Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 21
Filter
1.
J Pathol ; 234(2): 178-89, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24827423

ABSTRACT

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.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Centrioles/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Prostatic Neoplasms/metabolism , Animals , Cell Line , Centrosome/metabolism , Humans , Male , Mice, Inbred C57BL , Mitosis/physiology , Prostatic Neoplasms/pathology , Up-Regulation
2.
J Cell Sci ; 126(Pt 6): 1517-30, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23418359

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , MicroRNAs/metabolism , N-Acetylgalactosaminyltransferases/metabolism , PTEN Phosphohydrolase/metabolism , Vimentin/metabolism , Animals , Carcinogenesis/genetics , Enzyme Repression/genetics , Gene Expression Regulation, Neoplastic , Hep G2 Cells , Humans , Mice , Mice, Transgenic , MicroRNAs/genetics , N-Acetylgalactosaminyltransferases/genetics , PTEN Phosphohydrolase/genetics , Signal Transduction , Transgenes/genetics , Tumor Stem Cell Assay , Vimentin/genetics , Polypeptide N-acetylgalactosaminyltransferase
3.
FASEB J ; 27(3): 907-19, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23180826

ABSTRACT

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.


Subject(s)
3' Untranslated Regions , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , MicroRNAs/metabolism , Neoplasm Proteins/biosynthesis , RNA, Neoplasm/metabolism , Versicans/biosynthesis , Animals , Apoptosis/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Mice, Transgenic , MicroRNAs/genetics , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Proteins/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Neoplasm/genetics , Versicans/genetics
4.
BMJ Open ; 2(5)2012.
Article in English | MEDLINE | ID: mdl-23048060

ABSTRACT

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.

5.
Prostate ; 72(2): 201-8, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-21563194

ABSTRACT

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.


Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Neoplasm Recurrence, Local/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-fes/biosynthesis , Adenocarcinoma/genetics , Adenocarcinoma/surgery , Animals , Disease Models, Animal , Disease-Free Survival , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Mice , Mice, Transgenic , Neoplasm Recurrence, Local/metabolism , Predictive Value of Tests , Prostatic Neoplasms/genetics , Prostatic Neoplasms/surgery , Proto-Oncogene Mas , Proto-Oncogene Proteins c-fes/genetics , Retrospective Studies
6.
Mol Cancer Ther ; 10(7): 1229-40, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21593385

ABSTRACT

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.


Subject(s)
Cell Transformation, Neoplastic/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloproteins/genetics , Metalloproteins/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Animals , Antigens, Polyomavirus Transforming/genetics , Antigens, Polyomavirus Transforming/metabolism , Cell Cycle/genetics , Cell Line, Transformed , Cell Line, Tumor , Cell Proliferation , Disease Progression , Gene Knockdown Techniques , Gene Order , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mice, Transgenic , NIH 3T3 Cells , Neoplasms/pathology , Phosphorylation/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA, Messenger/genetics , Signal Transduction/genetics , Tripartite Motif Proteins
7.
Mol Imaging ; 8(4): 209-20, 2009.
Article in English | MEDLINE | ID: mdl-19728975

ABSTRACT

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.


Subject(s)
Biomarkers, Tumor/metabolism , Contrast Media/administration & dosage , Prostatic Neoplasms/diagnostic imaging , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Biomarkers, Tumor/analysis , Contrast Media/analysis , Contrast Media/metabolism , Drug Delivery Systems/methods , Early Detection of Cancer , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Neoplasm Metastasis , Neovascularization, Pathologic/diagnostic imaging , Prognosis , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Ultrasonography , Vascular Endothelial Growth Factor Receptor-2/analysis
8.
Nat Cell Biol ; 11(8): 1031-8, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19633662

ABSTRACT

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.


Subject(s)
Fibronectins/genetics , Gene Expression Regulation , MicroRNAs/genetics , Animals , Base Sequence , Cell Adhesion , Cell Line , Cell Line, Tumor , Cell Proliferation , Female , Flow Cytometry , Humans , Kidney/metabolism , Kidney/pathology , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Molecular Sequence Data , Myocardium/metabolism , Myocardium/pathology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Spleen/metabolism , Spleen/pathology , Transfection
9.
Development ; 135(4): 775-84, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18184727

ABSTRACT

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.


Subject(s)
Epithelial Cells/metabolism , Membrane Proteins/metabolism , Organogenesis , Prostate/growth & development , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Regeneration , Alleles , Androgens/pharmacology , Animals , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/pathology , Epithelium/metabolism , Epithelium/pathology , Gene Expression Regulation, Developmental/drug effects , Male , Membrane Proteins/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Organ Specificity/drug effects , Prostate/drug effects , Prostate/enzymology , Prostate/pathology , Regeneration/drug effects , Sexual Maturation/drug effects
10.
Cancer Res ; 67(6): 2830-9, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-17363606

ABSTRACT

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.


Subject(s)
Adenocarcinoma/blood supply , Adenocarcinoma/diagnostic imaging , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/diagnostic imaging , Ultrasonography, Doppler/methods , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Cell Growth Processes/physiology , Disease Models, Animal , Genetic Engineering , Image Processing, Computer-Assisted/methods , Male , Mice , Mice, Transgenic , Neovascularization, Pathologic/diagnostic imaging , Neovascularization, Pathologic/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology
11.
Development ; 134(4): 723-34, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17215304

ABSTRACT

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.


Subject(s)
Androgens/physiology , Morphogenesis , Prostate/growth & development , Receptor, Fibroblast Growth Factor, Type 2/physiology , Animals , Animals, Newborn , Embryo, Mammalian , Epithelial Cells , Homeostasis , Male , Mice , Mice, Knockout , Mice, Transgenic , Organogenesis , Prostate/cytology , Prostate/embryology , Prostate/ultrastructure , Receptor, Fibroblast Growth Factor, Type 2/deficiency
12.
Mol Cancer Res ; 4(12): 957-69, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17189386

ABSTRACT

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.


Subject(s)
Carcinogens/toxicity , Cell Transformation, Neoplastic , Protein Kinases/physiology , Simian virus 40/genetics , Animals , Disease Models, Animal , Humans , Male , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Phosphorylation , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/physiopathology , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Up-Regulation
13.
Clin Cancer Res ; 11(21): 7911-9, 2005 Nov 01.
Article in English | MEDLINE | ID: mdl-16278416

ABSTRACT

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.


Subject(s)
Biomarkers, Tumor/blood , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/blood , Prostatic Neoplasms/diagnosis , Animals , Blotting, Western , Clinical Trials as Topic , DNA, Complementary/metabolism , Disease Models, Animal , Down-Regulation , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Glycosylation , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Male , Mice , Mice, Transgenic , Prostate-Specific Antigen/biosynthesis , Prostatic Secretory Proteins/blood , Recombinant Proteins/chemistry , Time Factors
14.
Cancer Res ; 65(14): 6337-45, 2005 Jul 15.
Article in English | MEDLINE | ID: mdl-16024636

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Prostatic Neoplasms/diagnostic imaging , Adenocarcinoma/diagnostic imaging , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Image Processing, Computer-Assisted/methods , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Sensitivity and Specificity , Ultrasonography
15.
Urology ; 65(4): 719-23, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15833515

ABSTRACT

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.


Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/surgery , Neoplasm Recurrence, Local/metabolism , Prostatectomy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/surgery , Prostatic Secretory Proteins/biosynthesis , Adenocarcinoma/chemistry , Aged , Disease Progression , Follow-Up Studies , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/chemistry , Predictive Value of Tests , Prostatic Neoplasms/chemistry , Prostatic Secretory Proteins/analysis
16.
Mol Ther ; 11(3): 348-62, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15727931

ABSTRACT

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.


Subject(s)
Adenocarcinoma/genetics , Disease Models, Animal , Prostatic Neoplasms/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Humans , Liver Neoplasms/secondary , Lung Neoplasms/secondary , Male , Mice , Oligonucleotide Array Sequence Analysis , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Secretory Proteins/genetics , Prostatic Secretory Proteins/metabolism
17.
Oncogene ; 24(9): 1510-24, 2005 Feb 24.
Article in English | MEDLINE | ID: mdl-15674347

ABSTRACT

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.


Subject(s)
Adenocarcinoma/genetics , Antigens, Polyomavirus Transforming/genetics , Prostatic Neoplasms/genetics , Simian virus 40/genetics , Adenocarcinoma/pathology , Animals , Basement Membrane/pathology , Exons/genetics , Male , Mice , Mice, Transgenic , Neoplasm Metastasis , Orchiectomy , Prostatic Neoplasms/pathology , Prostatic Secretory Proteins/genetics
18.
Biochem J ; 385(Pt 1): 105-14, 2005 Jan 01.
Article in English | MEDLINE | ID: mdl-15344909

ABSTRACT

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.


Subject(s)
Blood Proteins/isolation & purification , Blood Proteins/metabolism , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Gene Expression Profiling , Glycoproteins/isolation & purification , Glycoproteins/metabolism , Prostatic Secretory Proteins/metabolism , Aged , Aged, 80 and over , Amino Acid Sequence , Amino Acids , Antibodies, Monoclonal/immunology , Blood Proteins/chemistry , Blood Proteins/genetics , Blotting, Western , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cloning, Molecular , Glycoproteins/chemistry , Glycoproteins/genetics , Glycosylation , Humans , Immunohistochemistry , Immunoprecipitation , Kinetics , Male , Middle Aged , Molecular Sequence Data , Molecular Weight , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/blood , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism
19.
Urology ; 61(4): 851-7, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12670588

ABSTRACT

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.


Subject(s)
Carcinoma, Transitional Cell/metabolism , Helix-Loop-Helix Motifs , Trans-Activators/analysis , Urinary Bladder Neoplasms/metabolism , Adult , Aged , Basic Helix-Loop-Helix Transcription Factors , Carcinoma, Transitional Cell/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , In Situ Hybridization , Male , Middle Aged , Neoplasm Proteins/metabolism , Neoplasm Staging , RNA, Messenger/analysis , Urinary Bladder/pathology , Urinary Bladder Neoplasms/pathology
20.
J Cell Biochem ; 88(5): 999-1011, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12616537

ABSTRACT

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.


Subject(s)
Prostate/metabolism , Prostatic Secretory Proteins/biosynthesis , Animals , Antibodies/immunology , Blotting, Western , Cross Reactions , Disease Models, Animal , Fixatives , Gene Expression , Gene Products, nef , Genetic Therapy , Genetic Vectors , Glutathione Transferase , Immunohistochemistry , Male , Mice , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Prostatic Secretory Proteins/genetics , Prostatic Secretory Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...