RESUMEN

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.

Asunto(s)

ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Humanos , Sitios de Unión , ADN/metabolismo , ADN/química , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Microscopía de Fuerza Atómica , Unión Proteica

RESUMEN

The base excision repair (BER) is the primary damage repair pathway for repairing most of the endogenous DNA damage including oxidative base lesions, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs) in the genome. Repair of these damages in cells relies on sequential recruitment and coordinated actions of multiple DNA repair enzymes, which include DNA glycosylases (such as OGG1), AP-endonucleases (APE1), DNA polymerases, and DNA ligases. APE1 plays a key role in the BER pathway by repairing the AP sites and SSBs in the genome. Several methods have been developed to generate a map of endogenous AP sites or SSBs in the genome and the binding of DNA repair proteins. In this chapter, we describe detailed approaches to map genome-wide occupancy or enrichment of APE1 in human cells using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). Further, we discuss standard bioinformatics approaches for analyzing ChIP-seq data to identify APE1 enrichment or binding peaks in the genome.

RESUMEN

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.

Asunto(s)

Carcinoma Ductal Pancreático , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , G-Cuádruplex , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Carcinoma Ductal Pancreático/genética , ADN/química , Endonucleasas/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Pancreáticas

RESUMEN

Genomic DNA in the nucleus is wrapped around nucleosomes, a repeating unit of chromatin. The nucleosome, consisting of octamer of core histones, is a barrier for several cellular processes that require access to the naked DNA. The FAcilitates Chromatin Transcription (FACT), a histone chaperone complex, is involved in nucleosome remodeling via eviction or assembly of histones during transcription, replication, and DNA repair. Increasing evidence suggests that FACT plays an important role in multiple DNA repair pathways including transcription-coupled nucleotide excision repair (TC-NER) of UV-induced damage, DNA single- and double-strand breaks (DSBs) repair, and base excision repair (BER) of oxidized or alkylated damaged bases. Further, studies have shown overexpression of FACT in multiple types of cancer and its association with drug resistance and patients' poor prognosis. In this review, we discuss how FACT is accumulated at the damage site and what functions it performs. We describe the known mechanisms by which FACT facilitates repair of different types of DNA damage. Further, we highlight the recent advances in a class of FACT inhibitors, called curaxins, which show promise as a new adjuvant therapy to sensitize multiple types of cancer to chemotherapy and radiation.

Asunto(s)

Ensamble y Desensamble de Cromatina , Reparación del ADN , Chaperonas de Histonas/metabolismo , Nucleosomas/metabolismo , Animales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética

RESUMEN

Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71 MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.

Asunto(s)

Cisplatino/administración & dosificación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Meduloblastoma/tratamiento farmacológico , Factores de Elongación Transcripcional/genética , Adolescente , Adulto , Animales , Carbazoles/administración & dosificación , Carbazoles/efectos adversos , Niño , Preescolar , Cisplatino/efectos adversos , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Xenoinjertos , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Chaperonas de Histonas/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/radioterapia , Ratones , Factores de Elongación Transcripcional/antagonistas & inhibidores , Adulto Joven

RESUMEN

BACKGROUND: Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) plays a critical role in DNA base excision repair (BER) pathway and has been reported to be overexpressed in multiple cancers. Previously, we have shown that histone chaperone FACT complex (Facilitates Chromatin Transcription, a heterodimer of SSRP1 and SPT16 proteins) facilitates the chromatin access and DNA repair function of APE1, and their expression levels are correlated with promoting drug resistance in cancer. FACT inhibitor has been introduced in phase I and II clinical trials for chemosensitization of advanced solid cancers. However, the expression profile and prognostic significance of APE1 and FACT complex in bladder cancer remains largely unknown. METHODS: Retrospectively, 69 bladder cancer samples were retrieved and submitted for immunohistochemical staining of APE1 and SSRP1. Expression profile including cytoplasmic and nuclear staining of APE1 and expression level of SSRP1 was examined and semi-quantified to render a H-score. The prognostic significance of APE1 and SSRP1 was evaluated by Kaplan-Meier survival analysis in our cohort and R2 database. RESULTS: APE1 expression is elevated in bladder cancer compared to normal adjacent tissues. Compared with low grade tumors, high grade tumors show a shift in the staining pattern including higher intensity and positive cytoplasmic staining. Carcinoma in situ has a similar staining pattern to high grade tumors. APE1 and SSRP1 staining intensity increases as tumor progresses with stage. There is a correlation between APE1 and SSRP1 staining in invasive bladder cancer (Spearman r = 0.5466, p < 0.0001). The increased expression of APE1 and SSRP1 is associated with poor survival in Kaplan-Meier analysis in our cohort and in R2-TCGA bladder cancer database. CONCLUSIONS: The expression levels of APE1 and SSRP1 are significantly elevated in bladder cancer as compared to normal adjacent tissues. APE1 correlates with SSRP1 expression in high grade tumors. Overexpression of APE1 and SSRP1 is associated with poor survival in bladder cancer. This suggests the usage of FACT inhibitor curaxins in muscle invasive bladder cancer to target FACT complex and APE1 to improve chemosensitization after further validation.

RESUMEN

Medulloblastoma (MB) is the most common malignant central nervous system tumor in pediatric patients. Mainstay of therapy remains surgical resection followed by craniospinal radiation and chemotherapy, although limitations to this therapy are applied in the youngest patients. Clinically, tumors are divided into average and high-risk status on the basis of age, metastasis at diagnosis, and extent of surgical resection. However, technological advances in high-throughput screening have facilitated the analysis of large transcriptomic datasets that have been used to generate the current classification system, dividing patients into four primary subgroups, i.e., WNT (wingless), SHH (sonic hedgehog), and the non-SHH/WNT subgroups 3 and 4. Each subgroup can further be subdivided on the basis of a combination of cytogenetic and epigenetic events, some in distinct signaling pathways, that activate specific phenotypes impacting patient prognosis. Here, we delve deeper into the genetic basis for each subgroup by reviewing the extent of cytogenetic events in key genes that trigger neoplastic transformation or that exhibit oncogenic properties. Each of these discussions is further centered on how these genetic aberrations can be exploited to generate novel targeted therapeutics for each subgroup along with a discussion on challenges that are currently faced in generating said therapies. Our future hope is that through better understanding of subgroup-specific cytogenetic events, the field may improve diagnosis, prognosis, and treatment to improve overall quality of life for these patients.

RESUMEN

BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved.

Asunto(s)

Fibroblastos , Polimorfismo de Nucleótido Simple , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Secuenciación del Exoma

RESUMEN

In addition to the key roles of reversible acetylation of histones in chromatin in epigenetic regulation of gene expression, acetylation of nonhistone proteins by histone acetyltransferases (HATs) p300 and CBP is involved in DNA transactions, including repair of base damages and strand breaks. We characterized acetylation of human NEIL1 DNA glycosylase and AP-endonuclease 1 (APE1), which initiate repair of oxidized bases and single-strand breaks (SSBs), respectively. Acetylation induces localized conformation change because of neutralization of the positive charge of specific acetyl-acceptor Lys residues, which are often present in clusters. Acetylation in NEIL1, APE1, and possibly other base excision repair (BER)/SSB repair (SSBR) enzymes by HATs, prebound to chromatin, induces assembly of active repair complexes on the chromatin. In this review, we discuss the roles of acetylation of NEIL1 and APE1 in modulating their activities and complex formation with other proteins for fine-tuning BER in chromatin. Further, the implications of promoter/enhancer-bound acetylated BER protein complexes in the regulation of transcriptional activation, mediated by complex interplay of acetylation and demethylation of histones are discussed.

Asunto(s)

ADN Glicosilasas/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , ADN/metabolismo , Daño del ADN , Histona Acetiltransferasas/metabolismo , Humanos

RESUMEN

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

Asunto(s)

Daño del ADN , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , G-Cuádruplex , Células A549 , Acetilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Expresión Génica , Genes myc , Genoma Humano , Guanina/química , Guanina/metabolismo , Células HCT116 , Humanos , Oxidación-Reducción , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Gene fusions that contribute to oncogenicity can be explored for identifying cancer biomarkers and potential drug targets. To investigate the nature and distribution of fusion transcripts in cancer, we examined the transcriptome data of about 9,000 primary tumors from 33 different cancers in TCGA (The Cancer Genome Atlas) along with cell line data from CCLE (Cancer Cell Line Encyclopedia) using ChimeRScope, a novel fusion detection algorithm. We identified several fusions with sense (canonical, 39%) or antisense (non-canonical, 61%) transcripts recurrent across cancers. The majority of the recurrent non-canonical fusions found in our study are novel, unexplored, and exhibited highly variable profiles across cancers, with breast cancer and glioblastoma having the highest and lowest rates, respectively. Overall, 4,344 recurrent fusions were identified from TCGA in this study, of which 70% were novel. Additional analysis of 802 tumor-derived cell line transcriptome data across 20 cancers revealed significant variability in recurrent fusion profiles between primary tumors and corresponding cell lines. A subset of canonical and non-canonical fusions was validated by examining the structural variation evidence in whole-genome sequencing (WGS) data or by Sanger sequencing of fusion junctions. Several recurrent fusion genes identified in our study show promise for drug repurposing in basket trials and present opportunities for mechanistic studies.

RESUMEN

Fluorouracil (5-FU) remains a first-line chemotherapeutic agent for colorectal cancer. However, a subset of colorectal cancer patients who have defective mismatch-repair (dMMR) pathway show resistance to 5-FU. Here, we demonstrate that the efficacy of 5-FU in dMMR colorectal cancer cells is largely dependent on the DNA base excision repair (BER) pathway. Downregulation of APE1, a key enzyme in the BER pathway, decreases IC50 of 5-FU in dMMR colorectal cancer cells by 10-fold. Furthermore, we discover that the facilitates chromatin transcription (FACT) complex facilitates 5-FU repair in DNA via promoting the recruitment and acetylation of APE1 (AcAPE1) to damage sites in chromatin. Downregulation of FACT affects 5-FU damage repair in DNA and sensitizes dMMR colorectal cancer cells to 5-FU. Targeting the FACT complex with curaxins, a class of small molecules, significantly improves the 5-FU efficacy in dMMR colorectal cancer in vitro (∼50-fold decrease in IC50) and in vivo xenograft models. We show that primary tumor tissues of colorectal cancer patients have higher FACT and AcAPE1 levels compared with adjacent nontumor tissues. Additionally, there is a strong clinical correlation of FACT and AcAPE1 levels with colorectal cancer patients' response to chemotherapy. Together, our study demonstrates that targeting FACT with curaxins is a promising strategy to overcome 5-FU resistance in dMMR colorectal cancer patients.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carbazoles/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas de Unión al ADN/antagonistas & inhibidores , Fluorouracilo/farmacología , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Factores de Elongación Transcripcional/antagonistas & inhibidores , Animales , Carbazoles/administración & dosificación , Cromatina/metabolismo , Neoplasias del Colon/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Fluorouracilo/administración & dosificación , Células HCT116 , Células HEK293 , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/antagonistas & inhibidores , Chaperonas de Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Factores de Elongación Transcripcional/metabolismo , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Protein posttranslational modifications (PTMs), including acetylation, have emerged as important regulators for controlling many cellular processes. DNA base excision repair (BER), a highly coordinated multistep cellular process, is primarily involved in the repair of both endogenous and drug-induced exogenous DNA base damages. BER relies on sequential recruitment and coordinated actions of multiple proteins. Increasing evidence suggests that acetylation of lysine residues of BER proteins facilitates fine-tuning of enzymatic activities, protein-protein interactions, and coordination of the steps in BER pathway. In this chapter, we describe detailed in vitro and in vivo approaches to examine the effect of acetylation on BER enzymes, focusing on the impact of acetylation of AP-endonuclease (APE1), a key enzyme in BER pathway, on its DNA damage repair activity, substrate-binding, and subcellular localization.

Asunto(s)

Bioensayo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Acetilación , Bioensayo/métodos , Catálisis , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Unión Proteica , Proteínas Recombinantes , Especificidad por Sustrato

RESUMEN

Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1's disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296-298 with neutral Ala loses nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites does not affect NEIL1's nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing the acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.

Asunto(s)

ADN Glicosilasas/metabolismo , Reparación del ADN , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Acetilación , Cromatina/metabolismo , ADN/metabolismo , ADN Glicosilasas/química , Humanos , Factores de Transcripción p300-CBP/metabolismo

RESUMEN

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , Chaperonas Moleculares/genética , Péptidos/farmacología , Proteínas Inhibidoras de STAT Activados/genética , Factor de Transcripción STAT3/genética , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cisplatino/farmacología , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Chaperonas Moleculares/metabolismo , Péptidos/síntesis química , Proteínas Inhibidoras de STAT Activados/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo

RESUMEN

The human apurinic/apyrimidinic endonuclease 1 (APE1) is a pleiotropic nuclear protein with roles in DNA base excision repair pathway as well as in regulation of transcription. Recently, the presence of extracellular plasma APE1 was reported in endotoxemic rats. However, the biological significance and the extracellular function of APE1 remain unclear. In this study, we found that monocytes secrete APE1 upon inflammatory challenges. Challenging the monocytic cells with extracellular APE1 resulted in the increased expression and secretion of the pro-inflammatory cytokine IL-6. Additionally, the extracellular APE1 treatment activated the transcription factor NF-κB, followed by its increased occupancy at the IL-6 promoter, resulting in the induction of IL-6 expression. APE1-induced IL-6 further served to elicit autocrine and paracrine cellular responses. Moreover, the extracellular IL-6 promoted the secretion of APE1, thus indicating a functional feedforward loop in this pathway. Furthermore, we show that APE1 is secreted through extracellular vesicles formation via endosomal sorting complex required for transport (ESCRT)-dependent pathway. Together, our study demonstrates a novel role of extracellular APE1 in IL-6-dependent cellular responses.

Asunto(s)

Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Regulación de la Expresión Génica , Interleucina-6/genética , Monocitos/metabolismo , FN-kappa B/metabolismo , Compuestos de Anilina/farmacología , Animales , Comunicación Autocrina/efectos de los fármacos , Compuestos de Bencilideno/farmacología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Células HCT116 , Humanos , Ratones , FN-kappa B/genética , Comunicación Paracrina/efectos de los fármacos , Cultivo Primario de Células , Células RAW 264.7 , Células THP-1

RESUMEN

Apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome, inhibit transcription and block replication. The primary enzyme that repairs AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Although the mechanism by which APE1 repairs AP sites in vitro has been extensively investigated, it is largely unknown how APE1 repairs AP sites in cells. Here, we show that APE1 is acetylated (AcAPE1) after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of the positive charges of the lysine residues in the N-terminal domain of APE1 induces a conformational change; this in turn enhances the AP endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Thus, mammalian cells, unlike Saccharomyces cerevisiae or Escherichia coli cells, require acetylation of APE1 for the efficient repair of AP sites and base damage in the genome. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity.

Asunto(s)

Cromatina/metabolismo , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Acetilación , Biocatálisis , Ciclo Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Genoma , Humanos , Lisina/metabolismo , Modelos Biológicos , Unión Proteica , Conformación Proteica

RESUMEN

Apurinic/apyrimidinic (AP) sites are frequently generated in the genome by spontaneous depurination/depyrimidination or after removal of oxidized/modified bases by DNA glycosylases during the base excision repair (BER) pathway. Unrepaired AP sites are mutagenic and block DNA replication and transcription. The primary enzyme to repair AP sites in mammalian cells is AP endonuclease (APE1), which plays a key role in this repair pathway. Although overexpression of APE1 in diverse cancer types and its association with chemotherapeutic resistance are well documented, alteration of posttranslational modification of APE1 and modulation of its functions during tumorigenesis are largely unknown. Here, we show that both classical histone deacetylase HDAC1 and NAD+-dependent deacetylase SIRT1 regulate acetylation level of APE1 and acetylation of APE1 enhances its AP-endonuclease activity both in vitro and in cells. Modulation of APE1 acetylation level in cells alters AP site repair capacity of the cell extracts in vitro. Primary tumor tissues of diverse cancer types have higher level of acetylated APE1 (AcAPE1) compared to adjacent non-tumor tissue and exhibit enhanced AP site repair capacity. Importantly, in the absence of APE1 acetylation, cells accumulate AP sites in the genome and show increased sensitivity to DNA damaging agents. Together, our study demonstrates that elevation of acetylation level of APE1 in tumor could be a novel mechanism by which cells handle the elevated levels of DNA damages in response to genotoxic stress and maintain sustained proliferation.

Asunto(s)

Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Acetilación , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/genética , Histona Desacetilasa 1/metabolismo , Humanos , NAD/metabolismo , Neoplasias/patología , Unión Proteica , Sirtuina 1/metabolismo

RESUMEN

Mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a ubiquitous and multifunctional protein, plays an essential role in the repair of both endogenous and drug-induced DNA damages in the genome. Unlike its E.coli counterpart Xth, mammalian APE1 has a unique N-terminal domain and possesses both DNA damage repair and transcriptional regulatory functions. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the cellular and molecular mechanisms that alter APE1 functions during tumorigenesis are largely unknown. Here, we show the presence of full-length APE1 and N-terminal truncated isoforms of APE1 in tumor tissue samples of various cancer types. However, primary tumor tissue has higher levels of acetylated APE1 (AcAPE1) as well as full-length APE1 compared to adjacent non-tumor tissue. We found that APE1 is proteolytically cleaved by an unknown serine protease at its N-terminus following residue lysine (Lys) Lys6 and/or Lys7 and after Lys27 and Lys31 or Lys32. Acetylation of these Lys residues in APE1 prevents this proteolysis. The N-terminal domain of APE1 and its acetylation are required for modulation of the expression of hundreds of genes. Importantly, we found that AcAPE1 is essential for sustained cell proliferation. Together, our study demonstrates that increased acetylation levels of APE1 in tumor cells inhibit the limited N-terminal proteolysis of APE1 and thereby maintain the functions of APE1 to promote tumor cells' sustained proliferation and survival.

Asunto(s)

Proliferación Celular/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias/metabolismo , Neoplasias/patología , Acetilación , Humanos , Proteolisis , Células Tumorales Cultivadas