Kurzfassung

The ability of premalignant and transformed cells to evade host immune surveillance in the hepatic niche is essential for initiation and progression of hepatocellular carcinoma (HCC). Presently, it is well recognized that epigenetic alterations can promote tumor cell adaptation leading to immune escape, but the exact mechanisms contributing to this immunosuppressive phenotype remain elusive. Both, modulation of DNA-methylation as well as histone modifications have a major impact on gene...The ability of premalignant and transformed cells to evade host immune surveillance in the hepatic niche is essential for initiation and progression of hepatocellular carcinoma (HCC). Presently, it is well recognized that epigenetic alterations can promote tumor cell adaptation leading to immune escape, but the exact mechanisms contributing to this immunosuppressive phenotype remain elusive. Both, modulation of DNA-methylation as well as histone modifications have a major impact on gene expression changes and, thus, may play an important role on tumor immunity.  In the proposed project, we will define the role of two key epigenetic regulators, the DNA methyltransferase DNMT1 and the histone deacetylase SIRT6 in cellular and molecular mechanisms of HCC immune escape. Alterations of these genes are frequently observed in HCC and induce genome-wide DNA hypomethylation as well as accumulation of active histone acetylation marks contributing to transcriptional de-regulation. Interestingly, both genes are known to regulate the aging process and their loss can induce a senescence associated secretory phenotype (SASP) that can modulate immune responses in the tumor micromilieu. Here, we will evaluate the potential of these two key epigenetic modifiers to affect the immune phenotype of neoplastic cells and thus the anti-
Project TP07156tumor response in HCC. To mechanistically dissect the relative importance of these genes for tumor cells and cells of the hepatic microenvironment as well as their role in promoting cancer progression a new syngeneic mouse transplantation HCC model will be utilized. In this model, we will characterize tumor growth, metastasis and inflammatory infiltration in the presence and absence of DNMT1/SIRT6 using confocal microscopy and FACS analysis. To determine the epigenetic alterations related to expression changes of transcriptional networks we will use genome-wide next-generation sequencing and array-based technologies (RNA-seq, ChIP-Seq, methylation arrays). Here we will focus on expression changes of immune-relevant genes including oncofetal antigens, apoptosis-related genes, and genes coding for costimulatory or immune-suppressive factors and will correlate DNA-methylation changes and alterations of SIRT6-dependent histone marks with the corresponding gene loci. This (epi)genetic analysis will be supplemented on the protein level by characterization of the spectrum of tumor-secreted cytokines, chemokines and other soluble molecules facilitating the escape.  Functional analysis will be done by co-culture experiments using murine and human hepatoma cell clones with CRISPR/Cas9 mediated SIRT6/DNMT1 deletion or overexpression and different immune cells (macrophages, cytotoxic T cells, natural killer cells). To confirm the relevance of our findings for human cancer, obtained results will be validated in human HCC samples available from our clinical registry unit comprising >2500 well-annotated HCC.» weiterlesen» einklappen