RESUMO
Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression that contribute to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells (ESC). In this study we interrogated three well-established mammary cell lines to model epigenetic programming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co-residing at the same gene promoter, in the MCF7 (ESR/PGR+) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in ESC. We validated the biological relevance of this "oncofetal epigenetic" signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment-resistant cancers. J. Cell. Physiol. 231: 2474-2481, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Epigênese Genética , Histonas/metabolismo , Lisina/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Genes Neoplásicos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-TraducionalRESUMO
The Runx1 transcription factor, known for its essential role in normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx1 increases concomitantly with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant downregulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mice and in human breast cancer cell lines MCF7 and triple-negative MDA-MB-231 that represent early- and late-stage diseases, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3' untranslated region (3'UTR) and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion, while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late-stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação para Baixo/genética , MicroRNAs/genética , Neoplasias de Mama Triplo Negativas/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Camundongos , Fenótipo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2. These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter.
Assuntos
Cromatina/química , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Sintenia , Fatores de Transcrição/metabolismoRESUMO
Long suspected, recently recognized, and increasingly studied, non protein-coding RNAs (ncRNAs) are emerging as key drivers of biological control and pathology. Since their discovery in 1993, microRNAs (miRNAs) have been the subject of intense research focus and investigations have revealed striking findings, establishing that these molecules can exert a substantial level of biological control in numerous tissues. More recently, long ncRNAs (lncRNAs), the lesser-studied siblings of miRNA, have been suggested to have a similar robust role in developmental and adult tissue regulation. Mesenchymal stem cells (MSCs) are an important source of multipotent cells for normal and therapeutic tissue repair. Much is known about the critical role of miRNAs in biogenesis and differentiation of MSCs however; recent studies have suggested lncRNAs may play an equally important role in the regulation of these cells. Here we highlight the role of lncRNAs in the regulation of mesenchymal stem cell lineages including adipocytes, chondrocytes, myoblasts, and osteoblasts. In addition, the potential for these noncoding RNAs to be used as biomarkers for disease or therapeutic targets is also discussed.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Adipócitos/citologia , Adipócitos/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Humanos , Mioblastos/citologia , Mioblastos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
BACKGROUND: Many leukemias result from chromosomal rearrangements. The t(8;21) chromosomal translocation produces AML1-ETO, an oncogenic fusion protein that compromises the function of AML1, a transcription factor critical for myeloid cell differentiation. Because of the pressing need for new therapies in the treatment of acute myleoid leukemia, we investigated the genome-wide occupancy of AML1-ETO in leukemic cells to discover novel regulatory mechanisms involving AML-ETO bound genes. RESULTS: We report the co-localization of AML1-ETO with the N-CoR co-repressor to be primarily on genomic regions distal to transcriptional start sites (TSSs). These regions exhibit over-representation of the motif for PU.1, a key hematopoietic regulator and member of the ETS family of transcription factors. A significant discovery of our study is that genes co-occupied by AML1-ETO and N-CoR (e.g., TYROBP and LAPTM5) are associated with the leukemic phenotype, as determined by analyses of gene ontology and by the observation that these genes are predominantly up-regulated upon AML1-ETO depletion. In contrast, the AML1-ETO/p300 gene network is less responsive to AML1-ETO depletion and less associated with the differentiation block characteristic of leukemic cells. Furthermore, a substantial fraction of AML1-ETO/p300 co-localization occurs near TSSs in promoter regions associated with transcriptionally active loci. CONCLUSIONS: Our findings establish a novel and dominant t(8;21) AML leukemia signature characterized by occupancy of AML1-ETO/N-CoR at promoter-distal genomic regions enriched in motifs for myeloid differentiation factors, thus providing mechanistic insight into the leukemic phenotype.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Genoma Humano , Leucemia Mieloide Aguda/genética , Correpressor 1 de Receptor Nuclear/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Correpressor 1 de Receptor Nuclear/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Análise de Sequência de DNARESUMO
Skeletal metastasis is a serious complication of many primary cancers. A common feature of tumor cells that metastasize to the bone marrow microenvironment is that they initiate a cascade of events, recruiting and presumably/potentially altering the phenotype of bone marrow mesenchymal stromal cells (MSC) to produce an environment that allows for tumor growth and in some cases, drug-resistant dormancy of latent cancer cells. Consequently the MSC population can contribute to metastatic disease through several distinct mechanisms by differentiating into cancer-associated fibroblasts (CAFs). Understanding the expression and epigenetic changes that occur as normal MSCs become associated with metastatic tumors would reveal possible therapeutic targets for treating skeletal metastasis.
Assuntos
Neoplasias Ósseas/genética , Células-Tronco Mesenquimais/metabolismo , Neoplasias/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Diferenciação Celular/genética , Fibroblastos/metabolismo , Humanos , Células-Tronco Mesenquimais/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Epigenetic regulation utilizes different mechanisms to convey heritable traits to progeny cells that are independent of DNA sequence, including DNA silencing, post-translational modifications of histone proteins, and the post-transcriptional modulation of RNA transcript levels by non-coding RNAs. Although long non-coding RNAs have recently emerged as important regulators of gene imprinting, their functions during osteogenesis are as yet unexplored. In contrast, microRNAs (miRNAs) are well characterized for their control of osteogenic and osteoclastic pathways; thus, further defining how gene regulatory networks essential for skeleton functions are coordinated and finely tuned through the activities of miRNAs. Roles of miRNAs are constantly expanding as new studies uncover associations with skeletal disorders. The distinct functions of epigenetic regulators and evidence for integrating their activities to control normal bone gene expression and bone disease will be presented. In addition, potential for using "signature miRNAs" to identify, manage, and therapeutically treat osteosarcoma will be discussed in this review.
Assuntos
Doenças Ósseas/fisiopatologia , Osso e Ossos/fisiologia , Epigênese Genética/fisiologia , Homeostase/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Ósseas/fisiopatologia , Humanos , MicroRNAs/fisiologia , Osteoblastos/fisiologia , Osteossarcoma/fisiopatologiaRESUMO
Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.
Assuntos
Osteogênese , Fatores de Transcrição , Osteogênese/genética , Fatores de Transcrição/metabolismo , Lisina/metabolismo , Proteínas Nucleares/genética , Diferenciação Celular/genética , Epigênese Genética , Osteoblastos/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
MicroRNAs (miRNAs) negatively and post-transcriptionally regulate expression of multiple target genes to support anabolic pathways for bone formation. Here, we show that miR-218 is induced during osteoblast differentiation and has potent osteogenic properties. miR-218 promotes commitment and differentiation of bone marrow stromal cells by activating a positive Wnt signaling loop. In a feed forward mechanism, miR-218 stimulates the Wnt pathway by down-regulating three Wnt signaling inhibitors during the process of osteogenesis: Sclerostin (SOST), Dickkopf2 (DKK2), and secreted frizzled-related protein2 (SFRP2). In turn, miR-218 expression is up-regulated in response to stimulated Wnt signaling and functionally drives Wnt-related transcription and osteoblast differentiation, thereby creating a positive feedback loop. Furthermore, in metastatic breast cancer cells but not in normal mammary epithelial cells, miR-218 enhances Wnt activity and abnormal expression of osteoblastic genes (osteomimicry) that contribute to homing and growth of cells metastatic to bone. Thus, miR-218/Wnt signaling circuit amplifies both the osteoblast phenotype and osteomimicry-related tumor activity.
Assuntos
Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , MicroRNAs/biossíntese , Osteoblastos/metabolismo , RNA Neoplásico/biossíntese , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Glicoproteínas/genética , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , MicroRNAs/genética , Metástase Neoplásica , Osteoblastos/patologia , RNA Neoplásico/genéticaRESUMO
Induced osteogenesis includes a program of microRNAs (miRs) to repress the translation of genes that act as inhibitors of bone formation. How expression of bone-related miRs is regulated remains a compelling question. Here we report that Runx2, a transcription factor essential for osteoblastogenesis, negatively regulates expression of the miR cluster 23aâ¼27aâ¼24-2. Overexpression, reporter, and chromatin immunoprecipitation assays established the presence of a functional Runx binding element that represses expression of these miRs. Consistent with this finding, exogenous expression of each of the miRs suppressed osteoblast differentiation, whereas antagomirs increased bone marker expression. The biological significance of Runx2 repression of this miR cluster is that each miR directly targets the 3' UTR of SATB2, which is known to synergize with Runx2 to facilitate bone formation. The findings suggest Runx2-negative regulation of multiple miRs by a feed-forward mechanism to cause derepression of SATB2 to promote differentiation. We find also that miR-23a represses Runx2 in the terminally differentiated osteocyte, representing a feedback mechanism to attenuate osteoblast maturation. We provide direct evidence for an interdependent relationship among transcriptional inhibition of the miR cluster by Runx2, translational repression of Runx2 and of SATB2 by the cluster miRs during progression of osteoblast differentiation. Furthermore, miR cluster gain of function (i.e., inhibition of osteogenesis) is rescued by the exogenous expression of SATB2. Taken together, we have established a regulatory network with a central role for the miR cluster 23aâ¼27aâ¼24-2 in both progression and maintenance of the osteocyte phenotype.
Assuntos
Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Redes Reguladoras de Genes , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/genética , Família Multigênica , Osteoblastos/citologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação para Baixo/genética , Retroalimentação Fisiológica , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Ligação Proteica , Ratos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genéticaRESUMO
The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.
Assuntos
Neoplasias da Mama , Células-Tronco Mesenquimais , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Transcriptoma , Microambiente Tumoral/genética , Humanos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genéticaRESUMO
The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.
Assuntos
Células-Tronco Mesenquimais , RNA Longo não Codificante , Humanos , Osteogênese/genética , Autorrenovação Celular , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Diferenciação Celular/genéticaRESUMO
Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.
RESUMO
The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants.
Assuntos
Osteoclastos , Titânio , Histonas/metabolismo , Metilação , Osteoblastos , Osteoclastos/metabolismo , Propriedades de Superfície , Titânio/farmacologiaRESUMO
Selective estrogen receptor modulators (SERMs), including the SERM/SERD bazedoxifene (BZA), are used to treat postmenopausal osteoporosis and may reduce breast cancer (BCa) risk. One of the most persistent unresolved questions regarding menopausal hormone therapy is compromised control of proliferation and phenotype because of short- or long-term administration of mixed-function estrogen receptor (ER) ligands. To gain insight into epigenetic effectors of the transcriptomes of hormone and BZA-treated BCa cells, we evaluated a panel of histone modifications. The impact of short-term hormone treatment and BZA on gene expression and genome-wide epigenetic profiles was examined in ERαneg mammary epithelial cells (MCF10A) and ERα+ luminal breast cancer cells (MCF7). We tested individual components and combinations of 17ß-estradiol (E2), estrogen compounds (EC10) and BZA. RNA-seq for gene expression and ChIP-seq for active (H3K4me3, H3K4ac, H3K27ac) and repressive (H3K27me3) histone modifications were performed. Our results show that the combination of BZA with E2 or EC10 reduces estrogen-mediated patterns of histone modifications and gene expression in MCF-7ERα+ cells. In contrast, BZA has minimal effects on these parameters in MCF10A mammary epithelial cells. BZA-induced changes in histone modifications in MCF7 cells are characterized by altered H3K4ac patterns, with changes at distal enhancers of ERα-target genes and at promoters of non-ERα bound proliferation-related genes. Notably, the ERα target gene GREB1 is the most sensitive to BZA treatment. Our findings provide direct mechanistic-based evidence that BZA induces epigenetic changes in E2 and EC10 mediated control of ERα regulatory programs to target distinctive proliferation gene pathways that restrain the potential for breast cancer development.
Assuntos
Neoplasias da Mama , Estrogênios Conjugados (USP) , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Epigênese Genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Estrogênios Conjugados (USP)/farmacologia , Feminino , Humanos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , TranscriptomaRESUMO
Bone formation requires osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) and lineage progression of committed osteoblast precursors. Osteogenic phenotype commitment is epigenetically controlled by genomic (chromatin) and non-genomic (non-coding RNA) mechanisms. Control of osteogenesis by long non-coding RNAs remains a largely unexplored molecular frontier. Here, we performed comprehensive transcriptome analysis at early stages of osteogenic cell fate determination in human MSCs, focusing on expression of lncRNAs. We identified a chromatin-bound lncRNA (MIR181A1HG) that is highly expressed in self-renewing MSCs. MIR181A1HG is down-regulated when MSCs become osteogenic lineage committed and is retained during adipogenic differentiation, suggesting lineage-related molecular functions. Consistent with a key role in human MSC proliferation and survival, we demonstrate that knockdown of MIR181A1HG in the absence of osteogenic stimuli impedes cell cycle progression. Loss of MIR181A1HG enhances differentiation into osteo-chondroprogenitors that produce multiple extracellular matrix proteins. RNA-seq analysis shows that loss of chromatin-bound MIR181A1HG alters expression and BMP2 responsiveness of skeletal gene networks (e.g., SOX5 and DLX5). We propose that MIR181A1HG is a novel epigenetic regulator of early stages of mesenchymal lineage commitment towards osteo-chondroprogenitors. This discovery permits consideration of MIR181A1HG and its associated regulatory pathways as targets for promoting new bone formation in skeletal disorders.
Assuntos
Osteogênese , RNA Longo não Codificante , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Osteoblastos/metabolismo , Osteogênese/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.
Assuntos
Calcificação Fisiológica/genética , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Complexos Multiproteicos/metabolismo , Osteogênese/genética , Fatores de Transcrição/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Células HEK293 , Proteínas Homeobox A10 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Complexos Multiproteicos/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp7 , Células Estromais/metabolismo , Fatores de Transcrição/genéticaRESUMO
We have developed a novel Ribonucleoprotein Immunoprecipitation (RNP-IP) method to isolate miR-RISC complexes, associated microRNAs and target mRNAs. Our method characterizes mRNAs present in immunoprecipitates containing miR-RISC complexes that were obtained using GW182 and AGO2 antibodies. MicroRNA bound transcripts were reverse transcribed and amplified using seed sequence and 3'UTR derived primers. This flexible IP-based assay is a straightforward method to identify miRs participating in gene regulation and their cognate mRNAs in real time.
Assuntos
MicroRNAs/metabolismo , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas , Células 3T3 , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Imunoprecipitação , CamundongosRESUMO
Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF.
Assuntos
Neoplasias da Mama/patologia , Adesões Focais , Neoplasias da Próstata/patologia , Sialoglicoproteínas/fisiologia , Transdução de Sinais , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Sialoproteína de Ligação à Integrina , Masculino , Metástase Neoplásica , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways.