Cluster analysis of immunohistochemical profiles delineates CK7, vimentin, S100A1 and C‐kit (CD117) as an optimal panel in the differential diagnosis of renal oncocytoma from its mimics

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106819/1/j.1365-2559.2011.03753.x.pd

Epidermal Growth Factor Receptor Expression and Mutational Analysis in Synovial Sarcomas and Malignant Peripheral Nerve Sheath Tumors

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140003/1/onco0459.pd

Histologic Alterations from Neoadjuvant Chemotherapy in High‐Grade Extremity Soft Tissue Sarcoma: Clinicopathological Correlation

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139918/1/onco0451.pd

Notch signaling regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5+ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5+ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.SynopsisThe Notch signaling pathway is required to maintain LGR5+ antral stem cells and epithelial cell homeostasis.Gastric antral stem cells display active Notch1 receptor signaling.Global Notch inhibition reduces stem and progenitor cell proliferation and increases differentiation of all lineages.Notch activation in LGR5+ stem cells increases stem and progenitor cell proliferation and inhibits differentiation.Notch activation enhances antral stem cell function, leading to tissue expansion via gland fission and tumor formation.The Notch signaling pathway is required to maintain LGR5+ antral stem cells and epithelial cell homeostasis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/1/embj201490583-sup-0002-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/2/embj201490583.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/3/embj201490583.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/4/embj201490583-sup-0001-Appendix.pd

Reduced selenium-binding protein 1 expression is associated with poor outcome in lung adenocarcinomas

The effects of selenium, an essential nutrient with anti-carcinogenic properties, are mediated by selenium-binding proteins. The protein expression status of human selenium-binding protein 1 (SBP1) in human tumours and the exact function of this protein are not known. In this study, quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used on 93 lung adenocarcinomas and ten uninvolved lung samples. Two likely isoforms of a 56 kD protein that showed a significantly decreased abundance in lung adenocarcinomas were observed. Tandem mass spectrometry and 2-D western blot analysis identified these two proteins as human SBP1. Tumour tissue microarrays were utilized to examine the cellular expression patterns of SBP1 using immunohistochemistry. The same tissue samples were examined for SBP1 mRNA expression using oligonucleotide microarrays. Two major SBP1 isoforms were detected, with an acidic isoform (457) being significantly down-regulated in lung adenocarcinomas compared with normal lung ( p = 0.02). Two additional more acidic SBP1 isoforms were only observed in normal lung. SBP1 protein isoforms and SBP1 mRNA levels were significantly decreased in poorly differentiated (versus moderately and well-differentiated), T2–T4 (versus T1), and bronchus-derived (versus bronchioloalveolar) tumours. Low levels of SBP1 protein (native form, 460) correlated significantly with poor survival ( p = 0.007). The lack of SBP1 expression was not due to gene deletion. Treatment of A549 lung adenocarcinoma cells with the methylation inhibitor 5-azacytidine did not affect expression of the SBP1 protein. Analysis of the tumour proliferation status using Ki-67 suggests that down-regulated expression of SBP1 may reflect increased cell proliferation and decreased differentiation in lung adenocarcinomas. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34487/1/1524_ftp.pd

Site-specific protein photochemical covalent attachment to carbon nanotube side walls and its electronic impact on single molecule function

Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of four different proteins, including the fluorescent protein GFP and a β-lactamase binding protein (BBP), to carbon nanotube side walls. AFM showed that on attachment BBP could still recognize and bind additional protein components. Single molecule fluorescence revealed that on attachment to SWCNTs function was retained and there was feedback to GFP in terms of fluorescence intensity and improved resistance to photobleaching; GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the chromophore having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein–CNT hybrid bioconjugates. It can be potentially applied to any protein of choice; the attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position

Abstract 3568: CYP3A4 epoxygenase activity mediates ER+ mammary tumor growth and angiogenesis, in part, through EET biosynthesis and is inhibited by biguanides

Abstract: While cytochrome P450 enzymes (CYPs) are implicated in tumor angiogenesis through biosynthesis of epoxyeicosatrienoic acids (EETs), little is known about breast cancer cell-intrinsic CYPs that exhibit epoxygenase activity, such as CYP3A4. In an orthotopic breast cancer model, silencing of epithelial CYP3A4 suppressed angiogenesis-related escape of ER+ breast tumors from dormancy. While the diabetes drug metformin inhibits mitochondrial complex I and inhibits tumor growth, how it does so is unknown. Metformin inhibited CYP epoxygenase activity and co-crystallized in the active site of CYP3A4, hydrogen bonding with arginine 212, allowing the development of hexyl-benzyl-biguanide (HBB) as a CYP3A4 inhibitor using molecular modeling. HBB exhibited more than 10-fold greater potency than metformin for inhibition of ER+ mammary tumor growth and inhibited associated tumor angiogenesis. HBB inhibited EET biosynthesis ∼40-fold more potently than metformin and was ∼40-fold more potent for activation of AMPK phosphorylation. EETs suppressed and CYP silencing promoted AMPK phosphorylation, linking CYPs with AMPK regulation in breast cancer. HBB depolarized mitochondria, reduced oxygen consumption rates and suppressed the Warburg effect, while EETs restored the mitochondrial membrane potential. CYP3A4 silencing and HBB treatment increased reactive oxygen species (ROS) production, suggesting that CYPs suppress cancer cell death, in part, through suppression of ROS. CYP3A4 silencing sensitized breast cancer cells to hormonal therapy and chemotherapy, abrogated by EETs. Because EETs are autocrine, paracrine and endocrine, these results implicate CYPs in tumor growth, in part, through cell-cell mediation of mitochondrial homeostasis and demonstrate the potential of CYP3A4 as a therapeutic target in breast cancer. Citation Format: Zhijun Guo, Irina F. Sevrioukova, Eric Hanse, Ilia Denisov, Xia Zhang, Ting-Lan Chiu, Daniel Swedien, Justin Stamschror, Juan Alvarez, William Marerro Ortiz, Monique Morgan, Michael Maher, Kathryn J. Chavez, Dafydd Thomas, Young Kyung Bae, Jonathan Henriksen, Beverly Norris, Robert J. Schumacher, Henry Wang, Robin Bliss, Haitao Chu, Rebecca Cuellar, Thomas L. Poulos, Stephen G. Sligar, William Atkins, Stephen Schmechel, Jorge Capdevila, John Falck, Ian Blair, Jeffrey P. Jones, Gunda Georg, Kalpna Gupta, Ameeta Kelekar, Elizabeth Amin, David A. Potter. CYP3A4 epoxygenase activity mediates ER+ mammary tumor growth and angiogenesis, in part, through EET biosynthesis and is inhibited by biguanides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3568. doi:10.1158/1538-7445.AM2015-356

Analogues of Marine Guanidine Alkaloids Are in Vitro Effective against Trypanosoma cruzi and Selectively Eliminate Leishmania (L.) infantum Intracellular Amastigotes

Synthetic analogues of marine sponge guanidine alkaloids showed in vitro antiparasitic activity against Leishmania (L.) infantum and Trypanosoma cruzi. Guanidines 10 and 11 presented the highest selectivity index when tested against Leishmania. The antiparasitic activity of 10 and 11 was investigated in host cells and in parasites. Both compounds induced depolarization of mitochondrial membrane potential, upregulation of reactive oxygen species levels, and increased plasma membrane permeability in Leishmania parasites. Immunomodulatory assays suggested an NO-independent effect of guanidines 10 and 11 on macrophages. The same compounds also promoted anti-inflammatory activity in L. (L.) infantum-infected macrophages cocultived with splenocytes, reducing the production of cytokines MCP-1 and IFN-γ. Guanidines 10 and 11 affect the bioenergetic metabolism of Leishmania, with selective elimination of parasites via a host-independent mechanism

The Role of Whole Blood Impedance Aggregometry and Its Utilisation in the Diagnosis and Prognosis of Patients with Systemic Inflammatory Response Syndrome and Sepsis in Acute Critical Illness

Objective: To assess the prognostic and diagnostic value of whole blood impedance aggregometry in patients with sepsis and SIRS and to compare with whole blood parameters (platelet count, haemoglobin, haematocrit and white cell count). Methods: We performed an observational, prospective study in the acute setting. Platelet function was determined using whole blood impedance aggregometry (multiplate) on admission to the Emergency Department or Intensive Care Unit and at 6 and 24 hours post admission. Platelet count, haemoglobin, haematocrit and white cell count were also determined. Results: 106 adult patients that met SIRS and sepsis criteria were included. Platelet aggregation was significantly reduced in patients with severe sepsis/septic shock when compared to SIRS/uncomplicated sepsis (ADP: 90.7±37.6 vs 61.4±40.6; p<0.001, Arachadonic Acid 99.9±48.3 vs 66.3±50.2; p = 0.001, Collagen 102.6±33.0 vs 79.1±38.8; p = 0.001; SD ± mean)). Furthermore platelet aggregation was significantly reduced in the 28 day mortality group when compared with the survival group (Arachadonic Acid 58.8±47.7 vs 91.1±50.9; p<0.05, Collagen 36.6±36.6 vs 98.0±35.1; p = 0.001; SD ± mean)). However haemoglobin, haematocrit and platelet count were more effective at distinguishing between subgroups and were equally effective indicators of prognosis. Significant positive correlations were observed between whole blood impedance aggregometry and platelet count (ADP 0.588 p<0.0001, Arachadonic Acid 0.611 p<0.0001, Collagen 0.599 p<0.0001 (Pearson correlation)). Conclusions: Reduced platelet aggregometry responses were not only significantly associated with morbidity and mortality in sepsis and SIRS patients, but also correlated with the different pathological groups. Whole blood aggregometry significantly correlated with platelet count, however, when we adjust for the different groups we investigated, the effect of platelet count appears to be non-significant

Recurrent Chromosomal Copy Number Alterations in Sporadic Chordomas

The molecular events in chordoma pathogenesis have not been fully delineated, particularly with respect to copy number changes. Understanding copy number alterations in chordoma may reveal critical disease mechanisms that could be exploited for tumor classification and therapy. We report the copy number analysis of 21 sporadic chordomas using array comparative genomic hybridization (CGH). Recurrent copy changes were further evaluated with immunohistochemistry, methylation specific PCR, and quantitative real-time PCR. Similar to previous findings, large copy number losses, involving chromosomes 1p, 3, 4, 9, 10, 13, 14, and 18, were more common than copy number gains. Loss of CDKN2A with or without loss of CDKN2B on 9p21.3 was observed in 16/20 (80%) unique cases of which six (30%) showed homozygous deletions ranging from 76 kilobases to 4.7 megabases. One copy loss of the 10q23.31 region which encodes PTEN was found in 16/20 (80%) cases. Loss of CDKN2A and PTEN expression in the majority of cases was not attributed to promoter methylation. Our sporadic chordoma cases did not show hotspot point mutations in some common cancer gene targets. Moreover, most of these sporadic tumors are not associated with T (brachyury) duplication or amplification. Deficiency of CDKN2A and PTEN expression, although shared across many other different types of tumors, likely represents a key aspect of chordoma pathogenesis. Sporadic chordomas may rely on mechanisms other than copy number gain if they indeed exploit T/ brachyury for proliferation