ABSTRACT
Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.
Subject(s)
Melanoma/metabolism , Positive Transcriptional Elongation Factor B/genetics , Pyrimidines/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Melanoma/genetics , Melanoma/pathology , Melanoma, Experimental , Oncogene Proteins/genetics , Transcription Factors , Transcription, Genetic , Tumor Suppressor Proteins/genetics , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Deciphering the genomic regulatory code of enhancers is a key challenge in biology because this code underlies cellular identity. A better understanding of how enhancers work will improve the interpretation of noncoding genome variation and empower the generation of cell type-specific drivers for gene therapy. Here, we explore the combination of deep learning and cross-species chromatin accessibility profiling to build explainable enhancer models. We apply this strategy to decipher the enhancer code in melanoma, a relevant case study owing to the presence of distinct melanoma cell states. We trained and validated a deep learning model, called DeepMEL, using chromatin accessibility data of 26 melanoma samples across six different species. We show the accuracy of DeepMEL predictions on the CAGI5 challenge, where it significantly outperforms existing models on the melanoma enhancer of IRF4 Next, we exploit DeepMEL to analyze enhancer architectures and identify accurate transcription factor binding sites for the core regulatory complexes in the two different melanoma states, with distinct roles for each transcription factor, in terms of nucleosome displacement or enhancer activation. Finally, DeepMEL identifies orthologous enhancers across distantly related species, where sequence alignment fails, and the model highlights specific nucleotide substitutions that underlie enhancer turnover. DeepMEL can be used from the Kipoi database to predict and optimize candidate enhancers and to prioritize enhancer mutations. In addition, our computational strategy can be applied to other cancer or normal cell types.
Subject(s)
Computational Biology/methods , Melanoma/genetics , Zebrafish/genetics , Animals , Deep Learning , Dogs , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Horses , Humans , Mice , SwineABSTRACT
OBJECTIVES: The aim of this article was to provide practical guidance in setting up patient registries to facilitate real-world data collection for health care decision making. METHODS: This guidance was based on our experiences and involvement in setting up patient registries in oncology in the Netherlands. All aspects were structured according to 1) mission and goals ("the Why"), 2) stakeholders and funding ("the Who"), 3) type and content ("the What"), and 4) identification and recruitment of patients, data handling, and pharmacovigilance ("the How"). RESULTS: The mission of most patient registries is improving patient health by improving the quality of patient care; monitoring and evaluating patient care is often the primary goal ("the Why"). It is important to align the objectives of the registry and agree on a clear and functional governance structure with all stakeholders ("the Who"). There is often a trade off between reliability, validity, and specificity of data elements and feasibility of data collection ("the What"). Patient privacy should be carefully protected, and address (inter-)national and local regulations. Patient registries can reveal unique safety information, but it can be challenging to comply with pharmacovigilance guidelines ("the How"). CONCLUSIONS: It is crucial to set up an efficient patient registry that serves its aims by collecting the right data of the right patient in the right way. It can be expected that patient registries will become the new standard alongside randomized controlled trials due to their unique value.
Subject(s)
Data Collection/methods , Decision Making , Health Services Research/methods , Medical Oncology/methods , Policy Making , Registries , Confidentiality , Data Accuracy , Data Collection/economics , Data Collection/standards , Guideline Adherence , Guidelines as Topic , Health Services Research/economics , Health Services Research/standards , Humans , Medical Oncology/economics , Medical Oncology/standards , Netherlands , Organizational Objectives , Pharmacovigilance , Registries/standards , Reproducibility of Results , Research Support as TopicABSTRACT
Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.
Subject(s)
Seminoma , Zebrafish , Animals , Genes, Tumor Suppressor , Genotype , Humans , Mutation , Zebrafish/geneticsABSTRACT
Biallelic mutations of the von Hippel-Lindau (VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma (RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product (pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer (AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC.
Subject(s)
Carcinoma, Renal Cell/mortality , E2F1 Transcription Factor/genetics , Gene Expression Regulation, Neoplastic/physiology , Kidney Neoplasms/mortality , Von Hippel-Lindau Tumor Suppressor Protein/physiology , Animals , Blotting, Western , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cellular Senescence , Disease Models, Animal , E2F1 Transcription Factor/metabolism , Female , Humans , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Male , Middle Aged , Organisms, Genetically Modified , Plasmids , Prognosis , Proliferating Cell Nuclear Antigen/metabolism , Proportional Hazards Models , Real-Time Polymerase Chain Reaction , Survival Rate , Transfection , Tumor Cells, Cultured , ZebrafishABSTRACT
Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel-Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells.
Subject(s)
Energy Metabolism , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological , Zebrafish/metabolism , Animals , Basal Metabolism , Cell Polarity , Embryo, Nonmammalian/enzymology , Embryo, Nonmammalian/pathology , Food Deprivation , Insulin-Like Growth Factor I/metabolism , Intestines/pathology , Larva/cytology , Larva/metabolism , Mutation/genetics , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Survival Analysis , TOR Serine-Threonine Kinases/metabolism , Zebrafish/embryology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response. The vhl mutants develop polycythemia with concomitantly increased epo/epor mRNA levels and erythropoietin signaling. In situ hybridizations reveal global up-regulation of both red and white hematopoietic lineages. Hematopoietic tissues are highly proliferative, with enlarged populations of c-myb(+) hematopoietic stem cells and circulating erythroid precursors. Chemical activation of hypoxia-inducible factor signaling recapitulated aspects of the vhl(-/-) phenotype. Furthermore, microarray expression analysis confirms the hypoxic response and hematopoietic phenotype observed in vhl(-/-) embryos. We conclude that VHL participates in regulating hematopoiesis and erythroid differentiation. Injections with human VHLp30 and R200W mutant mRNA demonstrate functional conservation of VHL between mammals and zebrafish at the amino acid level, indicating that vhl mutants are a powerful new tool to study genotype-phenotype correlations in human disease. Zebrafish vhl mutants are the first congenital embryonic viable systemic vertebrate animal model for VHL, representing the most accurate model for VHL-associated polycythemia to date. They will contribute to our understanding of hypoxic signaling, hematopoiesis, and VHL-associated disease progression.
Subject(s)
Disease Models, Animal , Hypoxia/genetics , Polycythemia/genetics , Tumor Suppressor Proteins/physiology , Zebrafish Proteins/physiology , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Lineage , Conserved Sequence , Gene Knockout Techniques , Germ-Line Mutation , Hematopoiesis/genetics , Humans , Hypoxia/physiopathology , Molecular Sequence Data , Point Mutation , Polycythemia/physiopathology , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , RNA, Messenger/pharmacology , Recombinant Fusion Proteins/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Synteny , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics , Von Hippel-Lindau Tumor Suppressor Protein/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/physiology , Zebrafish , Zebrafish Proteins/deficiency , Zebrafish Proteins/geneticsABSTRACT
A progressive increase in copy number variation (CNV) characterizes the natural history of cutaneous melanoma progression toward later disease stages, but our understanding of genetic drivers underlying chromosomal arm-level CNVs remains limited. To identify candidate progression drivers, we mined the TCGA SKCM dataset and identified HDGF as a recurrently amplified gene whose high mRNA expression correlates with poor patient survival. Using melanocyte-specific overexpression in the zebrafish BRAFV600E -driven MiniCoopR melanoma model, we show that HDGF accelerates melanoma development in vivo. Transcriptional analysis of HDGF compared to control EGFP tumors showed the activation of endothelial/angiogenic pathways. We validated this observation using an endothelial kdrl:mCherry reporter line which showed HDGF to increases tumor vasculature. HDGF is frequently co-altered with the established melanoma driver SETDB1. Both genes are located on chromosome 1q, and their co-amplification is observed in up to 13% of metastatic melanoma. TCGA patients with both genes amplified and/or overexpressed have a worse melanoma specific survival. We tested co-expression of HDGF and SETDB1 in the MiniCoopR model, which resulted in faster and more aggressive melanoma development than either gene individually. Our work identifies the co-amplification of HDGF and SETDB1 as a functional driver of melanoma progression and poor patient prognosis.
Subject(s)
Biomarkers, Tumor/genetics , Chromosomes, Human, Pair 1/genetics , Histone-Lysine N-Methyltransferase/genetics , Intercellular Signaling Peptides and Proteins/genetics , Melanoma/mortality , Mutation , Skin Neoplasms/mortality , Animals , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Humans , Melanoma/genetics , Melanoma/pathology , Prognosis , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Survival Rate , ZebrafishABSTRACT
Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , MTOR Inhibitors/administration & dosage , Melanoma/drug therapy , Neurofibromin 1/genetics , PTEN Phosphohydrolase/genetics , Pyrimidines/administration & dosage , Sulfonamides/administration & dosage , Thiophenes/administration & dosage , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Synergism , Everolimus/administration & dosage , Everolimus/pharmacology , Humans , Loss of Function Mutation , MTOR Inhibitors/pharmacology , Melanoma/genetics , Melanoma/pathology , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyrimidines/pharmacology , Sirolimus/administration & dosage , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Sulfonamides/pharmacology , Thiophenes/pharmacology , Xenograft Model Antitumor Assays , ZebrafishABSTRACT
Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.
Subject(s)
Drug Resistance, Neoplasm/genetics , Matrix Attachment Region Binding Proteins/metabolism , Melanoma/genetics , Neoplasm Invasiveness/genetics , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Animals , CRISPR-Cas Systems , Cell Line, Tumor , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Matrix Attachment Region Binding Proteins/genetics , Melanoma/drug therapy , Melanoma/metabolism , Neural Crest/cytology , Transcription Factors/genetics , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
Mutations in the von Hippel-Lindau (VHL) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types as well. Reports of VHL mutations are dispersed throughout original articles and databases that have not been recently updated. We compiled a comprehensive mutation table of 1,548 germline and somatic VHL mutations, derived from this protein of only 213 amino acids. We describe detailed phenotype and gene mutation information for 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). These data represent the most extensive catalog of germline VHL mutations to date. We also review VHL disease, known and theorized pathogenesis of common VHL manifestations, and genotype-phenotype correlations. Analysis of all VHL families, excluding germline mutations resulting in congenital polycythemias, describes the spectrum of mutation types: 52% missense, 13% frameshift, 11% nonsense, 6% in-frame deletions/insertions, 11% large/complete deletions, and 7% splice mutations. This easy-to-use compilation of VHL mutations is intended to facilitate research and function as a necessary adjunct for physicians when providing patient information.
Subject(s)
Genetic Association Studies , von Hippel-Lindau Disease/diagnosis , von Hippel-Lindau Disease/genetics , Frameshift Mutation , Germ-Line Mutation , Humans , Kidney Neoplasms/genetics , Mutation , Pedigree , von Hippel-Lindau Disease/pathologyABSTRACT
Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/ß-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890.
Subject(s)
Carcinogenesis/genetics , Gain of Function Mutation/genetics , Histocompatibility Antigens/genetics , Histone-Lysine N-Methyltransferase/genetics , Oncogenes/genetics , Cell Line, Tumor , Humans , MutationABSTRACT
Cilia perform essential motile and sensory functions central to many developmental and physiological processes. Disruption of their structure or function can have profound phenotypic consequences, and has been linked to left-right patterning and polycystic kidney disease. In a forward genetic screen for mutations affecting ciliary motility, we isolated zebrafish mutant hu255H. The mutation was found to disrupt an ortholog of the uncharacterized highly conserved human SDS22-like leucine-rich repeat(LRR)-containing protein LRRC50 (16q24.1) and Chlamydomonas Oda7p. Zebrafish lrrc50 is specifically expressed in all ciliated tissues. lrrc50(hu255H) mutants develop pronephric cysts with an increased proliferative index, severely reduced brush border, and disorganized pronephric cilia manifesting impaired localized fluid flow consistent with ciliary dysfunction. Electron microscopy analysis revealed ultrastructural irregularities of the dynein arms and misalignments of the outer-doublet microtubules on the ciliary axonemes, suggesting instability of the ciliary architecture in lrrc50(hu255H) mutants. TheSDS22-like leucine-rich repeats present in Lrrc50 are necessary for proper protein function, since injection of a deletion construct of the first LRR did not rescue the zebrafish mutant phenotype. Subcellular distribution of human LRRC50-EGFP in MDCK and HEK293T cells is diffusely cytoplasmic and concentrated at the mitotic spindle poles and cilium. LRRC50 RNAi knock-down in human proximal tubule HK-2 cells thoroughly recapitulated the zebrafish brush border and cilia phenotype, suggesting conservation of LRRC50 function between both species. In summary, we present the first genetic vertebrate model for lrrc50 function and propose LRRC50 to be a novel candidate gene for human cystic kidney disease, involved in regulation of microtubule-based cilia and actin-based brush border microvilli.
Subject(s)
Cilia , Mutation , Polycystic Kidney Diseases/etiology , Proteins/genetics , Animals , Humans , Leucine-Rich Repeat Proteins , Mice , ZebrafishABSTRACT
BACKGROUND: von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene. METHODS: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. RESULTS: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. CONCLUSIONS: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.
Subject(s)
Kidney Glomerulus/metabolism , Kidney Tubules, Proximal/metabolism , Tumor Suppressor Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/physiology , Animals , Embryonic Development/genetics , Kidney Glomerulus/abnormalities , Kidney Glomerulus/growth & development , Kidney Tubules, Proximal/abnormalities , Kidney Tubules, Proximal/growth & development , Larva , Mutation , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitorsABSTRACT
Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.
Subject(s)
Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Melanoma/genetics , Animals , Histones/metabolism , Humans , Lysine/genetics , Lysine/metabolism , Methylation , Mice, Nude , Promoter Regions, Genetic/geneticsABSTRACT
Melanoma is the most aggressive and deadliest form of skin cancer. A detailed knowledge of the cellular, molecular, and genetic events underlying melanoma progression is highly relevant to diagnosis, prognosis and risk stratification, and the development of new therapies. In the last decade, zebrafish have emerged as a valuable model system for the study of melanoma. Pathway conservation, coupled with the availability of robust genetic, transgenic, and chemical tools, has made the zebrafish a powerful model for identifying novel disease genes, visualizing cancer initiation, interrogating tumor-microenvironment interactions, and discovering new therapeutics that regulate melanocyte and melanoma development. In this review, we will give an overview of these studies, and highlight recent advancements that will help unravel melanoma pathogenesis and impact human disease.
Subject(s)
Melanoma/pathology , Melanoma/therapy , Translational Research, Biomedical , Zebrafish/physiology , Animals , Carcinogenesis/pathology , Disease Models, Animal , Humans , Melanocytes/pathologyABSTRACT
OBJECTIVE: Determine healthcare costs of upper-extremity surgical correction in children with spastic cerebral palsy (CP). METHOD: This cohort study included 39 children with spastic CP who had surgery for their upper extremity at a Dutch hospital. A retrospective cost analysis was performed including both hospital and rehabilitation costs. Hospital costs were determined using microcosting methodology. Rehabilitation costs were estimated using reference prices. RESULTS: Hospital costs averaged 6813 per child. Labor (50%), overheads (29%), and medical aids (15%) were important cost drivers. Rehabilitation costs were estimated at 3599 per child. CONCLUSIONS: Surgery of the upper extremity is an important contributor to the healthcare costs of children with CP. Our study shows that labor is the most important cost driver for hospital costs, owing to the multidisciplinary approach and patient-specific treatment plan. A remarkable finding was the substantial amount of rehabilitation costs.
Subject(s)
Cerebral Palsy/economics , Cerebral Palsy/surgery , Costs and Cost Analysis/economics , Upper Extremity/surgery , Adolescent , Child , Female , Humans , Male , Retrospective StudiesABSTRACT
The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.
Subject(s)
Genetic Vectors/administration & dosage , Lentivirus/genetics , Melanoma/genetics , Retroviridae/genetics , Transduction, Genetic , Zebrafish/embryology , Zebrafish/genetics , Animals , Melanoma/pathology , Membrane Glycoproteins/genetics , Tumor Cells, Cultured , Viral Envelope Proteins/genetics , Zebrafish/growth & developmentABSTRACT
We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.
Subject(s)
Bone Diseases, Developmental/etiology , Developmental Disabilities/etiology , Immunologic Deficiency Syndromes/etiology , Mutation , N-Acetylglucosaminyltransferases/genetics , Animals , Child, Preschool , Female , Heparitin Sulfate/physiology , Humans , Induced Pluripotent Stem Cells/cytology , Infant , Lymphocytes/physiology , ZebrafishABSTRACT
The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.