In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS61K in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation

Lineage-specific co-evolution of the Egf receptor/ligand signaling system

<p>Abstract</p> <p>Background</p> <p>The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system.</p> <p>Results</p> <p>We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated <it>egfr </it>genes, <it>egfra </it>and <it>egfrb</it>, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish <it>hbegf </it>being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of <it>egfra</it>, <it>egfrb </it>and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using <it>in vitro </it>analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution.</p> <p>Conclusions</p> <p>Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr ligands. This fish-specific occurrence of Egf receptor specialization offers unique opportunities to study the functions of different Egf receptor-ligand combinations and their biological outputs in vertebrates. Furthermore, our results strongly support the use of homologous ligands in future studies, as sufficient cross-specificity is very unlikely for this ligand/receptor system.</p

MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation

Contains fulltext : 87376.pdf (publisher's version ) (Open Access)BACKGROUND: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. RESULTS: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. CONCLUSIONS: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG

Slow-release L-cysteine capsule prevents gastric mucosa exposure to carcinogenic acetaldehyde : results of a randomised single-blinded, cross-over study of Helicobacter-associated atrophic gastritis

Introduction: Helicobacter-induced atrophic gastritis with a hypochlorhydric milieu is a risk factor for gastric cancer. Microbes colonising acid-free stomach oxidise ethanol to acetaldehyde, a recognised group 1 carcinogen. Objective: To assess gastric production of acetaldehyde and its inert condensation product, non-toxic 2-methyl-1,3-thiazolidine-4-carboxylic acid (MTCA), after alcohol intake under treatment with slow-release L-cysteine or placebo. Methods: Seven patients with biopsy-confirmed atrophic gastritis, low serum pepsinogen and high gastrin-17 were studied in a cross-over single-blinded design. On separate days, patients randomly received 200 mg slow-release L-cysteine or placebo with intragastric instillation of 15% (0.3 g/kg) ethanol. After intake, gastric concentrations of ethanol, acetaldehyde, L-cysteine and MTCA were analysed. Results: Administration of L-cysteine increased MTCA (p <.0004) and decreased gastric acetaldehyde concentrations by 68% (p <.0001). The peak L-cysteine level was 7552 +/- 2687 mu mol/L at 40 min and peak MTCA level 196 +/- 98 mu mol/L at 80 min after intake. Gastric L-cysteine and MTCA concentrations were maintained for 3 h. The AUC for MTCA was 11-fold higher than acetaldehyde, indicating gastric first-pass metabolism of ethanol. With placebo, acetaldehyde remained elevated also at low ethanol concentrations representing 'non-alcoholic' beverages and food items. Conclusions: After gastric ethanol instillation, slow-release L-cysteine eliminates acetaldehyde to form inactive MTCA, which remains in gastric juice for up to 3 h. High acetaldehyde levels indicate a marked gastric first-pass metabolism of ethanol resulting in gastric accumulation of carcinogenic acetaldehyde. Local exposure of the gastric mucosa to acetaldehyde can be mitigated by slow-release L-cysteine capsules.Peer reviewe

The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission

1Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs− parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs− parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

<p>Abstract</p> <p>Background</p> <p>Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the <it>Xiphophorus </it>melanoma model system, a mutated version of the EGF receptor Xmrk (<it>Xiphophorus </it>melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.</p> <p>Methods</p> <p>Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene <it>FOSL1 </it>was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.</p> <p>Results</p> <p>Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (<it>Fosl1</it>), early growth response 1 (<it>Egr1</it>), osteopontin (<it>Opn</it>), insulin-like growth factor binding protein 3 (<it>Igfbp3</it>), dual-specificity phosphatase 4 (<it>Dusp4</it>), and tumor-associated antigen L6 (<it>Taal6</it>). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that <it>FOSL1</it>, <it>OPN</it>, <it>IGFBP3</it>, <it>DUSP4</it>, and <it>TAAL6 </it>also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of <it>FOSL1 </it>in human melanoma cell lines reduced their proliferation and migration.</p> <p>Conclusion</p> <p>Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.</p

Predicting microenvironment in CXCR4- and FAP-positive solid tumors - a pan-cancer machine learning workflow for theranostic target structures

Simple Summary Imaging based on positron emission tomography (PET) is a crucial part of up-to-date cancer care. For this purpose, PET employs and marks target structures at the cellular surface. Recently, C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) emerged as clinically relevant PET targets. However, it is unclear whether high levels of CXCR4 and FAP represent distinct cancer states—especially in solid tumors. Therefore, we established a machine learning model based on 9242 samples from 29 different cancer entities. Our analysis revealed that—in most solid tumors—high levels of CXCR4 were associated with immune cells infiltrating these tumors. Instead, FAP-positive tumors were characterized by high amounts of tumor vessels. Our machine learning approach potentially can identify the Achilles’ heel of tumors in a non-invasive manner—by performing PET without having to obtain tumor tissue beforehand. Abstract (1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database—representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets

The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2

The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease

Effects of dexmedetomidine and MK-467 on plasma glucose, insulin and glucagon in a glibenclamide-induced canine hypoglycaemia model

The commonly used sedative α2-adrenoceptor agonist dexmedetomidine has adverse cardiovascular effects in dogs that can be prevented by concomitant administration of the peripherally acting α2-adrenoceptor antagonist MK-467. An ancillary effect of dexmedetomidine is to decrease insulin release from the pancreas, whereas MK-467 stimulates insulin release. This study assessed the effects of co-administered dexmedetomidine and MK-467 in a canine glibenclamide-induced hypoglycaemia model. In a randomised, cross-over experiment, eight beagle dogs received five intravenous treatments, comprising two administrations of saline, with dexmedetomidine or dexmedetomidine and MK-467, and three administrations of glibenclamide, with saline, dexmedetomidine or dexmedetomidine and MK-467. Plasma concentrations of glucose, lactate, insulin, glucagon and the test drugs were monitored. Administration of glibenclamide significantly increased insulin secretion and decreased blood glucose concentrations. Dexmedetomidine counteracted glibenclamide-evoked hypoglycaemia. This was opposed by the α2-adrenoceptor antagonist MK-467, but the glibenclamide-evoked hypoglycaemia was not potentiated by co-administration of dexmedetomidine and MK-467. None of the dogs developed uncontrolled hypoglycaemia. Thus, the combination of dexmedetomidine and MK-467 appeared to be safe in this canine hypoglycaemia model. Nevertheless, when MK-467 is used to alleviate the undesired cardiovascular effects of α2-adrenoceptor agonists in dogs, it should be used with caution in animals at risk for hypoglycaemia because of its insulin-releasing and hypoglycaemic effects.</p