Expression of the EWSR1-FLI1 fusion oncogene in pancreas cells drives pancreatic atrophy and lipomatosis.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) harbors mutant KRAS as the most common driver mutation. Studies on mouse models have uncovered the tumorigenic characteristics of the Kras oncogene driving pancreatic carcinogenesis. Similarly, Ewing sarcoma predominantly depends on the occurrence of the EWSR1-FLI1 fusion oncogene. The expression of EWSR1-FLI1 affects pro-tumorigenic pathways and induces cell transformation. In this study, we investigated whether mutant Kras could be exchanged by another potent oncogene, such as EWSR1-FLI1, to initiate pancreatic cancer development. METHODS: We generated two conditional mouse models expressing mutant KrasG12D (KC) or the EWSR1-FLI1 oncogene (E/F) in pancreas cells. Pancreatic tissue was collected from the mice at 4-6 weeks and 11-13 weeks of age as well as from survival cohorts to determine the development of spontaneous acinar-to-ductal metaplasia (ADM) and neoplastic lesions. Immunohistochemistry and immunofluorescence staining were performed to characterize and quantify changes in tissue morphology. RESULTS: The expression of the EWSR1-FLI1 fusion protein in pancreas cells was confirmed by positive FLI1 immunohistochemistry staining. Notably, the EWSR1-FLI1 expression in pancreas cells resulted in a strong depletion of the acinar cell mass and an extensive lipomatosis. Although the E/F mice exhibited spontaneous ADM formation and a shorter overall survival rate compared to KC mice, no development of neoplastic lesion was observed in aging E/F mice. CONCLUSIONS: The expression of the EWSR1-FLI1 oncogene leads to a strong pancreatic atrophy and lipomatosis. ADM formation indicates that pancreatic acinar cells are susceptible for EWSR1-FLI1-mediated oncogenic transformation to a limited extent. However, the EWSR1-FLI1 oncogene is insufficient to induce pancreatic cancer development.

Ring1b-dependent epigenetic remodelling is an essential prerequisite for pancreatic carcinogenesis.

BACKGROUND AND AIMS: Besides well-defined genetic alterations, the dedifferentiation of mature acinar cells is an important prerequisite for pancreatic carcinogenesis. Acinar-specific genes controlling cell homeostasis are extensively downregulated during cancer development; however, the underlying mechanisms are poorly understood. Now, we devised a novel in vitro strategy to determine genome-wide dynamics in the epigenetic landscape in pancreatic carcinogenesis. DESIGN: With our in vitro carcinogenic sequence, we performed global gene expression analysis and ChIP sequencing for the histone modifications H3K4me3, H3K27me3 and H2AK119ub. Followed by a comprehensive bioinformatic approach, we captured gene clusters with extensive epigenetic and transcriptional remodelling. Relevance of Ring1b-catalysed H2AK119ub in acinar cell reprogramming was studied in an inducible Ring1b knockout mouse model. CRISPR/Cas9-mediated Ring1b ablation as well as drug-induced Ring1b inhibition were functionally characterised in pancreatic cancer cells. RESULTS: The epigenome is vigorously modified during pancreatic carcinogenesis, defining cellular identity. Particularly, regulatory acinar cell transcription factors are epigenetically silenced by the Ring1b-catalysed histone modification H2AK119ub in acinar-to-ductal metaplasia and pancreatic cancer cells. Ring1b knockout mice showed greatly impaired acinar cell dedifferentiation and pancreatic tumour formation due to a retained expression of acinar differentiation genes. Depletion or drug-induced inhibition of Ring1b promoted tumour cell reprogramming towards a less aggressive phenotype. CONCLUSIONS: Our data provide substantial evidence that the epigenetic silencing of acinar cell fate genes is a mandatory event in the development and progression of pancreatic cancer. Targeting the epigenetic repressor Ring1b could offer new therapeutic options.

Altered histone acetylation patterns in pancreatic cancer cell lines induce subtype­specific transcriptomic and phenotypical changes.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced tumor stages with chemotherapy as the only treatment option. Transcriptomic analysis has defined a classical and basal­like PDAC subtype, which are regulated by epigenetic modification. The present study aimed to determine if drug­induced epigenetic reprogramming of pancreatic cancer cells affects PDAC subtype identity and chemosensitivity. Classical and basal­like PDAC cell lines PaTu­S, Capan­1, Capan­2, Colo357, PaTu­T, PANC­1 and MIAPaCa­2, were treated for a short (up to 96 h) and long (up to 30 weeks) period with histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. The cells were analyzed using gene expression approaches, immunoblot analysis, and various cell assays to assess cell characteristics, such as proliferation, colony formation, cell migration and sensitivity to chemotherapeutic drugs. Classical and basal­like PDAC cell lines showed pronounced epigenetic regulation of subtype­specific genes through acetylation of lysine 27 on Histone H3 (H3K27ac). Moreover, classical cell lines revealed a significantly decreased expression of HDAC2 and increased total levels of H3K27ac in comparison with the basal­like cell lines. Following HAT inhibitor treatment, classical cell lines exhibited a loss of epithelial marker gene expression, decreased chemotherapy response gene score and increased cell migration in vitro, indicating a tumor­promoting phenotype. HDAC inhibitor treatment, however, exerted minimal reprogramming effects in both subtypes. Epigenetic reprogramming of classical and basal­like tumor cells did not have a major impact on gemcitabine response, although the gemcitabine transporter gene SLC29A1 (solute carrier family 29 member 1) was epigenetically regulated.