Juan Fullá

Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche

The different prostate cancer (PCa) cell populations (bulk and cancer stem cells, CSCs) release exosomes that contain miRNAs that could modify the local or premetastatic niche. The analysis of the differential expression of miRNAs in exosomes allows evaluating the differential biological effect of both populations on the niche, and the identification of potential biomarkers and therapeutic targets. Five PCa primary cell cultures were established to originate bulk and CSCs cultures. From them, exosomes were purified by precipitation for miRNAs extraction to perform a comparative profile of miRNAs by next generation sequencing in an Illumina platform. 1839 miRNAs were identified in the exosomes. Of these 990 were known miRNAs, from which only 19 were significantly differentially expressed: 6 were overexpressed in CSCs and 13 in bulk cells exosomes. miR-100-5p and miR-21-5p were the most abundant miRNAs. Bioinformatics analysis indicated that differentially expressed miRNAs are highly related with PCa carcinogenesis, fibroblast proliferation, differentiation and migration, and angiogenesis. Besides, miRNAs from bulk cells affects osteoblast differentiation. Later, their effect was evaluated in normal prostate fibroblasts (WPMY-1) where transfection with miR-100-5p, miR-21-5p and miR-139-5p increased the expression of metalloproteinases (MMPs) -2, -9 and -13 and RANKL and fibroblast migration. The higher effect was achieved with miR21 transfection. As conclusion, miRNAs have a differential pattern between PCa bulk and CSCs exosomes that act collaboratively in PCa progression and metastasis. The most abundant miRNAs in PCa exosomes are interesting potential biomarkers and therapeutic targets.

Increased SNAIL expression and low syndecan levels are associated with high Gleason grade in prostate cancer

Prostate cancer (PC) is a leading male oncologic malignancy wideworld. During malignant transformation, normal epithelial cells undergo genetic and morphological changes known as epithelial-mesenchymal transition (EMT). Several regulatory genes and specific marker proteins are involved in PC EMT. Recently, syndecans have been associated with malignancy grade and Gleason score in PC. Considering that SNAIL is mainly a gene repressor increased in PC and that syndecan promoters have putative binding sites for this repressor, we propose that SNAIL might regulate syndecan expression during PC EMT. The aim of this study was to analyze immunochemically the expression of SNAIL, syndecans 1 and 2 and other EMT markers in a tissue microarray (TMA) of PC samples and PC cell lines. The TMAs included PC samples of different Gleason grade and benign prostatic hyperplasia (BPH) samples, as non-malignant controls. PC3 and LNCaP cell lines were used as models of PC representing different tumorigenic capacities. Semi-quantitative immunohistochemistry was performed on TMAs and fluorescence immunocytochemistry and western blot analysis were conducted on cell cultures. Results show that SNAIL exhibits increased expression in high Gleason specimens compared to low histological grade and BPH samples. Accordingly, PC3 cells show higher SNAIL expression levels compared to LNCaP cells. Conversely, syndecan 1, similarly to E-cadherin (a known marker of EMT), shows a decreased expression in high Gleason grades samples and PC3 cells. Interestingly, syndecan 2 shows no changes associated to histological grade. It is concluded that increased SNAIL levels in advanced PC are associated with low expression of syndecan 1. The mechanism by which SNAIL regulates the expression of syndecan 1 remains to be investigated.

Manejo endoscópico de mucoceles de senos paranasales: experiencia en 46 pacientes

OBJECTIVE The purpose of this study was to determine changes in the surgical treatment of patients with the diagnosis of paranasal mucoceles managed in a Latin American hospital. We hypothesised that endonasal endoscopic surgeries had emerged as the main treatment option for this disease in the last five years. METHODS A retrospective chart review of all patients who were diagnosed with paranasal sinus mucoceles and treated at the Otorhinolaryngology Head and Neck Department of our hospital from 2002 to 2010 was performed. Patient demographic data, mucoceles location, symptoms, surgical approach and complications were recorded. RESULTS A total of 46 patients were included (27 males; 19 females). This series include 29 patients (63%) with frontal or frontoethmoidal mucoceles, 14 (30.4%) with maxillary and 3 (6.5%) with sphenoid mucoceles. Ninety-five percent of the patients were treated with intranasal endoscopic surgery. Complications occurred only in 7 cases (15.2%). CONCLUSIONS This study confirms that over the last 9 years significant changes have occurred in the surgical treatment of paranasal mucocele in our hospital, as endoscopic surgeries increased from 34% to over 90% as the first option of treatment for mucoceles.

Altered expression patterns of syndecan-1 and -2 predict biochemical recurrence in prostate cancer

The clinical features of prostate cancer do not provide an accurate determination of patients undergoing biochemical relapse and are therefore not suitable as indicators of prognosis for recurrence. New molecular markers are needed for proper pre-treatment risk stratification of patients. Our aim was to assess the value of altered expression of syndecan-1 and -2 as a marker for predicting biochemical relapse in patients with clinically localized prostate cancer treated by radical prostatectomy. The expression of syndecan-1 and -2 was examined by immunohistochemical staining in a series of 60 paraffin-embedded tissue samples from patients with localized prostate cancer. Ten specimens from patients with benign prostatic hyperplasia were used as non-malignant controls. Semiquantitative analysis was performed to evaluate the staining patterns. To investigate the prognostic value, Kaplan-Meier survival curves were performed and compared by a log-rank test. In benign samples, syndecan-1 was expressed in basal and secretory epithelial cells with basolateral membrane localisation, whereas syndecan-2 was expressed preferentially in basal cells. In prostate cancer samples, the expression patterns of both syndecans shifted to granular-cytoplasmic localisation. Survival analysis showed a significant difference (P < 0.05) between normal and altered expression of syndecan-1 and -2 in free prostate-specific antigen recurrence survival curves. These data suggest that the expression of syndecan-1 and -2 can be used as a prognostic marker for patients with clinically localized prostate cancer, improving the prostate-specific antigen recurrence risk stratification.

Abstract 1562: miRNAs in exosomes from prostate cancer cells modify normal fibroblasts and osteoblasts favoring tumor spread and metastasis

Introduction: Exosomes from bulk and cancer stem cells (CSCs) from prostate cancer primary cultures present a differential pattern of miRNAs. The most abundant miRNA in all exosomes is miR-100, while miR-21 and miR-139 were the most abundant differentially expressed miRNAs in bulk and CSCs exosomes, respectively. Bioinformatics analysis suggested that these miRNAs were related with tumor progression. The functional effect of these miRNAs on targets related with changes at primary and pre-metastatic microenvironment was evaluated in prostatic stromal cells and osteoblasts. Methods: Normal human undifferentiated osteoblasts (hFOB1.19) and prostate fibroblasts (WPMY-1) cell lines were transfected, by separated, with 25 nM of the following miRNas: miR-100-5p, miR-21-5p, miR-139-5p. let7c was included as transfection control. After 48 hours, changes in the expression of metalloproteinases (MMP)-2, -9 and-13, RANKL, OPG and IL-8 at mRNAs and protein levels were evaluated by qPCR and western blot, respectively. As transfection control, expression of specific targets previously described for each miRNA was evaluated. Besides, the effect of miRNAs in fibroblast migration was evaluated by transwell assay. Results: In fibroblasts, transfection with miR-21, -100 and -139 increased significantly expression of RANKL, IL8 and all MMPs, with a higher effect in MMP-9. Besides, miR-21 increased fibroblast migration. In osteoblasts, transfection with miRNAs changed the balance that triggers vicious circle at the bone, increasing RANKL and diminishing OPG expression. miRNAs also increased MMPs expression. The greater effect was observed with miR-21 transfection. Conclusions: Through exosomes, prostate cancer cells recruit normal cells to favor their growth and spread. miRNAs in exosomes increase expression of proteins (in particular, MMPs and RANKL) and cell migration of fibroblasts that create a favorable microenvironment for tumor progression at the primary niche. Besides, through bloodstream, tumoral exosomes can reach the bone, preparing the premetastatic niche. miRNAs from these exosomes change the balance RANKL/OPG that triggers osteoclast recruitment facilitating metastases by activation of the bone vicious circle. miR-21, overexpressed in bulk cells, the main cellular component of tumor, has the higher effect in modify microenvironment, being a potential therapeutic target. (FONDECYT 11121525 and 1140417). Citation Format: Eliana Andahur, Enrique A. Castellon, Chistian Ramos, Juan A. Fulla, Catherine A. Sanchez. miRNAs in exosomes from prostate cancer cells modify normal fibroblasts and osteoblasts favoring tumor spread and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1562.