ABSTRACT
The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , NF-kappa B/metabolism , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Cytokines/metabolism , Gene Expression Regulation , HEK293 Cells , HumansABSTRACT
Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 8/physiology , Gene Expression Regulation, Neoplastic , Neoplasms/drug therapy , Animals , Cell Line, Tumor , Cell Nucleolus/metabolism , Cellular Senescence , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Genomics , Humans , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , S-Phase Kinase-Associated Proteins/metabolism , Transcription, Genetic , Treatment OutcomeABSTRACT
Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of coatomer protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform coatomer protein complex ζ2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.
Subject(s)
Coatomer Protein/genetics , Neoplasms/genetics , Apoptosis/genetics , Autophagy/genetics , Base Sequence , Cell Line, Tumor , DNA, Neoplasm/genetics , Female , Gene Knockdown Techniques , Gene Silencing , Golgi Apparatus/genetics , Golgi Apparatus/pathology , Humans , Male , MicroRNAs/genetics , Neoplasms/pathology , Protein Isoforms/genetics , RNA, Neoplasm/genetics , RNA, Small Interfering/genetics , Suppression, GeneticABSTRACT
Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.
Subject(s)
Cyclin-Dependent Kinase 8 , Cyclin-Dependent Kinases , Prostatic Neoplasms, Castration-Resistant , Protein Kinase Inhibitors , Xenograft Model Antitumor Assays , Male , Humans , Animals , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/enzymology , Mice , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinase 8/metabolism , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Tumor Microenvironment/drug effectsABSTRACT
Evaluation of gene co-regulation is a powerful approach for revealing regulatory associations between genes and predicting biological function, especially in genetically diverse samples. Here, we applied this strategy to identify transcripts that are co-regulated with unfolded protein response (UPR) genes in cultured fibroblasts from outbred deer mice. Our analyses showed that the transcriptome associated with RASSF1, a tumor suppressor involved in cell cycle regulation and not previously linked to UPR, is highly correlated with the transcriptome of several UPR-related genes, such as BiP/GRP78, DNAJB9, GRP94, ATF4, DNAJC3, and CHOP/DDIT3. Conversely, gene ontology analyses for genes co-regulated with RASSF1 predicted a previously unreported involvement in UPR-associated apoptosis. Bioinformatic analyses indicated the presence of ATF4-binding sites in the RASSF1 promoter, which were shown to be operational using chromatin immunoprecipitation. Reporter assays revealed that the RASSF1 promoter is responsive to ATF4, while ablation of RASSF1 mitigated the expression of the ATF4 effector BBC3 and abrogated tunicamycin-induced apoptosis. Collectively, these results implicate RASSF1 in the regulation of endoplasmic reticulum stress-associated apoptosis downstream of ATF4. They also illustrate the power of gene coordination analysis in predicting biological functions and revealing regulatory associations between genes.
Subject(s)
Activating Transcription Factor 4 , Endoplasmic Reticulum Stress , Tumor Suppressor Proteins , Unfolded Protein Response , Cell Cycle Proteins/genetics , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Gene Expression Regulation , Transcriptome/genetics , Unfolded Protein Response/genetics , Activating Transcription Factor 4/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvß3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations.
Subject(s)
Antineoplastic Agents/pharmacology , Models, Biological , Thyroxine/analogs & derivatives , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Culture Techniques/instrumentation , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Computational Biology , Drug Therapy, Combination , Female , Humans , Monte Carlo Method , Nanoparticles/administration & dosage , Resveratrol , Stilbenes/administration & dosage , Thyroxine/administration & dosage , Thyroxine/pharmacologyABSTRACT
The unfolded protein response (UPR) is involved in the pathogenesis of metabolic disorders, yet whether variations in the UPR among individuals influence the propensity for metabolic disease remains unexplored. Using outbred deer mice as a model, we show that the intensity of UPR in fibroblasts isolated early in life predicts the extent of body weight gain after high-fat diet (HFD) administration. Contrary to those with intense UPR, animals with moderate UPR in fibroblasts and therefore displaying compromised stress resolution did not gain body weight but developed inflammation, especially in the skin, after HFD administration. Fibroblasts emerged as potent modifiers of this differential responsiveness to HFD, as indicated by the comparison of the UPR profiles of fibroblasts responding to fatty acids in vitro, by correlation analyses between UPR and proinflammatory cytokine-associated transcriptomes, and by BiP (also known as HSPA5) immunolocalization in skin lesions from animals receiving HFD. These results suggest that the UPR operates as a modifier of an individual's propensity for body weight gain in a manner that, at least in part, involves the regulation of an inflammatory response by skin fibroblasts. This article has an associated First Person interview with the first author of the paper.
Subject(s)
Endoplasmic Reticulum Stress , Fibroblasts/pathology , Inflammation/pathology , Skin/pathology , Weight Gain , Animals , Biomarkers/blood , Cytokines/metabolism , Diet, High-Fat , Endoplasmic Reticulum Chaperone BiP/metabolism , Endoplasmic Reticulum Stress/drug effects , Fatty Acids/pharmacology , Fibroblasts/drug effects , Inflammation/blood , Leptin/blood , Models, Biological , Organ Size/drug effects , Peromyscus , Transcriptome/genetics , Unfolded Protein Response/drug effects , Weight Gain/drug effectsABSTRACT
Drug resistance is the main obstacle to achieving cures with both conventional and targeted anticancer drugs. The emergence of acquired drug resistance is initially mediated by non-genetic transcriptional changes, which occur at a much higher frequency than mutations and may involve population-scale transcriptomic adaptation. CDK8/19 kinases, through association with transcriptional Mediator complex, regulate transcriptional reprogramming by co-operating with different signal-responsive transcription factors. Here we tested if CDK8/19 inhibition could prevent adaptation to drugs acting on epidermal growth factor receptor (EGFR/ERBB1/HER1). The development of resistance was analyzed following long-term exposure of BT474 and SKBR3 breast cancer cells to EGFR-targeting small molecules (gefitinib, erlotinib) and of SW48 colon cancer cells to an anti-EGFR monoclonal antibody cetuximab. In all cases, treatment of small cell populations (~105 cells) with a single dose of the drug initially led to growth inhibition that was followed by the resumption of proliferation and development of drug resistance in the adapted populations. However, this adaptation was always prevented by the addition of selective CDK8/19 inhibitors, even though such inhibitors alone had only moderate or no effect on cell growth. These results indicate that combining EGFR-targeting drugs with CDK8/19 inhibitors may delay or prevent the development of tumor resistance to therapy.
Subject(s)
Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Resistance, Neoplasm , ErbB Receptors/metabolism , Molecular Targeted Therapy , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor , Cetuximab/pharmacology , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Drug Resistance, Neoplasm/drug effects , Erlotinib Hydrochloride/pharmacology , Gefitinib/pharmacology , Humans , Inhibitory Concentration 50ABSTRACT
CDK7, a transcriptional cyclin-dependent kinase, is emerging as a novel cancer target. Triple-negative breast cancers (TNBC) but not estrogen receptor-positive (ER+) breast cancers have been reported to be uniquely sensitive to the CDK7 inhibitor THZ1 due to the inhibition of a cluster of TNBC-specific genes. However, bioinformatic analysis indicates that CDK7 RNA expression is associated with negative prognosis in all the major subtypes of breast cancer. To further elucidate the effects of CDK7 inhibition in breast cancer, we profiled a panel of cell lines representing different breast cancer subtypes. THZ1 inhibited cell growth in all subtypes (TNBC, HER2+, ER+, and HER2+/ER+) with no apparent subtype selectivity. THZ1 inhibited CDK7 activity and induced G1 arrest and apoptosis in all the tested cell lines, but THZ1 sensitivity did not correlate with CDK7 inhibition or CDK7 expression levels. THZ1 sensitivity across the cell line panel did not correlate with TNBC-specific gene expression but it was found to correlate with the differential inhibition of three genes: CDKN1B, MYC and transcriptional coregulator CITED2. Response to THZ1 also correlated with basal CITED2 protein expression, a potential marker of CDK7 inhibitor sensitivity. Furthermore, all of the THZ1-inhibited genes examined were inducible by EGF but THZ1 prevented this induction. THZ1 had synergistic or additive effects when combined with the EGFR inhibitor erlotinib, with no outward selectivity for a particular subtype of breast cancer. These results suggest a potential broad utility for CDK7 inhibitors in breast cancer therapy and the potential for combining CDK7 and EGFR inhibitors.
Subject(s)
Breast Neoplasms/drug therapy , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/enzymology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/enzymology , Cell Line, Tumor , Cyclin-Dependent Kinases/biosynthesis , Drug Synergism , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride/administration & dosage , Erlotinib Hydrochloride/pharmacology , Female , Gene Expression/drug effects , Humans , MCF-7 Cells , Phenylenediamines/administration & dosage , Phenylenediamines/pharmacology , Protein Kinase Inhibitors/administration & dosage , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Survival Analysis , Trans-Activators/biosynthesis , Trans-Activators/genetics , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
: Unresectable hepatic metastases of colon cancer respond poorly to existing therapies and are a major cause of colon cancer lethality. In this study, we evaluated the therapeutic viability of targeting the mediator kinase CDK8, an early clinical stage drug target, as a means to suppress metastasis of colon cancer. CDK8 was amplified or overexpressed in many colon cancers and CDK8 expression correlated with shorter patient survival. Knockdown or inhibition of CDK8 had little effect on colon cancer cell growth but suppressed metastatic growth of mouse and human colon cancer cells in the liver. This effect was due in part to inhibition of already established hepatic metastases, indicating therapeutic potential of CDK8 inhibitors in the metastatic setting. In contrast, knockdown or inhibition of CDK8 had no significant effect on the growth of tumors implanted subcutaneously, intrasplenically, or orthotopically in the cecum. CDK8 mediated colon cancer growth in the liver through downregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 via TGFß/SMAD-driven expression of a TIMP3-targeting microRNA, miR-181b, along with induction of Mmp3 in murine or MMP9 in human colon cancer cells via Wnt/ß-catenin-driven transcription. These findings reveal a new mechanism for negative regulation of gene expression by CDK8 and a site-specific role for CDK8 in colon cancer hepatic metastasis. Our results indicate the utility of CDK8 inhibitors for the treatment of colon cancer metastases in the liver and suggest that CDK8 inhibitors may be considered in other therapeutic settings involving TGFß/SMAD or Wnt/ß-catenin pathway activation. SIGNIFICANCE: These findings demonstrate that inhibition of the transcription-regulating kinase CDK8 exerts a site-specific tumor-suppressive effect on colon cancer growth in the liver, representing a unique therapeutic opportunity for the treatment of advanced colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/23/6594/F1.large.jpg.
Subject(s)
Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin-Dependent Kinase 8/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Matrix Metalloproteinases/genetics , Tissue Inhibitor of Metalloproteinase-3/genetics , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase 8/genetics , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/mortality , Matrix Metalloproteinases/metabolism , Mice , MicroRNAs/genetics , RNA Interference , Tissue Inhibitor of Metalloproteinase-3/metabolism , Treatment Outcome , Wnt Signaling Pathway/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.
Subject(s)
Breast Neoplasms/pathology , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Estrogen Receptor alpha/metabolism , Estrogens/biosynthesis , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Breast Neoplasms/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Drug Resistance, Neoplasm/drug effects , Estradiol/analogs & derivatives , Estradiol/pharmacology , Female , Fulvestrant , Humans , Mice , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Protein Kinase Inhibitors/pharmacology , Transcription, Genetic , Transcriptome , Xenograft Model Antitumor AssaysABSTRACT
The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex (MRN) that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome (NBS), a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance of telomere stability.
Subject(s)
Cell Cycle Proteins/physiology , Cell Cycle/physiology , Chromosomal Instability , DNA Repair , Nuclear Proteins/physiology , Telomere/physiology , DNA Damage , Genes, cdc , Humans , Models, Biological , Phosphatidylinositol 3-Kinases/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Telomere/geneticsABSTRACT
Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases have been considered the primary activators of the cellular response to DNA damage. They belong to the protein kinase family, phosphoinositide 3-kinase-related kinase (PIKKs). In human beings, deficiency of these kinases leads to hereditary diseases, namely ataxia telangiectasia (AT) with ATM deficiency and ATR-Seckel with ATR deficiency. NBS1, a component of MRE11/RAD50/NBS1 (MRN) complex, is another important player in DNA damage response (DDR). Mutations of NBS1 are responsible for Nijmegen breakage syndrome (NBS), a human hereditary disease with the characteristics that almost encompassed those of AT and ATR-Seckel. NBS1 has been conventionally thought to be a downstream substrate of ATM and ATR in DDR; however, recent studies suggest that NBS1/MRN functions upstream of both ATM and ATR by recruiting them to the proximity of DNA damage sites and activating their functions. In this mini-review, we would emphasize the requirement of NBS1 as an upstream mediator for the modulation of PIKK family proteins ATM and ATR.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , DNA Damage/physiology , DNA-Binding Proteins/metabolism , Nuclear Proteins/physiology , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , DNA Repair/physiology , Humans , Models, BiologicalABSTRACT
Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A(L)). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59- and CD59+ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.
Subject(s)
DNA Mutational Analysis/methods , Flow Cytometry/methods , Mutagens/toxicity , Animals , CD59 Antigens/metabolism , CHO Cells , Chromosomes, Human, Pair 11 , Cricetinae , Cross-Linking Reagents/toxicity , Ethyl Methanesulfonate/toxicity , Humans , Mitomycin/toxicity , Organometallic Compounds/toxicity , Radiation, Ionizing , Time FactorsABSTRACT
Numerous extracellular stimuli activate SK1 (sphingosine kinase type 1) to catalyse the production of sphingosine 1-phosphate, a bioactive lipid that functions as both an extracellular ligand for a family of G-protein-linked receptors and as a putative intracellular messenger. Phorbol esters, calcium or immunoglobulin receptors stimulate SK1 by promoting its translocation to the plasma membrane, which brings it into proximity both to its substrate (i.e. sphingosine) and to activating acidic phospholipids (e.g. phosphatidylserine). To evaluate the consequence of SK translocation, we generated an SK1-derivative tagged with a myristoylation sequence (Myr-SK1) on its N-terminus and overexpressed the construct in 3T3-L1 fibroblasts using recombinant retrovirus. Myr-SK1 overexpression increased SK activity by more than 50-fold in crude membranes, while only stimulating cytoplasmic SK activity by 4-fold. In contrast, the overexpression of WT-SK1 (wild-type SK1), as well as that of a construct containing a false myristoylation sequence (A2-Myr-SK1), markedly increased SK activity in both membrane and cytoplasmic compartments. Immunofluorescence confirmed that Myr-SK1 preferentially localized at the plasma membrane, whereas WT-SK1 and A2-Myr-SK1 partitioned in cytoplasmic/perinuclear cellular regions. Surprisingly, Myr-SK1 overexpression significantly decreased the rates of cell proliferation by delaying exit from G0/G1 phase. Moreover, expression of Myr-SK1 but not WT-SK1 or A2-Myr-SK1 protected cells from apoptosis induced by serum withdrawal. Collectively, these findings reveal that altering the subcellular location of SK1 has marked effects on cell function, with plasma membrane-associated SK having a potent inhibitory effect on the G1-S phase transition.
Subject(s)
Apoptosis/physiology , Cell Membrane/enzymology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , 3T3-L1 Cells , Animals , Apoptosis/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Culture Media, Serum-Free/pharmacology , Fibroblasts/drug effects , Fibroblasts/enzymology , Gene Expression , Humans , Mice , Myristic Acid/metabolism , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/geneticsABSTRACT
Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.
Subject(s)
Androgens/deficiency , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Disease Progression , Gene Expression , Humans , Male , Promoter Regions, Genetic , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Receptors, Androgen/metabolism , Signal Transduction/geneticsABSTRACT
The mechanistic relevance of intergenic disease-associated genetic loci (IDAGL) containing highly statistically significant disease-linked SNPs remains unknown. Here, we present experimental and clinical evidence supporting the importantance of the role of IDAGL in human diseases. A targeted RT-PCR screen coupled with sequencing of purified PCR products detects widespread transcription at multiple IDAGL and identifies 96 small noncoding trans-regulatory RNAs of ~100-300 nt in length containing SNPs (snpRNAs) associated with 21 common disorders. Multiple independent lines of experimental evidence support functionality of snpRNAs by documenting their cell type-specific expression and evolutionary conservation of sequences, genomic coordinates and biological effects. Chromatin state signatures, expression profiling experiments and luciferase reporter assays demonstrate that many IDAGL are Polycomb-regulated long-range enhancers. Expression of snpRNAs in human and mouse cells markedly affects cellular behavior and induces allele-specific clinically relevant phenotypic changes: NLRP1-locus snpRNAs rs2670660 exert regulatory effects on monocyte/macrophage transdifferentiation, induce prostate cancer (PC) susceptibility snpRNAs and transform low-malignancy hormone-dependent human PC cells into highly malignant androgen-independent PC. Q-PCR analysis and luciferase reporter assays demonstrate that snpRNA sequences represent allele-specific "decoy" targets of microRNAs that function as SNP allele-specific modifiers of microRNA expression and activity. We demonstrate that trans-acting RNA molecules facilitating resistance to androgen depletion (RAD) in vitro and castration-resistant phenotype (CRP) in vivo of PC contain intergenic 8q24-locus SNP variants (rs1447295; rs16901979; rs6983267) that were recently linked with increased risk of PC. Q-PCR analysis of clinical samples reveals markedly increased and highly concordant (r = 0.896; p < 0.0001) snpRNA expression levels in tumor tissues compared with the adjacent normal prostate [122-fold and 45-fold in Gleason 7 tumors (p = 0.03); 370-fold and 127-fold in Gleason 8 tumors (p = 0.0001) for NLRP1-locus and 8q24-locus snpRNAs, respectively]. Our experiments indicate that RAD and CR phenotype of human PC cells can be triggered by ncRNA molecules transcribed from the NLRP1-locus intergenic enhancer at 17p13 and by downstream activation of the 8q24-locus snpRNAs. Our results define the IDAGL at 17p13 and 8q24 as candidate regulatory loci of RAD and CR phenotypes of PC, reveal previously unknown molecular links between the innate immunity/inflammasome system and development of hormone-independent PC and identify novel molecular and genetic targets with diagnostic and therapeutic potentials, exploration of which should be highly beneficial for personalized clinical management of PC.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 8/genetics , Gene Expression Regulation, Neoplastic/genetics , Phenotype , Polymorphism, Single Nucleotide/genetics , Prostatic Neoplasms/genetics , Regulatory Sequences, Ribonucleic Acid/genetics , Animals , Cell Differentiation/genetics , Cell Line, Tumor , Computational Biology , DNA, Intergenic/genetics , Flow Cytometry , Gene Expression Profiling , Humans , Luciferases , Male , Mice , Microarray Analysis , NLR Proteins , Prostatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIß, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIß and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G 2. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.
Subject(s)
Cell Cycle/physiology , DNA Topoisomerases, Type II/deficiency , DNA Topoisomerases, Type II/metabolism , Animals , Cell Line , Cells, Cultured , DNA Primers/genetics , Doxycycline , Flow Cytometry , Humans , Immunoenzyme Techniques , Indoles , Microscopy, Fluorescence , Time-Lapse ImagingABSTRACT
Meta-analysis of genomic coordinates of SNP variations identified in genome-wide association studies (GWAS) of up to 712,253 samples (comprising 221,158 disease cases, 322,862 controls, and 168,233 case/control subjects of obesity GWAS) reveals that 39% of SNPs associated with 22 common human disorders are located within intergenic regions. Chromatin-state maps based on H3K4me3-H3K36me3 signatures show that many intergenic disease-linked SNPs are located within the boundaries of the K4-K36 domains, suggesting that SNP-harboring genomic regions are transcribed. Here we report identification of 13 trans-regulatory RNAs (transRNAs) 100 to 200 nucleotides in length containing intergenic SNP sequences associated with Crohn's disease, rheumatoid arthritis, type 1 diabetes, vitiligo, hypertension and multiple types of epithelial malignancies (prostate, breast, ovarian and colorectal cancers). We demonstrate that NALP1 loci intergenic SNP sequence, rs2670660, is expressed in human cells and may contribute to clinical manifestations of autoimmune and autoimflammatory phenotypes by generating distinct allelic variants of transRNAs. Stable expression of allele-specific sense and anti-sense variants of transRNAs markedly alters cellular behavior, affect cell cycle progression, and interfere with monocyte/macrophage transdifferentiation. On a molecular level, forced expression of allele-specific sense and anti-sense variants of transRNAs asserts allele-specific genome-wide effects on abundance of hundreds microRNAs and mRNAs. Using lentiviral gene transfer, microarray and Q-RT-PCR technologies, we identify rs2670660 allele-specific gene expression signatures (GES) which appear useful for detecting the activated states of innate immunity/inflammasome pathways in approximately 700 clinical samples from 185 control subjects and 350 patients diagnosed with nine common human disorders, including Crohn's disease, ulcerative colitis, rheumatoid arthritis, Huntington disease, autism, Alzheimer disease, obesity, prostate and breast cancers. Microarray analysis of clinical samples demonstrates that rs2670660 allele-specific GES are engaged in patients' peripheral blood mononuclear cells (PBMC) which encounter pathological conditions in coherent tissues of a human body during immune surveillance and homeostasis monitoring. These data indicate that expression of transRNAs encoded by specific intergenic sequences can trigger activation of innate immunity/inflammasome pathways and contribute to clinical development of autoinflammatory and autoimmune syndromes. Documented in this work single-base substitution-driven molecular and biological antagonisms of intergenic SNP-containing transRNAs suggest a guiding mechanism of selection and retention of phenotype-compatible intergenic variations during evolution. According to this model, random genetic variations which generate transRNAs asserting antagonistic phenotype-altering effects compared to ancestral alleles will be selected and retained as SNP variants.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Polymorphism, Single Nucleotide , Regulatory Sequences, Ribonucleic Acid/genetics , Alleles , Autoimmune Diseases/genetics , Base Sequence , Cell Differentiation , Cell Division , Genetic Predisposition to Disease , Genome, Human , Humans , MicroRNAs/metabolism , Molecular Sequence Data , NLR Proteins , Oligonucleotide Array Sequence AnalysisABSTRACT
The effects of heat are strongly dependent on the time of heating at a given temperature. The relationship between treatment time and temperature for a biological isoeffect (the Arrhenius plot) has been confirmed for a variety of normal tissues and tumours. A marked change of slope occurs somewhere between 42-43 degrees C. Above this transition temperature the slope is constant for a variety of cells and tissues. Therefore, when defining thermal doses in hyperthermia studies, both the time and temperature of heating are equally important determinants. In this study, cell cycle progression and apoptosis were analysed in HL-60 cells after heating from 5-60 min at 45.0 degrees C and also heating with five different iso-dose time-temperature heat treatments. A heat shock of 5-15 min at 45.0 degrees C caused the accumulation of cells in G1 and G2/M phases after 12 h at 37 degrees C, whereas a heat shock of 20-60 min at 45.0 degrees C reduced the number of non-apoptotic cells in all phases because the number of apoptotic cells increased. The fraction of apoptotic cells followed a sigmoid curve as the heating time increased from 5-60 min at 45.0 degrees C. Cell cycle analysis showed that apoptosis occurred predominantly in S-phase cells for shorter heating times but in all phases at longer times. An isodose heat shock lower than 44.0 degrees C (42.0-43.0 degrees C) gave the same apoptotic index, while heat shock from 44.0-46.0 degrees C caused a greater than expected apoptotic index. Thus, there was a transition at 44.0 degrees C in HL-60 cells, above which apoptosis increased rapidly. These results indicate that isodose analysis based on clonogenic survival in fibroblast cells may not be relevant for cell types which readily undergo apoptosis. Clonogenic survival was also compared with apoptosis for HL-60 cells and an apoptotic-resistant derivative cell line, HWC-2, heated for various times at 45.0 degrees C. Survival based on a clonogenic assay was much lower than survival based only on apoptotic index at all times for HL-60 cells. HWC-2 cells did not undergo apoptosis and also had a higher clonogenic survival than HL-60 cells.