RESUMO
Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (Kras(LA2)). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras(LA2) model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras(LA2) model. By contrast, the Kras(LA2) tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.
Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Genes ras/genética , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Mutação/genética , Proteína Oncogênica p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Animais , Carcinógenos/toxicidade , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/genética , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Feminino , Instabilidade Genômica/genética , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Metilnitrosoureia/toxicidade , Camundongos , Modelos Genéticos , Mutação Puntual/genética , Uretana/toxicidadeRESUMO
Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16(INK4a) tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane [intraperitoneal (i.p.), 1 mg/g]. Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients.
Assuntos
Carcinógenos , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Hemangiossarcoma/prevenção & controle , Neoplasias Hepáticas/prevenção & controle , Uretana , Animais , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dosagem de Genes , Predisposição Genética para Doença , Hemangiossarcoma/induzido quimicamente , Hemangiossarcoma/genética , Hemangiossarcoma/metabolismo , Hemangiossarcoma/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais , Fatores de Tempo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Expression of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) is frequently reduced in human colorectal cancer, and this correlates with poor patient prognosis. To clarify the role of p27 in gastrointestinal (GI) cancer, we measured p27 expression, as well as the effect of germline deletion of p27, in 3 different mouse models of GI neoplasia. p27 expression was frequently reduced in GI tumors arising in 1,2-dimethylhydrazine (DMH) treated mice, and in Apc mutant Min/+ mice, but not in GI tumors arising in Smad3 mutant mice. Germline deletion of p27 resulted in accelerated tumor development and increased tumor cell proliferation in both DMH treated and Min/+ mice, but not in Smad3 mutant mice. p27 deficiency also led to increased adenoma to adenocarcinoma progression. These results indicate that reduction of p27 cooperates with mutations in Apc but not in Smad3 during GI tumorigenesis. Thus, tumor suppression by p27 is contingent on the specific oncogenic pathway that drives tumor development.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/genética , Neoplasias Gastrointestinais/patologia , Genes Supressores de Tumor/fisiologia , Transativadores/genética , Proteínas Supressoras de Tumor/fisiologia , 1,2-Dimetilidrazina , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Adenoma/induzido quimicamente , Adenoma/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas do Citoesqueleto/metabolismo , Neoplasias Gastrointestinais/induzido quimicamente , Neoplasias Gastrointestinais/genética , Perda de Heterozigosidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Associadas a Fase S , Transdução de Sinais , Proteínas Smad , Proteínas Supressoras de Tumor/genética , beta CateninaRESUMO
The oncogene Ras and the tumor suppressor gene p53 are frequently co-mutated in human cancer and mutations in Ras and p53 can cooperate to generate a more malignant cell state. To discover novel druggable targets for cancers carrying co-mutations in Ras and p53, we performed arrayed, kinome focused siRNA and oncology drug phenotypic screening utilizing a set of syngeneic Ras mutant squamous cell carcinoma (SCC) cell lines that also carried co-mutations in selected p53 pathway genes. These cell lines were derived from SCCs from carcinogen-treated inbred mice which harbored germline deletions or mutations in Trp53, p19Arf, Atm, or Prkdc. Both siRNA and drug phenotypic screening converge to implicate the phosphoinositol kinases, receptor tyrosine kinases, MAP kinases, as well as cell cycle and DNA damage response genes as targetable dependencies in SCC. Differences in functional kinome profiles between Ras mutant cell lines reflect incomplete penetrance of Ras synthetic lethal kinases and indicate that co-mutations cause a rewiring of survival pathways in Ras mutant tumors. This study describes the functional kinomic landscape of Ras/p53 mutant chemically-induced squamous cell carcinoma in both the baseline unperturbed state and following DNA damage and nominates candidate therapeutic targets, including the Nek4 kinase, for further development.
Assuntos
Carcinoma de Células Escamosas , Proteína Supressora de Tumor p53 , Proteínas ras , Animais , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Humanos , Camundongos , Mutação , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genéticaRESUMO
p53 is required for DNA damage-induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53-deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA-dependent protein kinase (DNA-PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation-induced apoptosis, whereas apoptosis in DNA-PK(cs)/p53, Ku80/p53 and Ku70/p53 double-null mice was quantitatively equivalent to that seen in wild-type mice. This p53-independent apoptotic response was specific to the loss of DNA-PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double-null mice. Furthermore, it was associated with an increase in phospho-checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA-PK, does not require p53.
Assuntos
Apoptose , Dano ao DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Regulação para Baixo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Camundongos , Camundongos Knockout , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Ovarian clear cell carcinoma (OCCC) is a rare, chemo-resistant subtype of ovarian cancer. To identify novel therapeutic targets and combination therapies for OCCC, we subjected a set of patient-derived ovarian cancer cell lines to arrayed high-throughput siRNA and drug screening. The results indicated OCCC cells are vulnerable to knockdown of epigenetic gene targets such as bromodomain and extra-terminal domain (BET) proteins BRD2 and BRD3. Subsequent RNA interference assays, as well as BET inhibitor treatments, validated these BET proteins as potential therapeutic targets. Because development of resistance to single targeted agents is common, we next performed sensitizer drug screens to identify potential combination therapies with the BET inhibitor CPI0610. Several PI3K or AKT inhibitors were among the top drug combinations identified and subsequent work showed CPI0610 synergized with alpelisib or MK2206 by inducing p53-independent apoptosis. We further verified synergy between CPI0610 and PI3K-AKT pathway inhibitors alpelisib, MK2206, or ipatasertib in tumor organoids obtained directly from patients with OCCC. These findings indicate further preclinical evaluation of BET inhibitors, alone or in combination with PI3K-AKT inhibitors for OCCC, is warranted.
Assuntos
Adenocarcinoma de Células Claras/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma de Células Claras/patologia , Feminino , Humanos , Neoplasias Ovarianas/patologia , TransfecçãoRESUMO
BACKGROUND: Homologous recombination deficiencies (HRD) are present in approximately half of epithelial ovarian cancers, for which PARP inhibitors (PARPi) are becoming a preferred treatment option. However, a considerable proportion of these carcinomas acquire resistance or harbour de novo resistance, posing a significant challenge to treatment. METHODS: To identify new combinatorial therapeutics to overcome resistance to PARPi, we employed high-throughput conditional RNAi and drug screening of patient-derived ovarian cancer cells. To prioritise clinically relevant drug combinations, we integrated empirical validation with analysis of The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) datasets to nominate candidate targets and drugs, reaching three main findings. FINDINGS: Firstly, we found that the PARPi rucaparib enhanced the effect of BET inhibitors (CPI-203 & CPI-0610) irrespective of clinical subtype or HRD status. Additional drug combination screens identified that dasatinib, a non-receptor tyrosine kinase inhibitor, augmented the effects of rucaparib and BET inhibitors, proposing a potential broadly applicable triple-drug combination for high-grade serous and clear cell ovarian carcinomas. Secondly, rucaparib synergised with the BCL2 family inhibitor navitoclax, with preferential activity in ovarian carcinomas that harbour alterations in BRCA1/2, BARD1, or MSH2/6. Thirdly, we identified potentially antagonistic drug combinations between the PARPi rucaparib and vinca alkaloids, anthracyclines, and antimetabolites, cautioning their use in the clinic. INTERPRETATION: These findings propose therapeutic strategies to address PARP inhibitor resistance using agents that are already approved or are in clinical development, with the potential for rapid translation to benefit a broad population of ovarian cancer patients.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidoresRESUMO
The cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) (p27) is a marker of prognosis in many cancers, including breast cancer. Low p27 expression correlates with poor prognosis, especially in hormone receptor positive breast tumors. This association suggests a role for p27 in hormone-dependent cancer. We used the Wnt-1 transgenic mouse model to further explore the role of p27 in hormone-driven breast cancer. We found that p27 deficiency did not alter breast cancer rate in either male or female Wnt-1 mice. However, we did find p27-/- females had reduced levels of serum progesterone (P) and increased variability in estradiol (E), which could have affected their cancer susceptibility. To equalize hormone levels, an additional cohort of Wnt-1 female mice was ovariectomized and implanted with slow release pellets of E and P. Although this treatment did not alter the breast cancer rate, it did accelerate the development of pituitary and gastric tumors in p27-/- mice. This study shows that while not a significant inhibitor of Wnt-1-driven breast cancer, p27 inhibits gastric tumors, whose latency is modulated by sex steroids.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Estradiol/sangue , Neoplasias Mamárias Experimentais/sangue , Neoplasias Hormônio-Dependentes/sangue , Progesterona/metabolismo , Proteína Wnt1/metabolismo , Animais , Inibidor de Quinase Dependente de Ciclina p27/genética , Estradiol/farmacologia , Feminino , Masculino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Neoplasias Hormônio-Dependentes/patologia , Ovariectomia , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Progesterona/farmacologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína Wnt1/genéticaRESUMO
Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage, and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in the suppression of Ras-driven tumors, we examined susceptibility to skin carcinogenesis in 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Atm- and p53-deficient mice and compared these results to previous studies on Arf-deficient mice. Mice with epidermal-specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared with wild-type littermates. In contrast, Atm-deficient mice showed no increase in papilloma number, growth, or malignant progression. gamma-H2AX and p53 levels were increased in both Atm(+/+) and Atm(-/-) papillomas, whereas Arf(-/-) papillomas showed much lower p53 expression. Thus, although there is evidence of DNA damage, signaling through Arf seems to regulate p53 in these Ras-driven tumors. In spontaneous and radiation-induced lymphoma models, tumor latency was accelerated in Atm(-/-)p53(-/-) compound mutant mice compared with the single mutant Atm(-/-) or p53(-/-) mice, indicating cooperation between loss of Atm and loss of p53. Although p53-mediated apoptosis was impaired in irradiated Atm(-/-) lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm(-/-) mice. In conclusion, in these models of oncogene- or DNA damage-induced tumors, p53 retains tumor suppressor activity in the absence of Atm.
Assuntos
Proteínas de Ligação a DNA/deficiência , Proteínas Serina-Treonina Quinases/deficiência , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/deficiência , 9,10-Dimetil-1,2-benzantraceno , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Perda de Heterozigosidade/genética , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/deficiência , Proteínas Supressoras de Tumor/metabolismoRESUMO
The ataxia-telangiectasia mutated (Atm) protein kinase is a central regulator of the cellular response to DNA damage. Although Atm can regulate p53, it is not known if this Atm function varies between tissues. Previous studies showed that the induction of p53 and apoptosis by whole-body ionizing radiation varies greatly between tissue and tumor types, so here we asked if Atm also had a tissue-specific role in the ionizing radiation response. Irradiated Atm-null mice showed impaired p53 induction and apoptosis in thymus, spleen, and brain. In contrast, radiation-induced p53, apoptosis, phosphorylation of Chk2, and G(2)-M cell cycle arrest were slightly delayed in Atm(-/-) epithelial cells of the small intestine but reached wild-type levels by 4 h. Radiation-induced p53 and apoptosis in Atm(-/-) hair follicle epithelial cells were not impaired at any of the time points examined. Thus, Atm is essential for radiation-induced apoptosis in lymphoid tissues but is largely dispensable in epithelial cells. This indicates that marked differences in DNA damage signaling pathways exist between tissues, which could explain some of the tissue-specific phenotypes, especially tumor suppression, associated with Atm deficiency.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epitélio/metabolismo , Epitélio/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Epiderme/metabolismo , Epiderme/patologia , Epiderme/efeitos da radiação , Epitélio/patologia , Feminino , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Folículo Piloso/efeitos da radiação , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Intestino Delgado/efeitos da radiação , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Tecido Linfoide/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Especificidade de Órgãos , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Irradiação Corporal TotalRESUMO
Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low-passage tumor cells derived from a patient with treatment-resistant HPV-negative HNSCC.Experimental Design: A tumor cell culture was established and subjected to whole-exome sequencing, RNA sequencing, comparative genome hybridization, and high-throughput phenotyping with a siRNA library covering the druggable genome and an oncology drug library. Secondary screens of candidate target genes were performed on the primary tumor cells and two nontumorigenic keratinocyte cell cultures for validation and to assess cancer specificity. siRNA screens of the kinome on two isogenic pairs of p53-mutated HNSCC cell lines were used to determine generalizability. Clinical utility was addressed by performing drug screens on two additional HNSCC cell cultures derived from patients enrolled in a clinical trial.Results: Many of the identified copy number aberrations and somatic mutations in the primary tumor were typical of HPV(-) HNSCC, but none pointed to obvious therapeutic choices. In contrast, siRNA profiling identified 391 candidate target genes, 35 of which were preferentially lethal to cancer cells, most of which were not genomically altered. Chemotherapies and targeted agents with strong tumor-specific activities corroborated the siRNA profiling results and included drugs that targeted the mitotic spindle, the proteasome, and G2-M kinases WEE1 and CHK1 We also show the feasibility of ex vivo drug profiling for patients enrolled in a clinical trial.Conclusions: High-throughput phenotyping with siRNA and drug libraries using patient-derived tumor cells prioritizes mutated driver genes and identifies novel drug targets not revealed by genomic profiling. Functional profiling is a promising adjunct to DNA sequencing for precision oncology. Clin Cancer Res; 24(12); 2828-43. ©2018 AACR.
Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Terapia de Alvo Molecular , Medicina de Precisão , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Hibridização Genômica Comparativa , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Genômica/métodos , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Mutação , Tomografia por Emissão de Pósitrons , Medicina de Precisão/métodos , RNA Interferente Pequeno/genética , Tomografia Computadorizada por Raios X , Transcriptoma , Sequenciamento do ExomaRESUMO
Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).
Assuntos
Genes ras , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/fisiologia , Proteína Supressora de Tumor p53/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Alelos , Animais , Carcinógenos , Carcinoma de Células Escamosas/patologia , Cruzamentos Genéticos , Inibidor p16 de Quinase Dependente de Ciclina , Progressão da Doença , Genótipo , Perda de Heterozigosidade , Metástase Linfática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Papiloma/genética , Papiloma/metabolismo , Papiloma/patologia , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Ionizing radiation (IR) is one of the most widely used treatments for cancer. However, acute damage to the gastrointestinal tract or gastrointestinal acute radiation syndrome (GI-ARS) is a major dose-limiting side effect, and the mechanisms that underlie this remain unclear. Here we use mouse models to explore the relative roles of DNA repair, apoptosis, and cell cycle arrest in radiation response. IR induces DNA double strand breaks and DNA-PK mutant Prkdcscid/scid mice are sensitive to GI-ARS due to an inability to repair these breaks. IR also activates the tumor suppressor p53 to trigger apoptotic cell death within intestinal crypt cells and p53 deficient mice are resistant to apoptosis. To determine if DNA-PK and p53 interact to govern radiosensitivity, we compared the response of single and compound mutant mice to 8 Gy IR. Compound mutant Prkdcscid/scid/Trp53-/-mice died earliest due to severe GI-ARS. While both Prkdcscid/scid and Prkdcscid/scid/Trp53-/-mutant mice had higher levels of IR-induced DNA damage, particularly within the stem cell compartment of the intestinal crypt, in Prkdcscid/scid/Trp53-/-mice these damaged cells abnormally progressed through the cell cycle resulting in mitotic cell death. This led to a loss of Paneth cells and a failure to regenerate the differentiated epithelial cells required for intestinal function. IR-induced apoptosis did not correlate with radiosensitivity. Overall, these data reveal that DNA repair, mediated by DNA-PK, and cell cycle arrest, mediated by p53, cooperate to protect the stem cell niche after DNA damage, suggesting combination approaches to modulate both pathways may be beneficial to reduce GI-ARS. As many cancers harbor p53 mutations, this also suggests targeting DNA-PK may be effective to enhance sensitivity of p53 mutant tumors to radiation.
Assuntos
Síndrome Aguda da Radiação/metabolismo , Síndrome Aguda da Radiação/patologia , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Trato Gastrointestinal/patologia , Proteínas Nucleares/metabolismo , Radiação Ionizante , Células-Tronco/metabolismo , Células-Tronco/patologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos da radiação , Proliferação de Células/efeitos da radiação , Dano ao DNA , Trato Gastrointestinal/efeitos da radiação , Histonas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos Endogâmicos C57BL , Mitose , Índice Mitótico , Celulas de Paneth/metabolismo , Fenótipo , Fosforilação/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Repressoras/metabolismo , Coloração e Rotulagem , Nicho de Células-Tronco/efeitos da radiação , Células-Tronco/efeitos da radiação , SurvivinaRESUMO
Connexin32 knockout mice (Cx32-KO) exhibit increased chemical- and radiation-induced liver and lung tumor formation with many lung tumors demonstrating decreased levels of the tumor suppressor p27KIP1. To determine if p27 deficiency alters Cx32-influenced tumorigenesis, we have generated a Cx32/p27 double-deficient mouse strain (DKO) and show here that exposure of these mice to X-ray radiation resulted in an increase or decrease in tumorigenesis depending on the tissue. Several tissues were highly sensitive to loss of p27 tumor suppressor function (intestine, adrenal, pituitary) resulting in an increased overall tumor burden in DKO mice compared to both wild-type (P<0.005) and Cx32-KO mice (P=0.066). However, additional deletion of p27 in a Cx32-KO background resulted in a statistically significant decrease in the liver tumor incidence suggesting that Cx32 and p27 pathways mechanistically interact. Immunohistochemical analysis revealed an increased percentage of Cx32-KO liver and lung tumors harboring active mitogen-activated protein kinase (Erk1, Erk2) pathways in contrast to lower percentages of activated wild-type (P<0.005) and DKO tumors (P=0.027). Increased MAPK activation in liver tumors did not correlate with Ha-ras codon-61 mutation status. This study demonstrates that tissues dependent on Cx32 tumor suppression, such as the liver and lung, exhibit altered tumorigenesis and tumor biology (MAPK pathway activation) related to p27 status.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Conexinas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/etiologia , Proteínas Supressoras de Tumor/fisiologia , Adenoma/etiologia , Animais , Conexinas/deficiência , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas do Citoesqueleto/genética , Ativação Enzimática , Feminino , Genes ras , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Pulmonares/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Induzidas por Radiação/etiologia , Especificidade de Órgãos , Neoplasias Hipofisárias/etiologia , Transativadores/genética , Proteínas Supressoras de Tumor/deficiência , Aumento de Peso , beta Catenina , Proteína beta-1 de Junções ComunicantesRESUMO
Mutations in p53 and reduced expression of the Cdk inhibitor p27Kip1 are frequently observed in a wide variety of human cancers, but it is not known whether alterations in these tumor suppressors interact to influence tumor progression. To address this, we measured tumor latency and spectrum in p53 and p27 single and compound mutant mice. p53-/- (null) mice developed T-cell lymphomas and soft-tissue sarcomas, while p27-/- mice developed adenomas of the pituitary and lung, but with much longer latency. The latency for tumor development in p53-/- p27-/- and p53-/- p27+/- compound mutant mice was significantly reduced, by 15-30%, compared to single mutant p53-/- mice. The tumor spectrum in the compound mutants was similar to that of p53-/- mice, and additional tumors of diverse histotypes. In tumors from p53-/- mice, p27 protein levels were reduced to a greater extent than were mRNA levels, indicating that p27 is downregulated in tumors at the transcriptional as well as post-transcriptional levels. In contrast, mice deficient in another Cdk inhibitor p21Cip1, which is also a transcriptional target and effector of p53, showed only a marginal increase in tumor predisposition in response to ENU treatment. Thus, downregulation of p27 is a common feature in p53-/- tumors. Germline deletion of one or both alleles of p27 accelerates tumor development and associated mortality in p53-/- mice, indicating potent synergy between loss of p27 and p53. Although p21 is functionally similar to both p53 and p27, it plays a lesser role in tumor suppression. These results further highlight the highly cooperative nature of p27 and its central role in tumor suppression.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclinas/fisiologia , Neoplasias Experimentais/patologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Primers do DNA , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/fisiopatologiaRESUMO
Since the groundbreaking studies in the middle part of the last century showing liver cancer in rodents exposed to aromatic amines, the liver has been widely used as a model target organ of chemical carcinogenesis. This protocol describes a method for inducing liver tumors by injecting mice with the widely used alkylating agents N-ethyl-N-nitrosourea (ENU) and N-nitrosodiethylamine (DEN). ENU does not require metabolic activation and readily induces tumors in a number of tissues, including the lungs, stomach, and ovaries, as well as inducing lymphomas. Mice injected with DEN can also develop other tumors, including those of the gastrointestinal tract, skin, lungs, and lymphocytes, but because DEN is metabolized in the liver, it is most effective at inducing liver tumors.
Assuntos
Carcinógenos/administração & dosagem , Dietilnitrosamina/administração & dosagem , Etilnitrosoureia/administração & dosagem , Neoplasias Hepáticas/induzido quimicamente , Animais , Injeções Intraperitoneais , CamundongosRESUMO
In this protocol, colon cancer is induced in mice through a series of injections with 1,2-dimethylhydrazine. Mice will develop primarily colon tumors starting at about 3 mo after the first injection. Tumors in the lung, uterus, and small intestine may also be seen, as well as lymphomas.
Assuntos
1,2-Dimetilidrazina/metabolismo , Neoplasias do Colo/induzido quimicamente , 1,2-Dimetilidrazina/administração & dosagem , Animais , Neoplasias do Colo/patologia , Injeções Subcutâneas , Camundongos , Fatores de TempoRESUMO
In this protocol, urethane (ethyl carbamate) is used to induce lung tumors in mice. The use of urethane as an experimental carcinogen is especially attractive as it is inexpensive, relatively safe to handle, stable, and water soluble, and the protocol involves simple intraperitoneal (i.p.) injections in young mice. Urethane typically induces bronchioalveolar adenomas and, to a lesser extent, adenocarcinomas that resemble the adenocarcinoma subtype of non-small cell lung carcinoma. On a sensitive genetic background such as A/J, mice develop multiple adenomas visible on the lung surface by 25 wk, followed by the appearance of adenocarcinomas by 40 wk. Less-sensitive strains such as B6/129 develop tumors with a longer latency.
Assuntos
Carcinógenos , Neoplasias Pulmonares/induzido quimicamente , Uretana , Adenocarcinoma Bronquioloalveolar/induzido quimicamente , Adenocarcinoma Bronquioloalveolar/patologia , Adenoma/induzido quimicamente , Adenoma/patologia , Animais , Injeções Intraperitoneais , Neoplasias Pulmonares/patologia , Camundongos , Fatores de TempoRESUMO
Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.
Assuntos
Metilação de DNA , Genes Supressores de Tumor , Neoplasias/genética , Proteínas Repressoras/genética , Animais , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Haploinsuficiência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neoplasias/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Análise de SobrevidaRESUMO
PURPOSE: To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. RESULTS: Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. CONCLUSIONS: WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer.