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Despite the enormous success of childhood prophylactic vaccination against diseases caused by pathogens, there is currently no similar preventive vaccine program against diseases confronted with age like breast cancer and ovarian cancer. With the exception of the annual influenza vaccine, current recommendations for adult vaccination are for either primary vaccines not received during childhood or for booster vaccinations to maintain the immunity against pathogens already induced during childhood. Here we describe a strategy to provide prophylactic pre-emptive immunity against the development of adult onset cancers not associated with any definitive etiopathogenic agent. We propose that safe and effective pre-emptive immunity may be induced in cancer-free subjects by vaccination against immunodominant tissue-specific self-proteins that are 'retired' from expression in normal tissues as part of the normal aging process but are expressed in tumors that emerge with age. Primary immunoprevention of adult onset cancers like breast cancer and ovarian cancer represents a great challenge and an even greater unmet need for our current healthcare.
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Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Neoplasias/imunologia , Neoplasias/prevenção & controle , Vacinação , Idade de Início , Autoantígenos/imunologia , Biomarcadores , Vacinas Anticâncer/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Imunidade , Neoplasias/diagnóstico , Neoplasias/epidemiologia , Especificidade de Órgãos/imunologia , Transdução de SinaisRESUMO
Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.
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Cromatina/metabolismo , Ciclina E/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Radiação Ionizante , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
INTRODUCTION: The outcome of recurrent/metastatic gynaecological malignancy has drastically improved with the introduction of poly(ADP-ribose) polymerase inhibitors and immunotherapy, but the use of these drugs in routine practice is complicated due to access barriers and their high cost in developing countries. The purpose of this study is to present the clinical response, outcome and safety of oral metronomic chemotherapy (OMCT) in resource-limited, financially constrained populations. METHODS: This is a retrospective study on patients with advanced gynaecological cancer treated at Chittaranjan National Cancer Institute, Kolkata, India, from 2021 to 2023. The patients were treated with one of these two regimens: a split-dose course of cyclophosphamide (50 mg orally once daily for 21 days) and capecitabine (500 mg twice daily continuous) or a fixed-dose combination (capecitabine 1800 mg and cyclophosphamide 80 mg orally for 14 days in every 21 days) until disease progression or unacceptable toxicities occurred. All data was captured from the hospital's medical records until June 2023. Toxicity data was reported per the Common Terminology Criteria for Adverse Events (CTCAE) v5.1, and progression-free survival (PFS) was estimated using Kaplan-Meier methods. RESULTS: Among 34 screened patients, 10 were excluded due to noncompliance. This study analysed 24 patients with a median age at diagnosis of 56 years (IQ range 44-75). Sixteen (67%) patients were at stage IV disease with an Eastern Cooperative Oncology Group (ECOG) performance status of 3. Ovarian and cervical cancers were 80% and 20%, respectively; among them, 16 (67%) patients were platinum-refractory. Forty-two per cent of patients received three lines of chemotherapy before OMCT. A split course versus fixed dose was given to 67% versus 33% of the population; the best responses per the Response Evaluation Criteria in Solid Tumours v1.1 were complete response in 12%, partial response in 67% and stable disease in 21%. The most common toxicities were grade I anaemia (54%), grade I chemotherapy-induced nausea and vomiting (46%), grade I fatigue (42%) and grade I neutropenia (21%). Twenty-five per cent of patients were offered next-line systemic therapy after progression. The entire cohort had a median PFS of nine months (95%, CI: 5.2-12.7). Cox regression analysis identified a median PFS of 12 months (95%, CI: 6.2-17.7) among platinum-refractory groups. CONCLUSION: OMCT was a well-tolerated, affordable regimen with durable clinical response and survival outcome (median PFS of nine months) in recurrent, refractory advanced gynaecological cancer and can be offered to patients at resource-limited centres.
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The antiapoptotic BCL-2 proteins regulate lymphocyte survival and are over-expressed in lymphoid malignancies, including chronic lymphocytic leukemia. The small molecule inhibitor ABT-737 binds with high affinity to BCL-2, BCL-XL, and BCL-W but with low affinity to MCL-1, BFL-1, and BCL-B. The active analog of ABT-737, navitoclax, has shown a high therapeutic index in lymphoid malignancies; developing a predictive marker for it would be clinically valuable for patient selection or choice of drug combinations. Here we used RT-PCR as a highly sensitive and quantitative assay to compare expression of antiapoptotic BCL-2 genes that are known to be targeted by ABT-737. Our findings reveal that the relative ratio of MCL-1 and BFL-1 to BCL-2 expression provides a highly significant linear correlation with ABT-737 sensitivity (r = 0.6, P < .001). In contrast, antiapoptotic transcript levels, used individually or in combination for high or low affinity ABT-737-binding proteins, could not predict ABT-737 sensitivity. The (MCL-1 + BFL-1)/BCL-2 ratio was validated in a panel of leukemic cell lines subjected to genetic and pharmacologic manipulations. Changes after ABT-737 treatment included increased expression of BFL-1 and BCL-B that may contribute to treatment resistance. This study defines a highly significant BCL-2 expression index for predicting the response of CLL to ABT-737.
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Compostos de Bifenilo/farmacologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Família Multigênica/efeitos dos fármacos , Família Multigênica/genética , Piperazinas/farmacologia , Cultura Primária de Células , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
[This corrects the article DOI: 10.18632/oncotarget.27585.].
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Purpose: Epithelial ovarian carcinoma (EOC) is the most lethal of all human gynecologic malignancies. We previously reported that vaccination of female mice with the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in complete Freund's adjuvant (CFA) generates AMHR2-ED specific immunoglobulin G (IgG) that provides prevention and therapy against murine EOCs. Although CFA is the "gold standard" adjuvant in animal studies, it is not approved for human use because it often induces painful granulomas and abscesses. Thus, the objective of this study is to identify an alternative adjuvant to CFA for use in our ovarian cancer vaccine clinical trials. Materials and Methods: Because it has been used successfully without serious adverse effects in numerous human clinical trials, we selected the IgG-inducing squalene-based adjuvant, AddaVax™, for evaluation of its ability to facilitate vaccine-induced prevention and treatment of EOC in mice. To this end, we immunized female C57BL/6 mice with recombinant mouse AMHR2-ED emulsified with either AddaVax or CFA as adjuvant and compared the results. Results: We found that formulation of the AMHR2-ED vaccine with AddaVax adjuvant induced high serum titers of IgG and significant inhibition of EOC growth with significantly enhanced overall survival of mice using both prevention and therapeutic protocols. These results were compared favorably with results obtained using CFA as an adjuvant in the AMHR2-ED vaccine. Conclusion: Our data indicate that the AMHR2-ED vaccine formulated with AddaVax may be used in human clinical trials and thereby serve as a novel and effective way to control human EOC.
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The cyclin E/Cdk2 complex plays an essential role in the G(1)/S cell cycle transition and DNA replication. Earlier we showed that in hematopoietic tumor cells, caspase-mediated cleavage of cyclin E generates p18-cyclin E, which is unable to interact with Cdk2 and therefore plays a role independent of the cell cycle. The expression of a cleavage-resistant cyclin E mutant greatly diminishes apoptosis, indicating the critical role of cyclin E cleavage. p18-cyclin E expression can induce apoptosis or sensitization to apoptotic stimuli in many cell types. Here we identify Ku70 as a specific p18-cyclin E-interacting partner. In hematopoietic tumor cell lines, the association of p18-cyclin E with Ku70 induces the dissociation of Bax from Ku70, followed by Bax activation. This mechanism of Bax activation leads to the amplification of the apoptosis signal in all tumor cell lines examined. N-terminal Ku70 deletion mutants are unable to bind to p18-cyclin E to regulate its apoptotic effect. p18-cyclin E-mediated amplification of apoptosis is dependent on Bax and Ku70 being greatly diminished in Ku70(-/-) and Bax(-/-) mouse embryo fibroblasts and in hematopoietic cells where Bax knockdown was achieved by short interfering RNA. The p18-cyclin E/Ku70 and Bax/Ku70 interactions provide a balance between apoptosis and the survival of cells exposed to genotoxic stress.
Assuntos
Antígenos Nucleares/metabolismo , Apoptose , Ciclina E/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antígenos Nucleares/genética , Caspase 2/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Ciclina E/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Autoantígeno Ku , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genéticaRESUMO
Epithelial ovarian carcinoma (EOC) is the most prevalent and lethal form of ovarian cancer. The low five-year overall survival after EOC diagnosis indicates an urgent need for more effective ways to control this disease. Anti-Müllerian hormone receptor 2 (AMHR2) is an ovarian protein overexpressed in the majority of human EOCs. We have previously found that vaccination against the ovarian-specific extracellular domain of AMHR2 (AMHR2-ED) significantly inhibits growth of murine EOCs through an IgG-mediated mechanism that agonizes receptor signaling of a Bax/caspase-3 dependent proapoptotic cascade. To determine if a single monoclonal antibody (mAb) could inhibit growth of human EOC, we generated a panel of mAbs specific for recombinant human AMHR2-ED and characterized a candidate mAb for humanization and use in clinical trials. We found that our candidate 4D12G1 mAb is an IgG1 that shows high affinity antigen-specific binding to the 7-mer 20KTLGELL26 sequence of AMHR2-ED that facilitates induction of programmed cell death in EOC cells. Most importantly, the 4D12G1 mAb significantly inhibits growth of primary human EOCs in patient-derived xenografts (PDXs) by inducing direct apoptosis of EOC tumors. Our results support the view that a humanized 4D12G1 mAb may be a much needed and effective reagent for passive immunotherapy of human EOC.
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A number of methods have been developed to examine the morphologic, biochemical, and molecular changes that happen during the DNA damage response that may ultimately lead to death of cells through various mechanisms that include apoptosis. When cells are exposed to ionizing radiation or chemical DNA-damaging agents, double-stranded DNA breaks (DSB) are generated that rapidly result in the phosphorylation of histone variant H2AX. Because phosphorylation of H2AX at Ser 139 correlates well with each DSB, phospho-H2AX is a sensitive marker to used to examine the DNA damage and its repair. Apoptotic cells are characterized on the basis of their reduced DNA content and morphologic changes, including nuclear condensation, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, or Hoechst staining. The appearance of phosphatidylserine on the plasma membrane with annexin V-fluorochrome conjugates indicates the changes in plasma membrane composition and function. By combining it with propidium iodide staining, this method can also be used to distinguish early versus late apoptotic or necrotic events. The activation of caspases is another well-known biochemical marker of apoptosis. Finally, the Bcl-2 family of proteins and the mitochondria that play a critical role in DNA damage-induced apoptosis can be examined by translocation of Bax and cytochrome c in and out of mitochondria. In this chapter, we discuss the most commonly used techniques used in our laboratory for determining the DNA damage response leading to apoptosis.
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Apoptose/fisiologia , Dano ao DNA/fisiologia , Anexina A5/metabolismo , Caspase 3/análise , Fracionamento Celular/métodos , Proliferação de Células , Sobrevivência Celular , Citocromos c/análise , Fragmentação do DNA , Citometria de Fluxo/métodos , Histonas , Humanos , Imuno-Histoquímica , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/análise , Proteína X Associada a bcl-2/análiseRESUMO
A number of methods have been developed to identify the cells that undergo apoptosis by analyzing the morphological, biochemical, and molecular changes that take place during this universal biological process. The best recognized biochemical hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the fluorescein-labeled CaspaTag pan-caspase in situ detection kit.
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Apoptose , Caspase 3/metabolismo , Dano ao DNA/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Biomarcadores Tumorais/metabolismo , Colorimetria/métodos , Ativação Enzimática/fisiologia , Fluoresceína/química , Humanos , Immunoblotting/métodos , Imuno-Histoquímica/métodosRESUMO
Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G(1) to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E(276-395) (where cyclin E(276-395) is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E(1-275), cyclin E(276-395) deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E(276-395), but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.
Assuntos
Apoptose , Quinases relacionadas a CDC2 e CDC28 , Ciclina E/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Processamento de Proteína Pós-Traducional , Apoptose/efeitos da radiação , Western Blotting , Caspases/metabolismo , Ciclina E/química , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Peso Molecular , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes , Células Tumorais CultivadasRESUMO
Epithelial ovarian carcinoma (EOC) is the most prevalent form of ovarian cancer in the United States, representing approximately 85% of all cases and causing more deaths than any other gynecologic malignancy. We propose that optimized control of EOC requires the incorporation of a vaccine capable of inducing safe and effective preemptive immunity in cancer-free women. In addition, we hypothesize that ovarian-specific self-proteins that are "retired" from autoimmune-inducing expression levels as ovaries age but are expressed at high levels in emerging EOC may serve as vaccine targets for mediating safe and effective primary immunoprevention. Here, we show that expression of the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in normal tissues is confined exclusively to the human ovary, drops to nonautoimmune inducing levels in postmenopausal ovaries, and is at high levels in approximately 90% of human EOC. We found that AMHR2-ED vaccination significantly inhibits growth of murine EOC and enhances overall survival without inducing oophoritis in aged female mice. The observed inhibition of EOC growth was mediated substantially by induction of AMHR2-ED-specific IgG antibodies that agonize receptor signaling of a Bax/caspase-3-dependent proapoptotic cascade. Our results indicate that AMHR2-ED vaccination may be particularly useful in providing safe and effective preemptive immunity against EOC in women at high genetic or familial risk who have the greatest need for a preventive vaccine and ultimately in cancer-free postmenopausal women who account for 75% of all EOC cases. Cancer Prev Res; 10(11); 612-24. ©2017 AACRSee related editorial by Shoemaker et al., p. 607.
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Vacinas Anticâncer/uso terapêutico , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/terapia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Idoso , Idoso de 80 Anos ou mais , Vacinas Anticâncer/imunologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Tolerância Imunológica/imunologia , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/patologia , Ooforite/epidemiologia , Ooforite/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ovário/imunologia , Ovário/patologia , Poliendocrinopatias Autoimunes/epidemiologia , Poliendocrinopatias Autoimunes/imunologia , Pós-Menopausa , Proteínas Serina-Treonina Quinases , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinação/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A large number of methods devoted to the identification of apoptotic cells and the analysis of the morphological, biochemical, and molecular changes that take place during this universal biological process have been developed. Apoptotic cells are recognized on the basis of their reduced DNA content and morphological changes that include nuclear condensation and which can be detected by flow cytometry (sub-G1 DNA content), Trypan Blue, or Hoechst staining. Changes in plasma membrane composition and function are detected by the appearance of phosphatidylserine on the plasma membrane, which reacts with Annexin V-fluorochrome conjugates. Combined with propidium iodide (PI) staining, this method can distinguish between the early and late apoptotic events. The best-recognized biochemical hallmarks of apoptosis are the activation of cysteine proteases (caspases), condensation of chromatin, and fragmentation of genomic DNA into nucleosomal fragments. Recognized by a variety of assays, activated caspases cleave many cellular proteins and the resulting fragments may serve as apoptosis markers. Finally, the mitochondria and the Bcl-2 family proteins play an important role in this process that can be recognized by translocation of apoptogenic factors, such as Bax and cytochrome c, in and out of mitochondria.
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Apoptose/fisiologia , Doenças Cardiovasculares/fisiopatologia , Técnicas Citológicas/métodos , Anexina A5 , Bisbenzimidazol , Caspase 3/análise , Fracionamento Celular , Membrana Celular/fisiologia , Colorimetria , Citocromos c/análise , Fragmentação do DNA , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Mitocôndrias/metabolismo , Sais de Tetrazólio , Azul Tripano , Proteína Killer-Antagonista Homóloga a bcl-2/análise , Proteína X Associada a bcl-2/análiseRESUMO
The retinoblastoma (pRb), p107, and p130 pocket proteins bind to the E2F transcription factors to control gene expression. E2F4 protein levels increased and accumulated in the nuclei of prostate carcinoma cells subjected to ionizing radiation (IR). The IR-induced increase of E2F4 levels led to an increase in E2F4 binding to p130 but had no effect on E2F4/p107 or E2F5/p130 complexes. The increase in E2F4/p130 association after IR was observed in prostate carcinoma cells regardless of their sensitivity to androgens, but not in breast carcinoma cells. E2F4/p130 complex formation was dependent on dissociation of p130 from cyclin-dependent kinase 2 and p130 dephosphorylation. Disruption of E2F4 through small interfering RNA prevented p130/E2F4 complex formation and sensitized cells to IR-induced apoptosis, leading to caspase-3 activation, cleavage of its substrate, poly(ADP-ribose) polymerase, and nuclear condensation. The E2F4/p130 pocket protein complex emerges as a new target of radiation in prostate carcinoma cells.
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Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Biossíntese de Proteínas , Fatores de Transcrição/biossíntese , Apoptose/genética , Apoptose/efeitos da radiação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Fator de Transcrição E2F4 , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/radioterapia , Neoplasias da Próstata/radioterapia , Proteínas/genética , Proteínas/metabolismo , Proteínas/efeitos da radiação , RNA Interferente Pequeno/genética , Proteína p130 Retinoblastoma-Like , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/efeitos da radiação , TransfecçãoRESUMO
We have proposed that safe and effective protection against the development of adult onset cancers may be achieved by vaccination against tissue-specific self-proteins that are "retired" from expression at immunogenic levels in normal tissues as we age, but are overexpressed in emerging tumors. α-Lactalbumin is an example of a "retired" self-protein because its expression in normal tissues is confined exclusively to the breast during late pregnancy and lactation, but is also expressed in the vast majority of human triple negative breast cancers (TNBC)-the most aggressive and lethal form of breast cancer and the predominant form that occurs in women at high genetic risk including those with mutated BRCA1 genes. In anticipation of upcoming clinical trials, here we provide preclinical data indicating that α-lactalbumin has the potential as a vaccine target for inducing safe and effective primary immunoprevention as well as immunotherapy against TNBC.
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Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E-/- mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors.
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Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Nitrofenóis/farmacologia , Fragmentos de Peptídeos , Fosforilação , Piperazinas/farmacologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/farmacologiaRESUMO
Anti-Müllerian hormone receptor, type II (AMHR2), is a differentiation protein expressed in 90% of primary epithelial ovarian carcinomas (EOCs), the most deadly gynecologic malignancy. We propose that AMHR2 may serve as a useful target for vaccination against EOC. To this end, we generated the recombinant 399-amino acid cytoplasmic domain of mouse AMHR2 (AMHR2-CD) and tested its efficacy as a vaccine target in inhibiting growth of the ID8 transplantable EOC cell line in C57BL/6 mice and in preventing growth of autochthonous EOCs that occur spontaneously in transgenic mice. We found that AMHR2-CD immunization of C57BL/6 females induced a prominent antigen-specific proinflammatory CD4+ T cell response that resulted in a mild transient autoimmune oophoritis that resolved rapidly with no detectable lingering adverse effects on ovarian function. AMHR2-CD vaccination significantly inhibited ID8 tumor growth when administered either prophylactically or therapeutically, and protection against EOC growth was passively transferred into naive recipients with AMHR2-CD-primed CD4+ T cells but not with primed B cells. In addition, prophylactic AMHR2-CD vaccination of TgMISIIR-TAg transgenic mice significantly inhibited growth of autochthonous EOCs and provided a 41.7% increase in mean overall survival. We conclude that AMHR2-CD vaccination provides effective immunotherapy of EOC with relatively benign autoimmune complications.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Carcinoma/imunologia , Ooforite/prevenção & controle , Neoplasias Ovarianas/imunologia , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/administração & dosagem , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/transplante , Vacinas Anticâncer/efeitos adversos , Carcinoma/genética , Carcinoma/prevenção & controle , Processos de Crescimento Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Epiteliais/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Transplante de Neoplasias , Ooforite/etiologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/prevenção & controle , Engenharia de Proteínas , Estrutura Terciária de Proteína/genética , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta/genéticaRESUMO
ABT-737 is a small molecule Bcl-2 homology (BH)-3 domain mimetic that binds to the Bcl-2 family proteins Bcl-2 and Bcl-xL and is currently under investigation in the clinic. In this study, we investigated potential mechanisms of resistance to ABT-737 in leukemia cell lines. Compared with parental cells, cells that have developed acquired resistance to ABT-737 showed increased expression of Mcl-1 in addition to posttranslational modifications that facilitated both Mcl-1 stabilization and its interaction with the BH3-only protein Bim. To sensitize resistant cells, Mcl-1 was targeted by two pan-Bcl-2 family inhibitors, obatoclax and gossypol. Although gossypol was effective only in resistant cells, obatoclax induced cell death in both parental and ABT-737-resistant cells. NOXA levels were increased substantially by treatment with gossypol and its expression was critical for the gossypol response. Mechanistically, the newly generated NOXA interacted with Mcl-1 and displaced Bim from the Mcl-1/Bim complex, freeing Bim to trigger the mitochondrial apoptotic pathway. Together, our findings indicate that NOXA and Mcl-1 are critical determinants for gossypol-mediated cell death in ABT-737-resistant cells. These data therefore reveal novel insight into mechanisms of acquired resistance to ABT-737.