[Show abstract][Hide abstract] ABSTRACT: The stimulatory NKG2D receptor on lymphocytes promotes tumor immune surveillance by targeting ligands selectively induced on cancer cells. Progressing tumors counteract by employing tactics to disable lymphocyte NKG2D. This negative dynamic is escalated as some human cancer cells co-opt expression of NKG2D, thereby complementing the presence of its ligands for autonomous stimulation of oncogenic signaling. Clinical association data imply relationships between cancer cell NKG2D and metastatic disease. Here we show that NKG2D promotes cancer cell plasticity by induction of phenotypic, molecular, and functional signatures diagnostic of the epithelial-mesenchymal transition, and of stem-like traits via induction of Sox9, a key transcriptional regulator of breast stem cell maintenance. These findings obtained with model breast tumor lines and xenotransplants were recapitulated by ex vivo cancer cells from primary invasive breast carcinomas. Thus, NKG2D may have the capacity to drive high malignancy traits underlying metastatic disease.
PLoS ONE 10/2014; 9(10):e108942. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Marrow stromal cells (MSCs) constitute a poorly defined heterogeneous population of cells, typically isolated only after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, both CD146+ and CD146- MSC subpopulations were shown by global DNase I hypersensitive site mapping to have a lineage association with marrow fibroblasts, but not with endothelial or hematopoietic cells. Gene expression profiles generated for the CD146+ and CD146- fibroblasts indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors; consequently the conditioned media (CM) from these fibroblasts induce different fates in peripheral blood monocytes. Monocytes incubated in CD146+ CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146- fibroblasts express markers associated with dendritic cells. Importantly when CD14+ monocytes are cultured in contact with CD146+ fibroblasts the combined cell populations show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast gene expression in co-cultures of monocytes and CD146- fibroblasts do not differ from genes expressed when monocytes are cultured with CM from CD146- fibroblasts. These in vitro results show that a specific subpopulation of stromal cells, the CD146+ fibroblasts in contact with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immune histochemistry of marrow biopsies showing juxtaposed CD146+ cells and CD68+ cells associated with these up-regulated proteins. These data suggest a major role for fibroblast-macrophage interactions in functional hematopoietic niches.
Stem cells and development 10/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential role of conventional and regulatory T cells (Tregs) in protection from HIV-1 infection remains unclear. To address this question, we analyzed samples from 129 HIV-1-exposed seronegative individuals (HESN) from a HIV-1-serodiscordant couples cohort. To assess the presence of HIV-specific T cell responses and Treg function, we measured the proliferation of T cells in response to HIV-1 peptide pools in PBMCs and PBMCs depleted of Tregs. We identified HIV-specific CD4+ and CD8+ T cell responses and surprisingly, the overall CD4+ and CD8+ T cell response rate was not increased when Tregs were removed from cell preparations. Of the 20 individuals that had HIV-1-specific CD4+ T cell responses, only eight had Tregs that could suppress this proliferation. When compared with individuals whose Tregs could suppress HIV-1-specific CD4+ T cell proliferation, individuals with Tregs unable to suppress showed a trend toward increased T cell activation and Treg frequency and a significant increase in HIV-1-specific production of MIP1β by CD4+ T cells, autocrine production of which has been shown to be protective in terms of HIV-1 infection of CD4+ T cells.
AIDS research and human retroviruses 07/2013; · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domain-containing protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.
[Show abstract][Hide abstract] ABSTRACT: To identify new candidate therapeutic targets for Glioblastoma multiforme (GBM), we combined functional genetics and GBM network modeling to identify kinases required for the growth of patient-derived brain tumor initiating cells (BTICs), but which are dispensable to proliferating human neural stem cells (NSCs). This approach yielded BUB1B/BUBR1, a critical mitotic spindle checkpoint player, as the top scoring GBM-lethal kinase. Knockdown of BUB1B inhibited expansion of BTIC isolates, both in vitro and in vivo, without affecting proliferation of NSCs or astrocytes. Mechanistic studies revealed that BUB1B's GLEBs domain activity is required to suppress lethal kinetochore-microtubule (KT-MT) attachment defects in GBM isolates and genetically transformed cells with altered sister KT dynamics, which likely favor KT-MT instability. These results indicate that GBM tumors have added requirement for BUB1B to suppress lethal consequences of altered KT function. They further suggest that sister KT measurements may predict cancer-specific sensitivity to BUB1B inhibition and perhaps other mitotic targets that affect KT-MT stability.
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7⁻/⁻; p53⁻/⁻ carcinomas exhibit a CIN⁺ phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations.
Molecular and Cellular Biology 04/2012; 32(11):2160-7. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tolerant self-antigen-specific CD8 T cells fail to proliferate in response to antigen, thereby preventing autoimmune disease. By using an in vivo mouse model, we show that tolerant T cells proliferate and become functional under lymphopenic conditions, even in a tolerogenic environment. However, T cell rescue is only transient, with tolerance reimposed upon lymphorepletion even in the absence of tolerogen (self-antigen), challenging the prevailing paradigm that continuous antigen exposure is critical to maintain tolerance. Genome-wide messenger RNA and microRNA profiling revealed that tolerant T cells have a tolerance-specific gene profile that can be temporarily overridden under lymphopenic conditions but is inevitably reimposed, which suggests epigenetic regulation. These insights into the regulatory mechanisms that maintain or break self-tolerance may lead to new strategies for the treatment of cancer and autoimmunity.
[Show abstract][Hide abstract] ABSTRACT: Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO-targeted ubiquitin ligases (STUbLs). STUbLs bind to SUMOylated proteins via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. In this study, we show that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho (Gro/transducin-like enhancer (TLE)) during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO-independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. Our findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes.
The EMBO Journal 02/2011; 30(7):1289-301. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleosome-free regions (NFRs) at the 5' and 3' ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5' and 3' ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA.
Molecular and Cellular Biology 11/2010; 30(21):5110-22. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Delta strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.
[Show abstract][Hide abstract] ABSTRACT: Disseminated epithelial cells can be isolated from the bone marrow of a far greater fraction of prostate-cancer patients than the fraction of patients who progress to metastatic disease. To provide a better understanding of these cells, we have characterized their genomic alterations. We first present an array comparative genomic hybridization method capable of detecting genomic changes in the small number of disseminated cells (10-20) that can typically be obtained from bone marrow aspirates of prostate-cancer patients. We show multiple regions of copy-number change, including alterations common in prostate cancer, such as 8p loss, 8q gain, and gain encompassing the androgen-receptor gene on Xq, in the disseminated cell pools from 11 metastatic patients. We found fewer and less striking genomic alterations in the 48 pools of disseminated cells from patients with organ-confined disease. However, we identify changes shared by these samples with their corresponding primary tumors and prostate-cancer alterations reported in the literature, evidence that these cells, like those in advanced disease, are disseminated tumor cells (DTC). We also show that DTCs from patients with advanced and localized disease share several abnormalities, including losses containing cell-adhesion genes and alterations reported to associate with progressive disease. These shared alterations might confer the capability to disseminate or establish secondary disease. Overall, the spectrum of genomic deviations is evidence for metastatic capacity in advanced-disease DTCs and for variation in that capacity in DTCs from localized disease. Our analysis lays the foundation for elucidation of the relationship between DTC genomic alterations and progressive prostate cancer.
Cancer Research 07/2008; 68(14):5599-608. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and represses expression of P21CIP1 as part of a heterodimeric complex with Miz1. In vivo, expression of ZBTB4 is downregulated in advanced stages of multiple human tumours. In cell culture, depletion of ZBTB4 promotes cell cycle arrest in response to activation of p53 and suppresses apoptosis through regulation of P21CIP1, thereby promoting long-term cell survival. Our data suggest that Zbtb4 is a critical determinant of the cellular response to p53 activation and reinforce the notion that p21Cip1 can provide an essential survival signal in cells with activated p53.
The EMBO Journal 07/2008; 27(11):1563-74. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm results in attenuated endoreplication and growth arrest early in larval development. Drosophila also contains a single ortholog of the mammalian Mad/Mnt transcriptional repressor proteins (dMnt), which is thought to antagonize dMyc function. Here we show that animals lacking both dMyc and dMnt display increased viability and grow significantly larger and develop further than dMyc single mutants. We observe increased endoreplication and growth of larval tissues in these double mutants and disproportionate growth of the imaginal discs. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis. The additional loss of dMnt partially rescues expression of a small number of dMyc and dMnt genes that are primarily involved in rRNA synthesis and processing. Our results indicate that dMnt repression is normally overridden by dMyc activation during larval development. Therefore the severity of the dm null phenotype is likely due to unopposed repression by dMnt on a subset of genes critical for cell and organismal growth. Surprisingly, considerable growth and development can occur in the absence of both dMyc and dMnt.
[Show abstract][Hide abstract] ABSTRACT: Persistent Rel/nuclear factor-kappaB (NF-kappaB) activity is a hallmark of many human cancers, and the Rel proteins are implicated in leukemia/lymphomagenesis but the mechanism is not fully understood. Microarray analysis to identify transformation-impacting genes regulated by NF-kappaB's oncogenic v-Rel and c-Rel proteins uncovered that Rel protein expression leads to transcriptional repression of key B-cell receptor (BCR) components and signaling molecules like B-cell linker (BLNK), the B-cell adaptor for phosphoinositide 3-kinase (BCAP) and immunoglobulin lambda light chain (Ig lambda), and is accompanied by a block in BCR-mediated activation of extracellular signal-regulated kinase, Akt, and c-Jun-NH(2)-kinase in response to anti-IgM. The BLNK and BCAP proteins were also down-regulated in lymphoid cells expressing a transformation-competent chimeric RelA/v-Rel protein, suggesting a correlation with the capacity of Rel proteins to transform lymphocytes. DNA-binding studies identified functional NF-kappaB-binding sites, and chromatin immunoprecipitation (ChIP) data showed binding of Rel to the endogenous blnk and bcap promoters in vivo. Importantly, restoration of either BLNK or BCAP expression strongly inhibited transformation of primary chicken lymphocytes by the potent NF-kappaB oncoprotein v-Rel. These findings are interesting because blnk and other BCR components and signaling molecules are down-regulated in primary mediastinal large B-cell lymphomas and Hodgkin's lymphomas, which depend on c-Rel for survival, and are consistent with the tumor suppressor function of BLNK. Overall, our results indicate that down-regulation of BLNK and BCAP is an important contributing factor to the malignant transformation of lymphocytes by Rel and suggest that gene repression may be as important as transcriptional activation for Rel's transforming activity.
Cancer Research 03/2008; 68(3):808-14. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perhaps the greatest barrier to translation of serum biomarker discoveries is the inability to evaluate putative biomarkers in high throughput validation studies. Here we report on the development, production, and implementation of a high-density antibody microarray used to evaluate large numbers of candidate ovarian cancer serum biomarkers. The platform was shown to be useful for evaluation of individual antibodies for comparative analysis, such as with disease classification, and biomarker validation and discovery. We demonstrate its performance by showing that known tumor markers behave as expected. We also identify several promising biomarkers from a candidate list and generate hypotheses to support new discovery studies.
[Show abstract][Hide abstract] ABSTRACT: Integration of patterning cues via transcriptional networks to coordinate gene expression is critical during morphogenesis and misregulated in cancer. Using DNA adenine methyltransferase (Dam)ID chromatin profiling, we identified a protein-protein interaction between the Drosophila Myc oncogene and the Groucho corepressor that regulates a subset of direct dMyc targets. Most of these shared targets affect fate or mitosis particularly during neurogenesis, suggesting the dMyc-Groucho complex may coordinate fate acquisition with mitotic capacity during development. We find an antagonistic relationship between dMyc and Groucho that mimics the antagonistic interactions found for EGF and Notch signaling: dMyc is required to specify neuronal fate and enhance neuroblast mitosis, whereas Groucho is required to maintain epithelial fate and inhibit mitosis. Our results suggest that the dMyc-Groucho complex defines a previously undescribed mechanism of Myc function and may serve as the transcriptional unit that integrates EGF and Notch inputs to regulate early neuronal development.
Proceedings of the National Academy of Sciences 11/2007; 104(40):15771-6. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the "intra-S phase checkpoint." Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.
Molecular and Cellular Biology 10/2007; 27(18):6396-406. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein modification by glycosylation occurs through an essential biochemical pathway that produces mannosyl side chain substrates, which are covalently attached to proteins in the endoplasmic reticulum. We used DNA microarray analysis to characterize the cellular response to a conditional defect (pmi40-101) in the protein glycosylation pathway. Expression profiles were obtained from DNA microarrays containing essentially every gene from Saccharomyces cerevisiae. We validated the microarray analysis by examining the expression patterns of induced genes using transcriptional lacZ fusions. The major class of genes differentially expressed in the glycosylation mutant overlapped significantly with that of a starvation response and included those required for gluconeogenesis, the tricarboxylic acid and glyoxylate cycles, and protein and amino acid biosynthesis. Two mitogen-activated protein (MAP) kinase pathways were also activated in the mutant, the filamentous growth and protein kinase C pathways. Taken together, our results suggest that a checkpoint is activated in response to a protein glycosylation defect, allowing the cell to mount an adaptive response by the activation of multiple MAP kinase pathways.
FEMS Yeast Research 01/2007; 6(8):1264-73. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic gains and losses resulting from DNA strand breakage by ionizing radiation have been demonstrated in vitro and suspected in radiation-associated thyroid cancer. We hypothesized that copy number deviations might be more prevalent, and/or occur in genomic patterns, in tumors associated with presumptive DNA strand breakage from radiation exposure than in their spontaneous counterparts. We used cDNA microarray-based comparative genome hybridization to obtain genome-wide, high-resolution copy number profiles at 14,573 genomic loci in 23 post-Chernobyl and 20 spontaneous thyroid cancers. The prevalence of DNA gains in tumors from cases in exposed individuals was two- to fourfold higher than for cases in unexposed individuals and up to 10-fold higher for the subset of recurrent gains. DNA losses for all cases were low and more prevalent in spontaneous cases. We identified unique patterns of copy variation (mostly gains) that depended on a history of radiation exposure. Exposed cases, especially the young, harbored more recurrent gains that covered more of the genome. The largest regions, spanning 1.2 to 4.9 Mbp, were located at 1p36.32-.33, 2p23.2-.3, 3p21.1-.31, 6p22.1-.2, 7q36.1, 8q24.3, 9q34.11, 9q34.3, 11p15.5, 11q13.2-12.3, 14q32.33, 16p13.3, 16p11.2, 16q21-q12.2, 17q25.1, 19p13.31-qter, 22q11.21 and 22q13.2. Copy number changes, particularly gains, in post-Chernobyl thyroid cancer are influenced by radiation exposure and age at exposure, in addition to the neoplastic process.
Radiation Research 10/2006; 166(3):519-31. · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The packaging of DNA into chromatin allows eukaryotic cells to organize and compact their genomes but also creates an environment that is generally repressive to nuclear processes that depend upon DNA accessibility. There are several classes of enzymes that modulate the primary structure of chromatin to regulate various DNA-dependent processes. The biochemical activities of the yeast Isw1 ATP-dependent chromatin-remodeling enzyme have been well characterized in vitro, but little is known about how these activities are utilized in vivo. In this work, we sought to discern genetic backgrounds that require Isw1 activity for normal growth. We identified a three-way genetic interaction among Isw1, the NuA4 histone acetyltransferase complex, and the Swr1 histone replacement complex. Transcription microarray analysis revealed parallel functions for these three chromatin-modifying factors in the regulation of TATA-containing genes, including the repression of a large number of stress-induced genes under normal growth conditions. In contrast to a recruitment-based model, we find that the NuA4 and Swr1 complexes act throughout the genome while only a specific subset of the genome shows alterations in transcription.
Molecular and Cellular Biology 09/2006; 26(16):6117-29. · 5.04 Impact Factor