RESUMEN
Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Animales , Animales Modificados Genéticamente , Carcinogénesis/genética , Pie , Mano , Humanos , Melanoma/patología , Uñas , Oncogenes/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Transcripción Genética , Pez Cebra/genética , Melanoma Cutáneo MalignoRESUMEN
We show how enhancers of macrophage-specific genes are rendered accessible in differentiating macrophages to allow their induction in mature cells in response to an appropriate stimulus. Using a lentiviral knockdown approach in primary differentiating macrophages from mouse bone marrow, we demonstrate that enhancers of Il12b and Il1a are kept relatively lowly occupied by nucleosomes and accessible through recruitment of the nucleosome remodeler BAF/PBAF. Our results using an inducible cell line that expresses an estrogen receptor fusion of the macrophage-specific transcription factor PU.1 (PUER) show that BAF/PBAF recruitment to these enhancers is a consequence of translocation of PUER to the nucleus in the presence of tamoxifen, and we speculate that remodeler recruitment may be directly mediated by PU.1. In the absence of BAF/PBAF recruitment, nucleosome occupancy at the enhancer of Il12b (and to a lesser extent at Il1a) reaches high levels in bone marrow-derived macrophages (BMDMs), and the enhancers are not fully cleared of nucleosomes upon LPS induction, resulting in impaired gene expression. Analysis of Il12b expression in single cells suggests that recruitment of the remodeler is necessary for high levels of transcription from the same promoter, and we propose that remodelers function by increasing nucleosome turnover to facilitate transcription factor over nucleosome binding in a process we have termed "remodeler-assisted competition."
Asunto(s)
Diferenciación Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos/fisiología , Macrófagos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Estrógenos/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Cromosómicas no Histona/genética , Humanos , Ratones , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Proto-Oncogénicas/genética , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Desmosomes are transmembrane protein complexes that contribute to cell-cell adhesion in epithelia and other tissues. Here, we report the discovery of frequent genetic alterations in the desmosome in human cancers, with the strongest signal seen in cutaneous melanoma where desmosomes are mutated in over 70% of cases. In primary but not metastatic melanoma biopsies, the burden of coding mutations on desmosome genes associates with a strong reduction in desmosome gene expression. Analysis by spatial transcriptomics suggests that these expression decreases occur in keratinocytes in the microenvironment rather than in primary melanoma tumor cells. In further support of a microenvironmental origin, we find that loss-of-function knockdowns of the desmosome in keratinocytes yield markedly increased proliferation of adjacent melanocytes in keratinocyte/melanocyte co-cultures. Thus, gradual accumulation of desmosome mutations in neighboring cells may prime melanocytes for neoplastic transformation.
RESUMEN
Oncogenes can initiate tumors only in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors in keratinocytes. Electron microscopy demonstrates specialized cell-cell junctions between keratinocytes and melanoma cells, and multielectrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte cocultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that GABAergic signaling across the skin microenvironment regulates the ability of oncogenes to initiate melanoma. SIGNIFICANCE: This study shows evidence of GABA-mediated regulation of electrical activity between melanoma cells and keratinocytes, providing a new mechanism by which the microenvironment promotes tumor initiation. This provides insights into the role of the skin microenvironment in early melanomas while identifying GABA as a potential therapeutic target in melanoma. See related commentary by Ceol, p. 2128. This article is featured in Selected Articles from This Issue, p. 2109.
Asunto(s)
Melanoma , Animales , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Pez Cebra , Melanocitos/patología , Piel , Queratinocitos , Transformación Celular Neoplásica/genética , Ácido gamma-Aminobutírico , Microambiente TumoralRESUMEN
Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.
Asunto(s)
Análisis por Conglomerados , Melanoma/metabolismo , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Animales , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/patología , Cresta Neural/patología , Pez CebraRESUMEN
Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cellderived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.
Asunto(s)
Carcinogénesis/genética , Carcinogénesis/patología , Cromatina/metabolismo , Melanoma/genética , Melanoma/patología , Cresta Neural/patología , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Animales , Animales Modificados Genéticamente , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Ratones , Neoplasias Experimentales , Células Madre Neoplásicas/patología , Cresta Neural/metabolismo , Células Madre Pluripotentes/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Transcripción Genética , Pez CebraRESUMEN
Transcriptional profiling of tumors has revealed a stress-like state among the cancer cells with the concerted expression of genes such as fos, jun, and heat-shock proteins, though this has been controversial given possible dissociation-effects associated with single-cell RNA sequencing. Here, we validate the existence of this state using a combination of zebrafish melanoma modeling, spatial transcriptomics, and human samples. We found that the stress-like subpopulation of cancer cells is present from the early stages of tumorigenesis. Comparing with previously reported single-cell RNA sequencing datasets from diverse cancer types, including triple-negative breast cancer, oligodendroglioma, and pancreatic adenocarcinoma, indicated the conservation of this state during tumorigenesis. We also provide evidence that this state has higher tumor-seeding capabilities and that its induction leads to increased growth under both MEK and BRAF inhibitors. Collectively, our study supports the stress-like cells as a cancer cell state expressing a coherent set of genes and exhibiting drug-resistance properties.
Asunto(s)
Carcinogénesis/patología , Melanoma/genética , Estrés Fisiológico/genética , Adenocarcinoma/genética , Animales , Carcinogénesis/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanoma/metabolismo , Melanoma/fisiopatología , Inhibidores de Proteínas Quinasas/farmacología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Pez CebraRESUMEN
Lineage-specific transcription factors (TFs) are important determinants of cellular identity, but their exact mode of action has remained unclear. Here we show using a macrophage differentiation system that the lineage-specific TF PU.1 keeps macrophage-specific genes accessible during differentiation by preventing Polycomb repressive complex 2 (PRC2) binding to transcriptional regulatory elements. We demonstrate that the distal enhancer of a gene becomes bound by PRC2 as cells differentiate in the absence of PU.1 binding and that the gene is wrapped into heterochromatin, which is characterized by increased nucleosome occupancy and H3K27 trimethylation. This renders the gene inaccessible to the transcriptional machinery and prevents induction of the gene in response to an external signal in mature cells. In contrast, if PU.1 is bound at the transcriptional regulatory region of a gene during differentiation, PRC2 is not recruited, nucleosome occupancy is kept low, and the gene can be induced in mature macrophages. Similar results were obtained at the enhancers of other macrophage-specific genes that fail to bind PU.1 as an estrogen receptor fusion (PUER) in this system. These results show that one role of PU.1 is to exclude PRC2 and to prevent heterochromatin formation at macrophage-specific genes.
Asunto(s)
Heterocromatina/genética , Macrófagos/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Transcripción Genética/genética , Animales , Diferenciación Celular , Línea Celular , Femenino , Histonas/metabolismo , Subunidad p40 de la Interleucina-12/biosíntesis , Subunidad p40 de la Interleucina-12/genética , Interleucina-1alfa/biosíntesis , Interleucina-1alfa/genética , Lipopolisacáridos , Macrófagos/citología , Metilación , Ratones , Ratones Endogámicos BALB C , Nucleosomas/genética , Células Madre Pluripotentes/citología , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tamoxifeno/farmacologíaRESUMEN
Chromatin is thought to act as a barrier for binding of cis-regulatory transcription factors (TFs) to their sites on DNA and recruitment of the transcriptional machinery. Here we have analyzed changes in nucleosome occupancy at the enhancers as well as at the promoters of three pro-inflammatory genes when they are induced by bacterial lipopolysaccharides (LPS) in primary mouse macrophages. We find that nucleosomes are removed from the distal enhancers of IL12B and IL1A, as well as from the distal and proximal enhancers of IFNB1, and that clearance of enhancers correlates with binding of various cis-regulatory TFs. We further show that for IFNB1 the degree of nucleosome removal correlates well with the level of induction of the gene under different conditions. Surprisingly, we find that nucleosome occupancy at the promoters of IL12B and IL1A does not change significantly when the genes are induced, and that a considerably fraction of the cells is occupied by nucleosomes at any given time. We hypothesize that competing nucleosomes at the promoters of IL12B and IL1A may play a role in limiting the size of transcriptional bursts in individual cells, which may be important for controlling cytokine production in a population of immune cells.