RESUMO
Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.
Assuntos
Quinase do Linfoma Anaplásico , Sistema Nervoso Central , Proteínas de Drosophila , Animais , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/genética , Larva/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Transdução de Sinais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Drosophila/genética , Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão GênicaRESUMO
Preservation of a small population of cancer stem cells (CSCs) within a heterogeneous carcinoma serves as a paradigm to understand how select cells in a tissue maintain their undifferentiated status. In both embryogenesis and cancer, Snail has been correlated with stemness, but the molecular underpinning of this phenomenon remains largely ill-defined. In models of cutaneous squamous cell carcinoma (cSCC), we discovered a non-epithelial-mesenchymal transition function for the transcription factor Snail in maintaining the stemness of epidermal keratinocytes. Snail-expressing cells secrete the matricellular protein Mindin, which functions in an autocrine fashion to activate a Src-STAT3 pathway to reinforce their stem/progenitor phenotype. This pathway is activated by the engagement of Mindin with the leukocyte-specific integrin, CD11b (ITGAM), which is also unexpectedly expressed by epidermal keratinocytes. Interestingly, disruption of this signaling module in human cSCC attenuates tumorigenesis, suggesting that targeting Mindin would be a promising therapeutic approach to hinder cancer recurrence.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Cutâneas , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular , Humanos , Integrinas/metabolismo , Proteínas de Neoplasias , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Activating anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system, we employed CRIPSR/Cas9, incorporating orthologs of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibited enhanced Alk signaling phenotypes, but unexpectedly depended on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains displayed hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We showed that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing, we identified perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of Alk-activating mutations to perturb specification and promote survival in neuronal lineages. This article has an associated First Person interview with the first author of the paper.
Assuntos
Quinase do Linfoma Anaplásico , Diferenciação Celular , Proteínas de Drosophila , Neurônios , Quinase do Linfoma Anaplásico/genética , Animais , Criança , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Hiperplasia , Mutação , Neurônios/citologia , Receptores Proteína Tirosina Quinases/genéticaRESUMO
High-risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high-risk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8-10% of primary NB patients are ALK-positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the "2p-gain" region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v-sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI-sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p-gain, may benefit from ALK TKI-based therapeutic intervention.
Assuntos
Citocinas/genética , Citocinas/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Regulação para Cima , Quinase do Linfoma Anaplásico/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Mutação com Ganho de Função , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Análise de Sequência de RNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, we conducted a genetic suppressor screen in Drosophila melanogaster. Our screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma. Many of the identified suppressors were also able to modulate signaling output from activated oncogenic variants of human ALK, suggesting that our screen identified targets likely relevant in a wide range of contexts. Interestingly, two misexpression alleles of wallenda (wnd, encoding a leucine zipper bearing kinase similar to human DLK and LZK) were among the strongest suppressors. We show that Alk expression leads to a growth advantage and induces cell death in surrounding cells. Our results suggest that Alk activity conveys a competitive advantage to cells, which can be reversed by over-expression of the JNK kinase kinase Wnd.