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1.
BMC Biol ; 22(1): 176, 2024 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-39183304

RESUMO

BACKGROUND: Casein kinase 1α (CK1α), expressed in both ovarian germ and somatic cells, is involved in the initial meiosis and primordial follicle formation of mouse oocytes. Using in vitro and in vivo experiments in this study, we explored the function and mechanism of CK1α in estrogen synthesis in mice ovarian granulosa cells. METHODS: A CK1α knockout (cKO) mouse model, targeted specifically to ovarian granulosa cells (GCs), was employed to establish the influence of CK1α on in vivo estrogen synthesis. The influence of CK1α deficiency on GCs was determined in vivo and in vitro by immunofluorescence analysis and Western blot assay. Transcriptome profiling, differentially expressed genes and gene functional enrichment analyses, and computation protein-protein docking, were further employed to assess the CK1α pathway. Furthermore, wild-type female mice were treated with the CK1α antagonist D4476 to elucidate the CK1α's role in estrogen regulation. RESULTS: Ovarian GCs CK1α deficiency impaired fertility and superovulation of female mice; also, the average litter size and the estradiol (E2) level in the serum of cKO female mice were decreased by 57.3% and 87.4% vs. control mice, respectively. This deficiency disrupted the estrous cycle and enhanced the apoptosis in the GCs. We observed that CK1α mediated the secretion of estradiol in mouse ovarian GCs via the cytochrome P450 subfamily 19 member 1 (CYP19A1). CONCLUSIONS: These findings improve the existing understanding of the regulation mechanism of female reproduction and estrogen synthesis. TRIAL REGISTRATION: Not applicable.


Assuntos
Aromatase , Estradiol , Células da Granulosa , Camundongos Knockout , Animais , Feminino , Camundongos , Aromatase/metabolismo , Aromatase/genética , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/genética , Estradiol/metabolismo , Células da Granulosa/metabolismo
2.
Open Biol ; 14(7): 240075, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39043225

RESUMO

Palmoplantar keratoderma (PPK) is a multi-faceted skin disorder characterized by the thickening of the epidermis and abrasions on the palms and soles of the feet. Among the genetic causes, biallelic pathogenic variants in the FAM83G gene have been associated with PPK in dogs and humans. Here, a novel homozygous variant (c.794G>C, p.Arg265Pro) in the FAM83G gene, identified by whole exome sequencing in a 60-year-old female patient with PPK, is reported. The patient exhibited alterations in the skin of both hands and feet, dystrophic nails, thin, curly and sparse hair, long upper eyelid eyelashes, and poor dental enamel. FAM83G activates WNT signalling through association with ser/thr protein kinase CK1α. When expressed in FAM83G-/- DLD1 colorectal cancer cells, the FAM83GR265P variant displayed poor stability, a loss of interaction with CK1α and attenuated WNT signalling response. These defects persisted in skin fibroblast cells derived from the patient. Our findings imply that the loss of FAM83G-CK1α interaction and subsequent attenuation of WNT signalling underlie the pathogenesis of PPK caused by the FAM83GR265P variant.


Assuntos
Caseína Quinase Ialfa , Ceratodermia Palmar e Plantar , Via de Sinalização Wnt , Humanos , Feminino , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/patologia , Pessoa de Meia-Idade , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/genética , Sequenciamento do Exoma , Ligação Proteica , Fibroblastos/metabolismo
3.
Biochem Biophys Res Commun ; 723: 150189, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-38852281

RESUMO

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.


Assuntos
Processamento Alternativo , Caseína Quinase Ialfa , Animais , Humanos , Camundongos , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Proliferação de Células , Células HCT116 , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia
4.
mBio ; 15(8): e0111724, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-38940554

RESUMO

Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an essential viral DNA replication protein, maintains viral persistence by interacting with host Skp1-Cullin 1-F-box (SCF) E3 ubiquitin ligase complexes, which subsequently induces LT's proteasomal degradation, restricting MCPyV DNA replication. SCF E3 ubiquitin ligases require their substrates to be phosphorylated to bind them, utilizing phosphorylated serine residues as docking sites. The MCPyV LT unique region (MUR) is highly phosphorylated and plays a role in multiple host protein interactions, including SCF E3 ubiquitin ligases. Therefore, this domain highly governs LT stability. Though much work has been conducted to identify host factors that restrict MCPyV LT protein expression, the kinase(s) that cooperates with the SCF E3 ligase remains unknown. Here, we demonstrate that casein kinase 1 alpha (CK1α) negatively regulates MCPyV LT stability and LT-mediated replication by modulating interactions with the SCF ß-TrCP. Specifically, we show that numerous CK1 isoforms (α, δ, ε) localize in close proximity to MCPyV LT through in situ proximity ligation assays (PLA) and CK1α overexpression mainly resulted in decreased MCPyV LT protein expression. Inhibition of CK1α using short hairpin RNA (shRNA) and treatment of a CK1α inhibitor or an mTOR inhibitor, TORKinib, resulted in decreased ß-TrCP interaction with LT, increased LT expression, and enhanced MCPyV replication. The expression level of the CSNK1A1 gene transcripts is higher in MCPyV-positive MCC, suggesting a vital role of CK1α in limiting MCPyV replication required for establishing persistent infection. IMPORTANCE: Merkel cell polyomavirus (MCPyV) large tumor antigen is a polyphosphoprotein and the phosphorylation event is required to modulate various functions of LT, including viral replication. Therefore, cellular kinase pathways are indispensable for governing MCPyV polyomavirus infection and life cycle in coordinating with the immunosuppression environment at disease onset. Understanding the regulation mechanisms of MCPyV replication by viral and cellular factors will guide proper prevention strategies with targeted inhibitors for MCPyV-associated Merkel cell carcinoma (MCC) patients, who currently lack therapies.


Assuntos
Antígenos Virais de Tumores , Caseína Quinase Ialfa , Poliomavírus das Células de Merkel , Proteínas Contendo Repetições de beta-Transducina , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/metabolismo , Humanos , Fosforilação , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Antígenos Virais de Tumores/metabolismo , Antígenos Virais de Tumores/genética , Interações Hospedeiro-Patógeno , Proteólise , Replicação Viral , Ligação Proteica , Antígenos Transformantes de Poliomavirus/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Infecções por Polyomavirus/virologia , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/genética
5.
Cancer Cell ; 41(4): 726-739.e11, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-36898380

RESUMO

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.


Assuntos
Caseína Quinase Ialfa , Leucemia Mieloide Aguda , Animais , Humanos , Camundongos , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Hematopoese , Fator de Transcrição Ikaros/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Fatores de Transcrição
6.
Am J Pathol ; 191(12): 2195-2202, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34809787

RESUMO

The present study aimed to explore the roles of casein kinase 1α (CK1α) in endometriosis and its underlying mechanisms. Endometrial specimen were collected from the patients and healthy volunteers. The expression patterns of CK1α, phosphatase and tensin homolog (PTEN), and autophagy-related proteins were determined using immunohistochemistry staining, Western blot analysis, and quantitative RT-PCR. Besides, the CK1α-overexpressing cells and PTEN knockdown cells were constructed in the endometrial stromal cells isolated from endometriosis patients. In addition, the cells were transfected with pcDNA3.1-CK1α or pcDNA3.1-CK1α plus siRNA- PTEN. The expressions of CK1α, PTEN, and autophagy-related proteins were determined using Western blot and quantitative RT-PCR. The expressions of CK1α and autophagy-related 7 (Atg7) were significantly decreased in the ectopic endometrium compared with the eutopic endometrium. Spearman rank correlation analysis revealed positive correlations between CK1α and PTEN, CK1α and Atg7, and PTEN and Atg7. In addition, CK1α, PTEN, and autophagy-related proteins were down-regulated in ectopic endometrium. Interestingly, overexpression of CK1α significantly increased the expressions of autophagy-related proteins, whereas the protein expression of autophagy-related proteins was decreased with PTEN knock-down. CK1α regulated PTEN/Atg7-mediated autophagy in endometriosis.


Assuntos
Autofagia/fisiologia , Caseína Quinase Ialfa/genética , Endometriose/genética , Doenças Uterinas/genética , Adulto , Autofagia/genética , Proteína 7 Relacionada à Autofagia/fisiologia , Estudos de Casos e Controles , Caseína Quinase Ialfa/fisiologia , Regulação para Baixo/genética , Endometriose/patologia , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , PTEN Fosfo-Hidrolase/fisiologia , Transdução de Sinais/genética , Doenças Uterinas/patologia , Adulto Jovem
7.
J Cell Mol Med ; 25(15): 7395-7406, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34216174

RESUMO

Glioblastoma multiforme (GBM), a fatal brain tumour with no available targeted therapies, has a poor prognosis. At present, radiotherapy is one of the main methods to treat glioma, but it leads to an obvious increase in inflammatory factors in the tumour microenvironment, especially IL-6 and CXCL1, which plays a role in tumour to resistance radiotherapy and tumorigenesis. Casein kinase 1 alpha 1 (CK1α) (encoded on chromosome 5q by Csnk1a1) is considered an attractive target for Tp53 wild-type acute myeloid leukaemia (AML) treatment. In this study, we evaluated the anti-tumour effect of Csnk1a1 suppression in GBM cells in vitro and in vivo. We found that down-regulation of Csnk1a1 or inhibition by D4476, a Csnk1a1 inhibitor, reduced GBM cell proliferation efficiently in both Tp53 wild-type and Tp53-mutant GBM cells. On the contrary, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Csnk1a1 inhibition improved the sensitivity to radiotherapy. Furthermore, down-regulation of Csnk1a1 reduced the production and secretion of pro-inflammatory factors. In the preclinical GBM model, treatment with D4476 significantly inhibited the increase in pro-inflammatory factors caused by radiotherapy and improved radiotherapy sensitivity, thus inhibiting tumour growth and prolonging animal survival time. These results suggest targeting Csnk1a1 exert an anti-tumour role as an inhibitor of inflammatory factors, providing a new strategy for the treatment of glioma.


Assuntos
Neoplasias Encefálicas/metabolismo , Caseína Quinase Ialfa/metabolismo , Glioma/metabolismo , Tolerância a Radiação , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Caseína Quinase Ialfa/antagonistas & inibidores , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Glioma/patologia , Glioma/radioterapia , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Supressora de Tumor p53/genética
8.
Oncol Rep ; 44(5): 1895-1904, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32901886

RESUMO

Enhancement of autophagy serves as a promising therapeutic strategy for cancer, including acute myeloid leukemia (AML). Casein kinase 1α (CK1α), encoded by CSNK1A1, regulates Wnt/ß­catenin, p53 and other key signaling pathways, and is critically involved in tumor progression. However, the relationship and mechanism of CK1α with autophagy in AML still remain unclear. In the present study, it was found that AML patients had higher expression of CSNK1A1 mRNA than healthy donors. Furthermore, we analyzed 163 cases of AML patients in the LAML database of TCGA and found that AML patients with high CSNK1A1 had shorter overall survival than those with low or medium CSNK1A1 expression. Furthermore, we demonstrated that CK1α was a negative regulator of autophagy and apoptosis. Pharmacologic inhibition of CK1α using D4476 or CK1α knockdown via lentivirus­mediated shRNA suppressed proliferation and the clone formation by enhancing autophagic flux and apoptosis in AML cell lines as well as in patient blast cells. Intriguingly, D4476­induced cell death was aggravated in combination with an autophagy inhibitor, Spautin­1, suggesting that autophagy may be a pro­survival signaling. CK1α interacted with murine double minute 2 (MDM2) and p53, and CK1α inhibitor D4476 significantly upregulated p53 and phosphorylated 5' AMP­activated protein kinase (AMPK), and substantially inhibited the phosphorylation of mammalian target of rapamycin (mTOR). Our findings indicate that CK1α promotes AML by suppressing p53 downstream of MDM2­mediated autophagy and apoptosis, suggesting that targeting CK1α provides a therapeutic opportunity to treat AML.


Assuntos
Caseína Quinase Ialfa/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Apoptose/fisiologia , Autofagia/fisiologia , Benzamidas/farmacologia , Caseína Quinase Ialfa/antagonistas & inibidores , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Humanos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
9.
Int J Mol Sci ; 21(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824859

RESUMO

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators.


Assuntos
Caseína Quinase Ialfa/metabolismo , Neoplasias/metabolismo , Via de Sinalização Wnt , Animais , Antineoplásicos/uso terapêutico , Caseína Quinase Ialfa/genética , Ativadores de Enzimas/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico
10.
Biochem J ; 477(18): 3583-3598, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32686824

RESUMO

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Human fetal, but not adult, livers express appreciable amounts of SULT1E1 protein, which is mimicked in human hepatoma-derived HepG2 cells cultured in high glucose (450 mg/dl) medium. Here, we have investigated this glucose signal that leads to phosphorylation of nuclear receptor RORα (NR1F1) at Ser100 and the transcription mechanism by which phosphorylated RORα transduces this signal to nuclear receptor HNF4α, activating the SULT1E1 promoter. The promoter is repressed by non-phosphorylated RORα which binds a distal enhancer (-943/-922 bp) and interacts with and represses HNF4α-mediated transcription. In response to high glucose, RORα becomes phosphorylated at Ser100 and reverses its repression of HNF4α promoter activation. Moreover, the casein kinase CK1α, which is identified in an enhancer-bound nuclear protein complex, phosphorylates Ser100 in in vitro kinase assays. During these dynamic processes, both RORα and HNF4α remain on the enhancer. Thus, RORα utilizes phosphorylation to integrate HNF4α and transduces the glucose signal to regulate the SULT1E1 gene in HepG2 cells and this phosphorylation-mediated mechanism may also regulate SULT1E1 expressions in the human liver.


Assuntos
Caseína Quinase Ialfa/metabolismo , Estrogênios/metabolismo , Glucose/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Transdução de Sinais , Sulfotransferases/metabolismo , Animais , Células COS , Caseína Quinase Ialfa/genética , Chlorocebus aethiops , Estrogênios/genética , Glucose/genética , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Fosforilação , Sulfotransferases/genética
11.
EMBO J ; 39(14): e104410, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32511789

RESUMO

Casein kinase 1 alpha (CK1α) is a serine/threonine kinase with numerous functions, including regulating the Wnt/ß-catenin and p53 pathways. CK1α has a well-established role in inhibiting the p53 tumor suppressor by binding to MDMX and stimulating MDMX-p53 interaction. MDMX purified from cells contains near-stoichiometric amounts of CK1α, suggesting that MDMX may in turn regulate CK1α function. We present evidence that MDMX is a potent competitive inhibitor of CK1α kinase activity (Ki  = 8 nM). Depletion of MDMX increases CK1α activity and ß-catenin S45 phosphorylation, whereas ectopic MDMX expression inhibits CK1α activity and ß-catenin phosphorylation. The MDMX acidic domain and zinc finger are necessary and sufficient for binding and inhibition of CK1α. P53 binding to MDMX disrupts an intramolecular auto-regulatory interaction and enhances its ability to inhibit CK1α. P53-null mice expressing the MDMXW200S/W201G mutant, defective in CK1α binding, exhibit reduced Wnt/ß-catenin target gene expression and delayed tumor development. Therefore, MDMX is a physiological inhibitor of CK1α and has a role in modulating cellular response to Wnt signaling. The MDMX-CK1α interaction may account for certain p53-independent functions of MDMX.


Assuntos
Caseína Quinase Ialfa/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Via de Sinalização Wnt , Células A549 , Animais , Caseína Quinase Ialfa/genética , Proteínas de Ciclo Celular/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
12.
Nat Commun ; 11(1): 1141, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111827

RESUMO

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.


Assuntos
Caseína Quinase Ialfa/metabolismo , Ligases/antagonistas & inibidores , Ligases/metabolismo , Osteossarcoma/patologia , Proteínas do Grupo Polycomb/antagonistas & inibidores , Proteínas do Grupo Polycomb/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligases/genética , Camundongos , Mutação , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas do Grupo Polycomb/genética , Regiões Promotoras Genéticas , Compostos de Pirvínio/farmacologia , Compostos de Pirvínio/uso terapêutico , Análise de Sobrevida , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
13.
Oncogene ; 39(1): 176-186, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31462704

RESUMO

Somatic missense mutations of the CSNK1A1 gene encoding casein kinase 1 alpha (CK1α) occur in a subset of myelodysplastic syndrome (MDS) with del(5q) karyotype. The chromosomal deletion causes CSNK1A1 haplo-insufficiency. CK1α mutations have also been observed in a variety of solid and hematopoietic tumors at low frequency. The functional consequence of CK1α mutation remains unknown. Here we show that tumor-associated CK1α mutations exclusively localize to the substrate-binding cleft. Functional analysis of recurrent mutants E98K and D140A revealed enhanced binding to the p53 inhibitor MDMX, increased ability to stimulate MDMX-p53 binding, and increased suppression of p21 expression. Furthermore, E98K and D140A mutants have reduced ability to promote phosphorylation of ß-catenin, resulting in enhanced Wnt signaling. The results suggest that the CK1α mutations observed in tumors cause gain-of-function in cooperating with MDMX and inhibiting p53, and partial loss-of-function in suppressing Wnt signaling. These functional changes may promote expansion of abnormal myeloid progenitors in del(5q) MDS, and in rare cases drive the progression of other tumors.


Assuntos
Caseína Quinase Ialfa/genética , Síndromes Mielodisplásicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Deleção Cromossômica , Haploinsuficiência/genética , Xenoenxertos , Humanos , Camundongos , Mutação de Sentido Incorreto/genética , Síndromes Mielodisplásicas/patologia , Fosforilação/genética , Ligação Proteica/genética , Via de Sinalização Wnt/genética , beta Catenina/genética
14.
Anticancer Drugs ; 30(7): e0747, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31305293

RESUMO

Pyrvinium tosylate (PT) is an anthelminthic drug that has recently been shown to suppress various human cancers. However, whether PT is effective in nasopharyngeal carcinoma (NPC) has not been determined to date. In this work, we show the selective efficacy of PT in NPC while sparing normal nasopharyngeal epithelial cells, and its ability to increase chemosensitivity. We show that PT at 100 and 500 nmol/l significantly inhibits growth and induces apoptosis in several NPC cell lines without affecting normal nasopharyngeal epithelial cells. Using cell culture and xenograft mouse models, PT markedly enhances cisplatin's efficacy in NPC and the combination leads to almost complete tumor inhibition. Mechanism studies show that PT suppresses active, nuclear ß-catenin level and activity and increases Axin level in NPC cells. ß-Catenin overexpression completely reverses the inhibitory effects of PT, confirming that ß-catenin is the molecular mechanism of PT's action in NPC. In addition, the effects of PT on ß-catenin and Axin levels and on Wnt signaling in NPC cells are mediated by its activation of casine kinase 1α. Our work is the first to suggest that Wnt/ß-catenin is a selective target for NPC treatment, and provides the preclinical evidence on the translational potential of PT as a useful addition to the treatment armamentarium for NPC.


Assuntos
Caseína Quinase Ialfa/metabolismo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Carcinoma Nasofaríngeo/tratamento farmacológico , Compostos de Pirvínio/farmacologia , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Caseína Quinase Ialfa/genética , Proliferação de Células , Cisplatino/farmacologia , Quimioterapia Combinada , Humanos , Camundongos , Camundongos SCID , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Autophagy ; 15(7): 1130-1149, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30686098

RESUMO

UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle's balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1.


Assuntos
Autofagossomos/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Motivos de Aminoácidos/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Enzimas Desubiquitinantes/metabolismo , Endopeptidases , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Fosforilação , Prognóstico , Processamento de Proteína Pós-Traducional/genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação/genética
16.
Clin Cancer Res ; 25(4): 1379-1388, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487124

RESUMO

PURPOSE: Although most children with medulloblastoma are cured of their disease, Sonic Hedgehog (SHH) subgroup medulloblastoma driven by TRP53 mutations is essentially lethal. Casein kinase 1α (CK1α) phosphorylates and destabilizes GLI transcription factors, thereby inhibiting the key effectors of SHH signaling. We therefore tested a second-generation CK1α activator against TRP53-mutant, MYCN-amplified medulloblastoma. EXPERIMENTAL DESIGN: The ability of this CK1α activator to block SHH signaling was determined in vitro using GLI reporter cells, granular precursor primary cultures, and PATCHED1 (PTCH1)-mutant sphere cultures. While in vivo efficacy was tested using 2 different medulloblastoma mouse models: PTCH1 and ND2:SMOA1. Finally, the clinical relevance of CK1α activators was demonstrated using a TRP53-mutant, MYCN-amplified patient-derived xenograft. RESULTS: SSTC3 inhibited SHH activity in vitro, acting downstream of the vismodegib target SMOOTHENED (SMO), and reduced the viability of sphere cultures derived from SHH medulloblastoma. SSTC3 accumulated in the brain, inhibited growth of SHH medulloblastoma tumors, and blocked metastases in a genetically engineered vismodegib-resistant mouse model of SHH medulloblastoma. Importantly, SSTC3 attenuated growth and metastasis of orthotopic patient-derived TRP53-mutant, MYCN-amplified, SHH subgroup medulloblastoma xenografts, increasing overall survival. CONCLUSIONS: Using a newly described small-molecule, SSTC3, we show that CK1a activators could address a significant unmet clinical need for patients with SMO inhibitor-resistant medulloblastoma, including those harboring mutations in TRP53.


Assuntos
Benzoatos/farmacologia , Caseína Quinase Ialfa/genética , Meduloblastoma/tratamento farmacológico , Receptor Smoothened/genética , Anilidas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Xenoenxertos , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Metástase Neoplásica , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína GLI1 em Dedos de Zinco/genética
17.
Blood ; 132(6): 577-586, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-29954751

RESUMO

Primary effusion lymphoma (PEL) is an aggressive cancer with few treatment options. The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been shown to kill PEL cell lines, and lenalidomide is in clinical trials against PEL. IMiDs bind to the CRL4CRBN E3 ubiquitin ligase complex, leading to the acquisition of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), casein kinase 1 α (CK1α), and zinc finger protein 91 (ZFP91) as neosubstrates. IMiDs are effective against multiple myeloma because of degradation of IKZF1 and IKZF3 and the consequent loss of interferon regulatory factor 4 (IRF4) and MYC expression. Lenalidomide is also effective in chromosome 5q deletion-associated myelodysplastic syndrome as a result of degradation of CK1α. An essential IKZF1-IRF4-MYC axis has recently been proposed to underlie the toxicity of IMiDs in PEL. Here, we further investigate IMiD effectors in PEL cell lines, based on genome-wide CRISPR/Cas9 screens for essential human genes. These screens and extensive validation experiments show that, of the 4 neosubstrates, only CK1α is essential for the survival of PEL cell lines. In contrast, IKZF1 and IKZF3 are dispensable, individually or in combination. IRF4 was critical in all 8 PEL cell lines tested, and surprisingly, IMiDs triggered downregulation of IRF4 expression independently of both IKZF1 and IKZF3. Reexpression of CK1α and/or IRF4 partially rescued PEL cell lines from IMiD-mediated toxicity. In conclusion, IMiD toxicity in PEL cell lines is independent of IKZF1 and IKZF3 but proceeds through degradation of the neosubstrate CK1α and downregulation of IRF4.


Assuntos
Caseína Quinase Ialfa/fisiologia , Fatores Imunológicos/farmacologia , Fatores Reguladores de Interferon/fisiologia , Lenalidomida/farmacologia , Linfoma de Efusão Primária/tratamento farmacológico , Proteínas de Neoplasias/fisiologia , Talidomida/análogos & derivados , Sistemas CRISPR-Cas , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Fator de Transcrição Ikaros/fisiologia , Fatores Imunológicos/uso terapêutico , Fatores Reguladores de Interferon/biossíntese , Fatores Reguladores de Interferon/genética , Lenalidomida/uso terapêutico , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/metabolismo , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais , Talidomida/farmacologia , Talidomida/uso terapêutico , Ubiquitina-Proteína Ligases/fisiologia
18.
Mol Med Rep ; 17(6): 7559-7566, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29620268

RESUMO

Hepatitis C virus (HCV)­infected liver cells sensitize host cells to tumor necrosis factor (TNF)­related apoptosis­inducing ligand (TRAIL)­induced cell apoptosis; however, the precise mechanisms are unknown. In the present study, flow cytometry demonstrated that the Annexin V­positive Huh­7 cell number was higher in groups transfected with core proteins when compared with the pcDNA3.1 group. The mRNA and protein expression levels of B­cell lymphoma 2 (Bcl­2) were negatively associated, while Bcl­2­associated X protein (Bax) were positively correlated, with cell apoptotic rate, which, were verified by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. There were no significant differences in the expressions of casein kinase 1 (CK1)­Îµ, CK1γ or CK1δ; however, the mRNA and protein levels of CK1α were markedly higher in groups transfected with the T (those derived from the HCV­J6 strain), NT (those derived from non­tumor tissues) and C191 (those derived from tumor tissues) HCV core proteins than in mock group. When compared with the Mock and Negative Control (control known­down) groups, the mRNA and protein levels of CK1α were lower in the CK1α known­down group, and there were no marked Huh­7 cell morphological changes among the 3 groups. There was more sensitivity to cell apoptosis in CK1α­silenced, however, not in non­CK1α­silenced, Huh­7 cells. BH3 interacting­domain death agonist (Bid) protein levels in CK1α­silenced Huh­7 cells were higher when compared with non­CK1α­silenced Huh­7 cells, and the level of p53 that translocated to the nucleus increased. Chromatin immunoprecipitation­PCR demonstrated that p53 bound to human Bid gene promoter. The level of the Bid promoter in CK1α­silenced Huh­7 cells was significantly higher than in the non­CK1α­silenced Huh­7 cells. Electron microscopy indicated that p53 knockdown decreased HCV core protein and TRAIL­induced cell apoptosis. Bid/caspase­8 protein levels in CK1α­silenced Huh­7 cells that were transfected with p53 siRNA were lower than in the control group. The present study demonstrated that HCV core proteins sensitize host cells to TRAIL­induced cell apoptosis by activating the CK1α­p53­Bid dependent pathway.


Assuntos
Apoptose , Caseína Quinase Ialfa/metabolismo , Hepacivirus/fisiologia , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteínas do Core Viral/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
19.
Am J Physiol Renal Physiol ; 315(1): F57-F73, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29537311

RESUMO

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.


Assuntos
Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase Ialfa/antagonistas & inibidores , Rim/efeitos dos fármacos , Doenças Renais Policísticas/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Piridinas/farmacologia , Roscovitina/farmacologia , Animais , Caseína Quinase 1 épsilon/genética , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Catálise , Cromatografia de Afinidade/métodos , Modelos Animais de Doenças , Humanos , Rim/enzimologia , Rim/patologia , Camundongos Transgênicos , Doenças Renais Policísticas/enzimologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Purinas/metabolismo , Piridinas/metabolismo , Roscovitina/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Nat Cell Biol ; 20(4): 465-478, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29593330

RESUMO

The contribution of autophagy to cancer development remains controversial, largely owing to the fact that autophagy can be tumour suppressive or oncogenic in different biological contexts. Here, we show that in non-small-cell lung cancer (NSCLC), casein kinase 1 alpha 1 (CK1α) suppresses tumour growth by functioning as an autophagy inducer to activate an autophagy-regulating, tumour-suppressive PTEN/AKT/FOXO3a/Atg7 axis. Specifically, CK1α bound the C-terminal tail of PTEN and enhanced both PTEN stability and activity by competitively antagonizing NEDD4-1-induced PTEN polyubiquitination and abrogating PTEN phosphorylation, thereby inhibiting AKT activity and activating FOXO3a-induced transcription of Atg7. Notably, blocking CK1α-induced Atg7-dependent autophagy cooperates with oncogenic HRasV12 to initiate tumorigenesis of lung epithelial cells. An association of a CK1α-modulated autophagic program with the anti-neoplastic activities of the CK1α/PTEN/FOXO3a/Atg7 axis was demonstrated in xenografted tumour models and human NSCLC specimens. This provides insights into the biological and potentially clinical significance of autophagy in NSCLC.


Assuntos
Autofagia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caseína Quinase Ialfa/metabolismo , Proliferação de Células , Neoplasias Pulmonares/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Células A549 , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Caseína Quinase Ialfa/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Estabilidade Enzimática , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes ras , Células HCT116 , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Ubiquitina-Proteína Ligases Nedd4/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Tempo , Carga Tumoral , Ubiquitinação
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