Abstract 2315: Demethylating therapy modulates expression of the RON tyrosine kinase receptor in pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction - The RON tyrosine kinase receptor is overexpressed in 80% of Pancreatic Ductal Adenocarcinomas (PDAC) and mediates invasiveness, resistance to apoptosis, and VEGF secretion. Though point mutations are rare a wide variety of RON protein isoforms have been identified, some of which are constitutively active and oncogenic. Previous studies of the DNA methylation patterns of pancreatic cancer specimens identified the gene for RON, MST1R, to be controlled by epigenetic modification. Methylation is one mechanism to regulate expression of RON, and we seek to investigate the effects of hypomethylating therapy on isoform expression and alteration of PDAC biology. Methods - Established pancreatic cancer cell lines (MiaPaca2, BxPC3, AsPC1, HPDE, Panc1) and 2 novel cell lines derived from patient tumor specimens (34E, 79E) were treated with 5-Aza-2′-deoxycytidine (Decitabine, DAC) for 96 hours. Genomic DNA was analyzed using the Methylflash (Epigentek) assay to determine genomic methylation percentage. Reverse Transcriptase PCR and western blot analysis was used to determine treatment effects on RON transcription and expression. Results - Genomic methylation analysis of pancreatic cancer cell lines revealed that compared to the control HPDE line (1.2% methylation) some lines were hypomethylated (ASPC1-1.13%, 34E-1.17%, Panc1-1.17%) while others were hypermethylated (BxPC3-1.6%, MiaPaca2-1.98%, 79E-2.06%). Hypomethylating therapy was effective in decreasing genomic methylation to below normal levels (0.4%-0.9%) in all cell lines and depleted DNA MethylTransferase 1 (DNMT1) protein levels. The effects of demethylating therapy on RON expression were differential. The amount of RON transcripts and protein expression in the RON expressing lines (79E and 34E) was decreased but increased in MiaPaca2 and Panc1. MiaPaca2 does not normally express RON, but demethylation induced RON expression. Transcripts specific for the Δ55 isoform of RON decreased in 34E, 79E, and HPDE cell lines but did not change in the others. Transcription and expression of other RON isoforms which are constitutively active decreased in 34E, 79E, ASPC1, and BxPC3 lines, with less exon 11 skipping and partial splicing of exon 5 and 6. Though RON protein expression was decreased overall in the expressing lines, phosphorylation was still detectable following DAC treatment. Conclusions - Genomic methylation of pancreatic cancer cell lines is variable and RON expression is modulated by the methylation status of its promoter. Demethylating therapy has variable effects on RON expression and is context specific. RON isoform expression was decreased in response to treatment, linking DNA methylation to alternative splicing events. These findings suggest that the consequences of demethylating therapy on PDAC biology are complex and additional studies are required to further elucidate definitive mechanisms. Citation Format: Jeffery Chakedis, Randall French, Dawn Jaquish, Evangeline Mose, Andrew Lowy. Demethylating therapy modulates expression of the RON tyrosine kinase receptor in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2315. doi:10.1158/1538-7445.AM2014-2315