Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy
for this disease. We investigated the role of mitogen-activated ...protein kinase kinase (MEK) and phosphatidylinositol 3-kinase
(PI3K) inhibitors as targeted therapies for basal-like breast cancer.
Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information
to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms
underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor
xenograft models.
Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity
to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showed that loss
of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation
of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal–regulated kinase signaling synergized
to potently impair the growth of basal-like breast cancer models in vitro and in vivo .
Conclusions: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with
intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both
intact and deleted PTEN.
A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant ...efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.
Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the ...peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Disease activity measurement is a key component of rheumatoid arthritis (RA) management. Biomarkers that capture the complex and heterogeneous biology of RA have the potential to complement clinical ...disease activity assessment.
To develop a multi-biomarker disease activity (MBDA) test for rheumatoid arthritis.
Candidate serum protein biomarkers were selected from extensive literature screens, bioinformatics databases, mRNA expression and protein microarray data. Quantitative assays were identified and optimized for measuring candidate biomarkers in RA patient sera. Biomarkers with qualifying assays were prioritized in a series of studies based on their correlations to RA clinical disease activity (e.g. the Disease Activity Score 28-C-Reactive Protein DAS28-CRP, a validated metric commonly used in clinical trials) and their contributions to multivariate models. Prioritized biomarkers were used to train an algorithm to measure disease activity, assessed by correlation to DAS and area under the receiver operating characteristic curve for classification of low vs. moderate/high disease activity. The effect of comorbidities on the MBDA score was evaluated using linear models with adjustment for multiple hypothesis testing.
130 candidate biomarkers were tested in feasibility studies and 25 were selected for algorithm training. Multi-biomarker statistical models outperformed individual biomarkers at estimating disease activity. Biomarker-based scores were significantly correlated with DAS28-CRP and could discriminate patients with low vs. moderate/high clinical disease activity. Such scores were also able to track changes in DAS28-CRP and were significantly associated with both joint inflammation measured by ultrasound and damage progression measured by radiography. The final MBDA algorithm uses 12 biomarkers to generate an MBDA score between 1 and 100. No significant effects on the MBDA score were found for common comorbidities.
We followed a stepwise approach to develop a quantitative serum-based measure of RA disease activity, based on 12-biomarkers, which was consistently associated with clinical disease activity levels.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rheumatoid arthritis (RA) is a systemic and incurable autoimmune disease characterized by chronic inflammation in synovial lining of joints. To identify the signaling pathways involved in RA, its ...disease activity, and treatment response, we adapted a systems immunology approach to simultaneously quantify 42 signaling nodes in 21 immune cell subsets (e.g., IFNα→p-STAT5 in B cells) in peripheral blood mononuclear cells (PBMC) from 194 patients with longstanding RA (including 98 patients before and after treatment), and 41 healthy controls (HC). We found multiple differences between patients with RA compared to HC, predominantly in cytokine-induced Jak/STAT signaling in many immune cell subsets, suggesting pathways that may be associated with susceptibility to RA. We also found that high RA disease activity, compared to low disease activity, was associated with decreased (e.g., IFNα→p-STAT5, IL-10→p-STAT1) or increased (e.g., IL-6→STAT3) response to stimuli in multiple cell subsets. Finally, we compared signaling in patients with established, refractory RA before and six months after initiation of methotrexate (MTX) or TNF inhibitors (TNFi). We noted significant changes from pre-treatment to post-treatment in IFNα→p-STAT5 signaling and IL-10→p-STAT1 signaling in multiple cell subsets; these changes brought the aberrant RA signaling profiles toward those of HC. This large, comprehensive functional signaling pathway study provides novel insights into the pathogenesis of RA and shows the potential of quantification of cytokine-induced signaling as a biomarker of disease activity or treatment response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular ...signalling pathways. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
Quantitative assessment of disease activity in rheumatoid arthritis (RA) is important for patient management, and additional objective information may aid rheumatologists in clinical ...decision making. We validated a recently developed multibiomarker disease activity (MBDA) test relative to clinical disease activity in diverse RA cohorts.
Methods
Serum samples were obtained from the Index for Rheumatoid Arthritis Measurement, Brigham and Women's Hospital Rheumatoid Arthritis Sequential Study, and Leiden Early Arthritis Clinic cohorts. Levels of 12 biomarkers were measured and combined according to a prespecified algorithm to generate the composite MBDA score. The relationship of the MBDA score to clinical disease activity was characterized separately in seropositive and seronegative patients using Pearson's correlations and the area under the receiver operating characteristic curve (AUROC) to discriminate between patients with low and moderate/high disease activity. Associations between changes in MBDA score and clinical responses 6–12 weeks after initiation of anti–tumor necrosis factor or methotrexate treatment were evaluated by the AUROC.
Results
The MBDA score was significantly associated with the Disease Activity Score in 28 joints using the C‐reactive protein level (DAS28‐CRP) in both seropositive (AUROC 0.77, P < 0.001) and seronegative (AUROC 0.70, P < 0.001) patients. In subgroups based on age, sex, body mass index, and treatment, the MBDA score was associated with the DAS28‐CRP (P < 0.05) in all seropositive and most seronegative subgroups. Changes in the MBDA score at 6–12 weeks could discriminate both American College of Rheumatology criteria for 50% improvement responses (P = 0.03) and DAS28‐CRP improvement (P = 0.002). Changes in the MBDA score at 2 weeks were also associated with subsequent DAS28‐CRP response (P = 0.02).
Conclusion
Our findings establish the criterion and discriminant validity of a novel multibiomarker test as an objective measure of RA disease activity to aid in the management of RA in patients with this condition.
Significant improvements in the outcome of non–small cell lung carcinoma (NSCLC) have been reported in patients treated with
the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. To ...discover biomarkers for the enrichment of patients who
might benefit from treatment, a pharmacogenomic approach was used to identify gene signatures that may predict erlotinib activity
using in vitro model systems. Erlotinib sensitivity in a panel of 42 NSCLC cell lines was determined by EGFR-mediated proliferative potential,
EGFR mutations, and/or EGFR gene amplification, thus supporting an underlying biological mechanism of receptor activation. A strong multigene signature
indicative of an epithelial to mesenchymal transition (EMT) was identified as a determinant of insensitivity to erlotinib
through both supervised and unsupervised gene expression approaches. This observation was further supported by expression
analysis of classic EMT marker proteins, including E-cadherin and vimentin. To investigate the clinical relevance of these
findings, we examined expression of the epithelial marker E-cadherin by immunohistochemistry on primary tumor samples from
subjects enrolled in a randomized NSCLC clinical trial in which erlotinib in combination with chemotherapy previously failed
to show clinical activity. The majority (75%) of the 87 subjects tested showed strong E-cadherin staining and exhibited a
significantly longer time to progression (hazard ratio, 0.37; log rank P = 0.0028) and a nonsignificant trend toward longer survival with erlotinib plus chemotherapy treatment versus chemotherapy
alone. These data support a potential role for EMT as a determinant of EGFR activity in NSCLC tumor cells and E-cadherin expression
as a novel biomarker predicting clinical activity of the EGFR inhibitor erlotinib in NSCLC patients.
Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs ...that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To evaluate a multi-biomarker disease activity (MBDA) score, a novel index based on 12 serum proteins, as a tool to guide management of RA patients.
A total of 125 patients with RA from the Behandel ...Strategieën study were studied. Clinical data and serum samples were available from 179 visits, 91 at baseline and 88 at year 1. In each serum sample, 12 biomarkers were measured by quantitative multiplex immunoassays and the concentrations were used as input to a pre-specified algorithm to calculate MBDA scores.
MBDA scores had significant correlation with DAS28-ESR (Spearman's ρ = 0.66, P < 0.0001) and also correlated with simplified disease activity index, clinical disease activity index and HAQ Disability Index (all P < 0.0001). Changes in MBDA between baseline and year 1 were also correlated with changes in DAS28-ESR (ρ = 0.55, P < 0.0001). Groups stratified by European League Against Rheumatism disease activity (DAS28-ESR ≤ 3.2, 3.2-5.1 and > 5.1) had significantly different MBDA scores (P < 0.0001) and MBDA score could discriminate ACR/EULAR Boolean remission with an area under the receiver operating characteristic curve of 0.83 (P < 0.0001).
The MBDA score reflects current clinical disease activity and can track changes in disease activity over time.
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