RESUMEN
Metastatic progression is the key feature of prostate cancer primarily responsible for mortality caused by this disease. RAD9 is an oncogene for prostate cancer, and the encoded protein enhances metastasis-related phenotypes. RAD9 is a transcription factor with a limited set of regulated target genes, but the complete list of downstream genes critical for prostate carcinogenesis is unknown. We used microarray gene expression profiling and chromatin immunoprecipitation in parallel to identify genes transcriptionally controlled by RAD9 that contribute to this cancer. We found expression of 44 genes altered in human prostate cancer DU145 cells when RAD9 is knocked down by siRNA, and all of them bind RAD9 at their genomic location. FOXP1 and NDRG1 were down regulated when RAD9 expression was reduced, and we evaluated them further. We demonstrate that reduced RAD9, FOXP1 or NDGR1 expression decreases cell proliferation, rapid migration, anchorage-independent growth, anoikis resistance, and aerobic glycolysis. Ectopic expression of FOXP1 or NDRG1 partially restored aerobic glycolysis to prostate cancer cells with reduced RAD9 abundance, but only FOXP1 significantly complemented the other deficiencies. We thus show, for the first time, that RAD9 regulates FOXP1 and NDRG1 expression, and they function differently as downstream effectors for RAD9-mediated prostate cancer cell activities.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias de la Próstata , Línea Celular Tumoral , Proliferación Celular , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/patología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Prostate cancer is the second most common type of cancer and the second leading cause of cancer death in American men. RAD9 stabilizes the genome, but prostate cancer cells and tumors often have high quantities of the protein. Reduction of RAD9 level within prostate cancer cells decreases tumorigenicity of nude mouse xenographs and metastasis phenotypes in culture, indicating that RAD9 overproduction is essential for the disease. In prostate cancer DU145 cells, CpG hypermethylation in a transcription suppressor site of RAD9 intron 2 causes high-level gene expression. Herein, we demonstrate that DNA methyltransferases DNMT1 and DNMT3B are highly abundant in prostate cancer cells DU145, CWR22, LNCaP and PC-3; yet, these DNMTs bind primarily to the transcription suppressor in DU145, the only cells where methylation is critical for RAD9 regulation. For DU145 cells, DNMT1 or DNMT3B shRNA reduced RAD9 level and tumorigenicity, and RAD9 ectopic expression restored this latter activity in the DNMT knockdown cells. High levels of RAD9, DNMT1, DNMT3B and RAD9 transcription suppressor hypermethylation were significantly correlated in prostate tumors, and not in normal prostate tissues. Based on these results, we propose a novel model where RAD9 is regulated epigenetically by DNMT1 and DNMT3B, via targeted hypermethylation, and that consequent RAD9 overproduction promotes prostate tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Neoplasias de la Próstata/genética , Animales , Línea Celular Tumoral , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Próstata/patología , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de Xenoinjerto , ADN Metiltransferasa 3BRESUMEN
Radiotherapy is commonly used to treat a variety of solid human tumors, including localized prostate cancer. However, treatment failure often ensues due to tumor intrinsic or acquired radioresistance. Here we find that the MEK5/ERK5 signaling pathway is associated with resistance to genotoxic stress in aggressive prostate cancer cells. MEK5 knockdown by RNA interference sensitizes prostate cancer cells to ionizing radiation (IR) and etoposide treatment, as assessed by clonogenic survival and short-term proliferation assays. Mechanistically, MEK5 downregulation impairs phosphorylation of the catalytic subunit of DNA-PK at serine 2056 in response to IR or etoposide treatment. Although MEK5 knockdown does not influence the initial appearance of radiation- and etoposide-induced γH2AX and 53BP1 foci, it markedly delays their resolution, indicating a DNA repair defect. A cell-based assay shows that nonhomologous end joining (NHEJ) is compromised in cells with ablated MEK5 protein expression. Finally, MEK5 silencing combined with focal irradiation causes strong inhibition of tumor growth in mouse xenografts, compared with MEK5 depletion or radiation alone. These findings reveal a convergence between MEK5 signaling and DNA repair by NHEJ in conferring resistance to genotoxic stress in advanced prostate cancer and suggest targeting MEK5 as an effective therapeutic intervention in the management of this disease.
Asunto(s)
Antineoplásicos/farmacología , Reparación del ADN por Unión de Extremidades , ADN de Neoplasias/efectos de los fármacos , Resistencia a Antineoplásicos/genética , MAP Quinasa Quinasa 5/genética , Mutágenos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Técnicas de Silenciamiento del Gen , Humanos , MAP Quinasa Quinasa 5/antagonistas & inhibidores , MAP Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Masculino , Ratones , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/radioterapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RAD9A plays an important role in prostate tumorigenesis and metastasis-related phenotypes. The protein classically functions as part of the RAD9A-HUS1-RAD1 complex but can also act independently. RAD9A can selectively transactivate multiple genes, including CDKN1A and NEIL1 by binding p53-consensus sequences in or near promoters. RAD9A is overexpressed in human prostate cancer specimens and cell lines; its expression correlates with tumor progression. Silencing RAD9A in prostate cancer cells impairs their ability to form tumors in vivo and migrate as well as grow anchorage independently in vitro. We demonstrate herein that RAD9A transcriptionally controls AGR2, a gene aberrantly overexpressed in patients with metastatic prostate cancer. Transient or stable knockdown of RAD9A in PC-3 cells caused downregulation of AGR2 protein abundance. Reduced AGR2 protein levels were due to lower abundance of AGR2 mRNA. The AGR2 genomic region upstream of the coding initiation site contains several p53 consensus sequences. RAD9A bound specifically to the 5'-untranslated region of AGR2 in PC-3 cells at a partial p53 consensus sequence at position +3136 downstream from the transcription start site, determined by chromatin immunoprecipitation, followed by PCR amplification. Binding of RAD9A to the p53 consensus sequence was sufficient to drive AGR2 gene transcription, shown by a luciferase reporter assay. In contrast, when the RAD9A-binding sequence on the AGR2 was mutated, no luciferase activity was detected. Knockdown of RAD9A in PC-3 cells impaired cell migration and anchorage-independent growth. However, ectopically expressed AGR2 in RAD9A-depleted PC-3 cells restored these phenotypes. Our results suggest RAD9A drives metastasis by controlling AGR2 abundance.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Neoplasias de la Próstata/patología , Proteínas/genética , Línea Celular Tumoral , Movimiento Celular , Humanos , Masculino , Mucoproteínas , Metástasis de la Neoplasia , Proteínas Oncogénicas , Fenotipo , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
Prostate cancer is a complex disease, with multiple subtypes and clinical presentations. Much progress has been made in recent years to understand the underlying genetic basis that drives prostate cancer. Such mechanistic information is useful for development of novel therapeutic targets, to identify biomarkers for early detection or to distinguish between aggressive and indolent disease, and to predict treatment outcome. Multiple tests have become available in recent years to address these clinical needs for prostate cancer. We describe several of these assays, summarizing test details, performance characteristics, and acknowledging their limitations. There is a pressing unmet need for novel biomarkers that can demonstrate improvement in these areas. We introduce one such candidate biomarker, RAD9, describe its functions in the DNA damage response, and detail why it can potentially fill this void. RAD9 has multiple roles in prostate carcinogenesis, making it potentially useful as a clinical tool for men with prostate cancer. RAD9 was originally identified as a radioresistance gene, and subsequent investigations revealed several key functions in the response of cells to DNA damage, including involvement in cell cycle checkpoint control, at least five DNA repair pathways, and apoptosis. Further studies indicated aberrant overexpression in approximately 45% of prostate tumors, with a strong correlation between RAD9 abundance and cancer stage. A causal relationship between RAD9 and prostate cancer was first demonstrated using a mouse model, where tumorigenicity of human prostate cancer cells after subcutaneous injection into nude mice was diminished when RNA interference was used to reduce the normally high levels of the protein. In addition to activity needed for the initial development of tumors, cell culture studies indicated roles for RAD9 in promoting prostate cancer progression by controlling cell migration and invasion through regulation of ITGB1 protein levels, and anoikis resistance by modulating AKT activation. Furthermore, RAD9 enhances the resistance of human prostate cancer cells to radiation in part by regulating ITGB1 protein abundance. RAD9 binds androgen receptor and inhibits androgen-induced androgen receptor's activity as a transcription factor. Moreover, RAD9 also acts as a gene-specific transcription factor, through binding p53 consensus sequences at target gene promoters, and this likely contributes to its oncogenic activity. Given these diverse and extensive activities, RAD9 plays important roles in the initiation and progression of prostate cancer and can potentially serve as a valuable biomarker useful in the management of patients with this disease.
RESUMEN
The way cells respond to DNA damage is important since inefficient repair or misrepair of lesions can have deleterious consequences, including mutation, genomic instability, neurodegenerative disorders, premature aging, cancer or death. Whether damage occurs spontaneously as a byproduct of normal metabolic processes, or after exposure to exogenous agents, cells muster a coordinated, complex DNA damage response (DDR) to mitigate potential harmful effects. A variety of activities are involved to promote cell survival, and include DNA repair, DNA damage tolerance, as well as transient cell cycle arrest to provide time for repair before entry into critical cell cycle phases, an event that could be lethal if traversal occurs while damage is present. When such damage is prolonged or not repairable, senescence, apoptosis or autophagy is induced. One major level of DDR regulation occurs via the orchestrated transcriptional control of select sets of genes encoding proteins that mediate the response. p53 is a transcription factor that transactivates specific DDR downstream genes through binding DNA consensus sequences usually in or near target gene promoter regions. The profile of p53-regulated genes activated at any given time varies, and is dependent upon type of DNA damage or stress experienced, exact composition of the consensus DNA binding sequence, presence of other DNA binding proteins, as well as cell context. RAD9 is another protein critical for the response of cells to DNA damage, and can also selectively regulate gene transcription. The limited studies addressing the role of RAD9 in transcription regulation indicate that the protein transactivates at least one of its target genes, p21/waf1/cip1, by binding to DNA sequences demonstrated to be a p53 response element. NEIL1 is also regulated by RAD9 through a similar DNA sequence, though not yet directly verified as a bonafide p53 response element. These findings suggest a novel pathway whereby p53 and RAD9 control the DDR through a shared mechanism involving an overlapping network of downstream target genes. Details and unresolved questions about how these proteins coordinate or compete to execute the DDR through transcriptional reprogramming, as well as biological implications, are discussed.
Asunto(s)
Proteínas de Ciclo Celular/genética , Daño del ADN , Redes Reguladoras de Genes , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Inestabilidad Genómica , Humanos , Transcripción GenéticaRESUMEN
RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Glicosilasas/metabolismo , Reparación del ADN , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , ADN Glicosilasas/biosíntesis , ADN Glicosilasas/genética , Células Madre Embrionarias/metabolismo , Masculino , Ratones , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Radiation induced bystander effects are an important component of the overall response of cells to irradiation and are associated with human health risks. The mechanism responsible includes intra-cellular and inter-cellular signaling by which the bystander response is propagated. However, details of the signaling mechanism are not well defined. METHODS: We measured the bystander response of Mrad9+/+ and Mrad9-/- mouse embryonic stem cells, as well as human H1299 cells with inherent or RNA interference-mediated reduced RAD9 levels after exposure to 1 Gy α particles, by scoring chromosomal aberrations and micronuclei formation, respectively. In addition, we used microarray gene expression analyses to profile the transcriptome of directly irradiated and bystander H1299 cells. RESULTS: We demonstrated that Mrad9 null enhances chromatid aberration frequency induced by radiation in bystander mouse embryonic stem cells. In addition, we found that H1299 cells with reduced RAD9 protein levels showed a higher frequency of radiation induced bystander micronuclei formation, compared with parental cells containing inherent levels of RAD9. The enhanced bystander response in human cells was associated with a unique transcriptomic profile. In unirradiated cells, RAD9 reduction broadly affected stress response pathways at the mRNA level; there was reduction in transcript levels corresponding to genes encoding multiple members of the UVA-MAPK and p38MAPK families, such as STAT1 and PARP1, suggesting that these signaling mechanisms may not function optimally when RAD9 is reduced. Using network analysis, we found that differential activation of the SP1 and NUPR1 transcriptional regulators was predicted in directly irradiated and bystander H1299 cells. Transcription factor prediction analysis also implied that HIF1α (Hypoxia induced factor 1 alpha) activation by protein stabilization in irradiated cells could be a negative predictor of the bystander response, suggesting that local hypoxic stress experienced by cells directly exposed to radiation may influence whether or not they will elicit a bystander response in neighboring cells.
Asunto(s)
Efecto Espectador/genética , Proteínas de Ciclo Celular/deficiencia , Daño del ADN/genética , Traumatismos Experimentales por Radiación/genética , Transcriptoma/efectos de la radiación , Animales , Efecto Espectador/efectos de la radiación , Proteínas de Ciclo Celular/genética , Línea Celular , Aberraciones Cromosómicas , Daño del ADN/efectos de la radiación , Células Madre Embrionarias/efectos de la radiación , Humanos , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Mouse embryonic stem cells null for Rad9 are sensitive to deleterious effects of ionizing radiation exposure. Likewise, integrin ß1 is a known radioprotective factor. Previously, we showed that RAD9 downregulation in human prostate cancer cells reduces integrin ß1 protein levels and ectopic expression of Mrad9 restores inherent high levels. METHODS: We used RNA interference to knockdown Rad9 expression in PC3 and DU145 prostate cancer cells. These cells were then exposed to ionizing radiation, and integrin ß1 protein levels were measured by immunoblotting. Survival of irradiated cells was measured by clonogenicity, cell cycle analysis, PARP-1 cleavage, and trypan blue exclusion. RESULTS: The function of RAD9 in controlling integrin ß1 expression is unique and not shared by the other members of the 9-1-1 complex, HUS1 and RAD1. RAD9 or integrin ß1 silencing sensitizes DU145 and PC3 cells to ionizing radiation. Irradiation of DU145 cells with low levels of RAD9 induces cleavage of PARP-1 protein. High levels of ionizing radiation have no effect on integrin ß1 protein levels. However, when RAD9 downregulation is combined with 10 Gy of ionizing radiation in DU145 or PC3 cells, there is an additional 50% downregulation of integrin ß1 compared with levels in unirradiated RAD9 knockdown cells. Finally, PC3 cells growing on fibronectin display increased radioresistance. However, PC3 cells with RAD9 knockdown are no longer protected by fibronectin after treatment with ionizing radiation. CONCLUSIONS: Downregulation of RAD9 when combined with ionizing radiation results in reduction of ITGB1 protein levels in prostate cancer cells, and increased lethality.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Integrina beta1/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Exonucleasas/genética , Exonucleasas/metabolismo , Fibronectinas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/genética , Tolerancia a RadiaciónRESUMEN
DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-)factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genome-wide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo.
Asunto(s)
Daño del ADN/genética , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Neoplasias/genética , Neoplasias/patología , Animales , Carcinogénesis/genética , Puntos de Control del Ciclo Celular/genética , Reparación del ADN/genética , HumanosRESUMEN
The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Exonucleasas/metabolismo , Meiosis , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Reparación del ADN , Fertilidad , Células Germinativas/metabolismo , Masculino , Ratones , Recombinación Genética , Transducción de SeñalRESUMEN
In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIß-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Fertilidad/genética , Profase Meiótica I/fisiología , Reparación del ADN por Recombinación/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis/genética , Proteínas Portadoras/metabolismo , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/biosíntesis , Histonas/biosíntesis , Histonas/metabolismo , Masculino , Profase Meiótica I/genética , Ratones , Ratones Transgénicos , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Proteínas de Unión a Fosfato , Eliminación de Secuencia/genética , Espermatocitos/citología , Espermatogénesis/genética , TestículoRESUMEN
Platinum compounds are the foundation of chemotherapy regimens for non-small cell lung cancer (NSCLC) despite poor response rates and limited response duration. It has been reported that tumor expression of excision repair cross-complementation group 1 (ERCC1), a key component in nucleotide excision repair, may correlate with clinical response to platinum agents. We found that most primary lung tumor specimens demonstrated a stronger protein expression of poly (adenosine diphosphate ribose) polymerases 1 (PARP1) than their normal counterparts. Therefore, we hypothesized that combining PARP inhibition with platinum compounds may be an approach to improve platinum-based therapy for NSCLC. Drug combination experiments revealed that two distinct PARP inhibitors, olaparib and veliparib, not only potentiated the cell killing by cisplatin but also conferred cytotoxicity as a single agent specifically in ERCC1-low HCC827 and PC9 but not in ERCC1-high A549 and H157 lung cancer cells. Moreover, small interfering RNA knockdown of ERCC1 in A549 and H157 cells increased their sensitivities to both cisplatin and olaparib in a synergistic manner in our model. Furthermore, mechanistic studies indicated that combined PARP inhibitor and cisplatin could lead to sustained DNA double-strand breaks, prolonged G2/M cell cycle arrest with distinct activation of checkpoint kinase 1 signaling and more pronounced apoptosis preferentially in lung cancer cells with low ERCC1 expression. Collectively, these data suggest that there is a synergistic relationship between PARP inhibition and low ERCC1 expression in NSCLC that could be exploited for novel therapeutic approaches in lung cancer therapy based on tumor ERCC1 expression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Proteínas de Unión al ADN/biosíntesis , Endonucleasas/biosíntesis , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Roturas del ADN de Doble Cadena/efectos de los fármacos , Proteínas de Unión al ADN/genética , Sinergismo Farmacológico , Endonucleasas/genética , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Proteínas Quinasas/metabolismo , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Rad9 as part of the Rad9-Hus1-Rad1 complex is known to participate in cell cycle checkpoint activation and DNA repair. However, Rad9 can act as a sequence-specific transcription factor, modulating expression of a number of genes. Importantly, Rad9 is up-regulated in prostate cancer cell lines and clinical specimens. Its expression correlates positively with advanced stage tumors and its down-regulation reduces tumor burden in mice. We show here that transient down-regulation of Rad9 by RNA interference reduces DU145 and PC3 prostate cancer cell proliferation and survival in vitro. In addition, transient or stable down-regulation of Rad9 impairs migration and invasion of the cells. Moreover, stable reduction of Rad9 renders DU145 cell growth anchorage-dependent. It also decreases expression of integrin ß1 protein and sensitizes DU145 and LNCaP cells to anoikis and impairs Akt activation. On the other hand, stable expression of Mrad9, the mouse homolog, in DU145/shRNA Rad9 cells restores migration, invasion, anchorage-independent growth, integrin ß1 expression, and anoikis resistance with a concomitant elevation of Akt activation. We thus demonstrate for the first time that Rad9 contributes to prostate tumorigenesis by increasing not only tumor proliferation and survival but also tumor migration and invasion, anoikis resistance, and anchorage-independent growth.
Asunto(s)
Anoicis , Proteínas de Ciclo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/patología , Adhesión Celular , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Reparación del ADN , Progresión de la Enfermedad , Regulación hacia Abajo , Silenciador del Gen , Humanos , Integrina beta1/biosíntesis , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , Interferencia de ARN , Transducción de SeñalRESUMEN
When cells are exposed to a dose of radiation large enough to cause chromosome aberrations, they become arrested at the G(2)/M checkpoint, facilitating DNA repair. Defects in checkpoint control genes can impart radiosensitivity. Arrest kinetics were monitored in mouse embryo fibroblasts at doses ranging from 10 mGy to 5.0 Gy of γ radiation over a time course of 0 to 12 h. We observe no significant checkpoint engagement at doses below 100 mGy. The checkpoint is only fully activated at doses where most of the cells are either bound for mitotic catastrophe or are reproductively dead. Atm null cells with ablated checkpoint function exhibited no robust arrest. Surprisingly, haploinsufficiency for ATM alone or in combination with other radioresistance genes did not alter checkpoint activation. We have shown previously that haploinsufficiency for several radioresistance genes imparts intermediate phenotypes for several end points including apoptosis, transformation and survival. These findings suggest that checkpoint control does not contribute toward these intermediate phenotypes and that different biological processes can be activated at high doses compared to low doses.
Asunto(s)
Fibroblastos/citología , Fibroblastos/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Haploinsuficiencia/genética , Puntos de Control de la Fase M del Ciclo Celular/genética , Puntos de Control de la Fase M del Ciclo Celular/efectos de la radiación , Animales , Relación Dosis-Respuesta en la Radiación , Embrión de Mamíferos , Femenino , Fibroblastos/metabolismo , Ratones , EmbarazoRESUMEN
Rad9 plays a crucial role in maintaining genomic stability by regulating cell cycle checkpoints, DNA repair, telomere stability, and apoptosis. Rad9 controls these processes mainly as part of the heterotrimeric 9-1-1 (Rad9-Hus1-Rad1) complex. However, in recent years it has been demonstrated that Rad9 can also act independently of the 9-1-1 complex as a transcriptional factor, participate in immunoglobulin class switch recombination, and show 3'-5' exonuclease activity. Aberrant Rad9 expression has been associated with prostate, breast, lung, skin, thyroid, and gastric cancers. High expression of Rad9 is causally related to, at least, human prostate cancer growth. On the other hand, deletion of Mrad9, the mouse homolog, is responsible for increased skin cancer incidence. These results reveal that Rad9 can act as an oncogene or tumor suppressor. Which of the many functions of Rad9 are causally related to initiation and progression of tumorigenesis and the mechanistic details by which Rad9 induces or suppresses tumorigenesis are presently not known, but are crucial for the development of targeted therapeutic interventions.