The Ets-Transcription Factor Etv1 Induces Epithelial-Mesenchymal Transition (Emt) And Invasion As Well As Expands The Stromal Compartment In Vivo

Introduction: The Ets-transcription factor Etv1 is involved in epithelial-mesenchymal interactions during pancreatic development and shows enhanced expression in PanIN as well as in pancreatic ductal adenocarcinoma (PDAC). Therefore, we aimed to identify the mechanistic roles of Etv1 in EMT and invasion in PanIN and PDAC. Methods: Cells were isolated from Pdx1Cre;Kras G12D/+ -mice (PanIN) and Pdx1Cre;Kras G12D/+ ;p53 R175H/+ -mice (PDAC) and lentiviral tranduction was used to induce overexpression or knockdown (shRNA) of Etv1, respectively. Cells were grown in 3D organotypic culture to analyze morphology and growth behavior. Invasion and sphere formation (self-renewal) assays were performed. FACS-sorting was used to isolate CD44+/CD24+ cells as a putative cancer stem cell population in PDAC and analyzed for Etv1 expression. To assess the role of Etv1 in PDAC in vivo, orthotopic pancreatic xenograft transplantation of Etv1 overexpressing PDAC cells was performed. Results: Etv1 overexpression in PanIN cells grown in 3D-organotypic culture induces a spindle shaped morphology and highly perturbed cyst architecture in contrast to control PanIN cells that form spheroid cysts that resemble normal pancreatic ductal structures. Concurrently and consistently, EMT-related genes (snail, twist, zeb1 and zeb2, as well as vimentin and N-cadherin) were found upregulated by >2-fold in PanIN-mEtv1 cells. Moreover, the expression of Mmp3 and Mmp9 was significantly increased. Functionally, the invasive capacity of PanIN-mEtv1 cells was more than twice that of control cells; knockdown of Etv1 in PDAC-cells significantly abrogated their invasive capacity. In sphere forming assays PanIN-mEtv1 cells revealed an increased self-renewal capacity, whereas sphere formation is reduced by knockdown of Etv1 in PanIN as well as in PDAC cells. FACS-sorting and subsequent expression analysis revealed increased Etv1 levels in the putative cancer stem cell population of CD44+/CD24+ PDAC-cells. Data from orthotopic xenografts showed significantly larger tumors, significantly increased stromal expansion measured by trichrome staining, and increased local tumor invasion in Etv1-overexpressing cells compared to controls. The increased stromal expansion significantly correlated to the increased tumor volume observed in Etv1 over-expressing xenografts. Conclusion: Our novel data indicate that Etv1 induces EMT as well as invasion both in vitro and in vivo and mediates self-renewal capacity of premalignant cells derived from PanIN- as well as PDAC-cells. Our in vivo data suggest a role for Etv1 in expanding the stromal compartment. These striking data suggest that Etv1 is biologically important in EMT. In vivo experiments with conditional knockout of Etv1 in Pdx1Cre;Kras G12D/+ ;p53 R175H/+ -mice are ongoing to further elucidate the role of Etv1 in PanIN and PDAC initiation and progression. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo. [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 A46.