ABSTRACT
Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.
Subject(s)
Antineoplastic Agents , Cell-Penetrating Peptides , G-Quadruplexes , G-Quadruplexes/drug effects , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Cell Line, Tumor , Peptides/chemistry , Peptides/pharmacology , Ligands , DNA/chemistry , DNA/metabolism , Shelterin Complex/metabolism , Shelterin Complex/chemistry , Protein BindingABSTRACT
Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells with strong immunosuppressive activity that promote tumor growth. In this study, we describe a mechanism by which cancer cells control MDSCs in human cancers by upregulating TRF2, a protein required for telomere stability. Specifically, we showed that the TRF2 upregulation in cancer cells has extratelomeric roles in activating the expression of a network of genes involved in the biosynthesis of heparan sulfate proteoglycan, leading to profound changes in glycocalyx length and stiffness, as revealed by atomic force microscopy. This TRF2-dependent regulation facilitated the recruitment of MDSCs, their activation via the TLR2/MyD88/IL-6/STAT3 pathway leading to the inhibition of natural killer recruitment and cytotoxicity, and ultimately tumor progression and metastasis. The clinical relevance of these findings is supported by our analysis of cancer cohorts, which showed a correlation between high TRF2 expression and MDSC infiltration, which was inversely correlated with overall patient survival.
Subject(s)
Glycocalyx/metabolism , Neoplasms/immunology , Neoplasms/pathology , Telomeric Repeat Binding Protein 2/physiology , Tumor Escape/physiology , Animals , Cells, Cultured , Female , Gene Expression Regulation, Neoplastic , Glycocalyx/genetics , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Myeloid-Derived Suppressor Cells/metabolism , Myeloid-Derived Suppressor Cells/physiology , NIH 3T3 Cells , Neoplasms/genetics , Neoplasms/mortality , Telomere/metabolism , Telomeric Repeat Binding Protein 2/genetics , Tumor Escape/geneticsABSTRACT
Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands. A preliminary screening with the G-quadruplex on the Controlled Pore Glass assay proved that, among the investigated compounds, Dicentrine is the most effective ligand of telomeric and oncogenic G-quadruplexes, also showing good G-quadruplex vs. duplex selectivity. In-depth studies in solution demonstrated the ability of Dicentrine to thermally stabilize telomeric and oncogenic G-quadruplexes without affecting the control duplex. Interestingly, it showed higher affinity for the investigated G-quadruplex structures over the control duplex (Kb~106 vs. 105 M-1), with some preference for the telomeric over the oncogenic G-quadruplex model. Molecular dynamics simulations indicated that Dicentrine preferentially binds the G-quadruplex groove or the outer G-tetrad for the telomeric and oncogenic G-quadruplexes, respectively. Finally, biological assays proved that Dicentrine is highly effective in promoting potent and selective anticancer activity by inducing cell cycle arrest through apoptosis, preferentially targeting G-quadruplex structures localized at telomeres. Taken together, these data validate Dicentrine as a putative anticancer candidate drug selectively targeting cancer-related G-quadruplex structures.
Subject(s)
Antineoplastic Agents , G-Quadruplexes , Neoplasms , Humans , Ligands , Molecular Dynamics Simulation , Antineoplastic Agents/pharmacology , Telomere/metabolismABSTRACT
HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
Subject(s)
G-Quadruplexes , HMGB1 Protein/genetics , Nucleic Acid Conformation , Telomere/genetics , DNA/chemistry , DNA/genetics , Escherichia coli/genetics , HMGB1 Protein/chemistry , Humans , Telomerase/chemistry , Telomerase/genetics , Telomere/chemistryABSTRACT
The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.
Subject(s)
Colonic Neoplasms/metabolism , Sulfotransferases/genetics , Telomeric Repeat Binding Protein 2/metabolism , Tumor Microenvironment , Vascular Endothelial Growth Factor A/metabolism , Animals , Cell Line, Tumor , Colonic Neoplasms/blood supply , Colonic Neoplasms/pathology , Heparan Sulfate Proteoglycans/chemistry , Heparan Sulfate Proteoglycans/metabolism , Heparin/metabolism , Humans , Male , Mice , Mice, Nude , Neoplasm Metastasis , Neovascularization, Pathologic , Sulfatases , Sulfotransferases/biosynthesis , Telomeric Repeat Binding Protein 2/deficiency , Xenograft Model Antitumor AssaysABSTRACT
Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Azo Compounds/chemistry , DNA/metabolism , G-Quadruplexes , Osteosarcoma/drug therapy , Pyridines/chemistry , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , DNA/chemistry , Humans , Osteosarcoma/pathology , Tumor Cells, CulturedABSTRACT
A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands-according to an affinity chromatography-based screening method named G4-CPG-were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC50 values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays' data.
Subject(s)
Antineoplastic Agents/chemistry , G-Quadruplexes/drug effects , Imines/chemistry , Naphthalenes/chemistry , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , DNA Damage , HeLa Cells , Humans , Imines/pharmacology , Ligands , Naphthalenes/pharmacology , Telomere/drug effectsABSTRACT
Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.
Subject(s)
DNA Damage , Sirtuins/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Acetylation , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , Cell Line , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Humans , Immunohistochemistry , Models, Molecular , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Conformation , Protein Stability , Proteolysis/drug effects , Recombinant Fusion Proteins/metabolism , Sirtuins/chemistry , Substrate Specificity , Telomeric Repeat Binding Protein 2/chemistry , Telomeric Repeat Binding Protein 2/genetics , UbiquitinationABSTRACT
Inflammatory responses are elicited through lipid products of phospholipase A2 activity that acts on the membrane phospholipids, including the phosphoinositides, to form the proinflammatory arachidonic acid and, in parallel, the glycerophosphoinositols. Here, we investigate the role of the glycerophosphoinositol in the inflammatory response. We show that it is part of a negative feedback loop that limits proinflammatory and prothrombotic responses in human monocytes stimulated with lipopolysaccharide. This inhibition is exerted both on the signaling cascade initiated by the lipopolysaccharide with the glycerophosphoinositol-dependent decrease in IκB kinase α/ß, p38, JNK, and Erk1/2 kinase phosphorylation and at the nuclear level with decreased NF-κB translocation and binding to inflammatory gene promoters. In a model of endotoxemia in the mouse, treatment with glycerophosphoinositol reduced TNF-α synthesis, which supports the concept that glycerophosphoinositol inhibits the de novo synthesis of proinflammatory and prothrombotic compounds and might thus have a role as an endogenous mediator in the resolution of inflammation. As indicated, this effect of glycerophosphoinositol can also be exploited in the treatment of manifestations of severe inflammation by exogenous administration of the compound.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Blood Coagulation/drug effects , Endotoxemia/drug therapy , Gene Expression Regulation/drug effects , Inositol Phosphates/therapeutic use , Monocytes/drug effects , Protein Processing, Post-Translational/drug effects , Active Transport, Cell Nucleus/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Biomarkers/blood , Biomarkers/metabolism , Cells, Cultured , Chromatin Immunoprecipitation , Dose-Response Relationship, Drug , Endotoxemia/immunology , Endotoxemia/metabolism , HeLa Cells , Humans , Inositol Phosphates/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/toxicity , Male , Mice, Inbred C57BL , Microscopy, Confocal , Monocytes/cytology , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/blood , NF-kappa B/metabolism , Phosphorylation/drug effectsABSTRACT
Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression.In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.
Subject(s)
Antigens, CD/genetics , G-Quadruplexes , Glycoproteins/genetics , Neoplasm Invasiveness/genetics , Neoplastic Stem Cells/metabolism , Peptides/genetics , AC133 Antigen , Antigens, CD/chemistry , Cell Line, Tumor , Cell Proliferation/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Glycoproteins/chemistry , Humans , Neoplastic Stem Cells/pathology , Peptides/chemistry , Protein BiosynthesisABSTRACT
BACKGROUND: During the last decade, guanine G-rich sequences folding into G-quadruplex (G4) structures have received a lot of attention and their biological role is now a matter of large debate. Rising amounts of experimental evidence have validated several G-rich motifs as molecular targets in cancer treatment. Despite that an increasing number of small molecules has been reported to possess excellent G4 stabilizing properties, none of them has progressed through the drug-development pipeline due to their poor drug-like properties. In this context, the identification of G4 ligands with more favorable pharmacological properties and with a well-defined target activity could be fruitful for anticancer therapy application. SCOPE OF REVIEW: This manuscript outlines the current state of knowledge regarding EMICORON, a G4-interactive molecule structurally and biologically similar, on the one side, to coronene and, on the other side, to a bay-monosubstituted perylene. MAJOR CONCLUSIONS: Overall this work evidences that EMICORON, a new promising G4 ligand, possesses a marked antitumoral activity both standing alone and in combination with chemotherapeutics. Moreover, EMICORON represents a good example of multimodal class of antitumoral drug, able to simultaneously affect multiple targets participating in several distinct signaling pathways, thus simplifying the treatment modalities and improving the selectivity against cancer cells. GENERAL SIGNIFICANCE: Due to the importance of G4 forming sequences in crucial biological processes participating in tumor progression, their successful targeting with small molecules could represent a very important innovation in the development of effective therapeutic strategies against cancer. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , DNA, Neoplasm/drug effects , Drug Design , G-Quadruplexes/drug effects , Guanosine/metabolism , Imides/pharmacology , Neoplasms/drug therapy , Piperidines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Binding Sites , Cell Proliferation/drug effects , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Guanosine/chemistry , Humans , Imides/chemical synthesis , Imides/metabolism , Ligands , Models, Molecular , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Piperidines/chemical synthesis , Piperidines/metabolism , Signal Transduction/drug effects , Structure-Activity Relationship , Telomere/chemistry , Telomere/drug effects , Telomere/metabolism , Tumor Burden/drug effectsABSTRACT
BACKGROUND: G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. METHODS: A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MTT assays. RESULTS: A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4-forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells. CONCLUSIONS: The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands. GENERAL SIGNIFICANCE: Expanding the repertoire of the available G4-targeting chemotypes with improved physico-chemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Subject(s)
Antineoplastic Agents/pharmacology , DNA, Neoplasm/drug effects , Drug Design , G-Quadruplexes/drug effects , Guanosine/metabolism , High-Throughput Screening Assays , Neoplasms/drug therapy , Telomere/drug effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Binding Sites , Cell Line, Transformed , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Dose-Response Relationship, Drug , Guanosine/chemistry , Humans , Ligands , Models, Molecular , Neoplasms/genetics , Neoplasms/pathology , Small Molecule Libraries , Structure-Activity Relationship , Telomere/chemistry , Telomere/genetics , Telomere/metabolism , Time FactorsABSTRACT
Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase of DNA damage response (DDR) markers throughout the genome from normal toward immortalized and transformed cells. More interestingly, sensitivity to G4 ligands strongly correlated with the presence of a basal level of DNA damage, including at the telomeres, where the chromosome ends were exposed to the DDR without concurrent induction of DNA repair activity, as revealed by the lack of 53BP1 recruitment and telomere aberrations. The link between telomere uncapping and the response to G4 stabilization was directly assessed by showing that a partial TRF2 depletion, causing a basal level of telomere localized DDR, rendered telomerized fibroblasts prone to G4-induced telomere damage and anti-proliferative defects. Taken together these data strongly indicate that the presence of a basal level of telomere-associated DDR is a determinant of susceptibility to G4 stabilization.
Subject(s)
DNA Damage , G-Quadruplexes/drug effects , Neoplasms/genetics , Telomere , Blotting, Western , Chromatin Immunoprecipitation , Humans , In Situ Hybridization, Fluorescence , Tumor Cells, CulturedABSTRACT
Tumor angiogenesis is mainly mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced by cancer cells and active on the endothelium through the VEGF receptor 2 (VEGFR-2). Here we identify a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G-quadruplex (G4) structure. This G4 structure can be efficiently stabilized by small molecules with the consequent inhibition of vegfr-2 expression. Functionally, the G4-mediated reduction of VEGFR-2 protein causes a switching off of signaling components that, converging on actin cytoskeleton, regulate the cellular events leading to endothelial cell proliferation, migration and differentiation. As a result of endothelial cell function impairment, angiogenic process is strongly inhibited by G4 ligands both in vitro and in vivo. Interestingly, the G4-mediated antiangiogenic effect seems to recapitulate that observed by using a specific interference RNA against vegfr-2, and it is strongly antagonized by overexpressing the vegfr-2 gene. In conclusion, we describe the evidence for the existence of G4 in the promoter of vegfr-2, whose expression and function can be markedly inhibited by G4 ligands, thereby revealing a new, and so far undescribed, way to block VEGFR-2 as target for anticancer therapy.
Subject(s)
G-Quadruplexes , Neoplasms/blood supply , Neovascularization, Pathologic , Promoter Regions, Genetic , Vascular Endothelial Growth Factor Receptor-2/genetics , Animals , Cell Line, Tumor , Cells, Cultured , Human Umbilical Vein Endothelial Cells/physiology , Humans , Ligands , Mice , Mice, Inbred C57BL , Mice, Nude , Neovascularization, Physiologic , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
The shelterin protein TRF2 has come to the limelight for its role in telomere maintenance and tumorigenesis. Herein, the application of rational design and synthesis allowed identifying the first TRF2TRFH binder able to elicit a marked DNA damage response in cancer cells. This work paves the way for the unprecedented employment of a chemical tool to finely tune specific mechanisms underlying telomere maintenance.
Subject(s)
Drug Design , Peptides, Cyclic/pharmacology , Telomeric Repeat Binding Protein 2/antagonists & inhibitors , Humans , Models, Molecular , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Telomeric Repeat Binding Protein 2/metabolismABSTRACT
Following the results we previously reported on a series of xanthene and xanthone derivatives as G-quadruplex stabilizing ligands, in order to obtain a more selective compound with respect to the previous generation of derivatives, we decided to modify the structure of the core ligand, specifically its aromatic extension. In particular, here we report the design, synthesis and activity data of a new compound obtained by dimerization of the xanthene core (HELIXA4C). The reported results show that extension of the aromatic core and the increase of the number of polar side chains led to a great enhancement of G-quadruplex selectivity and telomere damage capability, as derived using ESI-MS evaluation, in vitro cancer screening and specific immunofluorescence assays.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , G-Quadruplexes/drug effects , Telomere/drug effects , Xanthones/chemistry , Xanthones/pharmacology , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Dimerization , Humans , Molecular Docking Simulation , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Xanthones/chemical synthesisABSTRACT
G-quadruplexes (G4s) are guanine-rich non-canonical secondary structures of nucleic acids that were identified in vitro almost half a century ago. Starting from the early 1980s, these structures were also observed in eukaryotic cells, first at the telomeric level and later in regulatory regions of cancer-related genes, in regulatory RNAs and within specific cell compartments such as lysosomes, mitochondria, and ribosomes. Because of the involvement of these structures in a large number of biological processes and in the pathogenesis of several diseases, including cancer, the interest in G4 targeting has exponentially increased in the last few years, and a great number of novel G4 ligands have been developed. Notably, G4 ligands represent a large family of heterogeneous molecules that can exert their functions by recognizing, binding, and stabilizing G4 structures in multiple ways. Regarding anti-cancer activity, the efficacy of G4 ligands was originally attributed to the capability of these molecules to inhibit the activity of telomerase, an enzyme that elongates telomeres and promotes endless replication in cancer cells. Thereafter, novel mechanisms through which G4 ligands exert their antitumoral activities have been defined, including the induction of DNA damage, control of gene expression, and regulation of metabolic pathways, among others. Here, we provided a perspective on the structure and function of G4 ligands with particular emphasis on their potential role as antitumoral agents. In particular, we critically examined the problems associated with the clinical translation of these molecules, trying to highlight the main aspects that should be taken into account during the phases of drug design and development. Indeed, taking advantage of the successes and failures, and the more recent technological progresses in the field, it would be possible to hypothesize the development of these molecules in the future that would represent a valid option for those cancers still missing effective therapies.
ABSTRACT
BACKGROUND: Breast Cancer (BC) can be classified, due to its heterogeneity, into multiple subtypes that differ for prognosis and clinical management. Notably, triple negative breast cancer (TNBC) - the most aggressive BC form - is refractory to endocrine and most of the target therapies. In this view, taxane-based therapy still represents the elective strategy for the treatment of this tumor. However, due variability in patients' response, management of TNBC still represents an unmet medical need. Telomeric Binding Factor 2 (TRF2), a key regulator of telomere integrity that is over-expressed in several tumors, including TNBC, has been recently found to plays a role in regulating autophagy, a degradative process that is involved in drug detoxification. Based on these considerations, we pointed, here, at investigating if TRF2, regulating autophagy, can affect tumor sensitivity to therapy. METHODS: Human TNBC cell lines, over-expressing or not TRF2, were subjected to treatment with different taxanes and drug efficacy was tested in terms of autophagic response and cell proliferation. Autophagy was evaluated first biochemically, by measuring the levels of LC3, and then by immunofluorescence analysis of LC3-puncta positive cells. Concerning the proliferation, cells were subjected to colony formation assays associated with western blot and FACS analyses. The obtained results were then confirmed also in mouse models. Finally, the clinical relevance of our findings was established by retrospective analysis on a cohort of TNBC patients subjected to taxane-based neoadjuvant chemotherapy. RESULTS: This study demonstrated that TRF2, inhibiting autophagy, is able to increase the sensitivity of TNBC cells to taxanes. The data, first obtained in in vitro models, were then recapitulated in preclinical mouse models and in a cohort of TNBC patients, definitively demonstrating that TRF2 over-expression enhances the efficacy of taxane-based neoadjuvant therapy in reducing tumor growth and its recurrence upon surgical intervention. CONCLUSIONS: Based on our finding it is possible to conclude that TRF2, already known for its role in promoting tumor formation and progression, might represents an Achilles' heel for cancer. In this view, TRF2 might be exploited as a putative biomarker to predict the response of TNBC patients to taxane-based neoadjuvant chemotherapy.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Animals , Mice , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Retrospective Studies , Taxoids/pharmacology , Taxoids/therapeutic use , Bridged-Ring Compounds/pharmacology , Bridged-Ring Compounds/therapeutic use , Cell Line, TumorABSTRACT
Group IVα phospholipase A(2) (PLA(2)IVα) is a lipolytic enzyme that catalyzes the hydrolysis of membrane phospholipids to generate precursors of potent inflammatory lipid mediators. Here, the role of PLA(2)IVα in Fc receptor (FcR)-mediated phagocytosis was investigated, demonstrating that PLA(2)IVα is selectively activated upon FcR-mediated phagocytosis in macrophages and that it rapidly translocates to the site of the nascent phagosome. Moreover, pharmacological inhibition of PLA(2)IVα by pyrrophenone reduces particle internalization by up to 50%. In parallel, fibroblasts from PLA(2)IVα knock-out mice overexpressing FcγRIIA and able to internalize IgG-opsonized beads show 50% lower phagocytosis, compared with wild-type cells, and transfection of PLA(2)IVα fully recovers this impaired function. Interestingly, transfection of the catalytically inactive deleted PLA(2)IVα mutant (PLA(2)IVα(1-525)) and point mutant (PLA(2)IVα-S228C) also promotes recovery of this impaired function. Finally, transfection of the PLA(2)IVα C2 domain (which is directly involved in PLA(2)IVα membrane binding), but not of PLA(2)IVα-D43N (which cannot bind to membranes), rescues FcR-mediated phagocytosis. These data unveil a new mechanism of action for PLA(2)IVα, which demonstrates that the membrane binding, and not the enzymatic activity, is required for PLA(2)IVα modulation of FcR-mediated phagocytosis.
Subject(s)
Cell Membrane/enzymology , Group IV Phospholipases A2/metabolism , Macrophages/enzymology , Phagocytosis/physiology , Phagosomes/metabolism , Amino Acid Substitution , Animals , Cell Line, Transformed , Cell Membrane/genetics , Fibroblasts/cytology , Fibroblasts/enzymology , Group IV Phospholipases A2/genetics , Humans , Macrophages/cytology , Mice , Mice, Knockout , Mutation, Missense , Phagocytosis/drug effects , Phagosomes/genetics , Protein Structure, Tertiary , Protein Transport/drug effects , Protein Transport/physiology , Pyrrolidines/pharmacology , Receptors, IgG/genetics , Receptors, IgG/metabolismABSTRACT
TERF2/TRF2 is a pleiotropic telomeric protein that plays a crucial role in tumor formation and progression through several telomere-dependent and -independent mechanisms. Here, we uncovered a novel function for this protein in regulating the macroautophagic/autophagic process upon different stimuli. By using both biochemical and cell biology approaches, we found that TERF2 binds to the non-histone chromatin-associated protein HMGB1, and this interaction is functional to the nuclear/cytoplasmic protein localization. Specifically, silencing of TERF2 alters the redox status of the cells, further exacerbated upon EBSS nutrient starvation, promoting the cytosolic translocation and the autophagic activity of HMGB1. Conversely, overexpression of wild-type TERF2, but not the mutant unable to bind HMGB1, negatively affects the cytosolic translocation of HMGB1, counteracting the stimulatory effect of EBSS starvation. Moreover, genetic depletion of HMGB1 or treatment with inflachromene, a specific inhibitor of its cytosolic translocation, completely abolished the pro-autophagic activity of TERF2 silencing. In conclusion, our data highlighted a novel mechanism through which TERF2 modulates the autophagic process, thus demonstrating the key role of the telomeric protein in regulating a process that is fundamental, under both physiological and pathological conditions, in defining the fate of the cells.Abbreviations: ALs: autolysosomes; ALT: alternative lengthening of telomeres; ATG: autophagy related; ATM: ATM serine/threonine kinase; CQ: Chloroquine; DCFDA: 2',7'-dichlorofluorescein diacetate; DDR: DNA damage response; DHE: dihydroethidium; EBSS: Earle's balanced salt solution; FACS: fluorescence-activated cell sorting; GFP: green fluorescent protein; EGFP: enhanced green fluorescent protein; GSH: reduced glutathione; GSSG: oxidized glutathione; HMGB1: high mobility group box 1; ICM: inflachromene; IF: immunofluorescence; IP: immunoprecipitation; NAC: N-acetyl-L-cysteine; NHEJ: non-homologous end joining; PLA: proximity ligation assay; RFP: red fluorescent protein; ROS: reactive oxygen species; TIF: telomere-induced foci; TERF2/TRF2: telomeric repeat binding factor 2.