Druggable Nucleolin Identifies Breast Tumours Associated with Poor Prognosis That Exhibit Different Biological Processes

Background: Nucleolin (NCL) is a multifunctional protein with oncogenic properties. Anti-NCL drugs show strong cytotoxic effects, including in triple-negative breast cancer (TNBC) models, and are currently being evaluated in phase II clinical trials. However, few studies have investigated the clinical value of NCL and whether NCL stratified cancer patients. Here, we have investigated for the first time the association of NCL with clinical characteristics in breast cancers independently of the different subtypes. Methods: Using two independent series (n = 216; n = 661), we evaluated the prognostic value of NCL in non-metastatic breast cancers using univariate and/or multivariate Cox-regression analyses. Results: We reported that NCL mRNA expression levels are markers of poor survivals independently of tumour size and lymph node invasion status (n = 216). In addition, an association of NCL expression levels with poor survival was observed in TNBC (n = 40, overall survival (OS) p = 0.0287, disease-free survival (DFS) p = 0.0194). Transcriptomic analyses issued from The Cancer Genome Atlas (TCGA) database (n = 661) revealed that breast tumours expressing either low or high NCL mRNA expression levels exhibit different gene expression profiles. These data suggest that tumours expressing high NCL mRNA levels are different from those expressing low NCL mRNA levels. Conclusions: NCL is an independent marker of prognosis in breast cancers. We anticipated that anti-NCL is a promising therapeutic strategy that could rapidly be evaluated in high NCL-expressing tumours to improve breast cancer management

Ribosomal RNA 2′O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer

International audienceRecent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2′O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2′O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2′O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2′O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2′O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2′O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2′O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer

Low level of Fibrillarin, a ribosome biogenesis factor, is a new independent marker of poor outcome in breast cancer

International audienceBackground: A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. Methods: Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levelsrelated breast tumours. Results: We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. Conclusion: Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper-but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors

Disability, fatigue, pain and their associates in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study

Objectives; Our aim was to describe the burden of early dcSSc in terms of disability, fatigue and pain in the European Scleroderma Observational Study cohort, and to explore associated clinical features. Methods; Patients completed questionnaires at study entry, 12 and 24 months, including the HAQ disability index (HAQ-DI), the Cochin Hand Function Scale (CHFS), the Functional Assessment of Chronic Illness Therapy-fatigue and the Short Form 36 (SF36). Associates examined included the modified Rodnan skin score (mRSS), current digital ulcers and internal organ involvement. Correlations between 12-month changes were also examined. Results; The 326 patients recruited (median disease duration 11.9 months) displayed high levels of disability [mean (S.D.) HAQ-DI 1.1 (0.83)], with ‘grip’ and ‘activity’ being most affected. Of the 18 activities assessed in the CHFS, those involving fine finger movements were most affected. High HAQ-DI and CHFS scores were both associated with high mRSS (ρ = 0.34, P < 0.0001 and ρ = 0.35, P < 0.0001, respectively). HAQ-DI was higher in patients with digital ulcers (P = 0.004), pulmonary fibrosis (P = 0.005), cardiac (P = 0.005) and muscle involvement (P = 0.002). As anticipated, HAQ-DI, CHFS, the Functional Assessment of Chronic Illness Therapy and SF36 scores were all highly correlated, in particular the HAQ-DI with the CHFS (ρ = 0.84, P < 0.0001). Worsening HAQ-DI over 12 months was strongly associated with increasing mRSS (ρ = 0.40, P < 0.0001), decreasing hand function (ρ = 0.57, P < 0.0001) and increasing fatigue (ρ = −0.53, P < 0.0001). Conclusion; The European Scleroderma Observational Study highlights the burden of disability in early dcSSc, with high levels of disability and fatigue, associating with the degree of skin thickening (mRSS). Impaired hand function is a major contributor to overall disability

Treatment outcome in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study (ESOS).

OBJECTIVES: The rarity of early diffuse cutaneous systemic sclerosis (dcSSc) makes randomised controlled trials very difficult. We aimed to use an observational approach to compare effectiveness of currently used treatment approaches. METHODS: This was a prospective, observational cohort study of early dcSSc (within three years of onset of skin thickening). Clinicians selected one of four protocols for each patient: methotrexate, mycophenolate mofetil (MMF), cyclophosphamide or 'no immunosuppressant'. Patients were assessed three-monthly for up to 24 months. The primary outcome was the change in modified Rodnan skin score (mRSS). Confounding by indication at baseline was accounted for using inverse probability of treatment (IPT) weights. As a secondary outcome, an IPT-weighted Cox model was used to test for differences in survival. RESULTS: Of 326 patients recruited from 50 centres, 65 were prescribed methotrexate, 118 MMF, 87 cyclophosphamide and 56 no immunosuppressant. 276 (84.7%) patients completed 12 and 234 (71.7%) 24 months follow-up (or reached last visit date). There were statistically significant reductions in mRSS at 12 months in all groups: -4.0 (-5.2 to -2.7) units for methotrexate, -4.1 (-5.3 to -2.9) for MMF, -3.3 (-4.9 to -1.7) for cyclophosphamide and -2.2 (-4.0 to -0.3) for no immunosuppressant (p value for between-group differences=0.346). There were no statistically significant differences in survival between protocols before (p=0.389) or after weighting (p=0.440), but survival was poorest in the no immunosuppressant group (84.0%) at 24 months. CONCLUSIONS: These findings may support using immunosuppressants for early dcSSc but suggest that overall benefit is modest over 12 months and that better treatments are needed. TRIAL REGISTRATION NUMBER: NCT02339441

A Novel Role of SMG1 in Cholesterol Homeostasis That Depends Partially on p53 Alternative Splicing

SIMPLE SUMMARY: p53 isoforms have been reported in various tumor types. Both p53β and p53γ were recently reported to retain functionalities of full-length p53α. A role for p53 and p53 loss in cholesterol metabolism has also emerged. We show that SMG1, a phosphatidylinositol 3-kinase-related kinase, when inhibited in p53 wild-type MCF7 and HepG2 cells, significantly alters the expression of cholesterol pathway genes, with a net increase in intracellular cholesterol and an increased sensitivity to Fatostatin in MCF7. We confirm a prior report that SMG1 inhibition in MCF7 cells promotes expression of p53β and show the first evidence for increases in p53γ. Further, induced p53β expression, confirmed with antibody, explained the loss of SMG1 upregulation of the ABCA1 cholesterol exporter where p53γ had no effect on ABCA1. Additionally, upregulation of ABCA1 upon SMG1 knockdown was independent of upregulation of nonsense-mediated decay target RASSF1C, previously suggested to regulate ABCA1 via a “RASSF1C-miR33a-ABCA1” axis. ABSTRACT: SMG1, a phosphatidylinositol 3-kinase-related kinase (PIKK), essential in nonsense-mediated RNA decay (NMD), also regulates p53, including the alternative splicing of p53 isoforms reported to retain p53 functions. We confirm that SMG1 inhibition in MCF7 tumor cells induces p53β and show p53γ increase. Inhibiting SMG1, but not UPF1 (a core factor in NMD), upregulated several cholesterol pathway genes. SMG1 knockdown significantly increased ABCA1, a cholesterol efflux pump shown to be positively regulated by full-length p53 (p53α). An investigation of RASSF1C, an NMD target, increased following SMG1 inhibition and reported to inhibit miR-33a-5p, a canonical ABCA1-inhibiting miRNA, did not explain the ABCA1 results. ABCA1 upregulation following SMG1 knockdown was inhibited by p53β siRNA with greatest inhibition when p53α and p53β were jointly suppressed, while p53γ siRNA had no effect. In contrast, increased expression of MVD, a cholesterol synthesis gene upregulated in p53 deficient backgrounds, was sensitive to combined targeting of p53α and p53γ. Phenotypically, we observed increased intracellular cholesterol and enhanced sensitivity of MCF7 to growth inhibitory effects of cholesterol-lowering Fatostatin following SMG1 inhibition. Our results suggest deregulation of cholesterol pathway genes following SMG1 knockdown may involve alternative p53 programming, possibly resulting from differential effects of p53 isoforms on cholesterol gene expression

Comparison of numerical simulations to experiments for atomization in a jet nebulizer.

The development of jet nebulizers for medical purposes is an important challenge of aerosol therapy. The performance of a nebulizer is characterized by its output rate of droplets with a diameter under 5 µm. However the optimization of this parameter through experiments has reached a plateau. The purpose of this study is to design a numerical model simulating the nebulization process and to compare it with experimental data. Such a model could provide a better understanding of the atomization process and the parameters influencing the nebulizer output. A model based on the Updraft nebulizer (Hudson) was designed with ANSYS Workbench. Boundary conditions were set with experimental data then transient 3D calculations were run on a 4 µm mesh with ANSYS Fluent. Two air flow rate (2 L/min and 8 L/min, limits of the operating range) were considered to account for different turbulence regimes. Numerical and experimental results were compared according to phenomenology and droplet size. The behavior of the liquid was compared to images acquired through shadowgraphy with a CCD Camera. Three experimental methods, laser diffractometry, phase Doppler anemometry (PDA) and shadowgraphy were used to characterize the droplet size distributions. Camera images showed similar patterns as numerical results. Droplet sizes obtained numerically are overestimated in relation to PDA and diffractometry, which only consider spherical droplets. However, at both flow rates, size distributions extracted from numerical image processing were similar to distributions obtained from shadowgraphy image processing. The simulation then provides a good understanding and prediction of the phenomena involved in the fragmentation of droplets over 10 µm. The laws of dynamics apply to droplets down to 1 µm, so we can assume the continuity of the distribution and extrapolate the results for droplets between 1 and 10 µm. So, this model could help predicting nebulizer output with defined geometrical and physical parameters

PDA setup (Dantec).

<p>The setup described corresponds to the Dantec Dynamics PDA system. The nebulizer is placed at the crossing of the two emitted laser beams. The receiver is placed at 70° off-axis to analyze the interference fringes and extract the size distribution.</p

Particle size distributions at 8/min.

<p>The size distributions obtained with the numerical results and the experimental methods at 8/min are represented. These are number distributions with a size range going from 5 to 100 µm. Numerical and Shadowgraphy results are similar whereas Laser Diffraction and PDA provide much smaller diameter values.</p