RESUMO
Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.
Assuntos
Farmacogenética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Proto-Oncogênicas , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Criança , Resistencia a Medicamentos Antineoplásicos/genética , Variação Genética , Linhagem Celular Tumoral , Vincristina/uso terapêutico , Vincristina/farmacologia , Polimorfismo de Nucleotídeo Único , Alelos , Cromatina/metabolismo , Cromatina/genética , Transativadores/genética , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacosRESUMO
B cell lineage acute lymphoblastic leukemia (B-ALL) is composed of diverse molecular subtypes, and while transcriptional and DNA methylation profiling has been extensively examined, the chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq in primary B-ALL cells from 156 patients spanning ten molecular subtypes and present this dataset as a resource. Differential chromatin accessibility and transcription factor (TF) footprint profiling were employed and identified B-ALL cell of origin, TF-target gene interactions enriched in B-ALL, and key TFs associated with accessible chromatin sites preferentially active in B-ALL. We further identified over 20% of accessible chromatin sites exhibiting strong subtype enrichment and candidate TFs that maintain subtype-specific chromatin architectures. Over 9,000 genetic variants were uncovered, contributing to variability in chromatin accessibility among patient samples. Our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants that promote unique gene-regulatory networks.
Assuntos
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Via de Sinalização Wnt , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMO
Although acute lymphoblastic leukemia (ALL) is the most common childhood cancer, there is limited understanding of the contribution of inherited genetic variation on inter-individual differences in chemotherapy response. Defining genetic factors impacting therapy failure can help better predict response and identify drug resistance mechanisms. We therefore mapped inherited noncoding variants associated with chemotherapeutic drug resistance and/or treatment outcome to ALL cis-regulatory elements and investigated their gene regulatory potential and genomic connectivity using massively parallel reporter assays and promoter capture Hi-C, respectively. We identified 53 variants with reproducible allele-specific effects on transcription and high-confidence gene targets. Subsequent functional interrogation of the top variant (rs1247117) determined that it disrupted a PU.1 consensus motif and PU.1 binding affinity. Importantly, deletion of the genomic interval containing rs1247117 sensitized ALL cells to vincristine. Together, these data demonstrate that noncoding regulatory variation associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to chemotherapeutic agents in ALL.
RESUMO
B-cell lineage acute lymphoblastic leukemia (B-ALL) is comprised of diverse molecular subtypes and while transcriptional and DNA methylation profiling of B-ALL subtypes has been extensively examined, the accompanying chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq for 10 B-ALL molecular subtypes in primary ALL cells from 154 patients. Comparisons with B-cell progenitors identified candidate B-ALL cell-of-origin and AP-1-associated cis-regulatory rewiring in B-ALL. Cis-regulatory rewiring promoted B-ALL-specific gene regulatory networks impacting oncogenic signaling pathways that perturb normal B-cell development. We also identified that over 20% of B-ALL accessible chromatin sites exhibit strong subtype enrichment, with transcription factor (TF) footprint profiling identifying candidate TFs that maintain subtype-specific chromatin architectures. Over 9000 inherited genetic variants were further uncovered that contribute to variability in chromatin accessibility among individual patient samples. Overall, our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants which promote unique gene regulatory networks that contribute to transcriptional differences among B-ALL subtypes.
RESUMO
Glucocorticoids (GCs; i.e., steroids) are important chemotherapeutic agents in the treatment of B-cell precursor acute lymphoblastic leukemia (B-ALL) and de novo GC resistance predicts relapse and poor clinical outcome in patients. Glucocorticoids induce B-ALL cell apoptosis through activation of glucocorticoid receptor (GR), a ligand-induced nuclear receptor transcription factor (TF). We previously identified disruptions to glucocorticoid receptor (GR)-bound cis -regulatory elements controlling TLE1 expression in GC-resistant primary B-ALL cells from patients. TLE1 is a GC-response gene up-regulated by steroids and functions as a canonical Wnt signaling repressor. To better understand the mechanistic relationship between GC signaling and canonical Wnt signaling, we performed diverse functional analyses that identified extensive crosstalk and mutual antagonism between these two signaling pathways in B-ALL. We determined that crosstalk and antagonism was driven by the binding of GR and the canonical Wnt signaling TFs LEF1 and TCF7L2 to overlapping sets of cis -regulatory elements associated with genes impacting cell death and cell proliferation, and was further accompanied by overlapping and opposing transcriptional programs. Our data additionally suggest that cis -regulatory disruptions at TLE1 are linked to GC resistance through a dampening of the GC response and GC-mediated apoptosis via enhanced canonical Wnt signaling. As a result of the extensive genomic and gene regulatory connectivity between these two signaling pathways, our data supports the importance of canonical Wnt signaling in mediating GC resistance in B-ALL.
RESUMO
FLT3 is an attractive therapeutic target in acute lymphoblastic leukemia (ALL) but the mechanism for its activation in this cancer is incompletely understood. Profiling global gene expression in large ALL cohorts, we identify over-expression of FLT3 in ZNF384-rearranged ALL, consistently across cases harboring different fusion partners with ZNF384. Mechanistically, we discover an intergenic enhancer element at the FLT3 locus that is exclusively activated in ZNF384-rearranged ALL, with the enhancer-promoter looping directly mediated by the fusion protein. There is also a global enrichment of active enhancers within ZNF384 binding sites across the genome in ZNF384-rearranged ALL cells. Downregulation of ZNF384 blunts FLT3 activation and decreases ALL cell sensitivity to FLT3 inhibitor gilteritinib in vitro. In patient-derived xenograft models of ZNF384-rearranged ALL, gilteritinib exhibits significant anti-leukemia efficacy as a monotherapy in vivo. Collectively, our results provide insights into FLT3 regulation in ALL and point to potential genomics-guided targeted therapy for this patient population.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Transativadores , Compostos de Anilina , Epigênese Genética , Fusão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirazinas , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Glucocorticoids (GCs) are a mainstay of contemporary, multidrug chemotherapy in the treatment of childhood acute lymphoblastic leukemia (ALL), and resistance to GCs remains a major clinical concern. Resistance to GCs is predictive of ALL relapse and poor clinical outcome, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in resistance. To address this, we mapped the gene regulatory response to GCs in two ALL cell lines using functional genomics and high-throughput reporter assays and identified thousands of GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and a depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. By integrating our GC response maps with genetic and epigenetic datasets in primary ALL cells from patients, we further uncovered cis-regulatory disruptions at GC-responsive genes that impact GC resistance in childhood ALL. Overall, these data indicate that GCs initiate pervasive effects on the leukemia epigenome, and that alterations to the GC gene regulatory network contribute to GC resistance.
Assuntos
Glucocorticoides , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linhagem Celular Tumoral , Cromatina , Resistencia a Medicamentos Antineoplásicos/genética , Epigenômica , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Glucocorticoides/genética , Esteroides , Fator de Transcrição AP-1/genéticaRESUMO
Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.
Assuntos
Aspartato-Amônia Ligase , Leucemia-Linfoma Linfoblástico de Células Precursoras , Fator 4 Ativador da Transcrição/genética , Aminoácidos/uso terapêutico , Asparaginase/farmacologia , Asparaginase/uso terapêutico , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Linhagem Celular Tumoral , Criança , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
BACKGROUND: There is growing evidence for the inherited basis of susceptibility to childhood acute lymphoblastic leukemia (ALL). Genome-wide association studies have identified non-coding ALL risk variants at the ARID5B gene locus, but their exact functional effects and the molecular mechanism linking ARID5B to B-cell ALL leukemogenesis remain largely unknown. METHODS: We performed targeted sequencing of ARID5B in germline DNA of 5008 children with ALL. Variants were evaluated for association with ALL susceptibility using 3644 patients from the UK10K cohort as non-ALL controls, under an additive model. Cis-regulatory elements in ARID5B were systematically identified using dCas9-KRAB-mediated enhancer interference system enhancer screen in ALL cells. Disruption of transcription factor binding by ARID5B variant was predicted informatically and then confirmed using chromatin immunoprecipitation and coimmunoprecipitation. ARID5B variant association with hematological traits was examined using UK Biobank dataset. All statistical tests were 2-sided. RESULTS: We identified 54 common variants in ARID5B statistically significantly associated with leukemia risk, all of which were noncoding. Six cis-regulatory elements at the ARID5B locus were discovered using CRISPR-based high-throughput enhancer screening. Strikingly, the top ALL risk variant (rs7090445, P = 5.57 × 10-45) is located precisely within the strongest enhancer element, which is also distally tethered to the ARID5B promoter. The variant allele disrupts the MEF2C binding motif sequence, resulting in reduced MEF2C affinity and decreased local chromosome accessibility. MEF2C influences ARID5B expression in ALL, likely via a transcription factor complex with RUNX1. Using the UK Biobank dataset (n = 349â861), we showed that rs7090445 was also associated with lymphocyte percentage and count in the general population (P = 8.6 × 10-22 and 2.1 × 10-18, respectively). CONCLUSIONS: Our results indicate that ALL risk variants in ARID5B function by modulating cis-regulatory elements at this locus.
Assuntos
Predisposição Genética para Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras , Fatores de Transcrição/metabolismo , Criança , Proteínas de Ligação a DNA/genética , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Transcrição/genéticaRESUMO
Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy comprised of molecular subtypes largely characterized by aneuploidy or recurring chromosomal rearrangements. Despite extensive information on the ALL transcriptome and methylome, there is limited understanding of the ALL chromatin landscape. We therefore mapped accessible chromatin in 24 primary ALL cell biospecimens comprising three common molecular subtypes (DUX4/ERG, ETV6-RUNX1 and hyperdiploid) from patients treated at St. Jude Children's Research Hospital. Our findings highlight extensive chromatin reprogramming in ALL, including the identification ALL subtype-specific chromatin landscapes that are additionally modulated by genetic variation. Chromatin accessibility differences between ALL and normal B-cells implicate the activation of B-cell repressed chromatin domains and detail the disruption of normal B-cell development in ALL. Among ALL subtypes, we uncovered roles for basic helix-loop-helix, homeodomain and activator protein 1 transcription factors in promoting subtype-specific chromatin accessibility and distinct gene regulatory networks. In addition to chromatin subtype-specificity, we further identified over 3500 DNA sequence variants that alter the ALL chromatin landscape and contribute to inter-individual variability in chromatin accessibility. Collectively, our data suggest that subtype-specific chromatin landscapes and gene regulatory networks impact ALL biology and contribute to transcriptomic differences among ALL subtypes.
Assuntos
Cromatina/genética , Aberrações Cromossômicas , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Leucemia-Linfoma Linfoblástico de Células Precursoras/classificação , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Epigenômica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Fatores de Transcrição/genética , TranscriptomaRESUMO
Acute lymphoblastic leukemia (ALL) is the most common cancer in children. TCF3-PBX1 fusion defines a common molecular subtype of ALL with unique clinical features, but the molecular basis of its inherited susceptibility is unknown. In a genome-wide association study of 1494 ALL cases and 2057 non-ALL controls, we identified a germline risk locus located in an intergenic region between BCL11A and PAPOLG: rs2665658, P = 1.88 × 10-8 for TCF3-PBX1 ALL vs non-ALL, and P = 1.70 × 10-8 for TCF3-PBX1 ALL vs other-ALL. The lead variant was validated in a replication cohort, and conditional analyses pointed to a single causal variant with subtype-specific effect. The risk variant is located in a regulatory DNA element uniquely activated in ALL cells with the TCF3-PBX1 fusion and may distally modulate the transcription of the adjacent gene REL. Our results expand the understanding of subtype-specific ALL susceptibility and highlight plausible interplay between germline variants and somatic genomic abnormalities in ALL pathogenesis.
Assuntos
Estudo de Associação Genômica Ampla , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Doença Aguda , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Predisposição Genética para Doença , Humanos , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.
Assuntos
MicroRNAs , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Genômica , Glucocorticoides/farmacologia , Humanos , Camundongos , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Neoplasias , Fatores de Transcrição/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Camundongos , Camundongos SCID , Neoplasias/genética , Neoplasias/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcription factors are DNA-binding proteins that have key roles in gene regulation1,2. Genome-wide occupancy maps of transcriptional regulators are important for understanding gene regulation and its effects on diverse biological processes3-6. However, only a minority of the more than 1,600 transcription factors encoded in the human genome has been assayed. Here we present, as part of the ENCODE (Encyclopedia of DNA Elements) project, data and analyses from chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiments using the human HepG2 cell line for 208 chromatin-associated proteins (CAPs). These comprise 171 transcription factors and 37 transcriptional cofactors and chromatin regulator proteins, and represent nearly one-quarter of CAPs expressed in HepG2 cells. The binding profiles of these CAPs form major groups associated predominantly with promoters or enhancers, or with both. We confirm and expand the current catalogue of DNA sequence motifs for transcription factors, and describe motifs that correspond to other transcription factors that are co-enriched with the primary ChIP target. For example, FOX family motifs are enriched in ChIP-seq peaks of 37 other CAPs. We show that motif content and occupancy patterns can distinguish between promoters and enhancers. This catalogue reveals high-occupancy target regions at which many CAPs associate, although each contains motifs for only a minority of the numerous associated transcription factors. These analyses provide a more complete overview of the gene regulatory networks that define this cell type, and demonstrate the usefulness of the large-scale production efforts of the ENCODE Consortium.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos/genética , Células Hep G2 , Humanos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
The basic leucine zipper transcription factor activating transcription factor-like (Batf) contributes to transcriptional programming of multiple effector T cells and is required for T helper 17 (Th17) and T follicular helper (Tfh) cell development. Here, we examine mechanisms by which Batf initiates gene transcription in developing effector CD4 T cells. We find that, in addition to its pioneering function, Batf controls developmentally regulated recruitment of the architectural factor Ctcf to promote chromatin looping that is associated with lineage-specific gene transcription. The chromatin-organizing actions of Batf are largely dependent on Ets1, which appears to be indispensable for the Batf-dependent recruitment of Ctcf. Moreover, most of the Batf-dependent sites to which Ctcf is recruited lie outside of activating protein-1-interferon regulatory factor (Ap-1-Irf) composite elements (AICEs), indicating that direct involvement of Batf-Irf complexes is not required. These results identify a cooperative role for Batf, Ets1, and Ctcf in chromatin reorganization that underpins the transcriptional programming of effector T cells.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fator de Ligação a CCCTC/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Cromatina/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Genoma , Humanos , Fatores Reguladores de Interferon/metabolismo , Interleucinas/metabolismo , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Ligação Proteica , Regulação para Cima/genéticaRESUMO
Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as "triple negative breast cancer" (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis.
Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Fator de Transcrição STAT3/genética , Transcriptoma , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Ligação Proteica , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transfecção , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking. METHODS: To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns. RESULTS: Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points. CONCLUSIONS: Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Receptores X do Fígado/genética , PPAR gama/genética , Proliferação de Células , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Metabolismo Energético/genética , Glutationa/metabolismo , Células HT29 , Humanos , Receptores X do Fígado/metabolismo , Estresse Oxidativo/genética , PPAR gama/metabolismo , Transdução de SinaisRESUMO
Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type-specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites.