Silencing of RON receptor signaling promotes apoptosis and gemcitabine sensitivity in pancreatic cancers

Abstract

The RON receptor tyrosine kinase is overexpressed in premalignant pancreatic intraepithelial neoplasia (PanIN) and in the majority of pancreatic cancers. In pancreatic cells, RON is an important K-Ras effector and RON ligand can enhance migration/invasion and apoptotic resistance. However, the pathobiological significance of RON overexpression in pancreatic cancers has yet to be fully established. In this study, we demonstrate that RON signaling mediates a unique transcriptional program that is conserved between cultured cells derived from murine PanIN or human pancreatic cancer cells grown as subcutaneous tumor xenografts. In both systems, RON signaling regulates expression of genes implicated in cancer-cell survival, including Bcl-2 and the transcription factors signal transducer and activator of transcription 3 (STAT 3) and c-Jun. shRNA-mediated silencing of RON in pancreatic cancer xenografts inhibited their growth, primarily by increasing susceptibility to apoptosis and by sensitizing them to gemcitabine treatment. Escape from RON silencing was associated with re-expression of RON and/or expression of phosphorylated forms of the related c-Met or epidermal growth factor receptors. These findings indicate that RON signaling mediates cell survival and in vivo resistance to gemcitabine in pancreatic cancer, and they reveal mechanisms through which pancreatic cancer cells may circumvent RON-directed therapies.

Figure 1

Activation of the RON receptor results in 858 differentially expressed genes after 30 minutes and 12 hours. Affymetrix gene chip analyses were performed using PanIN cells treated with 400ng/ml for HGFL for 0 minutes (untreated), 30 minutes or 12 hours. 311 genes were differentially expressed at 30 minutes while 582 were altered at 12 hours. Red areas represent genes that were upregulated and blue areas represent genes that were downregulated.

Figure 2

RON downregulation suppresses tumor growth in human xenograft tumors. A) XPA-1 and FG-derived tumor xenografts were screened for RON expression by immunoblotting. U = untransfected parental cell line, N = non-silencing shRNA vector transfected cell line, S = RON-silenced cell line. B) Growth of FG-derived tumor xenografts. Tumors were measured twice weekly via calipers and growth was plotted. C,D) Mice were injected with BrdU 2 hours prior to sacrifice. Sectioned tumors were stained and scored for BrdU incorporation. No statistical difference was seen in the latency, growth, tumor volume, or proliferation in the RON-silenced tumors relative to controls, P > 0.05 for both XPA-1 and FG.

Figure 3

Downregulation of RON results in increased necrosis in pancreatic xenografts. A) H&E staining revealed an 85% increase in necrosis in RON-silenced tumors. n = necrotic areas of tumor (p = 0.001) B) Viable RFP-labeled XPA-1 cells were detected within the tumor xenografts using the IVIS^® lumina live animal imager to measure photon emission twice weekly. There was a 3-fold reduction in photon emission from RON-silenced tumors after 30 days of growth (p = 0.002).

Figure 4

The effect of RON expression on apoptosis in pancreatic tumor xenografts. A) As measured by TUNEL staining, downregulation of the RON receptor resulted in XPA-1 and FG tumors with 43% and 74% more apoptotic cells per μm² relative to RON-expressing tumors, respectively (p= 0.05). B) Tumor lysates from RON-silenced and RON expressing xenografts were immunoblotted for expression levels of RON, p-ERK, total ERK, p-AKT, total AKT, bcl-2, c-jun, stat3. U = tumors derived from untransfected parental cells, N = non-silenced transfected, S = RON-silenced transfected. For XPA-derived tumors, Expression in RON-silenced xenografts was reduced for p-ERK, pAKT, bcl-2, c-jun and stat3 by 50%, 66%, 70%, 53%, and 40%, respectively; while for FG tumors, levels of p-ERK, pAKT and stat-3 were reduced by 62%, 58% and 47%, p < 0.05. In both cases, there was no significant difference in total-ERK or total AKT expression. All results depict lysates from tumors harvested after 28 days of growth.

Figure 5

Downregulation of the RON-receptor sensitizes pancreatic tumor xenografts to gemcitabine treatment. A) Left - Tumor regression after treatment with gemcitabine was more profound in RON-silenced tumors than in RON-expressing controls. Right -representative photographs using the IVIS^® Lumina live-animal imager. B) RON-silenced, gemcitabine treated tumors took nearly twice as long to reach 1000mm³ as RON-expressing tumors and greater than 3× longer than untreated tumors regardless of RON expression (p < 0.05).

Figure 6

RON-silenced tumors demonstrate kinase switching after escape from gemcitabine-induced tumor regression. Prior to escape, there is no demonstrable presence of RON, p-EGFR, or p-met. Expression of pEGFR, p-met and total RON is observed in tumors after escape, whereas total met and total EGFR were expressed both pre-escape and post-escape.