Epigenetics including DNA and RNA modifications have always been the hotspot field of life sciences in the post-genome era. Since the first mapping of N6-methyladenosine (m6A) and the discovery of its widespread presence in mRNA, there are at least 160-170 RNA modifications have been discovered. These methylations occur in different RNA types, and their distribution is species-specific. 5-methylcytosine (m5C) has been found in mRNA, rRNA and tRNA of representative organisms from all kinds of species. As reversible epigenetic modifications, m5C modifications of RNA affect the fate of the modified RNA molecules and play important roles in various biological processes including RNA stability control, protein synthesis, and transcriptional regulation. Furthermore, accumulative evidence also implicates the role of RNA m5C in tumorigenesis. Here, we review the latest progresses in the biological roles of m5C modifications and how it is regulated by corresponding “writers”, “readers” and “erasers” proteins, as well as the potential molecular mechanism in tumorigenesis and cancer immunotherapy.

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelialmesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC.

In the past three decades, endothelin and endothelin receptor antagonists have received great scientific and clinical interest, leading to the publication of more than 27,000 scientific articles since its discovery. The Thirteenth International Conference on Endothelin (ET-13) was held on September 8-11, 2013, at Tokyo Campus of the University of Tsukuba in Japan. Close to 300 scientists from 25 countries from around the world came to Tokyo to celebrate the anniversary of the discovery of the endothelin peptide discovered 25 years ago at the University of Tsukuba. This article summarizes some of the highlights of the conference, the anniversary celebration ceremony, and particularly the participation of next generation of endothelin researchers in endothelin science and the anniversary celebration. As a particular highlight, next generation endothelin researchers wrote a haiku (a traditional form of Japanese poetry originating from consisting of no more than three short verses and 27 on, or Japanese phonetic units) to describe the magic of endothelin science which they presented to the conference audience at the anniversary ceremony. The text of each haiku - both in its original language together with the English translation - is part of this article providing in an exemplary fashion how poetry can be bridged with science. Finally, we give an outlook towards the next 25 years of endothelin research.

In colorectal carcinoma (CRC), the infiltration of tumor tissue by cytotoxic and T-helper 1 (Th1) T cells has been associated with an improved clinical outcome. This antitumor immune response is characterized by an elevated expression of interferon (IFN)-γ and IFN-stimulated genes such as IRF-1, CXCL-9, CXCL-10, caspase-1 or guanylate-binding protein 1 (GBP-1). Using GBP-1 as a marker of cellular response to IFN-γ in human CRC specimens, we observed that tumor cells, but not stroma cells, frequently lose GBP-1 expression in the context of a Th-1-dominated immune response. Similarly, 6 out of 11 colorectal carcinoma cell lines failed to express GBP-1 or other IFN-stimulated genes after treatment with IFN-γ, and were resistant to IFN-γ-induced apoptosis. This indicated that the loss of responsiveness to IFN-γ might represent a mechanism of immune escape in CRC tumor cells. We then investigated whether defects in RNA and protein expression of IFN-γ pathway genes (IFNGR1, IFNGR2, JAK1, JAK2, STAT1) might account for the loss of responsiveness to the cytokine. The most frequent deregulation observed was the downregulation of IFNGR1 expression at the RNA and protein level in 4 out of 6 resistant cell lines, where treatment with 5-Aza-deoxycytidine, a DNA methylation inhibitor, could restore IFNGR1 expression. In the two remaining resistant cell lines, IFNGR1 was expressed but the protein (IFNγRα) was mis-glycosylated and failed to locate at the plasma membrane. In addition, STAT1 protein expression was downregulated in two resistant cell lines. Using a Crispr/Cas9 silencing approach, we showed that the downregulation of IFNGR1 expression is sufficient to inhibit IFN-γ signaling in CRC cell lines. Moreover, long-term treatment of a sensitive cell line with IFN-γ resulted in the downregulation of IFNGR1, but not STAT1 expression. In human CRC samples, mRNA expression of IFNGR1 was decreased compared to normal tissue. Furthermore, low tumor expression of IFNGR1, but not STAT1, correlated with a reduced cancer-related survival in patients with CRC. Finally, mice harboring a specific knockout of the IFN-γ receptor in intestinal epithelial cells developed more tumors than control mice in a carcinogen-induced colon tumorigenesis model, indicating that the absence of IFN-γ receptor expression in intestinal epithelial cells fosters tumor growth. Altogether, our data suggest that the loss of IFN-γ pathway gene expression is a common event in CRC and represents an intrinsic mechanism of immune escape. Citation Format: Nathalie Britzen-Laurent, Julia Straube, Maximilian Waldner, Christoph Becker, Roland Croner, Christian Pilarsky, Susanne Merkel, Michael Stürzl. Loss of IFN-γ pathway gene expression in tumor cells as mechanism of immune escape of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4047.