Abstract 3019: Alternative transcription of the RON tyrosine kinase receptor in human pancreatic adenocarcinoma generates active protein isoforms.

Abstract

Abstract Introduction: The RON tyrosine kinase receptor is overexpressed in the majority of pancreatic duct adenocarcinomas (PDAC) and has been shown to promote pancreatic cancer cell migration, invasion and apoptotic resistance. The RON gene can be alternatively transcribed to produce protein isoforms which have variable activities. RON isoforms have been described previously in colon, breast, gastric, and lung cancer specimens and have been shown to be oncogenic. Several therapies directed against RON are in preclinical and Phase 1 trials, therefore determining biological significance of RON isoforms is of great importance. Here we report their identification in human PDAC and investigate their importance using pancreatic cancer cell lines created to overexpress the isoforms. Methods: Patient derived PDAC xenografts (PDXS) were established in the pancreata of NSG mice. Low passage (P4 or less) xenograft tumors were used to generate RNA and protein lysates for RT-PCR and Western blot analysis. Primers specific for exons 4-6 and 10-12 were created to assess alternative transcription and deletion of these exons. Results: 19 PDX's were analyzed and alternative transcription of the RON gene was identified in all but 1 tumor. Deletions of exons 5, 6, and 11 were common and resulted in transcripts for RONΔ165 (which was as common as the wild type transcript), RONΔ160, RONΔ155, and RONΔ90 isoforms. The RONΔ55 isoform (short-form RON, is created by transcription from an alternative start in exon 11 resulting in a truncated protein where the extra-cellular domain is deleted) was present in 15 of 19 tumors. Western blot analysis and Immunoprecipitation (IP) was used to confirm protein expression and determine RON activity. 7 of 9 fresh tumor samples showed RON phosphorylation. Protein isoforms were purified by IP with RON 8 antibody and then resolved using gel electrophoresis. Mass spectrometry identified the presence of RONΔ165, RON Δ170, and RONe5|6in isoforms with RONΔ165 being the most abundant. We further tested our hypothesis that the RONΔ55 isoform is active by tagging it with green fluorescent protein (GFP) and transfecting the fusion protein into 3 cell lines (Mia-Paca 2, BxPC3, and HPDE6/E7). Analysis of the novel cell lines showed constitutive phosphorylation of Δ55 and increased phosphorylation of ERK and AKT indicating enhanced signaling via the MAPK and PI3K pathways. Conclusions: Our findings show for the first time that RON is alternatively transcribed and produces biologically active isoforms in tissue derived from human pancreatic duct adenocarcinoma. We have confirmed the presence of these isoforms at the protein level and found that RONΔ165 is abundant. Further work to understand the biologic significance of these isoforms is required to guide future drug development and to validate RON as a rational target in the treatment of pancreatic cancer. Citation Format: Jeffery Chakedis, Randall French, Michele Babicky, Megan Harper, Dawn Jaquish, Evangeline Mose, Andrew Lowy. Alternative transcription of the RON tyrosine kinase receptor in human pancreatic adenocarcinoma generates active protein isoforms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3019. doi:10.1158/1538-7445.AM2013-3019