The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.

The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway. BRAF(V600E) mutations are also present in benign melanocytic naevi, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Uncharted Waters: Zebrafish Cancer Models Navigate a Course for Oncogene Discovery.

Over a decade has elapsed since the first genetically-engineered zebrafish cancer model was described. During this time remarkable progress has been made. Sophisticated genetic tools have been built to generate oncogene expressing cancers and characterize multiple models of solid and blood tumors. These models have led to unique insights into mechanisms of tumor initiation and progression. New drug targets have been identified, particularly through the functional analysis of cancer genomes. Now in the second decade, zebrafish cancer models are poised for even faster growth as they are used in high-throughput genetic analyses to elucidate key mechanisms underlying critical cancer phenotypes.

Microenvironmental GABA Signaling Regulates Melanomagenesis through Reciprocal Melanoma-Keratinocyte Communication.

SUMMARY: GABA signaling by melanoma cells was found by Tagore and colleagues to trigger keratinocyte-driven growth of melanomas. This study reveals new roles for nonneuronal signaling by a neurotransmitter in regulating tumor initiation and outgrowth. See related article by Tagore et al., p. 2270 (4).

Pigment cell progenitor heterogeneity and reiteration of developmental signaling underlie melanocyte regeneration in zebrafish.

Tissue-resident stem and progenitor cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem and progenitor cells are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified melanocyte progenitors in adult zebrafish skin. To elucidate mechanisms governing melanocyte progenitor renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for progenitors, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of melanocyte progenitor direct differentiation and asymmetric division. Our findings show how activation of different subpopulations of mitfa-positive cells underlies cellular transitions required to properly reconstitute the melanocyte pigmentary system following injury.

The Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) oncogene is upregulated in pre-cystic epithelial cells revealing a new target for polycystic kidney disease.

Polycystic kidney disease (PKD) is an important cause of end stage renal disease, but treatment options are limited. While later stages of the disease have been extensively studied, mechanisms driving the initial conversion of renal tubules into cysts are not understood. To identify factors that promote the initiation of cysts we deleted polycystin-2 ( Pkd2 ) in mice and surveyed transcriptional changes before and immediately after cysts developed. We identified 74 genes which we term cyst initiation candidates (CICs). To identify conserved changes with relevance to human disease we compared these murine CICs to single cell transcriptomic data derived from patients with PKD and from healthy controls. Tumor-associated calcium signal transducer 2 ( Tacstd2 ) stood out as an epithelial-expressed gene whose levels were elevated prior to cystic transformation and further increased with disease progression. Human tissue biopsies and organoids show that TACSTD2 protein is low in normal kidney cells but is elevated in cyst lining cells. While TACSTD2 has not been studied in PKD, it has been studied in cancer where it is highly expressed in solid tumors while showing minimal expression in normal tissue. This property is being exploited by antibody drug conjugates that target TACSTD2 for the delivery of cytotoxic drugs. Our finding that Tacstd2 is highly expressed in cysts, but not normal tissue, suggests that it should be explored as a candidate for drug development in PKD. More immediately, our work suggests that PKD patients undergoing TACSTD2 treatment for cancer should be monitored for kidney effects. One Sentence Summary: The oncogene, tumor-associated calcium signal transducer 2 (Tacstd2) mRNA increased in abundance shortly after Pkd2 loss and may be a driver of cyst initiation in polycystic kidney disease.

Research Techniques Made Simple: Zebrafish Models for Human Dermatologic Disease.

Skin diseases affect nearly one third of the world's population. Disease types range from oncologic to inflammatory, and outcomes can be as severe as death and disfigurement. Although many skin diseases have been modeled in murine models, the advantages of zebrafish models have led to recent increasing use in modeling human disease. Their rapid development, comparable skin architecture, tractable genetics, unparalleled optical properties, and straightforward drug screens make them an excellent model to study skin disease. In this review, we discuss the attributes of the zebrafish model system as well as current zebrafish models for dermatologic diseases, including melanoma, squamous cell carcinoma, vitiligo, epidermal bullosa, psoriasis, and wounding.

Oncogenic BRAF induces whole-genome doubling through suppression of cytokinesis.

Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAFV600E, the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in vitro and in a zebrafish melanoma model. Mechanistically, BRAFV600E causes decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAFV600E activity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAFV600E activity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis - amplified centrosomes and whole-genome doubling events - can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.

Inactivation of the Hippo tumor suppressor pathway promotes melanoma.

Melanoma is commonly driven by activating mutations in the MAP kinase BRAF; however, oncogenic BRAF alone is insufficient to promote melanomagenesis. Instead, its expression induces a transient proliferative burst that ultimately ceases with the development of benign nevi comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that restrain nevus melanocyte proliferation remain poorly understood. Here we utilize cell and murine models to demonstrate that oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway, both in melanocytes in vitro and nevus melanocytes in vivo. Mechanistically, we show that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome doubling events, which independently reduce RhoA activity to promote Hippo activation. We also demonstrate that functional impairment of the Hippo pathway enables oncogenic BRAF-expressing melanocytes to bypass nevus formation and rapidly form melanomas. Our data reveal that the Hippo pathway enforces the stable arrest of nevus melanocytes and represents a critical barrier to melanoma development.

Working together: Heterotypic clusters and collective cell migration in melanoma metastasis.

In this issue of Developmental Cell, Campbell et al. (2021) show that melanoma cells with distinct invasive or proliferative gene signatures can form heterotypic clusters that extravasate collectively and readily seed the growth of metastatic lesions. These findings highlight interactions between heterogenous tumor cells as being critical for metastasis.

Melanoma models for the next generation of therapies.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

From Tank to Treatment: Modeling Melanoma in Zebrafish.

Melanoma is the deadliest form of skin cancer and one of few cancers with a growing incidence. A thorough understanding of its pathogenesis is fundamental to developing new strategies to combat mortality and morbidity. Zebrafish-due in large part to their tractable genetics, conserved pathways, and optical properties-have emerged as an excellent system to model melanoma. Zebrafish have been used to study melanoma from a single tumor initiating cell, through metastasis, remission, and finally into relapse. In this review, we examine seminal zebrafish studies that have advanced our understanding of melanoma.

A new class of C. elegans synMuv genes implicates a Tip60/NuA4-like HAT complex as a negative regulator of Ras signaling.

The class A and class B synMuv genes are functionally redundant negative regulators of a Ras signaling pathway that induces C. elegans vulval development. A number of class B synMuv genes encode components of an Rb and histone deacetylase complex that likely acts to repress transcription of genes required for vulval induction. We discovered a new class of synMuv genes that acts redundantly with both the A and B classes of genes in vulval cell-fate determination. These new class C synMuv genes encode TRRAP, MYST family histone acetyltransferase, and Enhancer of Polycomb homologs, which form a putative C. elegans Tip60/NuA4-like histone acetyltransferase complex. A fourth gene with partial class C synMuv properties encodes a homolog of the mammalian SWI/SNF family ATPase p400. Our findings indicate that the coordinated action of two chromatin-modifying complexes, one with histone deacetylase and the other with histone acetyltransferase activity, is important in regulating Ras signaling and specifying cell fates during C. elegans development.

Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling.

Preventing terminal differentiation is important in the development and progression of many cancers including melanoma. Recent identification of the BMP ligand GDF6 as a novel melanoma oncogene showed GDF6-activated BMP signaling suppresses differentiation of melanoma cells. Previous studies have identified roles for GDF6 orthologs during early embryonic and neural crest development, but have not identified direct regulation of melanocyte development by GDF6. Here, we investigate the BMP ligand gdf6a, a zebrafish ortholog of human GDF6, during the development of melanocytes from the neural crest. We establish that the loss of gdf6a or inhibition of BMP signaling during neural crest development disrupts normal pigment cell development, leading to an increase in the number of melanocytes and a corresponding decrease in iridophores, another neural crest-derived pigment cell type in zebrafish. This shift occurs as pigment cells arise from the neural crest and depends on mitfa, an ortholog of MITF, a key regulator of melanocyte development that is also targeted by oncogenic BMP signaling. Together, these results indicate that the oncogenic role ligand-dependent BMP signaling plays in suppressing differentiation in melanoma is a reiteration of its physiological roles during melanocyte development.

Melanoma-associated GRM3 variants dysregulate melanosome trafficking and cAMP signaling.

Large-scale sequencing studies have revealed several genes that are recurrently mutated in melanomas. To annotate the melanoma genome, we have expressed tumor-associated variants of these genes in zebrafish and characterized their effects on melanocyte development and function. Here, we describe expression of tumor-associated variants of the recurrently mutated metabotropic glutamate receptor 3 (GRM3) gene. Unlike wild-type GRM3, tumor-associated GRM3 variants disrupted trafficking of melanosomes, causing their aggregation in the cell body. Melanosomes are trafficked in a cAMP-dependent manner, and drugs that directly or indirectly increased cAMP levels were able to suppress melanosome aggregation in mutant GRM3-expressing melanocytes. Our data show that oncogenic GRM3 variants dysregulate cAMP signaling, a heretofore unknown role for these oncogenes. cAMP signaling has been implicated in melanoma progression and drug resistance, and our data show that oncogenic properties of GRM3 could be mediated, at least in part, by alterations in cAMP signaling.

Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers.

Identification and classification of genes that act antagonistically to let-60 Ras signaling in Caenorhabditis elegans vulval development.

The synthetic multivulva (synMuv) genes negatively regulate Ras-mediated vulval induction in the nematode Caenorhabditis elegans. The synMuv genes define three classes, A, B, and C, such that double mutants carrying mutations in genes of any two classes are multivulva. The class B synMuv genes include lin-35, a homolog of the retinoblastoma (Rb) tumor suppressor gene, as well as homologs of genes that function with Rb in transcriptional regulation. We screened for additional synMuv mutations using a strategy different from that of previous synMuv genetic screens. Some of the mutations we recovered affect new synMuv genes. We present criteria for assigning synMuv mutations into different genetic classes. We also describe the molecular characterization of the class B synMuv gene lin-65.

Identification and characterization of T reg-like cells in zebrafish.

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg-deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg-like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling.

The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF- or NRAS-activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAFV600E); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAFV600E); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAFV600E-mutant human melanoma cell line. We found that pathway stimulation upstream of BRAFV600E could paradoxically reduce signaling downstream of BRAFV600E, and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAFV600E signaling. In vivo, expression of wild-type BRAF delayed melanoma onset, but only in a kit-dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAFV600E-driven melanoma formation. Cancer Res; 77(21); 5820-30. ©2017 AACR.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.