RESUMEN
Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
Asunto(s)
Enzimas Reparadoras del ADN/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Dominio Catalítico , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Daño del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Pirofosfatasas/antagonistas & inhibidores , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto , Hidrolasas NudixRESUMEN
The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Fosfatasas cdc25/metabolismo , Movimiento Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/genética , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Expresión Génica , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas , Puntos de Control de la Fase S del Ciclo Celular/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Fosfatasas cdc25/genéticaRESUMEN
BACKGROUND: Chromosomal rearrangements in the form of deletions, insertions, inversions and translocations are frequently observed in breast cancer genomes, and a subset of these rearrangements may play a crucial role in tumorigenesis. To identify novel somatic chromosomal rearrangements, we determined the genome structures of 15 hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing. RESULTS: We identified and validated 40 somatic structural alterations, including the recurring fusion between genes DDX10 and SKA3 and translocations involving the EPHA5 gene. Other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. RNA interference-mediated suppression of five candidate genes (DDX10, SKA3, EPHA5, CLTC and TNIK) led to inhibition of breast cancer cell growth. Moreover, downregulation of DDX10 in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology. CONCLUSIONS: Using whole genome mate pair sequencing and RNA interference assays, we have discovered a number of novel gene rearrangements in breast cancer genomes and identified DDX10, SKA3, EPHA5, CLTC and TNIK as potential cancer genes with impact on the growth and proliferation of breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Reordenamiento Génico , Análisis de Secuencia de ADN , Mama/citología , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Genes Relacionados con las Neoplasias/genética , Genómica , Humanos , Células MCF-7 , Reacción en Cadena de la Polimerasa , Interferencia de ARN , Receptores de EsteroidesRESUMEN
Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine--inhibiting ATM and ATR amongst other kinases--is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
Asunto(s)
Daño del ADN , Replicación del ADN , Proteínas Quinasas/metabolismo , Rayos Ultravioleta , Cafeína/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Transformada , Supervivencia Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Roturas del ADN de Doble Cadena , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/deficiencia , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de la radiaciónRESUMEN
Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 µM. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Ácidos Araquidónicos/farmacología , Próstata/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Taurina/análogos & derivados , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/química , Moduladores de Receptores de Cannabinoides/química , Moduladores de Receptores de Cannabinoides/farmacología , Recuento de Células , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Masculino , Oxazinas , Próstata/patología , Neoplasias de la Próstata/patología , Taurina/química , Taurina/farmacología , XantenosRESUMEN
Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.
Asunto(s)
Proteína BRCA2/deficiencia , Neoplasias del Colon/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Tioguanina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Antimetabolitos Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Disparidad de Par Base , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Reparación del ADN , Sinergismo Farmacológico , Genes BRCA2 , Células HCT116 , Humanos , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/genética , RatonesRESUMEN
Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.
Asunto(s)
Neoplasias/enzimología , Neoplasias/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Recombinación Genética , Animales , Línea Celular Tumoral , Cricetinae , Daño del ADN , Replicación del ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Activación Enzimática , Técnicas de Inactivación de Genes , Genes BRCA1 , Genes BRCA2 , Humanos , Mutación , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) PolimerasasRESUMEN
BACKGROUND: Colorectal cancer is one of the most common causes of cancer-related mortality. The disease is clinically and genetically heterogeneous though a strong hereditary component has been identified. However, only a small proportion of the inherited susceptibility can be ascribed to dominant syndromes, such as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) or Familial Adenomatous Polyposis (FAP). In an attempt to identify novel colorectal cancer predisposing genes, we have performed a genome-wide linkage analysis in 30 Swedish non-FAP/non-HNPCC families with a strong family history of colorectal cancer. METHODS: Statistical analysis was performed using multipoint parametric and nonparametric linkage. RESULTS: Parametric analysis under the assumption of locus homogeneity excluded any common susceptibility regions harbouring a predisposing gene for colorectal cancer. However, several loci on chromosomes 2q, 3q, 6q, and 7q with suggestive linkage were detected in the parametric analysis under the assumption of locus heterogeneity as well as in the nonparametric analysis. Among these loci, the locus on chromosome 3q21.1-q26.2 was the most consistent finding providing positive results in both parametric and nonparametric analyses Heterogeneity LOD score (HLOD) = 1.90, alpha = 0.45, Non-Parametric LOD score (NPL) = 2.1). CONCLUSION: The strongest evidence of linkage was seen for the region on chromosome 3. Interestingly, the same region has recently been reported as the most significant finding in a genome-wide analysis performed with SNP arrays; thus our results independently support the finding on chromosome 3q.
Asunto(s)
Cromosomas Humanos Par 3/genética , Neoplasias Colorrectales/genética , Ligamiento Genético/genética , Anciano , Femenino , Predisposición Genética a la Enfermedad , Genoma , Humanos , Masculino , Persona de Mediana Edad , SueciaRESUMEN
The SMARCA3 gene was recently found to be a common target for methylation in colon and gastric cancer, suggesting it has possible tumor suppressor activity. To determine whether SMARCA3 plays a role in colorectal and/or gastric cancer predisposition, a mutation screening of the gene was performed in affected index cases from 20 Swedish families with colorectal and/or gastric cancer. Notably, one family included in the screening exhibited suggestive linkage to the region on chromosome 3q that harbors the SMARCA3 gene. In addition to known polymorphisms, nine novel variants - none of them clearly pathogenic - were detected. Seven of these variants were further tested in an additional 287 patients with a family history of the disease, but their frequency was found to not be significantly different from that observed in the controls. In conclusion, although a very low effect of some variants could not be excluded, it seems that the SMARCA3 gene does not play an important role in the predisposition to colorectal and gastric cancer.
RESUMEN
Cornelia de Lange syndrome (CdLS; OMIM 122470) is a rare multiple congenital anomaly/mental retardation syndrome characterized by distinctive dysmorphic facial features, severe growth and developmental delay and abnormalities of the upper limbs. About 50% of CdLS patients have been found to have heterozygous mutations in the NIPBL gene and a few cases were recently found to be caused by mutations in the X-linked SMC1L1 gene. We performed a mutation screening of all NIPBL coding exons by direct sequencing in 11 patients (nine sporadic and two familial cases) diagnosed with CdLS in Sweden and detected mutations in seven of the cases. All were de novo, and six of the mutations have not been previously described. Four patients without identifiable NIPBL mutations were subsequently subjected to multiplex ligation-dependent probe amplification analysis to exclude whole exon deletions/duplications of NIPBL. In addition, mutation analysis of the 5' untranslated region (5' UTR) of NIPBL was performed. Tiling resolution array comparative genomic hybridization analysis was carried out on these four patients to detect cryptic chromosome imbalances and in addition the boys were screened for SMC1L1 mutations. We found a de novo 9p duplication with a size of 0.6 Mb in one of the patients with a CdLS-like phenotype but no mutations were detected in SMC1L1. So far, two genes (NIPBL and SMC1L1) have been identified causing CdLS or CdLS-like phenotypes. However, in a considerable proportion of individuals demonstrating the CdLS phenotype, mutations in any of these two genes are not found and other potential loci harboring additional CdLS-causing genes should be considered.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Síndrome de Cornelia de Lange/genética , Proteínas/genética , Regiones no Traducidas 5'/genética , Adolescente , Niño , Inestabilidad Cromosómica , Cromosomas Humanos Par 9/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Mutación , Hibridación de Ácido Nucleico , Fenotipo , SueciaRESUMEN
BACKGROUND: The cell cycle checkpoint kinase 2 (CHEK2) 1100delC variant has recently been identified at high frequency in families with both breast and colorectal cancer, suggesting the possible role of this variant in colorectal cancer predisposition. PATIENTS AND METHODS: To evaluate the role of CHEK2 ll00delC among Swedish colorectal cancer patients, the variant frequency was determined in 174 selected familial cases, 644 unselected cases and 760 controls, as well as in l8 families used in the genome-wide linkage analysis, where weak linkage was seen for the region harboring the CHEK2 gene. RESULTS: CHEK2 l100delC was found in 1.15% of familial and in 0.93% of unselected cases, compared to 0.66% of controls, showing no significant difference between groups. One out of 45 familial cases with a family history of breast cancer was shown to be a carrier. The variant was not identified in the 18 families included in the linkage analysis. CONCLUSION: The CHEK2 1100delC was not significantly increased in Swedish colorectal cancer patients, however, in order to determine the role of the variant in colorectal cancer families with the history of breast cancer a larger sample size is needed.
Asunto(s)
Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Proteínas Serina-Treonina Quinasas/genética , Estudios de Casos y Controles , Quinasa de Punto de Control 2 , Cromosomas Humanos Par 22 , Mutación de Línea Germinal , Humanos , SueciaRESUMEN
Over the last few decades it has become clear that highly penetrant disease genes are responsible for a minor proportion of colorectal cancer cases. Families with hereditary syndromes are today recognized and included in surveillance programs known to reduce morbidity and mortality in colorectal cancer. Colorectal cancer is preventable and screening strategies in whole populations are currently under debate. Colorectal cancer can be considered a complex disease, with a combination of predisposing genetic variants and environmental factors that contribute to the illness as a whole. The progress made in the genome project provides an opportunity to determine such genetic variants and environmental factors. This knowledge can be used to define a subpopulation at increased risk for colorectal cancer. It will be more feasible to design preventive strategies for this subgroup than for a whole population.