ABSTRACT
A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.
Subject(s)
Biomarkers , Eye Proteins/metabolism , Genomics , Lymphoid Progenitor Cells/metabolism , Single-Cell Analysis , Animals , Cells, Cultured , Computational Biology/methods , Eye Proteins/genetics , Gene Expression Profiling , Genomics/methods , Hematopoiesis/genetics , High-Throughput Nucleotide Sequencing , Lymphoid Progenitor Cells/cytology , Lymphoid Progenitor Cells/immunology , Mice , Mice, Knockout , Mice, Transgenic , Proteomics , Single-Cell Analysis/methodsABSTRACT
The p53 tumor suppressor can restrict malignant transformation by triggering cell-autonomous programs of cell-cycle arrest or apoptosis. p53 also promotes cellular senescence, a tumor-suppressive program that involves stable cell-cycle arrest and secretion of factors that modify the tissue microenvironment. In the presence of chronic liver damage, we show that ablation of a p53-dependent senescence program in hepatic stellate cells increases liver fibrosis and cirrhosis associated with reduced survival and enhances the transformation of adjacent epithelial cells into hepatocellular carcinoma. p53-expressing senescent stellate cells release factors that skew macrophage polarization toward a tumor-inhibiting M1-state capable of attacking senescent cells in culture, whereas proliferating p53-deficient stellate cells secrete factors that stimulate polarization of macrophages into a tumor-promoting M2-state and enhance the proliferation of premalignant cells. Hence, p53 can act non-cell autonomously to suppress tumorigenesis by promoting an antitumor microenvironment, in part, through secreted factors that modulate macrophage function.
Subject(s)
Cell Transformation, Neoplastic , Cellular Senescence , Hepatic Stellate Cells/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Animals , Cellular Microenvironment , Fibrosis/pathology , Hepatic Stellate Cells/cytology , Humans , Inflammation/metabolism , Kupffer Cells/metabolism , Kupffer Cells/pathology , Liver/cytology , Liver/pathology , Macrophages/metabolism , Macrophages/pathology , Mice , NF-kappa BABSTRACT
During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.
Subject(s)
Databases, Genetic , Gene Expression/genetics , Hematopoiesis/genetics , Transcriptome/genetics , Animals , Computational Biology , Hematopoietic Stem Cells/metabolism , High-Throughput Nucleotide Sequencing/trends , Humans , Mice , RNA-Seq , SoftwareABSTRACT
Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.
Subject(s)
Cellular Senescence/physiology , Drug Resistance/physiology , Phenotype , Transcription Factor RelA/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Survival/physiology , Cellular Senescence/drug effects , Cellular Senescence/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma/metabolism , Mice , Protein Synthesis Inhibitors/pharmacology , RNA, Small Interfering/metabolism , Tetracycline/pharmacology , Tumor Suppressor Protein p53/metabolismABSTRACT
The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.
Subject(s)
Carcinogenesis , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , MAP Kinase Signaling System/genetics , Neoplasms, Glandular and Epithelial/genetics , Nuclear Proteins/genetics , Animals , Cell Proliferation , Disease Models, Animal , Drosophila Proteins/metabolism , Eye Neoplasms/genetics , Eye Neoplasms/pathology , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Neoplasms, Glandular and Epithelial/pathology , Nuclear Proteins/metabolism , Oncogene Protein p65(gag-jun)/genetics , Oncogene Protein p65(gag-jun)/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Emu-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X(L) protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Emu-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.
Subject(s)
Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Lymphoma/metabolism , Animals , Apoptosis/drug effects , Cell Line, Tumor , Histone Deacetylases/metabolism , Humans , Indoles , Lymphoma/pathology , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Neoplasm Transplantation , Panobinostat , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Death Domain/metabolism , Signal Transduction/drug effects , Survival Rate , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Histone Deacetylase Inhibitors , Mammary Neoplasms, Experimental/drug therapy , Neoplasm Proteins/agonists , Neoplasm Proteins/antagonists & inhibitors , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Animals , Antibodies, Monoclonal/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/biosynthesis , Cell Line, Tumor , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/pharmacology , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Inbred BALB C , Mice, SCID , Neoplasm Proteins/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , VorinostatABSTRACT
Expression of the human HIN-200 family member IFI 16 has been reported to suppress cell growth and contribute to the onset of cellular senescence. However the molecular events involved in this process have not been fully characterised. We fused IFI 16 to the estrogen receptor ligand-binding domain to establish an inducible model for studying the molecular events that cause these phenomena. In cells induced to express the ER-IFI 16 within the nucleus there was a decrease in cellular proliferation and concomitant growth arrest in the G1 phase of the cell cycle. Unlike previous reports, this did not appear to involve the p53-p21(WAF1/CIP1)-cdk2-pRb pathway. Following nuclear expression of ER-IFI 16 we noted senescence-like morphological changes and expression of senescence-associated beta-galactosidase in growth arrested cells. Importantly, we also found a marked reduction in telomerase activity in arrested cells compared to controls. Moreover, IFI 16 and hTERT co-localised within the nucleus and these two proteins physically interacted in vivo and in vitro. Together, these data suggest that IFI 16 may act as an endogenous regulator of telomerase activity and, through its interaction with hTERT, contributes to the inhibition of proliferation and induces a senescence-like state.
Subject(s)
Cell Proliferation , Cellular Senescence/physiology , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Telomerase/metabolism , Blotting, Western , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Fluorescent Antibody Technique , Humans , ImmunoprecipitationABSTRACT
Histone deacetylases (HDACs) are enzymes involved in the remodelling of chromatin, and have a key role in the epigenetic regulation of gene expression. In addition, the activity of non-histone proteins can be regulated through HDAC-mediated hypo-acetylation. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and several classes of HDAC inhibitors have been found to have potent and specific anticancer activities in preclinical studies. However, such studies have also indicated that the effects of HDAC inhibitors could be considerably broader and more complicated than originally understood. Here we summarize recent advances in the understanding of the molecular events that underlie the anticancer effects of HDAC inhibitors, and discuss how such information could be used in optimizing the development and application of these agents in the clinic, either as monotherapies or in combination with other anticancer drugs.
Subject(s)
Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Histone Deacetylase Inhibitors , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Enzyme Inhibitors/pharmacology , Histone Deacetylases/metabolism , Humans , Neoplasms/enzymology , Neoplasms/physiopathologyABSTRACT
Eosinophils are important in fighting parasitic infections and are implicated in the pathogenesis of asthma and allergy. IL-5 is a critical regulator of eosinophil development, controlling proliferation, differentiation, and maturation of the lineage. Mice that constitutively express IL-5 have in excess of 10-fold more eosinophils in the hematopoietic organs than their wild type (WT) counterparts. We have identified that much of this expansion is in a population of Siglec-F high eosinophils, which are rare in WT mice. In this study, we assessed transcription in myeloid progenitors, eosinophil precursors, and Siglec-F medium and Siglec-F high eosinophils from IL-5 transgenic mice and in doing so have created a useful resource for eosinophil biologists. We have then utilized these populations to construct an eosinophil trajectory based on gene expression and to identify gene sets that are associated with eosinophil lineage progression. Cell cycle genes were significantly associated with the trajectory, and we experimentally demonstrate an increasing trend toward quiescence along the trajectory. Additionally, we found gene expression changes associated with constitutive IL-5 signaling in eosinophil progenitors, many of which were not observed in eosinophils.
Subject(s)
Eosinophils/immunology , Gene Expression Profiling , Interleukin-5/immunology , Animals , Cell Differentiation/immunology , Cell Lineage/immunology , Eosinophils/cytology , Eosinophils/metabolism , Interleukin-5/metabolism , Mice , Mice, Transgenic , Myeloid Progenitor Cells/cytology , Myeloid Progenitor Cells/immunology , Myeloid Progenitor Cells/metabolismABSTRACT
In recent years multi-parameter flow cytometry has enabled identification of cells at major stages in myeloid development; from pluripotent hematopoietic stem cells, through populations with increasingly limited developmental potential (common myeloid progenitors and granulocyte-macrophage progenitors), to terminally differentiated mature cells. Myeloid progenitors are heterogeneous, and the surface markers that define transition states from progenitors to mature cells are poorly characterized. Siglec-F is a surface glycoprotein frequently used in combination with IL-5 receptor alpha (IL5Rα) for the identification of murine eosinophils. Here, we describe a CD11b+ Siglec-F+ IL5Rα- myeloid population in the bone marrow of C57BL/6 mice. The CD11b+ Siglec-F+ IL5Rα- cells are retained in eosinophil deficient PHIL mice, and are not expanded upon overexpression of IL-5, indicating that they are upstream or independent of the eosinophil lineage. We show these cells to have GMP-like developmental potential in vitro and in vivo, and to be transcriptionally distinct from the classically described GMP population. The CD11b+ Siglec-F+ IL5Rα- population expands in the bone marrow of Myb mutant mice, which is potentially due to negative transcriptional regulation of Siglec-F by Myb. Lastly, we show that the role of Siglec-F may be, at least in part, to regulate GMP viability.
Subject(s)
Granulocyte-Macrophage Progenitor Cells/cytology , Granulocyte-Macrophage Progenitor Cells/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Animals , Cell Differentiation/physiology , Mice , Mice, Inbred C57BLABSTRACT
E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML.
Subject(s)
Inhibitor of Differentiation Protein 2/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Transcription Factor 7-Like 2 Protein/genetics , Translocation, Genetic , Animals , Cell Proliferation , Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 8/genetics , Gene Expression Regulation, Leukemic , Humans , Inhibitor of Differentiation Protein 2/metabolism , Leukemia, Myeloid, Acute/metabolism , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Neoplasms, Experimental , Oncogene Proteins, Fusion/metabolism , Prognosis , Stem Cells/cytology , Stem Cells/metabolism , Survival Analysis , Transcription Factor 7-Like 2 Protein/metabolismABSTRACT
UNLABELLED: Oncogene-induced senescence is a potent barrier to tumorigenesis that limits cellular expansion following certain oncogenic events. Senescent cells display a repressive chromatin configuration thought to stably silence proliferation-promoting genes while simultaneously activating an unusual form of immune surveillance involving a secretory program referred to as the senescence-associated secretory phenotype (SASP). Here, we demonstrate that senescence also involves a global remodeling of the enhancer landscape with recruitment of the chromatin reader BRD4 to newly activated super-enhancers adjacent to key SASP genes. Transcriptional profiling and functional studies indicate that BRD4 is required for the SASP and downstream paracrine signaling. Consequently, BRD4 inhibition disrupts immune cell-mediated targeting and elimination of premalignant senescent cells in vitro and in vivo Our results identify a critical role for BRD4-bound super-enhancers in senescence immune surveillance and in the proper execution of a tumor-suppressive program. SIGNIFICANCE: This study reveals how cells undergoing oncogene-induced senescence acquire a distinctive enhancer landscape that includes formation of super-enhancers adjacent to immune-modulatory genes required for paracrine immune activation. This process links BRD4 and super-enhancers to a tumor-suppressive immune surveillance program that can be disrupted by small molecule inhibitors of the bromo and extra terminal domain family of proteins. Cancer Discov; 6(6); 612-29. ©2016 AACR.See related commentary by Vizioli and Adams, p. 576This article is highlighted in the In This Issue feature, p. 561.
Subject(s)
Cellular Senescence/genetics , Chromatin Assembly and Disassembly , Enhancer Elements, Genetic , Immunologic Surveillance/genetics , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Cell Cycle/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , Chromatin Immunoprecipitation , Cluster Analysis , Computational Biology/methods , Fibroblasts , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Gene Expression Profiling , Gene Expression Regulation , Hepatocytes/metabolism , High-Throughput Nucleotide Sequencing , Histones/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Nucleotide Motifs , Oncogenes , Paracrine Communication , Position-Specific Scoring Matrices , Protein BindingABSTRACT
Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.
Subject(s)
Blood Cells/cytology , Blood Cells/metabolism , Computational Biology , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , Cluster Analysis , Computational Biology/methods , Gene Expression Profiling , Humans , Mice , Web BrowserABSTRACT
BET family proteins are novel therapeutic targets for cancer and inflammation and represent the first chromatin readers against which small-molecule inhibitors have been developed. First-generation BET inhibitors have shown therapeutic efficacy in preclinical models, but the consequences of sustained BET protein inhibition in normal tissues remain poorly characterized. Using an inducible and reversible transgenic RNAi mouse model, we show that strong suppression of the BET protein Brd4 in adult animals has dramatic effects in multiple tissues. Brd4-depleted mice display reversible epidermal hyperplasia, alopecia, and decreased cellular diversity and stem cell depletion in the small intestine. Furthermore, Brd4-suppressed intestines are sensitive to organ stress and show impaired regeneration following irradiation, suggesting that concurrent Brd4 suppression and certain cytotoxic therapies may induce undesirable synergistic effects. These findings provide important insight into Brd4 function in normal tissues and, importantly, predict several potential outcomes associated with potent and sustained BET protein inhibition.
Subject(s)
Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Azepines/pharmacology , Bone Marrow Cells/metabolism , Cell Line , Doxorubicin/toxicity , Gamma Rays , Hyperplasia/pathology , Intestinal Mucosa/metabolism , Intestines/drug effects , Intestines/radiation effects , Mice , Mice, Transgenic , MicroRNAs/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , RNA Interference , RNA, Small Interfering/metabolism , Receptors, G-Protein-Coupled/metabolism , Skin/pathology , Stem Cells/cytology , Stem Cells/metabolism , Thymus Gland/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Triazoles/pharmacologyABSTRACT
Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.
Subject(s)
Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Acetylation/drug effects , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Enzyme Activation/drug effects , Fusion Proteins, bcr-abl/metabolism , HSP90 Heat-Shock Proteins/metabolism , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 2/antagonists & inhibitors , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/administration & dosage , Hydroxamic Acids/pharmacology , Lymphoma/drug therapy , Lymphoma/metabolism , Lymphoma/mortality , Lymphoma/pathology , Mice , Vorinostat , Xenograft Model Antitumor AssaysABSTRACT
We have shown previously that mutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (Ras(ACT)) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with Ras(ACT) in tumorigenesis, we carried out a genome-wide screen for genes that when overexpressed throughout the developing Drosophila eye enhance Ras(ACT)-driven hyperplasia. Ras(ACT)-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1(ACT)), RhoGEF2, and pbl cooperated with Ras(ACT), resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or Rac1 with Ras(ACT) upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with Ras(ACT), while in the clonal setting, JNK upregulation was sufficient for Ras(ACT)-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of Ras(V12)-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2(+) human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, our genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with Ras(ACT) in the whole-tissue context. Collectively our analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with Ras(ACT).
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Genes, ras , Guanine Nucleotide Exchange Factors/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Precancerous Conditions/enzymology , rho GTP-Binding Proteins/metabolism , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Cell Shape , Cell Survival , Clone Cells , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Eye/cytology , Eye/growth & development , Eye/ultrastructure , Genes, Insect/genetics , Humans , MAP Kinase Signaling System , Precancerous Conditions/pathology , Protein Kinase C/metabolism , Reproducibility of Results , Rho Guanine Nucleotide Exchange Factors , Up-Regulation/geneticsABSTRACT
Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that have moved rapidly through clinical development and in 2006 vorinostat (SAHA, Zolinza) was given FDA approval for the treatment of cutaneous T cell lymphoma. Class I, II and IV HDACs that are targets for these compounds deacetylate histone proteins, resulting in chromatin remodelling and altered gene transcription. In addition, numerous non-histone proteins are modified by acetylation and the inhibition of HDAC activity can therefore affect various molecular processes. This broad effect on protein function may account for the pleiotropic anti-tumor responses elicited by HDACi that include induction of tumor cell apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune responses and altered angiogenesis. The ability of HDACi to selectively induce tumor cells to undergo apoptosis is important for the therapeutic efficacy observed in pre-clinical models. Moreover, HDACi can augment the apoptotic effects of other anti-cancer agents that have diverse molecular targets. While HDACi are promising anti-cancer drugs, particularly given the scope to combine HDACi with other agents, identifying the key molecular events that determine the biological response of cells to HDACi treatment remains a challenge. Herein we focus on HDACi-induced apoptosis and discuss the various proteins and pathways that are affected by HDACi to mediate this programmed cell death response. In addition, we highlight the ability of HDACi to synergise with other anti-cancer agents to potently kill tumor cells and discuss the possible molecular processes that underpin the combination effect.