Characterisation of mRNA binding proteins as potential therapeutic targets : models and tools

Cancer is one of the most common three causes of premature death worldwide. In past years, colorectal cancer and primary liver cancer are among the malignancies with the highest incidence and mortality rates. This study focuses on two RNA-binding proteins (RBPs) that are aberrantly expressed in those cancer species and contribute to cancer initiation and progression. The first chapter addresses the role of the RBP ZFP36 ring finger protein (TTP) in hepatocellular carcinoma. The effect of a liver-specific TTP-knockout on tumour initiation was investigated in vivo using the diethylnitrosamine hepatocarcinogenesis model in mice. Further, TTP was overexpressed in various hepatoma cells to study the impact on several hallmarks of cancer in vitro. It is shown that TTP exerts tumour-promoting actions during hepatocarcinogenesis and tumour-suppressive actions during hepatic tumour progression. In the second chapter, the RNA-editing method TRIBE is combined with hydrodynamic gene delivery to transfect murine liver cells and enable the identification of new RNA targets of the RBP insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) in vivo. In the third chapter, IMP2 is validated as target for cancer therapy by employing 2D and 3D cell culture models of CRISPR/Cas9-mediated IMP2 knockout cells in vitro as well as zebrafish embryo xenografts in vivo. Based on these results, hit compounds of the screening for small molecule inhibitors of IMP2 could be tested.Krebs ist eine der drei häufigsten Todesursachen weltweit, wobei Darmkrebs und primärer Leberkrebs mit die höchsten Inzidenz- und Sterberaten in den vergangenen Jahren aufwiesen. Diese Studie konzentriert sich auf zwei RNA-bindende Proteine (RBPs), deren abnormales Vorkommen in diesen Krebsarten zur Krebsentstehung und -fortschreitung beiträgt. Das erste Kapitel adressiert die Rolle des RBPs Ringfingerprotein ZFP36 (TTP) beim hepatozellulären Karzinom. Anhand des Diethylnitrosamin-Hepatokarzinogenesemodells in Mäusen wurde der Effekt des leberspezifischen Ausschaltens von TTP auf die Tumorentstehung in vivo untersucht. Außerdem wurde TTP in Leberkrebszellen überexprimiert, um Auswirkungen auf Krebsmerkmale in vitro zu studieren. Es wird gezeigt, dass TTP während der Krebsentstehung fördernd und während Tumorprogression unterdrückend wirkt. Im zweiten Kapitel wird die RNA-Editierungsmethode TRIBE mit dem hydrodynamischen Gentransfer kombiniert, um Leberzellen in Mäusen zu transfizieren und die Identifizierung neuer RNA-Ziele des RBPs Insulinähnlicher Wachstumsfaktor 2-mRNA-bindendes Protein 2 (IMP2) in vivo zu ermöglichen. Im dritten Kapitel wird IMP2 als Ziel für die Krebstherapie validiert; in vitro durch Verwendung von IMP2-K.o.-Zellen (generiert via CRISPR/Cas9) in 2D- und 3D-Zellkulturmodellen, sowie in vivo mittels Zebrafischembryo-Xenotransplantaten. Darauf basierend konnten Trefferverbindungen aus dem Screening nach niedermolekularen Hemmstoffen von IMP2 getestet werden

Kupffer cells are protective in alcoholic steatosis

Massive accumulation of lipids is a characteristic of alcoholic liver disease. Excess of hepatic fat activates Kupffer cells (KCs), which affect disease progression. Yet, KCs contribute to the resolution and advancement of liver injury. Aim of the present study was to evaluate the effect of KC depletion on markers of liver injury and the hepatic lipidome in liver steatosis (Lieber-DeCarli diet, LDC, female mice, mixed C57BL/6J and DBA/2J background). LDC increased the number of dead hepatocytes without changing the mRNA levels of inflammatory cytokines in the liver. Animals fed LDC accumulated elevated levels of almost all lipid classes. KC ablation normalized phosphatidylcholine and phosphatidylinositol levels in LDC livers, but had no effect in the controls. A modest decline of trigylceride and diglyceride levels upon KC loss was observed in both groups. Serum aminotransferases and hepatic ceramide were elevated in all animals upon KC depletion, and in particular, cytotoxic very long-chain ceramides increased in the LDC livers. Meta-biclustering revealed that eight lipid species occurred in more than 40% of the biclusters, and four of them were very long-chain ceramides. KC loss was further associated with excess free cholesterol levels in LDC livers. Expression of inflammatory cytokines did, however, not increase in parallel. In summary, the current study described a function of KCs in hepatic ceramide and cholesterol metabolism in an animal model of LDC liver steatosis. High abundance of cytotoxic ceramides and free cholesterol predispose the liver to disease progression suggesting a protective role of KCs in alcoholic liver diseases

The mRNA-binding Protein TTP/ZFP36 in Hepatocarcinogenesis and Hepatocellular Carcinoma

Hepatic lipid deposition and inflammation represent risk factors for hepatocellular carcinoma (HCC). The mRNA-binding protein tristetraprolin (TTP, gene name ZFP36) has been suggested as a tumor suppressor in several malignancies, but it increases insulin resistance. The aim of this study was to elucidate the role of TTP in hepatocarcinogenesis and HCC progression. Employing liver-specific TTP-knockout (lsTtp-KO) mice in the diethylnitrosamine (DEN) hepatocarcinogenesis model, we observed a significantly reduced tumor burden compared to wild-type animals. Upon short-term DEN treatment, modelling early inflammatory processes in hepatocarcinogenesis, lsTtp-KO mice exhibited a reduced monocyte/macrophage ratio as compared to wild-type mice. While short-term DEN strongly induced an abundance of saturated and poly-unsaturated hepatic fatty acids, lsTtp-KO mice did not show these changes. These findings suggested anti-carcinogenic actions of TTP deletion due to effects on inflammation and metabolism. Interestingly, though, investigating effects of TTP on different hallmarks of cancer suggested tumor-suppressing actions: TTP inhibited proliferation, attenuated migration, and slightly increased chemosensitivity. In line with a tumor-suppressing activity, we observed a reduced expression of several oncogenes in TTP-overexpressing cells. Accordingly, ZFP36 expression was downregulated in tumor tissues in three large human data sets. Taken together, this study suggests that hepatocytic TTP promotes hepatocarcinogenesis, while it shows tumor-suppressive actions during hepatic tumor progression

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

Aim Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. Methods Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipi‑ tation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. Results We identifed IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was signif‑ cantly associated with resistance to selumetinib, geftinib, and regorafenib in PDOs and to 5-fuorouracil and oxalipl‑ atin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabiliza‑ tion of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidifcation rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confrmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. Conclusions IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mito‑ chondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

Abstract Aim Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. Methods Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. Results We identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. Conclusions IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.Innovative Medicines Initiative Joint UndertakingWilhelm Sander-Stiftung http://dx.doi.org/10.13039/100008672Martin-Luther-Universität Halle-Wittenber

Kupffer cells are protective in alcoholic steatosis

Massive accumulation of lipids is a characteristic of alcoholic liver disease. Excess of hepatic fat activates Kupffer cells (KCs), which affect disease progression. Yet, KCs contribute to the resolution and advancement of liver injury. Aim of the present study was to evaluate the effect of KC depletion on markers of liver injury and the hepatic lipidome in liver steatosis (Lieber-DeCarli diet, LDC, female mice, mixed C57BL/6J and DBA/2J background). LDC increased the number of dead hepatocytes without changing the mRNA levels of inflammatory cytokines in the liver. Animals fed LDC accumulated elevated levels of almost all lipid classes. KC ablation normalized phosphatidylcholine and phosphatidylinositol levels in LDC livers, but had no effect in the controls. A modest decline of trigylceride and diglyceride levels upon KC loss was observed in both groups. Serum aminotransferases and hepatic ceramide were elevated in all animals upon KC depletion, and in particular, cytotoxic very long-chain ceramides increased in the LDC livers. Meta-biclustering revealed that eight lipid species occurred in more than 40% of the biclusters, and four of them were very long-chain ceramides. KC loss was further associated with excess free cholesterol levels in LDC livers. Expression of inflammatory cytokines did, however, not increase in parallel. In summary, the current study described a function of KCs in hepatic ceramide and cholesterol metabolism in an animal model of LDC liver steatosis. High abundance of cytotoxic ceramides and free cholesterol predispose the liver to disease progression suggesting a protective role of KCs in alcoholic liver diseases