RESUMEN
The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity (p < 10-16) but was not mechanistically involved in DEX sensitivity. Subcutaneous, orthotopic and metastatic NSCLC xenografts, biomarker-selected, STK11/LKB1 mutant patient derived xenografts, and genetically engineered mouse models with KRAS/LKB1 mutant lung adenocarcinomas all showed marked in vivo anti-tumor responses with the glucocorticoid dexamethasone as a single agent or in combination with cisplatin. Mechanistically, GR activation triggers G1/S cell cycle arrest in LKB1 mutant NSCLCs by inducing the expression of the cyclin-dependent kinase inhibitor, CDKN1C/p57(Kip2). All findings were confirmed with functional genomic experiments including CRISPR knockouts and exogenous expression. Importantly, DEX-GR mediated cell cycle arrest did not interfere with NSCLC radiotherapy, or platinum response in vitro or with platinum response in vivo. While DEX induced LKB1 mutant NSCLCs in vitro exhibit markers of cellular senescence and demonstrate impaired migration, in vivo DEX treatment of a patient derived xenograft (PDX) STK11/LKB1 mutant model resulted in expression of apoptosis markers. These findings identify a previously unknown GR mediated therapeutic vulnerability in STK11/LKB1 mutant NSCLCs caused by induction of p57(Kip2) expression with both STK11 mutation and high expression of CPS1 as precision medicine biomarkers of this vulnerability.
RESUMEN
We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.
Asunto(s)
Regiones no Traducidas 5' , Alelos , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Repeticiones de Microsatélite , Regiones Promotoras Genéticas , Receptores de Estrógenos/genética , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Genes Reporteros , Genotipo , Humanos , Datos de Secuencia Molecular , Polimorfismo Genético , Receptores de Estrógenos/metabolismoRESUMEN
Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.
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Codón de Terminación/fisiología , Neoplasias Colorrectales/genética , Mutación del Sistema de Lectura/genética , Regulación de la Expresión Génica/genética , Inestabilidad de Microsatélites , ARN Mensajero/genética , Regiones no Traducidas 3'/fisiología , Ácido Anhídrido Hidrolasas , Línea Celular Tumoral , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/biosíntesis , Proteína 3 Homóloga de MutS , Mutación Missense , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Proteínas de Neoplasias/análisis , Proteínas Serina-Treonina Quinasas/biosíntesis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Genómica/métodos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias Pulmonares/patología , Trombospondina 1/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores Citoplasmáticos y Nucleares , Trombospondina 1/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Neoplasias Pulmonares/metabolismo , Complejos Multiproteicos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Estructura Molecular , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Tolerancia a RadiaciónRESUMEN
Oncogene-specific changes in cellular signaling have been widely observed in lung cancer. Here, we investigated how these alterations could affect signaling heterogeneity and suggest novel therapeutic strategies. We compared signaling changes across six human bronchial epithelial cell (HBEC) strains that were systematically transformed with various combinations of TP53, KRAS, and MYC-oncogenic alterations commonly found in non-small cell lung cancer (NSCLC). We interrogated at single-cell resolution how these alterations could affect classic readouts (ß-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1, and phospho-ERK1/2) of key pathways commonly affected in NSCLC. All three oncogenic alterations were required concurrently to observe significant signaling changes, and significant heterogeneity arose in this condition. Unexpectedly, we found two mutually exclusive altered subpopulations: one with STAT3 upregulation and another with SMAD2/3 downregulation. Treatment with a STAT3 inhibitor eliminated the upregulated STAT3 subpopulation, but left a large surviving subpopulation with downregulated SMAD2/3. A bioinformatics search identified BCL6, a gene downstream of SMAD2/3, as a novel pharmacologically accessible target of our transformed HBECs. Combination treatment with STAT3 and BCL6 inhibitors across a panel of NSCLC cell lines and in xenografted tumors significantly reduced tumor cell growth. We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (STAT3 and BCL6) downstream of common oncogenes, and tumor suppressors may provide a potent way to defeat intratumor heterogeneity. Cancer Res; 77(11); 3070-81. ©2017 AACR.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Terapia Combinada/métodos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Línea Celular Tumoral , Humanos , Transducción de Señal , TransfecciónRESUMEN
UNLABELLED: Ionizing radiation (IR) is a key therapeutic regimen for many head and neck cancers (HNC). However, the 5-year overall survival rate for locally advanced HNCs is approximately 50% and better therapeutic efficacy is needed. NAD(P)H: quinone oxidoreductase 1 (NQO1) is overexpressed in many cancers, and ß-lapachone (ß-lap), a unique NQO1 bioactivatable drug, exploits this enzyme to release massive reactive oxygen species (ROS) that synergize with IR to kill by programmed necrosis. ß-Lap represents a novel therapeutic opportunity in HNC leading to tumor-selective lethality that will enhance the efficacy of IR. Immunohistochemical staining and Western blot assays were used to assess the expression levels of NQO1 in HNC cells and tumors. Forty-five percent of endogenous HNCs expressed elevated NQO1 levels. In addition, multiple HNC cell lines and tumors demonstrated elevated levels of NQO1 expression and activity and were tested for anticancer lethality and radiosensitization by ß-lap using long-term survival assays. The combination of nontoxic ß-lap doses and IR significantly enhanced NQO1-dependent tumor cell lethality, increased ROS, TUNEL-positive cells, DNA damage, NAD(+), and ATP consumption, and resulted in significant antitumor efficacy and prolonged survival in two xenograft murine HNC models, demonstrating ß-lap radiosensitization of HNCs through a NQO1-dependent mechanism. This translational study offers a potential biomarker-driven strategy using NQO1 expression to select tumors susceptible to ß-lap-induced radiosensitization. Mol Cancer Ther; 15(7); 1757-67. ©2016 AACR.
Asunto(s)
Expresión Génica , Neoplasias de Cabeza y Cuello/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , Tolerancia a Radiación/genética , Radiación Ionizante , Adenosina Trifosfato/metabolismo , Animales , Catalasa/genética , Catalasa/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Modelos Animales de Enfermedad , Activación Enzimática , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Especies Reactivas de Oxígeno/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Somatic frameshift mutations in some genes containing coding mononucleotide repeats (cMNRs) are well known characteristics of tumors with high microsatellite instability (MSI-H). We identified 22 novel and 11 known target genes containing cMNRs with a length of 10 or more nucleotides by using a systematic database search. Frameshift mutation analysis was performed with these 33 genes in 39 MSI-H and 24 microsatellite stable (MSS) colorectal carcinomas by assessing the mobility shifts of PCR products in gel electrophoresis and by sequencing. All the 39 MSI-H colorectal carcinomas, except one, showed mutations in more than one gene, while no mutations were found in 24 MSS colorectal carcinomas. Of these MSI-H tumors, 11 genes were mutated in more than 40%. The most frequently mutated novel genes were MARCKS (72%), FLJ11383 (74%) and TAF1B (82%). Biallelic inactivation in MARCKS and FLJ11383 was also frequent in MSI-H tumors. The observed mutation frequency of the 11 known target genes was compatible with that found by previous studies. The very high frequency of mutations, biallelic mutations and the predicted truncation of protein products suggests that mutations of MARCKS, FLJ11383 and TAF1B are selected, and play a role in the tumorigenesis of MSI-H colorectal carcinomas.
Asunto(s)
Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Repeticiones de Microsatélite/genética , Mutagénesis/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Secuencia de Bases , Proteínas de Unión al Calcio , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Bases de Datos de Ácidos Nucleicos , Mutación del Sistema de Lectura , Glucosidasas , Humanos , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Reacción en Cadena de la Polimerasa , Secuencias Repetidas Terminales , Células Tumorales CultivadasRESUMEN
Frameshift mutation at coding mononucleotide repeat sequences are common in tumors with microsatellite instability (MSI-H), but the incidences are different among the target genes. We analyzed the mutational profiles of 12 known target genes containing polydeoxyadenosine repeats in their coding sequences in 39 MSI-H colorectal carcinomas and 40 MSI-H gastric carcinomas by using polymerase chain reaction and sequencing, and compared the results with the clinicopathologic characteristics. Frameshift mutations of target genes in the MSI-H colorectal and gastric carcinomas are increased according to the length of the polydeoxyadenosine repeats in the target genes. The mean mutational rates of MSI-H colorectal carcinomas and MSI-H gastric carcinomas were 2.03 and 1.95 in the 4 genes containing (A)10 repeats, 1.23 and 0.73 in the 4 genes with (A)9 repeats and 0.61 and 0.48 in the 4 genes containing (A)8 repeats, respectively (p<0.001). Among the evaluated clinicopathologic findings, intestinal type gastric carcinomas had more frameshift mutations than the diffuse type carcinomas (3.5 vs. 1.9, p=0.01). These findings suggest that mutational rates of the target genes in MSI-H tumors are diverse, and higher mutational rates are related to the length of mononucleotide repeat sequences of the target genes and histologic type of tumors.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Mutación del Sistema de Lectura , Repeticiones de Microsatélite/genética , Proteínas de Neoplasias/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana Edad , Mucinas/metabolismo , Estadificación de Neoplasias , Sistemas de Lectura Abierta , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: To examine the antagonistic effects of anti-extracellular matrix metalloprotease inducer (anti-EMMPRIN) antibody when combined with chemotherapy using a hypovascular pancreatic tumor model. PROCEDURES: Severely compromised immunodeficient mice bearing orthotopic MIA PaCa-2 tumors were used (five to six animals per group). Dynamic contrast-enhanced magnetic resonance imaging was used to examine the relationship between tumor vascularity and size. Therapy was initiated when tumors were hypovascular. Treatments included: (1) gemcitabine alone, (2) anti-EMMPRIN antibody alone, and (3) combination, each for 2 weeks. Additionally, another treatment arm included ß-lapachone, an NAD(P)H/quinone 1 (NQO1) bioactivated agent. (18)F-fluoro-D-glucose-positron emission tomography/computed tomography imaging was used weekly to monitor therapeutic effects. RESULTS: Gemcitabine or anti-EMMPRIN monotherapy significantly delayed tumor growth, but the combination therapy showed an antagonistic effect. Similarly, tumor growth was significantly suppressed by ß-lapachone alone, and additive effects were noted when combined with gemcitabine, but the therapeutic efficacy was reduced when anti-EMMPRIN antibody was added. CONCLUSIONS: Anti-EMMPRIN antibody with chemotherapy in hypovascular tumors results in antagonistic effects.
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Anticuerpos Antineoplásicos/inmunología , Basigina/inmunología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Desoxiglucosa , Sistemas de Liberación de Medicamentos , Femenino , Ratones , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/inmunología , Tomografía de Emisión de Positrones , Carga Tumoral/efectos de los fármacos , GemcitabinaRESUMEN
AIMS: ß-Lapachone (ß-lap), a novel radiosensitizer with potent antitumor efficacy alone, selectively kills solid cancers that over-express NAD(P)H: quinone oxidoreductase 1 (NQO1). Since breast or other solid cancers have heterogeneous NQO1 expression, therapies that reduce the resistance (e.g., NQO1(low)) of tumor cells will have significant clinical advantages. We tested whether NQO1-proficient (NQO1(+)) cells generated sufficient hydrogen peroxide (H2O2) after ß-lap treatment to elicit bystander effects, DNA damage, and cell death in neighboring NQO1(low) cells. RESULTS: ß-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after ß-lap treatment. ß-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to ß-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (ß-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.
Asunto(s)
Efecto Espectador/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/farmacología , Quinonas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones Desnudos , NAD(P)H Deshidrogenasa (Quinona)/deficiencia , NAD(P)H Deshidrogenasa (Quinona)/genética , Oxidación-Reducción/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. ß-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of ß-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where â¼120 moles of superoxide were formed per mole of ß-lapachone in 2 minutes. ß-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of ß-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. ß-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of ß-lapachone and other NQO1 bioactivatable drugs.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/administración & dosificación , Poli(ADP-Ribosa) Polimerasas/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Catalasa/genética , Catalasa/metabolismo , Roturas del ADN de Cadena Simple/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , Necrosis/genética , Necrosis/patología , Poli(ADP-Ribosa) Polimerasa-1 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Agents, such as ß-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than ß-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with ß-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to ß-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.
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Antineoplásicos/farmacología , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Neoplasias/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasas/metabolismo , Quinonas/farmacología , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Humanos , NAD/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , Naftoquinonas/farmacología , Necrosis , Neoplasias/metabolismo , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismoRESUMEN
PURPOSE: Pancreatic cancer is the fourth leading cause of cancer-related deaths, in which the 5-year survival rate is less than 5%. Current standard of care therapies offer little selectivity and high toxicity. Novel, tumor-selective approaches are desperately needed. Although prior work suggested that ß-lapachone (ß-lap) could be used for the treatment of pancreatic cancers, the lack of knowledge of the compound's mechanism of action prevented optimal use of this agent. EXPERIMENTAL DESIGN: We examined the role of NAD(P)H:quinone oxidoreductase-1 (NQO1) in ß-lap-mediated antitumor activity, using a series of MIA PaCa-2 pancreatic cancer clones varying in NQO1 levels by stable shRNA knockdown. The antitumor efficacy of ß-lap was determined using an optimal hydroxypropyl-ß-cyclodextran (HPß-CD) vehicle formulation in metastatic pancreatic cancer models. RESULTS: ß-Lap-mediated cell death required â¼90 enzymatic units of NQO1. Essential downstream mediators of lethality were as follows: (i) reactive oxygen species (ROS); (ii) single-strand DNA breaks induced by ROS; (iii) poly(ADP-ribose)polymerase-1 (PARP1) hyperactivation; (iv) dramatic NAD(+)/ATP depletion; and (v) programmed necrosis. We showed that 1 regimen of ß-lap therapy (5 treatments every other day) efficaciously regressed and reduced human pancreatic tumor burden and dramatically extended the survival of athymic mice, using metastatic pancreatic cancer models. CONCLUSIONS: Because NQO1 enzyme activities are easily measured and commonly overexpressed (i.e., >70%) in pancreatic cancers 5- to 10-fold above normal tissue, strategies using ß-lap to efficaciously treat pancreatic cancers are indicated. On the basis of optimal drug formulation and efficacious antitumor efficacy, such a therapy should be extremely safe and not accompanied with normal tissue toxicity or hemolytic anemia.
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Antineoplásicos/uso terapéutico , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Muerte Celular , Línea Celular Tumoral , Daño del ADN , Evaluación Preclínica de Medicamentos , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Naftoquinonas/uso terapéutico , Neoplasias Pancreáticas/enzimología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The clinical experimental agent, ß-lapachone (ß-lap; Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether ß-lap may warrant clinical evaluation as a radiosensitizer. ß-Lap killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species, irreversible DNA single-strand breaks (SSB), poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, NAD(+)/ATP depletion, and µ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), ß-lap radiosensitized NQO1(+) prostate cancer cells under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSB level, γ-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of µ-calpain-induced programmed cell death. Radiosensitization by ß-lap was blocked by the NQO1 inhibitor dicoumarol or the PARP-1 inhibitor DPQ. In a mouse xenograft model of prostate cancer, ß-lap synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in â¼60% of human prostate tumors evaluated relative to adjacent normal tissue, where ß-lap might be efficacious alone or in combination with radiation. Our findings offer a rationale for the clinical utilization of ß-lap (Arq 501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation.
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Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Apoptosis , Muerte Celular , Ensayo de Unidades Formadoras de Colonias , Ensayo Cometa , Daño del ADN , ADN de Neoplasias/genética , Dicumarol/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Glutatión/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Desnudos , Naftoquinonas/uso terapéutico , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/efectos de la radiación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Análisis de RegresiónRESUMEN
The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.
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Antimetabolitos Antineoplásicos/farmacología , Reparación de la Incompatibilidad de ADN , Fluorouracilo/farmacología , Neoplasias/tratamiento farmacológico , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Fluorouracilo/metabolismo , Fluorouracilo/uso terapéutico , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Transducción de SeñalRESUMEN
Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Fase G2 , Humanos , Modelos Biológicos , Modelos Genéticos , Homólogo 1 de la Proteína MutL , Transducción de Señal , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.
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Reparación del ADN , Proteínas Proto-Oncogénicas c-abl/fisiología , Antineoplásicos/farmacología , Disparidad de Par Base , Benzamidas , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Relación Dosis-Respuesta a Droga , Fase G2 , Humanos , Mesilato de Imatinib , Metilnitronitrosoguanidina/farmacología , Modelos Biológicos , Proteínas Nucleares/metabolismo , Piperazinas , Proteínas Proto-Oncogénicas c-abl/metabolismo , Pirimidinas/farmacología , Transducción de SeñalRESUMEN
Recent molecular genetic studies have revealed that two major types of genomic instabilities, chromosomal instability and microsatellite instability, exist in the colorectal carcinomas. To clarify the relationship between chromosomal abnormalities and mismatch repair gene defects in colorectal carcinomas, we performed a chromosomal analysis on 39 colorectal carcinomas with high-microsatellite instability (MSI-H) and compared the results obtained with those of 20 right-sided microsatellite-stable (MSS) colorectal carcinomas. Chromosomal imbalances (CIs) in MSS colorectal carcinomas were more frequent than in MSI-H colon carcinomas by comparative genomic hybridization analysis (70% versus 31%, P = 0.004). The CI patterns of MSI-H and MSS carcinomas were different. Frequent CIs in MSI-H colon carcinomas were gains of 4q (15%) and 8q (8%), and losses of 9q (21%), 1p (18%), and 11q (18%). In contrast, frequent CIs in right-sided MSS colon carcinomas were gains of 8q (50%), 13q (35%), and 20q (25%), and losses of 18q (55%), 15q (35%), and 17p (30%). We compared the mutation status of 45 target genes and CIs in our MSI-H tumors. Among these 45 target genes, mutation of hRAD50, a member of the DNA repair genes, and FLJ11383 were significantly related to MSI-H colorectal carcinomas with CIs (P = 0.01 and P = 0.02, respectively). Our findings indicate that unique CIs were present in a subset of MSI-H colorectal carcinomas and that these CIs are related to the mutation of several target genes, especially of hRAD50.
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Desequilibrio Alélico , Cromosomas/genética , Neoplasias Colorrectales/genética , Enzimas Reparadoras del ADN , Ácido Anhídrido Hidrolasas , Neoplasias Colorrectales/patología , ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Mutación del Sistema de Lectura , Frecuencia de los Genes , Humanos , Masculino , Repeticiones de Microsatélite/genéticaRESUMEN
The histopathologic type of lung cancer is known to be correlated with tumor behavior and prognosis. However, this classification is subjective and no specific molecular markers have been identified. The aim of this study was to identify protein markers in different types of nonsmall cell lung cancers. Two-dimensional polyacrylamide gel electrophoresis analysis was performed with paired samples of three squamous cell carcinomas, three adenocarcinomas, four large cell carcinomas, and four basaloid carcinomas. We found that 25 proteins in 14 cases of lung cancer were differentially expressed compared to matched nontumorous lung tissues. Among these 25 proteins, 11 proteins were down-regulated and 14 were up-regulated in these four types of lung cancer. Alloalbumin venezia, selenium-binding protein 1, carbonic dehydratase, heat shock 20KD-like protein, and SM22 alpha protein were down-regulated in all 14 cases of lung cancer examined, whereas alpha enolase was consistently up-regulated. Supervised hierarchical cluster analysis based on the 25 differentially expressed proteins showed that basaloid carcinoma formed one independent group, whereas the other three cancer types were not uniquely classifiable. Our findings suggest that basaloid carcinoma is a unique subtype of nonsmall cell lung carcinoma.