RESUMO
Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers.
Assuntos
Morte Celular/genética , Células Epiteliais/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas ras/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B upregulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B upregulation is of broad oncological interest.
Assuntos
Citidina Desaminase/biossíntese , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/fisiologia , Antígenos de Histocompatibilidade Menor/biossíntese , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Linhagem Celular , Imunoprecipitação da Cromatina , Células Epiteliais/virologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteólise , Fatores de Transcrição de Domínio TEA , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG12V ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Queratinócitos/virologia , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/virologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos , Humanos , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Domínios PDZ/fisiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/patologiaRESUMO
Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.
Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de Replicação , Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/química , Cromatina/química , Replicação do DNA , Células HEK293 , Células HeLa , Humanos , Lamina Tipo B/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Proteínas Nucleares/metabolismoRESUMO
UNLABELLED: NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE: A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.
Assuntos
Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/metabolismo , Replicação Viral , Células Cultivadas , Papillomavirus Humano 16/fisiologia , Humanos , Queratinócitos/virologia , ProteóliseRESUMO
Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.
Assuntos
Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Oncogenes/fisiologia , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Animais , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Técnicas de Cultura de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação/genética , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Neoplasias PancreáticasRESUMO
Although nuclear actin and Arps (actin-related proteins) are often identified as components of multi-protein chromatin-modifying enzyme complexes, such as chromatin remodeling and histone acetyltransferase (HAT) complexes, their molecular functions still remain largely elusive. Here, we investigated the role of human Arp4 (BAF53, also known as actin-like protein 6A) in Brg1-containing chromatin remodeling complexes. Depletion of Arp4 by RNA interference impaired the integrity of these complexes and accelerated the degradation of Brg1, indicating a crucial role in their maintenance, at least in certain human cell lines. We further found that Arp4 can form a heterocomplex with ß-actin. Based on structural similarities between conventional actin and Arp4, and the assumption that actin-Arp4 binding might mimic actin-actin binding, we introduced a series of mutations in Arp4 that might be expected to impair its interaction with ß-actin. Some of them indeed caused reduced binding to ß-actin. Interestingly, such mutant Arp4 proteins also showed reduced incorporation into Brg1 complexes, and their interaction with Myc-associated complexes as well as Tip60 HAT complexes were also impaired. Based on these findings, we propose that ß-actin-Arp4 complex formation might be a crucial feature in some chromatin-modifying enzyme complexes, such as the Brg1 complex.
Assuntos
Actinas/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Actinas/química , Actinas/deficiência , Actinas/genética , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/genética , TransfecçãoRESUMO
Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16.
Assuntos
Genoma Viral , Papillomavirus Humano 16/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Replicação Viral , Linhagem Celular , Replicação do DNA , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogênicas Virais/genéticaRESUMO
Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/genética , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Colo do Útero , Carcinogênese/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genéticaRESUMO
Human papillomaviruses (HPVs) are the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 is considered to contribute to disease initiation. Recently, we have demonstrated that transduction of oncogenic HRAS (HRAS(G12V)) and MYC together with HPV16 E6E7 is sufficient for tumorigenic transformation of normal human cervical keratinocytes (HCKs). Here, we show that transduction of HRAS(G12V) on the background of E6E7 expression causes accumulation of MYC protein and tumorigenic transformation of not only normal HCKs but also other normal primary human cells, including tongue keratinocytes and bronchial epithelial cells as well as hTERT-immortalized foreskin fibroblasts. Subcutaneous transplantation of as few as 200 HCKs expressing E6E7 and HRAS(G12V) resulted in tumor formation within 2 months. Dissecting RAS signaling pathways, constitutively active forms of AKT1 or MEK1 did not result in tumor formation with E6E7, but tumorigenic transformation was induced with addition of MYC. Increased MYC expression endowed resistance to calcium- and serum-induced terminal differentiation and activated the mammalian target of rapamycin (mTOR) pathway. An mTOR inhibitor (Rapamycin) and MYC inhibition a level not affecting proliferation in culture both markedly suppressed tumor formation by HCKs expressing E6E7 and HRAS(G12V). These results suggest that a single mutation of HRAS could be oncogenic in the background of deregulated expression of E6E7 and MYC plays a critical role in cooperation with the RAS signaling pathways in tumorigenesis. Thus inhibition of MYC and/or the downstream mTOR pathway could be a therapeutic strategy not only for the MYC-altered but also RAS-activated cancers.
Assuntos
Transformação Celular Neoplásica , Papillomavirus Humano 16/fisiologia , Proteínas E7 de Papillomavirus/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Células Cultivadas , HumanosRESUMO
From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/fisiologia , Fase G1/fisiologia , Complexos Multiproteicos/metabolismo , Origem de Replicação/fisiologia , Fase S/fisiologia , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/genética , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Ligação ProteicaRESUMO
CDC6, a replication licensing protein, is partially exported to the cytoplasm in human cells through phosphorylation by Cdk during S phase, but a significant proportion remains in the nucleus. We report here that human CDC6 physically interacts with ATR, a crucial checkpoint kinase, in a manner that is stimulated by phosphorylation by Cdk. CDC6 silencing by siRNAs affected ATR-dependent inhibition of mitotic entry elicited by modest replication stress. Whereas a Cdk-phosphorylation-mimicking CDC6 mutant could rescue the checkpoint defect by CDC6 silencing, a phosphorylation-deficient mutant could not. Furthermore, we found that the CDC6-ATR interaction is conserved in Xenopus. We show that the presence of Xenopus CDC6 during S phase is essential for Xenopus ATR to bind to chromatin in response to replication inhibition. In addition, when human CDC6 amino acid fragment 180-220, which can bind to both human and Xenopus ATR, was added to Xenopus egg extracts after assembly of the pre-replication complex, Xenopus Chk1 phosphorylation was significantly reduced without lowering replication, probably through a sequestration of CDC6-mediated ATR-chromatin interaction. Thus, CDC6 might regulate replication-checkpoint activation through the interaction with ATR in higher eukaryotic cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Células Eucarióticas/enzimologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Extratos Celulares , Quinase 1 do Ponto de Checagem , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Células Eucarióticas/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Mutação/genética , Óvulo/citologia , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Estresse FisiológicoRESUMO
Infection by high-risk human papillomaviruses (hrHPVs), including HPV type 16 (HPV16), is a major risk factor for oral squamous cell carcinomas (OSCCs). However, the pathogenic mechanism by which hrHPVs promote oral carcinogenesis remains to be elucidated. Here, we demonstrated that the suppression of a transporter associated with the antigen-processing complex (TAPs; TAP1 and TAP2), which is a key molecule in the transportation of viral antigenic peptides into MHC class-I cells, is affected by the E6 protein of HPV16. Mechanistically, HPV-mediated immune evasion is principally mediated via the signal-transduction network of a lymphotoxin (LT) pathway, in particular LTα1ß2 and LTßR. Our analysis of transcriptomic data from an HNSCC cohort from the Cancer Genome Atlas (TCGA) indicated that expression of TAP genes, particularly TAP2, was downregulated in HPV-infected cases. We further demonstrated that LTα1ß2 and LTßR were upregulated, which was negatively correlated with TAP1 and TAP2 expression in HPV-positive clinical OSCC samples. Taken together, our findings imply that HPV16 E6 regulates the machinery of the antigenic peptide-loading system and helps to clarify the role of oncogenic viruses in the context of oral carcinoma.
RESUMO
The E6 protein of cervical cancer-associated human papillomaviruses (HPVs) is known to suppress keratinocyte differentiation through unidentified mechanisms. Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis. Here, we report that the Notch1 gene is a novel target of p53 and can be down-regulated by E6 through p53 degradation in normal human epithelial cells. Thus, inactivation of p53 by E6 or short-hairpin RNA (shRNA) resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. The expression of E6, p53 shRNA, or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and its induction upon DNA damage. Furthermore, the induction of Notch1 and differentiation makers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type but not in p53-deficient mouse skin. Together, our findings not only demonstrate a novel link between p53 and Notch1 in keratinocyte differentiation upon genotoxic stress but also suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors.
Assuntos
Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Receptor Notch1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Dano ao DNA , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , RNA/química , RNA/metabolismo , Receptor Notch1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
Ovarian surface epithelium (OSE) is considered to give rise to epithelial ovarian carcinomas (EOCs). To elucidate early processes contributing to the development of EOCs from the OSE, two batches of primary human OSE cells were transduced with non-viral human genes (mutant Cdk4, cyclinD1 and hTERT) so as to efficiently establish normal diploid OSE cells without chromosomal instability. Then defined genetic alterations frequently observed in EOCs were transduced into the OSE cells. A combination of p53 inactivation and oncogenic Kras transduction did not confer tumor-forming ability in immunodeficient mice, though additional transduction of Akt or combined transduction of c-myc with bcl-2 did result in tumor formation. In the latter case, tumors demonstrated phenotypes reminiscent of human EOCs, including cytokeratin expression, a highly aggressive phenotype, metastatic behavior and formation of ascites. These results indicate that inactivation of p53 and activation of the Ras pathway play critical roles in ovarian carcinogenesis in co-operation with the Akt or c-myc pathways. This first in vitro model system faithfully recapitulating the development of EOCs using normal human OSE cells should greatly facilitate further studies of EOCs.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Oncogenes , Neoplasias Ovarianas/metabolismo , Animais , Transformação Celular Neoplásica/patologia , Células Cultivadas , Instabilidade Cromossômica , Ciclina D1/biossíntese , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/biossíntese , Quinase 4 Dependente de Ciclina/genética , Células Epiteliais/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Telomerase/biossíntese , Telomerase/genética , Transdução Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The origin recognition complex (ORC) binds to replication origins to regulate the cell cycle-dependent assembly of pre-replication complexes (pre-RCs). We have found a novel link between pre-RC assembly regulation and telomere homeostasis in human cells. Biochemical analyses showed that human ORC binds to TRF2, a telomere sequence-binding protein that protects telomeres and functions in telomere length homeostasis, via the ORC1 subunit. Immunostaining further revealed that ORC and TRF2 partially co-localize in nuclei, whereas chromatin immunoprecipitation analyses confirmed that pre-RCs are assembled at telomeres in a cell cycle-dependent manner. Over-expression of TRF2 stimulated ORC and MCM binding to chromatin and RNAi-directed TRF2 silencing resulted in reduced ORC binding and pre-RC assembly at telomeres. As expected from previous reports, TRF2 silencing induced telomere elongation. Interestingly, ORC1 silencing by RNAi weakened the TRF2 binding as well as the pre-RC assembly at telomeres, suggesting that ORC and TRF2 interact with each other to achieve stable binding. Furthermore, ORC1 silencing also resulted in modest telomere elongation. These data suggest that ORC might be involved in telomere homeostasis in human cells.
Assuntos
Replicação do DNA , Complexo de Reconhecimento de Origem/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Fibroblastos/metabolismo , Células HeLa/metabolismo , Humanos , Telômero/genéticaRESUMO
The success rate of establishing human cancer cell lines is not satisfactory and the established cell lines often do not preserve the molecular and histological features of the original tissues. In this study, we developed a novel culture method which can support proliferation of almost all primary epithelial ovarian cancer cells, as well as primary normal human oviductal epithelial cells. Cancer cells from fresh or frozen specimens were enriched by the anti-EpCAM antibody-conjugated magnetic beads, plated on Matrigel-coated plate and cultivated under the optimized culture conditions. Seventeen newly established ovarian cancer cell lines, which included all four major histotypes of ovarian cancer, were confirmed to express histotype-specific markers in vitro. Some of the cell lines from all the four histotypes, except mucinous type, generated tumors in immune-deficient mice and the xenograft tumor tissues recapitulated the corresponding original tissues faithfully. Furthermore, with poorly tumorigenic cell lines including mucinous type, we developed a novel xenograft model which could reconstruct the original tissue architecture through forced expression of a set of oncogenes followed by its silencing. With combination of the novel culture method and cell-derived xenograft system, virtually every epithelial ovarian cancer can be reconstituted in mice in a timely fashion.
Assuntos
Carcinoma Epitelial do Ovário/patologia , Cistadenoma Mucinoso/patologia , Neoplasias Ovarianas/patologia , Cultura Primária de Células/métodos , Adulto , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Cistadenoma Mucinoso/genética , Expressão Ectópica do Gene , Feminino , Humanos , Pessoa de Meia-Idade , Oncogenes/genética , Neoplasias Ovarianas/genética , Ovário/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Over the last two decades since discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen, HPVs have been well characterized as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV-positive cervical cancer cells. Their gene products are known to inactivate the major tumor suppressors, p53 and pRB, respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalize human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi-step carcinogenesis. Indeed, it has been shown that only one or two genetic alterations in addition to expression of E6 and E7 are experimentally sufficient to confer tumorigenicity to normal human cervical keratinocytes. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti-cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV-associated malignancies. This review highlights the novel functions of E6 and E7 as well as the molecular mechanisms of HPV-induced carcinogenesis.
Assuntos
Papillomavirus Humano 16 , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Feminino , Inativação Gênica , Genes p53/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Humanos , Proteínas Oncogênicas Virais/fisiologia , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus , Proteínas Repressoras/fisiologia , Risco , Neoplasias do Colo do Útero/prevenção & controleRESUMO
Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27(Kip1) was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF(Skp2) ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF(Skp2) pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , DNA/metabolismo , Fase S , Proteínas de Ciclo Celular/genética , Linhagem Celular , Inativação Gênica , Instabilidade Genômica , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Proteólise , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de SinaisRESUMO
NOTCH plays essential roles in cell fate specification during embryonic development and in adult tissue maintenance. In keratinocytes, it is a key inducer of differentiation. ROCK, an effector of the small GTPase Rho, is also implicated in keratinocyte differentiation, and its inhibition efficiently potentiates immortalization of human keratinocytes and greatly improves survival of dissociated human pluripotent stem cells. However, the molecular basis for ROCK activation is not fully established in these contexts. Here we provide evidence that intracellular forms of NOTCH1 trigger the immediate activation of ROCK1 independent of its transcriptional activity, promoting differentiation and resulting in decreased clonogenicity of normal human keratinocytes. Knockdown of NOTCH1 abrogated ROCK1 activation and conferred sustained clonogenicity upon differentiation stimuli. Treatment with a ROCK inhibitor, Y-27632, or ROCK1 silencing substantially rescued the growth defect induced by activated NOTCH1. Furthermore, we revealed that impaired self-renewal of human induced pluripotent stem cells upon dissociation is, at least in part, attributable to NOTCH-dependent ROCK activation. Thus, the present study unveils a novel NOTCH-ROCK pathway critical for cellular differentiation and loss of self-renewal capacity in a subset of immature cells.