Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naive cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGF beta/SMAD (transforming growth factor-beta/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naive cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.Fapesp-grant number 2012/04194-1, 2013/05172-4, 2014/24400-0 and 2015/10821-7, CNPq-grant number 150447/2013-2 and 471512/2013-3 and PRODOC-grant no 3193-32/2010. Work in the lab of KS Smalley was supported by the National Institutes of Health grants R01 CA161107, R21 CA198550, and Skin SPORE grant P50 CA168536info:eu-repo/semantics/publishedVersio

Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Serratia marcescens bacteraemia outbreak in haemodialysis patients with tunnelled catheters due to colonisation of antiseptic solution. Experience at 4 hospitals

Introduction: The application of antiseptic solution for handling tunnelled catheters is recommended in patients undergoing haemodialysis. These routine antiseptic procedures in handling catheters are crucial to avoid complications. We report an outbreak of Serratia marcescens (S. marcescens) bacteraemia in numerous haemodialysis units of the Community of Madrid.Material and methods: The first cases of bacteraemia due to S. marcescens were isolated in December 2014. The Preventive Medicine Services were informed of the detection of an atypical pathogen in several patients, suspecting a probable nosocomial outbreak. Information from 4 centres with similar S. marcescens bacteraemia was analysed.Results: "Twenty-one cases of bacteraemia related to S. marcescens were identified. The mean age of affected patients was 72 +/- 10 years. The mean time on haemodialysis of affected patients was 33 +/- 13 months (range: 3-83 months), the median time of tunnelled catheter was 22 +/- 13 months. In 11 cases the clinical picture was similar, with hypotension and general malaise during the haemodialysis session. Fever was present in a further 7 cases. In 3 cases the presentation was asymptomatic and was detected by blood cultures. All patients had tunnelled catheters (12 patients with catheter in the right jugular vein, 5 in the left jugular, 2 in the right femoral artery and 2 in the left subclavian artery). Gentamicin intravenous doses (1 mg/kg) with catheter lock solution with ciprofloxacin post-dialysis were administered for 3 weeks in 6 patients. In 12 patients the treatment was ceftazidime (2 g IV) plus catheter lock solution with the same antibiotic, for 2 weeks. Four patients received oral ciprofloxacin for 2 weeks, in one case together with IV vancomycin. The patients were asymptomatic and without new episodes 48 hours after the treatment. No major complications were observed. The teams informed the health authorities of the situation, which then reported the presence of batches of antiseptic (chlorhexidine 0.05 and 2%) colonised by S. marcescens. Given the routine application of this antiseptic in handling catheters at these units, this was considered the source of contagion and new cases were not observed after the removal of the batches.Conclusions: The presence of bacteraemia due to unconventional germs should alert us to a potential outbreak. The application of a solution contaminated by S. marcescens in haemodialysis catheters was the source of bacteraemia. The intravenous antibiotic treatment and the catheter lock solution allowed an excellent survival of patients and catheters. (C) 2016 Sociedad Espanola de Nefrologia. Published by Elsevier Espana, S.L.U