Citations

Monitoring activity of single cells has high significance for basic science and diagnostic applications.

ABSTRACTEpithelial‐mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to epithelial ovarian carcinoma (EOC) risk have been based on small sample sizes and none have sought replication in an independent population. We screened 15,816 single‐nucleotide polymorphisms (SNPs) in 296 genes in a discovery phase using data from a genome‐wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT‐related genes that were nominally (P < 0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive‐cancer patients and 23,447 controls. A P‐value <0.05 and a false discovery rate (FDR) <0.2 were considered statistically significant. In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (odds ratio (OR) = 1.16, 95% CI = 1.07–1.25, P = 0.0003, FDR = 0.19), whereas F8 rs7053448 (OR = 1.69, 95% CI = 1.27–2.24, P = 0.0003, FDR = 0.12), F8 rs7058826 (OR = 1.69, 95% CI = 1.27–2.24, P = 0.0003, FDR = 0.12), and CAPN13 rs1983383 (OR = 0.79, 95% CI = 0.69–0.90, P = 0.0005, FDR = 0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC.

Abstract Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKα facilitates its interaction with RelB and IKKα, indicating that DBC1-mediated suppression of alternative NF-κB is regulated by IKKα. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation.

Cell therapy is a potential therapeutic method for cerebral ischemia, which remains a serious problem. In the search for more effective therapeutic methods, many kinds of stem cells from various tissues have been developed and tested as candidate therapeutic agents. Among them, human umbilical cord blood (hUCB)‐derived mesenchymal stem cells (MSCs) are widely used for cell therapy because of their genetic flexibility. To confirm that they are effective and understand how they affect ischemic neural cells, hUCB‐MSCs were directly administered ipsilaterally into an ischemic zone induced by middle cerebral artery occlusion (MCAO). We found that the neurobehavioral performance of the hUCB‐MSC group was significantly improved compared with that of the vehicle‐injected control group. The infarct was also remarkably smaller in the hUCB‐MSC group. Additionally, hUCB‐MSC transplantation resulted in a greater number of newly generated cells and angiogenic and tissue repair factors and a lower number of inflammatory events in the penumbra zone. To determine why these events occurred, hUCB‐MSCs were assayed under hypoxic and normoxic conditions in vitro. The results showed that hUCB‐MSCs exhibit higher expression levels of thrombospondin1, pantraxin3, and vascular endothelial growth factor under hypoxic conditions than under normoxic conditions. These results were found to be correlated with our in vivo immunofluorescent staining results. On the basis of these findings, we suggest that hUCB‐MSCs may have a beneficial effect on cerebral ischemia, especially through angiogenesis, neurogenesis, and anti‐inflammatory effects, and thus could be used as a therapeutic agent to treat neurological disorders such as cerebral ischemia. © 2015 Wiley Periodicals, Inc.

Abstract Methyl-lysine (Kme) recognition domains play a central role in epigenetic regulation during cellular differentiation, development, and gene transcription with more than 200 known “reader” domains in the human proteome. We describe our target-class approach to ligand design and discovery for three cancer relevant members of this large family: L3MBTL3 (a Malignant Brain Tumor (MBT) domain containing reader), 53BP1 (a tandem Tudor domain) and CBX7 (a chromo domain). The advantages of a small molecule driven approach to modulating chromatin biology are numerous: temporal resolution; mechanistic flexibility (targeting a specific activity of a protein as opposed to ablating them all with transgenic knock-outs or RNA-interference techniques); ease of delivery; and most significantly, when warranted, a small molecule tool has the potential to provide an immediate transition to a drug discovery effort, potentially cutting years off the time between target validation and therapeutic intervention. UNC1215 binds the MBT domains of L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides. This probe is greater than 50-fold selective versus other members of the human MBT family and also demonstrates selectivity against more than 200 other Kme reader domains examined. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. The potency, specificity, and cellular effects of UNC1215 establish it as the first cell-active antagonist of a Kme reader domain and a useful chemical probe for biological studies of the function of L3MBTL3 (James, L. I. et al. Nat Chem Biol 2013, 9, 184-191). 53BP1 is a Kme binding protein that plays a central biological role in DNA Damage Repair (DDR) pathway via its recruitment to sites of DNA double strand breaks (DSB). BRCA1 is a checkpoint and DNA damage repair gene that is required for maintenance of genomic integrity, and the inheritance of mutated BRCA1 is a major risk factor for breast and ovarian cancer. The BRCA1 knockout murine model is embryonic lethal and a conditional knockout in mammary glands results in low frequency and long latency of mammary tumor formation. It was recently reported that the BRCA1-null developmental phenotype is rescued when placed on a 53BP1-null background. Adult mice from this model, that are null for both the 53BP1 and BRCA1 genes, age normally and display a very low incidence of tumor formation. Genomic instability can be rescued in 53BP1 knockouts because the homologous recombination (HR) pathway is largely restored in cells lacking both BRCA1 and 53BP1 (Cao, L. et al. Molecular cell 2009, 35 (4), 534-41). Based on this data, small molecule antagonists of 53BP1's biological function could act as viable therapeutics to restore HR in the DDR response in humans with BRAC1 mutations. We will report the first small molecule, fragment-like ligands with selectivity for binding to 53BP1. CBX7 is a chromo domain containing Kme reader that, along with other CBX domains, recruits the PRC1 complex to histone 3, lysine 27 trimethylation sites (H3K27me3) and enables transcriptional silencing downstream of the PRC2 complex which ‘writes’ the H3K27me3 mark via the catalytic activity of EZH2. As EZH2 is a well validated drug target with inhibitors now entering the clinic for the treatment of various cancers, we hypothesize that antagonism of the PRC1 complex recognition of H3K27me3 will phenocopy and perhaps synergize with the activity of EZH2 inhibitors (Konze, K. D. et al. ACS Chemical Biology 2013, 8, 1324-1334). Progress towards a chemical probe to validate CBX7 as a therapeutic target will be presented. The pursuit of high quality chemical probes for KMe reader proteins represents an emerging area that will open new avenues of research in chromatin biology and may in time, translate to new therapeutic approaches. Antagonists of the readers of acetylated lysine (BRD4 bromo domains) derived from pioneering chemical probe efforts (Filippakopoulos, P. et al. Nature 468, 1067-73 (2010)) have recently entered the clinic for the treatment of various malignancies and we hope that our chemical probe work will help to validate Kme readers as novel and significant anti-tumor targets. Citation Format: Lindsey I. James, Jake I. Stuckey, Brandi M. Baughman, Michael T. Perfetti, Samantha G. Pattenden, Brad M. Dickson, Jacqueline L. Norris, Gaofeng Cui, Pavel Mader, Aiping Dong, Yunxiang Mu, Scott B. Rothbart, Brian D. Strahl, Jinrong Min, Peter J. Brown, Dmitri B. Kireev, William P. Janzen, Kevin M. McBride, Mark T. Bedford, Georges Mer, Cheryl H. Arrowsmith, Stephen V. Frye. Targeting chromatin regulation for cancer therapy: progress towards chemical probes for methyl-lysine readers. [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 SY08-03. doi:10.1158/1538-7445.AM2015-SY08-03

Abstract Introduction: Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults, and comprise 30% of all carcinoid malignancies. They are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors, and are characterized by neuroendocrine differentiation and the potential to metastasize. Method: We characterized genomic alterations in pulmonary carcinoid tumors using whole genome and exome sequencing in addition to mRNA expression and SNP genotyping from specimens of normal lung, typical and atypical carcinoid, and SCLC. Results: Analysis of the sequencing data identified novel mutations in the pulmonary neuroendocrine tumor spectrum, including KRAS, ABCA9, GALNT10, HSFX1, PTHLH, RANBP2, BRIP1, FXR1, BACE2, NCOR2, DGKI, HERC1, HERC5 and GLI3. Pathway analysis found 45% of the cases to harbor a mutation in genes that are involved in protein-protein interaction with ubiquitin c (UBC) and may therefore play a role in ubiquitin protein degradation pathway. We also identified genes differentially expressed in corresponding regions of copy number variation, 52 of which were linked to ubiquitination pathways in 88% of the samples genotyped. Genes amplified in 15 to 23% of the cases included ITM2A, MAOA, EGR2, HAGH, TSC22D3, NBEA, TCEAL2, and TSPYL2 whereas deleted regions in 15% of the cases implicated CFD, GDI1, IFITM1 and IFITM2. Conclusion: These results suggest that in addition to previously identified pathways, dysregulation of the ubiquitin pathway may contribute to lung carcinoid tumorigenesis and could be exploited for therapeutic purposes. Citation Format: Michael K. Asiedu, Charles L. Thomas, Jie Dong, Prasidda Khadka, Zhifu Sun, Farhad Kosari, Jin Jen, Julian Molina, George Vasmatzis, Marie Christine Aubry, Ping Yang, Dennis Wigle. Genomic alterations modify ubiquitination pathways in pulmonary carcinoid tumors. [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 3895. doi:10.1158/1538-7445.AM2015-3895

Abstract Background: Genome-wide association studies (GWAS) have so far reported 12 loci associated with serous epithelial ovarian cancer (EOC) risk. We hypothesized that some of these loci function through nearby transcription factor (TF) genes and that putative target genes of these TFs as identified by coexpression may also be enriched for additional EOC risk associations. Methods: We selected TF genes within 1 Mb of the top signal at the 12 genome-wide significant risk loci. Mutual information, a form of correlation, was used to build networks of genes strongly coexpressed with each selected TF gene in the unified microarray dataset of 489 serous EOC tumors from The Cancer Genome Atlas. Genes represented in this dataset were subsequently ranked using a gene-level test based on results for germline SNPs from a serous EOC GWAS meta-analysis (2,196 cases/4,396 controls). Results: Gene set enrichment analysis identified six networks centered on TF genes (HOXB2, HOXB5, HOXB6, HOXB7 at 17q21.32 and HOXD1, HOXD3 at 2q31) that were significantly enriched for genes from the risk-associated end of the ranked list (P &lt; 0.05 and FDR &lt; 0.05). These results were replicated (P &lt; 0.05) using an independent association study (7,035 cases/21,693 controls). Genes underlying enrichment in the six networks were pooled into a combined network. Conclusion: We identified a HOX-centric network associated with serous EOC risk containing several genes with known or emerging roles in serous EOC development. Impact: Network analysis integrating large, context-specific datasets has the potential to offer mechanistic insights into cancer susceptibility and prioritize genes for experimental characterization. Cancer Epidemiol Biomarkers Prev; 24(10); 1574–84. ©2015 AACR.

BackgroundThe ability of von Willebrand factor (VWF) to bind platelet GP Ib and promote platelet plug formation is measured in vitro using the ristocetin cofactor (VWF:RCo) assay. Automated assay systems make testing more accessible for diagnosis, but do not necessarily improve sensitivity and accuracy.ObjectiveWe assessed the performance of a modified automated VWF:RCo assay protocol for the Behring Coagulation System (BCS®) compared to other available assay methods.MethodsResults from different VWF:RCo assays in a number of specialized commercial and research testing laboratories were compared using plasma samples with varying VWF:RCo activities (0–1.2 IU mL−1). Samples were prepared by mixing VWF concentrate or plasma standard into VWF‐depleted plasma. Commercially available lyophilized standard human plasma was also studied. Emphasis was put on the low measuring range. VWF:RCo accuracy was calculated based on the expected values, whereas precision was obtained from repeated measurements.ResultsIn the physiological concentration range, most of the automated tests resulted in acceptable accuracy, with varying reproducibility dependent on the method. However, several assays were inaccurate in the low measuring range. Only the modified BCS protocol showed acceptable accuracy over the entire measuring range with improved reproducibility.ConclusionsA modified BCS® VWF:RCo method can improve sensitivity and thus enhances the measuring range. Furthermore, the modified BCS® assay displayed good precision. This study indicates that the specific modifications − namely the combination of increased ristocetin concentration, reduced platelet content, VWF‐depleted plasma as on‐board diluent and a two‐curve calculation mode − reduces the issues seen with current VWF:RCo activity assays.

Dysfunction of the type 1 cholecystokinin (CCK) receptor (CCK1R) as a result of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore the possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that, similar to the impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had a negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes but was protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant Y140A was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in a high-cholesterol environment. This mechanism may contribute to the beneficial effect of ursodeoxycholic acid in reducing cholesterol gallstone formation.