that occur in recurrent versus matched primary tumors.

Procedures: We used 16 primary-recurrent tumor pairs from patients with stage III/IV serous epithelial OC from the Duke Gynecologic Oncology Tissue Bank. Genomic DNA and total RNA were extracted from FFPE tissues using the Puregene tissue DNA kit (Qiagen) and Ambion Recover All Total Nucleic Acid Isolation kit (described in Gyn Onc, 2013 (129): 159-164), respectively. Illumina Infinium HumanMethylation450 BeadChip arrays were used to quantify DNA methylation and Affymetrix Human Genome U133A 2.0 arrays were used to quantify gene expression. Methylation data were analyzed using Illumina’s GenomeStudio software and beta values for each CG site were obtained. Pearson’s r values for the correlation between gene expression (log2 RMA normalized) and methylation beta values were generated using GraphPad Prism 6. Genes with significant correlation between expression and methylation (p&lt;0.05) were entered into GATHER to identify pathways enriched among this gene set. Gene expression was further analyzed by tumor stage using an independent dataset of serous OCs with one-way ANOVA analysis in R. Expression of Claudin genes in 42 OC cell lines following treatment with the DNA methyltransferase inhibitor decitabine was analyzed using Affymetrix U133A gene expression microarrays and quantitative real-time PCR.

Results: Methylation-expression relationships for sixty-seven tight junction genes, including multiple Claudin family genes (Claudins 1,4,5,7,9 and 10), emerged as significantly enriched in recurrent but not primary OCs (Bayes factor 7; p&lt;0.0001). These Claudins were also upregulated following decitabine treatment, supporting that DNA methylation regulates their expression. Claudin expression correlated with tumor stage and most showed higher expression in tumor tissues as compared to normal fallopian tube fimbriae and ovarian surface epithelia (p&lt;0.05), while Claudin1 showed the reverse (lower in tumor than normal, p&lt;0.05). Treatment with PIKfyve Inhibitor YM201636, which blocks continuous recycling of Claudin-1 and Claudin-2 to the cell membrane, prevented spheroid formation under stem cell culture conditions. These finding suggests that Claudins may be important for cancer stem cell survival, which is postulated as an underlying cause of tumor recurrence.

Conclusion: Our findings indicate that tight junctions and the methylation-regulated expression of Claudins are significantly associated with recurrent OC and suggest that changes in DNA methylation modulate emergence of these relationships. Claudin regulation may play an important role in tumor recurrence and may offer new therapeutic opportunities.

Citation Format: Zhiqing Huang, MD, PhD, Zachery Visco, Gregory Sfakianos, MD, Regina Whitaker, BS, Andrew Berchuck, MD, Susan Murphy, PhD. Temporal shifts in the epigenetic regulation of tight junctions from primary to recurrent ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1319.