A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells

Abstract

The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription domain. The splice variant is detected in 73% of xenografts derived from pancreatic adenocarcinoma patients and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirm translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing cells, activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target.

Figure 1

Discovery of a novel RON isoform in human pancreatic cancer. A, A novel RON isoform created by alternative splicing of exons 5 and 6 is discovered in a pancreatic cancer specimen. B, The mRNA sequence of this splice variant contains a partial 5 and partial 6 (P5P6) exon splicing. C, There are 195 nucleotides omitted in the final protein resulting in a 65 amino acid deletion of the first Immunoglobin-Plexin-Transcription (IPT) domain. The P5P6 protein contains the extracellular Sema domain and intracellular tyrosine kinase domain similar to the full length RON protein.

Figure 2

P5P6 is expressed in the majority of pancreatic cancers. A, PCR detects the expression of P5P6 specific transcript in PDX samples. B, The majority of PDX and pancreatic cancer cell lines express the isoform. C, Absolute quantification PCR determined the number of total RON and P5P6 specific transcript copies in each PDX and pancreatic cancer cell line. Both transcripts are overexpressed in all PDX compared to normal pancreas. P5P6 copy number is positively correlated with total RON expression, but with variable overall percentages.

Figure 3

Mass spectrometry confirms the P5P6 protein is expressed in pancreatic cancer. A, The β chain of RON is detected by western blot in a representative group of PDX. Pro-RON and full length RON (flRON) bands are prominent (black arrows), however there are different molecular weight bands which may correspond to expressed isoforms (grey and red arrows). B, After trypsin digestion RON isoforms are distinguished by unique peptides using mass spectrometry. C, We discovered a peptide in PDX 1713 which can only be produced by partial exon 5 and partial exon 6 splicing. D, Several other peptides were detected which are specific to RON but not specific to any isoform. This confirms protein expression of P5P6 in pancreatic cancer.

Figure 4

P5P6 expression in human pancreatic duct epithelial cells. A, The P5P6-eGFP fusion protein is expressed in the HPDE cell line. Live cell imaging shows there is no change in cell morphology but the HPDE P5P6e cell line has a heterogeneous mix of fluorescent cells. B, The P5P6e protein is most abundant in the cytoplasm but some cells display plasma membrane localization (blue arrows). C, P5P6e protein is expressed in all three cell lines and is cleaved from a pro– form into α and β subunits. The isoform is constitutively active in all cell lines, though most heavily phosphorylated in HPDE. In the HPDE cell line, P5P6e is expressed at such a high level that degradation products are detectable and phosphorylated (Red arrows). D, The expression of P5P6e protein in HPDE and HPNE is comparable to total RON expression in pancreatic cancer cell lines (AsPC1, Bx-PC3, FG) and 3 PDX specimens.

Figure 5

P5P6 induces oncogenic phenotypes in human pancreatic duct epithelial cells A, P5P6e expression increases AKT phosphorylation in the HPDE cell line and activates both AKT/MAPK in HPNE. B, Stimulation with the RON ligand, MSP (100 ng/mL for 30 minutes) activates full length RON in the HPDE cell line but did not increase P5P6 phosphorylation. MSP stimulation did not change P5P6 downstream signaling. C, Small molecule RON/MET inhibitor BMS-777607 completely inhibited phosphorylation of P5P6 (3 μmol/L for 1 hour). This inhibition could not be overcome with MSP stimulation (100 ng/mL for 30 minutes). There is decreased AKT activation by blocking P5P6 phosphorylation, but there is no effect on the MAPK pathway. D, Mechanism of P5P6e tumorigenesis is distinct from RONΔ160 as there is no accumulation of total β-catenin. E, P5P6 signaling does not activate STAT3 transcription factor pathway in any cell line unlike MSP stimulated full length RON activation.

Figure 6

P5P6 transforms human pancreatic duct epithelial cells. A, Cell motility is significantly increased as a result of P5P6 expression in the HPDE cell lines but not in Cos or MiaPaca2 (Supplemental Figure S2B). B, NIH 3T3 transforming assay showed transformation with expression of P5P6e with significantly more colonies formed than the empty vector. The transforming effect is not as strong as KRAS G12V mutation expression.

Figure 7

P5P6 is tumorigenic when expressed in vivo. A, HPDE and HPDE P5P6e cells were injected orthotopically and subcutaneously into NSG mice. Only the HPDE P5P6e expressing cell line can form a tumor. B, All tumors expressed the P5P6 eGFP fusion protein and are brightly fluorescent. C, HPDE P5P6e Orthotopic mean tumor volume and weight was statistically significant as 6 of 8 had tumor growth (p-value 0.03). D, H+E and immunohistochemistry shows isoform expressing cells (Green, GFP) are mixed with a large stromal component (Blue, DAPI). HPDE P5P6e cells maintain morphology seen in culture and express E-cadherin (Red). Clusters of residual host pancreas acini are interspersed throughout the tumor (white arrows).