New Insights into the Role of Polybromo-1 in Prostate Cancer.

The human protein Polybromo-1 (PBMR1/BAF180) is a component of the SWI/SNF chromatin-remodeling complex that has been reported to be deregulated in tumors. However, its role in prostate cancer (PCa) is largely unknown. In this study, we described the PBRM1 transcriptional levels and the protein expression/localization in tissues of PCa patients and in prostatic cell lines. Increased PBRM1 mRNA levels were found in PCa samples, when compared to benign disease, and were correlated with higher Gleason score. We also verified that only the nuclear localization of PBRM1 protein is correlated with a more aggressive disease and high Prostate-Specific Antigen (PSA) levels in tissue microarrays. Intriguing expression patterns of mRNA and protein were identified in the cell lines. Although PBRM1 protein was restricted to the nuclei, in tumor cell lines in non-neoplastic cells, it was also present in vesicular-like structures that were dispersed within the cytoplasm. We knocked-down PBRM1 in the castration-resistant PCa (CRPC) cell line PC-3 and we verified that PBRM1 promotes the expression of several markers of aggressiveness, including EpCAM, TGF-ß, and N-Cadherin. Therefore, our data supported the hypothesis that PBRM1 displays a pivotal role in the promotion and maintenance of the malignant behavior of PCa, especially in CRPC.

Characterization of oligonucleotide aptamers targeting the 5'-UTR from dengue virus.

AIM: The dengue virus is responsible for a high worldwide incidence of infections, aggravated by late diagnosis, and often confused with other tropical diseases. Results/methodology: Oligonucleotide aptamers binding to the 5'-UTR from dengue virus selected after eight rounds by systematic evolution of ligands by exponential enrichment technology were analyzed by dot-blot assay and in silico prediction of secondary structures, demonstrating the presence of stem-loops that may have the potential for interaction with the viral genome, which can lead to loss of their original conformation. CONCLUSION: This is the first description of RNA aptamers against functional RNA elements of the dengue virus genome with implications for disease control, which may have potential as tools in the future of antiviral therapies and for diagnostics.

Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection.

We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.

Dynamic dialog between cytokeratin 18 and annexin A1 in breast cancer: a transcriptional disequilibrium.

Cytokeratins (CKs) constitute the cytoskeletal network and are regulated by post-translational modifications, acting not only as a mechanical support, but also in cell signaling and regulatory processes. Signaling is mediated by CK-associated proteins, such as Annexin A1 (ANXA1), a ligand of the CK18/CK8 complex. ANXA1 has a pivotal role in cellular and immunological responses, and together with CK18 have been implicated in several processes related to malignant transformation in breast cancer (BC). Our aim was to demonstrate how their interaction might be linked to BC development. We investigated transcript levels, protein expression and distribution for both targets in breast tissues of 92 patients (42 BCs and 50 benign diseases) using qPCR and immunohistochemistry, respectively. ANXA1 and CK18 mRNAs were inversely correlated, and their ratio in each TNM stage significantly differentiated BC from benign diseases (OR=5.62). These differences did not mirror tissue protein levels, but a significant dichotomous protein distribution in tumor tissues was observed, differing from the expected co-localization observed during cell homeostasis. The disequilibrium of transcriptional levels between ANXA1/CK18 and alterations in their tissue distribution are present either in initial events or tumor progression, which suggest a critical event in BC. The broken dialog between ANXA1 and CK18 in normal breast tissues may play a critical role in BC development, and together may be used as combined targets for BC diagnostics.

Prostate cancer antigen 3 (PCA3) RNA detection in blood and tissue samples for prostate cancer diagnosis.

BACKGROUND: The non-coding prostate cancer antigen 3 (PCA3) RNA is currently the most specific biomarker for prostate cancer (PCa) diagnosis. Although its clinical value has been validated in a urine assay after intensive prostatic massage, few studies have been conducted to establish its diagnostic value in the peripheral blood (PBL). The aim of the present study was to examine the PCA3 expression in blood as a diagnostic tool, and to provide an additional strategy to improve PCa diagnosis. METHODS: PCA3 transcripts were detected by RT-PCR in PBL and prostatic tissues from patients. PBL sampling also included a group of young healthy volunteers. The relationship between the PCA3 RNA detection and clinical characteristics was analyzed. RESULTS: PCA3 detection in blood presented 94% specificity and 32% sensitivity, and its combined detection in tissues significantly improved diagnostic parameters. However, PCA3 RNA detection in blood was also associated with PSA levels ≥10 ng/mL, and their combination provided a sensitivity of 60% and specificity of 93%. CONCLUSIONS: Detection of the PCA3 RNA in patients' blood is an efficient tool for PCa diagnosis because it allows a routine collection procedure, which is also supported by the ongoing screening marker, prostate-specific antigen (PSA). We propose its combined use with PSA levels ≥10 ng/mL, which improves accuracy, and prevents overdiagnosis and overtreatment.

Gene expression profile in the peripheral blood of patients with prostate cancer and benign prostatic hyperplasia.

BACKGROUND: Prostate cancer consists of multifactorial and multifocal events, generating differential gene expression in tumor cells. METHODS: The molecular profile of 14 gene expression was analyzed through cDNA array in blood samples of patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH). RESULTS: Messenger RNA from patient's blood showed significant differences between PCa and BPH groups only for the NOS3 gene, with an occurrence chance for PCa5.8-fold higher than BPH disease. CONCLUSION: The NOS3 gene expression in the patient's blood may be used as a putative biomarker for prostate cancer.

The -786T>C promoter polymorphism of the NOS3 gene is associated with prostate cancer progression.

BACKGROUND: There is no biological or epidemiological data on the association between NOS3 promoter polymorphisms and prostate cancer. The polymorphisms in the promoter region of NOS3 gene may be responsible for variations in the plasma NO, which may promote cancer progression by providing a selective growth advantage to tumor cells by angiogenic stimulus and by direct DNA damage. METHODS: This study aimed evaluating the NOS3 promoter polymorphisms by PCR-SSCP and sequencing, associating genotypes and haplotypes with NOS3 expression levels through semi-quantitative RT-PCR, and with PCA3 mRNA detection, a specific tumor biomarker, in the peripheral blood of pre-surgical samples from 177 patients; 83 PCa and 94 BPH. RESULTS: Three novel SNPs were identified -764A>G, -714G>T and -649G>A in the NOS3 gene promoter region, which together with the -786T>C generated four haplotypes (N, T, C, A). NOS3 gene expression levels were affected by the -786T>C polymorphism, and there was a 2-fold increase in NOS3 levels favored by the incorporation of each C allele. NOS3 levels higher than 80% of the constitutive gene expression level (B2M) presented a 4-fold increase in PCa occurrence. CONCLUSION: The -786T>C polymorphism was the most important promoter alteration of the NOS3 gene that may affect the PCa progression, but not its occurrence, and the incorporation of the C allele is associated with increased levels of NOS3 transcripts. The NOS3 transcript levels presented a bimodal behavior in tumor development and may be used as a biomarker together with the PCA3 marker for molecular staging of the prostate cancer.

Combined analysis of multiple mRNA markers by RT-PCR assay for prostate cancer diagnosis.

OBJECTIVES: To develop a semi-quantitative method for prostate cancer diagnosis and to validate this technique in clinical protocols with the use of multiplex RT-PCR assays for five different biomarkers associated with carcinogenesis, including the PCA3 gene. DESIGN AND METHODS: AR, SRD5A2, KLK2, PSMA, and PCA3 transcripts were analyzed by multiplex RT-PCR assay in 73 prostatic tissue samples from patients with prostate cancer (PCa) and benign hyperplasia (BPH). RESULTS: Significant differences were observed between cancerous and hypertrophic tissues in the relative expression of these genes. AR, KLK2, PSMA, and PCA3 genes displayed increased transcriptional levels in the cancer specimens; on the other hand, SRD5A2 mRNA levels were higher in the BPH samples. CONCLUSIONS: Our results suggest that the most promising marker for PCa diagnosis was positive PCA3 detection associated with serum PSA levels, which showed 28-fold higher chances for cancer occurrence, with 92% specificity and 94% positive predictive value.

The endothelial nitric oxide synthase Glu-298-Asp polymorphism and its mRNA expression in the peripheral blood of patients with prostate cancer and benign prostatic hyperplasia.

BACKGROUND: The endothelial nitric oxide synthase (ecNOS) has an important role in vascular development and in the carcinogenesis process of prostate cancer (PCa). The nitric oxide (NO) production may promote cancer progression by providing a selective growth advantage to tumor cells, by angiogenic stimulus and by direct DNA damage. METHODS: The present study aimed at evaluating the ecNOS Glu-298-Asp polymorphism by the PCR-RFLP technique, associating genotypes with gene expression levels and the tumor biomarker, Prostate Cancer Antigen (DD3), through semi-quantitative RT-PCR. Pre-surgical peripheral blood samples from 160 patients were analyzed: 84 PCa, 11 prostate intraepithelial neoplasia (PIN) and 65 benign prostatic hyperplasia (BPH). RESULTS: The GG and GT Glu-298-Asp genotypes were associated with positive DD3 expression in the peripheral blood, presenting a 3.32-fold higher risk of PCa occurrence. There was no association between genotypes and ecNOS mRNA expression levels; however, the presence of the G allele is closely related to the hematogenous dissemination event of tumoral cells, as evidenced by the DD3 positivity. The higher G allele frequency among pT3 and pT4 staged PCa patients suggests that this would be associated with advanced phenotypes of the disease and may also be contributing to higher NO levels, causing cancer progression. CONCLUSIONS: The G allele may have a secondary influence on the prostate cancer predisposition, but an essential role on the event of tumor cells hematogenous dissemination, probably due to the angiogenic stimulus.

Differential expression of the KLK2 and KLK3 genes in peripheral blood and tissues of patients with prostate cancer

We used the multiplex semi-quantitative reverse-transcriptase PCR (RT-PCR) to investigate kallikrein 2 and 3 (KLK2 and KLK3) mRNA levels in prostate tissue from 42 prostate cancer patients, 33 of whom were also assessed for peripheral blood KLK2 expression by qualitative semi-nested RT-PCR. We found that KLK2 was an important tissue biomarker for distinguishing between prostate cancer patients and those with benign prostatic hyperplasia, particularly when KLK2 expression was > 60 percent of that of the beta2-microglobulin constitutive gene. Patients with an average relative expression value > 0.6 (cutoff value) had an eleven-fold higher chance of having prostate cancer. When one or two tissues samples were evaluated for KLK2 expression using the cutoff value the estimated chance of having prostate cancer was increased by seven times for one positive sample and 45 times for two positive samples. There was no significant correlation between KLK3 gene expression and prostate cancer diagnosis. Logistic regression for blood and tissue KLK2 expression successfully detected 92 percent of the prostate cancer cases. The detection of KLK2 in blood showed a sensitivity of 59 percent and a specificity of 82 percent. This study indicates that the KLK2 gene may be a useful molecular marker for the diagnosis of prostate cancer and that analysis of KLK2 expression in blood and tissues could provide a novel approach for the clinical investigation of this type of cancer.