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1.
Front Immunol ; 10: 2468, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708921

RESUMO

Somewhat counterintuitively, the tyrosine phosphatase SHP-2 (SH2 domain-containing protein tyrosine phosphatase-2) is crucial for the activation of extracellular signal-regulated kinase (ERK) downstream of various growth factor receptors, thereby exerting essential developmental functions. This phosphatase also deploys proto-oncogenic functions and specific inhibitors have recently been developed. With respect to the immune system, the role of SHP-2 in the signaling of cytokines relevant for myelopoiesis and myeloid malignancies has been intensively studied. The function of this phosphatase downstream of cytokines important for lymphocytes is less understood, though multiple lines of evidence suggest its importance. In addition, SHP-2 has been proposed to mediate the suppressive effects of inhibitory receptors (IRs) that sustain a dysfunctional state in anticancer T cells. Molecules involved in IR signaling are of potential pharmaceutical interest as blockade of these inhibitory circuits leads to remarkable clinical benefit. Here, we discuss the dichotomy in the functions ascribed to SHP-2 downstream of cytokine receptors and IRs, with a focus on T and NK lymphocytes. Further, we highlight the importance of broadening our understanding of SHP-2's relevance in lymphocytes, an essential step to inform on side effects and unanticipated benefits of its therapeutic blockade.

2.
Brain ; 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31665242

RESUMO

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/ß-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

3.
Cell Stem Cell ; 24(5): 753-768.e6, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982770

RESUMO

Cell-autonomous Wnt signaling has well-characterized functions in controlling stem cell activity, including in the prostate. While niche cells secrete Wnt ligands, the effects of Wnt signaling in niche cells per se are less understood. Here, we show that stromal cells in the proximal prostatic duct near the urethra, a mouse prostate stem cell niche, not only produce multiple Wnt ligands but also exhibit strong Wnt/ß-catenin activity. The non-canonical Wnt ligand Wnt5a, secreted by proximal stromal cells, directly inhibits proliefration of prostate epithelial stem or progenitor cells whereas stromal cell-autonomous canonical Wnt/ß-catenin signaling indirectly suppresses prostate stem or progenitor activity via the transforming growth factor ß (TGFß) pathway. Collectively, these pathways restrain the proliferative potential of epithelial cells in the proximal prostatic ducts. Human prostate likewise exhibits spatially restricted distribution of stromal Wnt/ß-catenin activity, suggesting a conserved mechanism for tissue patterning. Thus, this study shows how distinct stromal signaling mechanisms within the prostate cooperate to regulate tissue homeostasis.

4.
Nat Commun ; 10(1): 1444, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926899

RESUMO

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Matadoras Naturais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Interleucina-15/metabolismo , Animais , Antígenos Ly/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Integrases/metabolismo , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/fisiologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Serina-Treonina Quinases TOR/metabolismo
5.
Cell Rep ; 26(2): 415-428.e5, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625324

RESUMO

We identified a regulatory system that acts downstream of Wnt/ß-catenin signaling in salivary gland and head and neck carcinomas. We show in a mouse tumor model of K14-Cre-induced Wnt/ß-catenin gain-of-function and Bmpr1a loss-of-function mutations that tumor-propagating cells exhibit increased Mll1 activity and genome-wide increased H3K4 tri-methylation at promoters. Null mutations of Mll1 in tumor mice and in xenotransplanted human head and neck tumors resulted in loss of self-renewal of tumor-propagating cells and in block of tumor formation but did not alter normal tissue homeostasis. CRISPR/Cas9 mutagenesis and pharmacological interference of Mll1 at sequences that inhibit essential protein-protein interactions or the SET enzyme active site also blocked the self-renewal of mouse and human tumor-propagating cells. Our work provides strong genetic evidence for a crucial role of Mll1 in solid tumors. Moreover, inhibitors targeting specific Mll1 interactions might offer additional directions for therapies to treat these aggressive tumors.

6.
Life Sci Alliance ; 2(1): e201800173, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30599048

RESUMO

We explored the connection between C/EBPα (CCAAT/enhancer-binding protein α) and Wnt signaling in gut homeostasis and carcinogenesis. C/EBPα was expressed in human and murine intestinal epithelia in the transit-amplifying region of the crypts and was absent in intestinal stem cells and Paneth cells with activated Wnt signaling. In human colorectal cancer and murine APCMin/+ polyps, C/EBPα was absent in the nuclear ß-catenin-positive tumor cells. In chemically induced intestinal carcinogenesis, C/EBPα KO in murine gut epithelia increased tumor volume. C/EBPα deletion extended the S-phase cell zone in intestinal organoids and activated typical proliferation gene expression signatures, including that of Wnt target genes. Genetic activation of ß-catenin in organoids attenuated C/EBPα expression, and ectopic C/EBPα expression in HCT116 cells abrogated proliferation. C/EBPα expression accompanied differentiation of the colon cancer cell line Caco-2, whereas ß-catenin stabilization suppressed C/EBPα. These data suggest homeostatic and oncogenic suppressor functions of C/EBPα in the gut by restricting Wnt signaling.

7.
Nat Commun ; 9(1): 3259, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30108215

RESUMO

Uncontrolled activation of TGFß signaling is a common denominator of fibrotic tissue remodeling. Here we characterize the tyrosine phosphatase SHP2 as a molecular checkpoint for TGFß-induced JAK2/STAT3 signaling and as a potential target for the treatment of fibrosis. TGFß stimulates the phosphatase activity of SHP2, although this effect is in part counterbalanced by inhibitory effects on SHP2 expression. Stimulation with TGFß promotes recruitment of SHP2 to JAK2 in fibroblasts with subsequent dephosphorylation of JAK2 at Y570 and activation of STAT3. The effects of SHP2 on STAT3 activation translate into major regulatory effects of SHP2 on fibroblast activation and tissue fibrosis. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570, reduces JAK2/STAT3 signaling, inhibits TGFß-induced fibroblast activation and ameliorates dermal and pulmonary fibrosis. Given the availability of potent SHP2 inhibitors, SHP2 might thus be a potential target for the treatment of fibrosis.

8.
Cell Microbiol ; 20(11): e12891, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30009515

RESUMO

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1ß, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

9.
Nat Med ; 24(7): 954-960, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29808009

RESUMO

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers.

10.
Cell Rep ; 23(1): 39-49, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617671

RESUMO

In chronic infection and cancer, T cells acquire a dysfunctional state characterized by the expression of inhibitory receptors. In vitro studies implicated the phosphatase Shp-2 downstream of these receptors, including PD-1. However, whether Shp-2 is responsible in vivo for such dysfunctional responses remains elusive. To address this, we generated T cell-specific Shp-2-deficient mice. These mice did not show differences in controlling chronic viral infections. In this context, Shp-2-deleted CD8+ T lymphocytes expanded moderately better but were less polyfunctional than control cells. Mice with Shp-2-deficient T cells also showed no significant improvement in controlling immunogenic tumors and responded similarly to controls to α-PD-1 treatment. We therefore showed that Shp-2 is dispensable in T cells for globally establishing exhaustion and for PD-1 signaling in vivo. These results reveal the existence of redundant mechanisms downstream of inhibitory receptors and represent the foundation for defining these relevant molecular events.

11.
Sci Rep ; 7(1): 9000, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28827556

RESUMO

The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.

12.
Cancer Res ; 77(8): 2134-2147, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28202523

RESUMO

Many tumors display intracellular heterogeneity with subsets of cancer stem cells (CSC) that sustain tumor growth, recurrence, and therapy resistance. Cancer-associated fibroblasts (CAF) have been shown to support and regulate CSC function. Here, we investigate the interactions between CSCs and CAFs in mammary gland tumors driven by combined activation of Wnt/ß-catenin and Hgf/Met signaling in mouse mammary epithelial cells. In this setting, CSCs secrete the Hedgehog ligand SHH, which regulate CAFs via paracrine activation of Hedgehog signaling. CAFs subsequently secrete factors that promote expansion and self-renewal of CSCs. In vivo treatment of tumors with the Hedgehog inhibitor vismodegib reduce CAF and CSC expansion, resulting in an overall delay of tumor formation. Our results identify a novel intracellular signaling module that synergistically regulates CAFs and CSCs. Targeting CAFs with Hedgehog inhibitors may offer a novel therapeutic strategy against breast cancer. Cancer Res; 77(8); 2134-47. ©2017 AACR.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Proteínas Hedgehog/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Anilidas/farmacologia , Animais , Comunicação Celular/fisiologia , Feminino , Camundongos , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridinas/farmacologia , Transdução de Sinais , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
13.
Cell Rep ; 16(12): 3388-3400, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27653698

RESUMO

Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including ß-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate's phosphorylation, rather than SUMOylation, and binding to RNF4's arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation.


Assuntos
Proteínas Nucleares/metabolismo , Oncogenes/fisiologia , Estabilidade Proteica , Fatores de Transcrição/metabolismo , Humanos , Ubiquitinação
14.
J Biol Chem ; 291(37): 19618-30, 2016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-27484798

RESUMO

The A-kinase anchoring protein (AKAP) GSK3ß interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3ß (GSK3ß). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3ß are required for the regulation of ß-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets ß-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause ß-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the ß-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3ß facilitates control of the destabilizing phosphorylation of ß-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on ß-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with ß-catenin. The regulation of ß-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3ß, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3ß is a conserved GSK3ß interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3ß by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Proteínas de Ancoragem à Quinase A , Células A549 , Proteínas Quinases Dependentes de AMP Cíclico/genética , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Proteínas Repressoras/genética , beta Catenina/genética
15.
Nat Cell Biol ; 18(5): 463-5, 2016 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27117330

RESUMO

Wnt/ß-catenin signalling is an important regulator of liver development, zonation and regeneration. The cell surface complex RSPO-LGR4/5-ZNF3/RNF43 is now shown to direct Wnt/ß-catenin signalling in orchestrating the division of the liver into functionally distinct metabolic zones, providing insights into the mechanisms that influence organ development and regeneration.


Assuntos
Fígado/citologia , Transdução de Sinais , Humanos , Proteínas Wnt , beta Catenina
16.
Cancer Res ; 76(4): 891-901, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26645562

RESUMO

Wnt/ß-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/ß-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between ß-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/ß-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell-cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy.


Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia
17.
Gut ; 65(10): 1690-701, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26156959

RESUMO

OBJECTIVES: We have previously identified a 115-gene signature that characterises the metastatic potential of human primary colon cancers. The signature included the canonical Wnt target gene BAMBI, which promoted experimental metastasis in mice. Here, we identified three new direct Wnt target genes from the signature, and studied their functions in epithelial-mesenchymal transition (EMT), cell migration and experimental metastasis. DESIGN: We examined experimental liver metastases following injection of selected tumour cells into spleens of NOD/SCID mice. Molecular and cellular techniques were used to identify direct transcription target genes of Wnt/ß-catenin signals. Microarray analyses and experiments that interfered with cell migration through inhibitors were performed to characterise downstream signalling systems. RESULTS: Three new genes from the colorectal cancer (CRC) metastasis signature, BOP1, CKS2 and NFIL3, were identified as direct transcription targets of ß-catenin/TCF4. Overexpression and knocking down of these genes in CRC cells promoted and inhibited, respectively, experimental metastasis in mice, EMT and cell motility in culture. Cell migration was repressed by interfering with distinct signalling systems through inhibitors of PI3K, JNK, p38 mitogen-activated protein kinase and/or mTOR. Gene expression profiling identified a series of migration-promoting genes, which were induced by BOP1, CKS2 and NFIL3, and could be repressed by inhibitors that are specific to these pathways. CONCLUSIONS: We identified new direct Wnt/ß-catenin target genes, BOP1, CKS2 and NFIL3, which induced EMT, cell migration and experimental metastasis of CRC cells. These genes crosstalk with different downstream signalling systems, and activate migration-promoting genes. These pathways and downstream genes may serve as therapeutic targets in the treatment of CRC metastasis.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Movimento Celular/genética , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas , Proteínas Nucleares/genética , Via de Sinalização Wnt/genética , Animais , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas Experimentais , Camundongos , Metástase Neoplásica , Células Tumorais Cultivadas
18.
Cell Rep ; 13(3): 561-572, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26456821

RESUMO

Gab1 is a scaffold protein that acts downstream of receptor tyrosine kinases. Here, we produced conditional Gab1 mutant mice (by K14- and Krox20-cre) and show that Gab1 mediates crucial signals in the control of both the hair cycle and the self-renewal of hair follicle stem cells. Remarkably, mutant hair follicles do not enter catagen, the destructive phase of the hair cycle. Instead, hair follicle stem cells lose quiescence and become exhausted, and thus no stem cell niches are established in the bulges. Moreover, conditional sustained activation of Mapk signaling by expression of a gain-of-function Mek1(DD) allele (by Krox20-cre) rescues hair cycle deficits and restores quiescence of the stem cells. Our data thus demonstrate an essential role of Gab1 downstream of receptor tyrosine kinases and upstream of Shp2 and Mapk in the regulation of the hair cycle and the self-renewal of hair follicle stem cells.


Assuntos
Células-Tronco Adultas/metabolismo , Autorrenovação Celular , Folículo Piloso/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfoproteínas/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Células Cultivadas , Proteína Adaptadora GRB2/metabolismo , Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Queratinócitos/citologia , Queratinócitos/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Camundongos , Fosfoproteínas/genética , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
20.
ChemMedChem ; 10(5): 815-26, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25877780

RESUMO

Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-{(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazino}benzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71±15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.


Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/enzimologia , Neoplasias/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Estrutura Molecular , Neoplasias/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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