RESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cell fate via activation of a diverse set of genes. There are conflicting reports describing the role of AhR in cancer. AhR-knockout mice do not develop tumors spontaneously, yet the AhR can act as a tumor suppressor in certain contexts. Loss of tumor suppression by p53 is common in human cancer. To investigate AhR function in the absence of p53, we generated mice lacking both AhR and p53. Mice deficient for AhR and p53 had shortened lifespan, increased tumorigenesis, and an altered tumor spectrum relative to control mice lacking only p53. In addition, knockout of both AhR and p53 resulted in reduced embryonic survival and neonatal fitness. We also examined the consequences of loss of AhR in p53-heterozygous mice and observed a significantly reduced lifespan and enhanced tumor burden. These findings reveal an important role for the AhR as a tumor suppressor in the absence of p53 signaling and support the development of anti-cancer therapeutics that would promote the tumor suppressive actions of the AhR.
Asunto(s)
Carcinogénesis , Receptores de Hidrocarburo de Aril , Proteína p53 Supresora de Tumor , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carcinogénesis/genética , Transformación Celular Neoplásica , Ligandos , Ratones , Ratones Noqueados , Receptores de Hidrocarburo de Aril/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.
Asunto(s)
Neoplasias Pulmonares , Receptores de Hidrocarburo de Aril , Humanos , Proteínas de Ciclo Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , ARN , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The human DNA mismatch repair (MMR) protein MLH1 has essential roles in the correction of replication errors and the activation of cell cycle checkpoints and cytotoxic responses to DNA damage that contribute to suppression of cancer risk. MLH1 functions as a heterodimer with the PMS2 protein, and steady state levels of PMS2 are very low in MLH1-deficient cells. Unique to MLH1 among MutL-homolog proteins, and conserved in identified eukaryotic MLH1 proteins, is the so-called C-terminal homology domain (CTH). The function of these C-terminal 20-30 amino acids is not known. We investigated the effect of a C-terminal truncation of human MLH1 (MLH1-L749X) on mammalian MMR by testing its activity in MLH1-deficient cells. We found the CTH to be essential for suppression of spontaneous mutation, activation of a cytotoxic response to 6-thioguanine, and maintenance of normal steady state levels of PMS2. Co-expression in doubly mutant Mlh1-/-; Pms2-/- fibroblasts showed that MLH1-L749X was unable to stabilize PMS2. Over-expression of MLH1-L749X did not reduce stabilization of PMS2 mediated by wild-type MLH1, indicating that truncation of the CTH reduces the ability to compete with wild-type MLH1 for interaction with PMS2. Lack of PMS2 stabilization also was observed with a previously reported pathogenic truncation (MLH1-Y750X), but not with two different point mutations in the CTH. Biochemical assays demonstrated that truncation of the CTH reduced the stability of heterodimers, although MLH1-L749X retained significant capacity for interaction with PMS2. Thus, the CTH of human MLH1 is necessary for error correction, checkpoint signaling, and for promoting interaction with, and the stability of, PMS2. Analysis of the CTH role in stabilizing PMS2 was facilitated by a novel intracellular assay for MLH1-PMS2 interaction. This assay should prove useful for identifying additional amino acids in MLH1 and PMS2 necessary for interaction in cells, and for determining the functional consequences of MLH1 mutations identified in human cancers.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Disparidad de Par Base/genética , Proteínas Portadoras/farmacología , Proteínas Portadoras/fisiología , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/farmacología , Proteínas Nucleares/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Ratones , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Mutación , Proteínas Nucleares/genética , Homología de Secuencia de Aminoácido , Supresión Genética , Tioguanina/farmacologíaRESUMEN
Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.
Asunto(s)
Adenosina Trifosfatasas/genética , Daño del ADN , Reparación de la Incompatibilidad de ADN , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Mutación , Adenosina Trifosfatasas/metabolismo , Animales , Células Cultivadas , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Metilnitrosourea/toxicidad , Ratones , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , MutagénesisRESUMEN
Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We evaluated the effect of MMR status (Mlh1(+/+) versus Mlh1(-/-)) on the carcinogenic potential of the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in mice. PhIP exposure did not obviously increase lymphoma or small intestinal tumorigenesis in either Mlh1-deficient or -proficient mice. In contrast, the frequency of aberrant crypt foci (ACF), a preneoplastic biomarker for colon tumorigenesis, was increased by PhIP, and the increase due to PhIP was significantly greater in Mlh1(-/-) versus wild-type littermates. This apparent heightened susceptibility to induction of ACF parallels the previously reported hypermutability of Mlh1-deficient mice to PhIP and is consistent with the hypothesis that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.
Asunto(s)
Carcinógenos/toxicidad , Proteínas Portadoras/fisiología , Neoplasias del Colon/genética , Reparación de la Incompatibilidad de ADN , ADN de Neoplasias/fisiología , Imidazoles/toxicidad , Proteínas Nucleares/fisiología , Lesiones Precancerosas/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Colon/efectos de los fármacos , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/patología , Neoplasias Hematológicas/inducido químicamente , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Incidencia , Ratones , Ratones Noqueados , Homólogo 1 de la Proteína MutL , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , Tasa de SupervivenciaRESUMEN
Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status (Mlh1+/+ versus Mlh1-/-) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1+/+ littermates. Similar induction was seen in Mlh1-/- small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the "signature PhIP mutation", were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1-/- mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1-/- mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.
Asunto(s)
Carcinógenos/toxicidad , Proteínas Portadoras/fisiología , Imidazoles/toxicidad , Mutágenos/toxicidad , Mutación/efectos de los fármacos , Proteínas Nucleares/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Colon/fisiología , Reparación del ADN/fisiología , Genes Reporteros , Tamización de Portadores Genéticos , Intestino Delgado/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Virales/genéticaRESUMEN
MutLalpha, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLalpha and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLalpha proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLalpha demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLalpha activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.