RESUMO
To study the physiological role of a single microRNA (miRNA), we generated transgenic mice expressing the miRNA precursor miR-17 and found that the mature miR-17-5p and the passenger strand miR-17-3p were abundantly expressed. We showed that mature miR-17-5p and passenger strand miR-17-3p could synergistically induce the development of hepatocellular carcinoma. The mature miR-17-5p exerted this function by repressing the expression of PTEN. In contrast, the passenger strand miR-17-3p repressed expression of vimentin, an intermediate filament with the ability to modulate metabolism, and GalNT7, an enzyme that regulates metabolism of liver toxin galactosamine. Hepatocellular carcinoma cells, HepG2, transfected with miR-17 formed larger tumors with more blood vessels and less tumor cell death than mock-treated cells. Expression of miR-17 precursor modulated HepG2 proliferation, migration, survival, morphogenesis and colony formation and inhibited endothelial tube formation. Silencing of PTEN, vimentin or GalNT7 with their respective siRNAs enhanced proliferation and migration. Re-expressing these molecules reversed their roles in proliferation, migration and tumorigenesis. Further experiments indicated that these three molecules do not interact with each other, but appear to function in different signaling pathways. Our results demonstrated that a mature miRNA can function synergistically with its passenger strand leading to the same phenotype but by regulating different targets located in different signaling pathways. We anticipate that our assay will serve as a helpful model for studying miRNA regulation.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Vimentina/metabolismo , Animais , Carcinogênese/genética , Repressão Enzimática/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , N-Acetilgalactosaminiltransferases/genética , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais , Transgenes/genética , Ensaio Tumoral de Célula-Tronco , Vimentina/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Although spindle- and kinetochore-associated protein 1 (Ska1) has previously been identified as essential for proper chromosome segregation, it is unknown whether it plays a role in tumour development. Here, we report that Ska1 over-expression promotes prostate tumourigenesis. Immunohistochemistry and quantitative RT-PCR analysis revealed that Ska1 was over-expressed in human prostatic intra-epithelial neoplasia (PIN), the most likely prostate cancer precursor, and adenocarcinomas. Up-regulation of Ska1 protein was also found to be tumour-specific in breast, lung and other common human cancers. Importantly, prostate-specific up-regulation of Ska1 in a transgenic mouse model resulted in spontaneous tumourigenesis. Furthermore, in addition to its abundance in spindle microtubules and the outer kinetochore interface during mitosis, Ska1 was enriched at centrosomes in cultured cells. Depletion of Ska1 caused a failure of centrosome duplication, whilst Ska1 over-expression led to centrosome amplification in human prostate epithelial cells via the induction of centriole over-duplication. These epithelial cells harbouring extra centrosomes switched from a non-tumourigenic to a tumourigenic state in nude mice. Taken together, these data indicate that Ska1 over-expression promotes tumourigenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Centríolos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular , Centrossomo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mitose/fisiologia , Neoplasias da Próstata/patologia , Regulação para CimaRESUMO
This study was designed to explore the role of versican in the development of hepatocellular carcinoma (HCC). Ectopic expression of the versican 3'-untranslated region (3'-UTR) was studied as a competitive endogenous RNA for regulating miRNA functions. We used this approach to modulate the expression of versican and its related proteins in 3'-UTR transgenic mice and in the liver cancer cell line HepG2, stably transfected with the 3'-UTR or a control vector. We demonstrated that transgenic mice expressing the versican 3'-UTR developed HCC and increased expression of versican isoforms V0 and V1. HepG2 cells transfected with versican 3'-UTR displayed increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis. We found that versican 3'-UTR could bind to miRNAs miR-133a, miR-199a*, miR-144, and miR-431 and also interacted with CD34 and fibronectin. As a consequence, expression of versican, CD34, and fibronectin was up-regulated by ectopic transfection of the versican 3'-UTR, which was confirmed in HepG2 cells and in transgenic mice as compared with wild-type controls. Transfection with siRNAs targeting the versican 3'-UTR abolished the effects of the 3'-UTR. Taken together, these results demonstrate that versican V0 and V1 isoforms play important roles in HCC development and that versican mRNAs compete with endogenous RNAs in regulating miRNA functions.
Assuntos
Regiões 3' não Traduzidas , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/metabolismo , Versicanas/biossíntese , Animais , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Neoplásico/genética , Versicanas/genéticaRESUMO
BACKGROUND: c-Fes is a proto-oncogene encoded non-receptor protein-tyrosine kinase (PTK). However, genetic studies have indicated that it has anti-tumorigenic effects in certain cancers. The pathological and clinical significance of c-Fes in prostate cancer are unknown. METHODS: Expression of c-Fes was evaluated in normal glands, prostatic intraepithelial neoplasia (PIN), cancer cells in tissues of knock-in mouse adenocarcinoma prostate (KIMAP) model, and prostate cancer patients free of metastasis. Expression of c-Fes was analyzed by immunohistochemistry, and quantified by using the immunoreactivity score (IRS) (staining intensity × percentage of positive cells). Relationships between c-Fes expression and pT stage, Gleason's score (GS), and biochemical recurrence in patients who underwent radical surgery were also investigated. RESULTS: In KIMAP, the percentage in normal glands, PIN and cancer cells positive for c-Fes expression were 0 (0/7), 25.0 (2/8), and 100% (7/7), respectively. In human tissues, c-Fes expression was also significantly higher in cancer cells than in normal cells and PIN, and it correlated with pT stage (P < 0.001) and GS (P = 0.047). Multivariate analysis showed that c-Fes expression was an independent predictor of poor outcome poor prognosis (hazard ratio = 3.21, 95% confidence interval = 1.11-9.37, P = 0.032). CONCLUSION: The results suggested that c-Fes expression is a useful predictor of biochemical recurrence after radical surgery. The results also suggested that c-Fes is a potentially useful therapeutic target in prostate cancer and a predictor of biochemical recurrence after radical prostatectomy.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Recidiva Local de Neoplasia/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-fes/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Animais , Modelos Animais de Doenças , Intervalo Livre de Doença , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Transgênicos , Recidiva Local de Neoplasia/metabolismo , Valor Preditivo dos Testes , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fes/genética , Estudos RetrospectivosRESUMO
We report the first application of high-frequency three-dimensional power Doppler ultrasound imaging in a genetically engineered mouse (GEM) prostate cancer model. We show that the technology sensitively and specifically depicts functional neoangiogenic blood flow because little or no flow is measurable in normal prostate tissue or tumors smaller than 2-3 mm diameter, the neoangiogenesis "switch-on" size. Vascular structures depicted by power Doppler were verified using Microfil-enhanced micro-computed tomography (micro-CT) and by correlation with microvessel distributions measured by immunohistochemistry and enhanced vascularity visualized by confocal microscopy in two GEM models [transgenic adenocarcinoma of the mouse prostate (TRAMP) and PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP)]. Four distinct phases of neoangiogenesis in cancer development were observed, specifically, (a) an early latent phase; (b) establishment of a peripheral capsular vascular structure as a neoangiogenesis initiation site; (c) a peak in tumor vascularity that occurs before aggressive tumor growth; and (d) rapid tumor growth accompanied by decreasing vascularity. Microsurgical interventions mimicking local delivery of antiangiogenesis drugs were done by ligating arteries upstream from feeder vessels branching to the prostate. Microsurgery produced an immediate reduction of tumor blood flow, and flow remained low from 1 h to 2 weeks or longer after treatment. Power Doppler, in conjunction with micro-CT, showed that the tumors recruit secondary blood supplies from nearby vessels, which likely accounts for the continued growth of the tumors after surgery. The microsurgical model represents an advanced angiogenic prostate cancer stage in GEM mice corresponding to clinically defined hormone-refractory prostate cancer. Three-dimensional power Doppler imaging is completely noninvasive and will facilitate basic and preclinical research on neoangiogenesis in live animal models.
Assuntos
Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/diagnóstico por imagem , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/diagnóstico por imagem , Ultrassonografia Doppler/métodos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Processos de Crescimento Celular/fisiologia , Modelos Animais de Doenças , Engenharia Genética , Processamento de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by > or = 10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA-mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America.
Assuntos
Carcinógenos/toxicidade , Transformação Celular Neoplásica , Proteínas Quinases/fisiologia , Vírus 40 dos Símios/genética , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Regulação para CimaRESUMO
Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.
Assuntos
Modelos Animais de Doenças , Neoplasias da Próstata/diagnóstico por imagem , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Processamento de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Sensibilidade e Especificidade , UltrassonografiaRESUMO
Prostate cancer (CaP) is the most common cancer in adult men in North America. Since there is no naturally occurring prostate cancer in the mouse, preclinical studies stipulate for the establishment of a genetically manipulated mouse CaP model with features close to the human situation. In view of the limitations of transgenic technique-derived CaP models, herein we report the first application of knockin technology to establish a new mouse adenocarcinoma prostate model (PSP-KIMAP) by targeting of SV40 Tag to a prostate tissue-specific gene, PSP94 (prostate secretory protein of 94 amino acids). In order to demonstrate its novelty, we compared KIMAP to a PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP) model. The CaP development of the PSP-KIMAP mice started almost immediately after puberty at 10 weeks of age from mouse prostatic intraepithelial neoplasia (mPIN) with microinvasion to well-differentiated CaP, and demonstrated a close-to-human kinetics of prolonged tumor growth and a predominance of well and moderately differentiated tumors. The invasive nature of KIMAP model was demonstrated by multitissue metastases (lymph node, lung and liver etc) and also by immunohistochemical study of multiple invasive prostate tumor markers. PSP-KIMAP model is responsive to androgen deprivation (castration). The knockin technology in our KIMAP model demonstrates highly predictive CaP development procedures and many advantageous features, which the traditional transgenic technique-derived CaP models could not reach for both basic and clinical studies. These features include the high stability of both phenotype and genotype, highly synchronous prostate cancer development, high and precise prostate tissue targeting and with no founder line variation. The differences between the two CaP models were attributed to the introduction of a single endogenous knockin mutation, resulting in a CaP model self-regulated and controlled by a prostate gene promoter/enhancer of PSP94.
Assuntos
Adenocarcinoma/genética , Antígenos Transformantes de Poliomavirus/genética , Neoplasias da Próstata/genética , Vírus 40 dos Símios/genética , Adenocarcinoma/patologia , Animais , Membrana Basal/patologia , Éxons/genética , Masculino , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Orquiectomia , Neoplasias da Próstata/patologia , Proteínas Secretadas pela Próstata/genéticaRESUMO
Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.
Assuntos
Biomarcadores Tumorais/sangue , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Animais , Western Blotting , Ensaios Clínicos como Assunto , DNA Complementar/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Antígeno Prostático Específico/biossíntese , Proteínas Secretadas pela Próstata/sangue , Proteínas Recombinantes/química , Fatores de TempoRESUMO
PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94's action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94's functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Perfilação da Expressão Gênica , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Proteínas Secretadas pela Próstata/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Aminoácidos , Anticorpos Monoclonais/imunologia , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Imuno-Histoquímica , Imunoprecipitação , Cinética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/sangue , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Endothelial Per-ARNT-Sim (PAS) domain protein-1 (EPAS-1)/hypoxia-inducible factor-2alpha (HIF-2alpha) is a member of the basic helix-loop-helix/PAS domain protein family and is considered to be an endothelial-specific, hypoxia-inducible transcription factor. Because hypoxia is a fundamental element of tumor biology determining clinical outcome, we performed an immunohistochemical study of EPAS-1 expression in a cohort of bladder cancer cases and assessed the possible correlation of EPAS-1 expression with tumor hypoxia and growth. In the 67 cases (37 radical cystectomy and 30 transurethral resection) studied, overexpression of EPAS-1/HIF-2alpha protein was not found in cancer cells or in normal tissues but was mostly found in stroma around cancer cells, and strong positive staining was noted in perinecrotic regions. The perinecrotic/tumorous expression of EPAS-1/HIF-2alpha was correlated statistically with higher histological grade (P < 0.001), advanced pathological T stage (P < 0.001), and presence of necrosis (P < 0.001). A parallel immunohistochemical analysis of a marker gene of vascular endothelial growth factor demonstrated its positive correlation with tumor grade, stage, and EPAS-1/HIF-2alpha overexpression, supporting the correlation of EPAS-1/HIF-2alpha up-regulation with tumor angiogenesis. To further clarify the relationship between hypoxia and vascularity in the perinecrotic/tumorous area with EPAS-1/HIF-2alpha expression, tissue microvessel density (MVD) was assessed. No significant correlation (P = 0.442) was found between EPAS-1/HIF-2alpha expression and MVD if the 67 tumors of different stages were all included. However, EPAS-1/HIF-2alpha-positive cases had lower MVD than EPAS-1/HIF-2alpha-negative cases (P = 0.001) if only invasive cancer cases were analyzed. In addition, in all EPAS-1/HIF-2alpha-positive staining cases, EPAS-1/HIF-2alpha-positive foci had lower MVD than EPAS-1/HIF-2alpha-negative foci (P < 0.001). Finally, using serial sections, the location of EPAS-1/HIF 2alpha expression was identified mainly in tumor-associated macrophage (TAM) as well as in some fibroblast cells. Focal TAM infiltration was identified at a higher level in EPAS-1-positive cases than EPAS-1-negative cases (P < 0.001). This is the first clinical report suggesting that hypoxia-induced, perinecrotic EPAS-1/HIF-2alpha expression is correlated with tumor progression and angiogenesis at higher grade and stage through focal TAM infiltration in invasive bladder cancer.
Assuntos
Endotélio/metabolismo , Endotélio/patologia , Hipóxia , Necrose , Transativadores/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Movimento Celular , Progressão da Doença , Fatores de Crescimento Endotelial , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfocinas , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVES AND DESIGN: We identified a novel TRIM59 gene, as an early signal transducer in two (SV40Tag and Ras) oncogene pathways in murine prostate cancer (CaP) models. We explore its clinical applications as a multitumour marker detecting early tumorigenesis by immunohistochemistry (IHC). SETTING AND PARTICIPANTS: 88 CaP patients were from a tissue microarray (TMA) of radical prostatectomy specimen, 42 patients from a 35 multiple tumour TMA, 75 patients with renal cell carcinoma (RCC) and 92 patients from eight different tumour groups (breast, lung, parotid, gastrointestinal, female genital tract, bladder, kidney and prostate cancer). RESULTS: TRIM59 upregulation specifically in tumour area was determined by IHC in 291 cases of 37 tumour types. To demonstrate that TRIM59 upregulation is 'tumour-specific', we characterised a significant correlation of TRIM59 IHC signals with tumorigenesis and progression, while in control and normal area, TRIM59 IHC signal was all negative or significantly low. TRIM59 protein upregulation in prostate and kidney cancers was detectable in both intensity and extent in early tumorigenesis of prostate intraepithelial neoplasia (p<0.05) and grade 1 of RCC (p<0.05), and stopped until high grades cancer. The results of the correlation in these two large cohorts of tumour types confirmed and repeated murine CaP model studies. Enhanced TRIM59 expression was identified in most of the 37 different tumours, while the highest intensities were in lung, breast, liver, skin, tongue and mouth (squamous cell cancer) and endometrial cancers. Multiple tumour upregulation was further confirmed by comparing relative scores of TRIM59 IHC signals in eight tumours with a larger patient population; and by a mouse whole-mount embryo (14.5 days post conception) test on the origin of TRIM59 upregulation in epithelial cells. CONCLUSIONS: TRIM59 may be used a novel multiple tumour marker for immunohistochemical detecting early tumorigenesis and could direct a novel strategy for molecular-targeted diagnosis and therapy of cancer.
RESUMO
A novel TRIM family member, TRIM59 gene was characterized to be upregulated in SV40 Tag oncogene-directed transgenic and knockout mouse prostate cancer models as a signaling pathway effector. We identified two phosphorylated forms of TRIM59 (p53 and p55) and characterized them using purified TRIM59 proteins from mouse prostate cancer models at different stages with wild-type mice and NIH3T3 cells as controls. p53/p55-TRIM59 proteins possibly represent Ser/Thr and Tyr phosphorylation modifications, respectively. Quantitative measurements by ELISA showed that the p-Ser/Thr TRIM59 correlated with tumorigenesis, whereas the p-Tyr-TRIM59 protein correlated with advanced cancer of the prostate (CaP). The function of TRIM59 was elucidated using short hairpin RNA (shRNA)-mediated knockdown of the gene in human CaP cells, which caused S-phase cell-cycle arrest and cell growth retardation. A hit-and-run effect of TRIM59 shRNA knockdown was observed 24 hours posttransfection. Differential cDNA microarrray analysis was conducted, which showed that the initial and rapid knockdown occurred early in the Ras signaling pathway. To confirm the proto-oncogenic function of TRIM59 in the Ras signaling pathway, we generated a transgenic mouse model using a prostate tissue-specific gene (PSP94) to direct the upregulation of the TRIM59 gene. Restricted TRIM59 gene upregulation in the prostate revealed the full potential for inducing tumorigenesis, similar to the expression of SV40 Tag, and coincided with the upregulation of genes specific to the Ras signaling pathway and bridging genes for SV40 Tag-mediated oncogenesis. The finding of a possible novel oncogene in animal models will implicate a novel strategy for diagnosis, prognosis, and therapy for cancer.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Ciclo Celular/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Técnicas de Silenciamento de Genes , Ordem dos Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Neoplasias/patologia , Fosforilação/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/genética , Proteínas com Motivo TripartidoRESUMO
Molecular imaging techniques used to detect the initiation of disease have the potential to provide the best opportunity for early treatment and cure. This report aimed at testing the possibility that Flk1+ (vascular endothelial growth factor receptor 2), a crucial angiogenesis factor of most tumor cells, could be a molecular targeted imaging marker for the diagnosis and prognosis of cancer. We performed Flk1-targeted microbubble-enhanced ultrasound (US) imaging of prostate cancer in a genetically engineered mouse model with normal-appearing intact US (negative) prostates and with three different tumor sizes (small, medium, and large). Higher levels of Flk1+ molecular signals were identified in the intact US (negative) prostate group by US-targeted imaging and immunohistochemical analysis. The increase in Flk1+ expression occurred prior to the angiogenesis switch-on phase and vascularity peak. After this peak accumulation stage of Flk1+ molecules, lower and stabilized levels of Flk1+ signals were maintained together with tumor growth from small, to medium, to large size. In a longitudinal observation in a subset (n = 5) of mice with established tumors, elevated Flk1+ signals were observed in tissues surrounding the prostate cancer, for example, the ipsilateral boundary zones between two developing tumor lobes, new tumor blood vessel recruits, the urethra border, and the pelvic node basin. The potential of Flk1-targeted US imaging as a predictive imaging tool was confirmed by correlation studies of three-dimensional US B-mode imaging, gross pathology, and histology analyses. The results of the application in a genetically engineered mouse model with prostate cancer of molecular Flk1-targeted US imaging support the contention that Flk1 can be used as a molecular imaging marker for small tumors undetectable by microimaging and as a molecular diagnostic and prognosis marker for tumor metastasis and progression.
Assuntos
Biomarcadores Tumorais/metabolismo , Meios de Contraste/administração & dosagem , Neoplasias da Próstata/diagnóstico por imagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Biomarcadores Tumorais/análise , Meios de Contraste/análise , Meios de Contraste/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Detecção Precoce de Câncer , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Metástase Neoplásica , Neovascularização Patológica/diagnóstico por imagem , Prognóstico , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ultrassonografia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17~92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.
Assuntos
Fibronectinas/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Animais , Sequência de Bases , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Citometria de Fluxo , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Dados de Sequência Molecular , Miocárdio/metabolismo , Miocárdio/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Baço/metabolismo , Baço/patologia , TransfecçãoRESUMO
The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2alpha (FRS2alpha) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2alpha is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2alpha was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2alpha function in the prostate, the Frs2alpha alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2alpha disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2alpha had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2alpha inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2alpha-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2alpha(cn) mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Organogênese , Próstata/crescimento & desenvolvimento , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Regeneração , Alelos , Androgênios/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Epitélio/metabolismo , Epitélio/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/enzimologia , Próstata/patologia , Regeneração/efeitos dos fármacos , Maturidade Sexual/efeitos dos fármacosRESUMO
The fibroblast growth factor (FGF) family consists of 22 members and regulates a broad spectrum of biological activities by activating diverse isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and FGF10 have been implicated in the regulation of prostate development and prostate tissue homeostasis by signaling through the FGFR2 isoform. Using conditional gene ablation with the Cre-LoxP system in mice, we demonstrate a tissue-specific requirement for FGFR2 in urogenital epithelial cells--the precursors of prostatic epithelial cells--for prostatic branching morphogenesis and prostatic growth. Most Fgfr2 conditional null (Fgfr2(cn)) embryos developed only two dorsal prostatic (dp) and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate, which has two anterior prostatic (ap), two dp, two lp and two ventral prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well developed epithelial ductal networks, the Fgfr2(cn) prostates, despite retaining a compartmented tissue structure, exhibited a primitive epithelial architecture. Moreover, although Fgfr2(cn) prostates continued to produce secretory proteins in an androgen-dependent manner, they responded poorly to androgen with respect to tissue homeostasis. The results demonstrate that FGFR2 is important for prostate organogenesis and for the prostate to develop into a strictly androgen-dependent organ with respect to tissue homeostasis but not to the secretory function, implying that androgens may regulate tissue homeostasis and tissue function differently. Therefore, Fgfr2(cn) prostates provide a useful animal model for scrutinizing molecular mechanisms by which androgens regulate prostate growth, homeostasis and function, and may yield clues as to how advanced-tumor prostate cells escape strict androgen regulations.
Assuntos
Androgênios/fisiologia , Morfogênese , Próstata/crescimento & desenvolvimento , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/fisiologia , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Células Epiteliais , Homeostase , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Organogênese , Próstata/citologia , Próstata/embriologia , Próstata/ultraestrutura , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiênciaRESUMO
OBJECTIVES: To examine the impact of prostate secretory protein of 94 amino acids (PSP94) expression within the primary tumor on disease recurrence and progression using radical prostatectomy specimens from patients with adenocarcinoma of the prostate. METHODS: PSP94 immunohistochemistry was performed on 59 radical prostatectomy specimens. The degree of PSP94 expression was reported as either present (group 1) or absent (group 2). Clinical data, including survival outcome measures, were correlated with PSP94 expression. RESULTS: The time to disease progression for group 1 was shorter compared with that for group 2 (P = 0.042). Disease-free survival was also less in group 1 than in group 2 (P = 0.033). Multivariate analyses demonstrated that PSP94 expression was an independent prognostic factor for a shorter interval to disease progression (P = 0.046) and disease-free survival (P = 0.049). CONCLUSIONS: PSP94 expression in radical prostatectomy tumor specimens appears to be associated with worsened survival outcomes and may provide additional prognostic information.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/cirurgia , Recidiva Local de Neoplasia/metabolismo , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/cirurgia , Proteínas Secretadas pela Próstata/biossíntese , Adenocarcinoma/química , Idoso , Progressão da Doença , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/química , Valor Preditivo dos Testes , Neoplasias da Próstata/química , Proteínas Secretadas pela Próstata/análiseRESUMO
Preclinical studies of prostate cancer (CaP) have employed a genetically engineered mouse model, since there is no naturally occurring CaP in rodents. We have previously reported a new knock-in mouse adenocarcinoma prostate (KIMAP) model. In this study, we demonstrate that the new model possesses a tumor architecture of heterogeneity and multifocality similar to that of human CaP, by utilizing a new compound scoring system to compare with the PSP94 (approved gene symbol Msmb) gene-directed transgenic mouse CaP model (TGMAP). KIMAP mice showed a balanced distribution of tumor extent, which penetrated the prostate gland. Comparative studies on cDNA microarrays demonstrated that KIMAP tumors were upregulated with higher contents of immunoresponse genes, whereas PSP-TGMAP tumors had neuroendocrine (NE) differentiation. The majority of KIMAP mice did not progress to NE CaP, which was observed only at a very late stage and a low frequency. Several tumor marker genes characteristic of human CaP were uniquely identified in KIMAP tumors, including hepsin, maspin, Nkx3.1, CD10 and PSP94 (similar to PSA), etc. The differences between these two CaP models are attributed to the introduction of a single endogenous knock-in mutation. Due to the similarities between human CaP tumors and the PSP-KIMAP tumors, this preclinical model may supplement the current transgenic models to study CaP more accurately.
Assuntos
Adenocarcinoma/genética , Modelos Animais de Doenças , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Humanos , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Secretadas pela Próstata/genética , Proteínas Secretadas pela Próstata/metabolismoRESUMO
OBJECTIVES: Endothelial Per-ARNT-Sim (PAS) domain protein-1 (EPAS-1)/hypoxia-inducible factor (HIF)-2alpha is a recently identified, endothelial-specific, hypoxia-induced transcription factor and is reputed to have an important role in tumor angiogenesis and tumor progression. Only a few studies have reported on the clinical correlation with grade, stage, necrosis, and microvessel density in transitional cell carcinoma of the bladder. In vitro studies have reported no concordance of EPAS-1/HIF-2alpha mRNA and protein expression. We sought to elucidate these two issues. METHODS: Surgical specimens from 110 patients with transitional cell carcinoma comprised the study, including 51 from radical cystectomy and 59 from transurethral resection of bladder tumor. Immunohistochemistry and in situ hybridization were performed with antibodies against EPAS-1/HIF-2alpha, CD31, and a human EPAS-1/HIF-2alpha cDNA subclone probe. RESULTS: EPAS-1/HIF-2alpha protein expression was found almost exclusively in high-grade and high-stage tumors (P <0.0001). EPAS-1/HIF-2alpha mRNA expression was detectable only in high-grade (grade 3) and high-stage (pT3a or greater) cases with positive EPAS-1/HIF-2alpha protein expression (P = 0.0017). The presence of necrosis correlated with EPAS-1/HIF-2alpha expression, tumor grade, and tumor stage (P <0.0001). Microvessel density was much lower in invasive, larger tumor nodules in EPAS-1/HIF-2alpha-positive cases than in the negative cases (P <0.0001). CONCLUSIONS: EPAS-1/HIF-2alpha protein and mRNA levels correlate with higher grade and advanced bladder transitional cell carcinoma. In lower stage cases, no concordance was found between transcription and translation of EPAS-1/HIF-2alpha gene expression. Because EPAS-1/HIF-2alpha mRNA is only detectable in highly invasive cancer cases, it may serve as a therapeutic target (eg, by an antisense mRNA approach) for bladder cancer.