RESUMO
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Assuntos
Sistemas CRISPR-Cas , Hepatócitos , Neoplasias Hepáticas , Fígado , Metástase Neoplásica , Proteínas do Tecido Nervoso , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Elementos de DNA Transponíveis , Fluorescência , Hepatócitos/metabolismo , Hepatócitos/citologia , Hepatócitos/patologia , Fator 4 Semelhante a Kruppel/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/secundário , Melanoma/metabolismo , Melanoma/patologia , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Semaforinas/antagonistas & inibidores , Semaforinas/metabolismoRESUMO
Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.
Assuntos
Neurônios , Sequências Reguladoras de Ácido Ribonucleico , Regiões 3' não Traduzidas/genética , Animais , Humanos , Camundongos , Neurônios/metabolismo , Nucleotídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNARESUMO
RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.
Assuntos
Artrite/genética , Doenças Inflamatórias Intestinais/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Imunodeficiência Combinada Severa/genética , Alelos , Artrite/imunologia , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Linfopenia/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linhagem , Imunodeficiência Combinada Severa/imunologiaRESUMO
Mutations in genes encoding components of the immune system cause primary immunodeficiencies. Here, we study a patient with recurrent atypical mycobacterial infection and early-onset metastatic bladder carcinoma. Exome sequencing identified two homozygous missense germline mutations, P733L and P832S, in the JAK1 protein that mediates signalling from multiple cytokine receptors. Cells from this patient exhibit reduced JAK1 and STAT phosphorylation following cytokine stimulations, reduced induction of expression of interferon-regulated genes and dysregulated cytokine production; which are indicative of signalling defects in multiple immune response pathways including Interferon-γ production. Reconstitution experiments in the JAK1-deficient cells demonstrate that the impaired JAK1 function is mainly attributable to the effect of the P733L mutation. Further analyses of the mutant protein reveal a phosphorylation-independent role of JAK1 in signal transduction. These findings clarify JAK1 signalling mechanisms and demonstrate a critical function of JAK1 in protection against mycobacterial infection and possibly the immunological surveillance of cancer.
Assuntos
Alelos , Janus Quinase 1/genética , Mutação/genética , Infecções por Mycobacterium/enzimologia , Infecções por Mycobacterium/genética , Sequência de Aminoácidos , Sequência de Bases , Células Sanguíneas/metabolismo , Criança , Pré-Escolar , Citocinas/sangue , Suscetibilidade a Doenças , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Janus Quinase 1/química , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , TYK2 Quinase/metabolismo , Adulto JovemRESUMO
The tight coupling of protein folding pathways with disposal mechanisms promotes the efficacy of protein production in the endoplasmic reticulum (ER). It has been hypothesized that the ER-resident molecular chaperone glucose-regulated protein 94 (GRP94) is part of this quality control coupling because it supports folding of select client proteins yet also robustly associates with the lectin osteosarcoma amplified 9 (OS-9), a component involved in ER-associated degradation (ERAD). To explore this possibility, we investigated potential functions for the GRP94/OS-9 complex in ER quality control. Unexpectedly, GRP94 does not collaborate with OS-9 in ERAD of misfolded substrates, nor is the chaperone required directly for OS-9 folding. Instead, OS-9 binds preferentially to a subpopulation of GRP94 that is hyperglycosylated on cryptic N-linked glycan acceptor sites. Hyperglycosylated GRP94 forms have nonnative conformations and are less active. As a result, these species are degraded much faster than the major, monoglycosylated form of GRP94 in an OS-9-mediated, ERAD-independent, lysosomal-like mechanism. This study therefore clarifies the role of the GRP94/OS-9 complex and describes a novel pathway by which glycosylation of cryptic acceptor sites influences the function and fate of an ER-resident chaperone.
Assuntos
Lectinas/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional , Proteólise , Trifosfato de Adenosina/metabolismo , Degradação Associada com o Retículo Endoplasmático , Glicosilação , Células HEK293 , Humanos , Cinética , Lectinas/química , Lisossomos/metabolismo , Glicoproteínas de Membrana/química , Proteínas de Neoplasias/química , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Regulação Enzimológica da Expressão Gênica , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Proliferação de Células , Cisteína/química , Dissulfetos/química , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Inositol/química , Camundongos , Dados de Sequência Molecular , Oxigênio/química , Fosforilação , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.
Assuntos
Desenho de Fármacos , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Drosophila/efeitos dos fármacos , Drosophila/crescimento & desenvolvimento , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Toll-Like/metabolismoRESUMO
OBJECTIVE: Farnesyl diphosphate synthase (FPPs) produces FPP which is considered a branch-point intermediate in the synthesis of sterols and isoprenylated cellular metabolites. In this study we investigated whether detectable FPPs activity was present in human colorectal cancer (CRC), also evaluating in vitro the role of this enzyme in the growth and apoptosis of CRC cells by using Pamidronate (PAM), a FPPs activity inhibitor. METHODS: The activity level of FPPs was determined in CRC and the normal surrounding mucosa of 50 patients by radiochemical assay. The FPPs mRNA expression was investigated in 15 of 50 patients by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). K-ras mutation was evaluated using PCR and restriction enzyme analysis. Cell growth and apoptosis, after PAM treatment, in human CRC cell line DLD-1 were measured by MTT test and DNA fragmentation, respectively. RESULTS: FPPs activity was detectable in human CRC. FPPs activity and its mRNA were significantly more abundant in cancer samples than in normal mucosa. In vitro PAM resulted in a significant reduction of cell growth and also gave rise to a marked proapoptotic effect. CONCLUSIONS: This study provides the first evidence of the presence of FPPs activity in human CRC. Moreover, FPPs enzyme was found to play a significant role in colon cancer proliferation.