RESUMO
Aberrant activation or suppression of WNT/ß-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the ß-catenin-dependent and ß-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated ß-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and ß-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.
Assuntos
Caseína Quinase I , Via de Sinalização Wnt , beta Catenina , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fosforilação , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.
Assuntos
Fator 2 Relacionado a NF-E2 , Receptor EphA5 , Proteínas Quinases Ativadas por AMP/metabolismo , Mutação com Ganho de Função , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genéticaRESUMO
Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Tecido Adiposo Branco/patologia , Carcinogênese/genética , Modelos Animais de Doenças , Fator 2 Relacionado a NF-E2/genética , Lesões Pré-Cancerosas/genética , Trato Gastrointestinal Superior/patologia , Animais , Carcinoma de Células Escamosas/genética , Esôfago/patologia , Humanos , Hiperplasia/genética , Camundongos , Mutação , Língua/patologiaRESUMO
Human Pumilio proteins, PUM1 and PUM2, are sequence specific RNA-binding proteins that regulate protein expression. We used RNA-seq, rigorous statistical testing and an experimentally derived fold change cut-off to identify nearly 1000 target RNAs-including mRNAs and non-coding RNAs-that are functionally regulated by PUMs. Bioinformatic analysis defined a PUM Response Element (PRE) that was significantly enriched in transcripts that increased in abundance and matches the PUM RNA-binding consensus. We created a computational model that incorporates PRE position and frequency within an RNA relative to the magnitude of regulation. The model reveals significant correlation of PUM regulation with PREs in 3' untranslated regions (UTRs), coding sequences and non-coding RNAs, but not 5' UTRs. To define direct, high confidence PUM targets, we cross-referenced PUM-regulated RNAs with all PRE-containing RNAs and experimentally defined PUM-bound RNAs. The results define nearly 300 direct targets that include both PUM-repressed and, surprisingly, PUM-activated target RNAs. Annotation enrichment analysis reveal that PUMs regulate genes from multiple signaling pathways and developmental and neurological processes. Moreover, PUM target mRNAs impinge on human disease genes linked to cancer, neurological disorders and cardiovascular disease. These discoveries pave the way for determining how the PUM-dependent regulatory network impacts biological functions and disease states.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Ligação a RNA/genética , RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Ontologia Genética , Células HEK293 , Humanos , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Histone methyltransferases play essential roles in the organization and function of chromatin. They are also frequently mutated in human diseases including cancer1. One such often mutated methyltransferase, SETD2, associates co-transcriptionally with RNA polymerase II and catalyzes histone H3 lysine 36 trimethylation (H3K36me3) - a modification that contributes to gene transcription, splicing, and DNA repair2. While studies on SETD2 have largely focused on the consequences of its catalytic activity, the non-catalytic functions of SETD2 are largely unknown. Here we report a catalysis-independent function of SETD2 in maintaining nuclear lamina stability and genome integrity. We found that SETD2, via its intrinsically disordered N-terminus, associates with nuclear lamina proteins including lamin A/C, lamin B1, and emerin. Depletion of SETD2, or deletion of its N-terminus, resulted in widespread nuclear morphology abnormalities and genome stability defects that were reminiscent of a defective nuclear lamina. Mechanistically, the N-terminus of SETD2 facilitates the association of the mitotic kinase CDK1 with lamins, thereby promoting lamin phosphorylation and depolymerization required for nuclear envelope disassembly during mitosis. Taken together, our findings reveal an unanticipated link between the N-terminus of SETD2 and nuclear lamina organization that may underlie how SETD2 acts as a tumor suppressor.
RESUMO
Studies have shown that Nrf2E79Q/+ is one of the most common mutations found in human tumors. To elucidate how this genetic change contributes to lung cancer, we compared lung tumor development in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 loss, the most common mutations found in human lung tumors, in the presence or absence of Nrf2E79Q/+. Trp53/p16-deficient mice developed combined-small cell lung cancer (C-SCLC), a mixture of pure-SCLC (P-SCLC) and large cell neuroendocrine carcinoma. Mice possessing the LSL-Nrf2E79Q mutation showed no difference in the incidence or latency of C-SCLC compared with Nrf2+/+ mice. However, these tumors did not express NRF2 despite Cre-induced recombination of the LSL-Nrf2E79Q allele. Trp53/p16-deficient mice also developed P-SCLC, where activation of the NRF2E79Q mutation associated with a higher incidence of this tumor type. All C-SCLCs and P-SCLCs were positive for NE-markers, NKX1-2 (a lung cancer marker) and negative for P63 (a squamous cell marker), while only P-SCLC expressed NRF2 by immunohistochemistry. Analysis of a consensus NRF2 pathway signature in human NE+-lung tumors showed variable activation of NRF2 signaling. Our study characterizes the first GEMM that develops C-SCLC, a poorly-studied human cancer and implicates a role for NRF2 activation in SCLC development.
Assuntos
Carcinoma Neuroendócrino , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Carcinoma Neuroendócrino/patologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Incidência , Neoplasias Pulmonares/patologia , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Lung squamous carcinoma (LUSC) is a highly metastatic disease with a poor prognosis. Using an integrated screening approach, we found that miR-671-5p reduces LUSC metastasis by inhibiting a circular RNA (circRNA), CDR1as. Although the putative function of circRNA is through miRNA sponging, we found that miR-671-5p more potently silenced an axis of CDR1as and its antisense transcript, cerebellar degeneration related protein 1 (CDR1). Silencing of CDR1as or CDR1 significantly inhibited LUSC metastases and CDR1 was sufficient to promote migration and metastases. CDR1, which directly interacted with adaptor protein 1 (AP1) complex subunits and coatomer protein I (COPI) proteins, no longer promoted migration upon blockade of Golgi trafficking. Therapeutic inhibition of the CDR1as/CDR1 axis with miR-671-5p mimics reduced metastasis in vivo. This report demonstrates a novel role for CDR1 in promoting metastasis and Golgi trafficking. These findings reveal an miRNA/circRNA axis that regulates LUSC metastases through a previously unstudied protein, CDR1. SIGNIFICANCE: This study shows that circRNA, CDR1as, promotes lung squamous migration, metastasis, and Golgi trafficking through its complimentary transcript, CDR1.
Assuntos
Autoantígenos/metabolismo , Carcinoma de Células Escamosas/secundário , Complexo de Golgi/metabolismo , Neoplasias Pulmonares/patologia , Proteínas do Tecido Nervoso/metabolismo , RNA Circular/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Animais , Autoantígenos/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Humanos , Ácido Hialurônico/uso terapêutico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Nanopartículas/uso terapêutico , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genéticaRESUMO
Genomic amplification of the gene encoding and phosphorylation of the protein FADD (Fas-associated death domain) is associated with poor clinical outcome in lung cancer and in head and neck cancer. Activating mutations in the guanosine triphosphatase RAS promotes cell proliferation in various cancers. Increased abundance of phosphorylated FADD in patient-derived tumor samples predicts poor clinical outcome. Using immunohistochemistry analysis and in vivo imaging of conditional mouse models of KRAS(G12D)-driven lung cancer, we found that the deletion of the gene encoding FADD suppressed tumor growth, reduced the proliferative index of cells, and decreased the activation of downstream effectors of the RAS-MAPK (mitogen-activated protein kinase) pathway that promote the cell cycle, including retinoblastoma (RB) and cyclin D1. In mouse embryonic fibroblasts, the induction of mitosis upon activation of KRAS required FADD and the phosphorylation of FADD by CK1α (casein kinase 1α). Deleting the gene encoding CK1α in KRAS mutant mice abrogated the phosphorylation of FADD and suppressed lung cancer development. Phosphorylated FADD was most abundant during the G2/M phase of the cell cycle, and mass spectrometry revealed that phosphorylated FADD interacted with kinases that mediate the G2/M transition, including PLK1 (Polo-like kinase 1), AURKA (Aurora kinase A), and BUB1 (budding uninhibited by benzimidazoles 1). This interaction was decreased in cells treated with a CKI-7, a CK1α inhibitor. Therefore, as the kinase that phosphorylates FADD downstream of RAS, CK1α may be a therapeutic target for KRAS-driven lung cancer.
Assuntos
Caseína Quinase Ialfa/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Aurora Quinase A/metabolismo , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Primers do DNA/genética , Genótipo , Técnicas Histológicas , Imunoprecipitação , Medições Luminescentes , Espectrometria de Massas , Camundongos , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Microtomografia por Raio-X , Quinase 1 Polo-LikeRESUMO
PURPOSE: Ovarian cancer is the fifth leading cause of cancer-related deaths among American women. Platinum-based chemotherapy, such as cisplatin, represents the standard-of-care for ovarian cancer. However, toxicity and acquired resistance to cisplatin have proven challenging in the treatment of patients with ovarian cancer. EXPERIMENTAL DESIGN: Using a genetically engineered mouse model of ovarian endometrioid adenocarcinoma (OEA) in combination with molecular-imaging technologies, we studied the activation of the AKT serine/threonine kinase in response to long-term cisplatin therapy. RESULTS: Treatment of cells in culture and tumor-bearing animals with cisplatin resulted in activation of AKT, a key mediator of cell survival. On the basis of these results, we investigated the therapeutic use of AKT inhibition in combination with cisplatin, which resulted in enhanced and prolonged induction of apoptosis and in significantly improved tumor control as compared with either agent alone. CONCLUSION: These results provide an impetus for clinical trials using combination therapy. To facilitate these trials, we also show the use of diffusion-weighted MRI as an imaging biomarker for evaluation of therapeutic efficacy in OEA.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Endometrioide/metabolismo , Cisplatino/farmacologia , Imagem Molecular , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/genética , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Transplante Homólogo , Microambiente TumoralRESUMO
In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.