Regulation of Interferon-Induced Protein Kinase PKR: Modulation of P58IPK Inhibitory Function by a Novel Protein, P52rIPK

The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58^IPK, a cellular PKR inhibitor. P58^IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2α subunit (eIF-2α) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58^IPK-interacting protein. This protein, designated P52^rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52^rIPK and P58^IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58^IPK repressed PKR-mediated eIF-2α phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52^rIPK into these strains resulted in restoration of both PKR activity and eIF-2α phosphorylation, concomitant with growth suppression due to inhibition of P58^IPK function. Furthermore, P52^rIPK inhibited P58^IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58^IPK is inhibited through a direct interaction with P52^rIPK which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.     

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.Many human diseases are characterized by abnormalities in complex signaling pathways (1). The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting. Although these techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume. Furthermore, techniques such as Western blotting are not applicable in the routine clinical setting. Miniaturized parallel immunoassay techniques have been developed in recent years and have played a pivotal role in biomarker discovery (2). Antibody arrays enable multiple potential disease markers to be investigated in a single sample in parallel (3). Beyond this, Reverse Phase Protein Arrays (RPPA)¹ are sensitive high throughput tools that can quantify protein expression levels and activation status (posttranslational modifications such as phosphorylation) in multiple experimental samples simultaneously. The technique requires only minute amounts of samples, printed as lysate arrays onto slides, and hundreds of markers of interest can be investigated, array by array, in a miniaturized dot blot manner. Numerous reports have demonstrated that RPPA can be applied to various sources of cells and tissues to analyze protein profiles, signaling pathway networks, and for the identification of biomarkers (4–13). A recently published workshop report reviews the full potential and advances of RPPA for use in clinical, translational, and basic research (11).In oncology, the parallel profiling of multiple protein markers is particularly desirable to study tumor initiation and progression, to classify tumor disease states on the molecular level, and to discover and monitor biomarkers that can predict therapeutic response or tumor recurrence (14–16). The study of signaling response and analysis of pharmacodynamic (PD) markers upon treatment using in vitro and in vivo test systems (e.g. cell line or patient derived xenograft tumor models) is an established component of preclinical and early clinical drug development. These techniques can provide evidence of target pathway modulation for new therapeutic lead candidate compounds and provide valuable information on the drug mode of action (17), especially in the translational phase. Multiplex analyses of PD biomarkers by RPPA have been performed in vitro using cancer cell lines (18, 19) as well as in patient-derived tumor tissue and blood samples (20, 21) to assess response to treatment and target inhibition. A combination of RPPA signaling pathway mapping and functional PET imaging has recently been successfully evaluated in xenograft models as an early response PD marker for anti-cancer drug efficacy (13).Translating miniaturized multiple protein analysis platforms-such as RPPA - from preclinical to clinical applicability is highly desirable; however, issues such as the limited amount of available clinical samples and tumor heterogeneity must first be addressed. Furthermore, most studies of RPPA in tumor tissue to date have been conducted using proteins extracted from fresh-frozen (FF) tissue specimens; whereas, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation used in clinical pathology laboratories. FFPE yields excellent tissue architecture for histological assessment and enables analysis of individual proteins in situ by techniques such as immunohistochemistry. However, formalin fixation leads to extensive protein–protein and protein–nucleic acid cross-linking (22), which can hamper protein extraction and reduce both the overall yield of extracted protein and the profile of proteins detectable by proteomic techniques (23, 24). Furthermore, formalin-induced cross-linking induces conformational changes in protein structure that can alter the immunoreactivity of some proteins in situ by hiding or altering peptide epitopes (25, 26). Such artifacts are absent from snap-frozen tissue; therefore, protein profiles obtained from FF tissue are likely to reflect the in vivo biology of the tumor more closely. However, FF tumor tissue is not widely available because it is costly to collect and maintain in the clinical setting. FFPE tissue samples are routinely archived by nearly every hospital and offer a unique opportunity to study thousands of samples retrospectively with extensive clinical records and follow-up information.Several groups have now established protocols for retrieving cross-linked proteins from fixed tissues (27–33). These methods are mainly based on the use of concentrated ionic detergents and high temperature protocols closely related to the antigen retrieval methods developed for immunohistochemistry. These studies show that obtaining nondegraded, full-length proteins from FFPE tissues for multiplex analyses is feasible (27–33). More recently, protein extraction techniques optimized for fixed samples have been used to successfully conduct RPPA using FFPE tissue biopsies from different cancer types (34–40). Guo et al. systematically investigated several protein extraction methods and demonstrated that RPPA of FFPE materials is feasible, reproducible and can generate biologically relevant protein profiles (41). Other studies have confirmed the validity of this approach and shown that data generated from RPPA analyses of FFPE tissue demonstrate good concordance with traditional immunohistochemistry markers such as HER2 protein in breast cancer (34, 40). However, to date, analyses have been performed only for a limited set of protein markers.To evaluate whether analysis of a broader panel of protein markers is feasible and generates meaningful data from FFPE tumor tissue sections, we conducted RPPA on matched samples of FF and FFPE tissues using a set of 300 markers, the largest panel reported to date. Our aim was to identify markers that performed similarly when comparing the protein profiles measured in protein extracts from matched FF and FFPE tissue, using RPPA assays established for use in frozen tissues. Correlating selected markers and assays in such a way should qualify RPPA for further use with FFPE tissues of clinical relevance, e.g. in PD marker studies. In this paper, we have specifically focused on the technical issues relevant for using the RPPA platform in a clinical setting, and did not address the biology of the test systems used in detail. However, the models used have been pre-characterized to identify key signaling parameters in context of targeted drug treatment (42). We conducted a systematic comparison of RPPA protein profiles in matched FF and FFPE tumor tissues resected from three different xenograft models of human cancer, each treated with targeted therapeutic antibodies that have previously been shown to achieve tumor growth inhibition. Furthermore, we investigated the effect of targeted drug treatment on protein expression and activation status, and the concordance of matched FF and FFPE tissue RPPA profiles. Finally, with one of the applied tumor models, we compared a set of protein profiles measured with two different multiple assay platforms - the RPPA and the Luminex Bio-Plex system, and discuss their relevance with respect to the analysis of FFPE tissue.     

Determinants of Survival in Malignant Pleural Mesothelioma: A Surveillance,Epidemiology, and End Results (SEER) Study of 14,228 Patients

Introduction

Left untreated, malignant pleural mesothelioma (MPM) is associated with uniformly poor prognosis. Better survival has been reported with surgery-based multimodality therapy, but to date, no trial has demonstrated survival benefit of surgery over other therapies. We evaluated whether cancer-directed surgery influenced survival independently from other predictors in a large population-based dataset.

Methods

The SEER database was explored from 1973 to 2009 to identify all cases of pathologically-proven MPM. Age, sex, race, year of diagnosis, histology stage, cancer-directed surgery, radiation, and vital status were analyzed. The association between prognostic factors and survival was estimated using Cox regression and propensity matched analysis.

Results

There were 14,228 patients with pathologic diagnosis of MPM. On multivariable analysis, female gender, younger age, early stage, and treatment with surgery were independent predictors of longer survival. In comparison to no treatment, surgery alone was associated with significant improvement in survival [adjusted hazard ratio (adj HR) 0.64 (0.61–0.67)], but not radiation [adj HR 1.15 (1.08–1.23)]. Surgery and radiation combined had similar survival as surgery alone [adj HR 0.69 (0.64–0.76)]. Results were similar when cases diagnosed between 1973 and 1999 were compared to cases diagnosed between 2000 and 2009.

Conclusions

Despite developments in surgical and radiation techniques, the prognosis for MPM patients has not improved over the past 4 decades. Cancer-directed surgery is independently associated with better survival, suggesting that multimodal surgery-based therapy can benefit these patients. Further research in adjuvant treatment is necessary to improve prognosis in this challenging disease.     

High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes

Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes.     

Discovery of 2-substituted benzoxazole carboxamides as 5-HT3 receptor antagonists

A new class of 2-substituted benzoxazole carboxamides are presented as potent functional 5-HT(3) receptor antagonists. The chemical series possesses nanomolar in vitro activity against human 5-HT(3)A receptors. A chemistry optimization program was conducted and identified 2-aminobenzoxazoles as orally active 5-HT(3) receptor antagonists with good metabolic stability. These novel analogues possess drug-like characteristics and have potential utility for the treatment of diseases attributable to improper 5-HT(3) receptor function, especially diarrhea predominant irritable bowel syndrome (IBS-D).     

mTRAQ‐based quantification of potential endometrial carcinoma biomarkers from archived formalin‐fixed paraffin‐embedded tissues

Formalin‐fixed paraffin‐embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC–multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK‐M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK‐M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK‐M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.     

Modeling Nymphoides architecture: A morphological analysis of Nymphoides aquatica (Menyanthaceae)

? Premise of the study: Species in the aquatic genus Nymphoides have inflorescences that appear to arise from the petioles of floating leaves. The inflorescence-floating leaf complex can produce vegetative propagules and/or additional inflorescences and leaves. We analyzed the morphology of N. aquatica to determine how this complex relates to whole plant architecture and whether whole plant growth is sympodial or monopodial. ? Methods: We used dissections, measurements, and microscopic observations of field-collected plants and plants cultivated for 2 years in outdoor tanks in south Florida, USA. ? Key results: Nymphoides aquatica had a submerged plagiotropic rhizome that produced floating leaves in an alternate/spiral phyllotaxy. Rhizomes were composed of successive sympodial units that varied in the number of leaves produced before the apex terminated. The basic sympodial unit had a prophyll that subtended a renewal-shoot bud, a short-petioled leaf (SPL) with floating lamina, and an inflorescence; the SPL axillary bud expanded as a vegetative propagule. Plants produced either successive basic sympodial units or expanded sympodia that intercalated long-petioled leaves between the prophyll and the SPL. ? Conclusions: Nymphoides aquatica grows sympodially, forming a rhizome composed of successive basic sympodia and expanded sympodial units. Variations on these types of sympodial growth help explain the branching patterns and leaf morphologies described for other Nymphoides species. Monitoring how these two sympodial phases are affected by water depth provides an ecologically meaningful way to assess N. aquatica's responses to altered hydrology.