RESUMO
Detailed mechanisms involved in prostate cancer (CaP) development and progression are not well understood. Current experimental models used to study CaP are not well suited to address this issue. Previously, we have described the hormonal progression of non-tumorigenic human prostate epithelial cells (BPH1) into malignant cells via tissue recombination. Here, we describe a method to derive human cell lines from distinct stages of CaP that parallel cellular, genetic and epigenetic changes found in patients with cancers. This BPH1-derived Cancer Progression (BCaP) model represents different stages of cancer. Using diverse analytical strategies, we show that the BCaP model reproduces molecular characteristics of CaP in human patients. Furthermore, we demonstrate that BCaP cells have altered gene expression of shared pathways with human and transgenic mouse CaP data, as well as, increasing genomic instability with TMPRSS2-ERG fusion in advanced tumor cells. Together, these cell lines represent a unique model of human CaP progression providing a novel tool that will allow the discovery and experimental validation of mechanisms regulating human CaP development and progression. This BPH1-derived Cancer Progression (BCaP) model represents different stages of cancer. The BCaP model reproduces molecular characteristics of prostate cancer. The cells have altered gene expression with TMPRSS2-ERG fusion representing a unique model for prostate cancer progression.
Assuntos
Carcinogênese/genética , Proteínas de Fusão Oncogênica/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Carcinogênese/patologia , Linhagem Celular , Metilação de DNA , Conjuntos de Dados como Assunto , Progressão da Doença , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Regiões Promotoras Genéticas/genética , Próstata/citologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: CD147 is an MMP-inducing protein often implicated in cancer progression. The purpose of this study was to investigate the expression of CD147 in prostate cancer (PCa) progression and the prognostic ability of CD147 in predicting biochemical recurrence after prostatectomy. METHODS: Plasma membrane-localized CD147 protein expression was quantified in patient samples using immunohistochemistry and multispectral imaging, and expression was compared to clinico-pathological features (pathologic stage, Gleason score, tumor volume, preoperative PSA, lymph node status, surgical margins, biochemical recurrence status). CD147 specificity and expression were confirmed with immunoblotting of prostate cell lines, and CD147 mRNA expression was evaluated in public expression microarray datasets of patient prostate tumors. RESULTS: Expression of CD147 protein was significantly decreased in localized tumors (pT2; p = 0.02) and aggressive PCa (≥pT3; p = 0.004), and metastases (p = 0.001) compared to benign prostatic tissue. Decreased CD147 was associated with advanced pathologic stage (p = 0.009) and high Gleason score (p = 0.02), and low CD147 expression predicted biochemical recurrence (HR 0.55; 95 % CI 0.31-0.97; p = 0.04) independent of clinico-pathologic features. Immunoblot bands were detected at 44 kDa and 66 kDa, representing non-glycosylated and glycosylated forms of CD147 protein, and CD147 expression was lower in tumorigenic T10 cells than non-tumorigenic BPH-1 cells (p = 0.02). Decreased CD147 mRNA expression was associated with increased Gleason score and pathologic stage in patient tumors but is not associated with recurrence status. CONCLUSIONS: Membrane-associated CD147 expression is significantly decreased in PCa compared to non-malignant prostate tissue and is associated with tumor progression, and low CD147 expression predicts biochemical recurrence after prostatectomy independent of pathologic stage, Gleason score, lymph node status, surgical margins, and tumor volume in multivariable analysis.
Assuntos
Basigina/genética , Basigina/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Glicosilação , Humanos , Masculino , Gradação de Tumores , Metástase Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
BACKGROUND: The treatment of non-localized prostate cancer involves androgen deprivation (AD) therapy which results in tumor regression. Apoptosis has been implicated in the tumor response to AD, but constitutes a small fraction of the total tumor at any time. Cellular senescence is a response to sub-lethal stress in which cells are persistently growth arrested and develop distinct morphological and biochemical characteristics. The occurrence of senescence in prostate tumor tissue after AD therapy has not previously been investigated. METHODS: Phenotypic and molecular characteristics of senescence were examined in models of androgen-sensitive prostate cancer after AD and compared with androgen-intact controls. RESULTS: In vitro in LNCaP cells, AD induced elevated senescence-associated ß-galactosidase (SA-ß-gal) staining, decreased proliferation, and increased flow cytometric side scatter while minimally affecting cell viability. The increased expression of the senescence-related proteins Glb1, the cyclin-dependent kinase inhibitor p27(Kip1) and chromatin-regulating heterochromatin protein 1γ (HP1γ) were detected in LNCaP cells after AD in vitro by immunoblot and immunofluorescence microscopy. In mice bearing LuCaP xenograft tumors in vivo, surgical castration similarly increased SA-ß-gal staining, increased expression of p27(Kip1) and HP1γ, and decreased expression of the proliferation marker KI-67, with minimal induction of apoptosis identified by detection of cleaved caspase 3 and TUNEL. Immunohistochemical analysis of human prostate tumors removed after AD shows similar induction of Glb1, HP1γ and decreased KI-67. CONCLUSIONS: We conclude that AD induces characteristics consistent with cellular senescence in androgen-sensitive prostate cancer cells. This finding may explain incomplete tumor regression in response to AD.
Assuntos
Androgênios/deficiência , Senescência Celular/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Idoso , Androgênios/farmacologia , Animais , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The effective detection and management of muscle-invasive bladder Transition Cell Carcinoma (TCC) continues to be an urgent clinical challenge. While some differences of gene expression and function in papillary (Ta), superficial (T1) and muscle-invasive (≥T2) bladder cancers have been investigated, the understanding of mechanisms involved in the progression of bladder tumors remains incomplete. Statistical methods of pathway-enrichment, cluster analysis and text-mining can extract and help interpret functional information about gene expression patterns in large sets of genomic data. The public availability of patient-derived expression microarray data allows open access and analysis of large amounts of clinical data. Using these resources, we investigated gene expression differences associated with tumor progression and muscle-invasive TCC. Gene expression was calculated relative to Ta tumors to assess progression-associated differences, revealing a network of genes related to Ras/MAPK and PI3K signaling pathways with increased expression. Further, we identified genes within this network that are similarly expressed in superficial Ta and T1 stages but altered in muscle-invasive T2 tumors, finding 7 genes (COL3A1, COL5A1, COL11A1, FN1, ErbB3, MAPK10 and CDC25C) whose expression patterns in muscle-invasive tumors are consistent in 5 to 7 independent outside microarray studies. Further, we found increased expression of the fibrillar collagen proteins COL3A1 and COL5A1 in muscle-invasive tumor samples and metastatic T24 cells. Our results suggest that increased expression of genes involved in mitogenic signaling may support the progression of muscle-invasive bladder tumors that generally lack activating mutations in these pathways, while expression changes of fibrillar collagens, fibronectin and specific signaling proteins are associated with muscle-invasive disease. These results identify potential biomarkers and targets for TCC treatments, and provide an integrated systems-level perspective of TCC pathobiology to inform future studies.
Assuntos
Carcinoma de Células de Transição/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/patologia , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/genética , Proteínas ras/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma de Células de Transição/patologia , Biologia Computacional , Colágenos Fibrilares/metabolismo , Humanos , Imuno-Histoquímica , Análise em Microsséries , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Therapy-induced senescence (TIS), a cytostatic stress response in cancer cells, is induced inefficiently by current anticancer agents and radiation. The mechanisms that mediate TIS in cancer cells are not well defined. Herein, we characterize a robust senescence response both in vitro and in vivo to the quinone diaziquone (AZQ), previously identified in a high-throughput senescence-induction small-molecule screen. Using AZQ and several other agents that induce senescence, we screened a series of cyclin-dependent kinase inhibitors and found that p27(Kip1) was induced in all investigated prostate cancer cell lines. The ubiquitin-ligase Skp2 negatively regulates p27(Kip1) and, during TIS, is translocated to the cytoplasm before its expression is decreased in senescent cells. Overexpression of Skp2 blocks the effects of AZQ on senescence and p27(Kip1) induction. We also find that stable long-term short hairpin RNA knockdown of Skp2 decreases proliferation but does not generate the complete senescence phenotype. We conclude that Skp2 participates in regulating TIS but, alone, is insufficient to induce senescence in cancer cells.
RESUMO
The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models to determine the contributions of receptor dimerization and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: (1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, (2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and (3) receptor phosphorylation kinetics at the cell surface and early endosomes are comparable. We validated the last finding by measuring the EGFR dephosphorylation rates at various times following ligand addition both in whole cells and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. This study demonstrates that an iterative cycle of experiments and modeling can be used to gain mechanistic insight regarding complex cell signaling networks.
Assuntos
Receptores ErbB/metabolismo , Modelos Teóricos , Linhagem Celular , Células Epiteliais/metabolismo , Receptores ErbB/química , Humanos , Fosforilação , Multimerização ProteicaRESUMO
Cellular senescence is a response to nonlethal stress that results in persistent cytostasis with a distinct morphological and biochemical phenotype. The senescence phenotype, detected in tumors through the expression of mRNA and protein markers, can be generated in cancer cells lacking functional p53 and retinoblastoma protein. Current research suggests that therapy-induced senescence (TIS) represents a novel functional target that may improve cancer therapy. TIS can be induced in immortal and transformed cancer cells by selected anticancer compounds or radiation, and accumulating data indicate that TIS may produce reduced toxicity-related side effects and increased tumor-specific immune activity. This review examines the current status of TIS-regulated mechanisms, agents, and senescence biomarkers with the goal of encouraging further development of this approach to cancer therapy. Remaining hurdles include the lack of efficient senescence-inducing agents and incomplete biological data on tumor response. The identification of additional compounds and other targeted approaches to senescence induction will further the development of TIS in the clinical treatment of cancer.
Assuntos
Senescência Celular , Neoplasias/patologia , Neoplasias/terapia , Animais , Apoptose , Proliferação de Células , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Humanos , Estresse Oxidativo , Qualidade de Vida , Fatores de TempoRESUMO
Cellular senescence is a persistently growth-arrested phenotype in normal and transformed cells induced by noncytotoxic stress. Cytostasis as a method of cancer treatment has recently generated significant interest. Research into the induction of cellular senescence as cancer therapy has been hindered by a lack of compounds that efficiently induce this response. The authors describe a semiautomated high-throughput method to identify library compounds that induce senescence using prostate cancer cells cultured in 96-well plates. Primary hits are identified by low cell numbers after 3 days in culture, measured by Hoechst 33342 fluorescence. A secondary visual assessment of senescence-associated beta-galactosidase staining and cellular morphology in the same wells distinguishes senescence from quiescence, apoptosis, and other false positives. This method was used to screen a 4160-compound library of known bioactive compounds and natural products at a 10-microM dose. Candidate compounds were further selected based on persistent growth arrest after drug removal and increased expression of previously described senescence marker genes. Four lead compounds not previously associated with senescence were identified for further investigation. This is the first successful assay to identify novel agents from compound libraries based on senescence induction in cancer cells.
Assuntos
Senescência Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Benzimidazóis/metabolismo , Linhagem Celular Tumoral , Fluorescência , Humanos , Projetos Piloto , Reprodutibilidade dos TestesRESUMO
Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for Igf2, a paracrine growth factor, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.
Assuntos
Envelhecimento/genética , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Imunoprecipitação da Cromatina , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Imunofluorescência , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Próstata/metabolismo , RNA Longo não Codificante , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and approximately 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Our simulations predict that when EGFR is activated with TGF-alpha, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-alpha. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.
Assuntos
Endocitose/fisiologia , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Modelos Biológicos , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador alfa/metabolismo , Linhagem Celular , Simulação por Computador , Endotélio Vascular/metabolismo , Humanos , Modelos Estatísticos , Ligação ProteicaRESUMO
To investigate the intrinsic activities of the epidermal growth factor receptor and the role of its kinase domain in these functions within a cellular environment lacking endogenous ErbB protein expression, wild-type EGF receptor (WT-EGFR) and two kinase-impaired mutants, D813A and K721R, were expressed in 32D murine hematopoietic cells, a line which is normally dependent on interleukin 3 (IL3) for growth and survival. Addition of EGF in the absence of IL3 stimulates receptor autophosphorylation and, in the presence of serum, mitosis in cells expressing WT-EGFR, but not in cells expressing D813A or K721R. Unexpectedly, cells expressing WT-EGFR or K721R exhibited IL3-independent survival in the presence of fetal bovine serum; parental 32D cells and cells expressing D813A did not survive, apparently undergoing apoptosis in the absence of IL3, whether or not serum was present. Addition of EGF did not prevent the apoptosis of WT-EGFR or K721R cells in serum-free medium. Activation of Akt was not necessary to mediate the prosurvival activity of EGF receptor expression. These results suggest that the EGF receptor can mediate the prevention of apoptosis independently of both receptor-ligand binding and receptor kinase activity, and this activity is disrupted by the D813A mutation.