microRNA Expression Profiling in Young Prostate Cancer Patients

MicroRNAs (miRNAs) are small, non-coding RNA molecules with multiple roles in many biological processes. Few studies have shown the molecular characteristics in younger prostate cancer (PCa) patients. In this study, we performed miRNA profiling in young PCa (EO-PCa) cases compared with PCa arising in older men (LO-PCa).Experimental Design: Formalin-fixed, paraffin embedded tissue was used. miRNA was extracted for PCR array and NanoString methods. Relative miRNAs expression levels were obtained by comparing young vs older men, and young PCa tumor samples vs normal epithelium.Results: miRNA profiling showed a different expression pattern in PCa arising in younger men, and young PCa tumoral and its normal counterpart. Nine miRNAs (hsa-miR-140-5p, hsa-miR-146a, hsa-miR-29b, hsa-miR-9, hsa-miR-124-3p, hsa-let-7f-5p, hsa-miR-184, hsa-miR-373, hsa-miR-146b-5p) showed differences in the expression compared to LO-PCa. Fourteen miRNAs were significantly up-regulated (miR-1973, miR-663a, miR-575, miR-93-5p, miR-630, miR-600, miR-494, miR-150-5p, miR-137, miR-25-3p, miR-375, miR-489, miR-888-5p, miR-142-3p), while 9 were found down-regulated (miR-21-5p, miR-363-3p, miR-205-5p, miR-548ai, miR-3195, 145-5p, miR-143-3p, miR-222-3p, miR-221-3p) comparing young PCa tumoral tissue compared to normal counterpart. The higher expression of miR-600 and miR-137 were associated with high Gleason score, extraprostatic extension and lymphatic invasion.Conclusion: These results suggest that PCa in younger patients has a different expression profile compared to normal tissue and PCa arising in older man. Differentially expressed miRNAs provide insights of molecular mechanisms involve in this PCa subtype

Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-β signaling

<p>Abstract</p> <p>Background</p> <p>Germline mutations in the <it>FLCN </it>gene are responsible for the development of fibrofolliculomas, lung cysts and renal neoplasia in Birt-Hogg-Dube' (BHD) syndrome. The encoded protein folliculin (FLCN) is conserved across species but contains no classic motifs or domains and its function remains unknown. Somatic mutations or loss of heterozygosity in the remaining wild type copy of the <it>FLCN </it>gene have been found in renal tumors from BHD patients suggesting that <it>FLCN </it>is a classic tumor suppressor gene.</p> <p>Results</p> <p>To examine the tumor suppressor function of <it>FLCN</it>, wild-type or mutant <it>FLCN </it>(H255R) was stably expressed in a <it>FLCN-null </it>renal tumor cell line, UOK257, derived from a BHD patient. When these cells were injected into nude mice, tumor development was inversely dependent upon the level of wild-type <it>FLCN </it>expression. We identified genes that were differentially expressed in the cell lines with or without wild-type <it>FLCN</it>, many of which are involved in TGF-β signaling, including <it>TGF-β2 </it>(<it>TGFB2</it>)<it>, inhibin β A chain </it>(<it>INHBA</it>)<it>, thrombospondin 1 </it>(<it>THBS1</it>), <it>gremlin </it>(<it>GREM1</it>), and <it>SMAD3</it>. In support of the <it>in vitro </it>data, <it>TGFB2</it>, <it>INHBA</it>, <it>THBS1 </it>and <it>SMAD3 </it>expression levels were significantly lower in BHD-associated renal tumors compared with normal kidney tissue. Although receptor mediated SMAD phosphorylation was not affected, basal and maximal TGF-β-induced levels of <it>TGFB2</it>, <it>INHBA </it>and <it>SMAD7 </it>were dramatically reduced in <it>FLCN-null </it>cells compared with <it>FLCN</it>-restored cells. Secreted TGF-β2 and activin A (homo-dimer of INHBA) protein levels were also lower in <it>FLCN-null </it>cells compared with <it>FLCN</it>-restored cells. Consistent with a growth suppressive function, activin A (but not TGF-β2) completely suppressed anchorage-independent growth of <it>FLCN-null </it>UOK257 cells.</p> <p>Conclusions</p> <p>Our data demonstrate a role for <it>FLCN </it>in the regulation of key molecules in TGF-β signaling and confirm deregulation of their expression in BHD-associated renal tumors. Thus, deregulation of genes involved in TGF-β signaling by <it>FLCN </it>inactivation is likely to be an important step for tumorigenesis in BHD syndrome.</p

Inactivation of the FLCN Tumor Suppressor Gene Induces TFE3 Transcriptional Activity by Increasing Its Nuclear Localization

Germline mutations in a tumor suppressor gene FLCN lead to development of fibrofolliculomas, lung cysts and renal cell carcinoma (RCC) in Birt-Hogg-Dubé syndrome. TFE3 is a member of the MiTF/TFE transcription factor family and Xp11.2 translocations found in sporadic RCC involving TFE3 result in gene fusions and overexpression of chimeric fusion proteins that retain the C-terminal DNA binding domain of TFE3. We found that GPNMB expression, which is regulated by MiTF, was greatly elevated in renal cancer cells harboring either TFE3 translocations or FLCN inactivation. Since TFE3 is implicated in RCC, we hypothesized that elevated GPNMB expression was due to increased TFE3 activity resulting from the inactivation of FLCN.TFE3 knockdown reduced GPNMB expression in renal cancer cells harboring either TFE3 translocations or FLCN inactivation. Moreover, FLCN knockdown induced GPNMB expression in FLCN-restored renal cancer cells. Conversely, wildtype FLCN suppressed GPNMB expression in FLCN-null cells. FLCN inactivation was correlated with increased TFE3 transcriptional activity accompanied by its nuclear localization as revealed by elevated GPNMB mRNA and protein expression, and predominantly nuclear immunostaining of TFE3 in renal cancer cells, mouse embryo fibroblast cells, mouse kidneys and mouse and human renal tumors. Nuclear localization of TFE3 was associated with TFE3 post-translational modifications including decreased phosphorylation.Increased TFE3 activity is a downstream event induced by FLCN inactivation and is likely to be important for renal tumor development. This study provides an important novel mechanism for induction of TFE3 activity in addition to TFE3 overexpression resulting from Xp11.2 translocations, suggesting that TFE3 may be more broadly involved in tumorigenesis

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care

Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS.MethodsIn a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations.ResultsAll anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations.ConclusionKnowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.GENETICS in MEDICINE advance online publication, 4 January 2018; doi:10.1038/gim.2017.221

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Low VHL mRNA Expression is Associated with More Aggressive Tumor Features of Papillary Thyroid Carcinoma

Alterations of the von Hippel-Lindau (VHL) tumor suppressor gene can cause different hereditary tumors associated with VHL syndrome, but the potential role of the VHL gene in papillary thyroid carcinoma (PTC) has not been characterized. This study set out to investigate the relationship of VHL expression level with clinicopathological features of PTC in an ethnically and geographically homogenous group of 264 patients from Serbia, for the first time. Multivariate logistic regression analysis showed a strong correlation between low level of VHL expression and advanced clinical stage (OR55.78, 95% CI 3.17-10.53, P<0.0001), classical papillary morphology of the tumor (OR52.92, 95% CI 1.33-6.44, P=50.008) and multifocality (OR51.96, 95% CI 1.06-3.62, P=50.031). In disease-free survival analysis, low VHL expression had marginal significance (P=50.0502 by the log-rank test) but did not appear to be an independent predictor of the risk for chance of faster recurrence in a proportion hazards model. No somatic mutations or evidence of VHL downregulation via promoter hypermethylation in PTC were found. The results indicate that the decrease of VHL expression associates with tumor progression but the mechanism of downregulation remains to be elucidated

Phenotype and genotype of 87 patients with Mowat–Wilson syndrome and recommendations for care

Purpose: Mowat–Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype–phenotype correlations of MWS. Methods: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations. Results: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluati