ABSTRACT
BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.
Subject(s)
Colorectal Neoplasms , Tankyrases , Animals , Mice , Humans , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Tankyrases/genetics , Tankyrases/metabolism , Cell Line, Tumor , beta Catenin/genetics , beta Catenin/metabolism , Mice, Inbred NOD , Wnt Signaling Pathway/genetics , Biomarkers , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolismABSTRACT
Inspired by the privileged molecular skeletons of 14- and 15-membered antibiotics, we adopted a relatively unexplored synthetic approach that exploits alkaloidal macrocyclic scaffolds to generate modulators of protein-protein interactions (PPIs). As mimetics of hot-spot residues in the α-helices responsible for the transcriptional regulation, three hydrophobic sidechains were displayed on each of the four distinct macrocyclic scaffolds generating diversity of their spatial arrangements. Modular assembly of the building blocks followed by ring-closing olefin metathesis reaction and subsequent hydrogenation allowed concise and divergent synthesis of scaffolds 1-4. The 14-membered alkaloidal macrocycles 2-4 demonstrated similar inhibition of hypoxia-inducible factor (HIF)-1α transcriptional activities (IC50 between 8.7 and 10 µM), and 4 demonstrated the most potent inhibition of cell proliferation in vitro (IC50 = 12 µM against HTC116 colon cancer cell line). A docking model suggested that 4 could mimic the LLxxL motif in HIF-1α, in which the three sidechains are capable of matching the spatial arrangements of the protein hot-spot residues. Unlike most of the stapled peptides, the 14-membered alkaloidal scaffold has a similar size to the α-helix backbone and does not require additional atoms to induce α-helix mimetic structure. These experimental results underscore the potential of alkaloidal macrocyclic scaffolds featuring flexibly customizable skeletal, stereochemical, substitutional, and conformational properties for the development of non-peptidyl PPI modulators targeting α-helix-forming consensus sequences responsible for the transcriptional regulation.
Subject(s)
Alkaloids/pharmacology , Antineoplastic Agents/pharmacology , Drug Design , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Macrocyclic Compounds/pharmacology , Alkaloids/chemical synthesis , Alkaloids/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.
Subject(s)
Apoptosis , Diencephalon/embryology , Mesencephalon/embryology , Neural Tube/embryology , Animals , Apoptotic Protease-Activating Factor 1/genetics , Apoptotic Protease-Activating Factor 1/metabolism , Mice , Mice, Transgenic , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Installation of a nitrogen at the C6 position of artemisinin facilitates the addition of a functional unit on the cyclohexane moiety (C-ring). In this study, conjugation of an amphiphilic chain, composed of sequentially connected hydrophilic oligoethylene glycol, hydrophobic alkyl chain, urea, and 4,4'-disubstituted biphenyl linker, imparted self-assembling properties. The fully synthetic mid-molecular weight 6-aza-artemisinin 6 bearing the amphiphilic moiety formed aggregates (approx. 200 nm) at ambient temperature and exhibited increased in vitro anti-cancer activities compared to the N-benzylated aza-artemisinin 5.
Subject(s)
Antineoplastic Agents/pharmacology , Artemisinins/pharmacology , Surface-Active Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Artemisinins/chemistry , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Ethylene Glycol/chemistry , Ethylene Glycol/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Particle Size , Surface Properties , Surface-Active Agents/chemistry , Urea/chemistry , Urea/pharmacologyABSTRACT
Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/ß-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and ß-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.
Subject(s)
Colonic Neoplasms/drug therapy , Drug Design , Drug Discovery/methods , Enzyme Inhibitors/administration & dosage , Tankyrases/antagonists & inhibitors , Administration, Oral , Animals , Cell Line, Tumor , Colonic Neoplasms/enzymology , Enzyme Inhibitors/chemistry , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Protein Structure, Tertiary , Rats , Tankyrases/chemistry , Tankyrases/metabolism , Treatment Outcome , Xenograft Model Antitumor Assays/methodsABSTRACT
BACKGROUND: The timing of developmental events is tightly regulated along a time axis for normal development. Although the RNA-binding protein Lin28a plays a crucial role in the regulation of developmental timing in Caenorhabditis elegans, how the timing of Lin28a expression affects the rate and/or duration of developmental events during mammalian development remains to be addressed. RESULTS: In this study, we discovered that the timing and the duration of Lin28a expression affect embryonic growth. During the neurulation stage of mouse development, endogenous Lin28a levels start to drop. When Lin28a expression was maintained transiently using the inducible tetracycline-regulated gene expression (Tet-ON) system [doxycycline (Dox)-inducible Lin28a transgenic (iLin28a Tg) mice] with Dox administration at E8.5 and E9.5, it resulted in neonatal lethality, increased body weight (organomegaly), and an increased number of caudal vertebrae at birth. On the other hand, Lin28a induction only at E8.5 caused neonatal lethality and organomegaly, but did not affect the caudal vertebra number. Of note, although Dox treatment before or after neurulation still caused neonatal lethality, it neither caused organomegaly nor the increased caudal vertebra number in iLin28a Tg neonates. CONCLUSIONS: Temporal regulation of Lin28a expression during neurulation affects developmental events such as cessation of axial elongation and embryonic growth in mice.
Subject(s)
Body Size , Neurulation/physiology , RNA-Binding Proteins/physiology , Animals , Animals, Newborn , Doxycycline/pharmacology , Embryo, Mammalian , Gene Expression Regulation, Developmental , Mice , RNA-Binding Proteins/metabolism , Time FactorsABSTRACT
The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/ß-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/ß-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.
Subject(s)
Enzyme Inhibitors/pharmacology , Indoles/chemistry , Indoles/pharmacology , Spiro Compounds/pharmacology , Tankyrases/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , High-Throughput Screening Assays , Humans , Mice , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Spiro Compounds/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Telomere maintenance by telomerase activity supports the infinite growth of cancer cells. MST-312, a synthetic telomerase inhibitor, gradually shortens telomeres at non-acute lethal doses and eventually induces senescence and apoptosis of telomerase-positive cancer cells. Here we report that MST-312 at higher doses works as a dual inhibitor of telomerase and DNA topoisomerase II and exhibits acute anti-proliferative effects on cancer cells and xenografted tumours in vivo. Our cell-based chemical fingerprinting approach revealed that cancer cells with shorter telomeres and lower expression of lamin A, a nuclear architectural protein, exhibited higher sensitivity to the acute deleterious effects of MST-312, accompanied by formation of telomere dysfunction-induced foci and DNA double-strand breaks. Telomere elongation and lamin A overexpression attenuated telomeric and non-telomeric DNA damage, respectively, and both conferred resistance to apoptosis induced by MST-312 and other DNA damaging anticancer agents. These observations suggest that sufficient pools of telomeres and a nuclear lamina component contribute to the cellular robustness against DNA damage induced by therapeutic treatment in human cancer cells.
Subject(s)
Cell Proliferation , DNA Damage , DNA Repair , Lamin Type A/metabolism , Neoplasms/pathology , Telomere/metabolism , Artificial Cells , Benzamides/metabolism , Cell Line, Tumor , Enzyme Inhibitors/metabolism , Humans , Peptide Mapping , Sensitivity and Specificity , Telomerase/antagonists & inhibitors , Topoisomerase II Inhibitors/metabolismABSTRACT
Aberrant activation of Wnt/ß-catenin signaling causes tumorigenesis and promotes the proliferation of colorectal cancer cells. Porcupine inhibitors, which block secretion of Wnt ligands, may have only limited clinical impact for the treatment of colorectal cancer, because most colorectal cancer is caused by loss-of-function mutations of the tumor suppressor adenomatous polyposis coli (APC) downstream of Wnt ligands. Tankyrase poly(ADP-ribosyl)ates (PARylates) Axin, a negative regulator of ß-catenin. This post-translational modification causes ubiquitin-dependent degradation of Axin, resulting in ß-catenin accumulation. Tankyrase inhibitors downregulate ß-catenin and suppress the growth of APC-mutated colorectal cancer cells. Herein, we report a novel tankyrase-specific inhibitor RK-287107, which inhibits tankyrase-1 and -2 four- and eight-fold more potently, respectively, than G007-LK, a tankyrase inhibitor that has been previously reported as effective in mouse xenograft models. RK-287107 causes Axin2 accumulation and downregulates ß-catenin, T-cell factor/lymphoid enhancer factor reporter activity and the target gene expression in colorectal cancer cells harboring the shortly truncated APC mutations. Consistently, RK-287107 inhibits the growth of APC-mutated (ß-catenin-dependent) colorectal cancer COLO-320DM and SW403 cells but not the APC-wild (ß-catenin-independent) colorectal cancer RKO cells. Intraperitoneal or oral administration of RK-287107 suppresses COLO-320DM tumor growth in NOD-SCID mice. Rates of tumor growth inhibition showed good correlation with the behavior of pharmacodynamic biomarkers, such as Axin2 accumulation and MYC downregulation. These observations indicate that RK-287107 exerts a proof-of-concept antitumor effect, and thus may have potential for tankyrase-directed molecular cancer therapy.
Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Antineoplastic Agents/administration & dosage , Colorectal Neoplasms/drug therapy , Enzyme Inhibitors/administration & dosage , Tankyrases/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HCT116 Cells , Humans , Mice , Mutation , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Activation of Wnt/ß-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/ß-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/ß-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active ß-catenin was decreased and expression of ß-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/ß-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs.
Subject(s)
Colorectal Neoplasms/metabolism , Drug Resistance, Neoplasm , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Tankyrases/antagonists & inhibitors , Wnt Proteins/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Computational Biology/methods , Dose-Response Relationship, Drug , Humans , Wnt Signaling Pathway/drug effects , beta Catenin/metabolismABSTRACT
In most colorectal cancers, Wnt/ß-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli (APC) gene and plays a critical role in tumorigenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a negative regulator of ß-catenin, and upregulate ß-catenin signaling. Tankyrase inhibitors downregulate ß-catenin and are expected to be promising therapeutics for colorectal cancer. However, colorectal cancer cells are not always sensitive to tankyrase inhibitors, and predictive biomarkers for the drug sensitivity remain elusive. Here we demonstrate that the short-form APC mutations predict the sensitivity of colorectal cancer cells to tankyrase inhibitors. By using well-established colorectal cancer cell lines, we found that tankyrase inhibitors downregulated ß-catenin in the drug-sensitive, but not resistant, colorectal cancer cells. The drug-sensitive cells showed higher Tcf/LEF transcriptional activity than the resistant cells and possessed "short" truncated APCs lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs). In contrast, the drug-resistant cells possessed "long" APC retaining two or more 20-AARs. Knockdown of the long APCs with two 20-AARs increased ß-catenin, Tcf/LEF transcriptional activity and its target gene AXIN2 expression. Under these conditions, tankyrase inhibitors were able to downregulate ß-catenin in the resistant cells. These results indicate that the long APCs are hypomorphic mutants, whereas they exert a dominant-negative effect on Axin-dependent ß-catenin degradation caused by tankyrase inhibitors. Finally, we established 16 patient-derived colorectal cancer cells and confirmed that the tankyrase inhibitor-responsive cells harbor the short-form APC mutations. These observations exemplify the predictive importance of APC mutations, the most common genetic alteration in colorectal cancers, for molecular targeted therapeutics. Mol Cancer Ther; 16(4); 752-62. ©2017 AACR.
Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , Enzyme Inhibitors/pharmacology , Tankyrases/antagonists & inhibitors , Wnt Signaling Pathway/drug effects , Adenomatous Polyposis Coli Protein/metabolism , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/enzymology , HCT116 Cells , HT29 Cells , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Imides/pharmacology , Protein Binding , Quinolines/pharmacology , Sulfones/pharmacology , Triazoles/pharmacologyABSTRACT
Tankyrase, a PARP that promotes telomere elongation and Wnt/ß-catenin signaling, has various binding partners, suggesting that it has as-yet unidentified functions. Here, we report that the tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton and cancer cell invasion, which is closely associated with cancer progression. TNKS1BP1 colocalized with actin filaments and negatively regulated cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion, and lamellipodia ruffling were increased with activation of the ROCK/LIMK/cofilin pathway. TNKS1BP1 bound the actin-capping protein CapZA2. TNKS1BP1 depletion dissociated CapZA2 from the cytoskeleton, leading to cofilin phosphorylation and enhanced cell invasion. Tankyrase overexpression increased cofilin phosphorylation, dissociated CapZA2 from cytoskeleton, and enhanced cell invasion in a PARP activity-dependent manner. In clinical samples of pancreatic cancer, TNKS1BP1 expression was reduced in invasive regions. We propose that the tankyrase-TNKS1BP1 axis constitutes a posttranslational modulator of cell invasion whose aberration promotes cancer malignancy. Cancer Res; 77(9); 2328-38. ©2017 AACR.
Subject(s)
Actin Cytoskeleton/genetics , Neoplasm Invasiveness/genetics , Pancreatic Neoplasms/genetics , Telomeric Repeat Binding Protein 1/genetics , Actin Cytoskeleton/pathology , Actin Depolymerizing Factors/genetics , CapZ Actin Capping Protein/genetics , Cell Adhesion/genetics , Cell Line, Tumor , Female , Focal Adhesions/genetics , Gene Expression Regulation, Neoplastic , Humans , Lim Kinases/genetics , Male , Neoplasm Invasiveness/pathology , Pancreatic Neoplasms/pathology , Phosphorylation , Poly (ADP-Ribose) Polymerase-1/genetics , Telomeric Repeat Binding Protein 1/biosynthesis , Wnt Signaling Pathway , rho-Associated Kinases/geneticsABSTRACT
A cancer is a robustly evolving cell population originating from a normal diploid cell. Improper chromosome segregation causes aneuploidy, a driving force of cancer development and malignant progression. Telomeric repeat binding factor 1 (TRF1) has been established as a telomeric protein that negatively regulates telomere elongation by telomerase and promotes efficient DNA replication at telomeres. Intriguingly, overexpression of a mitotic kinase, Aurora-A, compromises efficient microtubule-kinetochore attachment in a TRF1-dependent manner. However, the precise role of TRF1 in mitosis remains elusive. Here we demonstrate that TRF1 is required for the centromeric function of Aurora-B, which ensures proper chromosome segregation. TRF1 depletion abolishes centromeric recruitment of Aurora-B and loosens sister centromere cohesion, resulting in the induction of merotelic kinetochore attachments, lagging chromosomes, and micronuclei. Accordingly, an absence of TRF1 in human and mouse diploid cells induces aneuploidy. These phenomena seem to be telomere independent, because a telomere-unbound TRF1 mutant can suppress the TRF1 knockdown phenotype. These observations indicate that TRF1 regulates the rigidity of the microtubule-kinetochore attachment, contributing to proper chromosome segregation and the maintenance of genomic integrity.
Subject(s)
Aurora Kinase B/metabolism , Centromere/metabolism , Chromosome Segregation , Kinetochores/chemistry , Microtubules/chemistry , Telomeric Repeat Binding Protein 1/metabolism , Aneuploidy , Animals , Aurora Kinase B/genetics , Cell Line , Cell Nucleus/genetics , Cell Nucleus/metabolism , HeLa Cells , Humans , Mice , Mitosis/genetics , RNA Interference , RNA, Small Interfering , Telomeric Repeat Binding Protein 1/geneticsABSTRACT
Limitless reproductive potential is one of the hallmarks of cancer cells. This ability is due to the maintenance of telomeres, erosion of which causes cellular senescence or death. While most cancer cells activate telomerase, a telomere-elongating enzyme, it remains elusive as to why cancer cells often maintain shorter telomeres than the cells in the surrounding normal tissues. Here, we show that forced telomere elongation in cancer cells promotes their differentiation in vivo. We elongated the telomeres of human prostate cancer cells that possess short telomeres by enhancing their telomerase activity. The resulting cells had long telomeres and retained the ability to form tumors in nude mice. Strikingly, these tumors exhibited many duct-like structures and reduced N-cadherin expression, reminiscent of well-differentiated adenocarcinoma. These changes were caused by telomere elongation and not by enhanced telomerase activity. Gene expression profiling revealed that tumor formation was accompanied by the expression of innate immune system-related genes, which have been implicated in maintaining tumor cells in an undifferentiated state and poor-prognosis cancers. In tumors derived from the telomere-elongated cells, upregulation of such gene sets is not observed. Our observations suggest a functional contribution of short telomeres to tumor malignancy by regulation of cancer cell differentiation.
Subject(s)
Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Telomerase/metabolism , Telomere/metabolism , Animals , Cell Differentiation , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Prostate/metabolism , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Telomerase/genetics , Up-RegulationABSTRACT
BACKGROUND: Extracts of Sasa senanensis Rehder are used in traditional Japanese medicine; however, little is known about the underlying mechanisms of their potential health benefits. METHODS: S. senanensis leaves were extracted with subcritical water. An active small-molecule was isolated using reversed-phase high-performance liquid chromatography (HPLC), and identified as 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde or PA). The effects of PA on the activity of histone demethylase, the Drosophila melanogaster lifespan and gene expression in Drosophila S2 cells were investigated. RESULTS: PA inhibited the activity of Jumonji domain-containing protein 2A (JMJD2A) histone demethylase in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 11.6 µM. However, there was no effect on lysine-specific demethylase 1 (LSD1), histone deacetylase 1 (HDAC1) or HDAC8. PA significantly extended the lifespan of female, but not male, Drosophila. In Drosophila S2 cells, the eukaryotic translation initiation factor 4E binding protein (4E-BP) was up-regulated by PA exposure. CONCLUSIONS: Our findings provide insight into the possible relationship between the pharmacological modulation of histone demethylation and lifespan extension by PA; they might also be important in the development of alternative therapies for age-related disorders.
Subject(s)
Benzaldehydes/pharmacology , Catechols/pharmacology , Drosophila melanogaster/drug effects , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Plant Extracts/pharmacology , Sasa/chemistry , Animals , Benzaldehydes/chemistry , Benzaldehydes/isolation & purification , Catechols/chemistry , Catechols/isolation & purification , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Female , Gene Expression/drug effects , Histone Demethylases/chemistry , Humans , Life Expectancy , Male , Models, Animal , Models, Molecular , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Leaves/chemistry , Species SpecificityABSTRACT
Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.
Subject(s)
Chromosome Segregation , Chromosomes, Fungal/metabolism , DNA Helicases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Schizosaccharomyces/genetics , Telomere-Binding Proteins/metabolism , Chromosome Segregation/drug effects , Exodeoxyribonucleases/metabolism , Gene Silencing/drug effects , HeLa Cells , Humans , Microbial Viability/drug effects , Mitosis/drug effects , Mutation/genetics , RecQ Helicases/metabolism , Recombination, Genetic/drug effects , Replication Protein A/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/drug effects , Shelterin Complex , Telomere/metabolism , Thiabendazole/pharmacology , Vinblastine/pharmacology , Werner Syndrome HelicaseABSTRACT
Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.
Subject(s)
Poly Adenosine Diphosphate Ribose/metabolism , Tankyrases/genetics , Telomere/metabolism , Telomeric Repeat Binding Protein 1/metabolism , Binding Sites , HeLa Cells , Humans , Mutant Proteins , Shelterin Complex , Tankyrases/metabolism , Telomere-Binding Proteins/metabolism , TransfectionABSTRACT
Telomeres protect chromosome ends from being recognized as DNA double-strand breaks. Telomere shortening, which occurs due to incomplete replication of DNA termini, limits the proliferative capacity of human somatic cells and contributes as a barrier to carcinogenesis. In most human cancer cells, telomerase maintains telomere length whereas TRF1, a telomeric protein, represses telomere access to telomerase. Tankyrase 1 is a PARP that dissociates TRF1 from telomeres by poly(ADP-ribosyl)ating TRF1. Thus, by reducing TRF1 loading on chromosome ends, tankyrase 1 enhances telomere access to telomerase and causes telomere elongation. Recent studies of knockout mice suggest that tankyrases may not regulate telomere length in mice (Mus musculus). Consistent with this idea is that mouse TRF1 has no canonical tankyrase-binding motif. However, the presence of such a motif is not a prerequisite to bind tankyrase 1 in certain species. Here, we found that, in mice, tankyrase 1 does not bind or poly(ADP-ribosyl)ate TRF1. Accordingly, mouse TRF1 was resistant to tankyrase 1-mediated release from telomeres. These observations indicate that telomeric function of tankyrase 1 is not conserved in mice. We also found that the canonical tankyrase 1-binding motif in TRF1 is conserved in several mammals but not in rats. Since mice and rats have much higher telomerase activity in their somatic tissues and much longer telomeres than those in other mammals, these rodent species might have evolved to resign the tankyrase 1-mediated telomere maintenance system. Meanwhile, PARP inhibitors induced non-telomeric tankyrase 1 foci in the nuclei, suggesting another function of tankyrase 1 at non-telomeric loci.
Subject(s)
Tankyrases/physiology , Telomere/physiology , Animals , Cell Nucleus/metabolism , Humans , Mice , Rats , Sequence Homology, Amino Acid , Species Specificity , Tankyrases/chemistry , Telomere/enzymology , Telomeric Repeat Binding Protein 1/metabolismABSTRACT
Telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase 1 poly(ADP-ribosyl)ates TRF1 and releases it from telomeres, allowing access of telomerase to telomeres. Here we demonstrate that tankyrase 1 inhibition in human cancer cells enhances telomere shortening by a telomerase inhibitor and hastens cell death. Conversely, either tankyrase 1 upregulation or telomere shortening, each of which decreased TRF1 loading on a chromosome end, attenuated the impact of telomerase inhibition. These results are consistent with the idea that telomeres having fewer TRF1s increase the efficiency of their elongation by telomerase. This study implies that both enzyme activity and accessibility to telomeres can be targets for telomerase inhibition.
Subject(s)
Neoplasms/drug therapy , Tankyrases/metabolism , Telomerase/metabolism , Telomere/metabolism , Apoptosis , Benzamides/metabolism , Benzamides/pharmacology , Catalytic Domain , Cell Line, Tumor , Cells, Cultured , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , In Situ Hybridization, Fluorescence , Neoplasms/metabolism , Nuclear Localization Signals , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Tankyrases/antagonists & inhibitors , Tankyrases/genetics , Telomerase/antagonists & inhibitors , Telomerase/genetics , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/metabolismABSTRACT
In human cells, telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase 1 binds TRF1 via its ankyrin domain and poly(ADP-ribosyl)ates it. Overexpression of tankyrase 1 in telomerase-positive cells releases TRF1 from telomeres, resulting in telomere elongation. The tankyrase 1 ankyrin domain is classified into five conserved subdomains, ARCs (ankyrin repeat clusters) I to V. Here, we investigated the biological significance of the ARCs. First, each ARC worked as an independent binding site for TRF1. Second, ARCs II to V recognized the N-terminal acidic domain of TRF1 whereas ARC I bound a discrete site between the homodimerization and the Myb-like domains of TRF1. Inactivation of TRF1 binding in the C-terminal ARC, ARC V, either by deletion or point mutation, significantly reduced the ability of tankyrase 1 to poly(ADP-ribosyl)ate TRF1, release TRF1 from telomeres, and elongate telomeres. In contrast, other ARCs, ARC II and/or IV, inactivated by point mutations still retained the biological function of tankyrase 1. On the other hand, ARC V per se was not sufficient for telomere elongation, suggesting a structural role for multiple ARCs. This work provides evidence that specific ARC-TRF1 interactions play roles in the essential catalytic function of tankyrase 1.