PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3.

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.

VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization.

Increased numbers of S100A4(+) cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4(+) cancer cells have been examined, the functional role of S100A4(+) stromal cells in metastasis is largely unknown. To study the contribution of S100A4(+) stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4(+) stromal cells. Depletion of S100A4(+) stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4(+) stromal cells are attributable to local non-bone marrow-derived S100A4(+) cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4(+) fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4(+) fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4(+) fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4(+) fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.

Germline DNA copy number variation in familial and early-onset breast cancer.

INTRODUCTION: Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease." METHODS: Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations. RESULTS: The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer. CONCLUSIONS: This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.

An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

No-match ORESTES explored as tumor markers.

Sequencing technologies and new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this objective by generating data from about 1.2 million expressed sequence tags. Approximately 30% of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3421 transcribed regions not associated with annotated transcripts, representing 83.3% of the platform. The total number of differentially expressed sequences was 1007. Also, 28% of analyzed sequences could represent noncoding RNAs. Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of three out of eight potentially tumor markers in prostate tissues. Lists of 1007 differentially expressed sequences, and the 291 potentially noncoding tumor markers were provided.

Molecular profiling of isolated histological components of wilms tumor implicates a common role for the Wnt signaling pathway in kidney and tumor development.

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common deregulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset.

Cellular prion protein expression in astrocytes modulates neuronal survival and differentiation.

The functions of cellular prion protein (PrP(C)) are under intense debate and PrP(C) loss of function has been implicated in the pathology of prion diseases. Neuronal PrP(C) engagement with stress-inducible protein-1 and laminin (LN) plays a key role in cell survival and differentiation. The present study evaluated whether PrP(C) expression in astrocytes modulates neuron-glia cross-talk that underlies neuronal survival and differentiation. Astrocytes from wild-type mice promoted a higher level neuritogenesis than astrocytes obtained from PrP(C)-null animals. Remarkably, neuritogenesis was greatly diminished in co-cultures combining PrP(C)-null astrocytes and neurons. LN secreted and deposited at the extracellular matrix by wild-type astrocytes presented a fibrillary pattern and was permissive for neuritogenesis. Conversely, LN coming from PrP(C)-null astrocytes displayed a punctate distribution, and did not support neuronal differentiation. Additionally, secreted soluble factors from PrP(C)-null astrocytes promoted lower levels of neuronal survival than those secreted by wild-type astrocytes. PrP(C) and stress-inducible protein-1 were characterized as soluble molecules secreted by astrocytes which participate in neuronal survival. Taken together, these data indicate that PrP(C) expression in astrocytes is critical for sustaining cell-to-cell interactions, the organization of the extracellular matrix, and the secretion of soluble factors, all of which are essential events for neuronal differentiation and survival.

Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways.

Understanding the physiological function of the cellular prion (PrPc) depends on the investigation of PrPc-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrPc ligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrPc interaction with STI1 in hippocampal neurons. Both PrPc and STI1 are abundantly expressed and highly colocalized in the hippocampus in situ, indicating that they can interact in vivo. Recombinant STI1 (His6-STI1) added to hippocampal cultures interacts with PrPc at the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrPc-null cells. This effect was abolished by antibodies against either PrPc or STI1 and was dependent on the STI1 domain that binds PrPc. Binding of these proteins induced the phosphorylation/activation of the mitogen-activated protein kinase, which was essential for STI1-promoted neuritogenesis. His6-STI1, but not its counterpart lacking the PrPc binding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrPc-STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.

SATR-1 hypomethylation is a common and early event in breast cancer.

Genome stability and normal gene expression are maintained by a fixed and predetermined DNA methylation pattern, which becomes abnormal in malignant cells. Hypomethylation of satellite DNA sequences is frequently found in tumors and has been associated with an increased frequency of DNA rearrangements and chromosome instability. In this work, we used methylation-sensitive arbitrarily primed polymerase chain reaction (MSAP-PCR) to identify differentially methylated DNA fragments in normal and tumor breast samples. We identified a novel differentially methylated fragment located on chromosome 5 with high similarity to a SATR-1 satellite sequence. This fragment was found to be hypomethylated in 63% of breast tumor cell lines and in 86% of breast tumors relative to normal breast tissue. We found that normal tissue adjacent to breast tumors displayed a variable decrease in methylation and that the decrease observed for most of these adjacent samples was higher than observed for normal breast tissue derived from reduction mammoplasty. The methylation decrease was, however, significantly higher in tumor samples than in adjacent tissue (chi2= 154, 1 df, P < 10(-4)), suggesting that SATR-1 hypomethylation frequently occurs in the early stages of tumor development. Our results highlight the importance of global DNA hypomethylation as a contributing factor in breast tumorigenesis.

Synaptosomal glutamate release and uptake in mice lacking the cellular prion protein.

Glutamate plays a central role in the fast excitatory synaptic transmission and is a key neurotransmitter involved in several neurophysiological processes. Glutamate levels on the synaptic cleft are related to neural excitability, neuroplasticity, and neuronal damage associated with excitotoxicity. Mice lacking the cellular prion protein (PrP(c)) gene (Prnp) present a decreased astrocytic glutamate uptake in cultures, higher neuronal excitability in vitro and sensitivity to pro-convulsant drugs in vivo, and age-dependent memory impairment. Here, we investigate if PrP(c) might be involved in neuronal uptake and release of glutamate. For this purpose, we compared synaptosomal preparations from the cerebral cortex, entorhinal cortex, hippocampus, cerebellum, and olfactory bulb of 3- or 9-month-old PrP(c) null mice and with respective wild-type controls. Although we observed differences in synaptosomal glutamate release and uptake regarding the age of mice and the brain structure studied, these differences were similar for PrP(c) null mice and their respective wild-type controls. Therefore, despite a possible correlation between neuronal glutamate transporters, excitability, and neuronal damage, our results suggest that PrP(c) expression is not critical for neuronal glutamate transport.

Gene expression arrays in cancer research: methods and applications.

During the last 5 years, the number of papers describing data obtained by microarray technology increased exponentially with about 3000 papers in 2003. Undoubtedly, cancer is by far the disease that received most of the attention as far as the amount of data generated. As array technology is rather new and highly dependent on bioinformatics, mathematics and statistics, a clear understanding of the knowledge and information derived from array-based experiments is not widely appreciated. We shall review herein some of the issues related to the construction of DNA arrays, quantities and heterogeneity of probes and targets, the consequences of the physical characteristics of the probes, data extraction and data analysis as well as the applications of array technology. Our goal is to bring to the general audience, some of the basics of array technology and its possible application in oncology. By discussing some of the basic aspects of the methodology, we hope to stimulate criticism concerning the conclusions proposed by authors, especially in the light of the very low degree of reproducibility already proven when commercially available platforms were compared . Regardless of its pitfalls, it is unquestionable that array technology will have a great impact in the management of cancer and its applications will range from the discovery of new drug targets, new molecular tools for diagnosis and prognosis as well as for a tailored treatment that will take into account the molecular determinants of a given tumor. Hence, we shall also highlight some of the already available and promising applications of array technology on the day-to-day practice of oncology.

Human gene discovery through experimental definition of transcribed regions of the human genome.

The sequencing of the human genome has failed to realize its primary goal: the identification of all human genes. We have learned that genes can only be identified with certainty within this vast and information-sparse structure by comparison with transcript sequences. Significantly more sequence data of this kind is required before we can claim to have deciphered our genetic blueprint.

Impaired exercise capacity, but unaltered mitochondrial respiration in skeletal or cardiac muscle of mice lacking cellular prion protein.

The studies of physiological roles for cellular prion protein (PrP(c)) have focused on possible functions of this protein in the CNS, where it is largely expressed. However, the observation that PrP(c) is expressed also in muscle tissue suggests that the physiological role of PrP(c) might not be limited to the central nervous system. In the present study, we investigated possible functions of PrP(c) in muscle using PrP(c) gene (Prnp) null mice (Prnp(0/0)). For this purpose, we submitted Prnp(0/0) animals to different protocols of exercise, and compared their performance to that of their respective wild-type controls. Prnp(0/0) mice showed an exercise-dependent impairment of locomotor activity. In searching for possible mechanisms associated with the impairment observed, we evaluated mitochondrial respiration (MR) in skeletal or cardiac muscle from these mice during resting or after different intensities of exercise. Baseline MR (states 3 and 4), respiratory control ratio (RCR) and mitochondrial membrane potential (DeltaPsi) were evaluated and were not different in skeletal or cardiac muscle tissue of Prnp(0/0) mice when compared with wild-type animals. We concluded that Prnp(0/0) mice show impairment of swimming capacity, perhaps reflecting impairment of muscular activity under more extreme exercise conditions. In spite of the mitochondrial abnormalities reported in Prnp(0/0) mice, our observation seems not to be related to MR. Our results indicate that further investigations should be conducted in order to improve our knowledge about the function of PrP(c) in muscle physiology and its possible role in several different neuromuscular pathologies.

Linear mRNA amplification approach for RNAseq from limited amount of RNA.

Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)(+) mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3' end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.

Cellular prion protein: on the road for functions.

Cellular prion (PrPc) is a plasma membrane glycosyphosphatidylinositol-anchored protein present in neurons but also in other cell types. Protein conservation among species suggests that PrPc may have important physiological roles. Cellular and molecular approaches have established several novel features of the regulation of PrPc expression, cellular trafficking as well as its participation in copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling and cell survival. It is therefore likely that PrPc plays pleiotropic roles in neuronal and non-neuronal cells, and as such the loss of function of PrPc may be an important component of various diseases.

Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus.

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.

Hippocampal synaptic plasticity in mice devoid of cellular prion protein.

The cellular prion protein plays a role in the etiology of transmissible and inherited spongiform encephalopathies. However, the physiological role of the cellular prion protein is still under debate. Results regarding the synaptic transmission using the same strain of animals where the cellular prion protein gene was ablated are controversial, and need further investigation. In this work, we have studied the hippocampal synaptic transmission in mice devoid of normal cellular prion protein, and have shown that these animals present an increased excitability in this area by the lower threshold (20 Hz) to generate long-term potentiation (LTP) in hippocampal dentate gyrus when compared to wild-type animals. The mice devoid of normal cellular prion protein are also more sensitive to the blocking effects of dizocilpine and 2-amino-5-phosphonopentanoic acid on the hippocampal long-term potentiation generation. In situ hydridization experiments demonstrated overexpression of the mRNAs for the N-methyl-D-aspartate (NMDA) receptor NR2A and NR2B subunits in mice devoid of normal cellular prion protein. Therefore, our results indicate that these animals have an increased hippocampal synaptic plasticity which can be explained by a facilitated glutamatergic transmission. The higher expression of specific N-methyl-d-aspartate receptor subunits may account for these effects.

Decreased hyperlocomotion induced by MK-801, but not amphetamine and caffeine in mice lacking cellular prion protein (PrP(C)).

The cellular prion protein (PrP(C)) has been involved in several neurodegenerative disorders however it has been proposed that it is also be implicated in psychotic disorders. We investigated the effect of three psychotropic drugs in locomotor activity of PrP(C) knockout (Prnp(O/O)) and wild-type mice. The NMDA receptor channel blocker MK-801 (0.25 mg/kg), the indirect dopamine agonist amphetamine (1 mg/kg) and the adenosine receptor antagonist caffeine (10 mg/kg) were administered i.p. after 60 min of habituation and locomotion was monitored for 3 h. Prnp(O/O) mice presented a diminished hyperlocomotor response to MK-801 treatment but normal response to amphetamine and caffeine compared to wild type mice. These results suggest that lack of PrP(C) leads to a functional alteration in the glutamatergic system, whereas the regulation of both dopaminergic and adenosinergic systems are preserved. Finally, lack of PrP(C) seems not to exacerbate the response to these psychotropic drugs, which modulate neurotransmitter systems possibly involved in schizophrenia and psychotic disorders.

Cellular prion protein ablation impairs behavior as a function of age.

Cellular prion protein (PrPc) has been associated with some physiological functions in recent reports. Here we investigate behavioral parameters in 3- and 9-month-old mice lacking PrPc protein (Prnp0/0) and in rats after intrahippocampal administration of affinity purified anti-PrPc IgG (0.09 microg/side). No differences were observed between 3-month-old animals. However, 9-month-old Prnp0/0 mice and rats infused with anti-PrPc antibody showed a clear impairment of short- and long-term memory retention of a step-down inhibitory avoidance task. A decreased locomotor activity during exploration of an open field was also observed. These results suggest that systems involved in memory formation become more susceptible to mechanisms that require PrPc between the ages of 3 and 9 months in both mice and rats.

High capacity and low cost detection of prion protein gene variant alleles by denaturing HPLC.

Mutations in the human prion protein gene (PRNP) are responsible for hereditary diseases called transmissible spongiform encephalopathies (TSE) and a polymorphic site at codon 129 determines sensitivity to infectious forms of these maladies. More recently, codon 129 has been related to cognition performance in the elderly, in Alzheimer disease (AD) and in Down syndrome. Furthermore, a rare polymorphism at codon 171 was described in 23% of patients with mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS), the most common form of surgically remediable epileptic syndrome. Thus, a method that permits fast and efficient screening of PRNP mutations and polymorphisms in patients, in high risk populations, and in family members is desirable. In the present study, we established the conditions for analysis of the PRNP open reading frame using denaturing high-performance liquid chromatography (DHPLC), whereby unpurified PCR products were subjected to denaturing and reannealing steps leading to heteroduplex formation. We described specific profiles for the PRNP polymorphisms at codons 129 (M/V), 117 (A/A silent), 219 (E/K), 171 (N/S), and the octarepeat deletion using amplified DNA from 562 samples. The chromatograms for TSE-associated mutations at codons 102 (P/L), 183 (T/A), and 210 (V/I) were also determined. Specificity of the DHPLC profile for each PRNP variant allele was confirmed in 100% of the samples by direct and cloned DNA sequencing in addition to endonuclease digestion when applicable. Therefore, the present study shows that DHPLC is a rapid, highly accurate and efficient technique for the detection of PRNP genetic variants.