RESUMO
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.
Assuntos
Melanoma , Animais , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Peixe-Zebra , Melanócitos/patologia , Pele , Queratinócitos , Transformação Celular Neoplásica/genética , Ácido gama-Aminobutírico , Microambiente TumoralRESUMO
Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the melanocytic cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved across human melanoma cell lines and patient tumors. This melanocytic state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon fatty acid oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt cell cycle progression and slow melanoma growth in vivo. Because the melanocytic cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle.
Assuntos
Gotículas Lipídicas , Melanoma , Animais , Humanos , Gotículas Lipídicas/metabolismo , Peixe-Zebra/genética , Melanoma/patologia , Melanócitos/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/genéticaRESUMO
Bioprinting is an additive manufacturing technique that focuses on developing living tissue constructs using bioinks. Bioink is crucial in determining the stability of printed patterns, which remains a major challenge in bioprinting. Thus, the choices of bioink composition, modifications, and cross-linking methods are being continuously researched to augment the clinical translation of bioprinted constructs. Hyaluronic acid (HA) is a naturally occurring polysaccharide with the repeating unit of N-acetyl-glucosamine and d-glucuronic acid disaccharides. It is present in the extracellular matrix (ECM) of tissues (skin, cartilage, nerve, muscle, etc.) with a wide range of molecular weights. Due to the nature of its chemical structure, HA could be easily subjected to chemical modifications and cross-linking that would enable better printability and stability. These interesting properties have made HA an ideal choice of bioinks for developing tissue constructs for regenerative medicine applications. In this Review, the physicochemical properties, reaction chemistry involved in various cross-linking strategies, and biomedical applications of HA have been elaborately discussed. Further, the features of HA bioinks, emerging strategies in HA bioink preparations, and their applications in 3D bioprinting have been highlighted. Finally, the current challenges and future perspectives in the clinical translation of HA-based bioinks are outlined.
Assuntos
Bioimpressão , Engenharia Tecidual , Engenharia Tecidual/métodos , Hidrogéis/química , Alicerces Teciduais/química , Ácido Hialurônico/farmacologia , Bioimpressão/métodosRESUMO
Obesity is a rising concern and associated with an increase in numerous cancers, often in a sex-specific manner. Preclinical models are needed to deconvolute the intersection between obesity, sex and melanoma. Here, we generated a zebrafish system that can be used as a platform for studying these factors. We studied how germline overexpression of Agrp along with a high-fat diet affects melanomas dependent on BRAFV600E and loss of p53. This revealed an increase in tumor incidence and area in male, but not female, obese fish, consistent with the clinical literature. We then determined whether this was further affected by additional somatic mutations in the clinically relevant genes rb1 or ptena/b. We found that the male obesogenic effect on melanoma was present with tumors generated with BRAF;p53;Rb1 but not BRAF;p53;Pten. These data indicate that both germline (Agrp) and somatic (BRAF, Rb1) mutations contribute to obesity-related effects in melanoma. Given the rapid genetic tools available in the zebrafish, this provides a high-throughput system to dissect the interactions of genetics, diet, sex and host factors in obesity-related cancers.
Assuntos
Melanoma , Peixe-Zebra , Animais , Feminino , Masculino , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteína Relacionada com Agouti/genética , Proteína Supressora de Tumor p53/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Melanoma/genética , Melanoma/patologia , Mutação/genética , Obesidade/complicações , Obesidade/genética , DietaRESUMO
In melanoma, predicting which tumors will ultimately metastasize guides treatment decisions. Transcriptional signatures of primary tumors have been utilized to predict metastasis, but which among these are driver or passenger events remains unclear. We used data from the adjuvant AVAST-M trial to identify a predictive gene signature in localized tumors that ultimately metastasized. Using a zebrafish model of primary melanoma, we interrogated the top genes from the AVAST-M signature in vivo. This identified GRAMD1B, a cholesterol transfer protein, as a bona fide metastasis suppressor, with a majority of knockout animals rapidly developing metastasis. Mechanistically, excess free cholesterol or its metabolite 27-hydroxycholesterol promotes invasiveness via activation of an AP-1 program, which is associated with increased metastasis in humans. Our data demonstrate that the transcriptional seeds of metastasis are embedded within localized tumors, suggesting that early targeting of these programs can be used to prevent metastatic relapse. SIGNIFICANCE: We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion. This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Melanoma , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Recidiva Local de Neoplasia/genética , Melanoma/patologia , Perfilação da Expressão Gênica , Metástase Neoplásica , Regulação Neoplásica da Expressão GênicaRESUMO
Cancer cells exist in a complex ecosystem with numerous other cell types in the tumor microenvironment (TME). The composition of this tumor/TME ecosystem will vary at each anatomic site and affects phenotypes such as initiation, metastasis, and drug resistance. A mechanistic understanding of the large number of cell-cell interactions between tumor and TME requires models that allow us to both characterize as well as genetically perturb this complexity. Zebrafish are a model system optimized for this problem, because of the large number of existing cell-type-specific drivers that can label nearly any cell in the TME. These include stromal cells, immune cells, and tissue resident normal cells. These cell-type-specific promoters/enhancers can be used to drive fluorophores to facilitate imaging and also CRISPR cassettes to facilitate perturbations. A major advantage of the zebrafish is the ease by which large numbers of TME cell types can be studied at once, within the same animal. While these features make the zebrafish well suited to investigate the TME, the model has important limitations, which we also discuss. In this review, we describe the existing toolset for studying the TME using zebrafish models of cancer and highlight unique biological insights that can be gained by leveraging this powerful resource.
Assuntos
Neoplasias , Peixe-Zebra , Animais , Microambiente Tumoral , Ecossistema , Neoplasias/genética , Neoplasias/patologia , Modelos BiológicosRESUMO
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.
Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Cromatina/metabolismo , Melanoma/genética , Melanoma/patologia , Crista Neural/patologia , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Animais Geneticamente Modificados , Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos , Neoplasias Experimentais , Células-Tronco Neoplásicas/patologia , Crista Neural/metabolismo , Células-Tronco Pluripotentes/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Transcrição Gênica , Peixe-ZebraRESUMO
Mechanisms that control translation play important roles in tumor progression and metastasis. Emerging evidence has revealed that dysregulated translation also impacts immune evasion in response to cellular or oncogenic stress. Here, we summarize current knowledge regarding the translational control of immune checkpoints and implications for cancer immunotherapies.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/imunologia , Biossíntese de Proteínas/imunologia , Evasão Tumoral/genética , Progressão da Doença , Fatores de Iniciação em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas de Checkpoint Imunológico/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Biossíntese de Proteínas/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacosRESUMO
Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.
Assuntos
Antígeno B7-H1 , Fatores de Iniciação em Eucariotos , Neoplasias Pulmonares , Antígeno B7-H1/genética , Fatores de Iniciação em Eucariotos/genética , Heme/biossíntese , Humanos , Neoplasias Pulmonares/genéticaRESUMO
Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability. This approach allows discovery of both gain- and loss-of-function mutations in the same screen. Transposon insertions sites are recovered by high-throughput sequencing. We recently applied this system to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells. Here we describe a method for performing ex vivo Sleeping Beauty-mediated mutagenesis screens in these cells, which may be adapted for the acquisition of many different phenotypes in distinct cell types.
Assuntos
Elementos de DNA Transponíveis , Testes Genéticos/métodos , Mutagênese , Proteínas de Neoplasias/genética , Neoplasias/genética , Genoma Humano , Humanos , Neoplasias/patologiaRESUMO
PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. Although PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types. Given that PCDH7 is a potentially targetable molecule on the surface of cancer cells, further investigation of its role in tumorigenesis in vivo is needed to evaluate the therapeutic potential of its inhibition. Here, we report the analysis of novel PCDH7 gain- and loss-of-function mouse models and provide compelling evidence that this cell-surface protein acts as a potent lung cancer driver. Employing a Cre-inducible transgenic allele, we demonstrated that enforced PCDH7 expression significantly accelerates KrasG12D -driven lung tumorigenesis and potentiates MAPK pathway activation. Furthermore, we performed in vivo somatic genome editing with CRISPR/Cas9 in KrasLSL-G12D ; Tp53fl/fl (KP) mice to assess the consequences of PCDH7 loss of function. Inactivation of PCDH7 in KP mice significantly reduced lung tumor development, prolonged survival, and diminished phospho-activation of ERK1/2. Together, these findings establish a critical oncogenic function for PCDH7 in vivo and highlight the therapeutic potential of PCDH7 inhibition for lung cancer. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the new inducible transgenic model described here provides a robust experimental system for broadly elucidating the effects of PCDH7 overexpression in vivo. IMPLICATIONS: In this study, we establish a critical oncogenic function for PCDH7 in vivo using novel mouse models and CRISPR/Cas9 genome editing, and we validate the therapeutic potential of PCDH7 inhibition for lung cancer.
Assuntos
Adenocarcinoma de Pulmão/genética , Caderinas/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Caderinas/deficiência , Caderinas/metabolismo , Carcinogênese , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Protocaderinas , Transdução de SinaisRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006650.].
RESUMO
Hepatocellular carcinoma (HCC) is the fifth most common solid tumor in the world and the third leading cause of cancer-associated deaths. A Sleeping Beauty-mediated transposon mutagenesis screen previously identified mutations that cooperate with MYC to accelerate liver tumorigenesis. This revealed a tumor suppressor role for Steroid Receptor Coactivator 2/Nuclear Receptor Coactivator 2 (Src-2/Ncoa2) in liver cancer. In contrast, SRC-2 promotes survival and metastasis in prostate cancer cells, suggesting a tissue-specific and context-dependent role for SRC-2 in tumorigenesis. To determine if genetic loss of SRC-2 is sufficient to accelerate MYC-mediated liver tumorigenesis, we bred Src-2-/- mice with a MYC-induced liver tumor model and observed a significant increase in liver tumor burden. RNA sequencing of liver tumors and in vivo chromatin immunoprecipitation assays revealed a set of direct target genes that are bound by SRC-2 and exhibit downregulated expression in Src-2-/- liver tumors. We demonstrate that activation of SHP (Small Heterodimer Partner), DKK4 (Dickkopf-4), and CADM4 (Cell Adhesion Molecule 4) by SRC-2 suppresses tumorigenesis in vitro and in vivo. These studies suggest that SRC-2 may exhibit oncogenic or tumor suppressor activity depending on the target genes and nuclear receptors that are expressed in distinct tissues and illuminate the mechanisms of tumor suppression by SRC-2 in liver.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Alelos , Animais , Antineoplásicos/química , Carcinogênese , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Imunoprecipitação da Cromatina , Elementos de DNA Transponíveis , Feminino , Deleção de Genes , Células Hep G2 , Humanos , Imunoglobulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Mutagênese , Metástase Neoplásica , Transplante de Neoplasias , Coativador 2 de Receptor Nuclear/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Análise de Sequência de RNARESUMO
Oral cancer is one of the most commonly occurring cancers worldwide, decreasing the patient's survival rate due to tumor recurrence and metastasis. Menadione (Vitamin K3) is known to exhibit cytotoxicity in various cancer cells but the present study focused on its effects on viability, apoptosis, epithelial to mesenchymal transition (EMT), anchorage independent growth and migration of oral cancer cells. The results show that menadione is more cytotoxic to SAS (oral squamous carcinoma) cells but not to non-tumorigenic HEK293 and HaCaT cells. Menadione treatment increased the expression of pro-apoptotic proteins, Bax and p53, with a concurrent decrease in anti-apoptotic proteins, Bcl-2 and p65. Menadione induced the expression of E-cadherin but reduced the expression of EMT markers, vimentin and fibronectin. Menadione also inhibited anchorage independent growth and migration in SAS cells. These findings reveal and confirm that menadione is a potential candidate in oral cancer therapy as it exhibits cytotoxic, antineoplastic and antimigratory effects besides effectively blocking EMT in oral cancer cells.