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African American (AA) women are often diagnosed with more aggressive breast cancers and have worse survival outcomes than their Caucasian American (CA) counterparts. However, a comprehensive understanding of this disparity remains unclear. In this study, we attempted to identify the race-specific non-invasive protein biomarkers that may particularly benefit interventions aimed at reducing the risk of recurrence and metastasis in breast cancers (BrCa). Our technical strategy has been to discover candidate protein biomarkers in patient sera using a high throughput antibody microarray platform. A total of 240 subjects were selected, composed of controls and all immunohistochemistry-based subtypes of breast cancer cases, subdivided by pre- and post-menopausal status and by race. A global Wilcoxon analysis comparing no-cancer controls and cancer patients identified Pyk2, SAPK/JNK, and phosphatase and tensin homolog as present in higher concentrations in cancer patient serum. A paired t-test revealed that c-kit and Rb are significantly over-represented in AA cancer serum when compared to CA cancer serum. Interestingly, VEGFR2, a protein linked to BrCa metastasis and poor prognosis, was significantly over-represented in AA cancer serum compared to AA controls; however, this was not found in CA cancer serum compared to CA controls, suggesting a possible explanation for the higher incidence of aggressive BrCa in AA versus CA patients. Through examining race-specific differences in the protein landscape of BrCa patient serum, the identified proteins could lay the groundwork for the development of an all-inclusive “liquid mammogram test.”

Reverse phase protein array (RPPA) technology introduced a miniaturized “antigen-down” or “dot-blot” immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.

The goldfish (Carassius auratus) is a widely studied vertebrate model organism for studying cell proliferation in the adult brain, and provide the experimental advantage of growing their body and brain throughout their ∼30-year life time. Cell proliferation occurs in the teleost brain in widespread proliferation zones. Increased cell proliferation in the brain has been linked to the actions of certain antidepressants, including tranylcypromine (TCP), which is used in the treatment of depression. We hypothesized that proliferation zones in the adult goldfish brain can be used to determine the antidepressant effects on cellular proliferation. Here, we report that bromodeoxyuridine (BrdU) labeling over a 24-hr period can be used to rapidly identify the proliferation zones throughout the goldfish brain, including the telencephalon, diencephalon, optic tectal lobes, cerebellum, and facial and vagal lobes. In the first 24 hr of BrdU administration, TCP caused an approximate and significant doubling of labeled cells in the combined brain regions examined, as detected by BrdU immunohistochemistry. TCP caused the greatest increase in cell proliferation in the cerebellum. The normal migratory paths of the proliferating cells within the cerebellum were not affected by TCP treatment. These results indicate that the goldfish provide significant advantages as a vertebrate model for rapidly investigating the effects of antidepressant drugs on cellular proliferation and migration in the normal and injured brain. Anat Rec, 297:1919–1926, 2014. © 2014 Wiley Periodicals, Inc.

Background: Quantitative estimation of circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. However, a simple, straightforward technique is unavailable to estimate the cfDNA in clinical labs. Moreover, the prognostic value of cfDNA in patients with breast cancer (BrCa) is currently under debate. The aim of this study was to develop a simple yet effective quantitative method for measuring the cfDNA in serum and to eventually investigate the relationship between cfDNA and the occurrence of recurrence in BrCa patients. Methods: A total of 240 patient cases (n=240) were selected and are comprised of different subtypes of breast cancer patients and control individuals. We selected 21 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. For the compare studies, each of the recurrent and non-recurrent serum samples was incubated with the SYBR Green I (2 μM). A standard graph was also made with known DNA concentration to calculate the amount of cfDNA in these recurrent and non-recurrent serum samples. Additionally, a comparative study was also performed with the serum of patients with non-recurrent BrCa versus healthy patients. Results: We develop a simple fluorescent based measuring technique which can easily estimate the cfDNA in one step. SYBR Green binds to DNA, and as a result, the fluorescence of SYBR Green increases substantially. Global Wilcoxon analyses were performed to compare the cfDNA amount between non-recurrent and recurrent patients. There is a significant difference in fluorescent intensities between recurrent patients' samples versus non-recurrent patients which are directly proportional to the cfDNA levels. The amount of cfDNA is higher in recurrent patient (ratio is 1.3 up; p= 0.03; AUC=0.76) compared to similar non-recurrent patients. While we compared the fluorescence data between normal/healthy patients versus non-recurrent is turned out as non-significant (healthy to non-recurrent ratio = 1.03; p= 0.20, AUC=0.61). Conclusion: In this current study, we developed a straightforward one-step technique to measure the amount of cfDNA in serum, which can easily translate into a clinical diagnostic tool. To the best of our knowledge, this is the first report which demonstrates serum cfDNA as an early detection marker for recurrent breast cancer patients. The relatively high level of cfDNA in the serum of recurrent breast cancer patients compared to non-recurrent breast cancer patients indicates an uncovered circulating genetic information which triggers the cancer recurrence pathway to relapse cancer in the near future. Citation Format: Bera A, Eidelman O, Russ E, Landa A, Karaian J, Eklund M, Hu H, Pollard HB, Shriver CD, Srivastava M. Circulating cell-free DNA in serum as a marker for the early detection of tumor recurrence in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-26.

Background/Aims: Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function. Methods: We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB. Results: We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation. Conclusion: We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR.

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity at 10q, are commonly observed in most human tumors, including prostate, breast, and kidney cancers. The ANXA7-GTPase is a tumor suppressor, which is frequently inactivated by genomic alterations at 10q21. In the last few years, considerable amounts of data have accumulated describing inactivation of ANXA7-GTPase in a variety of human malignancies and demonstrating the tumor suppressor potential of ANXA7-GTPase. ANXA7-GTPase contains a calcium binding domain that classifies it as a member of the annexin family. The cancer-specific expression of ANXA7-GTPase, coupled with its importance in regulating cell death, cell motility, and invasion, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Recently, emerging evidence suggests that ANXA7-GTPase is a critical factor associated with the metastatic state of several cancers and can be used as a risk biomarker for HER2 negative breast cancer patients. Cross talk between ANXA7, PTEN, and EGFR leads to constitutive activation of PI3K-AKT signaling, a central pathway of tumor cell survival and proliferation. This review focuses on the recent progress in understanding the tumor suppressor functions of ANXA7-GTPase emphasizing the role of this gene in Ca

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity at 10q, are commonly observed in most human tumors, including prostate, breast, and kidney cancers. The ANXA7-GTPase is a tumor suppressor, which is frequently inactivated by genomic alterations at 10q21. In the last few years, considerable amounts of data have accumulated describing inactivation of ANXA7-GTPase in a variety of human malignancies and demonstrating the tumor suppressor potential of ANXA7-GTPase. ANXA7-GTPase contains a calcium binding domain that classifies it as a member of the annexin family. The cancer-specific expression of ANXA7-GTPase, coupled with its importance in regulating cell death, cell motility, and invasion, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Recently, emerging evidence suggests that ANXA7-GTPase is a critical factor associated with the metastatic state of several cancers and can be used as a risk biomarker for HER2 negative breast cancer patients. Cross talk between ANXA7, PTEN, and EGFR leads to constitutive activation of PI3K-AKT signaling, a central pathway of tumor cell survival and proliferation. This review focuses on the recent progress in understanding the tumor suppressor functions of ANXA7-GTPase emphasizing the role of this gene in Ca2+ metabolism, and exploring opportunities for function as an example of a calcium binding GTPase acting as a tumor suppressor and opportunities for ANXA7-GTPase gene cancer therapy.

“Soldier's Heart,” is an American Civil War term linking post-traumatic stress disorder (PTSD) with increased propensity for cardiovascular disease (CVD). We have hypothesized that there might be a quantifiable genetic basis for this linkage. To test this hypothesis we identified a comprehensive set of candidate risk genes for PTSD, and tested whether any were also independent risk genes for CVD. A functional analysis algorithm was used to identify associated signaling networks. We identified 106 PTSD studies that report one or more polymorphic variants in 87 candidate genes in 83,463 subjects and controls. The top upstream drivers for these PTSD risk genes are predicted to be the glucocorticoid receptor (NR3C1) and Tumor Necrosis Factor alpha (TNFA). We find that 37 of the PTSD candidate risk genes are also candidate independent risk genes for CVD. The association between PTSD and CVD is significant by Fisher's Exact Test (P = 3 × 10−54). We also find 15 PTSD risk genes that are independently associated with Type 2 Diabetes Mellitus (T2DM; also significant by Fisher's Exact Test (P = 1.8 × 10−16). Our findings offer quantitative evidence for a genetic link between post-traumatic stress and cardiovascular disease, Computationally, the common mechanism for this linkage between PTSD and CVD is innate immunity and NFκB-mediated inflammation.

Cystic fibrosis (CF) is an autosomal recessive disease due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, F508del-CFTR being the most frequent mutation. The CF lung is characterized by a hyperinflammatory phenotype and is regulated by multiple factors that coordinate its pathophysiology. In CF the expression of CFTR as well as proinflammatory genes are regulated at the level of messenger RNA (mRNA) stability, which subsequently affect translation. These mechanisms are mediated by inflammatory RNA-binding proteins as well as small endogenous noncoding microRNAs, in coordination with cellular signaling pathways. These regulatory factors exhibit altered expression and function in vivo in the CF lung, and play a key role in the pathophysiology of CF lung disease. In this review, we have described the role of mRNA stability and associated regulatory mechanisms in CF lung disease. WIREs RNA 2017, 8:e1408. doi: 10.1002/wrna.1408 For further resources related to this article, please visit the WIREs website.

Objective This paper provides an overview of our study that was designed to assess the health impact of environmental exposures to open pit burning in deployed troops. Methods The rationale for the study and the structure of the research plan was laid out. An overview of each article in the supplement was provided. The cohort of deployed Service members was assessed for airborne exposure, relevant biomarkers, and health outcomes following deployment to Balad, Iraq, and/or Bagram, Afghanistan. Results Polycyclic aromatic hydrocarbon (PAH) exposures were elevated, and serum biomarkers were statistically different postdeployment. Associations were noted between PAHs and dioxins and microRNAs. Some health outcomes were evident in deployers compared with nondeployers. Conclusions Future research will examine the associations between demographic variables, smoking status, biomarker levels, and related health outcomes.

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

Insulin secretion from the pancreatic β-cell is controlled by changes in membrane potential and intracellular Ca(2+). The contribution of intracellular Ca(2+) stores to this process is poorly understood. We have previously shown that β-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP(3) receptors and defects in IP(3)-dependent Ca(2+) signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca(2+) stores in the β-cell, we measured the effects of Ca(2+) mobilizing agents on electrical activity, intracellular Ca(2+) and insulin secretion in control and mutant β-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) β-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca(2+) mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca(2+) stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating β-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.

Purpose: To determine whether curcumin would inhibit IκB kinase β (IKKβ) kinase activity and suppress expression of proinflammatory cytokines in head and neck squamous cell carcinoma cancer (HNSCC) patients. Experimental Design: Saliva was collected before and after subjects chewed curcumin tablets. Protein was extracted and IKKβ kinase activity measured. Interleukin (IL)-6 and IL-8 levels in the salivary supernatants were measured by ELISA. IL-6, IL-8, and other interleukin were also measured independently with ELISA to confirm the inhibitory effect of curcumin on expression and secretion of salivary cytokines. Results: Curcumin treatment led to a reduction in IKKβ kinase activity in the salivary cells of HNSCC patients (P < 0.05). Treatment of UM-SCC1 cells with curcumin as well as with post-curcumin salivary supernatant showed a reduction of IKKβ kinase activity. Significant reduction of IL-8 levels (P < 0.05) was seen in post-curcumin samples from patients with dental caries. Although there was reduced IL-8 expression in 8 of 21 post-curcumin samples of HNSCC patients, the data did not reach statistical significance. Saliva samples from HNSCC patients were also analyzed in a blinded fashion for expression of cytokines. IL-10, IFN-γ, IL-12p70, and IL-2 clustered together, and granulocyte macrophage colony stimulating factor and TNF-α clustered together. Log10 ratio analysis showed decrease in expression of all nine cytokines in both the salivary supernatant and salivary cells of curcumin-treated samples. Conclusions: Curcumin inhibited IKKβ kinase activity in the saliva of HNSCC patients, and this inhibition correlated with reduced expression of a number of cytokines. IKKβ kinase could be a useful biomarker for detecting the effect of curcumin in head and neck cancer. Clin Cancer Res; 17(18); 5953–61. ©2011 AACR.

Purpose—Kidney transplantation is the treatment of choice for end stage renal disease, with long term allograft loss being the major obstacle, and for which potential treatments are based on a histological diagnosis. The problem is that markers for predicting graft rejection are limited in number and invasive and quite non-specific. We have hypothesized that protein biomarkers might be discovered in the urine of patients when acute or chronic rejection might be occurring. Experimental design—We have established a workflow in which initial screening for candidate biomarkers is first performed using urine samples on large scale antibody microarrays. This approach generated several dozen candidates. The next step is to qualify some of the strongest signals using the high throughput Reverse Capture Protein Microarray platform. Results—Four top candidates including ANXA11, Integrin α3 and Integrin β3, and TNFα initially identified by the antibody microarray platform were all qualified using Reverse Capture Protein Microarrays. We also used Receiver Operating Condition (ROC) curves to independently quantify the specificity and sensitivity of these four analytes. Conclusions and clinical relevance—The present data suggest that these novel four analytes in the urine, together or independently, may contribute to a robust and quantitative urine proteomic signature for diagnosing acute or chronic rejection of renal allografts.

Neurological dysfunction after traumatic brain injury (TBI) is caused by both the primary injury and a secondary cascade of biochemical and metabolic events. Since TBI can be caused by a variety of mechanisms, numerous models have been developed to facilitate its study. The most prevalent models are controlled cortical impact and fluid percussion injury. Both typically use "sham" (craniotomy alone) animals as controls. However, the sham operation is objectively damaging, and we hypothesized that the craniotomy itself may cause a unique brain injury distinct from the impact injury. To test this hypothesis, 38 adult female rats were assigned to one of three groups: control (anesthesia only); craniotomy performed by manual trephine; or craniotomy performed by electric dental drill. The rats were then subjected to behavioral testing, imaging analysis, and quantification of cortical concentrations of cytokines. Both craniotomy methods generate visible MRI lesions that persist for 14 days. The initial lesion generated by the drill technique is significantly larger than that generated by the trephine. Behavioral data mirrored lesion volume. For example, drill rats have significantly impaired sensory and motor responses compared to trephine or naïve rats. Finally, of the seven tested cytokines, KC-GRO and IFN-γ showed significant increases in both craniotomy models compared to naïve rats. We conclude that the traditional sham operation as a control confers profound proinflammatory, morphological, and behavioral damage, which confounds interpretation of conventional experimental brain injury models. Any experimental design incorporating "sham" procedures should distinguish among sham, experimentally injured, and healthy/naïve animals, to help reduce confounding factors.

Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology.

Analysis of somatic protein-coding mutations, particularly via tumor exome sequencing, has driven discovery of novel cancer genes in recent years. However, ~97% of the human genome is non-coding and not addressable by exome-targeting methods. Tumor whole genome sequencing, by contrast, allows for genome-wide discovery of somatic events and may identify novel non-coding driver mutations. In conjunction, flexible tools employing principled statistical methods are needed for discovery of recurrent somatic driver mutations from tumor whole genomes. Here, we developed a computational toolset that addresses these needs and describe its application to a novel cohort of lung adenocarcinoma tumors generated by the APOLLO (Applied Proteogenomics OrganizationaL Learning and Outcomes) consortium. Our toolset flexibly computes feature-wise and hotspot somatic mutation enrichment statistics in both protein-coding and non-coding (e.g. promoters, enhancers) genomic regions. Additionally, we incorporated an affinity propagation-based clustering procedure that groups test regions by user-specified genomic covariates that can influence local mutation properties. We applied our toolset to several TCGA datasets of tumor exome sequencing. We found strong agreement between our results and those of other tools: among the top 15 genes called at FDR < 0.01 by MutSigCV, we observed a median overlap of 75.7% with genes ranked in the top 15 by our methods. We next applied our toolset to tumor whole genome sequencing data from our APOLLO lung adenocarcinoma cohort. During analysis, we simultaneously controlled for several genomic covariates, including GC/CpG content, replication timing, and proximal mRNA expression. Gene-centric analysis identified several significantly mutated genes, including KRAS, STK11, and TP53 (FDR < 0.005). We examined matched mRNA-seq data from this cohort and found significant over-expression of KRAS mRNA in samples possessing a non-silent coding mutation in this gene (p < 0.02). We additionally assessed somatic mutation enrichment in promoters, 3' UTRs, non-coding genes, and lung-specific enhancers. From these results, we found significant enrichment of somatic mutations in the body of the long non-coding RNA NEAT1. Among the tumors with somatic NEAT1 mutations, a single sample was hypermutated at this locus and corresponded with the lowest observed RNA expression for this lncRNA in our cohort (TPM of 12.2 vs. overall mean TPM of 84.2). In conclusion, we developed a flexible toolset for interrogating both coding and non-coding landscapes from tumor whole genomes. We applied our methods to a novel cohort of lung adenocarcinoma tumors and identified recurrently mutated genes and non-coding regions, including the NEAT1 lncRNA. Citation Format: Anthony R. Soltis, Coralie Viollet, Harvey B. Pollard, Christopher A. Moskaluk, Robert F. Browning, Clifton L. Dalgard, Craig D. Shriver, Matthew D. Wilkerson. Flexible discovery of recurrent coding and non-coding mutations in tumor whole genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2281.

The tumor suppressor role of annexin-A7 (ANXA7) was previously demonstrated by cancer susceptibility in Anxa7(+/-)-mice and by ANXA7 loss in human cancers, especially in hormone-resistant prostate tumors. To gain mechanistic insights into ANXA7 tumor suppression, we undertook an in vitro study in which we compared wild-type (WT)-ANXA7 and dominant-negative (DN)-ANXA7 effects to a conventional tumor suppressor p53 in prostate cancer cells with different androgen sensitivity. Unlike p53 (which caused cell growth arrest and apoptosis to a noticeable extent in benign PrEC), WT-ANXA7 demonstrated profound cytotoxicityin androgen-sensitive LNCaP as well as in the androgen-resistant DU145 and PC3 prostate cancer cells, but not in PrEC. In androgen-sensitive LNCaP, WT-ANXA7 decreased low-molecular-weight (LMW) AR protein forms and maintained higher retinoblastoma 1 (RB1)/phospho-RB1 ratio. In contrast, DN-ANXA7 (which lacks phosphatidylserine liposome aggregation properties) increased LMW-AR forms and hyperphosphorylated RB1 that was consistent with the lack of DN-ANXA7 cytotoxicity. According to the microarray-based Ingenuity Pathways Analysis, a major WT-ANXA7 effect in androgen-sensitive LNCaP constituted of upregulation of the RB1-binding transcription factor E2F1 along with its downstream proapoptotic targets such as ASK1 and ASPP2. These results suggested a reversal of the RBdependent repression of the proapoptotic E2F-mediated transcription. However, DN-ANXA7 increased RB1/2 (but not E2F1) expression and induced the proliferation-promoting ERK5, thereby maintaining the RB-dependent repression of E2F-mediated apoptosis in LNcaP. On the other hand, in androgen-resistant cells, WT-ANXA7 tumor suppressor effects involved PTEN and NFkB pathways. Thus, ANXA7 revived the RB-associated cell survival control and overcame androgen resistance and dysfunctional status of major tumor suppressors commonly mutated in prostate cancer. Published 2009 UICC.

The most common mutation in cystic fibrosis (CF) is DeltaF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked DeltaF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of DeltaF508-CFTR. The half-life of DeltaF508-CFTR is also dramatically enhanced. Dexamethasone-activated DeltaF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and DeltaF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects DeltaF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated DeltaF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.

Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (Abeta). We have hypothesized that the intrinsic Abeta calcium channel activity of the oligomeric Abeta polymer may be responsible for the neurotoxic properties of Abeta, and that Abeta channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the Abeta channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the Abeta channel and protect neurons from Abeta toxicity. Both block the Abeta channel with similar potency (approximately 500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery.

In a previous study of sodium 4-phenylbutyrate (4-PBA)-responsive proteins in cystic fibrosis (CF) IB3-1 bronchial epithelial cells, we identified 85 differentially expressed high abundance proteins from whole cellular lysate (Singh, O. V., Vij, N., Mogayzel, P. J., Jr., Jozwik, C., Pollard, H. B., and Zeitlin, P. L. (2006) Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells. J. Proteome Res. 5, 562–571). In the present work we hypothesize that a subset of heat shock proteins that interact with cystic fibrosis transmembrane conductance regulator (CFTR) in common during chemical rescue and genetic repair will identify therapeutic networks for targeted intervention. Immunocomplexes were generated from total cellular lysates, and three subcellular fractions (endoplasmic reticulum (ER), cytosol, and plasma membrane) with anti-CFTR polyclonal antibody from CF (IB3-1), chemically rescued CF (4-PBA-treated IB3-1), and genetically repaired CF (IB3-1/S9 daughter cells repaired by gene transfer with adeno-associated virus-(wild type) CFTR). CFTR-interacting proteins were analyzed on two-dimensional gels and identified by mass spectrometry. A set of 16 proteins known to act in ER-associated degradation were regulated in common and functionally connected to the protein processing, protein folding, and inflammatory response. Some of these proteins were modulated exclusively in ER, cytosol, or plasma membrane. A subset of 4-PBA-modulated ER-associated degradation chaperones (GRP94, HSP84, GRP78, GRP75, and GRP58) was observed to associate with the immature B form of CFTR in ER. HSP70 and HSC70 interacted with the C band (mature form) of CFTR at the cell surface. We conclude that chemically rescued CFTR associates with a specific set of HSP70 family proteins that mark therapeutic interactions and can be useful to correct both ion transport and inflammatory phenotypes in CF subjects.

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2-DE and/or MS need to be complemented by different kinds of technologies. The well-known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody-centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label-free detection technologies, human genome-scale numbers of analytes, and increases in analytic and bioinformatic speeds.

Tumor suppressor function of ubiquitously expressed Annexin-A7, ANXA7 (10q21) that is involved in exocytosis and membrane fusion was based on cancer prone phenotype in Anxa7(+/-) mice as well as ANXA7 role in human prostate and breast cancers. To clarify ANXA7 biomarker and tumor suppressor function, we analyzed its expression pattern in comparison to the prostate-specific biomarker NKX3.1. Immunohistochemistry-based ANXA7 and NKX3.1 protein expression was analyzed on human tissue microarrays of 4,061 specimens from a wide spectrum of the histopathologically well-characterized tumors in different stages compared to corresponding normal tissues. Decreased ANXA7 expression was mostly associated with high invasive potential in multiple tumors. Although some metastases retained relatively high ANXA7 rates compared to primary cancer tissues, the lymph node metastases from different sites (including prostate and breast) had decreased ANXA7 expression in comparison to the intact lymphatic tissues. Major ANXA7 downregulation pattern was deviated in tumors of glandular (especially neuroendocrine) origin. ANXA7 and NKX3.1 proteins were synexpressed in the male urogenital system and adrenal gland. Gene expression profiling in prostate and breast cancers (SMD) revealed distinct hormone-related profiles for NKX3.1 and ANXA7, where ANXA7 expression correlated with steroid sulfatase which has a pivotal role in steroidogenesis. Abundant protein presence in adrenal gland and its loss in hormone-refractory prostate cancer indicated that ANXA7 can be relevant to steroidogenesis and androgen sensitivity in particular. With tumor suppressor pattern validated in different tumors, ANXA7 can be an attractive diagnostic and therapeutic target associated with the hormone and/or neurotransmitter-mediated modulation of tumorigenesis.

Over the past 15 years, the emerging field of network science has revealed the key features of brain networks, which include small-world topology, the presence of highly connected hubs, and hierarchical modularity. The value of network studies of the brain is underscored by the range of network alterations that have been identified in neurological and psychiatric disorders, including epilepsy, depression, Alzheimer's disease, schizophrenia, and many others. Here we briefly summarize the concepts of graph theory that are used to quantify network properties and describe common experimental approaches for analysis of brain networks of structural and functional connectivity. These range from tract tracing to functional magnetic resonance imaging, diffusion tensor imaging, electroencephalography, and magnetoencephalography. We then summarize the major findings from the application of graph theory to nervous systems ranging from Caenorhabditis elegans to more complex primate brains, including man. Focusing, then, on studies involving the amygdala, a brain region that has attracted intense interest as a center for emotional processing, fear, and motivation, we discuss the features of the amygdala in brain networks for fear conditioning and emotional perception. Finally, to highlight the utility of graph theory for studying dysfunction of the amygdala in mental illness, we review data with regard to changes in the hub properties of the amygdala in brain networks of patients with depression. We suggest that network studies of the human brain may serve to focus attention on regions and connections that act as principal drivers and controllers of brain function in health and disease.

A central problem in cellular neurobiology is how the process of exocytosis of transmitters and hormones is regulated at the molecular level. Calcium is important in the process and may act by initiating the formation of fusion complexes of secretory granules to each other and to plasma membranes. We have discovered and isolated a new 47,000 MW protein (synexin) from adrenal medulla tissue that fuses chromaffin granule membranes only in the presence of calcium. Synexin activity was detected in a number of secretory tissues including human platelets and bovine brain, and the synexin molecule was found by immunofluorescent cytochemistry to be localized to the cytoplasm of chromaffin cells. Purified synexin molecules were found to self-associate in the presence of Ca++ to form paracrystalline arrays of 50 X 150 A rods, and the association was dependent on [Ca++] in an identical fashion to the Ca++ dependence of granule membrane fusion. On the basis of these data we suggest that synexin may be the intracellular receptor for calcium during exocytosis. However, the actual release event of 'fission' of the secretory vesicle-plasma membrane complex did not appear to be related to synexin action, and we have considered the hypothesis that the chemiosmotic mechanism for ATP, Cl--dependent chromaffin granule lysis might provide the necessary localized force. We have now shown that the granule model successfully predicts the secretory properties of human platelets, bovine parathyroid cells, and bovine chromaffin cells. Like the granule lysis system, secretion from these cells required specific anions in the medium was inhibited by anion transport blocking drugs and proton ionophores, and was suppressed by elevated osmotic strength. We suggest that secretory granules fused to plasma membranes in secreting cells, perhaps by synexin, may experience net solute uptake and subsequently undergo local, outwardly directed osmotic lysis, or exocytosis.

Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

The non-human primate (NHP) brain closely parallels the human brain in terms of both circuitry, and of the specific functional regions which form connections within the circuits. Using conventional manufacturing technology, we constructed a Brain Block Slicer tailored to the structure of the NHP brain, and used it to make precise and reproducible coronal slices for next generation sequencing-based transcriptome analysis. Here we use computed tomography to scan the original brain block, and to use additive manufacturing to produce a 3-D printed NHP Brain Slicer that is fully comparable to the original. The block is easily scalable to primate brains of different sizes, and different slicing geometries can be deployed in silico, including sagittal and horizontal, in addition to the coronal geometry shown in the present printed brain block.

A serum proteomics platform enabling expression Profiling in transplantation-associated clinical subsets gives an opportunity to identify non-invasive biomarkers that can accurately predict transplant outcome. In this study, we attempted to identify candidate serum biomarkers that could predict kidney allograft rejection/injury, regardless of its etiological and therapeutic heterogeneity. Using serum samples collected from kidney transplantation patients and healthy controls, we first employed Clontech-500 Ab microarrays to Profile acute rejection (AR) and chronic graft injury (CGI) versus stable graft function (SF) and normal kidneys (NK). Using GenePattern analysis of duplicate arrays on pooled samples, we identified gender-independent biomarkers PARP1, MAPK1, SRP54, DP1, and p57 (FDR ≈ 25%), the concordant downregulation of which represented a detrimental Profile common for both rejection/ injury types (AR-CGI). The reverse phase arrays qualified a 2-fold upregulation of PARP1 with an ROC of 0.87 in individual samples from patients with SF vs. AR-CGI rendering serum PARP1 as a biomarker for early prognosis. Ingenuity Pathways Analysis (IPA) connected PARP1 to some other markers (MAPK1), elucidating their possible interactions and connections to the immune response and graft-versus-host disease signaling. The downregulation of serum PARP1 in the damaged graft tissues, represents a perspective non-invasive marker, predicting the failing kidney graft, regardless of rejection/injury causes or gender. Thus, the successful identification of PARP1 as a bio-marker in limited patient cohorts demonstrates that serum proteomics platform empowered by the GenePattern- and IPA-based Bioinformatics algorithm can guarantee a successful development of the clinically applicable prognostic biomarker panel.

Tocopherol succinate (TS) has been shown to protect mice against acute radiation syndrome, however, its exact mechanism of action and its possible use in humans has not yet been evaluated. Our approach has been to test the radioprotectant properties of TS on CD34-positive stem cells from healthy volunteers. We hypothesize that a radioproteomics strategy can identify a drug-dependent, personalized proteomics signature for radioprotection. To directly test the radioproteomics hypothesis, we treated human CD34-positive stem cells with 20 μM TS for 24 h, and then exposed the cells to 2 Gy of cobalt-60 gamma-radiation. We isolated protein from all cultures and used a high throughput Antibody Microarray (AbMA) platform to measure concentrations of 725 low abundance proteins. As an in vivo control, we also tested mouse CD34-positive stem cells using the same preemptive TS paradigm on progenitor colony forming units. TS pretreatment of in vitro or in vivo CD34-positive stem cells rescued radiation-induced loss of colony-forming potential of progenitors. We identified 50 of 725 proteins that could be preemptively rescued from radiation-induced reduction by pretreatment with TS. Ingenuity Pathway Analysis (IPA) reveals that the modified proteins fall into categories dominated by epigenetic regulation, DNA repair, and inflammation. Our results suggest that radioproteomics can be used to develop personalized medicine for radioprotection using protein signatures from primary CD34-positive progenitors derived from the patient or victim prior to radiation exposure. The protective effect of TS may be due to its ability to preemptively activate epigenetic mechanisms relevant to radioprotection and to preemptively activate the programs for DNA repair and inflammation leading to cell survival.

Tumor suppressor function for Annexin A7 (ANXA7; 10q21) is based on cancer-prone phenotype in Anxa7(+/−) mouse and ANXA7 prognostic role in human cancers. Because ANXA7-caused liposome aggregation can be promoted by arachidonic acid (AA), we hypothesized that the phospholipid-binding tumor suppressor ANXA7 is associated with AA cascade. In a comparative study of ANXA7 versus canonical tumor suppressor p53 effects on AA lipoxygenation pathway in the p53-mutant and androgen-insensitive DU145 prostate cancer cells, both tumor suppressors altered gene expression of major 5-lipoxygenase (LOX) and 15-LOXs, including response to T helper 2 (Th2)-cytokine [interleukin-4 (IL-4)] and endogenous steroids (mimicked by dexamethasone). Wild-type and mutant ANXA7 distinctly affected expression of the dexamethasone-induced 15-LOX-2 (a prostate-specific endogenous tumor suppressor) as well as the IL-4-induced 15-LOX-1. On the other hand, wild-type p53 restored 5-LOX expression in DU145 to levels comparable to benign prostate epithelial cells. Using mass spectrometry of DNA affinity–enriched nuclear proteins, we detected different proteins that were bound to adjacent p53 and estrogen response elements in the 5-LOX promoter in DU145 cells introduced with ANXA7 versus p53. Sex hormone regulator 17-β hydroxysteroid dehydrogenase 4 was identified under p53 introduction, which induced the 5-LOX expression. Meantime, nuclear proteins bound to the same 5-LOX promoter site under introduction of ANXA7 (that was associated with the repressed 5-LOX) were identified as zinc finger proteins ZNF433 and Aiolos, pyrin domain–containing NALP10, and the p53-regulating DNA repair enzyme APEX1. Thus, ANXA7 and p53 can distinctly regulate LOX transcription that is potentially relevant to the AA-mediated cell growth control in tumor suppression. (Cancer Res 2006; 66(19): 9609-16)

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFkappaB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.

4-Phenylbutyrate (4-PBA) is an oral butyrate derivative that has recently been approved for treatment of urea cycle disorders and is under investigation in clinical trials of cancer, hemoglobinopathies, and cystic fibrosis (CF). We hypothesized that proteome profiling of IB3-1 cystic fibrosis bronchial epithelial cells treated with 4-PBA would identify butyrate-responsive cellular chaperones, protein processing enzymes, and cell trafficking molecules associated with the amelioration of the chloride transport defect in these cells. Protein profiles were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Over a pI range of 4-7 and molecular weight from 20 to 150 kDa a total of 85 differentially expressed proteins were detected. Most of the identified proteins were chaperones, catalytic enzymes, and proteins comprising structural elements, cellular defense, protein biosynthesis, trafficking activity, and ion transport. Subsets of these proteins were confirmed by immunoblot analysis. These data represent a first-draft of the pharmacoproteomics map of 4-PBA treated cystic fibrosis bronchial epithelial cells.

We have developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods (two-dimensional gel electrophoresis and mass spectrometry) to identify ORFs that when expressed are likely to yield high levels of soluble, purified protein. Because the method works on pools of ORFs, the procedures needed to subclone, express, purify, and assay protein expression for hundreds of clones are greatly simplified. Small scale expression and purification of 12 positive clones identified by POET from a pool of 688 Caenorhabditis elegans ORFs expressed in Escherichia coli yielded on average 6 times as much protein as 12 negative clones. Larger scale expression and purification of six of the positive clones yielded 47–374 mg of purified protein/liter. Using POET, pools of ORFs can be constructed, and the pools of the resulting proteins can be analyzed and manipulated to rapidly acquire information about the attributes of hundreds of proteins simultaneously.

Cystic fibrosis (CF) is a common, lethal genetic disease, which is due to mutations in the CFTR gene. The CF lung expresses a profoundly proinflammatory phenotype, due to constitutive hypersecretion of IL-8 from epithelial cells lining the airways. In a systematic search for candidate drugs that might be used therapeutically to suppress IL-8 secretion from these cells, we have identified a potent and efficacious series of amphiphilic pyridinium salts. The most potent of these salts is MRS2481, an (R)-1-phenylpropionic acid ester, with an IC50 of ca. 1microM. We have synthesized 21 analogues of MRS2481, which have proven sufficient to develop a preliminary structure-activity relationship (SAR). For optimal activity, we have found that the ester must be connected to the pyridinium derivative by an eight-carbon chain. An optical isomer of the lead compound, containing an (S)-1-phenylpropionic acid ester, has been found to be a much less active. The mechanism of action of MRS2481 appears to involve inhibition of signaling of the NF(kappa)B and AP-1 transcription factors to the IL-8 promoter. MRS2481 is a potent inhibitor of TNFalpha-induced phosphorylation and proteosomal destruction of I(kappa)B(alpha). Inasmuch as I(kappa)B(alpha) is the principal inhibitor of the NF(kappa)B signaling pathway, preservation of intact I(kappa)B(alpha) would serve to keep the IL-8 promoter silent. We also find that MRS2481 blocks TNF(alpha)-activated phosphorylation of JNK, the c-JUN kinase. The IL-8 promoter is also activated by an AP-1 site, which requires a phospho-c-JUN/c-FOS dimer for activity. We therefore interpret these data to suggest that the mechanism of MRS2481 action is to inhibit both NF(kappa)B and AP-1 signaling on the IL-8 promoter. Given the medicinally promising properties of water-solubility, potency in the low muM concentration range, and high efficacy, we anticipate that MRS2481, or a further optimized derivative, may find an important place in the armamentarium of pharmaceutical strategies yet to be arrayed against the inflammatory phenotype of the CF lung.

Protein profiles of cultured cystic fibrosis (CF) lung epithelial cells were analyzed by two-dimensional gel electrophoresis and mass spectrometry (MS). The analysis gave rise to a protein map over the pI range of 4-7, and a molecular weight range of ca. 100-10 kDa. The map contains 194 identified proteins, which were detectable by silver stain. All silver stained features were identified by matrix-assisted laser desorption/ionization-time of flight MS of tryptic peptides. Some proteins were found to be represented by multiple features on the 2-D gel. Among the high abundance proteins identified were sets of proteins associated with inflammation, including the classical NFkappaB, p65 (RelA) and NFkappaB, p65 (RelB). We suggest that this composite atlas of the high abundance CF lung epithelial proteome will serve as a reference database for future studies of candidate CF drugs, validating different approaches to CFTR gene therapy, and analogous investigations of other types of human lung disorders.

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.

Loss of heterozygosity (LOH) in the 10q21 region that harbors the tumor suppressor gene ANX7 -GTPase gene have been found in 35% of prostate tumors. Therefore, the rationale for this study is that this gene could also be implicated in breast pathogenesis as well. We investigated allelic losses in microsatellites of the 10q21 region, and their correlations with ANX7 status, estrogen receptor (ER) status, progesterone receptor (PR) status, Ki-67 status and pathological phenotype in 30 breast carcinomas with matched control specimens. The LOH analysis was performed by amplifying DNA by PCR, using four markers of the 10q21 region (AFMa299ya5, AFM220xe5, AFM 063xc5, AFM200wf4). LOH in at least one marker of the 10q21 region (AFM220xe5 marker close to ANX7) was found in 66% of the first set of informative tumors containing 10 pairs of specimens. Subsequent comparison between 20 carcinomas using AFM220xe5, with and without LOH in terms of pathological parameters showed significant associations with differences in age ( P =0.04), ER ( P =0.05), Ki-67 ( P =0.04) and PR ( P =0.01); a trend toward significance was found for tumor size ( P =0.06) and histological grade III ( P =0.14). These results suggest that the ANX7 gene, or other genes of the 10q21 region, could be functionally related to breast cancer, probably influencing the hormone receptor expression associated with poor prognosis during development.

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein IL-8, and the high levels of IL-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of IL-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-κB (IκBα). IκBα phosphorylation is a required step in the activation of the NF-κB signaling pathway and the subsequent expression of IL-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P< 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing IL-8-dependent lung inflammation in CF.

Purpose: ANX7-GTPase located on chromosome 10q21 is significantly altered and associated with hormone-refractory metastatic prostate cancers. Therefore, we investigated whether levels of ANX7 correlate with breast cancer progression and survival Experimental Design: A diagnostic tumor tissue microarray containing 525 human breast tissue specimens at different stages of the disease was assayed for ANX7 using immunocytochemical methods with ANX7 monoclonal antibody. A separate prognostic tumor tissue microarray containing 553 human breast tissue specimens annotated with clinicopathological parameters was assayed for ANX7, HER2, estrogen receptor, progesterone receptor, and p53 protein. Results: We report here for the first time that the expression of ANX7-GTPase is significantly enhanced and associated with the presence of metastatic disease (P < 0.0001) in the 525 human breast tissue specimens analyzed. Furthermore, using a separate 553 case retrospective prognostic tumor tissue microarray, we found that increased ANX7 expression is also significantly associated with poor overall patient survival (P < 0.014). This is particularly true when restricted to patients in whom the BRE clinical grade is 2 (P < 0.001) or for whom there is a lack of HER2 expression (P < 0.002). Finally, Cox regression analysis shows that as the expression of ANX7 rises, the probability of survival decreases by more than 10-fold for those patients with HER2-negative tumors. These latter patients represented 66% of the population affected with breast cancer in this study. Conclusions: High levels of ANX7 in tumor correlate strongly with poor survival of HER2-negative patients and the most aggressive forms of breast cancer. This is the first study to demonstrate that ANX7 antibody has the potential for development into an in vivo diagnostic and therapeutic tool. This simple and reliable immunohistochemical assay may therefore become an important biomarker for metastatic breast cancer diagnosis and management of HER2-negative breast tumor patients.

Annexin 7 (ANX7) acts as a tumor suppressor gene in prostate cancer, where loss of heterozygosity and reduction of ANX7 protein expression is associated with aggressive metastatic tumors. To investigate the mechanism by which this gene controls tumor development, we have developed an Anx7 (+/-) knockout mouse. As hypothesized, the Anx7 (+/-) mouse has a cancer-prone phenotype. The emerging tumors express low levels of Anx7 protein. Nonetheless, the wild-type Anx7 allele is detectable in laser-capture microdissection-derived tumor tissue cells. Genome array analysis of hepatocellular carcinoma tissue indicates that the Anx7(+/-) genotype is accompanied by profound reductions of expression of several other tumor suppressor genes, DNA repair genes, and apoptosis-related genes. In situ analysis by tissue imprinting from chromosomes in the primary tumor and spectral karyotyping analysis of derived cell lines identify chromosomal instability and clonal chromosomal aberrations. Furthermore, whereas 23% of the mutant mice develop spontaneous neoplasms, all mice exhibit growth anomalies, including gender-specific gigantism and organomegaly. We conclude that haploinsufficiency of Anx7 expression appears to drive disease progression to cancer because of genomic instability through a discrete signaling pathway involving other tumor suppressor genes, DNA-repair genes, and apoptosis-related genes.

A combination of immuno-electron microscopy and electron energy-loss spectrum-imaging was used to map the distributions of endocrine polypeptide hormones and proteins in mouse pancreatic islet of Langerhans. Tissue was analyzed from control animals and from mice that were heterozygous for the Anx7 gene, which defines a Ca2+/GTP-dependent membrane fusion and ion channel protein. The heterozygous Anx7 (+/-) mouse displays defects in IP3 receptor mediated Ca2+ signaling and insulin secretion. Therefore, information was obtained about the distributions of the hormones insulin and glucagon, as well as the proteins ANX7 and the IP3 receptor. Insulin secretion appears to be defective in the mutants. It was found from immunolabeling experiments that expression of the IP3 receptor is reduced in mutant islets compared to control islets. Subcellular distributions of sulfur and nitrogen obtained by electron energy-loss spectrum-imaging showed that the insulin concentrations of beta granules were essentially the same in control and mutant islets. By contrast, immunogold labeling of mutant islets shows more insulin immunoreactivity in the beta granules. It follows that insulin may be packaged differently in mutant islets, making antigenic determinants more available to the labeling antibody. The increased rate of insulin secretion in the hyperplastic mutant islets can be explained by compensatory increases in islet size, rather than by an increased insulin concentration in the beta cells. The results indicate that reduced ANX7 expression leads to defects in the IP3 receptor expression in the endocrine cells of the mutant mouse. Increased size of the islet or of adrenal medulla may be a compensatory mechanism for secretion defect by individual endocrine cells. Defects in IP3 receptor expression, and documented consequences of a Ca2+ signaling defect, lead to other changes in organelles such as the mitochondrial number in islet beta-cells. The effects and consequences of reduced ANX7 expression on mitochondria are evident in ultrastructural observations.

Background: Accurate detection of somatic mutations is critical for informing targeted therapy options. Prevalent non-cancer cell admixture complicates this detection in breast cancer. Conventional mutation detection relies on DNA sequencing; however in prior work, we demonstrated that combining RNA and DNA sequencing increases mutation signal strength, or mutant allele fraction (MAF). The ratio of RNA MAF versus DNA MAF (RNA:DNA MAF) was greatest in low purity breast tumors. We hypothesized that this elevation is biologically driven and would be conserved in a second, distinct tissue specimen of the same tumors. Here, we compare mutation characteristics between two tissue blocks in a cohort of breast tumors (n = 8) to evaluate possible preservation of RNA versus DNA mutation signal throughout the tumor. Methods: We selected four high purity and four low purity breast tumors (“Block1”) from The Cancer Genome Atlas (TCGA) cohort and associated ABSOLUTE purity analysis. For these tumors, we acquired a second tissue block (“Block2”) not analyzed by TCGA, cut sections, analyzed sections by H&E stains, and extracted nucleic acids. Whole genome DNA sequencing and mRNA sequencing was performed for Block2 specimens using Illumina X and NextSeq 500 sequencers, respectively. Somatic mutations in Block2 were detected using UNCeqR and compared to published UNCeqR somatic mutations from TCGA. We then evaluate MAF characteristics in the entire TCGA breast tumor cohort (n = 695). Results: Tumor purity estimates, determined by histology and by sequencing, were reduced in Block2 of the low purity tumor set versus the high purity tumor set, consistent with Block1 analysis. Molecular properties of genome-wide gene expression and somatic DNA copy number were highly similar between block-mated specimens (p < 0.01). We then identified expressed mutations present in Block1 and Block2 of the same tumor and compared the MAFs on these common mutations. DNA MAF and RNA MAF were each significantly correlated between Block1 and Block2 (p < 1e-12 in both cases). The average RNA:DNA MAF was 2.5 for the cohort, indicating that RNA mutation signal is greater than DNA in general. In Block2 specimens, the RNA:DNA MAFs were significantly greater in the low purity tumor set than the high purity tumor set (mean 2.7 versus 2.1, p < 6e-5), reflecting the same trend observed in Block1 specimens. Analyzing the entire TCGA cohort, RNA:DNA MAF was positively correlated with proliferation pathway gene expression (p < 3e-16 ) and was greatest in the Basal subtype versus other subtypes (p < 2e-9). Conclusion: Mutant allele fraction both of DNA and of RNA was conserved across breast tumor subsections. Low purity and basal subtype breast tumors had elevated RNA:DNA MAF supporting a relationship to underlying biology and identifying classes of tumors with pronounced benefit for DNA and RNA integrated mutation analysis. Citation Format: Jerez Te, Coralie Viollet, Xijun Zhang, Jatinder Singh, Jeffrey A. Hooke, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Clifton L. Dalgard, Matthew D. Wilkerson. Reproducible elevation of RNA versus DNA mutation signal in low purity breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2017-5403

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1β is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.