RESUMO
Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYC Antisense 1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.
Assuntos
Neoplasias Pulmonares , RNA Longo não Codificante , Animais , Humanos , Filogenia , Regulação Neoplásica da Expressão Gênica , RNA Antissenso/genética , Ciclo Celular/genética , Proliferação de Células/genética , Neoplasias Pulmonares/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Movimento Celular , Mamíferos/genética , Fator de Ligação a CCAAT/genéticaRESUMO
G-quadruplexes (G4s) are implicated in pathological processes such as cancer and infective diseases. Their targeting with G4-ligands has shown therapeutic capacity. Most of the current G4-ligands are planar molecules, do not discriminate among G4s, and have poor druglike properties. The available methods to identify compounds selective for one single G4 are often time-consuming. Here, we describe the development, validation, and application of an affinity-selection mass spectrometry method that employs unlabeled G4 oligonucleotides as targets and allows testing of up to 320 unmodified small molecules in a single tube. As a proof of concept, this method was applied to screen a library of 40â¯000 druglike molecules against two G4s, transcriptional regulators of the HIV-1 LTR promoter. We identified nonplanar pyrazolopyrimidines that selectively recognize and stabilize the major HIV-1 LTR G4 possibly by fitting and binding through H-bonding in its unique binding pocket. The compounds inhibit LTR promoter activity and HIV-1 replication. We propose this method to prompt the fast development of new G4-based therapeutics.
Assuntos
Quadruplex G , HIV-1 , HIV-1/genética , Ligantes , Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
Cancer development and progression are not solely cell-autonomous and genetically driven processes. Dynamic interaction of cancer cells with the surrounding microenvironment, intended as the chemical/physical conditions as well as the mixture of non-neoplastic cells of the tumor niche, drive epigenetic changes that are pivotal for the acquisition of malignant traits. Cancer-associated fibroblasts (CAF), namely fibroblasts that, corrupted by cancer cells, acquire a myofibroblast-like reactive phenotype, are able to sustain tumor features by the secretion of soluble paracrine signals and the delivery extracellular vesicles. In such diabolic liaison, a major role has been ascribed to noncoding RNAs. Defined as RNAs that are functional though not being translated into proteins, noncoding RNAs predominantly act as regulators of gene expression at both the transcriptional and post-transcriptional levels. In this review, we summarize the current knowledge of microRNAs and long noncoding RNAs that act intracellularly in either CAFs or cancer cells to sustain tumor-stroma interplay. We also report on the major role of extracellular noncoding RNAs that are bidirectionally transferred between either cell type. Upon presenting a comprehensive view of the existing literature, we provide our critical opinion regarding the possible clinical utility of tumor-stroma related noncoding RNAs as therapeutic target/tools or prognostic/predictive biomarkers.
RESUMO
Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.
Assuntos
Quadruplex G , Regulação Neoplásica da Expressão Gênica/genética , Lipossarcoma/terapia , Terapia de Alvo Molecular , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Neoplasias de Tecidos Moles/terapia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Ligantes , Modelos Genéticos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVES: The Unyvero molecular assay was tested for the clinical resolution of discordant results, evaluating its role in prosthetic joint infection diagnosis. METHODS: Multiplex PCR was performed on 45 samples from prosthesis treatment (either sonication or dithiothreitol). Analytical performance was compared to that of biofilm culture using Musculoskeletal Infection Society criteria as gold standard. RESULTS: Unyvero and biofilm culture showed similar agreement rates compared to the gold standard (34/43 and 32/43, respectively). Both methods showed six additional identifications compatible with true infection; five positive results from biofilm culture were deemed contaminations. CONCLUSIONS: The Unyvero system showed good performances and a significantly shorter turnaround time compared to cultural methods, presenting an added value to PJI diagnosis even when performed following a composite approach.
Assuntos
Artrite Infecciosa , Infecções Relacionadas à Prótese , Humanos , Reação em Cadeia da Polimerase Multiplex , Estudos Prospectivos , Infecções Relacionadas à Prótese/diagnóstico , Sensibilidade e Especificidade , SonicaçãoRESUMO
Targeting of G-quadruplexes, non-canonical conformations that form in G-rich regions of nucleic acids, has been proposed as a novel therapeutic strategy toward several diseases, including cancer and infections. The unavailability of highly selective molecules targeting a G-quadruplex of choice has hampered relevant applications. Herein, we describe a novel approach, based on naphthalene diimide (NDI)-peptide nucleic acid (PNA) conjugates, taking advantage of the cooperative interaction of the NDI with the G-quadruplex structure and hybridization of the PNA with the flanking region upstream or downstream the targeted G-quadruplex. By biophysical and biomolecular assays, we show that the NDI-PNA conjugates are able to specifically recognize the G-quadruplex of choice within the HIV-1 LTR region, consisting of overlapping and therefore mutually exclusive G-quadruplexes. Additionally, the conjugates can induce and stabilize the least populated G-quadruplex at the expenses of the more stable ones. The general and straightforward design and synthesis, which readily apply to any G4 target of choice, together with both the red-fluorescent emission and the possibility to introduce cellular localization signals, make the novel conjugates available to selectively control G-quadruplex folding over a wide range of applications.
Assuntos
Quadruplex G , Repetição Terminal Longa de HIV , Ácidos Nucleicos Peptídicos/química , DNA/química , HIV-1/genética , Células HeLa , Humanos , Imidas/química , Ligantes , Modelos Genéticos , Naftalenos/química , Ácidos Nucleicos Peptídicos/metabolismoRESUMO
Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of the non-canonical nucleic acid G-quadruplex structures within oncogene promoters has stood out as a promising approach to interfere with oncogenic signaling pathways in cancer cells, paving the way toward the development of G-quadruplex ligands as antitumor drugs. Here, we present the synthesis and screening of a library of differently functionalized core-extended naphthalene diimides for their activity against the BRAFV600E-mutant melanoma cell line. The most promising compound was able to stabilize G-quadruplexes that formed in the promoter regions of two target genes relevant to melanoma, KIT and BCL-2. This activity led to the suppression of protein expression and thus to interference with oncogenic signaling pathways involved in BRAF-mutant melanoma cell survival, apoptosis, and resistance to drugs. This G-quadruplex ligand thus represents a suitable candidate for the development of melanoma treatment options based on a new mechanism of action and could reveal particular significance in the context of resistance to targeted therapies of BRAF-mutant melanoma cells.
Assuntos
Imidas/uso terapêutico , Melanoma/metabolismo , Naftalenos/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dicroísmo Circular , Quadruplex G/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Melanoma/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais/genéticaRESUMO
Retroviruses infect almost all vertebrates, from humans to domestic and farm animals, from primates to wild animals, where they cause severe diseases, including immunodeficiencies, neurological disorders, and cancer. Nonhuman retroviruses have also been recently associated with human diseases. To date, no effective treatments are available; therefore, finding retrovirus-specific therapeutic targets is becoming an impelling issue. G-Quadruplexes are four-stranded nucleic acid structures that form in guanine-rich regions. Highly conserved G-quadruplexes located in the long-terminal-repeat (LTR) promoter of HIV-1 were shown to modulate the virus transcription machinery; moreover, the astonishingly high degree of conservation of G-quadruplex sequences in all primate lentiviruses corroborates the idea that these noncanonical nucleic acid structures are crucial elements in the lentiviral biology and thus have been selected for during evolution. In this work, we aimed at investigating the presence and conservation of G-quadruplexes in the Retroviridae family. Genomewide bioinformatics analysis showed that, despite their documented high genetic variability, most retroviruses contain highly conserved putative G-quadruplex-forming sequences in their promoter regions. Biophysical and biomolecular assays proved that these sequences actually fold into G-quadruplexes in physiological concentrations of relevant cations and that they are further stabilized by ligands. These results validate the relevance of G-quadruplexes in retroviruses and endorse the employment of G-quadruplex ligands as innovative antiretroviral drugs. This study indicates new possible pathways in the management of retroviral infections in humans and animal species. Moreover, it may shed light on the mechanism and functions of retrovirus genomes and derived transposable elements in the human genome.
Assuntos
Biologia Computacional/métodos , RNA Viral/química , Retroviridae/genética , Sequências Repetidas Terminais , Animais , Dicroísmo Circular , Quadruplex G , Humanos , Regiões Promotoras Genéticas , Retroviridae/química , Análise de Sequência de RNA , Sequenciamento Completo do GenomaRESUMO
Stabilization of the G-quadruplexes (G4s) within the androgen receptor (AR) gene promoter to block transcription may represent an innovative approach to interfere with aberrant AR signaling in castration resistant prostate cancer (CRPC). A library of differently functionalized naphthalene diimides (NDIs) was screened for their ability to stabilize AR G4s: the core-extended NDI (7) stood out as the most promising ligand. AR-positive cells were remarkably sensitive to 7 in comparison to AR-negative CRCP or normal prostate epithelial cells; 7 induced remarkable impairment of AR mRNA and protein amounts and significant perturbations in the expression levels of KLK3 and of genes involved in the activation of AR program via feedback mechanisms. Moreover, 7 synergistically interacted with Enzalutamide, an inhibitor of AR signaling used in second-line therapies. Overall, our data show that stabilization of AR G4s may represent an alternative treatment options for CRPC and other malignancies relying on aberrant androgen signaling.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Sinergismo Farmacológico , Quadruplex G , Imidas/química , Naftalenos/química , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/química , Antagonistas de Receptores de Andrógenos/química , Benzamidas , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Masculino , Nitrilas , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Transdução de Sinais , Células Tumorais CultivadasRESUMO
G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in order to reach the clinical use. So far, G4 ligands have been mainly identified through high-throughput screening methods or design of molecules with pre-set features. Here, we describe the development of new heterocyclic ligands through a fragment-based drug discovery (FBDD) approach. The ligands were designed against the major G4 present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), the stabilization of which has been shown to suppress viral gene expression and replication. Our method is based on the generation of molecular fragment small libraries, screened against the target to further elaborate them into lead compounds. We screened 150 small molecules, composed by structurally and chemically different fragments, selected from commercially available and in-house compounds; synthetic elaboration yielded several G4 ligands and two final G4 binders, both embedding an amidoxime moiety; one of these two compounds showed preferential binding for the HIV-1 LTR G4. This work presents the discovery of a novel potential pharmacophore and highlights the possibility to apply a fragment-based approach to develop G4 ligands with unexpected chemical features.
Assuntos
Repetição Terminal Longa de HIV/efeitos dos fármacos , Compostos Heterocíclicos/síntese química , Oximas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Quadruplex G , Compostos Heterocíclicos/química , Ligantes , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
BACKGROUND: Prosthetic joint infection (PJI) is among the most-severe complications of a total joint arthroplasty. Identification of the causal organism is of paramount importance for successful treatment, and sonication of implants may aid in this identification. Dithiothreitol (DTT) treatment has been proposed as an alternative to sonication to improve diagnosis, reduce costs, and improve reliability of the procedure, but its efficacy remains poorly characterized. QUESTIONS/PURPOSES: (1) Are DTT and sonication more sensitive and/or more specific than standard cultures of tissue samples for the diagnosis of PJI? (2) Which test (DTT or sonication) is more sensitive when the clinician does not suspect infection before surgery? (3) Which test (DTT or sonication) is more sensitive when the clinician suspects infection before surgery? METHODS: Two hundred thirty-two patients undergoing revision of a knee or hip arthroplasty were prospectively evaluated in this randomized study. Cultures were performed on five tissue samples from each patient and on fluid obtained by prosthesis treatment in patients randomly assigned to sonication (117 patients) or DTT (115 patients). The reference standard against which cultures (on tissue samples and on fluids from sonication or DTT) were compared was the Musculoskeletal Infection Society definition of PJI. RESULTS: Cultures on sonication and DTT fluids provided higher sensitivity (89% and 91%, respectively) than those on standard cultures of tissue samples (79%; p < 0.001). Among patients in whom infection was not suspected before surgery, the sensitivity of DTT was greater than that for sonication and cultures on tissue samples (100% versus 70% and 50%; p < 0.001). Among patients in whom infection was suspected before surgery, the sensitivity of DTT and sonication were not greater than that for standard cultures (89% and 94% versus 86%). CONCLUSIONS: In this randomized study, we found no difference in sensitivity between DTT and sonication for the detection of PJI, and both of those tests were more sensitive than standard tissue cultures. Thus, cultures of sonication or DTT fluid should be considered important additional tools to standard cultures for definition of PJI and should be considered together with other criteria, especially in settings where infection is not suspected before revision surgery.Level of Evidence Level I, diagnostic study.
Assuntos
Artroplastia de Quadril/efeitos adversos , Artroplastia do Joelho/efeitos adversos , Bactérias/isolamento & purificação , Técnicas Bacteriológicas , Ditiotreitol/administração & dosagem , Prótese de Quadril/efeitos adversos , Prótese do Joelho/efeitos adversos , Infecções Relacionadas à Prótese/diagnóstico , Sonicação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/instrumentação , Artroplastia do Joelho/instrumentação , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/cirurgia , Reoperação , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocortical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotoxicity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at concentrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater efficacy of combination therapy (mitotane plus chemotherapeutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.