RESUMEN
BACKGROUND: Metastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known. METHODS: Pull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein-protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays. RESULTS: We show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function. CONCLUSION: These results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer.
Asunto(s)
Proteínas del Tejido Nervioso , Neoplasias de la Próstata , Receptores de Superficie Celular , Humanos , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Mutación , Proteínas ras/genética , Proteínas ras/metabolismo , Metástasis de la Neoplasia , Animales , Fosforilación , Transducción de Señal , Ratones , Semaforinas/metabolismo , Semaforinas/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genéticaRESUMEN
Aberrant mitosis can result in aneuploidy and cancer. The small GTPase, Ran, is a key regulator of mitosis. B-type Plexins regulate Ran activity by acting as RanGTPase activating proteins (GAPs) and have been implicated in cancer progression. However, whether B-type plexins have a role in mitosis has not so far been investigated. We show here that PlexinB1 functions in the control of mitosis. Depletion of PlexinB1 affects mitotic spindle assembly, significantly delaying anaphase. This leads to mitotic catastrophe in some cells, and prolonged application of the spindle assembly checkpoint. PlexinB1 depletion also promoted acentrosomal microtubule nucleation and defects in spindle pole refocussing and increased the number of cells with multipolar or aberrant mitotic spindles. An increase in lagging chromosomes or chromosomal bridges at anaphase was also found upon PlexinB1 depletion. PlexinB1 localises to the mitotic spindle in dividing cells. The mitotic defects observed upon PlexinB1 depletion were rescued by an RCC1 inhibitor, indicating that PlexinB1 signals, via Ran, to affect mitosis. These errors in mitosis generated multinucleate cells, and nuclei of altered morphology and abnormal karyotype. Furthermore, Semaphorin4D-treatment increased the percentage of cells with micronuclei, precursors of chromothripsis. Implications: Defects in B-type plexins may contribute to the well-established role of plexins in cancer progression by inducing chromosomal instability.
RESUMEN
Resistance to anti-androgen therapy for metastatic prostate cancer is a major clinical problem. Sema3C promotes resistance to androgen withdrawal via its receptor, PlexinB1. Activation of PlexinB1 promotes the ligand-independent nuclear translocation of the androgen receptor (AR), which may contribute to resistance to androgen deprivation therapy. However, the mechanism by which PlexinB1 promotes nuclear translocation is unclear. We show here that PlexinB1 and B2 regulate nuclear import by acting as GTPase activating proteins (GAPs) for the small RasGTPase Ran, a key regulator of nuclear trafficking. Purified PlexinB1/B2 protein catalyses the hydrolysis of RanGTP, and mutations in the GAP domain of PlexinB1 inhibit this activity. Activation of PlexinB1/B2 with Sema4D decreases the levels of RanGTP, while PlexinB1 or B2 depletion increases the levels of activated Ran in the cell. Ran directly associates with B-type plexins in a GTP-dependent manner. Sema4D is internalised by endocytosis, and PlexinB1 and Ran display overlapping patterns of expression. Furthermore, Sema4D/PlexinB1-induced AR nuclear translocation is dependent on the GAP domain of PlexinB1 and is blocked by the expression of non-functional Ran mutants. Depletion of PlexinB1 decreases the nuclear/cytoplasmic ratio of Ran, indicative of a higher RanGTP/GDP ratio. Plexins may promote the growth of androgen-independent prostate cancer through their activity as RanGAPs.
Asunto(s)
Moléculas de Adhesión Celular , Neoplasias de la Próstata , Receptores Androgénicos , Masculino , Humanos , Transporte Activo de Núcleo Celular , Receptores Androgénicos/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Andrógenos , Antagonistas de Andrógenos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , GTP Fosfohidrolasas/metabolismoRESUMEN
Metastatic prostate cancer is essentially incurable and is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We show here that prostate epithelial cell-specific expression of a mutant form of Plexin-B1 (P1597L) which was identified in metastatic deposits in patients with prostate cancer, significantly increases metastasis, in particular metastasis to distant sites, in two transgenic mouse models of prostate cancer (PbCre+Ptenfl /flKrasG12V and PbCre+Ptenfl /flp53fl/ fl ). In contrast, prostate epithelial cell-specific expression of wild-type (WT) Plexin-B1 in PbCre+Ptenfl /flKrasG12V mice significantly decreases metastasis, showing that a single clinically relevant Pro1597Leu amino-acid change converts Plexin-B1 from a metastasis-suppressor to a metastasis-promoter. Furthermore, PLXNB1P1597L significantly increased invasion of tumor cells into the prostate stroma, while PLXNB1WT reduced invasion, suggesting that Plexin-B1 has a role in the initial stages of metastasis. Deletion of RhoA/C or PDZRhoGEF in Ptenfl /flKrasG12VPLXNB1P1597L mice suppressed metastasis, implicating the Rho/ROCK pathway in this phenotypic switch. Germline deletion of Plexin-B1, to model anti-Plexin-B1 therapy, significantly decreased invasion and metastasis in both models. Our results demonstrate that Plexin-B1 plays a complex yet significant role in metastasis in mouse models of prostate cancer and is a potential therapeutic target to block the lethal spread of the disease. Significance: Few therapeutic targets have been identified specifically for preventing locally invasive/oligometastatic prostate cancer from becoming more widely disseminated. Our findings suggest Plexin-B1 signaling, particularly from the clinically relevant P1597L mutant, is such a target.
Asunto(s)
Antineoplásicos , Neoplasias de la Próstata , Humanos , Masculino , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Neoplasias de la Próstata/genética , Ratones TransgénicosRESUMEN
Androgen receptor (AR) and glucocorticoid receptor (GR) are nuclear receptors whose function depends on their entry into the nucleus where they activate transcription of an overlapping set of genes. Both AR and GR have a role in resistance to androgen deprivation therapy (ADT), the mainstay of treatment for late stage prostate cancer. PlexinB1, a receptor for semaphorins, has been implicated in various cancers including prostate cancer and has a role in resistance to ADT. We show here that activation of PlexinB1 by Sema4D and Sema3C results in translocation of endogenous GR to the nucleus in prostate cancer cells, and that this effect is dependent on PlexinB1 expression. Sema4D/Sema3C promotes the translocation of GR-GFP to the nucleus and mutation of the nuclear localization sequence (NLS1) of GR abrogates this response. These findings implicate the importin α/ß system in the Sema4D/Sema3C-mediated nuclear import of GR. Knockdown of PlexinB1 in prostate cancer cells decreases the levels of glucocorticoid-responsive gene products and antagonizes the decrease in cell motility and cell area of prostate cancer cells upon dexamethasone treatment, demonstrating the functional significance of these findings. These results show that PlexinB1 activation has a role in the trafficking and activation of the nuclear receptor GR and thus may have a role in resistance to androgen deprivation therapy in late stage prostate cancer.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Tejido Nervioso/genética , Neoplasias de la Próstata/genética , Receptores de Superficie Celular/genética , Receptores de Glucocorticoides/metabolismo , Antígenos CD/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Células HeLa , Humanos , Masculino , Mutación , Proteínas del Tejido Nervioso/metabolismo , Señales de Localización Nuclear , Células PC-3 , Neoplasias de la Próstata/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Receptores de Glucocorticoides/genética , Semaforinas/metabolismo , Activación TranscripcionalRESUMEN
The aim of this study was to test the utility of AIMP3, an upstream regulator of DNA damage response following genotoxic stress, as a clinical biomarker in muscle-invasive bladder cancer (MIBC). AIMP3 was identified from a meta-analysis of a global gene-expression dataset. AIMP3 protein expression was determined by immunohistochemistry on a customised bladder cancer tissue-microarray (TMA). The mechanism of gene silencing was probed using methylation-specific PCR. The association between AIMP3 expression, Tp53 transactivity and genomic stability was analysed. In vitro AIMP3 translocation to the nucleus in response to ionising radiation was demonstrated using immunofluorescence. Radiosensitisation effects of siRNA-mediated AIMP3-knockdown were measured using colony forming assays. TMAs derived from patients enrolled in BCON, a Phase III multicentre radiotherapy trial in bladder cancer (ISRCTN45938399) were used to evaluate the association between AIMP3 expression and survival. The prognostic value of AIMP3 expression was determined in a TMA derived from patients treated by radical cystectomy. Loss of AIMP3 expression was frequent in MIBC and associated with impaired Tp53 transactivity and genomic instability. AIMP3-knockdown was associated with an increase in radioresistance. Loss of AIMP3 expression was associated with survival in MIBC patients following radiotherapy (HR = 0.53; 95% CI: 0.36 to 0.78, p = 0.002) but was not prognostic in the cystectomy set. In conclusion, AIMP3 expression is lost in a subset of bladder cancers and is significantly predictive of survival following radiotherapy in MIBC patients.
Asunto(s)
Genes Supresores de Tumor , Factores de Elongación de Péptidos/genética , Proteínas Supresoras de Tumor/genética , Neoplasias de la Vejiga Urinaria/radioterapia , Anciano , Anciano de 80 o más Años , Cistectomía , Femenino , Genes p53 , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso/patología , Invasividad Neoplásica , Factores de Elongación de Péptidos/fisiología , Análisis de Matrices Tisulares , Proteínas Supresoras de Tumor/fisiología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumors, indicating a role for plexinB1 in the pathogenesis of prostate cancer. However, the effect of semaphorin/plexin signaling is highly context dependent and whether plexinB1 acts as an inducer or inhibitor of prostate tumor progression in this context is not known. METHODS: The response of prostate cancer cell lines to plexinB1 activation was assessed in migration, invasion, proliferation and protein phosphorylation assays. Expression was assessed by quantitative RTPCR and immunoblotting. RESULTS: Different prostate cancer cell lines respond to Sema4D (the ligand for plexinB1) in diverse ways. Activation of endogenous plexinB1 enhances migration, invasion and anchorage-independent growth of LNCaP prostate cancer cells via activation of ErbB2 and Akt. In contrast, Sema4D-stimulation decreased the motility and proliferative capacity of PC3 cells. LNCaP has a missense mutation (Thr1697Ala) in the plexinB1 gene while LNCaP-LN3, a derivative of LNCaP, expresses high levels of wild-type plexinB1 only. Sema4D stimulation increases the motility and anchorage independent growth of both cell lines, showing that these responses are not dependent on the presence of the Thr1697Ala form of plexinB1. ErbB2 and plexinB1 are expressed in primary prostate epithelial cells. CONCLUSIONS: PlexinB1 signals via ErbB2 to increase the invasive phenotype of prostate cancer cells. Both wild-type and mutant forms of plexinB1 are potential targets for anti-cancer therapy in prostate tumors that express ErbB2.
Asunto(s)
Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Animales , Células COS , Chlorocebus aethiops , Humanos , Masculino , Neoplasias de la Próstata/fisiopatología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumours, indicating a role for plexinB1 in the pathogenesis of prostate cancer. RESULTS: Two specific mutations found in prostate cancer enhance RhoD binding and one other mutation results in loss of inhibition of Rac-dependent Pak1 phosphorylation and lamellipodia formation and in impairment of trafficking of plexinB1 to the membrane. None of the three characterised mutations affect PDZRhoGEF binding, RhoA activity, the interaction of plexinB1 with the oncogenes ErbB2 or c-Met or ErbB2 phosphorylation. The mutations have the net effect of increasing cell motility by blocking plexinB1-mediated inhibition of Rac while enhancing the interaction with RhoD, an anti-migratory factor. CONCLUSIONS: PlexinB1 mutations block plexinB1-mediated signalling pathways that inhibit cell motility.
Asunto(s)
Mutación , Proteínas del Tejido Nervioso/genética , Neoplasias de la Próstata/genética , Receptores de Superficie Celular/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Activación Enzimática/genética , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Melanoma arises through complex genetic and epigenetic changes, resulting in uncontrolled proliferation, invasion, and metastatic disease. Semaphorins regulate axon guidance through interaction with their receptors, plexins and neuropilins. Plexin B1, the semaphorin 4D receptor, activates oncogenic receptors c-Met and ErbB-2 in several cell types, suggesting it promotes tumor growth through stimulation of these receptors. A study by Argast et al. has shown that plexin B1 is a tumor-suppressor protein for melanoma metastasis in a mouse model. In this report, we show that plexin B1 is lost in metastatic and deeply invasive melanoma in patient samples in vivo. Unexpectedly, introduction of plexin B1 into human melanoma cell lines suppressed, rather than activated, the oncogenic receptor, c-Met, by its ligand hepatocyte growth factor (HGF). Plexin B1 also activated Akt in melanoma. Plexin B1 significantly abrogated cell migration in response to HGF but rendered cells resistant to apoptosis by cisplatin. Plexin B1 is predicted to function as a classic tumor-suppressor protein in melanoma, in part through suppression of c-Met signaling and c-Met-dependent migration. However, because plexin B1 activates Akt, a multifunctional protein involved in tumor progression in several cancers, plexin B1 may function as a tumor promoter in melanomas not driven by c-Met activation.
Asunto(s)
Melanoma/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Proteínas Proto-Oncogénicas c-met/fisiología , Receptores de Superficie Celular/fisiología , Neoplasias Cutáneas/fisiopatología , Proteínas Supresoras de Tumor/fisiología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Cisplatino/farmacología , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
A cure cannot be assured for all men with clinically localized prostate cancer undergoing radical treatment. Molecular markers would be invaluable if they could improve the prediction of occult metastatic disease. This study was carried out to investigate the expression of BCL-2, Ki-67, p53 and E-cadherin in radical prostatectomy specimens. We sought to assess their ability to predict early biochemical relapse in a specific therapeutic setting. Eighty-two patients comprising 41 case pairs were matched for pathological stage, Gleason grade and preoperative prostate-specific antigen (PSA) concentration. One patient in each pair had biochemical recurrence (defined as PSA >or= 0.2 ng mL(-1) within 2 years of surgery) and the other remained biochemically free of disease (defined as undetectable PSA at least 3 years after surgery). Immunohistochemical analysis was performed to assess marker expression on four replicate tissue microarrays constructed with benign and malignant tissue from each radical prostatectomy specimen. Ki-67, p53 and BCL-2, but not E-cadherin, were significantly upregulated in prostate adenocarcinoma compared with benign prostate tissue (P < 0.01). However, no significant differences in expression of any of the markers were observed when comparing patients who developed early biochemical relapse with patients who had no biochemical recurrence. This study showed that expression of p53, BCL-2 and Ki-67 was upregulated in clinically localized prostate cancer compared with benign prostate tissue, with no alteration in E-cadherin expression. Biomarker upregulation had no prognostic value for biochemical recurrence after radical prostatectomy, even after considering pathological stage, whole tumour Gleason grade and preoperative serum PSA level.
Asunto(s)
Adenocarcinoma/diagnóstico , Cadherinas/metabolismo , Perfilación de la Expresión Génica , Antígeno Ki-67/metabolismo , Neoplasias de la Próstata/diagnóstico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/cirugía , Anciano , Biomarcadores de Tumor/metabolismo , Cadherinas/genética , Estudios de Casos y Controles , Regulación Neoplásica de la Expresión Génica , Humanos , Antígeno Ki-67/genética , Masculino , Persona de Mediana Edad , Pronóstico , Próstata/metabolismo , Próstata/patología , Antígeno Prostático Específico/sangre , Prostatectomía , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/cirugía , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factores de Riesgo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Semaphorins are a large class of secreted or membrane-associated proteins that act as chemotactic cues for cell movement via their transmembrane receptors, plexins. We hypothesized that the function of the semaphorin signaling pathway in the control of cell migration could be harnessed by cancer cells during invasion and metastasis. We now report 13 somatic missense mutations in the cytoplasmic domain of the Plexin-B1 gene. Mutations were found in 89% (8 of 9) of prostate cancer bone metastases, in 41% (7 of 17) of lymph node metastases, and in 46% (41 of 89) of primary cancers. Forty percent of prostate cancers contained the same mutation. Overexpression of the Plexin-B1 protein was found in the majority of primary tumors. The mutations hinder Rac and R-Ras binding and R-RasGAP activity, resulting in an increase in cell motility, invasion, adhesion, and lamellipodia extension. These results identify a key role for Plexin-B1 and the semaphorin signaling pathway it mediates in prostate cancer.
Asunto(s)
Adenocarcinoma/genética , Mutación Missense , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Neoplasias de la Próstata/genética , Receptores de Superficie Celular/genética , Adenocarcinoma/patología , Adenocarcinoma/secundario , Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática/genética , Masculino , Invasividad Neoplásica/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/fisiología , Polimorfismo Conformacional Retorcido-Simple , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Seudópodos/ultraestructura , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismoRESUMEN
Chemokines are small, secreted proteins and are now the largest known cytokine family. They mediate their effects through a family of G-protein-coupled receptors and were initially recognized for their ability to act as chemo-attractants and activators of specific types of leucocytes in a variety of immune and inflammatory responses. However, during the past 5 years there has been a chemokine revolution in cancer and all scientists and clinicians in oncology-related fields are now aware of their crucial role at all stages of neoplastic transformation and progression. The most important chemokine ligand-receptor interaction is that of the CXCL12 (stromal cell-derived factor-1, SDF-1) ligand with its exclusive receptor CXCR4; this interaction has a pivotal role in the directional migration of cancer cells during the metastatic process. This has been demonstrated by in vitro and in vivo experiments in addition to retrospective clinical studies. These findings have exciting implications in the field of cancer therapeutics, with several small molecule CXCR4 antagonists having been developed, which may provide clinical benefit in the therapy of cancer metastasis. Interestingly, it is likely that the effect of the anti-HER2 antibody [trastuzumab (Herceptin] in breast cancer involves downregulation of the CXCR4 receptor. Unfortunately, a major problem is that chemokine receptors are expressed in other cells within the body, particularly those of the immune system and it is not clear what effects long-term CXCR4 antagonism could have on innate and adaptive immunity. However, there is little doubt that the great strides made in elucidating the complex relationship between chemokines and their role in cancer will soon translate into significant survival benefits for patients.
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Movimiento Celular/fisiología , Quimiocinas CXC/fisiología , Neoplasias/fisiopatología , Receptores CXCR4/fisiología , Quimiocina CXCL12 , HumanosRESUMEN
Morbidity and mortality due to prostate cancer are mainly a result of prostate cancer metastases. After the initial neoplastic transformation of cells, the process of metastasis involves a series of sequential steps, which involve neoangiogenesis and lymphangiogenesis, loss of adhesion with migration away from the primary tumour and entry into the systemic vasculature or lymphatics. Metastatic growth in sites such as lymph nodes and bone marrow then involves the specific non-random homing of prostate cancer cells. An appreciation and understanding of this metastatic cascade in relation to prostate cancer is clinically important in order to stratify men with prostate cancer into prognostic groups. Moreover, it is crucial in the future development of therapies that can prevent metastases.
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Neoplasias de la Próstata/patología , Cadherinas/metabolismo , Adhesión Celular , Humanos , Linfangiogénesis , Masculino , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Neovascularización Patológica , Neoplasias de la Próstata/irrigación sanguínea , Células del Estroma/patologíaRESUMEN
Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.
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Sondas de ADN/química , Compuestos Orgánicos/química , Reacción en Cadena de la Polimerasa/métodos , Benzotiazoles , Diaminas , Reacción en Cadena de la Polimerasa/instrumentación , Quinolinas , Sensibilidad y EspecificidadRESUMEN
AIM: Chemokines or chemotactic cytokines are known to be important in the directional migration or chemotaxis of leucocytes in conditions of homeostasis and in inflammatory or immunological responses. However, the role of chemokines is extending beyond their involvement in mediating leucocyte trafficking with an increasing body of evidence suggesting these proteins are intimately involved in many stages of tumour development and progression. Our aim was to study the role of the CXCL12:CXCR4 chemokine ligand:receptor complex in determining the organ-specific metastasis of prostate cancer. MATERIALS AND METHODS: CXCR4 mRNA expression was determined by RT-PCR in 3 metastatic prostate cancer cell lines DU145, LNCaP and PC3, the primary prostate cancer cell line 1542 CPT3X and the normal prostate epithelial cell lines 1542 NPTX and Pre 2.8. This was followed by Taqman quantitative PCR analysis of CXCR4 mRNA in these cell lines. Flow cytometry analysis was then used to measure the expression of the CXCR4 receptor protein on the cell surface. The influence of the receptor on cell migration was studied using Transwell, Migration Assays. Finally, Taqman quantitative PCR was performed on RNA obtained from laser microdissected fresh primary prostate tumour and benign tissue samples from patients. RESULTS: In DU145, LNCaP and PC3 CXCR4 mRNA expression was approximately 1000, 400 and 21 times respectively that of 1542 NPTX, Pre 2.8 and 1542 CPT3X. In patient primary tumour samples and patient benign tissue specimens CXCR4 mRNA expression was similar to that of the metastatic cell line DU145. Flow cytometry analysis showed that significantly higher levels of the CXCR4 receptor were present on the cell surface of the 3 metastatic cell lines. Migration studies revealed that chemotaxis of the metastatic cell lines PC3 and DU145 was enhanced by CXCL12 ligand and inhibited by antibody to CXCR4. CXCL12 did not influence the migration of the normal prostate epithelial cell line 1542 NPTX. CONCLUSIONS: We have demonstrated that human prostate cell lines derived from metastases express functional CXCR4 receptor and that CXCL12 ligand enhances their migratory capabilities. Also, laser microdissected primary patient tumours and patient benign tissue specimens express CXCR4 mRNA at high levels (it is suggested that post-transcriptional modification of the CXCR4 receptor plays a major role in regulating protein expression). These results suggest prostate cancers may be influenced by the CXCL12:CXCR4 pathway during metastasis. This pathway would provide a novel target for therapeutic intervention.
Asunto(s)
Quimiocinas CXC/fisiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores CXCR4/fisiología , Línea Celular Tumoral , Movimiento Celular , Quimiocina CXCL12 , Cartilla de ADN/farmacología , Relación Dosis-Respuesta a Droga , Etidio/farmacología , Citometría de Flujo , Humanos , Ligandos , Masculino , Metástasis de la Neoplasia , Oligonucleótidos Antisentido/farmacología , Procesamiento Proteico-Postraduccional , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A previous study of 34 nuclear pedigrees segregating juvenile myoclonic epilepsy (JME) gave significant evidence of linkage with heterogeneity to marker loci on chromosome 15q13-14 close to the candidate gene CHRNA7 (Hum. Mol. Genet. 6 (1997) 1329). The aim of this work was to further evaluate the putative aetiological role of CHRNA7 in JME within the 34 families originally described, and to assess the contribution of this locus to a broader phenotype of idiopathic generalised epilepsy (IGE). Multipoint linkage analysis and intrafamilial association studies were performed with microsatellite markers that encompass both CHRNA7 and its partial duplication (CHRFAM7A). A maximum HLOD of 3.45 [alpha=0.58; (Zall=2.88, P=0.0008)] was observed 8 cM distal to D15S1360, a CHRNA7 intragenic marker. Significant exclusion lod scores were obtained across the region in 12 mixed phenotype JME/IGE families. Mutation screening of the CHRNA7 gene (and consequently exons 5-10 of CHRFAM7A) and its putative promoter sequence identified a total of 13 sequence variants across 23 of 34 JME-affected families. Two variants (c.1354G>A and c.1466C>T) are predicted to result in amino acid changes and one (IVS9+5G>A) is predicted to result in aberrant transcript splicing. However, none of the variants alone appeared either necessary or sufficient to cause JME in the families in which they occurred. In conclusion, linkage analyses continue to support the existence of a locus on chromosome 15q13-14 that confers susceptibility to JME but not to a broader IGE phenotype. Causal sequence variants in the positional candidate CHRNA7 have not been identified but the presence of multiple segmental duplications in this region raises the possibility of undetected disease-causing genomic rearrangements.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 15/genética , Epilepsia Mioclónica Juvenil/genética , Receptores Nicotínicos/genética , Femenino , Ligamiento Genético , Variación Genética , Haplotipos , Humanos , Desequilibrio de Ligamiento , Masculino , Linaje , Regiones Promotoras Genéticas/genética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Several neuropsychiatric disorders map to human 15q13-q14, which contains a strong candidate in the alpha7-nicotinic acetylcholine receptor subunit gene (CHRNA7) and is partly duplicated, complicating further genetic analysis. We have shown that the partial duplication is in a hybrid (CHRFAM7A)between CHRNA7 and one of many copies of a novel gene (FAM7A). We have constructed a 3-Mb map of 15q13-q14 showing that CHRFAM7A is part of a large segmental duplication in the opposite orientation to CHRNA7 and revealing several other duplications. The data support a model of recent evolutionary events including duplications, at least one large deletion, and an inversion. We have identified two individuals with a structure that lacks CHRFAM7A and therefore predates many steps in this model, suggesting an unstable region with other intermediates possibly still in existence. This instability may be relevant to the many neuropsychiatric disorders that map in this region.