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1.
Oncogene ; 39(49): 7196-7208, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33037408

RESUMO

Metastasis is responsible for the death of most breast cancer patients. Robo1 has been implicated as a tumor suppressor for various cancers including breast cancer. However, it is not well understood how Robo1 expression is regulated during tumorigenesis. In this study, we uncovered that the transmembrane proline rich γ-carboxyglutamic acid protein 4 (PRRG4) promotes breast cancer metastasis by downregulating Robo1. Analysis of mRNA expression data in The Cancer Genome Atlas and immunohistochemistry assay on breast tumor samples showed that PRRG4 expression was higher in breast tumors than in normal breast tissues. Experiments with PRRG4 knockdown and overexpression revealed that PRRG4 promoted migration and invasion of breast cancer cells, and enhanced metastasis in an experimental metastasis model. Mechanistically, we found that PRRG4 via its LPSY and PPPY motifs recruited the E3 ubiquitin ligase NEDD4, which induced ubiquitination and degradation of Robo1, thus contributing to migration and invasion of breast cancer cells. In addition, PRRG4 interacted with and enhanced protein tyrosine kinase Src and FAK activation. Overall, our data support a model that PRRG4 via NEDD4 downregulates the Robo1, resulting in the activation of Src and FAK and promoting breast cancer metastasis. PRRG4 may be a novel target for treating metastatic breast cancer.

3.
J Biol Chem ; 295(40): 13838-13849, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-32753484

RESUMO

The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC50 of ∼2.41 µm in vitro Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.

4.
J Transl Med ; 18(1): 321, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831104

RESUMO

BACKGROUND: The outbreak of coronavirus disease (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its surface spike glycoprotein (S-protein) recognition on the receptor Angiotensin-converting enzyme 2 (ACE2) in humans. However, it remains unclear how genetic variations in ACE2 may affect its function and structure, and consequently alter the recognition by SARS-CoV-2. METHODS: We have systemically characterized missense variants in the gene ACE2 using data from the Genome Aggregation Database (gnomAD; N = 141,456). To investigate the putative deleterious role of missense variants, six existing functional prediction tools were applied to evaluate their impact. We further analyzed the structural flexibility of ACE2 and its protein-protein interface with the S-protein of SARS-CoV-2 using our developed Legion Interfaces Analysis (LiAn) program. RESULTS: Here, we characterized a total of 12 ACE2 putative deleterious missense variants. Of those 12 variants, we further showed that p.His378Arg could directly weaken the binding of catalytic metal atom to decrease ACE2 activity and p.Ser19Pro could distort the most important helix to the S-protein. Another seven missense variants may affect secondary structures (i.e. p.Gly211Arg; p.Asp206Gly; p.Arg219Cys; p.Arg219His, p.Lys341Arg, p.Ile468Val, and p.Ser547Cys), whereas p.Ile468Val with AF = 0.01 is only present in Asian. CONCLUSIONS: We provide strong evidence of putative deleterious missense variants in ACE2 that are present in specific populations, which could disrupt the function and structure of ACE2. These findings provide novel insight into the genetic variation in ACE2 which may affect the SARS-CoV-2 recognition and infection, and COVID-19 susceptibility and treatment.


Assuntos
Betacoronavirus/fisiologia , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/genética , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Betacoronavirus/metabolismo , Sítios de Ligação/genética , Infecções por Coronavirus/etnologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Predisposição Genética para Doença/etnologia , Variação Genética , Geografia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etnologia , Pneumonia Viral/genética , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Secundária de Proteína/genética , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
5.
Front Oncol ; 10: 1143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32766150

RESUMO

Chimeric Antigen Receptor (CAR)-T cells have great efficacy against CD19+ leukemia but little success for solid tumors. This study explored the effectiveness of third generation anti-HER2 CAR-T cells alone or in combination with anti-PD1 antibody on breast tumor cells expressing HER2 in vitro and in immune competent mouse model. The PDL1-positive mouse mammary tumor cell line 4T1 engineered to express luciferase and human HER2 was used as the target cell line (4T1-Luc-HER2). Anti-HER2 CAR-T cells were generated by transducing mouse spleen T cells with recombinant lentiviruses. ELISA analysis showed that IL-2 and IFN-γ secretion was increased in CAR-T cells co-cultured with the target cells, and the secretion of these two cytokines was increased further with the addition of anti-PD1 antibody. Lactate dehydrogenase assay revealed that CAR-T cells displayed a potent cytotoxicity against the target cells, and the addition of anti-PD1 antibody further enhanced the cytotoxicity. At the effector: target ratio of 16:1, cytotoxicity was 39.8% with CAR-T cells alone, and increased to 49.5% with the addition of anti-PD1 antibody. In immune competent syngeneic mouse model, CAR-T cells were found to be present in tumor stroma, inhibited tumor growth and increased tumor apoptosis significantly. Addition of anti-PD1 antibody further enhanced these anti-tumor activities. Twenty-one days after treatment, tumor weight was reduced by 50.0% and 73.3% in CAR-T group and CAR-T plus anti-PD1 group compared with blank T group. Our results indicate that anti-PD1 antibody can greatly increase the efficacy of anti-HER2 CAR-T against HER2-positive solid tumors.

6.
Cancer Sci ; 111(10): 3653-3664, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32713162

RESUMO

Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24-Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem-like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway-regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24-induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem-like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem-like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer.

7.
Cancer Cell ; 38(1): 79-96.e11, 2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32531268

RESUMO

Fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, plays oncogenic roles in various cancers, presenting an opportunity for the development of effective targeted therapeutics. Here, we report two potent small-molecule FTO inhibitors that exhibit strong anti-tumor effects in multiple types of cancers. We show that genetic depletion and pharmacological inhibition of FTO dramatically attenuate leukemia stem/initiating cell self-renewal and reprogram immune response by suppressing expression of immune checkpoint genes, especially LILRB4. FTO inhibition sensitizes leukemia cells to T cell cytotoxicity and overcomes hypomethylating agent-induced immune evasion. Our study demonstrates that FTO plays critical roles in cancer stem cell self-renewal and immune evasion and highlights the broad potential of targeting FTO for cancer therapy.

8.
Am J Cancer Res ; 10(2): 688-703, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32195036

RESUMO

Trastuzumab-resistance is still a major challenge in treating patients with HER2 positive breast cancer. In this study, we tried to overcome transtuzumab-resistance by examining the therapeutic efficacy of third generation anti-HER2 chimeric antigen receptor (CAR)-T cells alone and in combination with PD1 blockade against HER2 positive and trastuzumab-resistance breast cancer cells in vitro and xenograft model. The anti-HER2 CAR-T cells were generated by infecting CD3/CD28 activated peripheral blood mononuclear cells with lentivirus expressing third generation anti-HER2 CAR. Anti-HER2 CAR-T cells were specifically targeted to HER2 positive BT474 and trastuzumab resistant HCC1954 cells compared with HER2 negative breast cancer cells. Results from ELISA revealed that the secretion of IL-2 and IFN-γ was increased in anti-HER2 CAR-T cells after being co-cultured with HCC1954 cells, and was further increased with the addition of anti-PD1 antibody in the co-culture system. Furthermore, data from lactate dehydrogenase assay showed that anti-HER2 CAR-T cells displayed a potent cytotoxicity against HCC1954 and BT474 cells. Addition of anti-PD1 antibody further enhanced the cytotoxicity of anti-HER2 CAR-T cells against HCC1954 cells. Lastly, injection of anti-HER2 CAR-T cells significantly reduced the growth of HCC1954 xenograft tumors. Combining anti-HER2 CAR-T cells with anti-PD1 antibody further impaired the growth of HCC1954 tumors. The present results indicate that anti-HER2 CAR-T cells have therapeutic efficacy against trastuzumab resistant breast tumors and addition of the PD1 antibody can further enhance the therapeutic effect of anti-HER2 CAR-T cells. Thus, third generation anti-HER2 CAR-T cells along with PD1 blockade is a potential therapy to overcome trastuzumab resistance of breast cancer.

9.
Proc Natl Acad Sci U S A ; 117(7): 3621-3626, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32024762

RESUMO

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.


Assuntos
Reprogramação Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Dioxigenases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Domínio Catalítico , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/química , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
10.
Oncogene ; 39(6): 1246-1259, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31616062

RESUMO

Germline mutations of DNA double-strand break (DSB) response and repair genes that drive tumorigenesis could be a major cause of prostate cancer (PCa) heritability. In this study, we demonstrated the role of novel exonuclease 5 (EXO5) gene in androgen-induced double strand breaks repair via homology-directed repair pathway and prostate tumorigenesis. Using whole-exome sequencing of samples from 20 PCa families, with three or more siblings diagnosed with metastatic PCa, we identified mutations in 31 genes involved in DSB response and repair. Among them, the L151P mutation in the exonuclease 5 (EXO5) gene was present in all affected siblings in three PCa families. We found two other EXO5 SNPs significantly associated with risk of PCa in cases-controls study from databases of genotype and phenotype (dbGaP), which are in linkage disequilibrium (D' = 1) with Exo5 L151P found in PCa family. The L151 residue is conserved across diverse species and its mutation is deleterious for protein functions, as demonstrated by our bioinformatics analyses. The L151P mutation impairs the DNA repair function of EXO5 due to loss of nuclease activity, as well as failure of nuclear localization. CRISPR elimination of EXO5 in a PCa cell line impaired homology-directed recombination repair (HDR) and caused androgen-induced genomic instability, as indicated by frequent occurrence of the oncogenic fusion transcript TMPRSS2-ERG. Genetic and functional validation of the EXO5 mutations indicated that EXO5 is a risk gene for prostate tumorigenesis, likely due to its functions in HDR.


Assuntos
Androgênios/efeitos adversos , Carcinogênese/patologia , Reparo do DNA , Exonucleases/deficiência , Instabilidade Genômica , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Estudos de Casos e Controles , Quebras de DNA de Cadeia Dupla , Exonucleases/genética , Exonucleases/metabolismo , Seguimentos , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , Mutação , Neoplasias Hormônio-Dependentes/induzido quimicamente , Neoplasias Hormônio-Dependentes/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Prognóstico , Neoplasias da Próstata/induzido quimicamente , Neoplasias da Próstata/genética
11.
Oncol Rep ; 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31524276

RESUMO

Without effective treatment, glioblastoma is one of the deadliest cancers worldwide. The aim of the present study was to explore whether combinational immunotherapy is effective for treating malignant glioblastoma in vitro. The therapeutic efficacy of third generation anti­human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR)­T cells alone and in combination with PD1 blockade was investigated for the treatment of malignant glioblastoma cells in vitro. Anti­HER2 CAR­T cells were prepared by transducing activated primary human T cells with lentiviruses which expressed third generation anti­HER2 CAR. The CAR­positive cell ratio was detected using flow cytometry. The expression level of CAR was detected by western blot analysis. The binding of anti­HER2 CAR­T cells to HER2+ U251 glioblastoma cells was examined under a fluorescence microscope. The cytokine secretion of CAR­T cells induced by target cells was analyzed via ELISA. The cytotoxicity of anti­HER2 CAR­T cells alone or in combination with anti­programmed death­1 (PD1) antibody against HER2+/PDL1+ U251 cells was examined using an LDH assay. The CAR­positive cell ratio and expression level of CAR in prepared CAR­T cells were both high enough. Anti­HER2 CAR­T cells could specifically bind to U251 cells. The IL­2 and IFN­Î³ secretion of CAR­T cells increased after being co­cultured with U251 cells, and further increased in the presence of anti­PD1 antibody. Anti­HER2 CAR­T cells displayed a potent cytotoxicity against U251 cells. In addition, the presence of anti­PD1 antibody further enhanced the efficacy of anti­HER2 CAR­T cells against U251 cells. The present results indicated that blocking PD1 immuno­suppression can increase the activation of CAR­T cells after they are activated by a targeting antigen. Third generation anti­HER2 CAR­T cells along with PD1 blockade have a great therapeutic potential for combatting malignant glioblastoma.

12.
Ann Clin Transl Neurol ; 6(9): 1893-1899, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31478350

RESUMO

The maintenance of mitochondrial DNA (mtDNA) relies on proteins encoded by nuclear genes. Mutations in their coding sequences result in heterogenous clinical presentations featuring mtDNA instability in affected tissues. DNA2 is a multi-catalytic protein involved in the removal of single strand DNA during mtDNA replication or Long Patch Base Excision Repair pathway. We have previously described DNA2 mutations in adult patients affected with familial and sporadic forms of mitochondrial myopathy. Here we describe four novel probands presenting with limb weakness associated with novel DNA2 molecular defects. Biochemical assays were established to investigate the functional effects of these variants.


Assuntos
DNA Helicases/genética , DNA Mitocondrial/genética , Miopatias Mitocondriais/genética , Mutação , Idoso , Análise Mutacional de DNA , Replicação do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
13.
Eur J Pharmacol ; 861: 172590, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31401160

RESUMO

Increasing evidence displays that microRNAs (miRNAs) participate in the development of various human malignancies, including esophageal squamous cell carcinoma (ESCC). MicroRNA-33a-5p (miR-33a-5p) has recently been reported to function as a tumor suppressor in many human cancers. However, the expression and role of miR-33a-5p in ESCC remains largely unknown. Herein, we discovered that miR-33a-5p was down-regulated in both ESCC tissues and cell lines, compared with their normal counterparts, and decreased expression of miR-33a-5p was found to be closely associated with poor patient prognosis of ESCC. Moreover, functional experiments revealed that up-regulation of miR-33a-5p inhibited cell proliferation and metastasis of ESCC cells. In addition, the expression level of miR-33a-5p was found to be negatively correlated with the long non-coding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (DANCR), in both ESCC tissues and cell lines. Furthermore, zinc-finger-enhancer binding protein 1 (ZEB1) was predicted and confirmed to be a direct target of miR-33a-5p in ESCC. Further mechanistic investigation indicated that DANCR may function as a competing endogenous RNA (ceRNA) to sponge miR-33a-5p, and thereby up-regulate ZEB1 expression in ESCC. This study highlights the functions of miR-33a-5p in the progression of ESCC and suggests that the DANCR/miR-33a-5p/ZEB1 axis may be a potential prognostic and therapeutic target.


Assuntos
Progressão da Doença , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Carcinoma de Células Escamosas do Esôfago/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico
14.
Exp Cell Res ; 382(1): 111462, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31194976

RESUMO

Scaffolding adaptor Gab2 is overexpressed in a subset of high-grade ovarian cancer. Our published work shows that Gab2 via PI3K enhances migratory behaviors and epithelial to mesenchymal transition (EMT) features of ovarian cancer cells in vitro. However, it is still unclear how Gab2/PI3K pathway reuglates EMT characteristics and whether Gab2 promotes the growth of ovarian cancer stem cell (CSC)-like population and metastatic growth. In this study, we examined the effects of Gab2 expression on CSC-like cell growth using Aldefluor and tumorshpere assays commonly used for assessing ovarian cancer cells with CSC properties. Gab2 overexpression increased the number of ALDH+ cells and tumorsphere formation in two different ovarian cancer cell lines OVCAR5 and OVCAR8, whereas knockdown of Gab2 decreased the number of ALDH+ cells and tumorsphere formation in Caov-3 cells. Furthermore, Gab2 promoted metastatic tumor growth of OVCAR5 in nude mice. Mechanistically, we uncovered that Gab2 via PI3K specifically inhibited miR-200c expression. miR-200c downregulation contributed to the Gab2-enhanced cell migratory behaviors, EMT properties, and the expansion of ALDH+ cells and tumorspheres. Furthermore, Gab2 promoted CD44 expression and cell migration/invasion through miR-200c downregulation. Our findings support a model that Gab2-PI3K pathway via miR-200c downregulation promotes CD44 expression, EMT characteristics, and CSC-like cell growth. Therapies involving miR-200c or targeting CD44 should help treat ovarian cancer with high Gab2 expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/genética , RNA Neoplásico/fisiologia , Animais , Movimento Celular , Regulação para Baixo , Feminino , Xenoenxertos , Humanos , Receptores de Hialuronatos/antagonistas & inibidores , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/fisiologia , Interferência de RNA , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia
15.
Mol Ther Oncolytics ; 14: 149-158, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31211245

RESUMO

The immunosuppressive agent leflunomide has been used in the treatment of over 300,000 patients with rheumatoid arthritis. Its active metabolite, teriflunomide (Ter), directly inhibits dihydroorotate dehydrogenase (DHODH), an enzyme involved in nucleoside synthesis. We report that Ter not only shows in vitro anti-proliferative activity in pancreatic cancer (PC) cells as a single agent but also synergizes with the chemotherapeutic gemcitabine (Gem) in growth inhibition of PC cells. The growth-inhibitory effects of Ter are not solely caused by inhibition of DHODH. Through a kinase screening approach, we identified the PIM-3 serine-threonine kinase as a novel direct target. Subsequent dose-response kinase assays showed that Ter directly inhibited all three PIM family members, with the highest activities against PIM-3 and -1. The PIM-3 kinase was the PIM family member most often associated with PC oncogenesis and was also the kinase inhibited the most by Ter among more than 600 kinases investigated. Ter in PC cells induced changes in phosphorylation and expression of PIM downstream targets, consistent with the effects achieved by overexpression or downregulation of PIM-3. Finally, pharmacological inhibition of PIM proteins not only diminished PC cell proliferation, but also small-molecule pan-PIM and PIM-3 inhibitors synergized with Gem in growth inhibition of PC cells.

16.
Cancer Epidemiol Biomarkers Prev ; 28(8): 1308-1315, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31160347

RESUMO

BACKGROUND: Pathogenic variants in susceptibility genes lead to increased breast cancer risk. METHODS: To identify coding variants associated with breast cancer risk, we conducted whole-exome sequencing in genomic DNA samples from 831 breast cancer cases and 839 controls of Chinese women. We also genotyped samples, including 4,580 breast cancer cases and 6,695 controls, using whole exome-chip arrays. We further performed a replication study using a Multi-Ethnic Global Array in samples from 1,793 breast cases and 2,059 controls. A single marker analysis was performed using the Fisher exact test. RESULTS: We identified a missense variant (rs139379666, P2974L; AF = 0.09% for breast cancer cases, but none for controls) in the ATM gene for breast cancer risk using combing data from 7,204 breast cancer cases and 9,593 controls (P = 1.7 × 10-5). To investigate the functionality of the variant, we first silenced ATM and then transfected the overexpression vectors of ATM containing the risk alleles (TT) or reference alleles (CC) of the variant in U2OS and breast cancer SK-BR3 cells, respectively. Our results showed that compared with the reference allele, the risk allele significantly disrupts the activity of homologous recombination-mediated double-strand breaks repair efficiency. Our results further showed that the risk allele may play a defected regulation role in the activity of the ATM structure. CONCLUSIONS: Our findings identified a novel mutation that disrupts ATM function, conferring to breast cancer risk. IMPACT: Functional investigation of genetic association findings is necessary to discover a pathogenic variant for breast cancer risk.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Mutação de Sentido Incorreto , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Casos e Controles , China/epidemiologia , Estudos de Coortes , Feminino , Genótipo , Humanos , Fatores de Risco , Células Tumorais Cultivadas , Sequenciamento Completo do Exoma/métodos
18.
J Chem Inf Model ; 59(5): 1849-1857, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-30912940

RESUMO

Machine learning has exhibited powerful capabilities in many areas. However, machine learning models are mostly database dependent, requiring a new model if the database changes. Therefore, a universal model is highly desired to accommodate the widest variety of databases. Fortunately, this universality may be achieved by ensemble learning, which can integrate multiple learners to meet the demands of diversified databases. Therefore, we propose a general procedure for learning ensemble establishment based on noncovalent interactions (NCIs) databases. Additionally, accurate NCI computation is quite demanding for first-principles methods, for which a competent machine learning model can be an efficient solution to obtain high NCI accuracy with minimal computational resources. In regard to these aspects, multiple schemes of ensemble learning models (Bagging, Boosting, and Stacking frameworks), are explored in this study. The models are based on various low levels of density functional theory (DFT) calculations for the benchmark databases S66, S22, and X40. All NCIs computed by the DFT calculations can be improved to high-level accuracy (root-mean-square error RMSE = 0.22 kcal/mol in contrast to CCSD(T)/CBS benchmark) by established ensemble learning models. Compared with single machine learning models, ensemble models show better accuracy (RMSE of the best model is further lowered by ∼25%), robustness and goodness-of-fit according to evaluation parameters suggested by the OECD. Among ensemble learning models, heterogeneous Stacking ensemble models show the most valuable application potential. The standardized procedure of constructing learning ensembles has been well utilized on several NCI data sets, and this procedure may also be applicable for other chemical databases.


Assuntos
Teoria da Densidade Funcional , Aprendizado de Máquina , Química Computacional/métodos , Bases de Dados de Compostos Químicos , Modelos Lineares
19.
Mol Med Rep ; 19(3): 2180-2188, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30747211

RESUMO

MicroRNA­233­3p (miR­223­3p) is considered an important cancer­associated marker. The NACHT, LRR and PYD domains­containing protein 3 (NLRP3) inflammasome represents a novel potential target for the treatment of breast cancer. Therefore, it was hypothesized that miR­223­3p may affect tumor growth and immunosuppression in breast cancer by inhibiting the NLRP3 inflammasome. In the present study, an increased expression level of NLRP3 was detected in three breast cancer cell lines compared with normal mammary epithelial cells (HMEC). Suppressing the expression of NLRP3 in MCF­7 cell lines increased the apoptotic rate of breast cancer cells and reduced the proliferative capacity. NLRP3 was identified to be a direct target of miR­233­3p using a luciferase assay. In addition, miR­233­3p mimics inhibited the NLRP3­dependent processes in cancer cells by suppressing the NLRP3 expression level and the protein expression levels of its downstream factors, including PYD and CARD domain containing protein, interleukin­1ß and interleukin­18. In vivo experiments demonstrated the suppressive effect of miR­233­3p in tumor growth and immunosuppression. Collectively these findings suggested that the inactivation of the NLRP3 inflammasome driven by miR­223­3p reduced the growth and immunosuppression of breast cancer in vitro and in vivo, and may represent a novel therapeutic strategy in treating breast cancer.


Assuntos
Neoplasias da Mama/imunologia , Inflamassomos/imunologia , MicroRNAs/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade , Inflamassomos/genética , Camundongos , MicroRNAs/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
20.
Biochem Biophys Res Commun ; 508(4): 1227-1232, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30554655

RESUMO

Hepatocellular carcinoma (HCC) is the most prevalent malignancy in liver and a leading cause of cancer-related deaths. Despite the pressing need for treatment options, patients with HCC develop significant resistance and adverse side effects to current approved drugs that becomes a major barrier to effective treatment. A natural product Tetrandrine (TET) is a potential alternative treatment option for HCC, with demonstrated effectiveness and low toxicity. However, the mechanisms by which Tetrandrine inhibits HCC are unclear. In the current study, we identify Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) as a potential TET drug target through structural modeling. Screening of a panel of HCC cell lines reveal differential sensitivities toward TET treatment. Interestingly, IC50 of TET inhibition of HCC cell proliferation is positively correlated with CaMKIIδ expression level in these distinct HCC cells. Furthermore, TET treatment resulted in a marked reduction of CaMKIIδ phosphorylation level, and knockdown of CaMKIIδ reduced the sensitivity of HCC cells to TET. Most importantly, CaMKIIδ protein levels in high-grade human HCC samples were significantly elevated as compared to normal liver tissues. Taken together, our studies demonstrate that the natural compound TET targets CaMKIIδ in HCC cells, and that CaMKIIδ level is a potential biomarker to identify HCC patient populations sensitive to Tetrandrine treatment.


Assuntos
Benzilisoquinolinas/uso terapêutico , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Terapia de Alvo Molecular , Benzilisoquinolinas/química , Benzilisoquinolinas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Modelos Moleculares , Estadiamento de Neoplasias , Fosforilação/efeitos dos fármacos
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