Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, undergo acinar-ductal metaplasia and rapidly acquire increased growth potential. During this process several transcription factors from the basic helix-loop-helix (bHLH) family are downregulated while expression of their inhibitor Id3 is induced. Previously we showed that Id3 knockdown with siRNA resulted in growth arrest in PDA cells. Here we queried whether aggressive PDA cells can be reprogrammed to revert to their original quiescent acinar cell phenotype by shifting bHLH transcription programs. In order to mitigate Id3 and upregulate bHLH protein expression we engineered human PDA cells to stably express an inducible form of the bHLH factor E47. Remarkably, we found that E47 triggers both cell cycle arrest and re-activation of the acinar cell differentiation program and in PDA cells. E47 acted as a tumor suppressor of human pancreatic cancer cells in vitro and in vivo. In 4 human PDA cell lines E47 led to G0/G1 arrest within 48 hours and induction of E47 reduced tumor formation in vivo. Notably, even temporary induction of E47 in vitro prior to transplantation resulted in small tumors relative to controls, suggesting that E47 induces stable changes in cell growth. Mechanistically, we found that E47 induced arrest requires upregulation of the cyclin-dependent kinase inhibitor (cdki) p21. Simultaneous with cell cycle exit E47 expressing PDA cells exhibited restored acinar cell features. GSEA enrichment analysis of microarray data revealed significant induction of the exocrine gene signature by E47. These findings were confirmed by qPCR demonstrating up to 70,000 fold increases in expression of acinar digestive enzymes. Further, E47 induced feed forward expression of MIST1, the bHLH factor required for acinar maturation, which is profoundly downregulated in PDA. To determine whether induced MIST1 protein was functional in these cells, we examined expression of the MIST1 target genes Foxp2, Copz2, and Rab3d finding that they were significantly increased within 48 hours of inducing E47 activity. Further, PDA cells acquired MIST1-dependent Cx32 gap junctions, a hallmark of differentiated acinar cells. Collectively the data show that PDA cells retain a remarkable degree of plasticity which can be exploited to return them to a quiescent acinar-like state. The discovery that bHLH activity is the critical node regulating PDA cell fate offers new opportunities to manipulate transcription pathways for therapeutic advances in pancreatic cancer.

Citation Format: Sangwun Kim, Challeng Yang, Chelsea Riha, Reyhaneh Lamy, Brad L. Jakubison, Stephen F. Konieczny, Pamela Itkin-Ansari. Mechanisms of E47 induced quiescence and acinar cell differentiation in human pancreatic cancer cells. [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 A13.