ABSTRACT
The RUNX2 transcription factor was discovered as an essential transcriptional regulator for commitment to osteoblast lineage cells and bone formation. Expression of RUNX2 in other tissues, such as breast, prostate, and lung, has been linked to oncogenesis, cancer progression, and metastasis. In this study, we sought to determine the extent of RUNX2 involvement in other tumors using a pan-cancer analysis strategy. We correlated RUNX2 expression and clinical-pathological parameters in human cancers by interrogating publicly available multiparameter clinical data. Our analysis demonstrated that altered RUNX2 expression or function is associated with several cancer types from different tissues. We identified three tumor types associated with increased RUNX2 expression and four other tumor types associated with decreased RUNX2 expression. Our pan-cancer analysis for RUNX2 revealed numerous other discoveries for RUNX2 regulation of different cancers identified in each of the pan-cancer databases. Both up and down regulation of RUNX2 was observed during progression of specific types of cancers in promoting the distinct types of cancers.
Subject(s)
Core Binding Factor Alpha 1 Subunit , Gene Expression Regulation, Neoplastic , Neoplasms , Humans , Core Binding Factor Alpha 1 Subunit/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Prognosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolismABSTRACT
Objective criteria are required for prostate cancer (PCa) risk assessment, treatment decisions, evaluation of therapy, and initial indications of recurrence. Circulating microRNAs were utilized as biomarkers to distinguish PCa patients from cancer-free subjects or those encountering benign prostate hyperplasia. A panel of 60 microRNAs was developed with established roles in PCa initiation, progression, metastasis, and recurrence. Utilizing the FirePlex® platform for microRNA analysis, we demonstrated the efficacy and reproducibility of a rapid, high-throughput, serum-based assay for PCa biomarkers that circumvents the requirement for extraction and fractionation of patient specimens supporting feasibility for expanded clinical research and diagnostic applications.
Subject(s)
Biomarkers, Tumor , MicroRNAs , Prostatic Neoplasms , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/diagnosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , MicroRNAs/genetics , MicroRNAs/blood , Risk Assessment/methodsABSTRACT
Ductal carcinoma in situ (DCIS) constitutes an array of morphologically recognized intraductal neoplasms in the mammary ductal tree defined by an increased risk for subsequent invasive carcinomas at or near the site of biopsy detection. However, only 15-45% of untreated DCIS cases progress to invasive cancer, so understanding mechanisms that prevent progression is key to avoid overtreatment and provides a basis for alternative therapies and prevention. This study was designed to characterize the tumor microenvironment and molecular profile of high-risk DCIS that grew to a large size but remained as DCIS. All patients had DCIS lesions >5cm in size with at least one additional high-risk feature: young age (<45 years), high nuclear grade, hormone receptor negativity, HER2 positivity, the presence of comedonecrosis, or a palpable mass. The tumor immune microenvironment was characterized using multiplex immunofluorescence to identify immune cells and their spatial relationships within the ducts and stroma. Gene copy number analysis and whole exome DNA sequencing identified the mutational burden and driver mutations, and quantitative whole-transcriptome/gene expression analyses were performed. There was no association between the percent of the DCIS genome characterized by copy number variants (CNAs) and recurrence events (DCIS or invasive). Mutations, especially missense mutations, in the breast cancer driver genes PIK3CA and TP53 were common in this high-risk DCIS cohort (47% of evaluated lesions). Tumor infiltrating lymphocyte (TIL) density was higher in DCIS lesions with TP53 mutations (p=0.0079) compared to wildtype lesions, but not in lesions with PIK3CA mutations (p=0.44). Immune infiltrates were negatively associated with hormone receptor status and positively associated with HER2 expression. High levels of CD3+CD8- T cells were associated with good outcomes with respect to any subsequent recurrence (DCIS or invasive cancer), whereas high levels of CD3+Foxp3+ Treg cells were associated with poor outcomes. Spatial proximity analyses of immune cells and tumor cells demonstrated that close proximity of T cells with tumor cells was associated with good outcomes with respect to any recurrence as well as invasive recurrences. Interestingly, we found that myoepithelial continuity (distance between myoepithelial cells surrounding the involved ducts) was significantly lower in DCIS lesions compared to normal tissue (p=0.0002) or to atypical ductal hyperplasia (p=0.011). Gene set enrichment analysis identified several immune pathways associated with low myoepithelial continuity and a low myoepithelial continuity score was associated with better outcomes, suggesting that gaps in the myoepithelial layer may allow access/interactions between immune infiltrates and tumor cells. Our study demonstrates the immune microenvironment of DCIS, in particular the spatial proximity of tumor cells and T cells, and myoepithelial continuity are important determinants for progression of disease.
ABSTRACT
The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.
Subject(s)
Breast Neoplasms , NAD , Mice , Animals , Humans , Female , NAD/metabolism , Flavin-Adenine Dinucleotide/metabolism , Breast Neoplasms/diagnostic imaging , Lactic AcidABSTRACT
Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.
Subject(s)
RNA, Long Noncoding , Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Cell Survival , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Cell Proliferation/genetics , Bromodomain Containing Proteins , Cell Cycle Proteins/geneticsABSTRACT
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.
Subject(s)
Mesenchymal Stem Cells , RNA, Long Noncoding , Humans , Osteogenesis/genetics , Cell Self Renewal , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Cell Differentiation/geneticsABSTRACT
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 cell growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2,000 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.
ABSTRACT
TGF-ß signaling is a vital regulator for maintaining articular cartilage homeostasis. Runx transcription factors, downstream targets of TGF-ß signaling, have been studied in the context of osteoarthritis (OA). Although Runx partner core binding factor ß (Cbfß) is known to play a pivotal role in chondrocyte and osteoblast differentiation, the role of Cbfß in maintaining articular cartilage integrity remains obscure. This study investigated Cbfß as a novel anabolic modulator of TGF-ß signaling and determined its role in articular cartilage homeostasis. Cbfß significantly decreased in aged mouse articular cartilage and human OA cartilage. Articular chondrocyte-specific Cbfb-deficient mice (Cbfbâ³ac/â³ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfbâ³ac/â³ac mice showed enhanced OA progression under the surgically induced OA model in mice. Mechanistically, forced expression of Cbfß rescued Type II collagen (Col2α1) and Runx1 expression in Cbfß-deficient chondrocytes. TGF-ß1-mediated Col2α1 expression failed despite the p-Smad3 activation under TGF-ß1 treatment in Cbfß-deficient chondrocytes. Cbfß protected Runx1 from proteasomal degradation through Cbfß/Runx1 complex formation. These results indicate that Cbfß is a novel anabolic regulator for cartilage homeostasis, suggesting that Cbfß could protect OA development by maintaining the integrity of the TGF-ß signaling pathway in articular cartilage.
Subject(s)
Cartilage, Articular , Osteoarthritis , Mice , Animals , Humans , Cartilage, Articular/metabolism , Transforming Growth Factor beta1/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factor beta Subunit/metabolism , Signal Transduction , Osteoarthritis/metabolism , HomeostasisABSTRACT
Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.
Subject(s)
Chromatin , Histones , Histones/metabolism , Cyclin E/genetics , Cyclin E/metabolism , Nuclear Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle/genetics , Epigenesis, GeneticABSTRACT
Human Histone Locus Bodies (HLBs) are nuclear subdomains comprised of clustered histone genes that are coordinately regulated throughout the cell cycle. We addressed temporal-spatial higher-order genome organization for time-dependent chromatin remodeling at HLBs that supports control of cell proliferation. Proximity distances of specific genomic contacts within histone gene clusters exhibit subtle changes during the G1 phase in MCF10 breast cancer progression model cell lines. This approach directly demonstrates that the two principal histone gene regulatory proteins, HINFP (H4 gene regulator) and NPAT, localize at chromatin loop anchor-points, denoted by CTCF binding, supporting the stringent requirement for histone biosynthesis to package newly replicated DNA as chromatin. We identified a novel enhancer region located â¼ 2 MB distal to histone gene sub-clusters on chromosome 6 that consistently makes genomic contacts with HLB chromatin and is bound by NPAT. During G1 progression the first DNA loops form between one of three histone gene sub-clusters bound by HINFP and the distal enhancer region. Our findings are consistent with a model that the HINFP/NPAT complex controls the formation and dynamic remodeling of higher-order genomic organization of histone gene clusters at HLBs in early to late G1 phase to support transcription of histone mRNAs in S phase.
Subject(s)
Breast Neoplasms , Histones , Humans , Female , Histones/genetics , Histones/metabolism , Chromatin/genetics , Breast Neoplasms/genetics , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Nuclear Bodies , Multigene FamilyABSTRACT
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.
Subject(s)
Breast Neoplasms , Mesenchymal Stem Cells , Cell Line, Tumor , Cell Proliferation , Epithelial-Mesenchymal Transition/genetics , Mesenchymal Stem Cells/metabolism , Signal Transduction , Stromal Cells/metabolism , Transcriptome , Tumor Microenvironment/genetics , Humans , Breast Neoplasms/diagnosis , Breast Neoplasms/geneticsABSTRACT
BACKGROUND: Epigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types. RESULTS: We developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq. CONCLUSION: peaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.
Subject(s)
Chromatin Immunoprecipitation Sequencing , High-Throughput Nucleotide Sequencing , Chromatin Immunoprecipitation Sequencing/methods , Sequence Analysis, DNA/methods , Gene LibraryABSTRACT
The cell cycle is governed by stringent epigenetic mechanisms that, in response to intrinsic and extrinsic regulatory cues, support fidelity of DNA replication and cell division. We will focus on (1) the complex and interdependent processes that are obligatory for control of proliferation and compromised in cancer, (2) epigenetic and topological domains that are associated with distinct phases of the cell cycle that may be altered in cancer initiation and progression, and (3) the requirement for mitotic bookmarking to maintain intranuclear localization of transcriptional regulatory machinery to reinforce cell identity throughout the cell cycle to prevent malignant transformation.
Subject(s)
Epigenesis, Genetic , Neoplasms , Humans , Cell Cycle/genetics , Cell Division , Neoplasms/genetics , Neoplasms/pathology , Chromatin , Gene Expression RegulationABSTRACT
Epigenetic gene regulatory mechanisms play a central role in the biological control of cell and tissue structure, function, and phenotype. Identification of epigenetic dysregulation in cancer provides mechanistic into tumor initiation and progression and may prove valuable for a variety of clinical applications. We present an overview of epigenetically driven mechanisms that are obligatory for physiological regulation and parameters of epigenetic control that are modified in tumor cells. The interrelationship between nuclear structure and function is not mutually exclusive but synergistic. We explore concepts influencing the maintenance of chromatin structures, including phase separation, recognition signals, factors that mediate enhancer-promoter looping, and insulation and how these are altered during the cell cycle and in cancer. Understanding how these processes are altered in cancer provides a potential for advancing capabilities for the diagnosis and identification of novel therapeutic targets.
Subject(s)
Epigenesis, Genetic , Neoplasms , Humans , Phenotype , Neoplasms/genetics , Neoplasms/pathology , Gene Expression Regulation , ChromatinABSTRACT
Core facilities have a ubiquitous and increasingly valuable presence at research institutions. Although many shared cores were originally created to provide routine services and access to complex and expensive instrumentation for the research community, they are frequently called upon by investigators to design protocols and procedures to help answer complex research questions. For instance, shared microscopy resources are evolving from providing access to and training on complex imaging instruments to developing detailed innovative protocols and experimental strategies, including sample preparation techniques, staining, complex imaging parameters, and high-level image analyses. These approaches require close intellectual collaboration between core staff and research investigators to formulate and coordinate plans for protocol development suited to the research question. Herein, we provide an example of such coordinated collaboration between a shared microscopy facility and a team of scientists and clinician-investigators to approach a complex multiprobe immunostaining, imaging, and image analysis project investigating the tumor microenvironment from human breast cancer samples. Our hope is that this example may be used to convey to institute administrators the critical importance of the intellectual contributions of the scientific staff in core facilities to research endeavors.
Subject(s)
Microscopy , Research Personnel , Academies and Institutes , Health Facilities , Humans , Research DesignABSTRACT
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.
Subject(s)
Breast Neoplasms , Estrogens, Conjugated (USP) , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Epigenesis, Genetic , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens/pharmacology , Estrogens, Conjugated (USP)/pharmacology , Female , Humans , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Selective Estrogen Receptor Modulators/pharmacology , TranscriptomeABSTRACT
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.
Subject(s)
Osteogenesis , RNA, Long Noncoding , Cell Differentiation/genetics , Cell Lineage/genetics , Chromatin/genetics , Chromatin/metabolism , Epigenesis, Genetic , Osteoblasts/metabolism , Osteogenesis/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
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.
Subject(s)
Osteoclasts , Titanium , Histones/metabolism , Methylation , Osteoblasts , Osteoclasts/metabolism , Surface Properties , Titanium/pharmacologyABSTRACT
Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.