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1.
Scand J Immunol ; : e13391, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773691

RESUMO

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can be activated by microbial antigens and cytokines and are abundant in mucosal tissues including the colon. MAIT cells have cytotoxic and pro-inflammatory functions and have potentials for use as adoptive cell therapy. However, studies into their anti-cancer activity, including their role in colon cancer, are limited. Using an animal model of colon cancer, we showed that peritumoral injection of in vivo-expanded MAIT cells into RAG1-/- mice with MC38-derived tumours inhibits tumour growth compared to control. Multiplex cytokine analyses showed that tumours from the MAIT cell-treated group have higher expression of markers for eosinophil-activating cytokines, suggesting a potential association between eosinophil recruitment and tumour inhibition. In a human peripheral leukocyte co-culture model, we showed that leukocytes stimulated with MAIT ligand showed an increase in eotaxin-1 production and activation of eosinophils, associated with increased cancer cell killing. In conclusion, we showed that MAIT cells have a protective role in a murine colon cancer model, associated with modulation of the immune response to cancer, potentially involving eosinophil-associated mechanisms. Our results highlight the potential of MAIT cells for non-donor restricted colon cancer immunotherapy.

2.
Sci Adv ; 10(11): eadj6406, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38489355

RESUMO

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.


Assuntos
Mycobacterium tuberculosis , Neoplasias , Quinazolinas , Tiofenos , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Mycobacterium tuberculosis/metabolismo , Timidilato Sintase/metabolismo , Proteínas de Bactérias/metabolismo
3.
bioRxiv ; 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38293128

RESUMO

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can be activated by microbial antigens and cytokines and are abundant in mucosal tissues including the colon. MAIT cells have cytotoxic and pro-inflammatory functions and have potentials for use as adoptive cell therapy. However, studies into their anti-cancer activity, including their role in colon cancer, are limited. Using an animal model of colon cancer, we show that peritumoral injection of in vivo-expanded MAIT cells into RAG1-/- mice with MC38-derived tumors inhibits tumor growth compared to control. Multiplex cytokine analyses show that tumors from the MAIT cell-treated group have higher expression of markers for eosinophil-activating cytokines, suggesting an association between eosinophil recruitment and tumor inhibition. In a human peripheral leukocyte co-culture model, we show that leukocytes stimulated with MAIT ligand show an increase in eotaxin-1 production and activation of eosinophils, associated with increased cancer cell killing. In conclusion, we show that MAIT cells have a protective role in a murine colon cancer model, associated with modulation of the immune response to cancer, potentially involving eosinophil-associated mechanisms. Our results highlight the potential of MAIT cells for non-donor restricted colon cancer immunotherapy.

4.
bioRxiv ; 2023 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-37808753

RESUMO

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.

5.
J Transl Med ; 21(1): 428, 2023 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-37391777

RESUMO

BACKGROUND: Upregulation of an RNA-binding protein HuR has been implicated in glomerular diseases. Herein, we evaluated whether it is involved in renal tubular fibrosis. METHODS: HuR was firstly examined in human kidney biopsy tissue with tubular disease. Second, its expression and the effect of HuR inhibition with KH3 on tubular injury were further assessed in a mouse model induced by a unilateral renal ischemia/reperfusion (IR). KH3 (50 mg kg-1) was given daily via intraperitoneal injection from day 3 to 14 after IR. Last, one of HuR-targeted pathways was examined in cultured proximal tubular cells. RESULTS: HuR significantly increases at the site of tubular injury both in progressive CKD in patients and in IR-injured kidneys in mice, accompanied by upregulation of HuR targets that are involved in inflammation, profibrotic cytokines, oxidative stress, proliferation, apoptosis, tubular EMT process, matrix remodeling and fibrosis in renal tubulointerstitial fibrosis. KH3 treatment reduces the IR-induced tubular injury and fibrosis, accompanied by the remarkable amelioration in those involved pathways. A panel of mRNA array further revealed that 519 molecules in mouse kidney following IR injury changed their expression and 71.3% of them that are involved in 50 profibrotic pathways, were ameliorated when treated with KH3. In vitro, TGFß1 induced tubular HuR cytoplasmic translocation and subsequent tubular EMT, which were abrogated by KH3 administration in cultured HK-2 cells. CONCLUSIONS: These results suggest that excessive upregulation of HuR contributes to renal tubulointerstitial fibrosis by dysregulating genes involved in multiple profibrotic pathways and activating the TGFß1/HuR feedback circuit in tubular cells. Inhibition of HuR may have therapeutic potential for renal tubular fibrosis.


Assuntos
Nefropatias , Humanos , Animais , Camundongos , Rim , Apoptose , Citocinas , Citoplasma
6.
Mol Oncol ; 17(10): 1962-1980, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37357618

RESUMO

Chemotherapy remains the standard treatment for triple-negative breast cancer (TNBC); however, chemoresistance compromises its efficacy. The RNA-binding protein Hu antigen R (HuR) could be a potential therapeutic target to enhance the chemotherapy efficacy. HuR is known to mainly stabilize its target mRNAs, and/or promote the translation of encoded proteins, which are implicated in multiple cancer hallmarks, including chemoresistance. In this study, a docetaxel-resistant cell subline (231-TR) was established from the human TNBC cell line MDA-MB-231. Both the parental and resistant cell lines exhibited similar sensitivity to the small molecule functional inhibitor of HuR, KH-3. Docetaxel and KH-3 combination therapy synergistically inhibited cell proliferation in TNBC cells and tumor growth in three animal models. KH-3 downregulated the expression levels of HuR targets (e.g., ß-Catenin and BCL2) in a time- and dose-dependent manner. Moreover, KH-3 restored docetaxel's effects on activating Caspase-3 and cleaving PARP in 231-TR cells, induced apoptotic cell death, and caused S-phase cell cycle arrest. Together, our findings suggest that HuR is a critical mediator of docetaxel resistance and provide a rationale for combining HuR inhibitors and chemotherapeutic agents to enhance chemotherapy efficacy.


Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Humanos , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Docetaxel/farmacologia , Proteínas de Ligação a RNA , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
7.
J Med Chem ; 66(3): 2032-2053, 2023 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-36690437

RESUMO

The RNA-binding protein Hu antigen R (HuR) is a post-transcriptional regulator critical in several types of diseases, including cancer, making it a promising therapeutic target. We have identified small-molecule inhibitors of HuR through a screening approach used in combination with fragment analysis. A total of 36 new compounds originating from fragment linking or structural optimization were studied to establish structure-activity relationships in the set. Two top inhibitors, 1c and 7c, were further validated by binding assays and cellular functional assays. Both block HuR function by directly binding to the RNA-binding pocket, inhibit cancer cell growth dependence of HuR, and suppress cancer cell invasion. Intraperitoneal administration of inhibitor 1c inhibits tumor growth as a single agent and shows a synergistic effect in combination with chemotherapy docetaxel in breast cancer xenograft models. Mechanistically, 1c interferes with the HuR-TGFB/THBS1 axis.


Assuntos
Neoplasias , Humanos , Xenoenxertos , Transformação Celular Neoplásica , Linhagem Celular Tumoral
8.
Immunohorizons ; 7(1): 116-124, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36651819

RESUMO

Mucosal-associated invariant T (MAIT) cells are promising innate-like lymphocytes with potential for use in anti-tumor immunotherapy. Existing MAIT cell expansion protocols are associated with potentially decremental phenotypic changes, including increased frequency of CD4+ MAIT cells and higher inhibitory receptor expression. In this study, we compared the effect on expansion of human MAIT cells of a serum replacement, Physiologix XF SR (Phx), with traditional serum FBS for supplementing RPMI 1640 media. Using flow cytometry, we found that Phx supported a significantly higher proliferative capacity for MAIT cells and resulted in a lower frequency of CD4+ MAIT cells, which have been associated with reduced Th1 effector and cytolytic functions. We saw that culturing MAIT cells in Phx led to better survival of MAIT cells and lower frequency of PD-1+ MAIT cells than FBS-supplemented media. Functionally, we saw that Phx supplementation was associated with a higher frequency of IFN-γ+ MAIT cells after stimulation with Escherichia coli than FBS-supplemented RPMI. In conclusion, we show that MAIT cells cultured in Phx have higher proliferative capacity, lower expression of inhibitory receptors, and higher capacity to produce IFN-γ after E. coli stimulation than FBS-supplemented RPMI. This work shows that expanding MAIT cells with Phx compared with FBS-supplemented RPMI results in a more functionally desirable MAIT cell for future anti-tumor immunotherapy.


Assuntos
Células T Invariantes Associadas à Mucosa , Humanos , Células T Invariantes Associadas à Mucosa/metabolismo , Escherichia coli , Interferon gama/metabolismo , Citometria de Fluxo
9.
J Mol Cell Cardiol ; 174: 38-46, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36372279

RESUMO

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFß-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFß-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFß treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFß. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFß, in part through post-transcriptional regulation of Wisp1.


Assuntos
Proteínas de Sinalização Intercelular CCN , Proteína Semelhante a ELAV 1 , Miofibroblastos , Fator de Crescimento Transformador beta , Animais , Camundongos , Colágeno/metabolismo , Fibroblastos/metabolismo , Coração , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Proteínas de Sinalização Intercelular CCN/metabolismo
10.
J Med Chem ; 65(12): 8303-8331, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35696646

RESUMO

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure-activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial.


Assuntos
Núcleo Celular , Neoplasias , Biomarcadores/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Núcleo Celular/metabolismo , Humanos , Neoplasias/metabolismo , Pirimidinas , Pirróis
11.
J Immunol ; 208(5): 1042-1056, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35149530

RESUMO

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Mycobacterium tuberculosis/imunologia , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Granzimas/metabolismo , Homeostase/imunologia , Humanos , Interferon gama/metabolismo , Células T Invariantes Associadas à Mucosa/citologia , Receptores de Antígenos de Linfócitos T/imunologia , Ribitol/análogos & derivados , Ribitol/imunologia , Análise de Célula Única , Transcriptoma/genética , Fator de Necrose Tumoral alfa/metabolismo , Uracila/análogos & derivados , Uracila/imunologia
12.
Mol Cancer Ther ; 19(11): 2267-2277, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32879054

RESUMO

Pancreatic cancer has poor prognosis and treatment outcomes due to its highly metastatic nature and resistance to current treatments. The RNA-binding protein (RBP) Hu-antigen R (HuR) is a central player in posttranscriptional regulation of cancer-related gene expression, and contributes to tumorigenesis, tumor growth, metastasis, and drug resistance. HuR has been suggested to regulate pancreatic cancer epithelial-to-mesenchymal transition (EMT), but the mechanism was not well understood. Here, we further elucidated the role HuR plays in pancreatic cancer cell EMT, and developed a novel inhibitor specifically interrupting HuR-RNA binding. The data showed that HuR binds to the 3'-UTR of the mRNA of the transcription factor Snail, resulting in stabilization of Snail mRNA and enhanced Snail protein expression, thus promoted EMT, metastasis, and formation of stem-like cancer cells (CSC) in pancreatic cancer cells. siRNA silencing or CRISPR/Cas9 gene deletion of HuR inhibited pancreatic cancer cell EMT, migration, invasion, and inhibited CSCs. HuR knockout cells had dampened tumorigenicity in immunocompromised mice. A novel compound KH-3 interrupted HuR-RNA binding, and KH-3 inhibited pancreatic cancer cell viability, EMT, migration/invasion in vitro KH-3 showed HuR-dependent activity and inhibited HuR-positive tumor growth and metastasis in vivo.


Assuntos
Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Transição Epitelial-Mesenquimal/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Estabilidade de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Clin Sci (Lond) ; 134(12): 1433-1448, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32478392

RESUMO

Recent identification of an RNA-binding protein (HuR) that regulates mRNA turnover and translation of numerous transcripts via binding to an ARE in their 3'-UTR involved in inflammation and is abnormally elevated in varied kidney diseases offers a novel target for the treatment of renal inflammation and subsequent fibrosis. Thus, we hypothesized that treatment with a selective inhibition of HuR function with a small molecule, KH-3, would down-regulate HuR-targeted proinflammatory transcripts thereby improving glomerulosclerosis in experimental nephritis, where glomerular cellular HuR is elevated. Three experimental groups included normal and diseased rats treated with or without KH-3. Disease was induced by the monoclonal anti-Thy 1.1 antibody. KH-3 was given via daily intraperitoneal injection from day 1 after disease induction to day 5 at the dose of 50 mg/kg BW/day. At day 6, diseased animals treated with KH-3 showed significant reduction in glomerular HuR levels, proteinuria, podocyte injury determined by ameliorated podocyte loss and podocin expression, glomerular staining for periodic acid-Schiff positive extracellular matrix proteins, fibronectin and collagen IV and mRNA and protein levels of profibrotic markers, compared with untreated disease rats. KH-3 treatment also reduced disease-induced increases in renal TGFß1 and PAI-1 transcripts. Additionally, a marked increase in renal NF-κB-p65, Nox4, and glomerular macrophage cell infiltration observed in disease control group was largely reversed by KH-3 treatment. These results strongly support our hypothesis that down-regulation of HuR function with KH-3 has therapeutic potential for reversing glomerulosclerosis by reducing abundance of pro-inflammatory transcripts and related inflammation.


Assuntos
Proteína Semelhante a ELAV 1/antagonistas & inibidores , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Nefrite/metabolismo , Nefrite/patologia , Animais , Biomarcadores/metabolismo , Peso Corporal , Polaridade Celular , Colágeno/genética , Colágeno/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Humanos , Inflamação/patologia , Testes de Função Renal , Glomérulos Renais/fisiopatologia , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , NADPH Oxidase 4/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Antígenos Thy-1 , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
14.
Commun Biol ; 3(1): 193, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332873

RESUMO

Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is upregulated in breast cancer, and elevated cytoplasmic HuR correlates with high-grade tumors and poor clinical outcome of breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors, and cytokines that support major tumor hallmarks including invasion and metastasis. Here, we report a HuR inhibitor KH-3, which potently suppresses breast cancer cell growth and invasion. Furthermore, KH-3 inhibits breast cancer experimental lung metastasis, improves mouse survival, and reduces orthotopic tumor growth. Mechanistically, we identify FOXQ1 as a direct target of HuR. KH-3 disrupts HuR-FOXQ1 mRNA interaction, leading to inhibition of breast cancer invasion. Our study suggests that inhibiting HuR is a promising therapeutic strategy for lethal metastatic breast cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Pulmonares/prevenção & controle , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Med Chem ; 63(10): 5526-5567, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32342685

RESUMO

To identify novel D3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a ß-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 (20), potently promotes D3R-mediated ß-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.


Assuntos
Agonistas de Dopamina/química , Agonistas de Dopamina/farmacologia , Descoberta de Drogas/métodos , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/química , Animais , Células CHO , Cricetulus , Agonistas de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Secundária de Proteína , Receptores de Dopamina D3/metabolismo
16.
Cancers (Basel) ; 11(12)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847141

RESUMO

Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), ARID1A enhances DNA repair. Consequently, ARID1A has been proposed as a promising therapeutic target to sensitize cancer cells to chemotherapy and radiation. Here, we report that ARID1A is regulated by human antigen R (HuR), an RNA-binding protein that is highly expressed in a wide range of cancers and enables resistance to chemotherapy and radiation. Our results indicate that HuR binds ARID1A mRNA, thereby increasing its stability in breast cancer cells. We further find that ARID1A expression suppresses the accumulation of DNA double-strand breaks (DSBs) caused by radiation and can rescue the loss of radioresistance triggered by HuR inhibition, suggesting that ARID1A plays an important role in HuR-driven resistance to radiation. Taken together, our work shows that HuR and ARID1A form an important regulatory axis in radiation resistance that can be targeted to improve radiotherapy in breast cancer patients.

17.
Mol Cancer Ther ; 18(11): 2097-2110, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31395684

RESUMO

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell-like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine-based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.


Assuntos
Imidazóis/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/enzimologia , Neoplasias Pancreáticas/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Clin Invest ; 129(10): 4316-4331, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31310587

RESUMO

Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates interferon γ (IFNγ)-JAK-STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration and cancer immunity. IFNγ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased TH1-type chemokines and tumor-infiltrating lymphocytes (TILs) and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and TILs, enabling tumors to evade anti-tumor immunity and to resist anti-PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its co-factor, ascorbate/vitamin C, increased chemokine and TILs, leading to enhanced anti-tumor immunity and anti-PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFNγ-JAK-STAT-TET signaling pathway that mediates tumor response to anti-PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy and patient response to anti-PD-1/PD-L1 therapy, and stimulating TET activity as an adjuvant immunotherapy of solid tumors.


Assuntos
Proteínas de Ligação a DNA/imunologia , Imunoterapia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Proteínas Proto-Oncogênicas/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Proteínas de Ligação a DNA/genética , Dioxigenases , Humanos , Interferon gama/genética , Interferon gama/imunologia , Linfócitos do Interstício Tumoral/patologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células THP-1 , Células Th1/imunologia , Células Th1/patologia , Proteínas Supressoras de Tumor/genética
19.
ACS Infect Dis ; 5(8): 1433-1445, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31184461

RESUMO

The historical view of ß-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a ß-lactamase inhibitor. However, most antimycobacterial ß-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 ß-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a ß-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A ß-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent ß-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.


Assuntos
Antituberculosos/farmacologia , Cefalosporinas/farmacologia , Replicação do DNA , Mycobacterium tuberculosis/efeitos dos fármacos , Piridinas/farmacologia , Tionas/farmacologia , Administração Oral , Animais , Antituberculosos/administração & dosagem , Callithrix , Cefalosporinas/administração & dosagem , Descoberta de Drogas , Feminino , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mycobacterium tuberculosis/fisiologia , Piridinas/administração & dosagem , Tionas/administração & dosagem
20.
Sci Rep ; 9(1): 7374, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089242

RESUMO

Hu antigen R (HuR) is indeed one of the most studied RNA-binding protein (RBP) since its fundamental role both in tumorigenesis and cancer progression. For this reason, downregulation in HuR protein levels or inhibition of HuR biological function are, nowadays, attractive goals in cancer research. Here, we examined the antitumor effects of CMLD-2 in four thyroid cancer cell lines (SW1736, 8505 C, BCPAP and K1). Indeed, CMLD-2 competitively binds HuR protein disrupting its interaction with RNA-targets. 35 µM CLMD-2 produced a significant downregulation in thyroid cancer cell viability, coupled to an increase in apoptosis. Moreover, CMLD-2 treatment hindered both migration and colony formation ability. MAD2 is a microtubules-associated protein known to be greatly overexpressed in cancer and correlating with tumor aggressiveness. Furthermore, MAD2 is known to be a HuR target. CMLD-2 treatment induced a strong MAD2 downregulation and rescue experiments depicted it as a key effector in HuR-mediated in cancer. Altogether, these data contributed to foster HuR inhibition as valid antineoplastic treatment in thyroid cancer, highlighting MAD2 as a novel therapeutic target.


Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Mad2/genética , Pirrolidinas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Antineoplásicos/uso terapêutico , Benzopiranos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteína Semelhante a ELAV 1/metabolismo , Humanos , Pirrolidinas/uso terapêutico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia
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