Proteomic analysis of Alzheimer's disease cerebrospinal fluid reveals alterations associated with APOE ε4 and atomoxetine treatment.

Alzheimer's disease (AD) is currently defined by the aggregation of amyloid-ß (Aß) and tau proteins in the brain. Although biofluid biomarkers are available to measure Aß and tau pathology, few markers are available to measure the complex pathophysiology that is associated with these two cardinal neuropathologies. Here, we characterized the proteomic landscape of cerebrospinal fluid (CSF) changes associated with Aß and tau pathology in 300 individuals using two different proteomic technologies-tandem mass tag mass spectrometry and SomaScan. Integration of both data types allowed for generation of a robust protein coexpression network consisting of 34 modules derived from 5242 protein measurements, including disease-relevant modules associated with autophagy, ubiquitination, endocytosis, and glycolysis. Three modules strongly associated with the apolipoprotein E ε4 (APOE ε4) AD risk genotype mapped to oxidant detoxification, mitogen-associated protein kinase signaling, neddylation, and mitochondrial biology and overlapped with a previously described lipoprotein module in serum. Alterations of all three modules in blood were associated with dementia more than 20 years before diagnosis. Analysis of CSF samples from an AD phase 2 clinical trial of atomoxetine (ATX) demonstrated that abnormal elevations in the glycolysis CSF module-the network module most strongly correlated to cognitive function-were reduced by ATX treatment. Clustering of individuals based on their CSF proteomic profiles revealed heterogeneity of pathological changes not fully reflected by Aß and tau.

MicroRNA-190b Targets RFWD3 in Estrogen Receptor-Positive Breast Cancer.

Background: In the year 2020, breast cancer was the most common form of cancer worldwide. Roughly 70% of breast cancers are estrogen receptor-positive (ER+). MicroRNA-190b (miR-190b) has previously been reported to be upregulated in ER+ breast cancers. Previously, we have demonstrated that miR-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. Objectives: To further study the role of miR-190b in ER+ breast cancer and to identify its clinically relevant targets in breast cancer. Design: Patient cohort and cell line-based RNA-sequencing analysis. Methods: The Cancer Genome Atlas was used to obtain gene expression data and clinical information on patients with breast cancer. To identify messenger RNA (mRNA) targets for miR-190b, the ER+ breast cancer cell line T-47D was used to immunoprecipitate biotin-labeled miR-190b followed by RNA sequencing. Western blot was used to confirm miR-190b target. Patient survival based on miR-190b and selected target was studied using the Cancer Genome Atlas. Results: In this study, we confirm that miR-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of miR-190b results in more favorable outcomes in Luminal A patients, hazard ratio (HR) = 0.29, 95% confidence interval [CI] = 0.12-0.71, P = .0063. MicroRNA-190b target analysis identified RING finger and WD repeat domain 3 (RFWD3) as one of miR-190b regulatory targets in ER+ breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in patients with Luminal A breast cancer (HR = 2.22, 95% CI = 1.33-3.71, P = .002). Gene ontology analysis of our sequencing results indicates that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the ER+ subtypes, Luminal A, and Luminal B. Conclusions: We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in patients with Luminal A breast cancer but not in patients with Luminal B breast cancer. These findings provide novel insights into miR-190b role in breast cancer and that its clinical relevance is subtype specific.

MicroRNA-190b targets RFWD3 in ER-positive Breast Cancer Breast cancer is the most common diagnosed type of cancer worldwide. Most of them, or 70%, overexpressed the estrogen receptor (ER) which can be targeted with drugs. MicroRNA-190b (miR-190b) is known to be overexpressed in these types of breast cancers, and we have shown that loss of DNA methylation within the genomic region of miR-190b occurs in these ER+ cancers as well, which potentially is the cause for its overexpression. We, therefore, aimed at understanding miR-190b further. To do so, we used a technique called immunoprecipitation to capture miR-190b targets and performed RNA sequencing to identify potential targets. Of the targets, we identified RFWD3 and performed a western blot to confirm whether it was a true target. Finally, we performed survival analysis using data from the Cancer Genome Atlas to see whether RFWD3 was important for patient prognosis. In summary, we identified RFWD3 to be a target of miR-190b in ER+ breast cancers and that its expression is lower when miR-190b is elevated. We also saw that lower levels of RFWD3 are linked to better outcomes in a subgroup of ER+ breast cancers called Luminal A. These findings help in understanding miR-190b and its role in breast cancer and show that its clinical relevance is subgroup specific.

Proteomic associations with forced expiratory volume: a Mendelian randomisation study.

BACKGROUND: A decline in forced expiratory volume (FEV1) is a hallmark of respiratory diseases that are an important cause of morbidity among the elderly. While some data exist on biomarkers that are related to FEV1, we sought to do a systematic analysis of causal relations of biomarkers with FEV1. METHODS: Data from the population-based AGES-Reykjavik study were used. Serum proteomic measurements were done using 4782 DNA aptamers (SOMAmers). Data from 1479 participants with spirometric data were used to assess the association of SOMAmer measurements with FEV1 using linear regression. Bi-directional two-sample Mendelian randomisation (MR) analyses were done to assess causal relations of observationally associated SOMAmers with FEV1, using genotype and SOMAmer data from 5368 AGES-Reykjavik participants and genetic associations with FEV1 from a publicly available GWAS (n = 400,102). RESULTS: In observational analyses, 530 SOMAmers were associated with FEV1 after multiple testing adjustment (FDR < 0.05). The most significant were Retinoic Acid Receptor Responder 2 (RARRES2), R-Spondin 4 (RSPO4) and Alkaline Phosphatase, Placental Like 2 (ALPPL2). Of the 257 SOMAmers with genetic instruments available, eight were associated with FEV1 in MR analyses. Three were directionally consistent with the observational estimate, Thrombospondin 2 (THBS2), Endoplasmic Reticulum Oxidoreductase 1 Beta (ERO1B) and Apolipoprotein M (APOM). THBS2 was further supported by a colocalization analysis. Analyses in the reverse direction, testing whether changes in SOMAmer levels were caused by changes in FEV1, were performed but no significant associations were found after multiple testing adjustments. CONCLUSIONS: In summary, this large scale proteogenomic analyses of FEV1 reveals circulating protein markers of FEV1, as well as several proteins with potential causality to lung function.

Serum proteomics reveals APOE dependent and independent protein signatures in Alzheimer's disease.

The current demand for early intervention, prevention, and treatment of late onset Alzheimer's disease (LOAD) warrants deeper understanding of the underlying molecular processes which could contribute to biomarker and drug target discovery. Utilizing high-throughput proteomic measurements in serum from a prospective population-based cohort of older adults (n = 5,294), we identified 303 unique proteins associated with incident LOAD (median follow-up 12.8 years). Over 40% of these proteins were associated with LOAD independently of APOE-ε4 carrier status. These proteins were implicated in neuronal processes and overlapped with protein signatures of LOAD in brain and cerebrospinal fluid. We found 17 proteins which LOAD-association was strongly dependent on APOE-ε4 carrier status. Most of them showed consistent associations with LOAD in cerebrospinal fluid and a third had brain-specific gene expression. Remarkably, four proteins in this group (TBCA, ARL2, S100A13 and IRF6) were downregulated by APOE-ε4 yet upregulated as a consequence of LOAD as determined in a bi-directional Mendelian randomization analysis, reflecting a potential response to the disease onset. Accordingly, the direct association of these proteins to LOAD was reversed upon APOE-ε4 genotype adjustment, a finding which we replicate in an external cohort (n = 719). Our findings provide an insight into the dysregulated pathways that may lead to the development and early detection of LOAD, including those both independent and dependent on APOE-ε4. Importantly, many of the LOAD-associated proteins we find in the circulation have been found to be expressed - and have a direct link with AD - in brain tissue. Thus, the proteins identified here, and their upstream modulating pathways, provide a new source of circulating biomarker and therapeutic target candidates for LOAD.

Proteomic prediction of incident heart failure and its main subtypes.

AIM: To examine the ability of serum proteins in predicting future heart failure (HF) events, including HF with reduced or preserved ejection fraction (HFrEF or HFpEF), in relation to event time, and with or without considering established HF-associated clinical variables. METHODS AND RESULTS: In the prospective population-based Age, Gene/Environment Susceptibility Reykjavik Study (AGES-RS), 440 individuals developed HF after their first visit with a median follow-up of 5.45 years. Among them, 167 were diagnosed with HFrEF and 188 with HFpEF. A least absolute shrinkage and selection operator regression model with non-parametric bootstrap were used to select predictors from an analysis of 4782 serum proteins, and several pre-established clinical parameters linked to HF. A subset of 8-10 distinct or overlapping serum proteins predicted different future HF outcomes, and C-statistics were used to assess discrimination, revealing proteins combined with a C-index of 0.80 for all incident HF, 0.78 and 0.80 for incident HFpEF or HFrEF, respectively. In the AGES-RS, protein panels alone encompassed the risk contained in the clinical information and improved the performance characteristics of prediction models based on N-terminal pro-B-type natriuretic peptide and clinical risk factors. Finally, the protein predictors performed particularly well close to the time of an HF event, an outcome that was replicated in the Cardiovascular Health Study. CONCLUSION: A small number of circulating proteins accurately predicted future HF in the AGES-RS cohort of older adults, and they alone encompass the risk information found in a collection of clinical data. Incident HF events were predicted up to 8 years, with predictor performance significantly improving for events occurring less than 1 year ahead, a finding replicated in an external cohort study.

Proteomic Network Analysis of Alzheimer's Disease Cerebrospinal Fluid Reveals Alterations Associated with APOE ε4 Genotype and Atomoxetine Treatment.

Alzheimer's disease (AD) is currently defined at the research level by the aggregation of amyloid-ß (Aß) and tau proteins in brain. While biofluid biomarkers are available to measure Aß and tau pathology, few biomarkers are available to measure the complex pathophysiology that is associated with these two cardinal neuropathologies. Here we describe the proteomic landscape of cerebrospinal fluid (CSF) changes associated with Aß and tau pathology in 300 individuals as assessed by two different proteomic technologies-tandem mass tag (TMT) mass spectrometry and SomaScan. Harmonization and integration of both data types allowed for generation of a robust protein co-expression network consisting of 34 modules derived from 5242 protein measurements, including disease-relevant modules associated with autophagy, ubiquitination, endocytosis, and glycolysis. Three modules strongly associated with the apolipoprotein E ε4 (APOE ε4) AD risk genotype mapped to oxidant detoxification, mitogen associated protein kinase (MAPK) signaling, neddylation, and mitochondrial biology, and overlapped with a previously described lipoprotein module in serum. Neddylation and oxidant detoxification/MAPK signaling modules had a negative association with APOE ε4 whereas the mitochondrion module had a positive association with APOE ε4. The directions of association were consistent between CSF and blood in two independent longitudinal cohorts, and altered levels of all three modules in blood were associated with dementia over 20 years prior to diagnosis. Dual-proteomic platform analysis of CSF samples from an AD phase 2 clinical trial of atomoxetine (ATX) demonstrated that abnormal elevations in the glycolysis CSF module-the network module most strongly correlated to cognitive function-were reduced by ATX treatment. Individuals who had more severe glycolytic changes at baseline responded better to ATX. Clustering of individuals based on their CSF proteomic network profiles revealed ten groups that did not cleanly stratify by Aß and tau status, underscoring the heterogeneity of pathological changes not fully reflected by Aß and tau. AD biofluid proteomics holds promise for the development of biomarkers that reflect diverse pathologies for use in clinical trials and precision medicine.

A proteomic analysis of atrial fibrillation in a prospective longitudinal cohort (AGES-Reykjavik study).

AIMS: Atrial fibrillation (AF) is associated with high risk of comorbidities and mortality. Our aim was to examine causal and predictive relationships between 4137 serum proteins and incident AF in the prospective population-based Age, Gene/Environment Susceptibility-Reykjavik (AGES-Reykjavik) study. METHODS AND RESULTS: The study included 4765 participants, of whom 1172 developed AF. Cox proportional hazards regression models were fitted for 4137 baseline protein measurements adjusting for known risk factors. Protein associations were tested for replication in the Cardiovascular Health Study (CHS). Causal relationships were examined in a bidirectional, two-sample Mendelian randomization analysis. The time-dependent area under the receiver operating characteristic curve (AUC)-statistic was examined as protein levels and an AF-polygenic risk score (PRS) were added to clinical risk models. The proteomic signature of incident AF consisted of 76 proteins, of which 63 (83%) were novel and 29 (38%) were replicated in CHS. The signature included both N-terminal prohormone of brain natriuretic peptide (NT-proBNP)-dependent (e.g. CHST15, ATP1B1, and SVEP1) and independent components (e.g. ASPN, AKR1B, and LAMA1/LAMB1/LAMC1). Nine causal candidates were identified (TAGLN, WARS, CHST15, CHMP3, COL15A1, DUSP13, MANBA, QSOX2, and SRL). The reverse causal analysis suggested that most AF-associated proteins were affected by the genetic liability to AF. N-terminal prohormone of brain natriuretic peptide improved the prediction of incident AF events close to baseline with further improvements gained by the AF-PRS at all time points. CONCLUSION: The AF proteomic signature includes biologically relevant proteins, some of which may be causal. It mainly reflects an NT-proBNP-dependent consequence of the genetic liability to AF. N-terminal prohormone of brain natriuretic peptide is a promising marker for incident AF in the short term, but risk assessment incorporating a PRS may improve long-term risk assessment.

Serum proteomics reveals APOE dependent and independent protein signatures in Alzheimer's disease.

The current demand for early intervention, prevention, and treatment of late onset Alzheimer's disease (LOAD) warrants deeper understanding of the underlying molecular processes which could contribute to biomarker and drug target discovery. Utilizing high-throughput proteomic measurements in serum from a prospective population-based cohort of older adults (n=5,294), we identified 303 unique proteins associated with incident LOAD (median follow-up 12.8 years). Over 40% of these proteins were associated with LOAD independently of APOE-ε4 carrier status. These proteins were implicated in neuronal processes and overlapped with protein signatures of LOAD in brain and cerebrospinal fluid. We found 17 proteins which LOAD-association was strongly dependent on APOE-ε4 carrier status. Most of them showed consistent associations with LOAD in cerebrospinal fluid and a third had brain-specific gene expression. Remarkably, four proteins in this group (TBCA, ARL2, S100A13 and IRF6) were downregulated by APOE-ε4 yet upregulated as a consequence of LOAD as determined in a bi-directional Mendelian randomization analysis, reflecting a potential response to the disease onset. Accordingly, the direct association of these proteins to LOAD was reversed upon APOE-ε4 genotype adjustment, a finding which we replicate in an external cohort (n=719). Our findings provide an insight into the dysregulated pathways that may lead to the development and early detection of LOAD, including those both independent and dependent on APOE-ε4. Importantly, many of the LOAD-associated proteins we find in the circulation have been found to be expressed - and have a direct link with AD - in brain tissue. Thus, the proteins identified here, and their upstream modulating pathways, provide a new source of circulating biomarker and therapeutic target candidates for LOAD.

CpG promoter hypo-methylation and up-regulation of microRNA-190b in hormone receptor-positive breast cancer.

Estrogen receptor-positive breast cancer is subdivided into subtypes LuminalA and LuminalB, based on different expression patterns. MicroRNA-190b has been reported to be up-regulated in estrogen receptor-positive breast cancers. In this study we aimed to investigate the role of CpG promoter methylation in regulating miR-190b expression and its impact on clinical presentation and prognosis. DNA methylation analysis for the promotor of microRNA-190b was performed by pyrosequencing 549 primary breast tumors, of which 62 were carriers of the BRCA2 999del5 founder mutation, 71 proximal normal breast samples and 16 breast derived cell lines. MicroRNA-190b expression was analysed in 67 primary breast tumors, 14 paired normal breast samples and 16 breast derived cell lines. Tissue microarrays (TMAs) were available for ER (n = 436), PR (n = 436), HER-2 (N = 258) and Ki67 (n = 248). MiR-190b had reduced promoter methylation in estrogen receptor-positive breast cancers (P = 1.02e-12, Median values: ER+ 24.3, ER- 38.26) and miR-190b's expression was up-regulated in a correlative manner (P = 1.83e-06, Spearman's rho -0.62). Through breast cancer specific survival analysis, we demonstrated that LuminalA patients exhibiting miR-190b hypo-methylation had better survival than other patients (P = 0.034, HR = 0.29, 95% CI 0.09-0.91). We, furthermore, demonstrated that miR-190b hypo-methylation occurs less frequently in ER+ tumors from BRCA2 999del5 mutation carriers than in non-mutated individuals (P = 0.038, Χ 2 = 4.32, n = 335). Our results suggest that upregulation of miR-190b may occur through loss of promoter DNA methylation during the development of estrogen-receptor (ER) positive breast cancers, and that miR-190b hypo-methylation leads to increased breast cancer specific survival within the LuminalA- subtype but not LuminalB.