Abstract
In addition to defined genetic mutations, a deregulation of epigenetic remodelers highly contributes to pancreatic cancer formation in a setting of inflammatory acinar-to-ductal metaplasia (ADM). By analyzing an in vitro carcinogenesis model, consisting of isolated acinar cells, 3D cultured ADMs and tumor cells, we have already demonstrated that the epigenetic histone modifier Ring1b is reactivated during tumor development. As the catalytic subunit of the Polycomb Repressor Complex 1 (PRC1), Ring1b mono-ubiquitinates histone H2A at lysine 119 (H2AK119ub), leading to the transcriptional repression of specific target genes. Thus, we were able to show that its reactivation in ADMs and tumor cells accounts for the transcriptional silencing of the acinar cell fate genes Ptf1a and Rbpjl.
To prove that epigenetic changes catalyzed by Ring1b are an important prerequisite for ADM formation and pancreatic tumor progression in vivo, we now established an inducible Cre-mediated conditional Ring1b knockout (KO) mouse model. Ring1b KO mice were subjected to the cerulein-induced regeneration model and a KrasG12D-dependent tumorigenesis. Tissue was analyzed with the aid of immunohistochemistry, Western Blot, Chromatinimmunoprecipitation (ChIP) and qRT-PCR. Moreover, Ring1b was depleted in tumor cells via CRISPR/Cas9 and cells were functionally analyzed.
Interestingly, conditional Ring1b knockout mice undergoing cerulein-induced pancreatitis barely exhibited any tissue damage, ADMs and inflammation. Moreover, the formation of early precursor lesions was actually strongly impaired in Ring1b depleted KrasG12D mice. Here, expression analyses revealed significantly elevated expression levels of differentiation genes, such as Rbpjl, whereas the expression of the progenitor genes Rbpj and Sox9 was decreased in the knockout mice.
To describe the role of Ring1b in cancer cells, we generated CRISPR-mediated Ring1b knockout tumor cells. We found, that Ring1b KO cells are more sensitive to gemcitabine. After orthotopic injection into the pancreas of wild type mice, control cells established malignant, undifferentiated and highly proliferative tumors. In contrast, Ring1b knockout tumor clones formed none to very small and well differentiated ones.
Our results reveal that Ring1b is crucial for initiating ADM and pancreatic tumor formation. As an epigenetic remodeler, Ring1b causes repression of differentiation genes, which favors oncogenic transformation, tumor development and progression.
Citation Format: Simone Benitz, Sabrina Deubler, Jessica Guo, Katja Steiger, Susanne Raulefs, Bo Kong, Güralp Ceyhan, Irene Esposito, Christoph W. Michalski, Jörg Kleeff, Ivonne Regel.{Authors}. Epigenetic alterations mediated by Ring1b are crucial for acinar-to-ductal metaplasia and pancreatic carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B01.
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