RESUMO
Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.
Assuntos
Antineoplásicos , Carcinoma , Camundongos , Animais , Antineoplásicos/farmacologia , Transdução de Sinais , Ciclofosfamida/farmacologia , Células-Tronco Neoplásicas/metabolismo , Carcinoma/metabolismo , Linhagem Celular TumoralRESUMO
To fight cancer more efficiently with cell-based immunotherapy, more information about the cells of the immune system and their interaction with cancer cells in vivo is needed. Therefore paraffin wax embedded primary breast cancers from the syngeneic mouse WAP-T model and from xenografted tumors of breast, colon, melanoma, ovarian, neuroblastoma, pancreatic, prostate, and small cell lung cancer were investigated for the infiltration of immunocompetent cells by immunohistochemistry using antibodies against leukocyte markers. The following markers were used: CD45 as a pan-leukocyte marker, BSA-I as a dendritic cell marker, CD11b as an NK cell marker, and CD68 as a marker for macrophages. The labeled immune cells were attributed to the following locations: adjacent adipose tissue, tumor capsule, intra-tumoral septae, and cancer cells directly. In xenograft tumors, the highest score of CD45 and CD11b positive, NK, and dendritic cells were found in the adjacent adipose tissue, followed by lesser infiltration directly located at the cancer cells themselves. The detected numbers of CD45 positive cells differed between the tumor entities: few infiltrating cells in breast cancer, small cell lung cancer, neuroblastoma, a moderate infiltration in colon cancer, melanoma and ovarian cancer, strongest infiltration in prostate and pancreatic cancer. In the syngeneic tumors, the highest score of CD45 and CD11b positive, NK and dendritic cells were observed in the tumor capsule, followed by a lesser infiltration of the cancer tissue. Our findings argue for paying more attention to investigate how immune-competent cells can reach the tumor cells directly.
Assuntos
Imunidade Celular/fisiologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/fisiologia , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Hiperplasia/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Neoplasias Pulmonares , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Neuroblastoma/imunologia , Neoplasias Pancreáticas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The SV40 transgenic BALB/c mouse based WAP-T/WAP-TNP model for triple-negative breast cancer allows the analysis of parameters influencing immunotherapeutic approaches. Except for WAP-TNP tumors expressing the immune-dominant LCMV NP-epitope within SV40 T-antigen (T-AgNP) which is not expressed by T-Ag of WAP-T tumors, the tumors are extremely similar. Comparative anti-PD1/PD-L1 immunotherapy of WAP-T and WAP-TNP mice supported the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy, with highly immunogenic T-cell epitopes favoring rapid CTL exhaustion. Here we analyzed the immune response in NP8 mice during early times of tumor development. LCMV infection of lactating NP8 mice induced lifelong tumor protection by memory CTLs. Immunization with LCMV after involution and appearance of T-AgNP expressing parity-induced tumor progenitor cells could not cure the mice, as memory CTLs became exhausted. However, immunization significantly prolonged the time of tumor outgrowth. Elimination of exhausted CTLs and of immunosuppressive cells by sub-lethal γ-irradiation, followed by adoptive transfer of NP-epitope specific CTLs into NP8 tumor mice with early lesions, completely prevented tumor outgrowth, when lymphocytes obtained after injection of weakly immunogenic NP8 tumor-derived cells into BALB/c mice were transferred. Transfer of lymphocytes obtained after infection of BALB/c mice with highly immunogenic LCMV into such mice delayed tumor outgrowth for a significant period, but could not prevent it. We conclude that eliminating exhausted CTLs and immune-suppressive cells followed by transfer or generation of low-avidity tumor antigen-specific CTLs might be a promising approach for curative tumor immunotherapy.
RESUMO
Using the SV40 transgenic WAP-T/WAP-TNP mouse models for mammary carcinomas, we compared the response to immune checkpoint blockade therapy in tumor mice expressing either SV40 T-antigen containing the LCMV NP-epitope (T-AgNP in WAP-TNP mice), or the unmodified T-antigen (T-Ag in WAP-T mice). Specifically, we asked, whether the presence of the highly immunogenic NP-epitope in T-AgNP influences this response in comparison to the weakly immunogenic T-cell epitopes of T-Ag in WAP-T tumor mice. Treatment of WAP-TNP tumor mice with either anti-PD1 or anti-PD-L1 antibodies led to tumor regression, with anti-PD-L1 treatment being more effective. However, tumors had fully re-appeared after 21 days, indicating that CTL exhaustion had been rapidly re-established. Surprisingly, the same treatment applied to WAP-T tumor mice resulted in a significantly prolonged period of tumor regression. We provide evidence that in contrast to the weak antigenic stimuli exerted by T-cell epitopes of T-Ag, the strong antigenic stimulus of the NP-epitope in T-AgNP has a dual effect: (i) a rapid generation of active NP-specific CTLs, accompanied (ii) by accelerated CTL exhaustion. Our data support the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Neoplasias Mamárias Experimentais/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos Transformantes de Poliomavirus/genética , Antígeno B7-H1/imunologia , Epitopos de Linfócito T/genética , Feminino , Terapia de Imunossupressão , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Nucleoproteínas/genética , Fragmentos de Peptídeos/genética , Receptor de Morte Celular Programada 1/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Evasão TumoralRESUMO
We analyzed the molecular basis for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-controlled inhibition of epithelial-mesenchymal transition (EMT) in a mouse model for mammary adenocarcinoma (WAP-T mice). We demonstrate that silencing of CEACAM1 in WAP-T tumor-derived G-2 cells induces epithelial-mesenchymal plasticity (EMP), as evidenced by typical changes of gene expression, morphology and increased invasion. In contrast, reintroduction of CEACAM1 into G-2 cells reversed up-regulation of genes imposing mesenchymal transition, as well as cellular invasion. We identified the Wnt-pathway as target for CEACAM1-mediated repression of EMT. Importantly, ß-catenin phosphorylation status and transcriptional activity strongly depend on CEACAM1 expression: CEACAM1high G-2 cells displayed enhanced phosphorylation of ß-catenin at S33/S37/T41 and decreased phosphorylation at Y86, thereby inhibiting canonical Wnt/ß-catenin signaling. We identified Src-homology 2 domain-containing phosphatase 2 (SHP-2) as a critical binding partner of CEACAM1 that could modulate ß-catenin Y86 phosphorylation. Hence, CEACAM1 serves as a scaffold that controls membrane proximal ß-catenin signaling. In vivo, mammary tumors of WAP-T/CEACAM1null mice displayed increased nuclear translocation of ß-catenin and a dramatically enhanced metastasis rate compared to WAP-T mice. Hence, CEACAM1 controls EMT in vitro and in vivo by site-specific regulation of ß-catenin phosphorylation. Survival analyses of human mammary carcinoma patients corroborated these data, indicating that CEACAM1 is a prognostic marker for breast cancer survival.
Assuntos
Neoplasias da Mama/metabolismo , Antígeno Carcinoembrionário/metabolismo , Carcinoma/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/metabolismo , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Técnicas In Vitro , Neoplasias Mamárias Experimentais/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais , Regulação para Cima , beta Catenina/metabolismoRESUMO
In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cell responses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these 'endogenous' NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-TNP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-TNP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Neoplasias Mamárias Experimentais/imunologia , Animais , Antígenos de Neoplasias/imunologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos TransgênicosRESUMO
BACKGROUND: Experimental analysis of the metastatic cascade requires suitable model systems which allow tracing of disseminated tumor cells and the identification of factors leading to metastatic outgrowth in distant organs. Such models, especially models using immune-competent mice, are rather scarce. We here analyze tumor cell dissemination and metastasis in an immune-competent transplantable mouse mammary tumor model, based on the SV40 transgenic WAP-T mouse mammary carcinoma model. METHODS: We orthotopically transplanted into immune-competent WAP-T mice two tumor cell lines (H8N8, moderately metastatic, and G-2, non-metastatic), developed from primary WAP-T tumors. G-2 and H8N8 cells exhibit stem cell characteristics, form homeostatic, heterotypic tumor cell systems in vitro, and closely mimic endogenous primary tumors after orthotopic transplantation into syngeneic, immune-competent WAP-T mice. Tumor cell transgene-specific PCR allows monitoring of tumor cell dissemination into distinct organs, and immunohistochemistry for SV40 T-antigen tracing of single disseminated tumor cells (DTC). RESULTS: While only H8N8 cell-derived tumors developed metastases, tumors induced with both cell lines disseminated into a variety of organs with similar efficiency and similar organ distribution. H8N8 metastases arose only in lungs, indicating that organ-specific metastatic outgrowth depends on the ability of DTC to re-establish a tumor cell system rather than on invasion per se. Resection of small tumors (0.5 cm(3)) prevented metastasis of H8N8-derived tumors, most likely due to the rather short half-life of DTC, and thus to shorter exposure of the mice to DTC. In experimental metastasis by tail vein injection, G-2 and H8N8 cells both were able to form lung metastases with similar efficiency. However, after injection of sorted "mesenchymal" and "epithelial" G-2 cell subpopulations, only the "epithelial" subpopulation formed lung metastases. CONCLUSIONS: We demonstrate the utility of our mouse model to analyze factors influencing tumor cell dissemination and metastasis. We suggest that the different metastatic capacity of G-2 and H8N8 cells is due to their different degrees of epithelial-mesenchymal plasticity (EMP), and thus the ability of the respective disseminated cells to revert from a "mesenchymal" to an "epithelial" differentiation state.
Assuntos
Neoplasias da Mama/patologia , Carcinogênese , Neoplasias Mamárias Animais/patologia , Metástase Neoplásica/patologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/genéticaRESUMO
In this study, the effects of the standard chemotherapy, cyclophosphamide/adriamycin/5-fluorouracil (CAF) on tumor growth, dissemination and recurrence after orthotopic implantation of murine G-2 cells were analyzed in the syngeneic immunocompetent whey acidic protein-T mouse model (Wegwitz et al., PLoS One 2010; 5:e12103; Schulze-Garg et al., Oncogene 2000; 19:1028-37). Single-dose CAF treatment reduced tumor size significantly, but was not able to eradicate all tumor cells, as recurrent tumor growth was observed 4 weeks after CAF treatment. Nine days after CAF treatment, residual tumors showed features of regressive alterations and were composed of mesenchymal-like tumor cells, infiltrating immune cells and some tumor-associated fibroblasts with an intense deposition of collagen. Recurrent tumors were characterized by coagulative necrosis and less tumor cell differentiation compared with untreated tumors, suggesting a more aggressive tumor phenotype. In support, tumor cell dissemination was strongly enhanced in mice that had developed recurrent tumors in comparison with untreated controls, although only few disseminated tumor cells could be detected in various organs 9 days after CAF application. In vitro experiments revealed that CAF treatment of G-2 cells eliminates the vast majority of epithelial tumor cells, whereas tumor cells with a mesenchymal phenotype survive. These results together with the in vivo findings suggest that tumor cells that underwent epithelial-mesenchymal transition and/or exhibit stem-cell-like properties are difficult to eliminate using one round of CAF chemotherapy. The model system described here provides a valuable tool for the characterization of the effects of chemotherapeutic regimens on recurrent tumor growth and on tumor cell dissemination, thereby enabling the development and preclinical evaluation of novel therapeutic strategies to target mammary carcinomas.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclofosfamida/administração & dosagem , Ciclofosfamida/farmacologia , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Transplante de NeoplasiasRESUMO
To study the postulated mutant p53 (mutp53) "gain of function" effects in mammary tumor development, progression and metastasis, we crossed SV40 transgenic WAP-T mice with mutant p53 transgenic WAP-mutp53 mice. Compared to tumors in monotransgenic WAP-T mice, tumors in bitransgenic WAP-T x WAP-mutp53 mice showed higher tumor grading, enhanced vascularization, and significantly increased metastasis. Bitransgenic tumors revealed a gene signature associated with the oncogenic epithelial-mesenchymal transition pathway (EMT gene signature). In cultures of WAP-T tumor-derived G-2 cancer cells, which are comprised of subpopulations displaying "mesenchymal" and "epithelial" phenotypes, this EMT gene signature was associated with the "mesenchymal" compartment. Furthermore, ectopic expression of mutp53 in G-2 cells sufficed to induce a strong EMT phenotype. In contrast to these in vitro effects, monotransgenic and bitransgenic tumors were phenotypically similar suggesting that in vivo the tumor cell phenotype might be under control of the tumor microenvironment. In support, orthotopic transplantation of G-2 cells as well as of G-2 cells expressing ectopic mutp53 into syngeneic mice resulted in tumors with a predominantly epithelial phenotype, closely similar to that of endogenous primary tumors. We conclude that induction of an EMT gene signature by mutp53 in bitransgenic tumors primarily promotes tumor cell plasticity, that is, the probability of tumor cells to undergo EMT processes under appropriate stimuli, thereby possibly increasing their potential to disseminate and metastasize.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Mamárias Experimentais/patologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Mutação , Metástase Neoplásica , Microambiente TumoralRESUMO
BACKGROUND: The WAP-T mouse model is an established clinically relevant model of breast cancer. Lectins have been used to study malignant progression in clinical studies. We investigated lectin binding sites to test for the clinical relevance of this model. MATERIALS AND METHODS: Samples of the WAP-T mouse mammary tissues, from normal tissues to undifferentiated higher tumor grades were stained using an indirect technique with nine different lectins for intensity of lectin binding. RESULTS: HPA bound to the luminal epithelium in higher tumor grades in a similar pattern to that in human breast cancer. BSA-IB4 bound to luminal epithelium in hyperplasia and increased towards higher grades, comparable to previous clinical studies. PHA-L-binding to myoepithelium and luminal epithelium increased from hyperplasia to higher grades, comparable to findings in human breast cancer. CONCLUSION: The results of our study support the hypothesis that lectin binding sites change similarly in WAP-T and human breast cancer, stressing the similarity of this model with the clinical setting.
Assuntos
Neoplasias da Mama/metabolismo , Lectinas/metabolismo , Neoplasias Mamárias Animais/metabolismo , Animais , Sítios de Ligação , Neoplasias da Mama/patologia , Corantes , Feminino , Glicoconjugados , Humanos , Hiperplasia , Neoplasias Mamárias Animais/patologia , Lectinas de Ligação a Manose/metabolismo , Camundongos , Gradação de Tumores , Fito-Hemaglutininas/metabolismo , Lectinas de Plantas/metabolismo , Ligação Proteica , Proteínas Inativadoras de Ribossomos/metabolismo , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
WAP-T1 transgenic mice express SV40-TAg under control of the whey acidic protein (WAP) promoter, which directs activity of this strong viral oncogene to luminal cells of the mammary gland. Resting uniparous WAP-T1 glands develop hyperplasia composed of TAg positive cells prior to appearance of advanced tumor stages. We show that cells in hyperplasia display markers of alveolar differentiation, suggesting that TAg targets differentiating cells of the alveolar compartment. The glands show significant expression of Elf5 and milk genes (Lalba, Csn2, and Wap). TAg expressing cells largely co-stain with antibodies to Elf5, lack the epithelial marker Sca1, and are hormone receptor negative. High expression levels of Elf5 but not of milk genes are also seen in resting glands of normal BALB/c mice. This indicates that expression of Elf5 in resting WAP-T1 glands is not specifically induced by TAg. CK6a positive luminal cells lack TAg. These cells co-express the markers prominin-1, CK6a, and Sca1, and are positive for hormone receptors. These hormone sensitive cells localize to ducts and seem not to be targeted by TAg. Despite reaching an advanced stage in alveolar differentiation, the cells in hyperplasia do not exit the cell cycle. Thus, expression of TAg in conjunction with regular morphogenetic processes of alveologenesis seem to provide the basis for a hormone independent, unscheduled proliferation of differentiating cells in resting glands of WAP-T1 transgenic mice, leading to the formation of hyperplastic lesions.
RESUMO
Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.
Assuntos
DNA Super-Helicoidal/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , DNA Super-Helicoidal/química , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-ß expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-ß activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-ß to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-ß are involved in TERT gene regulation during carcinogenesis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Mamárias Experimentais/patologia , Proteínas do Leite/genética , Regiões Promotoras Genéticas/genética , Telomerase/genética , Animais , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Proteínas do Leite/genética , Proteínas Proto-Oncogênicas c-met/fisiologia , Animais , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Feminino , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Gradação de Tumores , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-met/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Mouse models are important tools to decipher the molecular mechanisms of mammary carcinogenesis and to mimic the respective human disease. Despite sharing common phenotypic and genetic features, the proper translation of murine models to human breast cancer remains a challenging task. In a previous study we showed that in the SV40 transgenic WAP-T mice an active Met-pathway and epithelial-mesenchymal characteristics distinguish low- and high-grade mammary carcinoma. To assign these murine tumors to corresponding human tumors we here incorporated the analysis of expression of transcription factor (TF) coding genes and show that thereby a more accurate interspecies translation can be achieved. We describe a novel cross-species translation procedure and demonstrate that expression of unsupervised selected TFs, such as ELF5, HOXA5 and TFCP2L1, can clearly distinguish between the human molecular breast cancer subtypes--or as, for example, expression of TFAP2B between yet unclassified subgroups. By integrating different levels of information like histology, gene set enrichment, expression of differentiation markers and TFs we conclude that tumors in WAP-T mice exhibit similarities to both, human basal-like and non-basal-like subtypes. We furthermore suggest that the low- and high-grade WAP-T tumor phenotypes might arise from distinct cells of tumor origin. Our results underscore the importance of TFs as common cross-species denominators in the regulatory networks underlying mammary carcinogenesis.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Fatores de Transcrição/fisiologia , Animais , Neoplasias da Mama/etiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/fisiologia , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , FenótipoRESUMO
BACKGROUND: Skin derived precursors (SKP) comprise a subset of specialized dermal cells that can be distinguished from fibroblast by their capacity for spheroidal growth. Recent investigations have shown that hair follicles constitute a niche for this cell type, but their localization and their definite function in non-follicular skin remains largely unknown. OBJECTIVE: To identify the dermal niche of non-follicular SKPs and to analyze whether functional aspects correlate with this localization. METHODS: SKPs were isolated from separate anatomical regions of human abdominal skin. Fluorescence activated cell sorting then was used to obtain a pure population of non-follicular SKPs. Functional characterization of these cells was performed applying differentiation and proliferation assays. Information on specific in vivo functions was derived from histological evaluation of quantity and localization patterns. RESULTS: Sphere forming capacity and differentiation assays show that SKPs reside in the papillary part of the dermis. Further delineation revealed that the dermal capillaries represent a niche for these cells which subsequently could be isolated by FACS utilizing a perivascular marker. Whereas functional properties described for follicular SKPs could also be detected in the perivascular SKP population, histological analyses additionally point to a cross-talk with epidermal stem cells and a reduction during chronological aging. CONCLUSION: Our data show that SKPs isolated from non-follicular skin originate from a perivascular niche. Compared to their follicular counterparts, no functional differences could be observed upon cultivation, but ex vivo analyses also point to unique functions and a contribution to the phenotype of aged skin.
Assuntos
Células-Tronco Adultas/citologia , Derme/citologia , Fibroblastos/citologia , Folículo Piloso/citologia , Pericitos/citologia , Nicho de Células-Tronco/fisiologia , Abdome , Adipogenia/fisiologia , Adulto , Antígeno CD146/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Humanos , Osteogênese/fisiologia , Pericitos/metabolismo , Envelhecimento da Pele/fisiologiaAssuntos
Diferenciação Celular/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Queratinócitos/metabolismo , Linhagem Celular , Proliferação de Células , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Queratina-10/metabolismo , Queratinócitos/citologia , RNA Interferente Pequeno , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição GênicaRESUMO
Human recombinant, baculovirus-expressed p53 protein focuses on 2D gels in multiple spots in the narrow pI range. Re-electrophoresis of the individual spots resulted in the appearance of multiple spots. The strings of spots were neither species specific, nor characteristic for baculovirus-expressed p53. Moreover, mutant p53 did not deviate from wild-type p53, indicating that this is an inherent property of p53. Okadaic acid treatment of insect cells, phosphate substitution reaction of purified p53, and individual analysis of all spots by mass spectrometry revealed that only a fraction of the recombinant p53 is phosphorylated. This finding excluded that the individual p53 spots in 2D gels reflect charge isomers generated by phosphorylation, but rather suggest that they are due to conformational flexibility of urea-denatured monomeric p53 molecules or deamidation of asparagine and glutamine residues. The latter possibility was confirmed by NanoLC-ESI MS/MS analysis. Our data provide a putative hint for a novel regulatory level for function and stability of p53, particularly the long-lived mutant p53 overexpressed in diverse tumor types.
Assuntos
Focalização Isoelétrica/métodos , Proteínas Recombinantes/química , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Linhagem Celular , Eletroforese em Gel Bidimensional , Humanos , Camundongos , Dados de Sequência Molecular , Mariposas , Fosforilação , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Supressora de Tumor p53/genéticaRESUMO
The molecular mechanisms underlying mutant p53 (mutp53) "gain-of-function" (GOF) are still insufficiently understood, but there is evidence that mutp53 is a transcriptional regulator that is recruited by specialized transcription factors. Here we analyzed the binding sites of mutp53 and the epigenetic status of mutp53-regulated genes that had been identified by global expression profiling upon depletion of endogenous mutp53 (R273H) expression in U251 glioblastoma cells. We found that mutp53 preferentially and autonomously binds to G/C-rich DNA around transcription start sites (TSS) of many genes characterized by active chromatin marks (H3K4me3) and frequently associated with transcription-competent RNA polymerase II. Mutp53-bound regions overlap predominantly with CpG islands and are enriched in G4-motifs that are prone to form G-quadruplex structures. In line, mutp53 binds and stabilizes a well-characterized G-quadruplex structure in vitro. Hence, we assume that binding of mutp53 to G/C-rich DNA regions associated with a large set of cancer-relevant genes is an initial step in their regulation by mutp53. Using GAS1 and HTR2A as model genes, we show that mutp53 affects several parameters of active transcription. Finally, we discuss a dual mode model of mutp53 GOF, which includes both stochastic and deterministic components.