Abstract 4347: Epigenetic targeting of DNMT1 in adipocytes inhibits high-grade serous ovarian cancer cell migration and invasion through TIMP3 upregulation
Authors: Tang Jessica , Fang Fang , Buechlein Aaron , Taverna Pietro , Nephew Kenneth P. 
Published: July 2017

Ovarian cancer frequently metastasizes to the omentum and adipocytes play a significant role in tumor progression. As methylation levels in obese adipose tissue are increased due to increased DNMT1 levels and activity, it was of interest to test the hypothesis that inhibiting DNMT1 would reverse adipocyte methylation, alter adipokine secretion, and decrease migration and invasion of ovarian cancer cells towards adipocytes. Human adipocytes were seeded in a 24-well plate and treated with low-dose guadecitabine (100nM daily for 3 days). Ovarian cancer cells (SKOV3, Kuramochi, OVCAR4, OVCAR8) were seeded in the Boyden chamber and allowed to migrate or invade toward adipocytes for 8 and 16h, respectively. Expression of epithelial-mesenchymal transiton (EMT) markers (SLUG, FN1, TWIST1) were assessed by qRT-PCR. Adipocyte-conditioned media was used to culture ovarian cancer cells in clonogenicity assay and a human adipokine array (R&D Systems) was used to assess changes in cytokine secretion. DNMT1 protein levels in adipocytes were determined by western blot. To determine a possible mechanism, DNA and RNA from guadecitabine-treated adipocytes were subjected to methylcapture-sequencing (MBD-seq) and RNA-seq, respectively. Guadecitabine treatment of adipocytes decreased (P&lt;0.05) migration of OVCAR4 and OVCAR8 (35% and 40%, respectively, compared to control), and a 50% decrease (P&lt;0.05) in invasion towards adipocytes after guadecitabine treatment was observed for OVCAR4, OVCAR8, and Kuramochi cells. Expression of EMT markers SLUG, FN1 and TWIST1 decreased (P&lt;0.05) after guadecitabine treatment. Conditioned media from guadecitabine-treated adipocytes decreased (P&lt;0.05) clonogenic survival by 18% compared to control. Adipokine array results revealed increased secretion of LIF (lipoprotein lipase inhibitor) and TIMP3 (metalloproteinase inhibitor) after guadecitabine treatment (1.6- and 1.8-fold increase, respectively; verified by qRT-PCR). Treatment with recombinant TIMP3 (50nM) decreased invasion of OVCAR8 (63%, P&lt;0.001) and OVCAR4 cells (73%, P&lt;0.05). DNMT1 protein levels in adipocytes decreased in the presence of guadecitabine, despite the presence of a DNA synthesis inhibitor. Ingenuity Pathway Analysis (IPA) of MBD-seq data showed significant changes in cell-to-cell signaling and interaction pathways (FC &gt;10; FDR &lt;0.05). RNA-seq demonstrated increased expression of matrix metalloprotease inhibitors (THSB2, TFPI2, and NDRG4) and IPA analysis revealed a significant change in regulation of EMT pathway (FC &gt; 1.5; FDR &lt;0.05). Guadecitabine treatment of adipocytes alters adipokine secretion resulting in decreased cancer cell migration and invasion. In addition to direct effects on ovarian cancer cells, hypomethylating agents may impact the tumor microenvironment to alter adipokine secretion leading to decreased metastasis.

Citation Format: Jessica Tang, Fang Fang, Aaron Buechlein, Pietro Taverna, Kenneth P. Nephew. Epigenetic targeting of DNMT1 in adipocytes inhibits high-grade serous ovarian cancer cell migration and invasion through TIMP3 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4347. doi:10.1158/1538-7445.AM2017-4347