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Supplementary Data from Protein Expression Profiling in Esophageal Adenocarcinoma Patients Indicates Association of Heat-Shock Protein 27 Expression and Chemotherapy Response

Purpose: To identify pretherapeutic predictive biomarkers in tumor biopsies of patients with locally advanced esophageal adenocarcinomas treated with neoadjuvant chemotherapy, we used an explorative proteomic approach to correlate pretherapeutic protein expression profiles with tumor response to neoadjuvant chemotherapy.Experimental Design: Thirty-four patients with locally advanced esophageal adenocarcinomas who received neoadjuvant platin/5-fluorouracil–based chemotherapy before surgical resection were enrolled in this study. Response to chemotherapy was determined (a) by the amount of decline of [18F]fluorodeoxyglucose tumor uptake 2 weeks after the start of chemotherapy measured by positron emission tomography and (b) by histopathologic evaluation of tumor regression after surgical resection. Explorative quantitative and qualitative protein expression analysis was done through a quantitative differential protein expression analysis that used dual-isotope radioactive labeling of protein extracts. Selected identified biomarkers were validated by immunohistochemistry and quantitative real time reverse transcription-PCR.Results: Proteomic analysis revealed four cellular stress response–associated proteins [heat-shock protein (HSP) 27, HSP60, glucose-regulated protein (GRP) 94, GRP78] and a number of cytoskeletal proteins whose pretherapeutic abundance was significantly different (P < 0.001) between responders and nonresponders. Immunohistochemistry and gene expression analysis confirmed these data, showing a significant association between low HSP27 expression and nonresponse to neoadjuvant chemotherapy (P = 0.049 and P = 0.032, respectively).Conclusions: Albeit preliminary, our encouraging data suggest that protein expression profiling may distinguish cancers with a different response to chemotherapy. Our results suggest that response to chemotherapy may be related to a different activation of stress response and inflammatory biology in general. Moreover, the potential of HSPs and GRPs as biomarkers of chemotherapy response warrants further validation.

Essence Apparatus is a collection of fiction short stories rooted in poetry and the traditions of ‘the uncanny' and ‘weird' writing. Included are also three expository essays relating to my creative work and its contextual and theoretical underpinnings, going in-depth on my relationship with the genre of science fiction and its literary device ‘cognitive estrangement', the tradition of writing the uncanny and the weird, and my Scandinavian and South African sources of inspiration. The short story collection Essence Apparatus feature zombies in Finland; an animalistic metamorphosis on the archipelago of Svalbard; the Chernobyl accident; humans living on Mars returning to their origins on Earth, but also interpersonal relationships between a daughter and her father, two sisters, and lovers. The stories explore the hard problem of consciousness and other philosophical questions; the search for knowledge and a feeling of belonging in a nature not fully understood, and the mechanisms of translocation and transfiguration as a fundamental part of being human.

FAHD1, a member of the FAH superfamily of enzymes, was identified in a proteomic screen for mitochondrial proteins with differential expression in young versus senescent human endothelial cells. FAHD1 acts as oxaloacetate decarboxylase, and recent observations suggest that FAHD1 plays an important role in regulating mitochondrial function. Thus, mutation of the nematode homolog, fahd-1, impairs mitochondrial function in Caenorhabditis elegans. When FAHD1 gene expression was silenced in human cells, activity of the mitochondrial electron transport (ETC) system was reduced and the cells entered premature senescence-like growth arrest. These findings suggest a model where FAHD1 regulates mitochondrial function and in consequence senescence. These findings are discussed here in the context of a new concept where senescence is divided into deep senescence and less severe forms of senescence. We propose that genetic inactivation of FAHD1 in human cells induces a specific form of cellular senescence, which we term senescence light and discuss it in the context of mitochondrial dysfunction associated senescence (MiDAS) described by others. Together these findings suggest the existence of a continuum of cellular senescence phenotypes, which may be at least in part reversible.

Endothelial cells of the blood–brain barrier form a structural and functional barrier maintaining brain homeostasis via paracellular tight junctions and specific transporters such as P-glycoprotein. The blood–brain barrier is responsible for negligible bioavailability of many neuroprotective drugs. In Alzheimer’s disease, current treatment approaches include inhibitors of BACE-1 (β-site of amyloid precursor protein cleaving enzyme), a proteinase generating neurotoxic β-amyloid. It is known that BACE-1 is highly expressed in endosomes and membranes of neurons and glia. We now provide evidence that BACE-1 is expressed in blood–brain barrier endothelial cells of human, mouse, and bovine origin. We further show its predominant membrane localization by 3D- dSTORM super-resolution microscopy, and by biochemical fractionation that further shows an abluminal distribution of BACE-1 in brain microvessels. We confirm its functionality in processing APP in primary mouse brain endothelial cells. In an Alzheimer’s disease mouse model we show that BACE-1 is upregulated at the blood–brain barrier compared to healthy controls. We therefore suggest a critical role for BACE-1 at the blood–brain barrier in β-amyloid generation and in vascular aspects of Alzheimer’s disease, particularly in the development of cerebral amyloid angiopathy.

Anna K. Bal-Price et al., © 2015 Informa Healthcare USA. The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

Previously we demonstrated that systemically administered neuregulin-1-β1, a nerve growth and differentiation factor, passed the blood-brain barrier and accumulated in brain areas with expression of its receptor ErbB4. In substantia nigra (SN), neuregulin-1-β1 phosphorylated ErbB4 and protected dopaminergic neurons in a toxin-based mouse model of Parkinson disease (PD). We studied ErbB4 in the context of human midbrain dopaminergic degeneration in vivo and in vitro. Post-mortem ventral midbrain tissue sections of neuropsychiatric healthy individuals and PD patients (matched for age, gender and post-mortem delay) were immunostained for ErbB4. Cultured Lund human mesencephalic (LUHMES) post-mitotic dopaminergic neurons were treated with dopaminergic toxins and analyzed for ErbB4 expression. In control individuals, 85.0±5.0% of dopaminergic neurons, containing cytoplasmic neuromelanin, expressed ErbB4 in the SN. In PD cases, the percentage of ErbB4-positive nigral dopaminergic neurons was increased to 94.9±2.5%. The mean ErbB4 immunoreactivity of melanized neurons was higher in PD than controls. LUHMES neurons upregulated ErbB4 when exposed to toxins 1-methyl-4-phenylpyridinium and 6-hydroxydopamine. Increased rate of ErbB4-positive dopaminergic neurons in PD may either reflect a better survival of ErbB4-positive neurons or an increased expression of ErbB4 by remaining neurons to seek trophic support. Enhanced ErbB4 expression in human in vitro toxin-based PD models supports the latter interpretation. Thus, dopaminergic neurons in SN might be susceptible to neuregulin-1 treatment in PD.

Neuregulin-1 (NRG1) belongs to a large family of growth and differentiation factors with a key role in the development and maintenance of the brain. Genetic association of NRG1 within brain disorders such as Alzheimer's disease, schizophrenia and neuroprotective properties of certain NRG1 isoforms have led to a variety of studies in corresponding disease models. In the present work, we investigated NRG1 with regard to its peripheral and central biodistribution after systemic application. We first-time radiolabeled the entire biologically active extracellular domain of NRG1 isotype-β1 (NRG1-β1 ECD; aa 2-246) with iodine-125 and administered it peripherally to healthy adult C57Bl6 mice. Blood kinetics and relative organ distribution of (125)I-labeled NRG1-β1 ECD were determined. The blood level of NRG1-β1 ECD peaked within the first hour after intraperitoneal (i.p.) application. The brain-blood ratios of (125)I-labeled NRG1-β1 ECD were time-dependently 150-370% higher compared to the brain impermeable control, (131)I-labeled bovine serum albumin. Autoradiographs of brain slices demonstrated that (125)I-labeled NRG1-β1 ECD accumulated in several regions of the brain e.g. frontal cortex, striatum and ventral midbrain containing the substantia nigra. In addition we found histochemical and biochemical evidence that phosphorylation of the NRG1 prototype receptor ErbB4 was increased in these regions after systemic application of NRG1-β1 ECD. Our data suggest that NRG1-β1 ECD passes the blood-brain barrier and activates cerebral ErbB4 receptors.

The existence of a small subpopulation of tumourigenic cancer stem cells in the bulk of human head and neck squamous cancers (SCC) has been recognised in recent reports. This subpopulation has self-renewal properties and is responsible for the production of differentiated daughter cells that form the bulk of the tumour. Stem cells in head and neck SCC can be identified functionally using their self-renewal properties, or by their characteristic surface markers. As their resistance to contemporary cancer treatments may eventually lead to the failure of treatment there is an urgent need to better understand their biology with the ultimate goal of developing new diagnostic markers and curative cancer treatments.

Neuregulin-1 (Nrg1) is genetically linked to schizophrenia, a disease caused by neurodevelopmental imbalance in dopaminergic function. The Nrg1 receptor ErbB4 is abundantly expressed on midbrain dopaminergic neurons. Nrg1 has been shown to penetrate blood-brain barrier, and peripherally administered Nrg1 activates ErbB4 and leads to a persistent hyperdopaminergic state in neonatal mice. These data prompted us to study the effect of peripheral administration of Nrg1 in the context of Parkinson's disease, a neurodegenerative disorder affecting the dopaminergic system in the adult brain. We observed that systemic injections of the extracellular domain of Nrg1s(1) (Nrg1s(1) -ECD) increased dopamine levels in the substantia nigra and striatum of adult mice. Nrg1s(1) -ECD injections also significantly protected the mouse nigrostriatal dopaminergic system morphologically and functionally against 6-hydroxydopamine-induced toxicity in vivo. Moreover, Nrg1s(1) -ECD also protected human dopaminergic neurons in vitro against 6-hydroxydopamine. In conclusion, we have identified Nrg1s(1) -ECD as a neurotrophic factor for adult mouse and human midbrain dopaminergic neurons with peripheral administratability, warranting further investigation as therapeutic option for PD patients. (Less)

Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.

There is an urgent need for surrogate biomarkers in clinical diagnostics but also preclinical in toxicology of chemicals and efficacy testing of pharmaceuticals. On the background of the emerging fields of systems biology and theranostics, the importance of time scales and the synchronization of complex biological readouts become increasingly obvious. Systemic effects such as responses to stimuli, medical intervention, cellular stress, or toxicity elicit immediate molecular changes on the protein level. Early events include phosphorylation and oxidation of proteins. These posttranslational modifications have a direct impact on enzyme activities and protein-protein interactions. Only at downstream stages is gene transcription activated. Here we outline the analytical and statistical requirements dealing with complex patterns of protein expression and the extraction of protein surrogate biomarkers defining specific early stages of biological responses. We also present successful examples from research on aging and discuss prerequisites and necessary considerations while moving candidates to validation.

Purpose: To identify pretherapeutic predictive biomarkers in tumor biopsies of patients with locally advanced esophageal adenocarcinomas treated with neoadjuvant chemotherapy, we used an explorative proteomic approach to correlate pretherapeutic protein expression profiles with tumor response to neoadjuvant chemotherapy. Experimental Design: Thirty-four patients with locally advanced esophageal adenocarcinomas who received neoadjuvant platin/5-fluorouracil–based chemotherapy before surgical resection were enrolled in this study. Response to chemotherapy was determined (a) by the amount of decline of [18F]fluorodeoxyglucose tumor uptake 2 weeks after the start of chemotherapy measured by positron emission tomography and (b) by histopathologic evaluation of tumor regression after surgical resection. Explorative quantitative and qualitative protein expression analysis was done through a quantitative differential protein expression analysis that used dual-isotope radioactive labeling of protein extracts. Selected identified biomarkers were validated by immunohistochemistry and quantitative real time reverse transcription-PCR. Results: Proteomic analysis revealed four cellular stress response–associated proteins [heat-shock protein (HSP) 27, HSP60, glucose-regulated protein (GRP) 94, GRP78] and a number of cytoskeletal proteins whose pretherapeutic abundance was significantly different (P < 0.001) between responders and nonresponders. Immunohistochemistry and gene expression analysis confirmed these data, showing a significant association between low HSP27 expression and nonresponse to neoadjuvant chemotherapy (P = 0.049 and P = 0.032, respectively). Conclusions: Albeit preliminary, our encouraging data suggest that protein expression profiling may distinguish cancers with a different response to chemotherapy. Our results suggest that response to chemotherapy may be related to a different activation of stress response and inflammatory biology in general. Moreover, the potential of HSPs and GRPs as biomarkers of chemotherapy response warrants further validation.

It is controversial whether DNA methylation plays a functional role in Drosophila . We have studied testis DNA of Drosophila melanogaster Meigen, 1830 with antisera against 5-methylcytosine (5mC) and found no evidence for the presence of significant amounts of 5mC. Reactions occur only with 1 of 3 5mC antisera, but they are restricted to nuclear regions without detectable amounts of DNA. The antisera apparently cross-react with other nuclear components. If the murine de novo DNA methyltransferases, DNMT3A and DNMT3B, are expressed under the control of the spermatocyte-specific β2-tubulin promoter in testes, DNA methylation is not increased and no effects on the fertility of the fly are seen. DNA methylation has, therefore, no functional relevance in the male germ line of Drosophila.