Metabolic reprogramming is a hallmark of cancer. Understanding cancer metabolism is instrumental to devise innovative therapeutic approaches. Anabolic metabolism, including the induction of lipogenic enzymes, is a key feature of proliferating cells. Here, we report a novel tumor suppressive function for adipose triglyceride lipase (ATGL), the rate limiting enzyme in the triglyceride hydrolysis cascade.In immunohistochemical analysis, non-small cell lung cancers, pancreatic adenocarcinoma as well as leiomyosarcoma showed significantly reduced levels of ATGL protein compared to corresponding normal tissues. The ATGL gene was frequently deleted in various forms of cancers. Low levels of ATGL mRNA correlated with significantly reduced survival in patients with ovarian, breast, gastric and non-small cell lung cancers. Remarkably, pulmonary neoplasia including invasive adenocarcinoma developed spontaneously in mice lacking ATGL pointing to an important role for this lipase in controlling tumor development.Loss of ATGL, as detected in several forms of human cancer, induces spontaneous development of pulmonary neoplasia in a mouse model. Our results, therefore, suggest a novel tumor suppressor function for ATGL and contribute to the understanding of cancer metabolism. We propose to evaluate loss of ATGL protein expression for the diagnosis of malignant tumors. Finally, modulation of the lipolytic pathway may represent a novel therapeutic approach in the treatment of human cancer.

Blockade of the interleukin-6 receptor (IL-6R) is a successful therapeutic strategy in various inflammatory diseases. IL-6 can signal via membrane-bound (classic signaling) and soluble forms (sIL-6R, trans-signaling) of the IL-6R. Trans-signaling is causative for the pro-inflammatory properties of IL-6, and the selective inhibition of this pathway holds the promise to cause less side effects than the global blockade of IL-6 signaling. We have recently shown that the majority of sIL-6R in humans is generated by proteolytic cleavage of the membrane-bound IL-6R, but whether this process is influenced by therapeutic blockade of the IL-6R is unknown. In this study, we show that the monoclonal antibody tocilizumab and a single chain antibody directed against the IL-6R efficiently block IL-6 signaling, but do not prevent the proteolytic generation of sIL-6R.

The nuclear orphan receptor NR4A1 functions as tumour suppressor in aggressive lymphomas by pro-apoptotic genomic and non-genomic effects. Here, we immunohistochemically studied the clinico-pathological relevance of NR4A1 protein expression patterns in a cohort of 60 diffuse large B cell lymphoma (DLBCL) patients and non-neoplastic lymph nodes. We observed a significant association between high cytoplasmic NR4A1 and favourable cancer-specific survival and the germinal centre B cell-like subtype, respectively. Moreover, the percentage of lymphoma cells exhibiting cytoplasmic NR4A1 significantly correlated to those showing cleaved caspase 3. Complementary, functional profiling using gene set enrichment of Reactome pathways based on publicly available microarray data was applied to determine pathways potentially implicated in cytoplasmic localization of NR4A1 and validated by means of semi quantitative real-time PCR. The pathway analysis revealed changes in the ERK1/2 pathway, and this was corroborated by the finding that high cytoplasmic NR4A1 was associated with higher expression of ERK1/2 targets in our cohort. These data indicate that high cytoplasmic NR4A1 is associated with a favourable lymphoma-specific survival and highlights the importance of NR4A1 expression patterns as potential prognostic marker for risk assessment in aggressive lymphomas.

Non-coding RNAs and especially microRNAs have been discovered to act as master regulators of cancer initiation and progression. The aim of our study was to discover and characterize the function of yet functionally uncharacterized microRNAs in human breast carcinogenesis.

Single circulating tumor cells (CTCs) or circulating tumor microemboli (CTMs) are potential biomarkers of renal cell cancer (RCC), however studies of CTCs/CTMs in RCC are limited. In this pilot study we aimed to evaluate a novel blood filtration technique suited for cytomorphological classification, immunocytochemical and molecular characterization of filtered, so called circulating non-hematologic cells (CNHCs) - putative CTCs/CTMs - in patients with RCC.

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

Several risk factors play the role in the development of hepatocellular carcinoma (HCC) from which chronic hepatitis B and C infections are the most important ones. DNA integration of hepatitis viruses alters the function of critical genes promoting malignant transformation of virus-infected liver cells.

We have isolated a microsporidium from a laboratory stock of the European grape vine moth, Lobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae). Screening of this stock showed an infection rate of more than 90%, whereas field collected larvae from three different locations in Rhineland-Palatinate (Germany) did not demonstrate any signs of infection. Light and electron microscopic investigations of infected insects showed that gross pathology, morphology, and ultrastructure of the microsporidium are similar to those described earlier for Pleistophora legeri. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods showed that our isolate was closely related to Cystosporogenes operophterae. Based on our morphological and molecular investigations we propose to rename this species Cystosporogenes legeri nov. comb.

Whilst the role of eukaryotic translation initiation factors (eIFs) has already been investigated in several human cancers, their role in endometrial cancer (EC) is relatively unknown. In the present retrospective study, 279 patients with EC (1180 samples) were included (mean age: 63.0 years, mean follow-up: 6.1 years). Samples were analysed for expression of 7 eIFs subunits (eIF2α, eIF3c, eIF3h, eIF4e, eIF4g, eIF5, eIF6) through immunohistochemistry and western blotting. Fifteen samples of healthy endometrium served as controls. Density and intensity were assessed and mean combined scores (CS) calculated for each patient. Upon immunohistochemistry, median eIF5 CS were significantly higher in EC as compared with non-neoplastic tissue (NNT, p < 0.001), whilst median eIF6 CS were significantly lower in EC (p < 0.001). Moreover, eIF5 (p = 0.002), eIF6 (p = 0.032) and eIF4g CS (p = 0.014) were significantly different when comparing NNT with EC grading types. Median eIF4g CS was higher in type II EC (p = 0.034). Upon western blot analysis, eIF4g (p < 0.001), peIF2α (p < 0.001) and eIF3h (p < 0.05) were significantly overexpressed in EC, while expression of eIF3c was significantly reduced in EC as compared with NNT (p < 0.001). The remaining eIFs were non-significant. Besides tumour stage (p < 0.001) and patient's age (p < 0.001), high eIF4g CS-levels were independently associated with poor prognosis (HR: 1.604, 95%CI: 1.037-2.483, p = 0.034). The other eIFs had no prognostic significance. Notably, the independent prognostic significance of eIF4g was lost when adding tumour type. Considering the difficulties in differentiating EC type I and II, eIF4g may serve as a novel prognostic marker indicating patient outcome.

Targeted inhibition of the c-Jun N-terminal kinases (JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)-related tumorigenesis. However, the cell-type-specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO)hepatocyte-specific knockout (Δhepa) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMOΔhepa/JNK1Δhepa mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMOΔhepa mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMOΔhepa (NEMOΔhepa/JNKΔhepa) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMOΔhepa/JNK1Δhepa mice recapitulated the phenotype of NEMOΔhepa/JNKΔhepa mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMOΔhepa mice. We found that NEMOΔhepa/JNKΔhepa livers exhibited overexpression of the interleukin-6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)-rapidly accelerated fibrosarcoma (Raf)-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B-2 (ErbB2) and EGFR signaling, to NEMOΔhepa/JNKΔhepa mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR-Raf-MEK-ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival.

Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels.

Head and neck cancer etiology and architecture is quite diverse and complex, impeding the prediction whether a patient could respond to a particular cancer immunotherapy or combination treatment. A concomitantly arising caveat is obviously the translation from pre-clinical, cell based in vitro systems as well as syngeneic murine tumor models towards the heterogeneous architecture of the human tumor ecosystems. To bridge this gap, we have established and employed a patient-derived HNSCC (head and neck squamous cell carcinoma) slice culturing system to assess immunomodulatory effects as well as permissivity and oncolytic virus (OV) action. The heterogeneous contexture of the human tumor ecosystem including tumor cells, cancer-associated fibroblasts and immune cells was preserved in our HNSCC slice culturing approach. Importantly, the immune cell compartment remained to be functional and cytotoxic T-cells could be activated by immunostimulatory antibodies. In addition, we uncovered that a high proportion of the patient-derived HNSCC slice cultures were susceptible to the OV VSV-GP. More specifically, VSV-GP infects a broad spectrum of tumor-associated lineages including epithelial and stromal cells and can induce apoptosis. In sum, this human tumor ex vivo platform might complement pre-clinical studies to eventually propel cancer immune-related drug discovery and ease the translation to the clinics.

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and in tissues.

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.

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy.