Cell Context-dependent Manner Research Articles

Abstract 2958: The novel protein kinase STK17A is a direct p53 target gene that mediates response to genotoxic and nutritional stress in a cancer cell context-dependent manner

Abstract STK17A (DRAK1) is a largely uncharacterized serine/threonine kinase that belongs to the death-associated protein kinase family. Prior microarray studies demonstrated that STK17A is induced with cisplatin in testicular cancer-derived human embryonal carcinoma (EC) cells. We now demonstrate the STK17A in a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner in a number of different cell contexts. A consensus and functional p53 response element was found upstream of the STK17A promoter and endogenous p53 was shown to bind to this site in a cisplatin-dependent manner. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with up-regulation of detoxifying and antioxidant genes and reduction in reactive oxygen species (ROS), whereas overexpression of STK17A increased ROS levels in EC cells. Interestingly, we found that STK17A is highly overexpressed in clinical glioblastoma (GBM) and cell lines compared to its expression in normal brain tissue and other cancers. High STK17A expression in GBM patients correlated with poor clinical outcome and decreased survival. STK17A knockdown in GBM cells decreased clonagenic cell growth, soft agar tumorigenicity, and survival in response to genotoxic and nutritional stress. Preliminary data will be presented implicating STK17A in the regulation of autophagy and the proximal autophagy component, ULK1. These findings indicate that STK17A may be added to a growing list of direct p53 target genes involved in autophagy that includes TIGAR, Sestrins, DAPK1 and DRAM and that by impacting autophagy STK17A may modulate response to genotoxic and nutritional stress in a cell context-depend manner. This study also suggests that STK17A is a potential novel prognostic maker and therapeutic target for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2958. doi:1538-7445.AM2012-2958

Genome-Wide Analysis of N1ICD/RBPJ Targets In Vivo Reveals Direct Transcriptional Regulation of Wnt, SHH, and Hippo Pathway Effectors by Notch1

The Notch pathway plays a pivotal role in regulating cell fate decisions in many stem cell systems. However, the full repertoire of Notch target genes in vivo and the mechanisms through which this pathway activity is integrated with other signaling pathways are largely unknown. Here, we report a transgenic mouse in which the activation of the Notch pathway massively expands the neural stem cell (NSC) pool in a cell context-dependent manner. Using this in vivo system, we identify direct targets of RBPJ/N1ICD in cortical NSCs at a genome-wide level through combined ChIP-Seq and transcriptome analyses. Through a highly conservative analysis of these datasets, we identified 98 genes that are directly regulated by N1ICD/RPBJ in vivo. These include many transcription factors that are known to be critical for NSC self-renewal (Sox2, Pax6, Tlx, and Id4) and the transcriptional effectors of the Wnt, SHH, and Hippo pathways, TCF4, Gli2, Gli3, Yap1, and Tead2. Since little is known about the function of the Hippo-Yap pathway in NSCs, we analyzed Yap1 expression and function in NSCs. We show that Yap1 expression is restricted to the stem cell compartment in the developing forebrain and that its expression is sufficient to rescue Notch pathway inhibition in NSC self-renewal assays. Together, results of this study reveal a previously underappreciated complexity and breadth of Notch1 targets in vivo and show direct interaction between Notch and Hippo-Yap pathways in NSCs.

Dynamic and Ligand-Selective Interactions of Vitamin D Receptor with Retinoid X Receptor and Cofactors in Living Cells

The vitamin D receptor (VDR) mediates vitamin D signaling in numerous physiological and pharmacological processes, including bone and calcium metabolism, cellular growth and differentiation, immunity, and cardiovascular function. Although transcriptional regulation by VDR has been investigated intensively, an understanding of ligand-selective dynamic VDR conformations remains elusive. Here, we examined ligand-dependent dynamic interactions of VDR with retinoid X receptor (RXR), steroid receptor coactivator 1 (SRC-1), and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) in cells using fluorescence resonance energy transfer (FRET) and chromatin immunoprecipitation (ChIP) assays. We compared the effects of 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], lithocholic acid (LCA), and (25R)-25-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,26,27-trinor-20-epivitamin D(3) (ADTT), a partial agonist/antagonist vitamin D derivative. In the absence of ligand, VDR homodimers were preferred to RXR heterodimers and were associated with SMRT. 1,25(OH)(2)D(3) induced heterodimerization with RXR, dissociation of SMRT, and association of SRC-1. LCA and ADTT induced those effects to a lesser extent at concentrations that did not induce expression of the VDR target gene CYP24A1 in human embryonic kidney (HEK) 293 cells. Unlike in HEK293 cells, ADTT increased CYP24A1 expression in HCT116 cells and increased the association of VDR and SMRT on the CYP24A1 promoter. The results indicate that ligand-selective conformation may lead to unique cofactor complex formation in a cell context-dependent manner. The combination of FRET and ChIP assays is a powerful tool useful in understanding ligand-selective dynamic VDR conformations and the development of selective VDR modulators.

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes.

KAI1 Gene Is Engaged in NDRG1 Gene-mediated Metastasis Suppression through the ATF3-NFκB Complex in Human Prostate Cancer

KAI1 Gene Is Engaged in NDRG1 Gene-mediated Metastasis Suppression through the ATF3-NFκB Complex in Human Prostate Cancer

BRAFV600E and Microenvironment in Thyroid Cancer: A Functional Link to Drive Cancer Progression

Papillary thyroid cancer (PTC) rates continue to increase in the United States and Europe, and, although most patients do well, some recur and die of their disease. Patients with PTC harboring the BRAF(V600E) mutation seem to display a more aggressive clinical behavior, but little is known about the role of this mutation in crucial processes in the tumor microenvironment, such as tumor adhesion, migration, invasion, and metastasis. The extracellular matrix (ECM) microenvironment is not merely a structural scaffold for the cellular elements of the epithelial and stromal microenvironment, but it also elicits a profound influence on cell behavior affecting viability, proliferation, adhesion, and motility. The effects of BRAF(V600E) on cell surface receptors (i.e., integrins) and ECM noncellular components [i.e., thrombospondin-1 (TSP-1) and fibronectin (FN)] seem to trigger different pathologic biological processes in a cell context-dependent manner. This review focuses on the recent progress in understanding the role of BRAF(V600E) in the regulation of some ECM noncellular components and trans-membrane receptors of the microenvironment in PTC in order to design novel targeted therapies directed at the BRAF(V600E) multifaceted signaling cascades. Some of these targeted therapeutics, such as ATP-competitive BRAF(V600E) inhibitors (i.e., orally bioavailable PLX4720 and PLX4032 compounds), are already under investigation.

∆N-p63 functions as an invasion, migration, and metastasis suppressor

AbstractThe tumor suppressor p53 homologues, TA-p73, and p63 have been shown to function as tumor suppressors. However, how they function as tumor suppressors remains elusive. Expression of p63 is controlled by two distinct promoters. Consequently, this results in two different gene products such as TA-(transactivation domain containing NH2 terminus) p63 and ∆N-(lacks NH2 terminus) p63. It is generally thought that the TA-p63 functions as a tumor suppressor, while the ∆N-p73 functions as a proto-oncogene. However, careful interpretation of the data concerning ∆N-p63 suggests that it could function as an invasion and metastasis/tumor suppressor in a cell context dependent manner.

∆N-p63 functions as an invasion, migration, and metastasis suppressor

AbstractThe tumor suppressor p53 homologues, TA-p73, and p63 have been shown to function as tumor suppressors. However, how they function as tumor suppressors remains elusive. Expression of p63 is controlled by two distinct promoters. Consequently, this results in two different gene products such as TA-(transactivation domain containing NH2 terminus) p63 and ∆N-(lacks NH2 terminus) p63. It is generally thought that the TA-p63 functions as a tumor suppressor, while the ∆N-p73 functions as a proto-oncogene. However, careful interpretation of the data concerning ∆N-p63 suggests that it could function as an invasion and metastasis/tumor suppressor in a cell context dependent manner.

Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

BackgroundNotch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues.Methodology/Principal FindingsTo analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis.Conclusion/SignificanceWe suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context.

Abstract 130: Molecular basis of autophagy-mediated resistance to radiation and Apo2L/TRAIL therapy of prostate cancer

Abstract Cell death and survival signaling plays an important role in the response to irradiation (IR) and antitumor chemotherapy. IR and other apoptotic insults, such as the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL), are used as prostate cancer therapeutics, however cellular resistance may hinder their effectiveness. PC3 cells were more sensitive to Apo2L/TRAIL-mediated cell death compared to LNCaP-derived C4-2 prostate cancer cells. The cell death observed showed both apoptotic as well as nonapoptotic features, raising the possibility that apart from classical apoptosis, autophagy might also contribute to cytotoxicity. Following Apo2L/TRAIL treatment of PC3 cells, microtubule associated protein 1 light chain LC3 (ATG8), a classical marker for autophagy, was recruited into autophagosomes following its cleavage (LC3 I) and lipid modification (LC3 II). In contrast, LC3 failed to associate with the autophagosomes in C4-2 cells. IR also resulted in increased LC3 II in PC3 cells whereas in C4-2 LC3 was predominantly unmodified. Inhibition of autophagosome and lysosome fusion with chloroquine resulted in accumulation of LC3 II in both PC3 and C4-2 cells suggesting that lipid modification of LC3 is intact in C4-2 cells. To address the rate of autophagic flux in detail we ectopically overexpressed GFP-mCherry-LC3 fusion protein and observed its localization upon Apo2L/TRAIL and IR treatment. C4-2 cells showed more LC3 II in the lysosomal compartment compared to autophagosomes, whereas PC3 had predominantly autophagosomal LC3 II. This indicates that the rate of autophagic outflux is higher in C4-2 cells upon Apo2L/TRAIL treatment. We next examined the expression and sub-cellular distribution of other autophagy related proteins. ATG5 is decreased in the membrane fraction of PC3 cells treated with Apo2L/TRAIL and IR. In C4-2 cells ATG5 is increased in the soluble and decreased in the membrane fractions upon IR while decreased in both cellular fractions upon Apo2L/TRAIL treatment. p62/SQSTM1 levels increased in poly-ubiquitinated aggregates in PC3 but not in C4-2 cells upon Apo2L/TRAIL treatment. Moreover, we observed differences in ATG5 and p62 associated native protein complexes among PC3 and C4-2 cells upon Apo2L/TRAIL and IR treatment. Characterization of these complexes would give new insights into autophagic signaling and Apo2L/TRAIL response. Our results suggest that in Apo2L/TRAIL or IR-sensitive PC3 cells accumulation of autophagosomes triggers apoptosis resulting in effective cell death whereas a more efficient autophagic response in C4-2 cells might be the reason for their resistance against Apo2L/TRAIL or IR. Further studying function of autophagy regulatory genes will help to understand their role in mediating cell death or survival outcomes in a cell context dependent manner, which may lead to the design of better therapies for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 130.

Abstract 3398: Double-faced Sema3E-PlexinD1 signaling inhibits tumor growth but promotes metastatic spreading

Abstract Semaphorins comprise a wide family of secreted and membrane-bound signals, originally found in neural development, and then implicated in a range of functions: from angiogenesis, to the immune response, to cancer progression. We have previously shown that high-affinity semaphorin receptors are found in the families of the plexins and of the neuropilins. Moreover, additional molecules interact with semaphorin receptors in cell-context dependent manner, featuring a complex scenario of multiple potential signaling pathways. The role of semaphorin signaling in tumor progression is currently under close scrutiny; it is known that cancer cells release semaphorins regulating their own behavior as well as that of cells in the tumor microenvironment, such as endothelial cells and recruited leukocytes. In this work, we focused on the function of Semaphorin 3E (Sema3E) and its high affinity receptor PlexinD1, and on their potential role to regulate cancer progression. Here we found that Sema3E and PlexinD1 are highly expressed in human metastatic tumors. Moreover, we provided experimental evidences that Sema3E released by tumor cells is a double-faced signal, acting via two distinctive signaling pathways. In paracrine manner, Sema3E-PlexinD1 signaling can elicit endothelial cell repulsion, thereby reducing vessel density and tumor growth. Conversely, Sema3E autocrine signaling in tumor cells thrusts their invasiveness, trans-endothelial migration, and metastatic spreading, through the trans-activation of PlexinD1-associated oncogenic kinases. Notably, by silencing the endogenous expression of either Sema3E or PlexinD1 the metastatic potential of cancer cells is significantly reduced, identifying this pathway as a major driver of the metastatic spreading. In sum, Sema3E autocrine signaling in cancer cells is crucially implicated to induce a metastatic behavior, and it appears as a promising target for strategies aimed at blocking tumor metastasis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3398.

Selective unresponsiveness to the inhibition of p38 MAPK activation by cAMP helps L929 fibroblastoma cells escape TNF-α-induced cell death

BackgroundThe cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress cell death, in a cell context-dependent manner. Our previous study has shown that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of mitogen-activated protein kinase (MAPK) p38 activation in fibroblasts, which leads to suppression of NF-κB activity and promotion of tumor necrosis factor-α (TNF-α)-induced cell death. However, it remains unknown whether this regulation is also applicable to fibroblastoma cells.MethodsIntracellular cAMP was determined in L929 fibroblastoma cells after treatment of the cells with various cAMP elevation agents. Effects of cAMP in the presence or absence of the RNA synthesis inhibitor actinomycin D or small interfering RNAs (siRNAs) against CREB on TNF-α-induced cell death in L929 cells were measured by propidium iodide (PI) staining and subsequent flow cytomety. The activation of p38 and c-Jun N-terminal protein kinase (JNK), another member of MAPK superfamily, was analyzed by immunoblotting. JNK selective inhibitor D-JNKi1 and p38 selective inhibitor SB203580 were included to examine the roles of JNK and p38 in this process. The expression of DLC or other mediators of cAMP was analyzed by immunoblotting. After ectopic expression of DLC with a transfection marker GFP, effects of cAMP on TNF-α-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.ResultsElevation of cAMP suppressed TNF-α-induced necrotic cell death in L929 fibroblastoma cells via CREB-mediated transcription. The pro-survival role of cAMP was associated with selective unresponsiveness of L929 cells to the inhibition of p38 activation by cAMP, even though cAMP significantly inhibited the activation of JNK under the same conditions. Further exploration revealed that the induction of DLC, the major mediator of p38 inhibition by cAMP, was impaired in L929 cells. Enforced inhibition of p38 activation by using p38 specific inhibitor or ectopic expression of DLC reversed the protection of L929 cells by cAMP from TNF-α-induced cell death.ConclusionThese data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.

Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

Polycomb protein EZH2-mediated gene silencing is implicated in breast tumorigenesis through methylation of histone H3 on Lysine 27 (H3K27). We have previously shown that S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A can modulate histone methylation and disrupt EZH2 complex. Here, we used 3-deazaneplanocin A, together with other chromatin remodeling agents, as well as RNA interference-mediated EZH2 depletion, to probe the role of EZH2 in coordination with other epigenetic components in gene regulation in breast cancer cells. Through genome-wide gene expression analysis, coupled with extensive chromatin immunoprecipitation analysis of histone modifications, we have identified a variety of gene sets that are regulated either by EZH2 alone or through the coordinated action of EZH2 with HDAC and/or DNA methylation. We further found that tumor antigen GAGEs were regulated by distinct epigenetic mechanisms in a cell context-dependent manner, possibly reflecting mechanistic heterogeneity in breast cancer. Intriguingly, we found that EZH2 regulates a remarkable cohort of genes whose functions are highly enriched in immunoresponse and autocrine inflammation network, and that their transcriptional activation upon EZH2 perturbation is cancer specific, revealing a potential novel role of EZH2 in regulating cancer immunity. These findings show the complexity and diversity of epigenetic regulation in human cancer and underscore the importance for developing combinatorial pharmacologic approaches for effective epigenetic gene reactivation.

Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.

Abstract 1497Poster Board I-520KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Identification and characterization of estrogen receptor-related receptor alpha and gamma in human glioma and astrocytoma cells

The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRalpha and ERRgamma proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC(50) and IC(50) values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRalpha and ERRgamma are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.

Glucocorticoid-regulated microRNAs and mirtrons in acute lymphoblastic leukemia

Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lymphoblastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are ~22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15 ~ 16 clusters. In an in vitro model, overexpression of miR15b ~ 16 mimics increased and silencing by miR15b ~ 16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner.

Menin, Histone H3 Methyltransferases, and Regulation of Cell Proliferation: Current Knowledge and Perspective

Menin is a tumor suppressor encoded by the MEN1 gene that is mutated in patients with an inherited syndrome, multiple endocrine neoplasia type 1 (MEN1). Loss of menin has potent impact on proliferation of endocrine and non-endocrine cells. However, until recently little has been known as to how menin regulates cell proliferation. Rapid research progress in the past several years suggests that menin represses proliferation of endocrine cells yet promotes proliferation in certain types of leukemia cells via interacting with various transcriptional regulators. Menin interacts with histone H3 methyltransferases such as MLL (mixed lineage leukemia) protein. Increasing evidence has linked the biological function of menin to epigenetic histone modifications, control of the pattern of gene expression, and regulation of cell proliferation in a cell type-specific manner. In light of these recent findings, an emerging model suggests that menin is a crucial regulator of histone modifiers by acting as a scaffold protein to coordinate gene transcription and cell proliferation in a cell context-dependent manner. This recent progress unravels the coordinating role of menin in epigenetics and regulation of cell cycle, providing novel insights into understanding regulation of beta cell functions and diabetes, as well as the development and therapy of endocrine tumors and leukemia.

Potential use of an estrogen–glucocorticoid receptor chimera as a drug screen for tissue selective estrogenic activity

SERMs act as ER agonists in bone despite their antagonistic properties in other tissues. As well as inhibiting bone remodelling, this effect may involve stimulation of osteoblast activity, in light of evidence from recent in vivo studies. However, progress in exploring this action has been hampered by a lack of accurate in vitro models. For example, ER antagonists are reported to stimulate reporter assays based on estrogen target genes in osteoblasts, contrary to their inhibitory effects in vivo. We examined whether evaluating global aspects of ER function provides a more accurate reflection of ER activation in osteoblasts, based on the use of morphological and/or transcriptional read-outs with green fluorescent protein (GFP)-receptor chimeras. Osteoblast-like (ROS and U2OS) and breast cancer (MCF7) cells were transfected with a human ERα–GFP fusion protein, and treated with ER agonists (17β-estradiol, and dienestrol), antagonists (ICI 182,780 and ZK 164015) and SERMs (tamoxifen, raloxifene, 4-hydroxytamoxifen (4-HT) and hexestrol). We investigated cellular compartmentalisation of these constructs by fluorescence microscopy, nuclear mobility by fluorescence recovery after photobleaching (FRAP), and global activation of estrogenic transcription using a ERE-luc reporter. SERMs caused a modest increase in ERE-luc activity in osteoblast-like cells (but not in breast cells), and a reduction in nuclear mobility in breast (but not osteoblast-like) cells. These studies were then repeated using a GFP chimera where the human GR ligand binding domain (LBD) was replaced by the human ERα LBD (ERGR–GFP), combined with a GRE-luc reporter. Interestingly, SERMs increased both cytoplasmic to nuclear translocation of ERGR–GFP, and GRE-luc reporter activity, in osteoblast-like (but not breast) cells. Indeed, transcriptional responses to SERMs in osteoblast-like cells were considerably greater with the ERGR/GRE-luc than the ERα/ERE-luc system, 4-HT inducing 300 and 25% increases in reporter activity respectively. ER antagonists were entirely without effect. We conclude that evaluation of global estrogenic activity, as opposed to activation of a specific target gene, provides a more accurate read-out for osteoblast stimulation. In particular, ERGR-mediated GRE-luc activity provides a high signal response to estrogen agonists and SERMs, in a cell context dependent manner closely resembling that observed in vivo. Further studies utilising this system are justified to explore the mechanistic basis for estrogenic stimulation of osteoblast activity, and to identify newer SERMs capable of targeting this activity.

BCL6-mediated Attenuation of DNA Damage Sensing Triggers Growth Arrest and Senescence through a p53-dependent Pathway in a Cell Context-dependent Manner

The BCL6 oncogenic transcriptional repressor is required for development of germinal center centroblasts, which undergo simultaneous genetic recombination and massive clonal expansion. Although BCL6 is required for survival of centroblasts, its expression in earlier B-cells is toxic. Understanding these opposing effects could provide critical insight into normal B-cell biology and lymphomagenesis. We examined the transcriptional and biological effects of BCL6 in various primary cells. BCL6 repression of ATR was previously shown to play a critical role in the centroblast phenotype. Likewise, we found that BCL6 could impose an ATR-dependent phenotype of attenuated DNA damage sensing and repair in primary fibroblasts and B-cells. BCL6 induced true genomic instability because DNA repair was delayed and was qualitatively impaired, which could be critical for BCL6-induced lymphomagenesis. Although BCL6 can directly repress TP53 in centroblasts, BCL6 induced TP53 expression in primary fibroblasts and B-cells, and these cells underwent p53-dependent growth arrest and senescence in the presence of physiological levels of BCL6. This differential ability to trigger a functional p53 response explains at least in part the different biological response to BCL6 expression in centroblasts versus other cells. The data suggest that targeted re-activation of TP53 could be of therapeutic value in centroblast-derived lymphomas.

Derepression in the Desert: The Third Workshop on Clinical Translation of Epigenetics in Cancer Therapeutics

In the past decade, the scientific and medical community has witnessed dramatic progress in epigenetics and has begun to understand some of the epigenetic mechanisms that contribute to human disease, particularly cancer. In January 2007, the third biannual workshop on Clinical Translation of