RESUMEN

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 Cebra

RESUMEN

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

Asunto(s)

Envejecimiento/genética , Electroporación , Transgenes , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Embrión no Mamífero/metabolismo , Técnicas de Transferencia de Gen , Melanoma/patología , Plásmidos/genética , Regiones Promotoras Genéticas , Pez Cebra/embriología

RESUMEN

Advanced, metastatic melanomas frequently grow in subcutaneous tissues and portend a poor prognosis. Though subcutaneous tissues are largely composed of adipocytes, the mechanisms by which adipocytes influence melanoma are poorly understood. Using in vitro and in vivo models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. Additionally, adipocytes directly transfer lipids to melanoma cells, which alters tumor cell metabolism. Adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAFV600E in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. Pharmacologic blockade of FATPs with the small-molecule inhibitor Lipofermata abrogates lipid transport into melanoma cells and reduces melanoma growth and invasion. These data demonstrate that stromal adipocytes can drive melanoma progression through FATP lipid transporters and represent a new target aimed at interrupting adipocyte-melanoma cross-talk.Significance: We demonstrate that stromal adipocytes are donors of lipids that mediate melanoma progression. Adipocyte-derived lipids are taken up by FATP proteins that are aberrantly expressed in melanoma. Inhibition of FATPs decreases melanoma lipid uptake, invasion, and growth. We provide a mechanism for how stromal adipocytes drive tumor progression and demonstrate a novel microenvironmental therapeutic target. Cancer Discov; 8(8); 1006-25. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.

Asunto(s)

Adipocitos/citología , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/genética , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Mutación , Invasividad Neoplásica , Trasplante de Neoplasias , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/farmacología , Tiadiazoles/administración & dosificación , Tiadiazoles/farmacología , Microambiente Tumoral , Regulación hacia Arriba , Pez Cebra

RESUMEN

Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.

Asunto(s)

Plasticidad de la Célula , Melanoma/patología , Microambiente Tumoral , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Plasticidad de la Célula/genética , Proliferación Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Modelos Biológicos , Metástasis de la Neoplasia , Fenotipo , Microambiente Tumoral/genética , Pez Cebra , Proteínas de Pez Cebra/metabolismo