RESUMO
Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.
Assuntos
Carcinoma de Células Escamosas/genética , Epiderme/metabolismo , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Proteína Supressora de Tumor p53/genética , Animais , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Células Cultivadas , Epiderme/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Queratinócitos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Situations in which epidermal mutant mice display early lethality after birth are rather frequent. This condition precludes any kind of analysis of adult or even newborn mice tissues. We propose the in vivo embryonic skin transplantation as an alternative to solve this problem. This method allows the generation of a stable epidermal tissue that mimics the specific mutant adult epidermis. It also reproduces the phenotypic consequences and susceptibility to tumorigenesis, enabling multiple studies. Moreover, different recipient mice can help in determining problems such as different inflammation processes or the contribution of dermal cells to the different phenotypes.
Assuntos
Epiderme/embriologia , Epiderme/transplante , Transplante de Pele/métodos , Animais , Diferenciação Celular , Embrião de Mamíferos/metabolismo , Feminino , Genótipo , Camundongos , Fenótipo , Engenharia TecidualRESUMO
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia, of poor prognosis and survival, which frequently displays Akt overactivation. Previously, we reported that mice expressing high levels of constitutively Akt activity (myrAkt) in oral epithelia develop lesions and tumors in the oral cavity. MATERIALS AND METHODS: Functional genomics of primary keratinocytes from different transgenic mouse lines and immunostaining of mouse and human samples were performed in order to identify and validate putative biomarkers of oral cancer progression. RESULTS: The expression of KLF4 was found to be increased only in tumor prone samples from mice bearing overactivation of Akt. Such increased expression was confirmed in oral dysplasias and tumors arising in those mice. Tissue microarray analysis of human samples confirmed the association between active Akt and increased KLF4 expression. CONCLUSION: These data support the notion that KLF4 is potentially a reliable marker of HNSCC, and that myrAkt transgenic mice are valuable tools for preclinical research of HNSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Mucosa Bucal/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Mucosa Bucal/patologia , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Regulação para CimaRESUMO
BACKGROUND: The specific ablation of Trp53 gene in mouse epidermis leads to the spontaneous development of aggressive squamous cell carcinoma, a process that is accelerated by the subsequent loss of Rb gene. MATERIALS AND METHODS: The possible mechanisms leading to spontaneous tumor formation in epidermis in the absence of Trp53 were studied focusing on hair cycle defects, inflammation and possible chromosomal instability (CIN). RESULTS: Loss of p53 induces tumorigenesis primarily by mediating early CIN and, to a minor extent, nuclear factor kappaB activation. Notably, CIN occurs not only in p53-deficient skin, but also in epidermis lacking both Rb and Tp53 tumor suppressors, indicating a predominant role of this process in spontaneous tumorigenesis. CONCLUSION: These data identify CIN as a major mechanism in tumorigenesis originated by Trp53 loss in stratified epithelia and imply that therapies aimed to counterbalance CIN might be of relevance for the treatment of human cancer bearing impaired p53 functions.
Assuntos
Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/patologia , Instabilidade Cromossômica , Inflamação , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Animais Recém-Nascidos , Perfilação da Expressão Gênica , Integrases/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Proteína do Retinoblastoma/fisiologiaRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia with poor prognosis and survival that frequently displays Akt overactivation. Here we show that mice displaying constitutive Akt activity (myrAkt) in combination with Trp53 loss in stratified epithelia develop oral cavity tumors that phenocopy human HNSCC. The myrAkt mice develop oral lesions, making it a possible model of human oral dysplasia. The malignant conversion of these lesions, which is hampered due to the induction of premature senescence, is achieved by the subsequent ablation of Trp53 gene in the same cells in vivo. Importantly, mouse oral tumors can be followed by in vivo imaging, show metastatic spreading to regional lymph nodes, and display activation of nuclear factor-kappaB and signal transducer and activator of transcription-3 pathways and decreased transforming growth factor-beta type II receptor expression, thus resembling human counterparts. In addition, malignant conversion is associated with increased number of putative tumor stem cells. These data identify activation of Akt and p53 loss as a major mechanism of oral tumorigenesis in vivo and suggest that blocking these signaling pathways could have therapeutic implications for the management of HNSCC.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Ativação Enzimática , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Mucosa Bucal/enzimologia , Mucosa Bucal/fisiologiaRESUMO
Functional inactivation of the pRb-dependent pathway is a general feature of human cancer. However, only a reduced spectrum of tumors displays inactivation of the Rb gene. This can be attributed, at least partially, to the possible overlapping functions carried out by the related retinoblastoma family members p107 and p130. We observed that loss of pRb in epidermis, using the Cre/LoxP technology, results in proliferation and differentiation defects. These alterations are partially compensated by the elevation in the levels of p107. Moreover, epidermis lacking pRb and p107, but not pRb alone, develops spontaneous tumors, and double deficient primary keratinocytes are highly susceptible to Ha-ras-induced transformation. Two-stage chemical carcinogenesis experiments in mice lacking pRb in epidermis revealed a reduced susceptibility in papilloma formation and an increase in the malignant conversion. We have now explored whether the loss of one p107 allele, inducing a decrease in the levels of p107 up to normal levels could restore the susceptibility of pRb-deficient skin to two-stage protocol. We observed partial restoration in the incidence, number, and size of tumors. However, there is no increased malignancy despite sustained p53 activation. We also observed a partial reduction in the levels of proapoptotic proteins in benign papillomas. These data confirm our previous suggestions on the role of p107 as a tumor suppressor in epidermis in the absence of pRb.
Assuntos
Carcinógenos/toxicidade , Epiderme/metabolismo , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Alelos , Animais , Apoptose , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Predisposição Genética para Doença , Camundongos , Camundongos Knockout , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
Squamous cell carcinomas (SCC) represent the most aggressive type of nonmelanoma skin cancer. Although little is known about the causal alterations of SCCs, in organ-transplanted patients the E7 and E6 oncogenes of human papillomavirus, targeting the p53- and pRb-dependent pathways, have been widely involved. Here, we report the functional consequences of the simultaneous elimination of Trp53 and retinoblastoma (Rb) genes in epidermis using Cre-loxP system. Loss of p53, but not pRb, produces spontaneous tumor development, indicating that p53 is the predominant tumor suppressor acting in mouse epidermis. Although the simultaneous inactivation of pRb and p53 does not aggravate the phenotype observed in Rb-deficient epidermis in terms of proliferation and/or differentiation, spontaneous SCC development is severely accelerated in doubly deficient mice. The tumors are aggressive and undifferentiated and display a hair follicle origin. Detailed analysis indicates that the acceleration is mediated by premature activation of the epidermal growth factor receptor/Akt pathway, resulting in increased proliferation in normal and dysplastic hair follicles and augmented tumor angiogenesis. The molecular characteristics of this model provide valuable tools to understand epidermal tumor formation and may ultimately contribute to the development of therapies for the treatment of aggressive squamous cancer.
Assuntos
Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Genes do Retinoblastoma , Proteína do Retinoblastoma/genética , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/metabolismo , Inativação Gênica , Predisposição Genética para Doença , Folículo Piloso/patologia , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteína Oncogênica v-akt/metabolismo , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/deficiência , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/deficiênciaRESUMO
The specific deletion of Rb gene in epidermis leads to altered proliferation and differentiation, but not to the development of spontaneous tumors. Our previous data have demonstrated the existence of a functional compensation of Rb loss by Rbl1 (p107) in as the phenotypic differences with respect to controls are intensified. However, the possible evolution of this aggravated phenotype, in particular in relationship with tumorigenesis, has not been evaluated due to the premature death of the double deficient mice. We have now investigated whether p107 can also act as a tumor suppressor in pRb-deficient epidermis using different experimental approaches. We found spontaneous tumor development in doubly-deficient skin grafts. Moreover, Rb-deficient keratinocytes are susceptible to Ha-ras-induced transformation, and this susceptibility is enhanced by p107 loss. Further functional analyses, including microarray gene expression profiling, indicated that the loss of p107, in the absence of pRb, produces the reduction of p53-dependent pro-apoptotic signals. Overall, our data demonstrate that p107 behaves as a tumor suppressor in epidermis in the absence of pRb and suggest novel tumor-suppressive roles for p107 in the context of functional p53 and activated Ras.
Assuntos
Genes do Retinoblastoma , Proteína p107 Retinoblastoma-Like/fisiologia , Neoplasias Cutâneas/genética , Pele/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Células Cultivadas , Feminino , Deleção de Genes , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Proteína p107 Retinoblastoma-Like/genéticaRESUMO
Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Akt(wt)), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ectoderma/anormalidades , Ectoderma/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Ectoderma/patologia , Ativação Enzimática , Epiderme/enzimologia , Epiderme/patologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Enzimológica da Expressão Gênica , Cabelo/anormalidades , Cabelo/ultraestrutura , Homeostase , Camundongos , Camundongos Transgênicos , Unhas Malformadas/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco/citologia , Células-Tronco/enzimologia , Anormalidades Dentárias/enzimologiaRESUMO
Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.