Genome-wide analysis of therapeutic response uncovers molecular pathways governing tamoxifen resistance in ER+ breast cancer.

BACKGROUND: Prioritization of breast cancer patients based on the risk of resistance to tamoxifen plays a significant role in personalized therapeutic planning and improving disease course and outcomes. METHODS: In this work, we demonstrate that a genome-wide pathway-centric computational framework elucidates molecular pathways as markers of tamoxifen resistance in ER+ breast cancer patients. In particular, we associated activity levels of molecular pathways with a wide spectrum of response to tamoxifen, which defined markers of tamoxifen resistance in patients with ER+ breast cancer. FINDINGS: We identified five biological pathways as markers of tamoxifen failure and demonstrated their ability to predict the risk of tamoxifen resistance in two independent patient cohorts (Test cohort1: log-rank p-value = 0.02, adjusted HR = 3.11; Test cohort2: log-rank p-value = 0.01, adjusted HR = 4.24). We have shown that these pathways are not markers of aggressiveness and outperform known markers of tamoxifen response. Furthermore, for adoption into clinic, we derived a list of pathway read-out genes and their associated scoring system, which assigns a risk of tamoxifen resistance for new incoming patients. INTERPRETATION: We propose that the identified pathways and their read-out genes can be utilized to prioritize patients who would benefit from tamoxifen treatment and patients at risk of tamoxifen resistance that should be offered alternative regimens. FUNDING: This work was supported by the Rutgers SHP Dean's research grant, Rutgers start-up funds, Libyan Ministry of Higher Education and Scientific Research, and Katrina Kehlet Graduate Award from The NJ Chapter of the Healthcare Information Management Systems Society.

Physician Review of a Celiac Disease Risk Estimation and Decision-Making Expert System.

Objective: Celiac disease is a genetic disease affecting people of all ages, resulting in small intestine enteropathy. It is considered to be a clinical chameleon. Average prevalence of celiac disease is 1 out of 100 people with data indicating the risk may be as high as 22% for those with first-degree relatives with the disease. Eighty-three percent of people with celiac disease may be undiagnosed. Average duration to diagnosis is 10 years. Data indicate that there is a lack of consensus regarding diagnostics and symptomatology.Method: A clinical decision support system (CDSS) was developed using Exsys Corvid for expert analysis (CD-CDSS). The CD-CDSS was divided into symptoms and manifestations with 80 points of navigation, and a serology section, and was validated by 13 experts in the field of celiac disease using a 10-statement 5-point Likert scale.Results: This scale was analyzed using Cronbach's alpha reliability coefficient, which was calculated using SPSS and revealed good internal consistency and reliability with a result of 0.813. One hundred percent of experts agreed that the CD-CDSS is capable of guiding a health care professional through the diagnostic process, contains an accurate list of symptoms based on the clinical literature, and can foster improved awareness and education about celiac disease and that there is a need for this system.Conclusions: A celiac disease risk estimation and decision-making expert system was successfully developed and evaluated by medical professionals, with 100% agreeing that this CD-CDSS is medically accurate and can guide health care professionals through the diagnostic process.

Three-Dimensional Cellular Raman Analysis: Evidence of Highly Ordered Lipids Within Cell Nuclei.

Striking levels of spatial organization exist among and within interphase cell chromosomes, raising the possibility that other nuclear molecular components may also be organized in ways that facilitate nuclear function. To further examine molecular distributions and organization within cell nuclei, we utilized Raman spectroscopy to map distributions of molecular components, with a focus on cellular lipids. Although the vast majority of cellular lipids are associated with membranes, mapping the 2870/2850 cm-1 lipid peak ratios revealed that the most highly ordered lipids within interphase cells are found within cell nuclei. This finding was seen in cells from multiple tissue types, noncancerous cells, and in cancer cell lines of different metastatic potential. These highly ordered lipids colocalize with nuclear chromatin, are present throughout the nuclear volume, and remain colocalized with chromatin through mitosis, when the nuclear envelope has dissociated. Phosphatidylinositol is a major component of the highly ordered lipids. The presence of phosphatidylinositol and other lipids in the nuclear interior is well established, but their highly ordered packing has not been reported and represents a unique finding. The molecular interactions involved in the formation and maintenance of these highly ordered lipids, and their potential effects on nuclear activities, remain to be discovered.

Structural basis for chemokine recognition by a G protein-coupled receptor and implications for receptor activation.

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein-dependent and ß-arrestin-dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein-dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance-based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical "two-site model" for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

A perspective on digital and computational pathology.

The digitization of images has not only led to increasingly sophisticated methods of quantitating information from those images themselves, but also to the development of new physics-based techniques for extracting information from the original specimen and presenting this as visual data in both two and three-dimensional (3D) forms. This evolution of an image-based discipline has reached maturity in Radiology, but it is only just beginning in Pathology. An historical perspective is provided both on the current state of computational imaging in pathology and of the factors that are impeding further progress in the development and application of these approaches. Emphasis is placed on barriers to the dissemination of information in this area. The value of computational imaging in basic and translational research is clear. However, while there are many examples of "virtual diagnostics" in Radiology, there are only relatively few in Pathology. Nevertheless, we can do cellular level analysis of lesions accessible by endoscopic or catheterization procedures, and a number of steps have been taken toward real-time imaging as adjuncts to traditional biopsies. Progress in computational imaging will greatly expand the role of pathologists in clinical medicine as well as research.

MIF inhibits monocytic movement through a non-canonical receptor and disruption of temporal Rho GTPase activities in U-937 cells.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that was initially identified by its ability to inhibit the movement of macrophages. Cell migration is a highly complex process involving changes to the cytoskeleton and cell adhesion molecules, and is regulated by the Rho GTPases. A simple model using human monocytic U-937 cells to elicit the classic MIF response was implemented to examine the mechanism of MIF-induced migration inhibition. Our results demonstrate that MIF inhibits migration of these U-937 cells through a non-canonical receptor, CXCR4, in the absence of the putative primary MIF receptor CD74. Migration inhibition is dependent upon a series of temporal perturbations of the activities of the Rho GTPases: initial activation followed by subsequent inactivation of RhoA, inactivation of Rac1, and cyclic activation of Cdc42. MIF-mediated changes in the activities of the Rho GTPases jointly contributed to migration inhibition in these cells. Collectively, these data suggest that the MIF-mediated migration inhibition is mediated by the outcome of G-protein signaling, and in less adherent cells such as those of the monocyte/macrophage lineage, RhoA directly affects net translocation through its ability to induce cell body contraction. These findings demonstrate that CXCR4 can mediate MIF signaling in the absence of CD74 in addition to serving as a MIF co-receptor along with CD74. These results correlate MIF activity to specific and sequential Rho GTPase activity perturbations, and given that CXCR4 functions in numerous processes, suggests potential roles for the modulation of cell movement in those events including development, cell survival and viral infection.

Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

The glycoprotein YKL-40 (CHI3L1) is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing ß-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and trans-differentiated phenotypes.

Impedance measurements in the biomedical sciences.

Biological organisms and their component organs, tissues and cells have unique electrical impedance properties. Impedance properties often change with changes in structure, composition, and metabolism, and can be indicative of the onset and progression of disease states. Over the past 100 years, instruments and analytical methods have been developed to measure the impedance properties of biological specimens and to utilize these measurements in both clinical and basic science settings. This chapter will review the applications of impedance measurements in the biomedical sciences, from whole body analysis to impedance measurements of single cells and cell monolayers, and how cellular impedance measuring instruments can now be used in high throughput screening applications.

Impedance measurements in the biomedical sciences.

Biological organisms and their component organs, tissues and cells have unique electrical impedance properties. Impedance properties often change with changes in structure, composition, and metabolism, and can be indicative of the onset and progression of disease states. Over the past 100 years, instruments and analytical methods have been developed to measure the impedance properties of biological specimens and to utilize these measurements in both clinical and basic science settings. This chapter will review the applications of impedance measurements in the biomedical sciences, from whole body analysis to impedance measurements of single cells and cell monolayers, and how cellular impedance measuring instruments can now be used in high throughput screening applications.

Localization of ORC1 during the cell cycle in human leukemia cells.

The interaction of the origin recognition complex (ORC) with replication origins is a critical parameter in eukaryotic replication initiation. In mammals the ORC remains bound except during mitosis, thus the localization of ORC complexes allows localization of origins. A monoclonal antibody that recognizes human ORC1 was used to localize ORC complexes in populations of human MOLT-4 cells separated by cell cycle position using centrifugal elutriation. ORC1 staining in cells in early G1 is diffuse and primarily peripheral. As the cells traverse G1, ORC1 accumulates and becomes more localized towards the center of the nucleus, however around the G1/S boundary the staining pattern changes and ORC1 appears peripheral. By mid to late S phase ORC1 immunofluorescence is again concentrated at the nuclear center. During anaphase, ORC1 staining is localized mainly in the pericentriolar regions. These findings suggest that concerted movements of origin DNA sequences in addition to the previously documented assembly and disassembly of protein complexes are an important aspect of replication initiation loci in eukaryotes.

Correcting miR-15a/16 genetic defect in New Zealand Black mouse model of CLL enhances drug sensitivity.

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G(1) in non-New Zealand Black B cell and New Zealand Black-derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G(1) accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G(1)/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3' untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease.

The splicing factor SF2/ASF binds to ARS homologs in a human rDNA replication origin.

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.

Chitinase 3-Like-1 (CHI3L1): a putative disease marker at the interface of proteomics and glycomics.

Chitinase 3-Like-1 (CHI3L1) is a secreted 40 kDa glycoprotein that is upregulated in a number of human cancers and in non-neoplastic disease states characterized by chronic inflammation and tissue remodeling. Increased serum levels of CHI3L1 parallel disease severity, poorer prognosis, and shorter survival in many human neoplasias, including cancers of the breast, colon, prostate, ovaries, brain, thyroid, lung, and liver. Increased serum CHI3L1 also correlates with disease severity in rheumatoid arthritis, osteoarthritis, liver fibrosis, inflammatory bowel disease, and bacterial septicemia. CHI3L1 is a rheumatoid arthritis (RA) autoantigen, and MHC complexes containing specific CHI3L1 peptides have been found in RA patients; however, intranasal introduction of these same CHI3L1 peptides can induce tolerance towards them. CHI3L1 is a nonhydrolytic member of the human chitinase family that binds chitin tightly and heparin at lower affinity. Interactions with type I collagen, CHI3L1's only known protein-binding partner, helps regulate collagen fibril formation. The principal sources of CHI3L1 are activated macrophages and chondrocytes, neutrophils, and some tissue and tumor cells. CHI3L1 can act as a fibroblast mitogen and can activate several signaling pathways, however, no cell surface-binding partner for CHI3L1 has been identified. The ability of CHI3L1 to bind both proteins and carbohydrates allows potential interactions with a variety of cell-surface and extracellular-matrix proteins, proteoglycans, and polysaccharides, and thus CHI3L1 can interface between proteomics and glycomics.

Multiple initiation sites within the human ribosomal RNA gene.

Numerous studies have demonstrated that DNA replication initiates within the 30 kB non-transcribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series of closely spaced primer pairs to measure nascent leading strand abundance in mid and late S phase cells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site and two less utilized sites within a 6 kb region of the NTS. The initiation sites colocalize with significant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-like sequences. An intrinsic DNA bending site was localized by circular permutation analysis to within several hundred base pairs of one initiation site. While DUE and MAR elements occur elsewhere throughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occurs only near replication initiation sites, a juxtaposition also seen in other well-studied mammalian replication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elements in mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNA genes, contributions from these sequence-associated properties may be more significant to initiation sites associated with transcriptionally inactive genes, than to initiation sites associated with transcriptionally active genes.

DNA replication initiates at different sites in early and late S phase within human ribosomal RNA genes.

Metazoan replication origins often contain multiple potential initiation sites, and the selection of which of the potential sites are used appears to be dependent upon multiple factors, including the state of differentiation, cell metabolism, and local transcriptional activity. Numerous studies have shown that a replication origin exists within the non-transcribed spacer region of the human ribosomal RNA gene. We here analyze nascent leading strand DNA from S phase human lymphoid cells, and find that while the majority of rDNA replicates in mid- and late S phase and preferentially initiates replication 6 kbp from the transcription start site, in very early S phase the preferred initiation site is much closer to the transcription start site and may involve rDNA promoter sequences. This early site is coincident with a minimum GC skew value, diagnostic for replication origins in bacteria and yeast. These results suggest that replication timing can influence initiation site selection. The timing and nucleolar localization of rDNA further suggest that this site likely participates in the small number of perinucleolar initiation foci observed in very early S phase cells that represent the beginning of cellular DNA replication.