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BACKGROUND: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. METHODS: Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. RESULTS: Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). CONCLUSIONS: This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.
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INTRODUCTION: Generating high levels of immunosuppressive adenosine in the tumor microenvironment contributes to cancer immune evasion. CD39 and CD73 hydrolyze adenosine triphosphate into adenosine; thus, efforts have been made to target this pathway for cancer immunotherapy. Our objective was optimizing a multiplex immunofluorescence (mIF) panel to explore the role of CD39 and CD73 within the tumor microenvironment. MATERIALS AND METHODS: In three-time points, a small cohort (n=8 ) of colorectal and pancreatic adenocarcinomas were automated staining using an mIF panel against CK, CD3, CD8, CD20, CD39, CD73 and CD68 to compare them with individual markers immunohistochemistry (IHC) for internal panel validation. Densities of immune cells and distances from different tumor-associated immune cells to tumor cells were exploratory assessment and compared with clinicopathologic variables and outcomes. RESULTS: Comparing the three-time points and individual IHC staining results, we demonstrated high reproducibility of the mIF panel. CD39 and CD73 expression was low in malignant cells; the exploratory analysis showed higher densities of CD39 expression by various cells, predominantly stromal cells, followed by T cells, macrophages, and B cells. No expression of CD73 by B cells or macrophages was detected. Distance analysis revealed proximity of cytotoxic T cells, macrophages, and T cells expressing CD39 to malignant cells, suggesting a close regulatory signal driven by this adenosine marker. CONCLUSIONS: We optimized an mIF panel for detection of markers in the adenosine pathway, an emerging clinically relevant pathway. The densities and spatial distribution demonstrated that this pathway may modulate aspects of the tumor immune microenvironment.
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In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.
Assuntos
Hemaglutininas Virais/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H9N2/genética , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/transmissão , Vírus Reordenados/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Cães , Ensaio de Imunoadsorção Enzimática , Furões , Hemaglutininas Virais/genética , Imuno-Histoquímica , Neuraminidase/genética , Infecções por Orthomyxoviridae/genética , Proteínas Virais/genéticaRESUMO
Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-ß type II receptor, bolstering its activity in the TGF-ß-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-ß-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.
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Neoplasias da Próstata , Receptores de Antígenos Quiméricos , Masculino , Humanos , Camundongos , Animais , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva , Neoplasias da Próstata/patologia , Linfócitos T , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Microambiente Tumoral , Oxirredutases/metabolismoRESUMO
Loss of PTEN is one of the most common mutations in prostate cancer, and loss of wild-type TP53 is associated with prostate cancer progression and castrate resistance. Modeling prostate cancer in the mouse has shown that while Pten deletion in prostate epithelial cells leads to adenocarcinoma, combined loss of Pten and TP53 results in rapidly developing disease with greater tumor burden and early death. TP53 contributes significantly to the regulation of stem cell self-renewal, and we hypothesized that loss of Pten/TP53 would result in measurable changes in prostate cancer stem/progenitor cell properties. Clonogenic assays that isolate progenitor function in primary prostate epithelial cells were used to measure self-renewal, differentiation, and tumorigenic potential. Pten/TP53 null as compared with wild-type protospheres showed increased self-renewal activity and modified lineage commitment. Orthotopic transplantation of Pten/TP53 null cells derived from protospheres produced invasive Prostatic Intraepithelial Neoplasia (PIN)/adenocarcinoma, recapitulating the pathology seen in primary tumors. Pten/TP53 null progenitors relative to wild type also demonstrated increased dependence on the AKT/mammalian target of rapamycin complex 1 (mTORC1) and androgen receptor (AR) pathways for clonogenic and tumorigenic growth. These data demonstrate roles for Pten/TP53 in prostate epithelial stem/progenitor cell function, and moreover, as seen in patients with castrate-resistant prostate cancer, suggest for the involvement of an AR-dependent axis in the clonogenic expansion of prostate cancer stem cells.
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Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/deficiência , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteína Supressora de Tumor p53/deficiência , Animais , Linhagem da Célula , Proliferação de Células , Separação Celular , Forma Celular , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Deleção de Genes , Imunofenotipagem , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , PTEN Fosfo-Hidrolase/metabolismo , Próstata/metabolismo , Próstata/patologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Esferoides Celulares/patologia , Serina-Treonina Quinases TOR , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais , Animais , Antineoplásicos/química , Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Proteína Glial Fibrilar Ácida , Glioblastoma/tratamento farmacológico , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Primary prostate cancer almost always has a luminal phenotype. However, little is known about the stem/progenitor properties of transformed cells within tumors. Using the aggressive Pten/Tp53-null mouse model of prostate cancer, we show that two classes of luminal progenitors exist within a tumor. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. Luminal cells, sorted directly from tumors or grown as organoids, initiated tumors of adenocarcinoma or multilineage histological phenotypes, which is consistent with luminal and multipotent differentiation potentials, respectively. Moreover, using organoids we show that the ability of luminal-committed progenitors to self-renew is a tumor-specific property, absent in benign luminal cells. Finally, a significant fraction of luminal progenitors survived in vivo castration. In all, these data reveal two luminal tumor populations with different stem/progenitor cell capacities, providing insight into prostate cancer cells that initiate tumors and can influence treatment response.
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Adenocarcinoma/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Animais , Linhagem da Célula , Separação Celular , Modelos Animais de Doenças , Células Epiteliais/patologia , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Organoides , FenótipoRESUMO
The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1-deficient tumors and development of relevant biomarkers.
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Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Aloenxertos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Análise por Conglomerados , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Modelos Animais de Doenças , Progressão da Doença , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutação , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Carga Tumoral/efeitos dos fármacosRESUMO
Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution.
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Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Queratina-18/biossíntese , Queratina-18/genética , Queratina-19/biossíntese , Queratina-19/genética , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos , Proteína do Retinoblastoma/genéticaRESUMO
The majority of human high-grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer.
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Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário , Modelos Animais de Doenças , Feminino , Deleção de Genes , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Mutação , Metástase Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1(hi)/EpCAM(+) basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.
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Regulação Neoplásica da Expressão Gênica , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/fisiologia , Transativadores/genética , Androgênios/fisiologia , Animais , Proliferação de Células , Cromossomos Artificiais Bacterianos/genética , Epitélio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Orquiectomia , Regiões Promotoras Genéticas , Próstata/metabolismo , Próstata/patologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Transativadores/metabolismo , Regulador Transcricional ERG , Células Tumorais CultivadasRESUMO
Patients with lung cancer with activating mutations in the EGF receptor (EGFR) kinase, who are treated long-term with tyrosine kinase inhibitors (TKI), often develop secondary mutations in EGFR associated with resistance. Mice engineered to develop lung adenocarcinomas driven by the human EGFR T790M resistance mutation are similarly resistant to the EGFR TKI erlotinib. By tumor volume endpoint analysis, these mouse tumors respond to BIBW 2992 (an irreversible EGFR/HER2 TKI) and rapamycin combination therapy. To correlate EGFR-driven changes in the lung with response to drug treatment, we conducted an integrative analysis of global transcriptome and metabolite profiling compared with quantitative imaging and histopathology at several time points during tumor progression and treatment. Responses to single-drug treatments were temporary, whereas combination therapy elicited a sustained response. During tumor development, metabolomic signatures indicated a shift to high anabolic activity and suppression of antitumor programs with 11 metabolites consistently present in both lung tissue and blood. Combination drug treatment reversed many of the molecular changes found in tumored lung. Data integration linking cancer signaling networks with metabolic activity identified key pathways such as glutamine and glutathione metabolism that signified response to single or dual treatments. Results from combination drug treatment suggest that metabolic transcriptional control through C-MYC and SREBP, as well as ELK1, NRF1, and NRF2, depends on both EGFR and mTORC1 signaling. Our findings establish the importance of kinetic therapeutic studies in preclinical assessment and provide in vivo evidence that TKI-mediated antiproliferative effects also manifest in specific metabolic regulation.
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Adenocarcinoma/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Afatinib , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Processos de Crescimento Celular/fisiologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/administração & dosagem , Sirolimo/administração & dosagem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Activation of Ras signaling in cardiomyocytes has been linked to pathogenic myocardial hypertrophy progression and subsequent heart failure. Whether cardiomyopathy can regress once initiated needs to be established more fully. A 'tet-off' system was used to regulate expression of H-Ras-G12V in myocardium to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling in vivo. Ras activation at weaning for 2 wk caused hypertrophy, whereas activation for 4 to 8 wk led to cardiomyopathy and heart failure. Discontinuing H-Ras-G12V transgene expression after cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart:body weight ratios, demonstrating the reversibility of early pathogenic hypertrophy. Activation of Ras and downstream ERK 1/2 was associated with elevated expression of proliferating cell nuclear antigen and cyclins B1 and D1, indicating cell-cycle activation and reentry. Coordinate elevation of broad-spectrum cyclin-dependent kinase inhibitors (p21, p27, and p57) and Tyr15 phosphorylation of cdc2 signified the activation of cell-cycle checkpoints; absence of cell-cycle completion and cardiomyocyte replication were documented by using immunohistochemistry for mitosis and cytokinesis markers. After resolution of cardiomyopathy, cell-cycle activators and inhibitors examined returned to basal levels, a change that we interpreted as exit from the cell cycle. Cardiac cell-cycle regulation plays a role in recovery from pathogenic hypertrophy. The model we present provides a means to further explore the underlying mechanisms governing cell-cycle capacity in cardiomyocytes, as well as progression and regression of pathogenic cardiomyocyte hypertrophy.
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Cardiomegalia/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas ras/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Cardiomegalia/enzimologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/patologia , Transgenes , Proteínas ras/genéticaRESUMO
Peroxisome proliferator-activated receptorδ (PPARδ) regulates a multiplicity of physiological processes associated with glucose and lipid metabolism, inflammation, and proliferation. One or more of these processes likely create risk factors associated with the ability of PPARδ agonists to promote tumorigenesis in some organs. In the present study, we describe a new gastric tumor mouse model that is dependent on the potent and highly selective PPARδ agonist GW501516 following carcinogen administration. The progression of gastric tumorigenesis was rapid as determined by magnetic resonance imaging and resulted in highly metastatic squamous cell carcinomas of the forestomach within two months. Tumorigenesis was associated with gene expression signatures indicative of cell adhesion, invasion, inflammation, and metabolism. Increased PPARδ expression in tumors correlated with increased PDK1, Akt, ß-catenin, and S100A9 expression. The rapid development of metastatic gastric tumors in this model will be useful for evaluating preventive and therapeutic interventions in this disease.