We recently demonstrated that increased expression of the RNA-binding protein RBM3 is associated with a favourable prognosis in breast cancer. The aim of this study was to examine the prognostic value of RBM3 mRNA and protein expression in epithelial ovarian cancer (EOC) and the cisplatin response upon RBM3 depletion in a cisplatin-sensitive ovarian cancer cell line.

Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials.

To increase our understanding of skin, it is important to define the molecular constituents of the cell types and epidermal layers that signify normal skin. We have combined a genome-wide transcriptomics analysis, using deep sequencing of mRNA from skin biopsies, with immunohistochemistry-based protein profiling to characterize the landscape of gene and protein expression in normal human skin. The transcriptomics and protein expression data of skin were compared to 26 (RNA) and 44 (protein) other normal tissue types. All 20,050 putative protein-coding genes were classified into categories based on patterns of expression. We found that 417 genes showed elevated expression in skin, with 106 genes expressed at least five-fold higher than that in other tissues. The 106 genes categorized as skin enriched encoded for well-known proteins involved in epidermal differentiation and proteins with unknown functions and expression patterns in skin, including the C1orf68 protein, which showed the highest relative enrichment in skin. In conclusion, we have applied a genome-wide analysis to identify the human skin-specific proteome and map the precise localization of the corresponding proteins in different compartments of the skin, to facilitate further functional studies to explore the molecular repertoire of normal skin and to identify biomarkers related to various skin diseases.

Cyclooxygenase-2 (COX-2) is an enzyme that has been extensively investigated as a prognostic marker in cancer. In non-small cell lung cancer (NSCLC) previous results regarding the prognostic impact of COX-2 expression are inconsistent. Therefore we evaluated the association between transcript levels and overall survival in nine publicly available gene expression data sets (total n = 1337) and determined in situ compartment-specific tumor and stromal cell protein expression in two independent cohorts (n = 616). Gene expression did not show any correlation with clinical parameters or with overall survival. Protein expression in tumor and stromal cells did not correlate with any clinical parameter or with overall survival in one of the analyzed cohorts, while a significant association of high stromal expression with longer survival was observed in both univariate and multivariate analysis in the other cohort. Stromal expression of COX-2 has not been separately evaluated in NSCLC previously and may be a subject of further investigation, whereas the presented findings from this comprehensive compartment specific evaluation clearly reject the hypothesis of COX-2 tumor cell expression having a prognostic value in NSCLC.

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC).

Anillin (ANLN), an actin-binding protein required for cytokinesis, has recently been presented as part of a prognostic marker panel in breast cancer. The objective of the current study was to further explore the prognostic and functional value of ANLN as a single biomarker in breast cancer.

Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links transcription termination to exosomal RNA degradation through CBCN.

Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer.

To better understand prostate function and disease, it is important to define and explore the molecular constituents that signify the prostate gland. The aim of this study was to define the prostate specific transcriptome and proteome, in comparison to 26 other human tissues. Deep sequencing of mRNA (RNA-seq) and immunohistochemistry-based protein profiling were combined to identify prostate specific gene expression patterns and to explore tissue biomarkers for potential clinical use in prostate cancer diagnostics. We identified 203 genes with elevated expression in the prostate, 22 of which showed more than five-fold higher expression levels compared to all other tissue types. In addition to previously well-known proteins we identified two poorly characterized proteins, TMEM79 and ACOXL, with potential to differentiate between benign and cancerous prostatic glands in tissue biopsies. In conclusion, we have applied a genome-wide analysis to identify the prostate specific proteome using transcriptomics and antibody-based protein profiling to identify genes with elevated expression in the prostate. Our data provides a starting point for further functional studies to explore the molecular repertoire of normal and diseased prostate including potential prostate cancer markers such as TMEM79 and ACOXL.

Identification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data.

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues.

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the CNS and is present in high concentrations in presynaptic terminals of neuronal cells. More recently, GABA has been ascribed a more widespread role in the control of cell proliferation during development where low concentrations of extrasynaptic GABA induce a tonic activation of GABA receptors. The GABA-A receptor consists of a ligand-gated chloride channel, formed by five subunits that are selected from 19 different subunit isoforms. The functional and pharmacological properties of the GABA-A channels are dictated by their subunit composition. Here we used qRT-PCR to compare mRNA levels of all 19 GABA-A channel subunits in samples of human glioma (n = 29) and peri-tumoral tissue (n = 5). All subunits except the ρ1 and ρ3 subunit were consistently detected. Lowest mRNA levels were found in glioblastoma compared to gliomas of lower malignancy, except for the θ subunit. The expression and cellular distribution of the α1, γ1, ρ2 and θ subunit proteins was investigated by immunohistochemistry on tissue microarrays containing 87 gliomas grade II. We found a strong co-expression of ρ2 and θ subunits in both astrocytomas (r = 0.86, p<0.0001) and oligodendroglial tumors (r = 0.66, p<0.0001). Kaplan-Meier analysis and Cox proportional hazards modeling to estimate the impact of GABA-A channel subunit expression on survival identified the ρ2 subunit (p = 0.043) but not the θ subunit (p = 0.64) as an independent predictor of improved survival in astrocytomas, together with established prognostic factors. Our data give support for the presence of distinct GABA-A channel subtypes in gliomas and provide the first link between specific composition of the A-channel and patient survival.

Blood-based proteomic profiling may aid and expand our understanding of diseases and their different phenotypes. The aim of the presented study was to profile serum samples from patients with malignant melanoma using affinity proteomic assays to describe proteins in the blood stream that are associated to stage or recurrence of melanoma.

Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.

The mammalian brain is a complex organ composed of many specialized cells, harboring sets of both common, widely distributed, as well as specialized and discretely localized proteins. Here we focus on the human brain, utilizing transcriptomics and public available Human Protein Atlas (HPA) data to analyze brain-enriched (frontal cortex) polyadenylated messenger RNA and long non-coding RNA and generate a genome-wide draft of global and cellular expression patterns of the brain. Based on transcriptomics analysis of altogether 27 tissues, we have estimated that approximately 3% (n=571) of all protein coding genes and 13% (n=87) of the long non-coding genes expressed in the human brain are enriched, having at least five times higher expression levels in brain as compared to any of the other analyzed peripheral tissues. Based on gene ontology analysis and detailed annotation using antibody-based tissue micro array analysis of the corresponding proteins, we found the majority of brain-enriched protein coding genes to be expressed in astrocytes, oligodendrocytes or in neurons with molecular properties linked to synaptic transmission and brain development. Detailed analysis of the transcripts and the genetic landscape of brain-enriched coding and non-coding genes revealed brain-enriched splice variants. Several clusters of neighboring brain-enriched genes were also identified, suggesting regulation of gene expression on the chromatin level. This multi-angle approach uncovered the brain-enriched transcriptome and linked genes to cell types and functions, providing novel insights into the molecular foundation of this highly specialized organ.

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.

In endometrioid endometrial adenocarcinoma (EEA), the currently established prognostic factors in clinical guidelines are stage and grade. Many guidelines include lymphovascular invasion (LVI) and tumor size as prognostic factors. Although several studies have associated lack of estrogen (ER) and progesterone receptor (PR) expression with reduced outcome, the prognostic use of these markers is uncommon. Better prognostication of clinical behavior would be useful in patients with early stage (I-II) disease. In this study we evaluated ER and PR as prognostic factors in EEA, and compared their expression with other potential biomarkers and clinical parameters.