RESUMO
Lung cancer has a significant incidence among the population and, unfortunately, has an unfavourable prognosis in most cases. The World Health Organization (WHO) classifies lung tumours into two subtypes based on their phenotype: the Non-Small Cell Lung Cancer (NSCLC) and the Small Cell Lung Cancer (SCLC). SCLC treatment, despite advances in chemotherapy and radiotherapy, is often unsuccessful for cancer recurrence highlighting the need to develop novel therapeutic strategies. In this review, we describe the genetic landscape and tumour microenvironment that characterize the pathological processes of SCLC and how they are responsible for tumour immune evasion. The immunosuppressive mechanisms engaged in SCLC are critical factors to understand the failure of immunotherapy in SCLC and, conversely, suggest that new signalling pathways, such as cGAS/STING, should be investigated as possible targets to stimulate an innate immune response in this subtype of lung cancer. The full comprehension of the innate immunity of cancer cells is thus crucial to open new challenges for successful immunotherapy in treating SCLC and improving patient outcomes.
RESUMO
G-quadruplexes (G4s) are non-canonical nucleic acid structures involved in fundamental biological processes. As G4s are promising anticancer targets, in past decades the search for effective anticancer G4 binders aimed at the discovery of more cytotoxic ligands interfering with specific G4 structures at oncogenes or telomeres. Here, we have instead observed a significant activation of innate immune genes by two unrelated ligands at non-cytotoxic concentrations. The studied G4 binders (pyridostatin and PhenDC3) can induce an increase of micronuclei triggering the activation of the cytoplasmic STING (stimulator of interferon response cGAMP interactor 1) signaling pathway in human and murine cancer cells. Ligand activity can then lead to type I interferon production and innate immune gene activation. Moreover, specific gene expression patterns mediated by a G4 binder in cancer cells correlate with immunological hot features and better survival in human TCGA (The Cancer Genome Atlas) breast tumors. The findings open to the development of cytostatic G4 binders as effective immunomodulators for combination immunotherapies in unresponsive tumors.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Citostáticos/farmacologia , Quadruplex G/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Feminino , Compostos de Anéis Fundidos/farmacologia , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Células MCF-7 , Melanoma Experimental/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Micronúcleos com Defeito Cromossômico , Nucleotidiltransferases/metabolismo , Ativação TranscricionalRESUMO
G-quadruplex (G4) binders have been investigated to discover new anticancer drugs worldwide in past decades. As these ligands are generally not highly cytotoxic, the discovery rational was mainly based on increasing the cell-killing potency. Nevertheless, no G4 binder has been shown yet to be effective in cancer patients. Here, G4 binder activity at low dosages will be discussed as a critical feature to discover ligands with therapeutic effects in cancer patients. Specific effects of G4 binders al low doses have been reported to occur in cancer and normal cells. Among them, genome instability and the stimulation of cytoplasmic processes related to autophagy and innate immune response open to the use of G4 binders as immune-stimulating agents. Thus, we propose a new rational of drug discovery, which is not based on cytotoxic potency but rather on immune gene activation at non-cytotoxic dosage.
Assuntos
Antineoplásicos , Quadruplex G , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Instabilidade Genômica , Humanos , Ligantes , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.
Assuntos
DNA/química , Quadruplex G , Genoma Humano , Estruturas R-Loop , RNA/química , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Pareamento de Bases , DNA/genética , DNA/metabolismo , Quadruplex G/efeitos dos fármacos , Instabilidade Genômica , Guanina/química , Guanina/metabolismo , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologia , Regiões Promotoras Genéticas , Estruturas R-Loop/efeitos dos fármacos , RNA/genética , RNA/metabolismo , Telômero/efeitos dos fármacos , Telômero/metabolismo , Telômero/ultraestrutura , Homeostase do TelômeroRESUMO
KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.
Assuntos
Quadruplex G , Guanina/análogos & derivados , Proteínas Proto-Oncogênicas p21(ras)/química , Sítio de Iniciação de Transcrição , Transcrição Gênica , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA/metabolismo , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Guanina/química , Guanina/metabolismo , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Oxirredução , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Datasets reporting microRNA expression profiles in normal and cancer cells show that miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance. We prepared variants of ss-miR-216b mimics with and without a 5' phosphate group. Both variants strongly suppressed oncogenic KRAS in PDAC cells and inhibited colony formation in pancreatic cancer cells. We observed that the designed ss-miR-216b mimics engaged AGO2 to promote the silencing of KRAS. We also tested a new delivery strategy based on the use of palmityl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with ss-miR-216b conjugated with two palmityl chains and a lipid-modified cell penetrating peptide (TAT). These versatile nanoparticles suppressed oncogenic KRAS in PDAC cells.
Assuntos
Proteínas Argonautas/genética , Carcinoma Ductal Pancreático/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Regiões 3' não Traduzidas/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lipídeos/química , Lipídeos/farmacologia , Lipossomos/química , Lipossomos/farmacologia , MicroRNAs/química , MicroRNAs/farmacologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Pâncreas/metabolismo , Pâncreas/patologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidoresRESUMO
A large variety of non-B secondary structures can be formed between DNA and RNA. In this chapter, we focus on G-quadruplexes (G4) and R-loops, which can have a close structural interplay. In recent years, increasing evidence pointed to the fact that they can strongly influence each other in vivo, both having physiological and pathological roles in normal and cancer cells. Here, we detail specific and accurate methods for purification of BG4 and S9.6 antibodies, and their subsequent use in immunofluorescence microscopy, enabling single-cell analysis of extent and localization of G4s and R-loops.
Assuntos
Quadruplex G , Estruturas R-Loop , DNA/química , RNA/química , Microscopia de FluorescênciaRESUMO
G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.
Assuntos
Antineoplásicos , Citostáticos , Quadruplex G , Neoplasias , Antineoplásicos/farmacologia , Instabilidade Genômica , Humanos , Hidrazonas/farmacologia , Interferon beta/genética , Ligantes , Neoplasias/genéticaRESUMO
Targeting G-quadruplex structures is currently viewed as a promising anticancer strategy. Searching for potent and selective G-quadruplex binders, here we describe a small series of new monohydrazone derivatives designed as analogues of a lead which was proved to stabilize G-quadruplex structures and increase R loop levels in human cancer cells. To investigate the G-quadruplex binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G-quadruplex-forming sequences. The obtained results allowed the identification of a highly selective G-quadruplex ligand that, when studied in human cancer cells, proved to be able to stabilize both G-quadruplexes and R loops and showed a potent cell killing activity associated with the formation of micronuclei, a clear sign of genome instability.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Hidrazonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Genoma/efeitos dos fármacos , Humanos , Hidrazonas/síntese química , Hidrazonas/metabolismo , Ligantes , Estruturas R-Loop/efeitos dos fármacosRESUMO
The human KRAS transcript contains a G-rich 5'-UTR sequence (77% GC) harboring several G4 motifs capable to form stable RNA G-quadruplex (RG4) structures that can serve as targets for small molecules. A biotin-streptavidin pull-down assay showed that 4,11-bis(2-aminoethylamino)anthra[2,3-b]furan-5,10-dione (2a) binds to RG4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays demonstrated that 2a and its analogue 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione (2b) repress translation in a dose-dependent manner. The effect of the G4-ligands on Panc-1 cancer cells has also been examined. Both 2a and 2b efficiently penetrate the cells, suppressing protein p21KRAS to <10% of the control. The KRAS down-regulation induces apoptosis together with a dramatic reduction of cell growth and colony formation. In summary, we report a strategy to suppress the KRAS oncogene in pancreatic cancer cells by means of small molecules binding to RG4s in the 5'-UTR of mRNA.
Assuntos
Regiões 5' não Traduzidas/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Tiofenos/química , Tiofenos/farmacologiaRESUMO
HRAS is regulated by two neighbouring quadruplex-forming GC-elements (hras-1 and hras-2), located upstream of the major transcription start sites (doi: 10.1093/nar/gku 5784). In this study we demonstrate that the C-rich strands of hras-1 and hras-2 fold into i-motif conformations (iMs) characterized under crowding conditions (PEG-300, 40% w/v) by semi-transitions at pH 6.3 and 6.7, respectively. Nondenaturing PAGE shows that the HRAS C-rich sequences migrate at both pH 5 and 7 as folded intramolecular structures. Chromatin immunoprecipitation shows that hnRNP A1 is associated under in vivo conditions to the GC-elements, while EMSA proves that hnRNP A1 binds tightly to the iMs. FRET and CD show that hnRNP A1 unfolds the iM structures upon binding. Furthermore, when hnRNP A1 is knocked out in T24 bladder cancer cells by a specific shRNA, the HRAS transcript level drops to 44 ± 5% of the control, suggesting that hnRNP A1 is necessary for gene activation. The sequestration by decoy oligonucleotides of the proteins (hnRNP A1 and others) binding to the HRAS iMs causes a significant inhibition of HRAS transcription. All these outcomes suggest that HRAS is regulated by a G-quadruplex/i-motif switch interacting with proteins that recognize non B-DNA conformations.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Desdobramento de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Sequência Rica em GC/genética , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos/genética , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Sequências Reguladoras de Ácido Nucleico/genética , Termodinâmica , Temperatura de TransiçãoRESUMO
In a previous study we have demonstrated that two neighboring G-quadruplexes, hras-1 and hras-2, located immediately upstream of the major transcription start site of HRAS, bind MAZ, a nuclear factor that activates transcription (Cogoi, S.; et al. Nucl. Acid Res. 2014, 42, 8379). For the present study we have designed G4 oligonucleotides with anthraquinone insertions and locked nucleic acids (LNA) modifications mimicking quadruplex hras-1. Luciferase, qRT-PCR, and Western blot data demonstrate that these constructs efficiently down regulate HRAS in T24 bladder cancer cells. The inhibitory efficiency of the G4 oligonucleotides correlates with their nuclease resistance in the cell environment. By chromatin immunoprecipitation we show that the association of MAZ to the HRAS promoter is strongly attenuated by the designed G4 oligonucleotides, thus suggesting that these constructs behave with a decoy mechanism.