RESUMO
Cervical cancer is the second most common malignancy in women, and the novel therapeutic treatment is needed. Abemaciclib is a FDA-approved drug for breast cancer treatment. In this work, we identified that abemaciclib has potent anti-cervical cancer activity. We demonstrate that abemaciclib is the most effective drug against human papillomavirus (HPV)-negative cervical cancer cells compared to ribociclib and palbociclib, with its IC50 at nanomolar concentration range. This is achieved by the inhibition of proliferation and induction of apoptosis, through specifically suppressing CDK4/6-Rb-E2F and mTOR pathways by abemaciclib in HPV-negative cervical cancer cells. Of note, the combination of abemaciclib with paclitaxel and cisplatin at sublethal concentration results in much greater efficacy than chemotherapy alone. In addition, we confirm the efficacy of abemaciclib and its combination with paclitaxel or cisplatin at the doses that are not toxic to mice in HPV-negative cervical cancer xenograft mouse model. Interestingly, we show that abemaciclib and other CDK4/6 inhibitors are not effective in targeting HPV-positive cervical cancer cells, and this is likely to be associated with the high p16 and low Rb expression in HPV-positive cervical cancer cells. Our work is the first to provide the preclinical evidence to demonstrate the potential of abemaciclib for the treatment of HPV-negative cervical cancer. The mechanism analysis highlights the therapeutic value of inhibiting CDK4/6 in HPV-negative but not HPV-positive cervical cancer.
Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Fatores de Transcrição E2F/antagonistas & inibidores , Proteína do Retinoblastoma/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Alphapapillomavirus/isolamento & purificação , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neoplasias do Colo do Útero/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Neoplasias Hepáticas/metabolismo , Proteína do Retinoblastoma/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Pontos de Checagem do Ciclo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Fosforilação , Prognóstico , Proteína do Retinoblastoma/antagonistas & inibidores , Transdução de Sinais , Análise Serial de TecidosRESUMO
PURPOSE: The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC. METHODS: The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA). RESULTS: We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated. CONCLUSIONS: From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.
Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Neoplasias Hepáticas/metabolismo , Purinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/mortalidade , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Sorafenibe/farmacologiaRESUMO
A third of patients with triple negative breast cancer (TNBC) have relapsed disease within 2-5 years from initial diagnosis, leaving an unmet need for therapeutic targets. TNBC frequently harbors alterations of the PI3K/AKT/mTOR pathway, but single agent PI3K/AKT/mTOR inhibitors have not shown marked efficacy. In this study, we investigated a strategy to improve efficacy of PI3K-α inhibitor BYL719 (alpelisib) in TNBC. While BYL719 is effective at inhibiting cell proliferation in T47D, a triple positive cell line, it had limited activity in TNBC. This may be partially due to persistent phosphorylation of RB, and incomplete inhibition of p-S6 in TNBC, since the inhibitory effect of BYL719 on p-RB and p-S6 was significantly reduced in TNBC compared to T47D cells. Addition of the CDK4/6 inhibitor LEE011 to BYL719 caused a simultaneous reduction of p-RB and p-S6, and a more complete inhibition of p-S6, leading to decreased expression of the pro-survival protein MCL-1, an induction of apoptosis, and an enhanced reduction of tumor growth in a PDX model of TNBC. These findings suggest that inhibition of p-RB and p-S6 is important for an effective response to the treatment of TNBC, and provides a strong rationale for clinical development of combination therapy with BYL719 and LEE011 for treatment of metastatic TNBC with intact RB.Presentation: This study was presented in part as an abstract at the 2016 San Antonio Breast Cancer Symposium (P3-03-15) and the 2018 Cancer Research and Targeted Therapy in London.
Assuntos
Aminopiridinas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Purinas/administração & dosagem , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Tiazóis/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Feminino , Humanos , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Understanding the underlying mechanisms of cell cycle regulation is essential to address how normal development and tissue homeostasis are achieved. Here, we present a novel cell cycle regulator Caliban (Clbn), the Drosophila ortholog of human Serologically defined colon cancer antigen 1 (SDCCAG1) gene. We show that ionizing radiation induces expression of clbn, and over-expression of clbn blocks G1-to-S cell cycle transition in Drosophila, while flies loss of clbn have defective S phase checkpoint in response to irradiation. Mechanistically, induced expression of clbn suppressed E2F1 activity and down-regulates the DNA replication and expression of its downstream target cyclin E, a key regulator of G1-to-S transition. Meanwhile, clbn over-expression leads to upregulation of the CDK inhibitor Dacapo (Dap), and upregulated Dap is decreased when e2f1 is over-expressed. Furthermore, expression of clbn is down-regulated in cells with e2f1 over-expression or rbf1 knockdown, indicating that Clbn and E2F1 act antagonistically in mediating G1-to-S transition. Thus we provide genetic evidence that Clbn works together with E2F1 in regulating cell cycle progression, and Clbn is required for S phase cell cycle checkpoint in response to DNA damage.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Radiação Ionizante , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Proteínas Supressoras de Tumor/metabolismo , Animais , Ciclina E/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.
Assuntos
Reprogramação Celular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Antígenos de Superfície/metabolismo , Linhagem Celular , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fosforilação , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
At this time, no molecular targeted therapies exist for treatment of retinoblastoma. This can be, in part, attributed to the lack of animal models that allow for both rapid identification of novel therapeutic targets and hypothesis driven drug testing. Within this scope, we have recently reported the first genuine genetic nonmammalian retinoblastoma cancer model within the aquatic model organism Xenopus tropicalis (Naert et al., Sci Rep 6: 35263, 2016). Here we describe the methods to generate rb1 mosaic mutant Xenopus tropicalis by employing the CRISPR/Cas9 technology. In depth, we discuss short guide RNA (sgRNA) design parameters, generation, quality control, quantification, and delivery followed by several methods for assessing genome editing efficiencies. As such the reader should be capable, by minor changes to the methods described here, to (co-) target rb1 or any one or multiple gene(s) within the Xenopus tropicalis genome by multiplex CRISPR/Cas9 methodology.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes/métodos , Genoma , Proteína do Retinoblastoma/antagonistas & inibidores , Xenopus/genética , Animais , Proteína do Retinoblastoma/genética , Xenopus/classificaçãoRESUMO
Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb.
Assuntos
NF-kappa B/fisiologia , Vírus do Orf/fisiologia , Vírus do Orf/patogenicidade , Proteína do Retinoblastoma/fisiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Ectima Contagioso/etiologia , Técnicas de Silenciamento de Genes , Genes Virais , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Quinase I-kappa B/metabolismo , Imunidade Inata , Mutação , NF-kappa B/antagonistas & inibidores , Vírus do Orf/genética , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Ovinos , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Virulência/genética , Virulência/imunologia , Virulência/fisiologiaRESUMO
Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Pan-genomic analyses identified p53/Rb and WNT/ß-catenin signaling pathways as main contributors to the disease. However, isolated ß-catenin constitutive activation failed to induce malignant progression in mouse adrenocortical tumors. Therefore, there still was a need for a relevant animal model to study ACC pathogenesis and to test new therapeutic approaches. Here, we have developed a transgenic mice model with adrenocortical specific expression of SV40 large T-antigen (AdTAg mice), to test the oncogenic potential of p53/Rb inhibition in the adrenal gland. All AdTAg mice develop large adrenal carcinomas that eventually metastasize to the liver and lungs, resulting in decreased overall survival. Consistent with ACC in patients, adrenal tumors in AdTAg mice autonomously produce large amounts of glucocorticoids and spontaneously activate WNT/ß-catenin signaling pathway during malignant progression. We show that this activation is associated with downregulation of secreted frizzled related proteins (Sfrp) and Znrf3 that act as inhibitors of the WNT signaling. We also show that mTORC1 pathway activation is an early event during neoplasia expansion and further demonstrate that mTORC1 pathway is activated in ACC patients. Preclinical inhibition of mTORC1 activity induces a marked reduction in tumor size, associated with induction of apoptosis and inhibition of proliferation that results in normalization of corticosterone plasma levels in AdTAg mice. Altogether, these data establish AdTAg mice as the first preclinical model for metastatic ACC.
Assuntos
Carcinoma Adrenocortical/patologia , Antígenos Transformantes de Poliomavirus/genética , Proteína do Retinoblastoma/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/fisiologia , Metástase Neoplásica , Proteína do Retinoblastoma/antagonistas & inibidores , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/fisiologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Via de Sinalização Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
The extracellular signal-regulated kinase (ERK) is one of the most important molecular targets for cancer that controls diverse cellular processes such as proliferation, survival, differentiation and motility. Similarly, the Rb (retinoblastoma protein) is a tumor suppressor protein and its function is to prevent excessive cell growth by inhibiting cell cycle progression. When the cell is ready to divide, pRb is phosphorylated, becomes inactive and allows cell cycle progression. Herein, we discovered a new series of tetrahydrocarbazoles as dual inhibitors of pERK and pRb phosphorylation. The in-house small molecule library was screened for inhibition of pERK and pRb phosphorylation, which led to the discovery of tetrahydrocarbazole series of compounds as potential leads. N-(3-methylcyclopentyl)-6-nitro-2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-amine (1) is the dual inhibitor lead identified through screening, displaying inhibition of pERK and pRb phosphorylation with IC50 values of 5.5 and 4.8 µM, respectively. A short structure-activity relationship (SAR) study has been performed, which identified another dual inhibitor 9-methyl-N-(4-methylbenzyl)-2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-amine (16) with IC50 values 4.4 and 3.5 µM for inhibition of pERK and pRb phosphorylation, respectively. This compound has a potential for further lead optimization to discover promising molecularly-targeted anticancer agents.
Assuntos
Carbazóis/química , Carbazóis/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteína do Retinoblastoma/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Descoberta de Drogas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína do Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
The tumour suppressor gene (Rb1) is necessary for the maintenance of telomere integrity in osteoblastic cells. We now show that the compaction of telomeric chromatin and the appropriate histone modifications of telomeric DNA are both dependent upon Rb1-mediated transcription of the telomere-derived long non-coding RNA TERRA. Expression of TERRA was reduced in Rb1 haploinsufficient cells, and further decreased by shRNA-mediated reduction of residual Rb1 expression. Restoration of Rb1 levels through lentiviral transduction was sufficient to reestablish both transcription of TERRA and condensation of telomeric chromatin. The human chromosome 15q TERRA promoter contains predicted retinoblastoma control elements, and was able to confer Rb1-dependent transcription upon a promoterless reporter gene. Chromatin immunoprecipitation revealed preferential binding of phosphorylated over non-phosphorylated Rb1 at the TERRA promoter. As Rb1-deficient cells show increased genomic instability we suggest that this novel non-canonical action of Rb1 may contribute to the tumour suppressive actions of Rb1.
Assuntos
Heterocromatina/ultraestrutura , Histonas/genética , Osteoblastos/metabolismo , RNA Longo não Codificante/genética , Proteína do Retinoblastoma/genética , Telômero/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Cromossomos Humanos Par 15 , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Instabilidade Genômica , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Fosforilação , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Telômero/ultraestrutura , Transcrição GênicaRESUMO
Ovarian epithelial carcinoma is the leading cause of deaths from gynecologic malignancy. New reagents with therapeutic potentials against ovarian cancer, especially the drug-resistant cases, are required for better treatment of ovarian cancer patients. Epigenetic events such as changes in DNA methylation and histone modification, through their effects on DNA-protein interaction, chromatin conformation, and gene expression, affect cell function, cancer behavior, clinical manifestations, and outcomes. Previous studies have shown that histone deacetylase (HDAC) inhibitors have strong cytostatic and apoptotic activities in hematologic and some solid cancer cells. Oxamflatin, a compound containing the aromatic sulfonamide and hydroxamic acid groups, is known to be a potent HDAC inhibitor capable of inhibiting the growth of mouse and human cancer cell lines. In this study we found that oxamflatin in the nM range induced morphological changes in OVCAR-5 and SKOV-3 ovarian cancer cell lines. Treatment with oxamflatin also led to decreased cell viability. Moreover, results of BrdU incorporation assay, cell counting, and Ki-67 immunostaining indicated that oxamflatin was able to significantly inhibit DNA synthesis and cell proliferation. Using real-time PCR and Western blot analyses we demonstrated that oxamflatin was capable of downregulating the expression of c-Myc, CDK4, E2F1, and the phosphorylation levels of Rb protein, but upregulating p21. These findings pave the way to examine if oxamflatin along with or in combination with other reagents could deliver anticancer effects against ovarian cancers in vivo.
Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/agonistas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Epigênese Genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Histona Desacetilases/metabolismo , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de SinaisRESUMO
This study describes the characterization of a novel kinase inhibitor, ON123300, which inhibits CDK4/6 (cyclin-dependent kinases 4 and 6) and phosphatidylinositol 3 kinase-δ (PI3K-δ) and exhibits potent activity against mantle cell lymphomas (MCLs) both in vitro and in vivo. We examined the effects of PD0332991 and ON123300 on cell cycle progression, modulation of the retinoblastoma (Rb) and PI3K/AKT pathways, and the induction of apoptosis in MCL cell lines and patient-derived samples. When Granta 519 and Z138C cells were incubated with PD0332991 and ON123300, both compounds were equally efficient in their ability to inhibit the phosphorylation of Rb family proteins. However, only ON123300 inhibited the phosphorylation of proteins associated with the PI3K/AKT pathway. Cells treated with PD0332991 rapidly accumulated in the G0/G1 phase of cell cycle as a function of increasing concentration. Although ON123300-treated cells arrested similarly at lower concentrations, higher concentrations resulted in the induction of apoptosis, which was not observed in PD0332991-treated samples. Mouse xenograft assays also showed a strong inhibition of MCL tumor growth in ON123300-treated animals. Finally, treatment of ibrutinib-sensitive and -resistant patient-derived MCLs with ON123300 also triggered apoptosis and inhibition of the Rb and PI3K/AKT pathways, suggesting that this compound might be an effective agent in MCL, including ibrutinib-resistant forms of the disease.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Linfoma de Célula do Manto/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Linfoma de Célula do Manto/patologia , Camundongos , NF-kappa B/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteína do Retinoblastoma/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Circulating endothelial progenitor cells (EPCs) have multiple protective effects that facilitate repair of damage to tissues and organs. However, while various stressors are known to impair EPC function, the mechanisms of oxidative stress-induced EPC senescence remains unknown. We demonstrated that B2 receptor (B2R) expression on circulating CD34(+) cells was significantly reduced in patients with diabetes mellitus (DM) as compared to healthy controls. Furthermore, CD34(+) cell B2R expression in patients with DM was inversely correlated with plasma myeloperoxidase concentrations. Bradykinin (BK) treatment decreased human EPC (hEPC) senescence and intracellular oxygen radical production, resulting in reduced retinoblastoma 1 (RB) RNA expression in H2O2-induced senescent hEPCs and a reversal of the B2R downregulation that is normally observed in senescent cells. Furthermore, BK treatment of H2O2-exposed cells leads to elevated phosphorylation of RB, AKT, and cyclin D1 compared with H2O2-treatment alone. Antagonists of B2R, PI3K, and EGFR signaling pathways and B2R siRNA blocked BK protective effects. In summary, this study demonstrates that BK significantly inhibits oxidative stress-induced hEPC senescence though B2R-mediated activation of PI3K and EGFR signaling pathways.
Assuntos
Antioxidantes/farmacologia , Bradicinina/farmacologia , Senescência Celular/efeitos dos fármacos , Diabetes Mellitus/enzimologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Receptores ErbB/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor B2 da Bradicinina/agonistas , Proteína do Retinoblastoma/metabolismo , Antígenos CD34/metabolismo , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Citoproteção , Diabetes Mellitus/sangue , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Células Progenitoras Endoteliais/enzimologia , Células Progenitoras Endoteliais/patologia , Receptores ErbB/antagonistas & inibidores , Humanos , Oxidantes/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Interferência de RNA , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Transdução de Sinais , TransfecçãoRESUMO
Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) detects intracellular DNA and signals through the adapter protein STING to initiate the antiviral response to DNA viruses. Whether DNA viruses can prevent activation of the cGAS-STING pathway remains largely unknown. Here, we identify the oncogenes of the DNA tumor viruses, including E7 from human papillomavirus (HPV) and E1A from adenovirus, as potent and specific inhibitors of the cGAS-STING pathway. We show that the LXCXE motif of these oncoproteins, which is essential for blockade of the retinoblastoma tumor suppressor, is also important for antagonizing DNA sensing. E1A and E7 bind to STING, and silencing of these oncogenes in human tumor cells restores the cGAS-STING pathway. Our findings reveal a host-virus conflict that may have shaped the evolution of viral oncogenes.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Vírus de DNA Tumorais/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Nucleotídeos Cíclicos/antagonistas & inibidores , Proteínas Oncogênicas Virais/metabolismo , Evasão Tumoral , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , DNA de Neoplasias/imunologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Evolução Molecular , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Redes e Vias Metabólicas , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteína do Retinoblastoma/antagonistas & inibidoresRESUMO
Cyclin-dependent kinase 2 (CDK2) has been reported to be overexpressed in human colorectal cancer; it is responsible for the G1toSphase transition in the cell cycle and its deregulation is a hallmark of cancer. The present study was the first to use idock, a free and opensource proteinligand docking software developed by our group, to identify potential CDK2 inhibitors from 4,311 US Food and Drug Administrationapproved small molecular drugs with a repurposing strategy. Among the top compounds identified by idock score, nine were selected for further study. Among them, adapalene (ADA; CD271,6[3(1adamantyl)4methoxyphenyl]2naphtoic acid) exhibited the highest antiproliferative effects in LOVO and DLD1 human colon cancer cell lines. Consistent with the expected properties of CDK2 inhibitors, the present study demonstrated that ADA significantly increased the G1phase population and decreased the expression of CDK2, cyclin E and retinoblastoma protein (Rb), as well as the phosphorylation of CDK2 (on Thr160) and Rb (on Ser795). Furthermore, the anticancer effects of ADA were examined in vivo on xenograft tumors derived from DLD1 human colorectal cancer cells subcutaneously inoculated in BALB/C nude mice. ADA (20 mg/kg orally) exhibited marked antitumor activity, comparable to that of oxaliplatin (40 mg/kg), and dosedependently inhibited tumor growth (P<0.05), while combined administration of ADA and oxaliplatin produced the highest therapeutic effect. To the best of our knowledge, the present study was the first to indicate that ADA inhibits CDK2 and is a potential candidate drug for the treatment of human colorectal cancer.
Assuntos
Adapaleno/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Adapaleno/química , Administração Oral , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ciclina E/antagonistas & inibidores , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Combinação de Medicamentos , Feminino , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Fosforilação/efeitos dos fármacos , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Retinoblastoma is the most common primary intraocular malignancy in children. Although significant advances in treatment have decreased mortality in recent years, morbidity continues to be associated with these therapies, and therefore, there is a pressing need for new therapeutic options. Transgenic mouse models are popular for testing new therapeutics as well as studying the pathophysiology of retinoblastoma. The T-antigen retinoblastoma (TAg-RB) model has close molecular and histological resemblance to human retinoblastoma tumors; these mice inactivate pRB by retinal-specific expression of the Simian Virus 40 T-antigens. Here, we evaluated whether optical coherence tomography (OCT) imaging could be used to document tumor growth in the TAg-RB model from the earliest stages of tumor development. METHODS: The Micron III rodent imaging system was used to obtain fundus photographs and OCT images of both eyes of TAg-RB mice weekly from 2 to 12 weeks of age and at 16 and 20 weeks of age to document tumor development. Tumor morphology was confirmed with histological analysis. RESULTS: Before being visible on funduscopy, hyperreflective masses arising in the inner nuclear layer were evident at 2 weeks of age with OCT imaging. After most of these hyperreflective cell clusters disappeared around 4 weeks of age, the first tumors became visible on OCT and funduscopy by 6 weeks. The masses grew into discrete, discoid tumors, preferentially in the periphery, that developed more irregular morphology over time, eventually merging and displacing the inner retinal layers into the vitreous. CONCLUSIONS: OCT is a non-invasive imaging modality for tracking early TAg-RB tumor growth in vivo. Using OCT, we characterized TAg-positive cells as early as 2 weeks, corresponding to the earliest stages at which tumors are histologically evident, and well before they are evident with funduscopy. Tracking tumor growth from its earliest stages will allow better analysis of the efficacy of novel therapeutics and genetic factors tested in this powerful mouse model.
Assuntos
Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Neoplasias da Retina/etiologia , Retinoblastoma/etiologia , Tomografia de Coerência Óptica , Animais , Modelos Animais de Doenças , Fundo de Olho , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Neoplasias da Retina/patologia , Neoplasias da Retina/fisiopatologia , Retinoblastoma/patologia , Retinoblastoma/fisiopatologia , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genéticaRESUMO
Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Clonagem Molecular/métodos , Vetores Genéticos , Lentivirus/genética , Animais , Apoptose , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doxiciclina/farmacologia , Resistência a Medicamentos/genética , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Camundongos , Camundongos SCID , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , RNA Interferente Pequeno/genética , Recombinação Genética , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Sarcoma Experimental/genética , Sarcoma Experimental/terapia , Transdução Genética , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Viruses are obligate intracellular parasites and need to reprogram host cells to establish long-term persistent infection and/or to produce viral progeny. Cellular changes initiated by the virus trigger cellular defense responses to cripple viral replication, and viruses have evolved countermeasures to neutralize them. Established models have suggested that human papillomaviruses target the retinoblastoma (RB1) and TP53 tumor suppressor networks to usurp cellular replication, which drives carcinogenesis. More recent studies, however, suggest that modulating the activity of the Polycomb family of transcriptional repressors and the resulting changes in epigenetic regulation are proximal steps in the rewiring of cellular signaling circuits. Consequently, RB1 inactivation evolved to tolerate the resulting cellular alterations. Therefore, epigenetic reprograming results in cellular "addictions" to pathways for survival. Inhibition of such a pathway could cause "synthetic lethality" in adapted cells while not markedly affecting normal cells and could prove to be an effective therapeutic approach.
Assuntos
Transformação Celular Neoplásica , Interações Hospedeiro-Patógeno , Papillomaviridae/imunologia , Papillomaviridae/fisiologia , Proteína do Retinoblastoma/metabolismo , Morte Celular , Sobrevivência Celular , Regulação da Expressão Gênica , Humanos , Proteínas do Grupo Polycomb/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Lunasin, a soybean bioactive peptide, has both chemopreventive and chemotherapeutic activities. The aim of this study was to determine the chemotherapeutic potential of lunasin against human lung cancer. Treatment of non-small cell lung cancer (NSCLC) cells with highly purified soybean-derived lunasin caused limited, cell-line specific anti-proliferative effects on anchorage-dependent growth whereas two normal bronchial epithelial cell lines were unaffected. Lunasin's antiproliferative effects were potentiated upon utilization of anchorage-independent conditions. Furthermore, NSCLC cell lines that were unaffected by lunasin in anchorage-dependent assays exhibited a dose-dependent inhibition in colony formation or colony size. Mouse xenograft studies revealed that 30 mg lunasin/kg body weight per day decreased NSCLC H1299 tumor volume by 63.0% at day 32. Mechanistic studies using cultured NSCLC H661 cells showed that lunasin inhibited cell cycle progression at the G1/S phase interface without inducing apoptosis. Immunoblot analyses of key cell-cycle proteins demonstrated that lunasin altered the expression of the G1 specific cyclin-dependent kinase complex components, increased levels of p27Kip1, reduced levels of phosphorylated Akt, and ultimately inhibited the sequential phosphorylation of the retinoblastoma protein (RB). These results establish for the first time that lunasin can inhibit NSCLC proliferation by suppressing cell-cycle dependent phosphorylation of RB.