A MicroRNA Approach to Evaluating Elevated Prostate Cancer Risk in Cancer-Free Men.

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.

Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

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.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

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.

LINC01638 Sustains Human Mesenchymal Stem Cell Self-Renewal and Competency for Osteogenic Cell Fate.

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.

Cbfß Is a Novel Modulator against Osteoarthritis by Maintaining Articular Cartilage Homeostasis through TGF-ß Signaling.

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 (Cbfb△ac/△ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfb△ac/△ac 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.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

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.

Spatiotemporal higher-order chromatin landscape of human histone gene clusters at histone locus bodies during the cell cycle in breast cancer progression.

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 âˆ¼ 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.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Fidelity of Mechanisms Governing the Cell Cycle.

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.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Cell and Tissue Structure, Function, and Phenotype.

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.

Sustained Morphine Delivery Suppresses Bone Formation and Alters Metabolic and Circulating miRNA Profiles in Male C57BL/6J Mice.

Opioid use is detrimental to bone health, causing both indirect and direct effects on bone turnover. Although the mechanisms of these effects are not entirely clear, recent studies have linked chronic opioid use to alterations in circulating miRNAs. Here, we developed a model of opioid-induced bone loss to understand bone turnover and identify candidate miRNA-mediated regulatory mechanisms. We evaluated the effects of sustained morphine treatment on male and female C57BL/6J mice by treating with vehicle (0.9% saline) or morphine (17 mg/kg) using subcutaneous osmotic minipumps for 25 days. Morphine-treated mice had higher energy expenditure and respiratory quotient, indicating a shift toward carbohydrate metabolism. Micro-computed tomography (µCT) analysis indicated a sex difference in the bone outcome, where male mice treated with morphine had reduced trabecular bone volume fraction (Tb.BV/TV) (15%) and trabecular bone mineral density (BMD) (14%) in the distal femur compared with vehicle. Conversely, bone microarchitecture was not changed in females after morphine treatment. Histomorphometric analysis demonstrated that in males, morphine reduced bone formation rate compared with vehicle, but osteoclast parameters were not different. Furthermore, morphine reduced bone formation marker gene expression in the tibia of males (Bglap and Dmp1). Circulating miRNA profile changes were evident in males, with 14 differentially expressed miRNAs associated with morphine treatment compared with two differentially expressed miRNAs in females. In males, target analysis indicated hypoxia-inducible factor (HIF) signaling pathway was targeted by miR-223-3p and fatty acid metabolism by miR-484, -223-3p, and -328-3p. Consequently, expression of miR-223-3p targets, including Igf1r and Stat3, was lower in morphine-treated bone. In summary, we have established a model where morphine leads to a lower trabecular bone formation in males and identified potential mediating miRNAs. Understanding the sex-specific mechanisms of bone loss from opioids will be important for improving management of the adverse effects of opioids on the skeleton. © 2022 American Society for Bone and Mineral Research (ASBMR).

MicroRNA-101a enhances trabecular bone accrual in male mice.

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis.

Epigenetic and transcriptome responsiveness to ER modulation by tissue selective estrogen complexes in breast epithelial and breast cancer cells.

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.

Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation.

The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22's functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest that the motor domain α2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of α2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.

LncMIR181A1HG is a novel chromatin-bound epigenetic suppressor of early stage osteogenic lineage commitment.

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.

Titanium with nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by regulating histone methylation.

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.

Hypoxia-inducible factor 2α is a novel inhibitor of chondrocyte maturation.

Hypoxic environment is essential for chondrocyte maturation and longitudinal bone growth. Although hypoxia-inducible factor 1 alpha (Hif-1α) has been known as a key player for chondrocyte survival and function, the function of Hif-2α in cartilage is mechanistically and clinically relevant but remains unknown. Here we demonstrated that Hif-2α was a novel inhibitor of chondrocyte maturation through downregulation of Runx2 stability. Mechanistically, Hif-2α binding to Runx2 inhibited chondrocyte maturation by Runx2 degradation through disrupting Runx2/Cbfß complex formation. The Hif-2α-mediated-Runx2 degradation could be rescued by Cbfß transfection due to the increase of Runx2/Cbfß complex formation. Consistently, mesenchymal cells derived from Hif-2α heterozygous mice were more rapidly differentiated into hypertrophic chondrocytes than those of wild-type mice in a micromass culture system. Collectively, these findings demonstrate that Hif-2α is a novel inhibitor for chondrocyte maturation by disrupting Runx2/Cbfß complex formation and consequential regulatory activity.

Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation.

Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and  protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-ß/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.

Inhibition of the RUNX1-CBFß transcription factor complex compromises mammary epithelial cell identity: a phenotype potentially stabilized by mitotic gene bookmarking.

RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report that interaction between RUNX1 and its heterodimeric partner CBFß is essential for sustaining mammary epithelial cell identity. Disruption of RUNX1-CBFß interaction, DNA binding, and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotype-related gene expression. During interphase, RUNX1 is organized as punctate, predominantly nuclear, foci that are dynamically redistributed during mitosis, with a subset localized to mitotic chromosomes. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 associates with RNA Pol II-transcribed protein coding and long non-coding RNA genes and RNA Pol I-transcribed ribosomal genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. A subset of these genes remains occupied by the protein during the mitosis to G1 transition. Together, these findings establish that the RUNX1-CBFß complex is required for maintenance of the normal mammary epithelial phenotype and its disruption leads to EMT. Importantly, our results suggest, for the first time, that RUNX1 mitotic bookmarking of a subset of epithelial-related genes may be an important epigenetic mechanism that contributes to stabilization of the mammary epithelial cell identity.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFß1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFß for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.