The G Protein-Coupled Receptor RAI3 Is an Independent Prognostic Factor for Pancreatic Cancer Survival and Regulates Proliferation via STAT3 Phosphorylation

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest tumors worldwide. Understanding the function of gene expression alterations is a prerequisite for developing new strategies in diagnostic and therapy. GPRC5A (RAI3), coding for a seven transmembrane G protein-coupled receptor is known to be overexpressed in pancreatic cancer and might be an interesting candidate for therapeutic intervention. Expression levels of RAI3 were compared using a tissue microarray of 435 resected patients with pancreatic cancer as well as 209 samples from chronic pancreatitis (CP), intra-ductal papillary mucinous neoplasm (IPMN) and normal pancreatic tissue. To elucidate the function of RAI3 overexpression, siRNA based knock-down was used and transfected cells were analyzed using proliferation and migration assays. Pancreatic cancer patients showed a statistically significant overexpression of RAI3 in comparison to normal and chronic pancreatitis tissue. Especially the loss of apical RAI3 expression represents an independent prognostic parameter for overall survival of patients with pancreatic cancer. Suppression of GPRC5a results in decreased cell growth, proliferation and migration in pancreatic cancer cell lines via a STAT3 modulated pathway, independent from ERK activation

Effect of siRNA based knock-down on the colony formation and migration of pancreatic cancer cell lines.

<p>A: Examples of the results of typical colony forming assays over 7 days after siRNA-treatment; B: Growth area compared to NC1 (statistically significant difference between RAI3 and NC2 is demonstrated by * for P<0.01 and ** for P<0.001 performed by t-test); C: Typical images of migrated cells in negative control (NC) and knock-down with GPRC5a siRNA (RAI3) in different cell lines; D: Number of migrated cells after 48 hour siRNA-treatment and subsequently 16 hours migration assay (* indicates statistically significant differences (P<0.05) in t-test between the negative control with non-sense-siRNA (NC) and the knock-down with GPRC5a-siRNA (RAI3)). (n = 3).</p

Univariate analysis of clinico pathological variables of the 376 patients with pancreatic cancer analyzed on the TMA.

<p>Univariate analysis of clinico pathological variables of the 376 patients with pancreatic cancer analyzed on the TMA.</p

Multivariate Cox proportional hazard model for different variables.

<p>Multivariate Cox proportional hazard model for different variables.</p

RAI3 immunohistochemistry as diagnostic and prognostic instrument.

<p>A: Definition and calculation of a diagnostic score for the presence of a pancreatic ductal adenocarcinoma (PDAC); B: Receiver Operating Characteristic (ROC) curve for the diagnostic score using for distinction of PDAC from Normal or Chronic Pancreatitis (CP) tissue (Sensitivity and specificity were calculated for two exemplary cut-off-points); C: Univariate Kaplan-Meier analysis (statistically significant differences between apical expression over 30% and less (p = 0.017) according to Log-Rank-test).</p

Expression of RAI3 in human tissue and cell lines.

<p>TMA—Immunochemistry with RAI3-Antibody in different types of pancreatic tissue. Presentation in 20-fold magnification of A-C: Normal; D: Chronic pancreatits (CP), E: Intraductal Papillary Mucinous Neoplasm (IPMN); F—H: Pancreatic Ductal Adenocarcinoma (PDAC). Three parameters were considered: RAI3-Intensity (I), RAI3-Expression (Ex) and Apical RAI3-Expression (aEx); I,J: Differential expression of RAI3 mRNA respective protein in common pancreatic cell lines and primary pancreatic cell lines measured by qRT-PCR (I) and Western Blot (J) with a conspicuously low expression in BxPc3 and PaCaDD165 in comparison to the other cell lines in the group. (n = 3).</p

Effect of treatment with GPRC5a-siRNA over 72 hours.

<p>A: Number of living cells in negative controls (NC; 1: Medium, 2: non-sense-siRNA), positive control (PC; eg5-siRNA) and knock-down with two different GPRC5a-siRNA (RAI3_A and RAI3_B). Statistically significant differences in t-test are marked with * for P-Value < 0.001 and ** for P-Value < 0.05; B: Western blot and quantitative real time PCR with RAI3 antibody confirms the successful knock-down using siRNA (for functional analysis the GPRC5A.1 siRNA was chosen) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170390#pone.0170390.s001" target="_blank">S1 Fig</a>). (n = 5).</p