Transcript Clusters Research Articles

Abstract IA28: Targeting stress-induced molecular chaperones CLU and Hsp27 to inhibit EMT and metastases

Abstract Stress-activated adaptive pathways help drive cancer cell survival, metastases, and acquired treatment resistance. Heat shock and other molecular chaperones play central roles in these stress responses, regulating protein homeostasis (proteostasis) as well as many signaling and transcriptional survival networks to facilitate cell survival and treatment resistance. Clusterin (CLU) and Hsp27 are stress-activated chaperones that treatment-induced and associated with metastases and treatment resistance in many cancers, functioning to protect cells from many varied therapeutic stressors that induce apoptosis. Both CLU and HSP27 levels increase after androgen deprivation and become highly expressed in metastatic CRPC. While studies have defined CLU and Hsp27 as key mediators of adaptive survival pathways and treatment resistance, they also regulate epithelial plasticity and epithelial-to-mesenchymal transition (EMT). Defining mechanistic linkages between treatment stress and effectors of EMT will yield new insights into metastasis and treatment strategies. EMT endows malignant cells with enhanced migratory and survival attributes that facilitate cancer metastases. EMT can be initiated by a wide variety of signals in the tumor environment that activate the transcription factors Twist and Snail, leading to repression of epithelial cell markers like E-cadherin and cytokeratins, and gain of mesenchymal markers like N-cadherin and vimentin. We recently defined roles for both CLU and Hsp27, downstream of TGF-β and IL-6, respectively, as key mediators of EMT and metastases. We found that factors such as TGF-β and IL-6 known to induce EMT, up-regulate molecular chaperones CLU and Hsp27. We then set out to define molecular mechanisms by which TGF-β and IL-6 regulate CLU and Hsp27 expression, respectively, and whether CLU and Hsp27 mediate TGF-β and IL-6 regulated EMT and metastasis. CLU is a mediator of TGF-β-induced EMT. Our data show that the transcription factor Twist1 mediates TGF-β-induced CLU expression, binding to E-boxes in the distal promoter region of CLU gene to regulate basal and TGF-β-induced CLU transcription. TGF-β induced stimulation of Twist1 as well as CLU transcript expression was potently suppressed by Twist1 silencing. CLU silencing reduced TGF-β induction of the mesenchymal markers N-cadherin and fibronectin, and inhibited migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, these findings identify CLU as a mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression. Hsp27 facilitates IL-6 mediated EMT via modulation of STAT3/Twist. In addition to a role for CLU in EMT, we found that Hsp27 overexpression drives, while its knockdown reverses, EMT, with congruent effects on cell migration, invasion and matrix metalloprotease activity. Mechanistically, silencing Hsp27 decreased IL-6 dependent STAT3 phosphorylation, nuclear translocation, and abrogated STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6 mediated EMT via modulation of STAT3/Twist signaling. Elevated expression of Hsp27 and Twist were also found in high grade PCa tumors. Overall, these data define a role of Hsp27 as a critical regulator of IL-6 dependent and independent EMT and highlights Hsp27 as an attractive anti-cancer target for metastatic CRPC. CLU and Hsp27 as Anti-Cancer Targets. Since Hsp27 and CLU are ATP-independent chaperones and not amenable to inhibition by small molecules, antisense strategies have been used to therapeutically target this chaperones, with evidence of anti-cancer activity in clinical trials. We developed OGX-011 as a CLU inhibitor and demonstrated pre-clinical activity and dose-dependent target suppression in Phase I studies. Moreover, we led a randomized Phase II trial that reported a 7-month-survival benefit when the CLU inhibitor, OGX-011, was combined with docetaxel in patients with CRPC; OGX-011 is now in phase III trials in metastatic CRPC and NSCLC. We also developed OGX-427 as an Hsp27 inhibitor and demonstrated pre-clinical activity and tolerability in Phase I studies. A randomized phase II study of OGX-427 plus Prednisone (P) vs. P alone enrolled 64 mCRPC patients. At 12 weeks, the proportion of patients progression free was 71% in OGX-427+P treated patients and 40% in patients on P. A ≥50% PSA decline occurred in 50% of patients on OGX-427+P and 20% patients treated with P. Measurable disease response occurred in 44% on OGX-427+P and 0 patients on P. CTC conversion from ≥5 to <5/7.5 ml occurred in 55% evaluable patients on OGX-427+P and 41% treated with P. These results confirm, for the first time, single agent activity for an Hsp27 inhibitor in cancer, and Phase II trials in metastatic bladder and CRPC are ongoing. Citation Format: Martin Gleave, Amina Zoubeidi, Kim Chi. Targeting stress-induced molecular chaperones CLU and Hsp27 to inhibit EMT and metastases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA28.

Transcriptome profiling of mice testes following low dose irradiation

BackgroundRadiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis.MethodsTranscriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi).ResultsWe identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster.ConclusionsThe five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

Clusterin Expression and Localization Are Affected by Rituximab and Doxorubicin Treatment in Non-Hodgkin Lymphoma Cells and B Lymphocytes

AbstractAbstract 1318 Introduction.Diffuse large B-cell lymphoma (DLBCL) is the most frequently aggressive Non-Hodgkin’s lymphoma (NHL). DLBCL first line therapy consists of R-CHOP regimen, which combines classical chemotherapeutical drugs and corticosteroids with Rituximab. Rituximab is a chimeric anti-CD20 monoclonal antibody which targets the CD20 surface antigen expressed on both normal and malignant B cells. Rituximab triggers antibody- and complement-dependent cytotoxicity, growth inhibition and apoptosis. Rituximab inhibits the expression of the anti-apoptotic proteins Bcl-2/Bcl-XL, down-modulating different survival pathways. Clusterin (CLU) is an ubiquitary protein involved in many physiological and pathological processes such as apoptosis and cancer. CLU has different transcriptional isoforms, which share exons from 2 to 9 but have a unique exon 1 and at least two protein forms: The cytoplasmatic precursor of about 60 kDa is secreted outside the cell after post-traslational modifications (sCLU), whereas a truncated protein (50–55 kDa) is produced only in particular conditions and localizes in the nucleus (nCLU). CLU expression is disregulated in almost all cancers and its role has been deeply investigated in solid tumors. Only limited information is currently available about its function in lymphomas. Methods.Toledo, a DLBCL-derived cell line and normal B lymphocytes were chosen as experimental models. B lymphocytes were purified and immuno-magnetically sorted from peripheral blood of 4 healthy volunteers. Toledo and B lymphocytes were treated with 10 or 100 μg/ml Rituximab or doxorubicin (from 0.05 to 1 μM) for 48 hours. Phenotype analysis was performed by flow cytometry; cell viability was assessed by Trypan blue exclusion; apoptosis was evaluated by Annexin V/propidium iodide. CLU expression and localization were evaluated by real time PCR, Western Blot (WB) and immunocytochemistry (ICC). Primers for the real time PCR were design in order to distinguish the unique exon 1 of each CLU transcriptional isoforms or exons common to all variants (CLU3-4). Results.Rituximab treatment doesn’t significantly inhibit Toledo proliferation and does not induce apoptosis despite the fact that these cells are CD20+. This suggests that Toledo are resistant to Rituximab. As expected, doxorubicin causes a marked growth inhibition by necrosis and apoptosis. Toledo cells have a very low basal level of the 60 kDa CLU protein form. Both Rituximab and doxorubicin up-regulate the 60 kDa form with 1 μM doxorubicin causing the most dramatic increase. The analysis of transcriptional isoforms by real-time PCR shows that CLU mRNA is almost undetectable in Toledo. CLU transcriptional variant 2 (CLU2) and regions common to all CLU transcripts (CLU3-4) are significantly up-regulated only after 1 μM doxorubicin but not after Rituximab. Fluorescence microscopy clearly shows a cytoplasmic localization of CLU under normal growth conditions. Interestingly, the treatment with both Rituximab and doxorubicin causes the appearance of a nuclear staining. This fact might be consistent with the onset of a process of cell death. 10 and 100 μg/ml Rituximab inhibits B lymphocytes cell survival in a dose-dependent manner (39.7% and 44% respectively after 48 hours). Interestingly, B lymphocytes clearly express high basal levels of the 60 kDa CLU and produce an additional form of about 55 kDa only after Rituximab but not after doxorubicin. Furthermore Rituximab-treated B lymphocytes display a Bcl-2 protein decrease while Toledo cells show high expression of Bcl-2 that doesn’t seem to change in response to Rituximab. Conclusions.Toledo are resistant to Rituximab up to 100 μg/ml and express very low levels of CLU under normal growth conditions. 60 kDa CLU is partially up-regulated and Bcl-2 expression is not significantly affected by Rituximab treatment. Doxorubicin strongly up-regulates the 60 kDa CLU isoform. At the transcriptional level CLU2 shows the same expression profile of the 60 kDa protein form. On the contrary, Rituximab changes the CLU profile of responsive B lymphocytes in that leads to the appearance of a 55 kDa form that is not visible after doxorubicin treatment. Rituximab also causes a reduction in the expression of the antiapoptotic protein Bcl-2. The biological role of CLU during the response of peripheral B lymphocytes to Rituximab treatment is yet to be determined and is currently being investigated. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice1

Beyond Mendelian inheritance, an understanding of the complexities and consequences of the transfer of nonhereditary information to successive generations is at an early stage. Such epigenetic functionality is exemplified by DNA methylation and, as genome-wide high-throughput methodologies emerge, is increasingly being considered in the context of conserved intragenic and intergenic CpG islands that function as alternate sites of transcription initiation. Here we characterize an intragenic CpG island in exon 2 of the protein-coding mouse Klf1 gene, from which clustered transcription initiation sites yield positive-strand, severely truncated, capped and spliced RNAs. Expression from this CpG island in the testis begins between Postnatal Days 14-20, increases during development, and is temporally correlated with the maturation of secondary spermatocytes as they become the dominant cell population in the seminiferous epithelium. Only full-length KLF1-encoding mRNAs are detected in the hematopoietic tissue, spleen; thus, expression from the exon 2 CpG island is both developmentally regulated and tissue restricted. DNA methylation analysis indicates that spatiotemporal expression from the Klf1 CpG island is not associated with hypermethylation. Finally, our computational analysis from multiple species confirms intragenic transcription initiation and indicates that the KLF1 CpG island is evolutionarily conserved. Currently we have no evidence that these truncated RNAs can be translated via nonconventional mechanisms such as in-frame, conserved non-AUG-dependent Kozak consensus sequences; however, high-quality carboxyl-terminal antibodies will more effectively address this issue.

UAP56 Couples piRNA Clusters to the Perinuclear Transposon Silencing Machinery

piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

Clusterin Mediates TGF-β–Induced Epithelial–Mesenchymal Transition and Metastasis via Twist1 in Prostate Cancer Cells

TGF-β promotes epithelial-mesenchymal transition (EMT) and induces clusterin (CLU) expression, linking these genes to cancer metastasis. CLU is a pleiotropic molecular chaperone that confers survival and proliferative advantage to cancer cells. However, the molecular mechanisms by which TGF-β regulates CLU expression and CLU affects metastasis remain unknown. In this study, we report that the transcription factor Twist1 mediates TGF-β-induced CLU expression. By binding to E-boxes in the distal promoter region of CLU gene, Twist1 regulated basal and TGF-β-induced CLU transcription. In addition, CLU reduction reduced TGF-β induction of the mesenchymal markers, N-cadherin and fibronectin, thereby inhibiting the migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, our findings indicate that CLU is an important mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression.

The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis

PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism that provides an essential protection for germ cell genomes against the activity of mobile genetic elements1. piRNA populations comprise a molecular definition of transposons that permits them to be distinguished from host genes and selectively silenced. piRNAs can be generated in two distinct ways. Primary piRNAs emanate from discrete genomic loci, termed piRNA clusters, and appear to be derived from long, single-stranded precursors2. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are likely formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner1,3. Secondary piRNAs arise during the adaptive ping-pong cycle, with their 5' termini being formed by the activity of PIWIs themselves2,4. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Zucchini, is a member of the phospholipase D family of phosphodiesterases, which includes both phospholipases and nucleases5–7. We have produced a dimeric, soluble fragment of the mouse Zucchini homolog (mZuc/PLD6) and have shown that it possesses single strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to PLD-family nucleases than to phospholipases. Considered together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs.

The AT-rich DNA-binding Protein SATB2 Promotes Expression and Physical Association of Human Gγ- and Aγ-Globin Genes

Matrix attachment region (MAR)-binding protein (MARBP) has profound influence on gene transcriptional control by tethering genes to the nuclear scaffold. MARBP SATB2 is recently known as a versatile regulator functioning in the differentiation of multiple cell types including embryonic stem cells, osteoblasts and immunocytes. Roles of SATB2 in erythroid cells and its working mechanism in orchestrating target gene expression are largely unexplored. We show here that SATB2 is expressed in erythroid cells and activates γ-globin genes by binding to MARs in their promoters and recruiting histone acetylase PCAF. Further analysis in higher-order chromatin structure shows that SATB2 affects physical proximity of human (G)γ- and (A)γ-globin promoters via self-association. We also found that SATB2 interacts with SATB1, which specifically activates ε-globin gene expression. Our results establish SATB2 as a novel γ-globin gene regulator and provide a glimpse of the differential and cooperative roles of SATB family proteins in modulating clustered genes transcription and mediating higher-order chromatin structures.

Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing

MicroRNAs control gene expression either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. How this regulation is achieved and maintained is still largely unknown. MiRNAs that reside on clustered or polycistronic transcripts represent a more complex case where individual miRNAs from a cluster are processed with different efficiencies despite being cotranscribed. To shed light on the regulatory mechanisms that might be operating in these cases, we considered the long polycistronic primary miRNA transcript pri-miR-17-92a that contains six miRNAs with diverse functions. The six miRNA domains on this cluster are differentially processed to produce varying amounts of resultant mature miRNAs in different tissues. How this is achieved is not known. We show, using various biochemical and biophysical methods coupled with mutational studies, that pri-miR-17-92a adopts a specific three-dimensional architecture that poses a kinetic barrier to its own processing. This tertiary structure could create suboptimal protein recognition sites on the pri-miRNA cluster due to higher-order structure formation.

Evolutionary conservation of histone modifications in mammals.

Histone modification is an important mechanism of gene regulation in eukaryotes. Why many histone modifications can be stably maintained in the midst of genetic and environmental changes is a fundamental question in evolutionary biology. We obtained genome-wide profiles of three histone marks, H3 lysine 4 tri-methylation (H3K4me3), H3 lysine 4 mono-methylation (H3K4me1), and H3 lysine 27 acetylation (H3K27ac), for several cell types from human and mouse. We identified histone modifications that were stable among different cell types in human and histone modifications that were evolutionarily conserved between mouse and human in the same cell type. We found that histone modifications that were stable among cell types were also likely to be conserved between species. This trend was consistently observed in promoter, intronic, and intergenic regions for all of the histone marks tested. Importantly, the trend was observed regardless of the expression breadth of the nearby gene, indicating that slow evolution of housekeeping genes was not the major reason for the correlation. These regions showed distinct genetic and epigenetic properties, such as clustered transcription factor binding sites (TFBSs), high GC content, and CTCF binding at flanking sides. Based on our observations, we proposed that TFBS clustering in or near a histone modification plays a significant role in stabilizing and conserving the histone modification because TFBS clustering promotes TFBS conservation, which in turn promotes histone modification conservation. In summary, the results of this study support the view that in mammalian genomes a common mechanism maintains histone modifications against both genetic and environmental (cellular) changes.

ProTRAC - a software for probabilistic piRNA cluster detection, visualization and analysis

BackgroundThroughout the metazoan lineage, typically gonadal expressed Piwi proteins and their guiding piRNAs (~26-32nt in length) form a protective mechanism of RNA interference directed against the propagation of transposable elements (TEs). Most piRNAs are generated from genomic piRNA clusters. Annotation of experimentally obtained piRNAs from small RNA/cDNA-libraries and detection of genomic piRNA clusters are crucial for a thorough understanding of the still enigmatic piRNA pathway, especially in an evolutionary context. Currently, detection of piRNA clusters relies on bioinformatics rather than detection and sequencing of primary piRNA cluster transcripts and the stringency of the methods applied in different studies differs considerably. Additionally, not all important piRNA cluster characteristics were taken into account during bioinformatic processing. Depending on the applied method this can lead to: i) an accidentally underrepresentation of TE related piRNAs, ii) overlook duplicated clusters harboring few or no single-copy loci and iii) false positive annotation of clusters that are in fact just accumulations of multi-copy loci corresponding to frequently mapped reads, but are not transcribed to piRNA precursors.ResultsWe developed a software which detects and analyses piRNA clusters (proTRAC, probabilistic TRacking and Analysis of Clusters) based on quantifiable deviations from a hypothetical uniform distribution regarding the decisive piRNA cluster characteristics. We used piRNA sequences from human, macaque, mouse and rat to identify piRNA clusters in the respective species with proTRAC and compared the obtained results with piRNA cluster annotation from piRNABank and the results generated by different hitherto applied methods.proTRAC identified clusters not annotated at piRNABank and rejected annotated clusters based on the absence of important features like strand asymmetry. We further show, that proTRAC detects clusters that are passed over if a minimum number of single-copy piRNA loci are required and that proTRAC assigns more sequence reads per cluster since it does not preclude frequently mapped reads from the analysis.ConclusionsWith proTRAC we provide a reliable tool for detection, visualization and analysis of piRNA clusters. Detected clusters are well supported by comprehensible probabilistic parameters and retain a maximum amount of information, thus overcoming the present conflict of sensitivity and specificity in piRNA cluster detection.

The tudor domain protein Kumo is required to assemble the nuage and to generate germline piRNAs inDrosophila

In Drosophila ovaries, distinct Piwi-interacting RNA (piRNA) pathways defend against transposons in somatic and germline cells. Germline piRNAs predominantly arise from bidirectional clusters and are amplified by the ping-pong cycle. In this study, we characterize a novel Drosophila gene, kumo and show that it encodes a conserved germline piRNA pathway component. Kumo contains five tudor domains and localizes to nuage, a unique structure present in animal germline cells, which is considered to be the processing site for germline piRNAs. Transposons targeted by the germline piRNA pathway are derepressed in kumo mutant females. Moreover, germline piRNA production is significantly reduced in mutant ovaries, thereby indicating that kumo is required to generate germline piRNAs. Kumo localizes to the nuage as well as to nucleus early female germ cells, where it is required to maintain cluster transcript levels. Our data suggest that kumo facilitates germline piRNA production by promoting piRNA cluster transcription in the nucleus and piRNA processing at the nuage.

Highlights of This Issue

Epithelial-mesenchymal transition (EMT) has been implicated in the initial stages of metastasis. What remains unclear, however, is how it is maintained in late metastatic cells and whether it also regulates the subsequent dormant state of micrometastases. Tran and colleagues detail a mechanistic link between EMT and cancer dormancy in which Snail1 and Twist1, two key EMT inducers, cooperate with each other to initiate and maintain EMT, respectively, and in so doing Twist1 contributes directly to the cancer dormancy program. Therefore, Twist1 represents a potential therapeutic target to manipulate tumor dormancy for clinical benefits.The transmembrane protein Trop2 has been reported to be overexpressed in carcinomas, but its function remains largely unknown. In an effort to elucidate its role in tumorigenesis, Wang and colleagues generated Trop2−/− animals, which lack an obvious tumor phenotype. However, when mated into an Arf-null background and exposed to carcinogens, Trop2−/− mice develop tumors with both squamous and spindle cell histology. Accordingly, immortalized cells from Trop2−/− Arf−/− mice show hyperactivation of MAPK and Src pathways. Additionally, poorly differentiated head and neck cancers exhibit low Trop2 levels. These results identify a tumor suppressor role and highlight unexpected signaling properties for this protein.In this study, Azad and colleagues present the first report of DNA-dependent protein kinase (DNA-PK) blockade leading to accelerated senescence in irradiated human cancer cells. The study shows that DNAPK inhibition modulates repair of therapeutically induced DNA doublestrand breaks and thus promotes p53-dependent accelerated senescence in irradiated cells. Significantly, this report notes the lack of clinical trials using novel pharmacological inhibitors of DNA-PK in conjunction with ionizing radiation. Thus, these data provide a sound rationale for both preclinical and clinical evaluation of novel DNA-PK inhibitors in combination with ionizing radiation and other anticancer agents that induce doublestrand breaks or inhibit double-strand break repair.Many strategies used to kill cancer cells induce stress-responses that inhibit treatment-induced cell death and promote emergence of a treatment resistant phenotype. Whereas both Y-box binding protein-1 (YB-1) and clusterin are known to confer taxane resistance in cancer, their mechanistic interrelationships are undefined. Shiota and colleagues found that many varied anticancer therapeutic agents including taxanes induce endoplasmic reticulum stress with activation of YB-1. YB-1 was shown to be a critical regulator of stress-induced clusterin transcription and expression, and clusterin was shown to play a dominant downstream mediator of YB-1 in stressinduced cytoprotection and paclitaxel resistance in prostate cancer cells.

Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats

We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

Global analysis of parental imprinting in human parthenogenetic induced pluripotent stem cells

To study the role of parental imprinting in human embryogenesis, we generated parthenogenetic human induced pluripotent stem cells (iPSCs) with a homozygote diploid karyotype. Global gene expression and DNA methylation analyses of the parthenogenetic cells enabled the identification of the entire repertoire of paternally expressed genes. We thus demonstrated that the gene U5D, encoding a variant of the U5 small RNA component of the spliceosome, is an imprinted gene. Introduction of the U5D gene into parthenogenetic cells partially corrected their molecular phenotype. Our analysis also uncovered multiple miRNAs existing as imprinted clustered transcripts, whose putative targets we then studied further. Examination of the consequences of parthenogenesis on human development identified marked effects on the differentiation of extraembryonic trophectoderm and embryonic liver and muscle tissues. This analysis suggests that distinct regulatory imprinted small RNAs and their targets have substantial roles in human development.

Abstract 3421: Twist1 and clusterin regulate epithelial-mesenchymal transition

Abstract INTRODUCTION AND OBJECTIVE: Clusterin (CLU) is a stress-activated, cytoprotective molecular chaperone, and is highly expressed in cells surviving apoptotic stimuli and in advanced and treatment-resistant cancers. In addition, CLU has recently been reported to be implicated in epithelial-mesenchymal transition (EMT). Similarly, oncogenic transcription factor Twist1 which binds to E-box (core sequence, 5’-CANNTG-3’) is known to be a master regulator of EMT. Although CLU is known to be transcriptionally activated by heat-shock factor 1, the mechanism CLU expression in EMT is not fully elucidated. This study begins to define mechanism of CLU expression and relevance in EMT of prostate cancer. METHODS: Human prostate cancer PC-3 and LNCaP cells were used to examine whether the manipulation of Twist1 expression affected CLU expression using quantitative real-time PCR, Western blotting and luciferase reporter assay using CLU promoter region. Chromatin-immunoprecipitation (ChIP) assays were used to determine whether Twist1 binds to CLU promoter region. We also examined markers of EMT after treatment with TGF-β1 and manipulation of Twist1/CLU expression. RESULTS: Twist1 overexpression was shown to upregulate CLU expression whereas Twist1 knockdown reduced CLU expression. ChIP assay and luciferase reporter assay revealed that Twist1 was bound to CLU promoter region and regulated CLU transcription. Treatment with TGF-β1, which is known to be an EMT-inducing cytokine, upregulated Twist1 expression followed by CLU expression. Furthermore, CLU knockdown ameliorated EMT induced by TGF-β1 and Twist1. CONCLUSIONS: This study indicates that CLU regulates EMT induced by TGF-β1 and Twist1 in prostate cancer cells. Modulation of CLU expression may be a useful strategy for developing novel therapeutics regulating EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3421. doi:10.1158/1538-7445.AM2011-3421

Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

Diversity in the Interactions of Isoforms Linked to Clustered Transcripts: A Systematic Literature Analysis

Existing protein-protein interactions databases cover only a portion of the interactome and interaction information on protein isoforms is underrepresented. This leads to a lack of information on the functional similarity of protein isoforms and the effects of transcript diversity on the protein interaction networks. We present a comprehensive automated literature analysis that extracts interactions involving human protein isoforms linked to clusters of transcripts with high sequence similarity and deliver them in a database called TBIID for knowledge discovery. We measure the interaction variability of the isoforms from the clustered transcripts by analysing the distribution of their interaction partners in TBIID. Almost all clusters analyzed (99%) contain isoforms with unique partners indicating that isoforms are specialized towards forming unique interactions and thus achieving functional diversity, which is similar to the results from public resources. TBIID is available at http://tbiid.emu.edu.tr containing most relevant candidates for future experiments focusing on understanding the isoform interaction networks and the resulting functional implications.

Human SMG-1 is Involved in Gemcitabine-Induced Primary microRNA-155/BIC Up-Regulation in Human Pancreatic Cancer PANC-1 Cells

Human primary microRNA-155/B-cell integration cluster (BIC) transcript is the precursor of microRNA-155. The overexpression of them has been widely observed in the progression of various types of tumors. Our objective was to investigate the effect of anticancer agents on the expression of BIC and possible signal pathways that involved in. Quantitative real-time reverse transcriptase polymerase chain reaction was used to measure the expression of BIC. Chemical inhibitors against c-Jun N-terminal kinase 1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2, protein kinase C, checkpoint kinase 1, and phosphatidylinositol 3 kinase (PI3K) were used for the evaluation of involved signal pathways. RNA interference was used to knock down the expression of ataxia-telangiectasia mutated, ataxia-telangiectasia and Rad3 related, and suppressor of morphogenesis in genitalia-1 (SMG-1), and Western blot was carried out to evaluate the knockdown effect. B-cell integration cluster expression was induced by a representative anti-pancreatic cancer drug, gemcitabine, in human pancreatic cancer PANC-1 cells. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and c-Jun N-terminal kinase inhibitors, but not the checkpoint kinase 1 and protein kinase C inhibitors, suppressed the up-regulation of BIC. B-cell integration cluster up-regulation was also significantly inhibited by the PI3K inhibitor wortmannin. RNA interference studies showed that wortmannin-sensitive SMG-1 but not ataxia-telangiectasia mutated or ataxia-telangiectasia and Rad3 related was involved in the up-regulation. Our results show that multiple pathways can be involved in the up-regulation of BIC. Furthermore, we demonstrate for the first time that PI3K SMG-1 is required for gemcitabine-induced up-regulation of BIC transcript.

Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.

Tobacco (Nicotiana tabacum) synthesizes nicotine and related pyridine alkaloids in the root, and their synthesis increases upon herbivory on the leaf via a jasmonate-mediated signaling cascade. Regulatory NIC loci that positively regulate nicotine biosynthesis have been genetically identified, and their mutant alleles have been used to breed low-nicotine tobacco varieties. Here, we report that the NIC2 locus, originally called locus B, comprises clustered transcription factor genes of an ethylene response factor (ERF) subfamily; in the nic2 mutant, at least seven ERF genes are deleted altogether. Overexpression, suppression, and dominant repression experiments using transgenic tobacco roots showed both functional redundancy and divergence among the NIC2-locus ERF genes. These transcription factors recognized a GCC-box element in the promoter of a nicotine pathway gene and specifically activated all known structural genes in the pathway. The NIC2-locus ERF genes are expressed in the root and upregulated by jasmonate with kinetics that are distinct among the members. Thus, gene duplication events generated a cluster of highly homologous transcription factor genes with transcriptional and functional diversity. The NIC2-locus ERFs are close homologs of ORCA3, a jasmonate-responsive transcriptional activator of indole alkaloid biosynthesis in Catharanthus roseus, indicating that the NIC2/ORCA3 ERF subfamily was recruited independently to regulate jasmonate-inducible secondary metabolism in distinct plant lineages.