RESUMO
Misregulated ß-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of ß-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear ß-catenin. We show that these inhibitors efficiently block Wnt/ß-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling.
Assuntos
Antineoplásicos/química , Descoberta de Drogas/métodos , Interferência de RNA , Transcrição Gênica/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster , Ensaios de Seleção de Medicamentos Antitumorais , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Oxazóis/química , Oxazóis/isolamento & purificação , Oxazóis/uso terapêutico , Bibliotecas de Moléculas Pequenas , Proteínas Wnt/genética , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. Here we report the first identification of a dominant-active Stat92E protein, Stat92E(DeltaNDeltaC), which lacks both N- and C-termini. Mis-expression of Stat92E(DeltaNDeltaC)in vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y(711) and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R(442P) substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E(R442P) translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R(442) is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R(442) may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. We conclude that the N- and C- termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E(R442) has a nuclear function by altering dimer:DNA binding.
Assuntos
Drosophila/metabolismo , Transativadores/genética , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Drosophila/genética , Fosforilação , Proteínas/genética , Proteínas/metabolismo , Transativadores/metabolismoRESUMO
Although the JAK/STAT pathway regulates numerous processes in vertebrates and invertebrates through modulating transcription, its functionally relevant transcriptional targets remain largely unknown. With one jak and one stat (stat92E), Drosophila provides a powerful system for finding new JAK/STAT target genes. Genome-wide expression profiling on eye discs in which Stat92E is hyperactivated, revealed 584 differentially regulated genes, including known targets domeless, socs36E, and wingless. Other differentially regulated genes (chinmo, lama, Mo25, Imp-L2, Serrate, Delta) were validated and may represent new Stat92E targets. Genetic experiments revealed that Stat92E cell-autonomously represses Serrate, which encodes a Notch ligand. Loss of Stat92E led to de-repression of Serrate in the dorsal eye, resulting in ectopic Notch signaling and aberrant eye growth there. Thus, our micro-array documents a new Stat92E target gene and a previously unidentified inhibitory action of Stat92E on Notch signaling. These data suggest that this study will be a useful resource for the identification of additional Stat92E targets.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/genética , Drosophila/metabolismo , Janus Quinases/fisiologia , Receptores Notch/metabolismo , Fatores de Transcrição STAT/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Animais , Biologia Computacional , Proteínas de Drosophila/genética , Olho/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Hibridização In Situ , Janus Quinases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Receptores Notch/genética , Fatores de Transcrição STAT/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
The Drosophila STAT transcription factor Stat92E regulates diverse functions, including organ development and stem cell self-renewal. However, the Stat92E functional effectors that mediate these processes are largely unknown. Here we show that chinmo is a cell-autonomous, downstream mediator of Stat92E that shares numerous functions with this protein. Loss of either gene results in malformed eyes and head capsules due to defects in eye progenitor cells. Hyperactivation of Stat92E or misexpression of Chinmo results in blood cell tumors. Both proteins are expressed in germline (GSCs) and cyst stem cells (CySCs) in the testis. While Stat92E is required for the self-renewal of both populations, chinmo is only required in CySCs, indicating that Stat92E regulates self-renewal in different stem cells through independent effectors. Like hyperactivated Stat92E, Chinmo misexpression in CySCs is sufficient to maintain GSCs nonautonomously. Chinmo is therefore a key effector of JAK/STAT signaling in a variety of developmental and pathological contexts.
Assuntos
Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Janus Quinase 1/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Fatores de Transcrição STAT/metabolismo , Células-Tronco/citologia , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Masculino , Microscopia de Fluorescência/métodos , Modelos Biológicos , Modelos Genéticos , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Transdução de Sinais , Testículo/metabolismoRESUMO
JAK/STAT signaling is thought to control growth and proliferation. However, here we show a novel role for this pathway in the patterning of Drosophila appendages. Loss of Stat92E function results mainly in ventralizations and multiplications of the proximo-distal axis in leg and antenna, primarily through the ectopic misexpression of wingless. We also show that the pathway ligand Unpaired is expressed in two domains in leg and antenna that abuts those of wingless and decapentaplegic. We report that JAK/STAT signaling represses both wingless and decapentaplegic, restricting them to their respective domains in leg and antenna. In a reciprocal manner, we show that wingless and decapentaplegic restrict unpaired to its two domains. Thus, a main function of the JAK/STAT pathway in leg and antennal development is to promote the formation of a single proximo-distal axis per disc by constraining the intersection of wingless and decapentaplegic to the center of the disc.
Assuntos
Padronização Corporal , Drosophila melanogaster/crescimento & desenvolvimento , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Animais , Padronização Corporal/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Janus Quinases/genética , Fatores de Transcrição STAT/genética , Transdução de Sinais , Transcrição GênicaRESUMO
During development, a small number of conserved signaling molecules regulate regional specification, in which uniform populations of cells acquire differences and ultimately give rise to distinct organs. In the Drosophila eye imaginal disc, Wingless (Wg) signaling defines the region that gives rise to head tissue. JAK/STAT signaling was thought to regulate growth of the eye disc but not pattern formation. However, we show that the JAK/STAT pathway plays an important role in patterning the eye disc: it promotes formation of the eye field through repression of the wg gene. Overexpression of the JAK/STAT activating ligand Unpaired in the eye leads to loss of wg expression and ectopic morphogenetic furrow initiation from the lateral margins. Conversely, tissue lacking stat92E, which cannot transduce JAK/STAT signals, is transformed from retinal tissue into head cuticle, a phenotype that is also observed with ectopic Wg signaling. Consistent with this, cells lacking stat92E exhibit ectopic wg expression. Conversely, wg is autonomously repressed in cells with hyperactivated Stat92E. Furthermore, we show that the JAK/STAT pathway regulates a small enhancer in the wg 3' cis genomic region. As this enhancer is devoid of Stat92E-binding elements, we conclude that Stat92E represses wg through another, as yet unidentified factor that is probably a direct target of Stat92E. Taken together, our study is the first to demonstrate a role for the JAK/STAT pathway in regional specification by acting antagonistically to wg.