Publications by authors named "J Ramalho-Carvalho"

26 Publications

Circulating MicroRNAs as Biomarkers for Prostate Cancer Detection and Metastasis Development Prediction.

Front Oncol 2019 11;9:900. Epub 2019 Sep 11.

Cancer Biology and Epigenetics Group, IPO Porto Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal.

Prostate Cancer (PCa) overdiagnosis and overtreatment, as a consequence of the limited specificity of current detection and prognostication methods, remains a major challenge in clinical practice. Therefore, development and validation of new molecular biomarkers amenable of detecting clinically significant disease is crucial. MicroRNAs (miRNA) deregulation is common in cancer, constituting potential non-invasive biomarkers for PCa detection and prognostication. Herein, we evaluated the screening and prognostic biomarker potential of two onco-microRNAs (miR-182-5p and miR-375-3p) in liquid biopsies (plasma) of PCa patients with clinically localized disease undergoing curative-intent treatment. A first cohort of 98 PCa and 15 normal prostates were used to assess PCa-specificity of miR-182-5p in tissues. A cohort composed of PCa 252 patients and 52 asymptomatic controls allowed for assessment of diagnostic and prognostic value in plasmas. After RNA extraction from tissue and plasma samples, cDNA synthesis specific for miRNAs was performed followed by measurement of miR-182-5p and miR-375-3p relative expression by RT-qPCR, using U6 snRNA gene as reference. MiR-182-5p was significantly overexpressed in PCa tissues ( < 0.0001) and in plasma of PCa patients ( = 0.0020), compared to respective controls. Moreover, miR-182-5p expression identified PCa with AUC = 0.81 (95% CI: 0.725-0.892, = 0.0001) in tissue and with 77% specificity and 99% NPV (AUC = 0.64, 95% CI: 0.561-0.709, = 0.0021) in plasma. Both circulating miR-182-5p and miR-375-3p levels associated with more advanced pathologic stage and the former was significantly higher in patients that developed metastasis ( = 0.0145). Indeed, at the time of diagnosis, circulating miR-375-3p levels predicted which patients would develop metastasis, with almost 50% sensitivity, 76% specificity, and a NPV of 89% (AUC = 0.62, 95% CI: 0.529-0.713, = 0.0149). We conclude that these two circulating miRNAs might be clinical useful as non-invasive biomarkers for detection and prediction of metastasis development at the diagnosis together with clinical variables used in routine practice.
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September 2019

Histone variant MacroH2A1 is downregulated in prostate cancer and influences malignant cell phenotype.

Cancer Cell Int 2019 29;19:112. Epub 2019 Apr 29.

Cancer Biology & Epigenetics Group, Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Research Center-LAB 3, F Bdg., 1st Floor, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal.

Background: Prostate cancer (PCa), a major cause of cancer-related morbidity and mortality worldwide and mostly asymptomatic at earliest stages, is characterized by disruption of genetic and epigenetic balance. A better understanding of how those mechanisms orchestrate disease might improve diagnostic and prognostic tools, allowing for improvements in treatment efficacy. Replacement of canonical histones, an epigenetic mechanism, is highly conserved among species and altered expression of histones variants (e.g., MacroH2A1) has been associated with tumorigenesis. H2AFY gene encodes two isoforms of H2A histone variant MacroH2A1: MacroH2A1.1 and MacroH2A1.2. Specifically, MacroH2A1.1 isoform inhibits cell proliferation and promotes cellular differentiation. Because the contribution of this histone variant to carcinogenesis has been reported in several cancer types, but not for PCa, we aimed to investigate the contribution of MacroH2A1 for prostate carcinogenesis.

Methods: MacroH2A1, MacroH2A1.1 and MacroH2A1.2 isoforms and the corresponding splicing regulators transcript levels were evaluated by RT-qPCR, in a tissue cohort composed by PCa, prostatic intraepithelial neoplasia (PIN) and normal prostate cases. Knockdown for MacroH2A1 and MacroH2A1.1 was performed through lentiviral transduction in DU145 cells, and MacroH2A1.1 overexpression was achieved in LNCaP cells by plasmid transfection, followed by functional assays. Biological and/or experimental replicates were performed when necessary, and specific statistical tests were applied to perform data analysis.

Results: MacroH2A1.1 transcript levels were downregulated in PIN and primary PCa compared to normal prostate tissues. The same was found for QKI, a MacroH2A1.1's splicing regulator. Moreover, lower MacroH2A1.1 and QKI expression levels associated with less differentiated tumors (Gleason score ≥ 7). Interestingly, MacroH2A1.1, but more impressively DDX17 (AUC = 0.93; p < 0.0001) and QKI (AUC = 0.94; p < 0.0001), accurately discriminated cancerous from noncancerous prostate tissues. Furthermore, in PCa cell lines, total MacroH2A1 knockdown augmented malignant features, whereas MacroH2A1.1 overexpression impressively attenuated the malignant phenotype.

Conclusions: Overall, our data, derived from primary PCa tissues and cell lines, anticipate a tumor suppressive role for MacroH2A1, particularly for the MacroH2A1.1 isoform, in prostate carcinogenesis.
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April 2019

Hybrid oncocytic/chromophobe renal cell tumor: An integrated genetic and epigenetic characterization of a case.

Exp Mol Pathol 2018 12 18;105(3):352-356. Epub 2018 Oct 18.

Department of Pathology, Portuguese Oncology Institute of Porto, Portugal; Cancer Biology & Epigenetics Group, Research Center, Portuguese Oncology Institute of Porto, Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Portugal. Electronic address:

Introduction: Hybrid oncocytic/chromophobe tumor (HOCT) is a renal cell neoplasm displaying overlapping cellular and architectural features of both renal oncocytoma (RO) and chromophobe renal cell carcinoma (chRCC). It has been described in the context of oncocytosis, Birt-Hogg-Dubé syndrome, and also sporadically. Thus far, HOCT immunohischemical profile and cytogenetic alterations have been reported, but not epigenetic alterations. Herein, we characterize a HOCT case, including microRNA expression, comparing it to sporadic RO and chRCC.

Methods: An HOCT was entirely submitted. Representative paraffin blocks were selected for histochemical, immunohistochemical and FISH analyses. MicroRNAs were extracted from the two components separately and selected microRNA expression was performed.

Results: This 4 cm HOCT, from a 69 year-old female, was composed mainly by oncocytic cells with an insular distribution (RO-like) and areas of larger clarified cells (chRCC-like). The two areas displayed different features: RO-like areas showed negative colloidal iron staining, multifocal CD15, negative CK7, focal multiple tetrasomies, and higher miR21 expression; chRCC-like areas showed colloidal iron diffuse staining with moderate intensity, focal CD15 and CK7, no numeric chromosomic alterations, and higher miR141 and miR200b expression.

Conclusion: microRNA expression in the two HOCT components is similar to its sporadic tumors counterparts. Morphologic, immunohistochemical, cytogenetic and epigenetic data on this case suggest either two independent pathogenic pathways or an early pathogenic divergence for RO-like and chRCC-like components of HOCT.
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December 2018

A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting .

Clin Epigenetics 2018 27;10:40. Epub 2018 Mar 27.

1Cancer Biology & Epigenetics Group - Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), F Bdg, 1st floor, Rua Dr António Bernardino de Almeida, 4200-072 Porto, Portugal.

Background: Prostate cancer (PCa) is a major cause of morbidity and mortality in men worldwide. MicroRNAs are globally downregulated in PCa, especially in poorly differentiated tumors. Nonetheless, the underlying mechanisms are still elusive. Herein, using combined analysis of microRNAs expression and genomewide DNA methylation, we aimed to identify epigenetically downregulated microRNAs in PCa.

Results: We found that miR-152-3p was underexpressed in PCa and that lower expression levels were associated with promoter hypermethylation in accordance with TCGA dataset analysis. Functional in vitro assays suggest that miR-152-3p suppresses cell viability and invasion potential, whereas it promotes cell cycle arrest at S and G2/M phases. Additionally, miR-152-3p expression was associated with longer disease-free survival in PCa patients from TCGA. Finally, , which is overexpressed in PCa, was identified as a novel miR-152-3p target gene.

Conclusions: Our findings demonstrate the advantages of using a combinatory approach to identify microRNAs downregulated due to aberrant promoter methylation. MiR-152-3p downregulation and promoter methylation was found to be prevalent in primary PCa, which impairs its role in control of cell viability, cell cycle regulation and invasion.
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February 2019

Methylation-Specific PCR.

Methods Mol Biol 2018 ;1708:447-472

Cancer Biology and Epigenetics Group; Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal.

Cytosine methylation is a DNA modification generally associated with transcriptional silencing. Factors that regulate methylation have been linked to human disease, yet how they contribute to malignancies remains largely unknown. Methylation of DNA can change the functional state of regulatory regions, but does not change the Watson-Crick base pairing of cytosine. Moreover, sequence symmetry of CpGs enables propagation of the methyl mark through cell division. This potential for inheritance coupled with the fact that DNA methylation patterns change during development and disease partially explains the interest in DNA methylation as a memory module. DNA methylation analysis also provides an opportunity to discover new and more powerful biomarkers that can help in clinical practice.Methylation-Specific PCR (MSP) is likely the most widely used technique to study DNA methylation of a locus of interest. MSP can rapidly detect the methylation status of any group of CpG sites within a CpG island, not requiring methylation-sensitive restriction enzymes. It also requires minute amounts of DNA, is very sensitive as it can detect <0.1% of methylated alleles in a specific locus, and can be used in different samples, including bodily fluids, and paraffin-embedded samples.
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July 2018