RESUMO
The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
[This corrects the article DOI: 10.1371/journal.pcbi.1006840.].
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Drug resistance in breast cancer cell populations has been shown to arise through phenotypic transition of cancer cells to a drug-tolerant state, for example through epithelial-to-mesenchymal transition or transition to a cancer stem cell state. However, many breast tumors are a heterogeneous mixture of cell types with numerous epigenetic states in addition to stem-like and mesenchymal phenotypes, and the dynamic behavior of this heterogeneous mixture in response to drug treatment is not well-understood. Recently, we showed that plasticity between differentiation states, as identified with intracellular markers such as cytokeratins, is linked to resistance to specific targeted therapeutics. Understanding the dynamics of differentiation-state transitions in this context could facilitate the development of more effective treatments for cancers that exhibit phenotypic heterogeneity and plasticity. In this work, we develop computational models of a drug-treated, phenotypically heterogeneous triple-negative breast cancer (TNBC) cell line to elucidate the feasibility of differentiation-state transition as a mechanism for therapeutic escape in this tumor subtype. Specifically, we use modeling to predict the changes in differentiation-state transitions that underlie specific therapy-induced changes in differentiation-state marker expression that we recently observed in the HCC1143 cell line. We report several statistically significant therapy-induced changes in transition rates between basal, luminal, mesenchymal, and non-basal/non-luminal/non-mesenchymal differentiation states in HCC1143 cell populations. Moreover, we validate model predictions on cell division and cell death empirically, and we test our models on an independent data set. Overall, we demonstrate that changes in differentiation-state transition rates induced by targeted therapy can provoke distinct differentiation-state aggregations of drug-resistant cells, which may be fundamental to the design of improved therapeutic regimens for cancers with phenotypic heterogeneity.
Assuntos
Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Morte Celular , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular Tumoral , Dimetil Sulfóxido/farmacologia , Transição Epitelial-Mesenquimal , Feminino , Humanos , Imidazóis/farmacologia , Modelos Biológicos , Piridonas/farmacologia , Pirimidinonas/farmacologia , Quinolinas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50-60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.
Assuntos
Autoantígenos/biossíntese , Neoplasias da Mama/metabolismo , Inibidores Enzimáticos/farmacologia , Chaperonas de Histonas/biossíntese , Proteínas de Membrana/biossíntese , Proteína Fosfatase 2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/biossíntese , Autoantígenos/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Sistemas de Liberação de Medicamentos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Chaperonas de Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genéticaRESUMO
Myeloid ecotropic viral integration site 1 (Meis1) forms a heterodimer with Pbx1 that augments Hox-dependent gene expression and is associated with leukemogenesis and HSC self-renewal. Here we identified 2 independent actions of Meis1 in hematopoietic development: one regulating cellular proliferation and the other involved in megakaryocyte lineage development. First, we found that endogenous Mesp1 indirectly induces Meis1 and Meis2 in endothelial cells derived from embryonic stem cells. Overexpression of Meis1 and Meis2 greatly enhanced the formation of hematopoietic colonies from embryonic stem cells, with the exception of erythroid colonies, by maintaining hematopoietic progenitor cells in a state of proliferation. Second, overexpression of Meis1 repressed the development of early erythroid progenitors, acting in vivo at the megakaryocyte-erythroid progenitor stage to skew development away from erythroid generation and toward megakaryocyte development. This previously unrecognized action of Meis1 may explain the embryonic lethality observed in Meis1(-/-) mice that arises from failure of lymphatic-venous separation and can result as a consequence of defective platelet generation. These results show that Meis1 exerts 2 independent functions, with its role in proliferation of hematopoietic progenitors acting earlier in development from its influence on the fate choice at the megakaryocyte-erythroid progenitor between megakaryocytic and erythroid development.
Assuntos
Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas de Neoplasias/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem Celular , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Eritropoese/genética , Eritropoese/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Hematopoese/fisiologia , Proteínas de Homeodomínio/genética , Megacariócitos/citologia , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Proteína Meis1 , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transdução de SinaisRESUMO
For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.
RESUMO
Pancreatic cancer remains a formidable challenge due to limited treatment options and its aggressive nature. In recent years, the naturally occurring anticancer compound juglone has emerged as a potential therapeutic candidate, showing promising results in inhibiting tumor growth and inducing cancer cell apoptosis. However, concerns over its toxicity have hampered juglone's clinical application. To address this issue, we have explored the use of polymeric micelles as a delivery system for juglone in pancreatic cancer treatment. These micelles, formulated using Poloxamer 407 and D-α-Tocopherol polyethylene glycol 1000 succinate, offer an innovative solution to enhance juglone's therapeutic potential while minimizing toxicity. In-vitro studies have demonstrated that micelle-formulated juglone (JM) effectively decreases proliferation and migration and increases apoptosis in pancreatic cancer cell lines. Importantly, in-vivo, JM exhibited no toxicity, allowing for increased dosing frequency compared to free drug administration. In mice, JM significantly reduced tumor growth in subcutaneous xenograft and orthotopic pancreatic cancer models. Beyond its direct antitumor effects, JM treatment also influenced the tumor microenvironment. In immunocompetent mice, JM increased immune cell infiltration and decreased stromal deposition and activation markers, suggesting an immunomodulatory role. To understand JM's mechanism of action, we conducted RNA sequencing and subsequent differential expression analysis on tumors that were treated with JM. The administration of JM treatment reduced the expression levels of the oncogenic protein MYC, thereby emphasizing its potential as a focused, therapeutic intervention. In conclusion, the polymeric micelles-mediated delivery of juglone holds excellent promise in pancreatic cancer therapy. This approach offers improved drug delivery, reduced toxicity, and enhanced therapeutic efficacy.
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Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response.
Assuntos
Neoplasias Mamárias Animais , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Agressão , Modelos Animais de Doenças , Mutação , PTEN Fosfo-Hidrolase/genética , Neoplasias de Mama Triplo Negativas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The reprogramming of somatic cells to inducible pluripotent stem cells requires a mesenchymal-to-epithelial transition. While differentiating ESCs can undergo the reverse process or epithelial-to-mesenchymal transition (EMT), little is known about the role of EMT in ESC differentiation and fate commitment. Here, we show that Snail homolog 1 (Snail) is expressed during ESC differentiation and is capable of inducing EMT on day 2 of ESC differentiation. Induction of EMT by Snail promotes mesoderm commitment while repressing markers of the primitive ectoderm and epiblast. Snail's impact on differentiation can be partly explained through its regulation of a number of ESC-associated microRNAs, including the microRNA-200 (miR-200) family. The miR-200 family is normally expressed in ESCs but is downregulated in a Wnt-dependent manner during EMT. Maintenance of miR-200 expression stalls differentiating ESCs at the epiblast-like stem cell (EpiSC) stage. Consistent with a role for activin in maintaining the EpiSC state, we find that inhibition of activin signaling decreases miR-200 expression and allows EMT to proceed with a bias toward neuroectoderm commitment. Furthermore, miR-200 requires activin to efficiently maintain cells at the epiblast stage. Together, these findings demonstrate that Snail and miR-200 act in opposition to regulate EMT and exit from the EpiSC stage toward induction of germ layer fates. By modulating expression levels of Snail, activin, and miR-200, we are able to control the order in which cells undergo EMT and transition out of the EpiSC state.
Assuntos
Desdiferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Animais , Desdiferenciação Celular/genética , Transição Epitelial-Mesenquimal/genética , Citometria de Fluxo , Camundongos , MicroRNAs/genética , Microscopia de Fluorescência , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
PIN1 is a phosphorylation-directed member of the peptidyl-prolyl cis/trans isomerase (PPIase) family that facilitates conformational changes in phosphorylated targets such as c-MYC (MYC). Following signaling events that mediate phosphorylation of MYC at Serine 62, PIN1 establishes structurally distinct pools of MYC through its trans-cis and cis-trans isomerization activity at Proline 63. Through these isomerization steps, PIN1 functionally regulates MYC's stability, the molecular timing of its DNA binding and transcriptional activity, and its subnuclear localization. Recently, our group showed that Serine 62 phosphorylated MYC can associate with the inner basket of the nuclear pore (NP) in a PIN1-dependent manner. The poised euchromatin at the NP basket enables rapid cellular response to environmental signals and cell stress, and PIN1-mediated trafficking of MYC calibrates this response. In this perspective, we describe the molecular aspects of PIN1 target recognition and PIN1's function in the context of its temporal and spatial regulation of MYC.
RESUMO
cMYC (MYC) is a potent oncoprotein that is subject to post-translational modifications that affect its stability and activity. Here, we show that Serine 62 phosphorylation, which increases MYC stability and oncogenic activity, is elevated while Threonine 58 phosphorylation, which targets MYC for degradation, is decreased in squamous cell carcinoma (SCC). The oncogenic role of MYC in the development of SCC is unclear since studies have shown in normal skin that wild-type MYC overexpression can drive loss of stem cells and epidermal differentiation. To investigate whether and how altered MYC phosphorylation might affect SCC development, progression, and metastasis, we generated mice with inducible expression of MYCWT or MYCT58A in the basal layer of the skin epidermis. In the T58A mutant, MYC is stabilized with constitutive S62 phosphorylation. When challenged with DMBA/TPA-mediated carcinogenesis, MYCT58A mice had accelerated development of papillomas, increased conversion to malignant lesions, and increased metastasis as compared to MYCWT mice. In addition, MYCT58A-driven SCC displayed stem cell gene expression not observed with MYCWT, including increased expression of Lgr6, Sox2, and CD34. In support of MYCT58A enhancing stem cell phenotypes, its expression was associated with an increased number of BrdU long-term label-retaining cells, increased CD34 expression in hair follicles, and increased colony formation from neonatal keratinocytes. Together, these results indicate that altering MYC phosphorylation changes its oncogenic activity-instead of diminishing establishment and/or maintenance of epidermal stem cell populations like wild-type MYC, pS62-MYC enhances these populations and, under carcinogenic conditions, pS62-MYC expression results in aggressive tumor phenotypes.
RESUMO
The c-MYC (MYC) oncoprotein is often overexpressed in human breast cancer; however, its role in driving disease phenotypes is poorly understood. Here, we investigate the role of MYC in HER2+ disease, examining the relationship between HER2 expression and MYC phosphorylation in HER2+ patient tumors and characterizing the functional effects of deregulating MYC expression in the murine NeuNT model of amplified-HER2 breast cancer. Deregulated MYC alone was not tumorigenic, but coexpression with NeuNT resulted in increased MYC Ser62 phosphorylation and accelerated tumorigenesis. The resulting tumors were metastatic and associated with decreased survival compared with NeuNT alone. MYC;NeuNT tumors had increased intertumoral heterogeneity including a subtype of tumors not observed in NeuNT tumors, which showed distinct metaplastic histology and worse survival. The distinct subtypes of MYC;NeuNT tumors match existing subtypes of amplified-HER2, estrogen receptor-negative human tumors by molecular expression, identifying the preclinical utility of this murine model to interrogate subtype-specific differences in amplified-HER2 breast cancer. We show that these subtypes have differential sensitivity to clinical HER2/EGFR-targeted therapeutics, but small-molecule activators of PP2A, the phosphatase that regulates MYC Ser62 phosphorylation, circumvents these subtype-specific differences and ubiquitously suppresses tumor growth, demonstrating the therapeutic utility of this approach in targeting deregulated MYC breast cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptor ErbB-2/genéticaRESUMO
The SNAG repression domain is comprised of a highly conserved 21-amino acid sequence, is named for its presence in the Snail/growth factor independence-1 class of zinc finger transcription factors, and is present in a variety of proto-oncogenic transcription factors and developmental regulators. The prototype SNAG domain containing oncogene, growth factor independence-1, is responsible for the development of T cell thymomas. The SNAIL proteins also encode the SNAG domain and play key roles in epithelial mesenchymal differentiation events during development and metastasis. Significantly, these oncogenic functions require a functional SNAG domain. The molecular mechanisms of SNAG domain-mediated transcriptional repression are largely unknown. Using a yeast two-hybrid strategy, we identified Ajuba, a multiple LIM domain protein that can function as a corepressor for the SNAG domain. Ajuba interacts with the SNAG domain in vitro and in vivo, colocalizes with it, and enhances SNAG-mediated transcriptional repression. Ajuba shuttles between the cytoplasm and the nucleus and may form a novel intracellular signaling system. Using an integrated reporter gene combined with chromatin immunoprecipitation, we observed rapid, SNAG-dependent assembly of a multiprotein complex that included Ajuba, SNAG, and histone modifications consistent with the repressed state. Thus, SNAG domain proteins may bind Ajuba, trapping it in the nucleus where it functions as an adapter or molecular scaffold for the assembly of macromolecular repression complexes at target promoters.
Assuntos
Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Homeodomínio/genética , Proteínas com Domínio LIM , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments.
Assuntos
Bioimpressão/métodos , Humanos , Fenótipo , Microambiente TumoralRESUMO
Intratumoral heterogeneity in cancers arises from genomic instability and epigenomic plasticity and is associated with resistance to cytotoxic and targeted therapies. We show here that cell-state heterogeneity, defined by differentiation-state marker expression, is high in triple-negative and basal-like breast cancer subtypes, and that drug tolerant persister (DTP) cell populations with altered marker expression emerge during treatment with a wide range of pathway-targeted therapeutic compounds. We show that MEK and PI3K/mTOR inhibitor-driven DTP states arise through distinct cell-state transitions rather than by Darwinian selection of preexisting subpopulations, and that these transitions involve dynamic remodeling of open chromatin architecture. Increased activity of many chromatin modifier enzymes, including BRD4, is observed in DTP cells. Co-treatment with the PI3K/mTOR inhibitor BEZ235 and the BET inhibitor JQ1 prevents changes to the open chromatin architecture, inhibits the acquisition of a DTP state, and results in robust cell death in vitro and xenograft regression in vivo.
Assuntos
Neoplasias da Mama/patologia , Diferenciação Celular , Plasticidade Celular , Resistencia a Medicamentos Antineoplásicos , Animais , Antineoplásicos/uso terapêutico , Azepinas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Cromatina/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Protein phosphatase 2A (PP2A) is a ubiquitously expressed Serine-Threonine phosphatase mediating 30-50% of protein phosphatase activity. PP2A functions as a heterotrimeric complex, with the B subunits directing target specificity to regulate the activity of many key pathways that control cellular phenotypes. PP2A-B56α has been shown to play a tumor suppressor role and to negatively control c-MYC stability and activity. Loss of B56α promotes cellular transformation, likely at least in part through its regulation of c-MYC. Here we report generation of a B56α hypomorph mouse with very low B56α expression that we used to study the physiologic activity of the PP2A-B56α phosphatase. The predominant phenotype we observed in mice with B56α deficiency in the whole body was spontaneous skin lesion formation with hyperproliferation of the epidermis, hair follicles and sebaceous glands. Increased levels of c-MYC phosphorylation on Serine62 and c-MYC activity were observed in the skin lesions of the B56αhm/hm mice. B56α deficiency was found to increase the number of skin stem cells, and consistent with this, papilloma initiation was accelerated in a carcinogenesis model. Further analysis of additional tissues revealed increased inflammation in spleen, liver, lung, and intestinal lymph nodes as well as in the skin lesions, resembling elevated extramedullary hematopoiesis phenotypes in the B56αhm/hm mice. We also observed an increase in the clonogenicity of bone marrow stem cells in B56αhm/hm mice. Overall, this model suggests that B56α is important for stem cells to maintain homeostasis and that B56α loss leading to increased activity of important oncogenes, including c-MYC, can result in aberrant cell growth and increased stem cells that can contribute to the initiation of malignancy.
Assuntos
Neoplasias Experimentais/patologia , Células-Tronco Neoplásicas/patologia , Proteína Fosfatase 2/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/enzimologiaRESUMO
Expression of the transcription factor Snail is required for normal vasculogenesis in the developing mouse embryo. In addition, tumors expressing Snail have been associated with a more malignant phenotype, with both increased invasive properties and angiogenesis. Although the relationship between Snail and vasculogenesis has been noted, no mechanistic analysis has been elucidated. Here, we show that in addition to inducing an epithelial mesenchymal transition, Snail promotes the cell-autonomous induction of Flk1(+) endothelial cells in an early subset of differentiating mouse embryonic stem (ES) cells. Cells that become Flk1+ in response to Snail have a transcriptional profile specific to Gata6+primitive endoderm, but not the early Nanog+epiblast. We further show that Snail's ability to promote Flk1(+) endothelium depends on fibroblast growth factor signaling as well as the repression of the microRNA-200 (miR-200) family, which directly targets the 3' UTRs of Flk1 and Ets1. Together, our results show that Snail is capable of inducing Flk1+ lineage commitment in a subset of differentiating ES cells through the down-regulation of the miR-200 family. We hypothesize that this mechanism of Snail-induced vasculogenesis may be conserved in both the early developing embryo and malignant cancers.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas , MicroRNAs/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Transição Epitelial-Mesenquimal , Citometria de Fluxo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Genes Reporter , Camadas Germinativas/irrigação sanguínea , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Luciferases , Camundongos , MicroRNAs/genética , Neovascularização Fisiológica , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAIL- and AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sequência de Bases , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Primers do DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Domínio LIM , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos , Regiões Promotoras Genéticas , Ligação Proteica , Proteína-Arginina N-Metiltransferases , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , TransfecçãoRESUMO
Snail family transcriptional repressors regulate epithelial mesenchymal transitions during physiological and pathological processes. A conserved SNAG repression domain present in all vertebrate Snail proteins is necessary for repressor complex assembly. Here, we identify the Ajuba family of LIM proteins as functional corepressors of the Snail family via an interaction with the SNAG domain. Ajuba LIM proteins interact with Snail in the nucleus on endogenous E-cadherin promoters and contribute to Snail-dependent repression of E-cadherin. Using Xenopus neural crest as a model of in vivo Snail- or Slug-induced EMT, we demonstrate that Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function. Because Ajuba LIM proteins are also components of adherens junctions and contribute to their assembly or stability, their functional interaction with Snail proteins in the nucleus suggests that Ajuba LIM proteins are important regulators of epithelia dynamics communicating surface events with nuclear responses.
Assuntos
Proteínas de Homeodomínio/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Sobrevivência Celular , Epitélio/embriologia , Epitélio/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Mesoderma/embriologia , Mesoderma/metabolismo , Modelos Neurológicos , Dados de Sequência Molecular , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Transfecção , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Wnt signaling is required for development of mesoderm-derived lineages and expression of transcription factors associated with the primitive streak. In a functional screen, we examined the mesoderm-inducing capacity of transcription factors whose expression was Wnt-dependent in differentiating ESCs. In contrast to many inactive factors, we found that mesoderm posterior 1 (Mesp1) promoted mesoderm development independently of Wnt signaling. Transient Mesp1 expression in ESCs promotes changes associated with epithelial-mesenchymal transition (EMT) and induction of Snai1, consistent with a role in gastrulation. Mesp1 expression also restricted the potential fates derived from ESCs, generating mesoderm progenitors with cardiovascular, but not hematopoietic, potential. Thus, in addition to its effects on EMT, Mesp1 may be capable of generating the recently identified multipotent cardiovascular progenitor from ESCs in vitro.