Abstract A71: The Prrx1 homeodomain transcription factor plays a central role in pancreatic regeneration and carcinogenesis.

Introduction: Embryonic development, regeneration and carcinogenesis share regulatory mechanisms in the pancreas. We sought to identify a transcriptional program in the pancreas that regulates these processes within a distinct cell population of pancreatic duct/duct-like cells. We have identified the paired-related homeodomain transcription factor Prrx1 as a potential master transcriptional factor in this context. Methods: Duct cells, acinar-to-ductal metaplastic cells during acute pancreatitis as well as PanIN cells in KrasG12D-driven early carcinogenesis ( Pdx1-cre;Kras G12D/+ ) were isolated using DBA lectin labeling and magnetic beads separation. We purified RNA from DBA lectin positive cells at critical days of pancreatic ductal development (E14.5, E15.5, E16.5, P1), days 0, 1, 3 and 5 of cerulein-induced acute pancreatitis and from mice in which Kras G12D genetically has been targeted to the pancreas at 10 and 20 weeks of age. The RNA samples were subjected to microarrays and bioinformatic analysis. Functional studies were performed. Results: We were particularly interested in genes that are downregulated during embryonic development as differentiation progresses, genes that are transiently upregulated during acute pancreatitis and subsequent regeneration as well as genes that are upregulated during early carcinogenesis (PanIN). This approach left us with 76 genes common to all three processes. Of these, Prrx1 was one the most regulated genes with known transcriptional activity. Initially, we validated Prrx1 expression by immunostaining in mouse/human tissues of pancreatitis and PanIN/PDAC. Interestingly, the Prrx1b transcript variant is strongly upregulated during cerulein-induced acute pancreatitis whereas the Prrx1a variant remains unchanged. In cancer, however, both variants are upregulated in a stepwise fashion depending upon the genetic alterations that have been introduced (wt vs. Kras G12D/+ vs. Kras G12D/+ ;p53 R172H/+ ). Most notably, circulating pancreatic cells express both variants significantly higher than in pancreatic cells still residing within the primary tumor. In order to assess Prrx1’s role in self-renewal we performed pancreato-sphere assays with pancreatic ductal cells derived from Pdx1-cre;Kras G12D/ + animals. Prrx1b overexpression led to significantly more and larger spheres. Prrx1a overexpression had no effect. Conversely, when we knockdown Prrx1b, but not Prrx1a, by RNAi sphere formation is decreased. In order to investigate the role of Prrx1 in an in vivo setting, we utilized the Prrx1CreER T2 GFP mouse model in which Prrx1 expressing cells are GFP positive. We sorted cells according to DBA lectin (D) and GFP (G) status by FACS. Of all four populations (D+G+, D+G, D-G+ and D-G-), it is the D+G+ population that has the greatest capacity to form spheroids. Remarkably, this population expands approximately 10-fold upon mild chronic injury. Finally, ChIP-seq revealed, for Prrx1a, the most enriched genes are related to cancer progression whereas Prrx1b appears to regulate focal adhesion and ErbB signaling. Conclusion: We have identified Prrx1b as a novel transcription factor that regulates pancreatic regeneration/self-renewal whereas both Prrx1a and Prrx1b appear to be important in pancreatic cancer progression and metastasis. Citation Format: Maximilian Reichert, Anil K. Rustgi, Shigetsugu Takano, Johannes A. von Burstin, Steffen Heeg, Gregory P. Botta, Ju-Seog Lee, Gunter Schneider, Andrew D. Rhim, Ben Z. Stanger. The Prrx1 homeodomain transcription factor plays a central role in pancreatic regeneration and carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A71.