RESUMO
BACKGROUND: Cancer arises through accumulation of somatically acquired genetic mutations. An important question is to delineate the temporal order of somatic mutations during carcinogenesis, which contributes to better understanding of cancer biology and facilitates identification of new therapeutic targets. Although a number of statistical and computational methods have been proposed to estimate the temporal order of mutations, they do not account for the differences in the functional impacts of mutations and thus are likely to be obscured by the presence of passenger mutations that do not contribute to cancer progression. In addition, many methods infer the order of mutations at the gene level, which have limited power due to the low mutation rate in most genes. RESULTS: In this paper, we develop a Probabilistic Approach for estimating the Temporal Order of Pathway mutations by leveraging functional Annotations of mutations (PATOPA). PATOPA infers the order of mutations at the pathway level, wherein it uses a probabilistic method to characterize the likelihood of mutational events from different pathways occurring in a certain order. The functional impact of each mutation is incorporated to weigh more on a mutation that is more integral to tumor development. A maximum likelihood method is used to estimate parameters and infer the probability of one pathway being mutated prior to another. Simulation studies and analysis of whole exome sequencing data from The Cancer Genome Atlas (TCGA) demonstrate that PATOPA is able to accurately estimate the temporal order of pathway mutations and provides new biological insights on carcinogenesis of colorectal and lung cancers. CONCLUSIONS: PATOPA provides a useful tool to estimate temporal order of mutations at the pathway level while leveraging functional annotations of mutations.
Assuntos
Carcinogênese/genética , Anotação de Sequência Molecular , Mutação/genética , Probabilidade , Transdução de Sinais/genética , Simulação por Computador , Bases de Dados Genéticas , Humanos , Taxa de Mutação , Neoplasias/genética , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Mobile crowdsourcing has been exploited to collect enough fingerprints for fingerprinting-based localization. Since the construction of a fingerprint database is time consuming, mobile users should be well motivated to participate in fingerprint collection task. To this end, a Walrasian equilibrium-based incentive mechanism is proposed in this paper to motivate mobile users. The proposed mechanism can eliminate the monopoly of the crowdsourcer, balance the supply and demand of fingerprint data, and maximize the benefit of all participators. In order to reach the Walrasian equilibrium, firstly, the social welfare maximization problem is constructed. To solve the original optimization problem, a dual decomposition method is employed. The maximization of social welfare is decomposed into the triple benefit optimization among the crowdsourcer, mobile users, and the whole system. Accordingly, a distributed iterative algorithm is designed. Through the simulation, the performance of the proposed incentive scheme is verified and analyzed. Simulation results demonstrated that the proposed iterative algorithm satisfies the convergence and optimality. Moreover, the self-reconstruction ability of the proposed incentive scheme was also verified, indicating that the system has strong robustness and scalability.
RESUMO
Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17ß-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Isoflavonas/farmacologia , Proteína Multifuncional do Peroxissomo-2/antagonistas & inibidores , Alcaloides/química , Antineoplásicos/síntese química , Antineoplásicos/química , Azocinas/química , Azocinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Isoflavonas/síntese química , Isoflavonas/química , Estrutura Molecular , Proteína Multifuncional do Peroxissomo-2/metabolismo , Quinolizinas/química , Quinolizinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Wnt signaling plays an important role in colorectal cancer (CRC). Although the mechanisms of ß-catenin degradation have been well studied, the mechanism by which ß-catenin activates transcription is still not fully understood. While screening a panel of DNA demethylases, we found that thymine DNA glycosylase (TDG) up-regulated Wnt signaling. TDG interacts with the transcription factor TCF4 and coactivator CREB-binding protein/p300 in the Wnt pathway. Knocking down TDG by shRNAs inhibited the proliferation of CRC cells in vitro and in vivo. In CRC patients, TDG levels were significantly higher in tumor tissues than in the adjacent normal tissues. These results suggest that TDG warrants consideration as a potential biomarker for CRC and as a target for CRC treatment.
Assuntos
Neoplasias Colorretais/patologia , Timina DNA Glicosilase/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Sialoglicoproteínas/metabolismo , Sumoilação , Timina DNA Glicosilase/química , Timina DNA Glicosilase/genética , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear ß-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/ß-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras/biossíntese , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Histonas , Humanos , Intestinos/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Células-Tronco/metabolismo , Células-Tronco/patologia , Transplante Heterólogo , Ubiquitinação/genética , beta Catenina/genéticaRESUMO
Kruppel-like factor 4 (KLF4) is a transcription factor that is highly expressed in differentiated epithelial cells including that of the skin. It is critical for specification or function of differentiated epithelial cells. Moreover, KLF4 functions either as a tumor suppressor or an oncogene depending on different cellular contexts. However, the role of KLF4 in skin tumorigenesis remains controversial. To address this issue, we first examined KLF4 expression using a cohort of samples from patients with skin squamous cell carcinoma and basal cell carcinoma and found that in 21 of 24 tumor tissues (87.5%), KLF4 expression as assayed by immunohistochemistry was absent when compared with that in normal tissues. In addition, knockdown of KLF4 in human epidermal squamous cell carcinoma SCC13 cells was accompanied by increased cell growth. Further analysis revealed that KLF4 deficiency promoted cell migration and adhesion, which are the important properties of tumor cells. These observations were supported by the effect upon overexpression of KLF4 in SCC13 cells. Furthermore, we generated a novel tamoxifen-inducible KLF4/CreER and KLF4(flox) double transgenic mouse model to examine the role of KLF4 in skin cancer development. Consistent with in vitro studies, KLF4 deficiency increased the ability of migration and adhesion of mouse primary skin keratinocytes. Moreover, KLF4 knockout led to increased cell proliferation and skin carcinogenesis in a classical DMBA/TPA mouse skin cancer model. Taken together, our data suggest that KLF4 inhibits cell proliferation, migration and adhesion and that loss of KLF4 promotes skin tumorigenesis.
Assuntos
Carcinoma Basocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Adesão Celular/genética , Diferenciação Celular , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Interferência de RNA , RNA Interferente Pequeno , Neoplasias Cutâneas/induzido quimicamente , Tamoxifeno/farmacologia , Acetato de TetradecanoilforbolRESUMO
A central question in Wnt signaling is the regulation of beta-catenin phosphorylation and degradation. Multiple kinases, including CKI alpha and GSK3, are involved in beta-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of beta-catenin. However, which phosphatase dephosphorylates beta-catenin in vivo and how the specificity of beta-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates beta-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/beta-catenin signaling in Drosophila. Moreover, we have identified PR55 alpha as the regulatory subunit of PP2A that controls beta-catenin phosphorylation and degradation. PR55 alpha, but not the catalytic subunit, PP2Ac, directly interacts with beta-catenin. RNA interference knockdown of PR55 alpha elevates beta-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55 alpha enhances Wnt signaling. Taken together, our results suggest that PR55 alpha specifically regulates PP2A-mediated beta-catenin dephosphorylation and plays an essential role in Wnt signaling.
Assuntos
Proteína Fosfatase 2/metabolismo , beta Catenina/metabolismo , Animais , Proteína Axina , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Drosophila melanogaster , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Imunoprecipitação , Cloreto de Lítio/farmacologia , Ácido Okadáico/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Subunidades Proteicas , Piranos/farmacologia , RNA Interferente Pequeno , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Compostos de Espiro/farmacologiaRESUMO
Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (ß-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.
RESUMO
The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target ß-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target ß-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Encéfalo/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Mitocôndrias/efeitos dos fármacos , Ureia/farmacologia , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Metabolismo Energético , Ativação Enzimática , Ativadores de Enzimas/química , Regulação Neoplásica da Expressão Gênica , Humanos , Hidrocarbonetos Fluorados/química , Mitocôndrias/metabolismo , Consumo de Oxigênio , Sulfonamidas/química , Sulfonamidas/farmacologia , Células Tumorais Cultivadas , Ureia/análogos & derivados , Ureia/químicaRESUMO
Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.
Assuntos
Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Neoplasias da Próstata , Multimerização Proteica/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Peixe-Zebra/metabolismo , Animais , Benzofuranos/síntese química , Benzofuranos/química , Benzofuranos/farmacologia , Sítios de Ligação , Colchicina , Humanos , Masculino , Camundongos , Camundongos Nus , Células PC-3 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Lung cancer is the leading cause of cancer mortality in the United States (U.S.). Squamous cell carcinoma (SQCC) represents 22.6% of all lung cancers nationally, and 26.4% in Appalachian Kentucky (AppKY), where death from lung cancer is exceptionally high. The Cancer Genome Atlas (TCGA) characterized genetic alterations in lung SQCC, but this cohort did not focus on AppKY residents. METHODS: Whole-exome sequencing was performed on tumor and normal DNA samples from 51 lung SQCC subjects from AppKY. Somatic genomic alterations were compared between the AppKY and TCGA SQCC cohorts. RESULTS: From this AppKY cohort, we identified an average of 237 nonsilent mutations per patient and, in comparison with TCGA, we found that PCMTD1 (18%) and IDH1 (12%) were more commonly altered in AppKY versus TCGA. Using IDH1 as a starting point, we identified a mutually exclusive mutational pattern (IDH1, KDM6A, KDM4E, JMJD1C) involving functionally related genes. We also found actionable mutations (10%) and/or intermediate or high-tumor mutation burden (65%), indicating potential therapeutic targets in 65% of subjects. CONCLUSIONS: This study has identified an increased percentage of IDH1 and PCMTD1 mutations in SQCC arising in the AppKY residents versus TCGA, with population-specific implications for the personalized treatment of this disease. IMPACT: Our study is the first report to characterize genomic alterations in lung SQCC from AppKY. These findings suggest population differences in the genetics of lung SQCC between AppKY and U.S. populations, highlighting the importance of the relevant population when developing personalized treatment approaches for this disease.
Assuntos
Carcinoma de Células Escamosas/genética , Isocitrato Desidrogenase/genética , Neoplasias Pulmonares/genética , Mutação , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Região dos Apalaches , Carcinoma de Células Escamosas/metabolismo , Feminino , Genômica , Humanos , Kentucky , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , População Branca/genética , Sequenciamento do ExomaRESUMO
Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of ß-catenin. Phosphorylation of ß-catenin by CK1α and GSK3 promotes ß-catenin binding to ß-TrCP, leading to ß-catenin degradation through the proteasome. The phosphorylation and ubiquitination of ß-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents ß-catenin ubiquitination. Inactivation of USP47 by RNAi increased ß-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas ß-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression.
Assuntos
Proteínas de Drosophila/fisiologia , Ubiquitina Tiolesterase/fisiologia , Proteases Específicas de Ubiquitina/fisiologia , Ubiquitinação , Via de Sinalização Wnt , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Drosophila melanogaster , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteólise , Asas de Animais/enzimologia , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure-activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Metionina Adenosiltransferase/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Estilbenos/farmacologia , Compostos de Anilina/síntese química , Compostos de Anilina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Biocatálise/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Metionina Adenosiltransferase/metabolismo , Estrutura Molecular , Subunidades Proteicas/efeitos dos fármacos , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Estilbenos/síntese química , Estilbenos/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells in vitro and in vivo. Using a biotinylated FIDAS analogue, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC cell growth. A novel FIDAS agent delivered orally repressed CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment.
Assuntos
Neoplasias do Colo/prevenção & controle , Metionina Adenosiltransferase/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Biocatálise , Neoplasias do Colo/enzimologia , Flúor/química , Humanos , Metionina Adenosiltransferase/metabolismo , Camundongos , Modelos Moleculares , Estilbenos/química , Transplante HeterólogoRESUMO
Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.
Assuntos
Células Epiteliais/citologia , Regulação Neoplásica da Expressão Gênica , Intestinos/citologia , Fatores de Transcrição Kruppel-Like/fisiologia , Animais , Células CACO-2 , Cromossomos Artificiais Bacterianos , Neoplasias do Colo/metabolismo , DNA Complementar/metabolismo , Homeostase , Humanos , Imuno-Histoquímica/métodos , Neoplasias Intestinais/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC is initiated by mutations of the tumor suppressor gene, adenomatous polyposis coli (APC), or ß-catenin gene. These mutations stabilize ß-catenin and constitutively activate Wnt/ß-catenin target genes, such as c-Myc and cyclin D1, ultimately leading to cancer. Naturally occurring stilbene derivatives, resveratrol and pterostilbene, inhibit Wnt signaling and repress CRC cell proliferation but are ineffective at concentrations less than 10 µM. To understand the structure--activity relationship within these stilbene derivatives and to develop more efficacious Wnt inhibitors than these natural products, we synthesized and evaluated a panel of fluorinated N,N-dialkylaminostilbenes. Among this panel, (E)-4-(2,6-difluorostyryl)-N,N-dimethylaniline (4r) inhibits Wnt signaling at nanomolar levels and inhibits the growth of human CRC cell xenografts in athymic nude mice at a dosage of 20 mg/kg. These fluorinated N,N-dialkylaminostilbenes appear to inhibit Wnt signaling downstream of ß-catenin, probably at the transcriptional level.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Estilbenos/síntese química , Proteínas Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Estilbenos/química , Estilbenos/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.
Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Dano ao DNA , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Mitocôndrias/metabolismoRESUMO
Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.