RESUMO
In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.
Assuntos
Imunidade Adaptativa , Mitofagia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Azoximetano/toxicidade , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Permeabilidade da Membrana Celular , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Compostos Ferrosos/metabolismo , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitofagia/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Taxa de SobrevidaRESUMO
Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/ß-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of ß-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, ß-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted ß-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This ß-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by ß-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Autorrenovação Celular/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , SARS-CoV-2/imunologia , Biomarcadores , COVID-19/metabolismo , Citocinas/metabolismo , Suscetibilidade a Doenças/imunologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Transdução de SinaisRESUMO
For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.
Assuntos
Desdiferenciação Celular , Folículo Piloso , Melanócitos , Nicho de Células-Tronco , Células-Tronco , Animais , Humanos , Camundongos , Folículo Piloso/citologia , Melanócitos/citologia , Células-Tronco/citologia , Microambiente Celular , Linhagem da Célula , Envelhecimento , Homeostase , Cor de Cabelo/fisiologiaRESUMO
Melanocyte stem cells (McSCs) intimately interact with epithelial stem cells (EpSCs) in the hair follicle bulge and secondary hair germ (sHG). Together, they undergo activation and differentiation to regenerate pigmented hair. However, the mechanisms behind this coordinated stem cell behavior have not been elucidated. Here, we identified Wnt signaling as a key pathway that couples the behavior of the two stem cells. EpSCs and McSCs coordinately activate Wnt signaling at the onset of hair follicle regeneration within the sHG. Using genetic mouse models that specifically target either EpSCs or McSCs, we show that Wnt activation in McSCs drives their differentiation into pigment-producing melanocytes, while EpSC Wnt signaling not only dictates hair follicle formation but also regulates McSC proliferation during hair regeneration. Our data define a role for Wnt signaling in the regulation of McSCs and also illustrate a mechanism for regeneration of complex organs through collaboration between heterotypic stem cell populations.
Assuntos
Células Epiteliais/citologia , Cabelo/fisiologia , Melanócitos/citologia , Pigmentação , Fenômenos Fisiológicos da Pele , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Cabelo/citologia , Doenças do Cabelo/metabolismo , Doenças do Cabelo/patologia , Folículo Piloso/citologia , Humanos , Camundongos , Regeneração , Transdução de Sinais , Pele/citologia , beta Catenina/metabolismoRESUMO
We tested cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/-KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.
Assuntos
Neoplasias Colorretais/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Indutores da Angiogênese/metabolismo , Animais , Anticorpos/imunologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Células Mieloides/metabolismo , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Endotélio Vascular/metabolismo , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Adulto , Idoso , Animais , Linhagem Celular , Endotélio Vascular/patologia , Feminino , Humanos , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Adulto Jovem , beta Catenina/metabolismoRESUMO
Growth factor signaling is involved in the development of various organs, but how signaling regulates organ morphogenesis and differentiation in a coordinated manner remains to be clarified. Here, we show how WNT signaling controls epithelial morphogenetic changes and differentiation using the salivary gland as a model. Experiments using genetically manipulated mice and organ cultures revealed that WNT signaling at an early stage (E12-E15) of submandibular salivary gland (SMG) development inhibits end bud morphogenesis and differentiation into proacini by suppressing Kit expression through the upregulation of the transcription factor MYB, and concomitantly increasing the expression of distal progenitor markers. In addition, WNT signaling at the early stage of SMG development promoted end bud cell proliferation, leading to duct formation. WNT signaling reduction at a late stage (E16-E18) of SMG development promoted end bud maturation and suppressed duct formation. Thus, WNT signaling controls the timing of SMG organogenesis by keeping end bud cells in an undifferentiated bipotent state.
Assuntos
Células Acinares/citologia , Diferenciação Celular , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/embriologia , Via de Sinalização Wnt , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glândula Submandibular/efeitos dos fármacos , Fatores de Tempo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
The neural crest (NC) is crucial for the evolutionary diversification of vertebrates. NC cells are induced at the neural plate border by the coordinated action of several signaling pathways, including Wnt/ß-catenin. NC cells are normally generated in the posterior neural plate border, whereas the anterior neural fold is devoid of NC cells. Using the mouse model, we show here that active repression of Wnt/ß-catenin signaling is required for maintenance of neuroepithelial identity in the anterior neural fold and for inhibition of NC induction. Conditional inactivation of Tcf7l1, a transcriptional repressor of Wnt target genes, leads to aberrant activation of Wnt/ß-catenin signaling in the anterior neuroectoderm and its conversion into NC. This reduces the developing prosencephalon without affecting the anterior-posterior neural character. Thus, Tcf7l1 defines the border between the NC and the prospective forebrain via restriction of the Wnt/ß-catenin signaling gradient.
Assuntos
Linhagem da Célula , Crista Neural/citologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Biomarcadores/metabolismo , Transdiferenciação Celular , Deleção de Genes , Humanos , Integrases/metabolismo , Camundongos Transgênicos , Crista Neural/metabolismo , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Fenótipo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição AP-2/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , beta Catenina/metabolismoRESUMO
The tips of mammalian digits can regenerate after amputation, like those of amphibians. It is unknown why this capacity is limited to the area associated with the nail. Here we show that nail stem cells (NSCs) reside in the proximal nail matrix and that the mechanisms governing NSC differentiation are coupled directly with their ability to orchestrate digit regeneration. Early nail progenitors undergo Wnt-dependent differentiation into the nail. After amputation, this Wnt activation is required for nail regeneration and also for attracting nerves that promote mesenchymal blastema growth, leading to the regeneration of the digit. Amputations proximal to the Wnt-active nail progenitors result in failure to regenerate the nail or digit. Nevertheless, ß-catenin stabilization in the NSC region induced their regeneration. These results establish a link between NSC differentiation and digit regeneration, and suggest that NSCs may have the potential to contribute to the development of novel treatments for amputees.
Assuntos
Extremidades/fisiologia , Casco e Garras/crescimento & desenvolvimento , Regeneração/fisiologia , Proteínas Wnt/metabolismo , Amputação Cirúrgica , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular , Células Cultivadas , Epitélio/metabolismo , Extremidades/crescimento & desenvolvimento , Extremidades/inervação , Casco e Garras/citologia , Casco e Garras/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Androgen, beta-catenin (CTNNB1), and estrogen pathways stimulate proliferative growth of developing mouse prostate but how these pathways interact is not fully understood. We previously found that androgens induce CTNNB1 signaling in mouse urogenital sinus (UGS) epithelium from which prostatic ductal epithelium derives. Others have shown that low estradiol concentrations induce UGS epithelial proliferative growth. Here, we found that CTNNB1 signaling overlaps cyclin D1 (CCND1) expression in prostatic buds and we used a genetic approach to test whether CTNNB1 signaling induces CCND1 expression. We observed an unexpected sexually dimorphic response to hyperactive CCNTB1 signaling: in male mouse UGS it increased Ccnd1 mRNA abundance without increasing its protein abundance but in female UGS it increased Ccnd1 mRNA and protein abundance, suggesting a potential role for estrogens in stabilizing CCND1 protein. Treating wild type male UGS explants with androgen and either 17ß-estradiol or a proteasome inhibitor increased CCND1 protein and KI67 labeling in prostatic bud epithelium. Together, our results are consistent with an epithelial proliferative growth mechanism linking CTNNB1-driven Ccnd1 transcription and estrogen-mediated CCND1 protein stabilization.
Assuntos
Ciclina D1/genética , Desenvolvimento Embrionário/genética , Estrogênios/genética , beta Catenina/genética , Androgênios/genética , Animais , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Estrogênios/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Organogênese , Próstata , RNA Mensageiro/genética , beta Catenina/metabolismoRESUMO
The TGF-ß and Wnt/ß-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF-ß has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF-ß signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF-ß type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF-ß receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF-ß receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/ß-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF-ß receptor impaired ß-catenin activity in vitro and in vivo Genetically restoring ß-catenin activity in proximal tubules lacking the TGF-ß receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF-ß receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of ß-catenin activity or an indirect effect of ß-catenin interacting with other growth factors. In conclusion, blocking TGF-ß and ß-catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Assuntos
Falência Renal Crônica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/metabolismo , Animais , Ácidos Aristolóquicos/química , Núcleo Celular/metabolismo , Colágeno/química , Cruzamentos Genéticos , Epitélio/metabolismo , Feminino , Deleção de Genes , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidoresRESUMO
A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration-resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR-defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium-specific inactivation of Aes in Ptenflox/flox mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways.
Assuntos
Movimento Celular/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Idoso , Animais , Western Blotting , Linhagem Celular Tumoral , Proteínas Correpressoras , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Metástase Neoplásica , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Breakdown in immunological tolerance to self-Ags or uncontrolled inflammation results in autoimmune disorders. Dendritic cells (DCs) play an important role in regulating the balance between inflammatory and regulatory responses in the periphery. However, factors in the tissue microenvironment and the signaling networks critical for programming DCs to control chronic inflammation and promote tolerance are unknown. In this study, we show that wnt ligand-mediated activation of ß-catenin signaling in DCs is critical for promoting tolerance and limiting neuroinflammation. DC-specific deletion of key upstream (lipoprotein receptor-related protein [LRP]5/6) or downstream (ß-catenin) mediators of canonical wnt signaling in mice exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of LRP5/6-ß-catenin-mediated signaling in DCs led to an increased Th1/Th17 cell differentiation but reduced regulatory T cell response. This was due to increased production of proinflammatory cytokines and decreased production of anti-inflammatory cytokines such as IL-10 and IL-27 by DCs lacking LRP5/6-ß-catenin signaling. Consistent with these findings, pharmacological activation of canonical wnt/ß-catenin signaling delayed experimental autoimmune encephalomyelitis onset and diminished CNS pathology. Thus, the activation of canonical wnt signaling in DCs limits effector T cell responses and represents a potential therapeutic approach to control autoimmune neuroinflammation.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt , Animais , Diferenciação Celular , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Deleção de Genes , Técnicas de Inativação de Genes , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/citologia , Células Th1/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMO
We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino-terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter-enhancer is in a typical CpG island, and contains a Yin-Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy-number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG-island promoter enhancer, and it is regulated transcriptionally.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metástase Neoplásica/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Correpressoras , Metilação de DNA/genética , Regulação para Baixo , Genes Supressores de Tumor , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Camundongos , Mutação/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Elementos de Resposta/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Fator de Transcrição YY1/metabolismoRESUMO
The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta-catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta-catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta-catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta-catenin is conditionally stabilized in the Shh+domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta-catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta-catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia Nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta-catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta-catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson's disease.
Assuntos
Dopamina/metabolismo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Mesencéfalo/citologia , Neurogênese/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Fatores Etários , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/genética , Embrião de Mamíferos , Feminino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Mesencéfalo/embriologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1âß-cateninâFgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.
Assuntos
Membro Posterior/embriologia , Anormalidades Maxilomandibulares/embriologia , Proteínas com Homeodomínio LIM/metabolismo , Osteogênese/genética , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Região Branquial/embriologia , Linhagem da Célula/genética , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Fosfatase 6 de Especificidade Dupla/biossíntese , Embrião de Mamíferos/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Fator 8 de Crescimento de Fibroblasto/biossíntese , Fator 8 de Crescimento de Fibroblasto/deficiência , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Membro Posterior/anormalidades , Proteínas de Homeodomínio/biossíntese , Anormalidades Maxilomandibulares/genética , Fatores de Transcrição Kruppel-Like/biossíntese , Proteínas com Homeodomínio LIM/genética , Mandíbula/embriologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Transdução de Sinais/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima , Proteína Gli3 com Dedos de Zinco , beta Catenina/genéticaRESUMO
In most cell types, nuclear ß-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of ß-catenin but critically depended on GSK3ß for effective ß-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that ß-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, ß-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive ß-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3ß small molecule inhibitors to subvert ß-catenin degradation. Clinically approved GSK3ß-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of ß-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3ß-inhibitors for targeted engagement of lymphoid-specific ß-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTS: Unlike other cell lineages, B-cells express nuclear ß-catenin protein at low baseline levels and depend on GSK3ß for its degradation.In B-cells, ß-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. CRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed ß-catenin-dependent Myc repression and induction of cell death. The discovery of GSK3ß-dependent degradation of ß-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3ß-inhibitors for the treatment of refractory B-cell malignancies. GRAPHICAL ABSTRACT: Abundant nuclear ß-cateninß-catenin pairs with TCF7 factors for transcriptional activation of MYCB-cells rely on efficient degradation of ß-catenin by GSK3ßB-cell-specific expression of Ikaros factors Unique vulnerability in B-cell tumors: GSK3ß-inhibitors induce nuclear accumulation of ß-catenin.ß-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC.
RESUMO
Organ formation requires integrating signals to coordinate proliferation, specify cell fates, and shape tissue. Tracing these events and signals remains a challenge, as intermediate states across many critical transitions are unresolvable over real time and space. Here, we designed a unique computational approach to decompose a non-linear differentiation process into key components to resolve the signals and cell behaviors that drive a rapid transition, using the hair follicle dermal condensate as a model. Combining scRNA sequencing with genetic perturbation, we reveal that proliferative Dkk1+ progenitors transiently amplify to become quiescent dermal condensate cells by the mere spatiotemporal patterning of Wnt/ß-catenin and SHH signaling gradients. Together, they deterministically coordinate a rapid transition from proliferation to quiescence, cell fate specification, and morphogenesis. Moreover, genetically repatterning these gradients reproduces these events autonomously in "slow motion" across more intermediates that resolve the process. This analysis unravels two morphogen gradients that intersect to coordinate events of organogenesis.
Assuntos
Transdução de Sinais , Pele , Diferenciação Celular , Folículo Piloso , Proteínas Hedgehog/genética , Morfogênese , Transdução de Sinais/genéticaRESUMO
The choroid plexus secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the choroid plexus epithelium arises from the Wnt- expressing cortical hem. Canonical Wnt signaling pathway molecules such as nuclear ß-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human ß-CATENIN are known to result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of choroid plexus epithelial identity and an apparent transformation of this tissue to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell derived organoids. The choroid plexus is also disrupted when ß-Catenin is conditionally inactivated. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal choroid plexus development in the mammalian brain.
Assuntos
Plexo Corióideo/metabolismo , Epitélio/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Plexo Corióideo/patologia , Feminino , Humanos , Masculino , Camundongos , Telencéfalo/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Tumor-infiltrating lymphocytes (TIL), which include tumor-specific T lymphocytes with frequency, are used for adoptive cell transfer therapy (ACT) in clinical practice. The optimization of TIL preparation has been investigated to reduce the senescence and increase the abundance of TIL, as both the quality and quantity of the transferred cells have great influence on the outcome of TIL-based ACT (TIL-ACT). Considering the effects of cell reprogramming on senescence, we expected that the anti-tumor effect could be enhanced by TIL regeneration. To confirm this hypothesis, we established tumor-specific TIL-derived iPS cells (TIL-iPSC) with human colorectal cancer specimens. T cells differentiated from TIL-iPSC (TIL-iPS-T) retained not only intrinsic T cell functions and tumor specificity, but also exhibited improved proliferation capacity and additional killing activity. Moreover, less differentiated profiles and prolonged persistency were seen in TIL-iPS-T compared with primary cells. Our findings imply that iPSC technology has great potential for TIL-ACT.