Abstract A72: Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce acinar to ductal metaplasia
Authors: Chakedis Jeffery M. , French Randall , Babicky Michele , Jaquish Dawn , Howard Haleigh , Mose Evangeline , Miyamoto Jaclyn , Walterscheid Zakk , Holman Patrick , Lowy Andrew M. 
Published: October 2015

Introduction:The RON tyrosine kinase receptor is increasingly overexpressed during progression to Pancreatic Intraepithelial Neoplasia (PanIN) and pancreatic duct adenocarcinoma (PDAC). RON expression in these neoplastic cells is associated with a highly invasive phenotype and resistance to gemcitabine. Mutations in the gene for RON, MST1R, are rare but alternative splicing produces a wide variety of protein isoforms. Exon skipping is the most common and a skipping of exon 11 produces RONΔ165. Alternative transcription start in intron 10 produces short-form RON (sfRON) which promotes metastasis in breast cancer. Eight RON isoforms have previously been reported in different epithelial cancer types (Colon, Breast, Stomach) and we initially sought to characterize their expression in PDAC. Using low passage patient derived xenografts (PDXs), we found that the known RON isoforms Δ165 and sfRON were expressed in the majority of PDX’s.

Results: This study revealed a novel RON isoform resulting from partial splicing of exons 5 and 6 (P5P6) to produce a protein that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. Peptides specific to RON P5P6 were detected in pancreatic cancer specimens by mass spectrometry which confirms translation of the protein. Using end point PCR we determined that RON is overexpressed in 90% of PDX and P5P6 expressed in 73%. In PDXs absolute quantification PCR determined RON isoforms P5P6, sfRON, and RONΔ165 make up 15 – 50% of total RON transcripts. This puts total isoform expression at up to 500 times more than the full length protein in normal pancreas. To determine function of these isoforms we overexpressed sfRON, RON P5P6, and RONΔ165 in a Human Pancreatic Ductal Epithelial (HPDE) cell line using a lentiviral vector. We found that all isoforms are constitutively phosphorylated, present in the cytoplasm in a reticular pattern, and RON P5P6 traffics to the plasma membrane. RON P5P6 is spliced into α and β subunits and can bind its ligand Macrophage Stimulating Protein similar to full length RON. Expression of P5P6 in HPDE cells activates downstream AKT and MAPK signaling pathways and increases cell motility, VEGF secretion, and markers of EMT. HPDE cells expressing P5P6 and short form RON were injected orthotopically into NOD SCID Gamma mouse pancreata and are tumorigenic while the parental cell line is not. P5P6 and short form RON tumors induce a denser stroma not characteristically observed in cell line derived xenografts. In HPDE sfRON tumors, host mouse acini adjacent to the tumor undergo acinar to ductal metaplasia. As the tumor invades into the mouse pancreas these newly formed ducts become increasingly dysplastic and begin to express mesenchymal markers.

Conclusion: We have identified protein isoforms of the RON tyrosine kinase receptor which are expressed in human pancreatic cancer. A novel isoform RON P5P6 and short form RON transform human pancreatic ductal epithelial cells to become tumorigenic. Expression of short form RON in these tumors causes host mouse pancreas to undergo acinar to ductal metaplasia. Ultimately these findings demonstrate that RON isoform expression by pancreatic cancer cells has significant biologic consequences and promotes interaction with the adjacent normal pancreas. Further work to characterize the soluble factors responsible for RON paracrine signaling is currently underway.

Citation Format: Jeffery M. Chakedis, Randall French, Michele Babicky, Dawn Jaquish, Haleigh Howard, Evangeline Mose, Jaclyn Miyamoto, Zakk Walterscheid, Patrick Holman, Andrew M. Lowy. Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A72.