RUNX1-dependent mechanisms in biological control and dysregulation in cancer.

The RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context-dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial-to-mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context-dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.

Selective expression of long non-coding RNAs in a breast cancer cell progression model.

Long non-coding RNAs (lncRNAs) are acknowledged as regulators of cancer biology and pathology. Our goal was to perform a stringent profiling of breast cancer cell lines that represent disease progression. We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (pre-malignant), and MCF-10CA1a (malignant) cells, to perform transcriptome wide sequencing. From these data, we have identified 346 lncRNAs with dysregulated expression across the progression series. By comparing lncRNAs from these datasets to those from an additional set of cell lines that represent different disease stages and subtypes, MCF-7 (early stage, luminal), and MDA-MB-231 (late stage, basal), 61 lncRNAs that are associated with breast cancer progression were identified. Querying breast cancer patient data from The Cancer Genome Atlas, we selected a lncRNA, IGF-like family member 2 antisense RNA 1 (IGFL2-AS1), of potential clinical relevance for functional characterization. Among the 61 lncRNAs, IGFL2-AS1 was the most significantly decreased. Our results indicate that this lncRNA plays a role in downregulating its nearest neighbor, IGFL1, and affects migration of breast cancer cells. Furthermore, the lncRNAs we identified provide a valuable resource to mechanistically and clinically understand the contribution of lncRNAs in breast cancer progression.

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression.

Regulation of osteogenesis by long noncoding RNAs: An epigenetic mechanism contributing to bone formation.

Long noncoding RNAs (lncRNAs) have recently emerged as novel regulators of lineage commitment, differentiation, development, viability, and disease progression. Few studies have examined their role in osteogenesis; however, given their critical and wide-ranging roles in other tissues, lncRNAs are most likely vital regulators of osteogenesis. In this study, we extensively characterized lncRNA expression in mesenchymal cells during commitment and differentiation to the osteoblast lineage using a whole transcriptome sequencing approach (RNA-Seq). Using mouse primary mesenchymal stromal cells (mMSC), we identified 1438 annotated lncRNAs expressed during MSC differentiation, 462 of which are differentially expressed. We performed guilt-by-association analysis using lncRNA and mRNA expression profiles to identify lncRNAs influencing MSC commitment and differentiation. These findings open novel dimensions for exploring lncRNAs in regulating normal bone formation and in skeletal disorders.

p38α MAPK is required for tooth morphogenesis and enamel secretion.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and ß4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.

Could lncRNAs be the missing links in control of mesenchymal stem cell differentiation?

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.

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.

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.

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.

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.

Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals.

Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in HA (hydroxyapatite) nucleation and growth. BSP (bone sialoprotein) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1-100 and 133-205 of the rat sequence were phosphorylated with CK2 (protein kinase CK2). Phosphorylation increased the nucleating activity of BSP and BSP-(133-205), but not BSP-(1-100). MS analysis revealed that the major site phosphorylated within BSP-(133-205) was Ser136, a site adjacent to the series of contiguous glutamate residues previously implicated in HA nucleation. The critical role of phosphorylated Ser136 in HA nucleation was confirmed by site-directed mutagenesis and functional analyses. Furthermore, peptides corresponding to the 133-148 sequence of rat BSP were synthesized with or without a phosphate group on Ser136. As expected, the phosphopeptide was a more potent nucleator. The mechanism of nucleation was investigated using molecular-dynamics simulations analysing BSP-(133-148) interacting with the {100} crystal face of HA. Both phosphorylated and non-phosphorylated sequences adsorbed to HA in extended conformations with alternating residues in contact with and facing away from the crystal face. However, this alternating-residue pattern was more pronounced when Ser136 was phosphorylated. These studies demonstrate a critical role for Ser136 phosphorylation in BSP-mediated HA nucleation and identify a unique mode of interaction between the nucleating site of the protein and the {100} face of HA.

Lysosomal protease expression in mature enamel.

The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

Transforming growth factor-beta1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis.

Transforming growth factor-beta1 (TGF-beta1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-beta1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-beta1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-beta1-mediated growth arrest and are prone to TGF-beta1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-beta1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-beta1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-beta1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.

Mitotically-Associated lncRNA (MANCR) Affects Genomic Stability and Cell Division in Aggressive Breast Cancer.

Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therapies; therefore, it is imperative to identify novel, targetable regulators of progression. Long non-coding RNAs (lncRNA) are important regulators in breast cancer and have great potential as therapeutic targets; however, little is known about how the majority of lncRNAs function within breast cancer. This study characterizes a novel lncRNA, MANCR (mitotically-associated long noncoding RNA; LINC00704), which is upregulated in breast cancer patient specimens and cells. Depletion of MANCR in triple-negative breast cancer cells significantly decreases cell proliferation and viability, with concomitant increases in DNA damage. Transcriptome analysis, based on RNA sequencing, following MANCR knockdown reveals significant differences in the expression of >2,000 transcripts, and gene set enrichment analysis identifies changes in multiple categories related to cell-cycle regulation. Furthermore, MANCR expression is highest in mitotic cells by both RT-qPCR and RNA in situ hybridization. Consistent with a role in cell-cycle regulation, MANCR-depleted cells have a lower mitotic index and higher incidences of defective cytokinesis and cell death. Taken together, these data reveal a role for the novel lncRNA, MANCR, in genomic stability of aggressive breast cancer, and identify it as a potential therapeutic target.Implications: The novel lncRNA, MANCR (LINC00704), is upregulated in breast cancer and is functionally linked with cell proliferation, viability, and genomic stability. Mol Cancer Res; 16(4); 587-98. ©2018 AACR.

Nuclear organization mediates cancer-compromised genetic and epigenetic control.

Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expression for biological control and is modified in cancer. Nuclear organization supports cell growth and phenotypic properties of normal and cancer cells by facilitating physiologically responsive interactions of chromosomes, genes and regulatory complexes at dynamic three-dimensional microenvironments. We will review nuclear structure/function relationships that include: 1. Epigenetic bookmarking of genes by phenotypic transcription factors to control fidelity and plasticity of gene expression as cells enter and exit mitosis; 2. Contributions of chromatin remodeling to breast cancer nuclear morphology, metabolism and effectiveness of chemotherapy; 3. Relationships between fidelity of nuclear organization and metastasis of breast cancer to bone; 4. Dynamic modifications of higher-order inter- and intra-chromosomal interactions in breast cancer cells; 5. Coordinate control of cell growth and phenotype by tissue-specific transcription factors; 6. Oncofetal epigenetic control by bivalent histone modifications that are functionally related to sustaining the stem cell phenotype; and 7. Noncoding RNA-mediated regulation in the onset and progression of breast cancer. The discovery of components to nuclear organization that are functionally related to cancer and compromise gene expression have the potential for translation to innovative cancer diagnosis and targeted therapy.

Bone sialoprotein expression enhances osteoblast differentiation and matrix mineralization in vitro.

Bone sialoprotein (BSP) is an acidic, noncollagenous glycoprotein abundantly expressed in mineralized tissues. Although BSP is frequently used as a marker of osteoblast differentiation, the role of the protein in osteoblast function is unclear. BSP belongs to the SIBLING (Small Integrin-binding LIgand N-linked Glycoprotein) family of RGD-containing matrix proteins, several members of which have been shown to affect cell differentiation. The normal levels of BSP expression in osteoblasts were specifically altered by CMV-mediated adenoviral overexpression in primary osteoblasts or inhibition by an RNA interference-based strategy in the MC3T3E1 cell line. Alternatively, osteoblast cultures were supplemented with recombinant BSP protein. Quantitative real-time PCR was used to monitor the mRNA levels of the osteoblast-related transcription factors Osterix and Runx2 as well as the osteoblast-specific gene osteocalcin. As markers of osteoblast differentiation, alkaline phosphatase enzyme activity, Runx2-luciferase reporter activity and calcein incorporation into mineralized cultures were also measured. The overexpression of BSP increased osteoblast-related gene expression as well as calcium incorporation and nodule formation by osteoblast cultures. Similarly, supplementation of osteoblast cultures with recombinant BSP increased several markers of osteoblast differentiation. Conversely, suppression of BSP expression by small-hairpin RNA-encoding plasmids inhibited expression of osteoblast markers and nodule formation. Overexpression of several functional-domain mutants of BSP demonstrated that increases in osteoblast-related gene expression and matrix mineralization observed in BSP overexpression models are mediated by the integrin-binding RGD motif found near the C-terminus of the protein. These results demonstrate that BSP may serve as a matrix-associated signal directly promoting osteoblast differentiation resulting in the increased production of a mineralized matrix.

In vivo functional analysis of polyglutamic acid domains in recombinant bone sialoprotein.

Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues that binds to hydroxyapatite and nucleates its formation in vitro. Two polyglutamic acid regions (poly [E]) are believed to participate in these activities. The aim of this study was to evaluate the contribution of these acidic regions to the binding of prokaryote recombinant BSP (prBSP(E)) within an actual in vivo environment. Full-length prBSP(E) and prBSP(E) in which the poly [E] domains were replaced by polyalanine (prBSP(A)) were tagged with dinitrophenol (DNP). Tagged preparations comprised intact molecules and some fragmented forms. They were infused through a surgically created hole in the bone of rat hemimandibles and detected using immunogold labeling with anti-DNP antibodies. prBSP(E)-DNP was consistently immunodetected along exposed mineralized bone surfaces and osteocyte canaliculi at the surgical site. Few gold particles were observed on these surfaces when prBSP(A)-DNP was infused. Quantitative analyses showed significant differences in labeling between prBSP(E)-DNP (5.04 +/- 0.73 particles/micro m2) and prBSP(A)-DNP (1.37 +/- 0.35 particles/micro m2). These results indicate that poly [E] domains influence binding of prBSP(E) to surfaces presenting a mixture of mineral and proteins bathed by tissue fluids and suggest that they may similarly mediate the interaction of native BSP in the bone environment.

Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition.

Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFß signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFß and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

Histone H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes.

The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways.