Higher-order chromatin structure is often perturbed in cancer and other pathological states. Alth... more Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines. Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways...
The transcription factor RUNX1 is one of the most frequently mutated genes in breast tumors. Howe... more The transcription factor RUNX1 is one of the most frequently mutated genes in breast tumors. However, the role of RUNX1 in the mammary gland is understudied. Previously, our group established that RUNX1 has tumor suppressor activities in both normal mammary epithelial and breast cancer cells, including inhibition of epithelial to mesenchymal transition (EMT), migration, and invasion. To better understand the cellular consequences mediated by RUNX1 loss-of-function in mammary epithelial cells, we performed global gene expression profiling (RNA-seq) in Runx1-depleted MCF10A cells. Upon loss of RUNX1, the expression of 1806 genes was significantly altered (with 2-fold change cutoff). Pathway analysis on these differentially expressed genes revealed that RUNX1 is involved in many cellular activities, including lipid metabolism, cell growth and cell cycle control, which were not previously reported. Consistent with expression profiling results, flow cytometry analysis showed the mitotic population in RUNX1 depleted cells was significantly decreased, indicating RUNX1 is important for proper cell division. To investigate the mechanisms by which RUNX1 controls expression of those differentially expressed genes, RUNX1 genome-wide occupancy analysis (ChIP-seq) was performed in MCF10A cells. We found that although RUNX1 binding is enriched at transcription start sites (TSS), RUNX1 also binds to loci distal to TSSs and multiple other regulatory elements. Therefore, RUNX1 has the capability to utilize multiple mechanisms to control target gene expression. Our results highlight crucial roles for RUNX1 in controlling normal growth of mammary epithelial cells, consistent with its tumor suppressor activities. Loss of RUNX1 leads to improper gene regulation and cell cycle defects, which may contribute to tumor progression in breast cancer. These RUNX1-mediated mechanisms point to novel intervention strategies for early stage breast cancer. Citation Format: Deli Hong, Andrew J. Fritz, Coralee E. Tye, Natalie A. Page, Joseph R. Boyd, Jane B. Lian, Janet L. Stein, Gary S. Stein. RUNX1 global binding and gene regulation in mammary epithelial cells revealed novel Runx1 mediated cellular activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4462.
Introduction: More than 80% of men with aggressive prostate cancer (PCa) develop metastatic disea... more Introduction: More than 80% of men with aggressive prostate cancer (PCa) develop metastatic disease and skeletal complications leading to a dramatic reduction in quality of life. There is a deficit in effective biomarkers predicting disease progression, response to treatment, or metastasis at early stages when intervention would be possible. A critical need exists for novel therapeutic targets to prevent PCa metastasis to bone. MicroRNAs (miRNA) are informative biomarkers, as they circulate, are easily detected in human serum, and are coupled to targets reflecting deregulated pathways. Secreted miRNAs may be bound to proteins, membrane-encapsulated, or free RNA molecules and can act as intercellular signaling molecules. Here we tested the hypothesis that miRNAs secreted from prostate cancer cells in the primary tumor function locally and at distal sites to alter the normal tissue environment and promote PCa. Objective: Use global miRNA and gene expression analysis to 1) define cohorts of miRNAs secreted from prostate cancer cells representing different disease stages, 2) identify expression levels of their target mRNAs, and 3) provide insight into mechanisms of secreted miRNA activity in promoting prostate cancer tumor growth and metastasis. Methods: RNA was isolated from normal prostate epithelial (RWPE-1), early stage, androgen responsive PCa (LNCaP), and late stage, androgen insensitive PCa (PC-3) cell lines and from their conditioned media. Global expression of both secreted and extracellular miRNAs were interrogated using Affymetrix miRNA v4.0 microarrays (miRBase v20). Stranded RNA-seq was performed in parallel to identify differential expression of miRNA target mRNAs. We utilized the RNA-seq data to limit predicted mRNAs to those having reciprocal expression changes to their targeting miRNA. Enriched biological pathways of candidate miRNAs were identified for these predicted mRNA targets. Results: To identify secreted miRNAs that are candidates for promoting prostatic tumor growth, cancer progression, and metastatic bone disease, we compared differentially expressed miRNAs between PCa cell lines and their conditioned media. While numerous miRNAs are found to be both secreted and expressed robustly in the cell layer, we find unique subsets of miRNAs in each cell line that are either preferentially secreted or retained within the cell. We examined putative mRNA targets of these miRNAs in our gene expression data and established reciprocal mRNA-miRNA relationships. The miRNAs were found to be associated with pathways known to play a role in PCa and metastatic bone disease including ErbB, Wnt, TGF-beta, and MAPK signaling, as well as previously unassociated biological processes. Functional in vitro assays were performed for several candidate miRNAs that were overexpressed or inhibited in normal prostate epithelial RWPE-1 and highly metastatic PC-3 cell lines. Phenotypic changes, including motility, invasion, migration, and proliferation, were monitored. As an example, inhibition of miR-30a-5p, an established tumor suppressor, increases the rate at which RWPE-1 and PC-3 cells migrate to heal a cell layer scratch while overexpression prevents wound closure in RWPE-1 cells. In vivo orthotopic experiments to assay the ability of candidate miRNAs to promote local and metastatic PCa tumor growth are ongoing. Conclusion: Our combined data identify, for the first time, miRNAs secreted from prostate tumor cells that promote metastatic events of prostate cancer. Several of these miRNAs have been reported to be elevated in PCa patient serum and can provide diagnostic/prognostic biomarkers of tumor development, response to treatment, and recurrence. Our findings link these miRNAs to deregulated cellular pathways for potential intervention of prostate cancer progression. Citation Format: Nicholas H. Farina, Cody J. Callahan, Coralee E. Tye, Joseph R. Boyd, Gary S. Stein, Janet L. Stein, Jane B. Lian. Secreted microRNAs from prostate cancer cells: Novel therapeutic targets. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B34.
The RUNX1 transcription factor has recently been shown to be obligatory for normal development. R... more 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.
Long noncoding RNAs (lncRNAs) have recently emerged as novel regulators of lineage commitment, di... more 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.
Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expressi... more 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 ph...
Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therap... more 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...
Long non-coding RNAs (lncRNAs) are acknowledged as regulators of cancer biology and pathology. Ou... more 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. Amon...
Alterations in nuclear morphology are common in cancer progression. However, the degree to which ... more 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, su...
Runx1 is a well characterized transcription factor essential for hematopoietic differentiation an... more 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 TGFβ 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 TGFβ 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.
The onset and progression of breast cancer are linked to genetic and epigenetic changes that alte... more 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 ...
Mmp20 null mice have a striking enamel phenotype where the enamel is thin, has an abnormal rod pa... more Mmp20 null mice have a striking enamel phenotype where the enamel is thin, has an abnormal rod pattern, abrades from the dentin and has a deteriorating tooth morphology as enamel development progresses. Objective: We sought to determine if introduction of a transgene expressing Mmp20 would revert, or partially revert, the severe enamel phenotype observed in the Mmp20 null mouse. Methods: An expression construct was designed such that the mouse amelogenin promoter drove expression of the mouse Mmp20 cDNA. This construct was microinjected into the pronuclei of fertilized mouse eggs. The resulting transgenic mice were assessed by qPCR for transgene expression and offspring of founders expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Transgenic and control mice were then assessed for enamel quality. Results: The Mmp20 transgenes brought the enamel back to full thickness. No significant difference in enamel thickness was observed a...
Non-coding RNAs (ncRNAs) have evolved in eukaryotes as epigenetic regulators of gene expression. ... more Non-coding RNAs (ncRNAs) have evolved in eukaryotes as epigenetic regulators of gene expression. The most abundant regulatory ncRNAs are the 20-24nt small microRNAs (miRNAs) and long non-coding RNAs (lncRNAs, <200nt). Each class of ncRNAs operates through distinct mechanisms, but their pathways to regulating gene expression are interrelated in ways that are just being recognized. While the importance of lncRNAs in epigenetic control of transcription, developmental processes and human traits is emerging, the identity of lncRNAs in skeletal biology is scarcely known. However, since the first profiling studies of miRNA at stages during osteoblast and osteoclast differentiation, over 1100 publications related to bone biology and pathologies can be found, as well as many recent comprehensive reviews summarizing miRNA in skeletal cells. Delineating the activities and targets of specific miRNAs regulating differentiation of osteogenic and resorptive bone cells, coupled with in vivo gain- and loss-of-function studies, discovered unique mechanisms that support bone development and bone homeostasis in adults. We present here "guiding principles" for addressing biological control of bone tissue formation by ncRNAs. This review emphasizes recent advances in understanding regulation of the process of miRNA biogenesis that impact on osteogenic lineage commitment, transcription factors and signaling pathways. Also discussed are the approaches to be pursued for an understanding of the role of lncRNAs in bone and the challenges in addressing their multiple and complex functions. Based on new knowledge of epigenetic control of gene expression to be gained for ncRNA regulation of the skeleton, new directions for translating the miRNAs and lncRNAs into therapeutic targets for skeletal disorders are possible. This article is part of a Special Issue entitled Epigenetics and Bone.
Higher-order chromatin structure is often perturbed in cancer and other pathological states. Alth... more Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines. Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways...
The transcription factor RUNX1 is one of the most frequently mutated genes in breast tumors. Howe... more The transcription factor RUNX1 is one of the most frequently mutated genes in breast tumors. However, the role of RUNX1 in the mammary gland is understudied. Previously, our group established that RUNX1 has tumor suppressor activities in both normal mammary epithelial and breast cancer cells, including inhibition of epithelial to mesenchymal transition (EMT), migration, and invasion. To better understand the cellular consequences mediated by RUNX1 loss-of-function in mammary epithelial cells, we performed global gene expression profiling (RNA-seq) in Runx1-depleted MCF10A cells. Upon loss of RUNX1, the expression of 1806 genes was significantly altered (with 2-fold change cutoff). Pathway analysis on these differentially expressed genes revealed that RUNX1 is involved in many cellular activities, including lipid metabolism, cell growth and cell cycle control, which were not previously reported. Consistent with expression profiling results, flow cytometry analysis showed the mitotic population in RUNX1 depleted cells was significantly decreased, indicating RUNX1 is important for proper cell division. To investigate the mechanisms by which RUNX1 controls expression of those differentially expressed genes, RUNX1 genome-wide occupancy analysis (ChIP-seq) was performed in MCF10A cells. We found that although RUNX1 binding is enriched at transcription start sites (TSS), RUNX1 also binds to loci distal to TSSs and multiple other regulatory elements. Therefore, RUNX1 has the capability to utilize multiple mechanisms to control target gene expression. Our results highlight crucial roles for RUNX1 in controlling normal growth of mammary epithelial cells, consistent with its tumor suppressor activities. Loss of RUNX1 leads to improper gene regulation and cell cycle defects, which may contribute to tumor progression in breast cancer. These RUNX1-mediated mechanisms point to novel intervention strategies for early stage breast cancer. Citation Format: Deli Hong, Andrew J. Fritz, Coralee E. Tye, Natalie A. Page, Joseph R. Boyd, Jane B. Lian, Janet L. Stein, Gary S. Stein. RUNX1 global binding and gene regulation in mammary epithelial cells revealed novel Runx1 mediated cellular activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4462.
Introduction: More than 80% of men with aggressive prostate cancer (PCa) develop metastatic disea... more Introduction: More than 80% of men with aggressive prostate cancer (PCa) develop metastatic disease and skeletal complications leading to a dramatic reduction in quality of life. There is a deficit in effective biomarkers predicting disease progression, response to treatment, or metastasis at early stages when intervention would be possible. A critical need exists for novel therapeutic targets to prevent PCa metastasis to bone. MicroRNAs (miRNA) are informative biomarkers, as they circulate, are easily detected in human serum, and are coupled to targets reflecting deregulated pathways. Secreted miRNAs may be bound to proteins, membrane-encapsulated, or free RNA molecules and can act as intercellular signaling molecules. Here we tested the hypothesis that miRNAs secreted from prostate cancer cells in the primary tumor function locally and at distal sites to alter the normal tissue environment and promote PCa. Objective: Use global miRNA and gene expression analysis to 1) define cohorts of miRNAs secreted from prostate cancer cells representing different disease stages, 2) identify expression levels of their target mRNAs, and 3) provide insight into mechanisms of secreted miRNA activity in promoting prostate cancer tumor growth and metastasis. Methods: RNA was isolated from normal prostate epithelial (RWPE-1), early stage, androgen responsive PCa (LNCaP), and late stage, androgen insensitive PCa (PC-3) cell lines and from their conditioned media. Global expression of both secreted and extracellular miRNAs were interrogated using Affymetrix miRNA v4.0 microarrays (miRBase v20). Stranded RNA-seq was performed in parallel to identify differential expression of miRNA target mRNAs. We utilized the RNA-seq data to limit predicted mRNAs to those having reciprocal expression changes to their targeting miRNA. Enriched biological pathways of candidate miRNAs were identified for these predicted mRNA targets. Results: To identify secreted miRNAs that are candidates for promoting prostatic tumor growth, cancer progression, and metastatic bone disease, we compared differentially expressed miRNAs between PCa cell lines and their conditioned media. While numerous miRNAs are found to be both secreted and expressed robustly in the cell layer, we find unique subsets of miRNAs in each cell line that are either preferentially secreted or retained within the cell. We examined putative mRNA targets of these miRNAs in our gene expression data and established reciprocal mRNA-miRNA relationships. The miRNAs were found to be associated with pathways known to play a role in PCa and metastatic bone disease including ErbB, Wnt, TGF-beta, and MAPK signaling, as well as previously unassociated biological processes. Functional in vitro assays were performed for several candidate miRNAs that were overexpressed or inhibited in normal prostate epithelial RWPE-1 and highly metastatic PC-3 cell lines. Phenotypic changes, including motility, invasion, migration, and proliferation, were monitored. As an example, inhibition of miR-30a-5p, an established tumor suppressor, increases the rate at which RWPE-1 and PC-3 cells migrate to heal a cell layer scratch while overexpression prevents wound closure in RWPE-1 cells. In vivo orthotopic experiments to assay the ability of candidate miRNAs to promote local and metastatic PCa tumor growth are ongoing. Conclusion: Our combined data identify, for the first time, miRNAs secreted from prostate tumor cells that promote metastatic events of prostate cancer. Several of these miRNAs have been reported to be elevated in PCa patient serum and can provide diagnostic/prognostic biomarkers of tumor development, response to treatment, and recurrence. Our findings link these miRNAs to deregulated cellular pathways for potential intervention of prostate cancer progression. Citation Format: Nicholas H. Farina, Cody J. Callahan, Coralee E. Tye, Joseph R. Boyd, Gary S. Stein, Janet L. Stein, Jane B. Lian. Secreted microRNAs from prostate cancer cells: Novel therapeutic targets. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B34.
The RUNX1 transcription factor has recently been shown to be obligatory for normal development. R... more 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.
Long noncoding RNAs (lncRNAs) have recently emerged as novel regulators of lineage commitment, di... more 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.
Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expressi... more 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 ph...
Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therap... more 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...
Long non-coding RNAs (lncRNAs) are acknowledged as regulators of cancer biology and pathology. Ou... more 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. Amon...
Alterations in nuclear morphology are common in cancer progression. However, the degree to which ... more 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, su...
Runx1 is a well characterized transcription factor essential for hematopoietic differentiation an... more 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 TGFβ 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 TGFβ 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.
The onset and progression of breast cancer are linked to genetic and epigenetic changes that alte... more 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 ...
Mmp20 null mice have a striking enamel phenotype where the enamel is thin, has an abnormal rod pa... more Mmp20 null mice have a striking enamel phenotype where the enamel is thin, has an abnormal rod pattern, abrades from the dentin and has a deteriorating tooth morphology as enamel development progresses. Objective: We sought to determine if introduction of a transgene expressing Mmp20 would revert, or partially revert, the severe enamel phenotype observed in the Mmp20 null mouse. Methods: An expression construct was designed such that the mouse amelogenin promoter drove expression of the mouse Mmp20 cDNA. This construct was microinjected into the pronuclei of fertilized mouse eggs. The resulting transgenic mice were assessed by qPCR for transgene expression and offspring of founders expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Transgenic and control mice were then assessed for enamel quality. Results: The Mmp20 transgenes brought the enamel back to full thickness. No significant difference in enamel thickness was observed a...
Non-coding RNAs (ncRNAs) have evolved in eukaryotes as epigenetic regulators of gene expression. ... more Non-coding RNAs (ncRNAs) have evolved in eukaryotes as epigenetic regulators of gene expression. The most abundant regulatory ncRNAs are the 20-24nt small microRNAs (miRNAs) and long non-coding RNAs (lncRNAs, <200nt). Each class of ncRNAs operates through distinct mechanisms, but their pathways to regulating gene expression are interrelated in ways that are just being recognized. While the importance of lncRNAs in epigenetic control of transcription, developmental processes and human traits is emerging, the identity of lncRNAs in skeletal biology is scarcely known. However, since the first profiling studies of miRNA at stages during osteoblast and osteoclast differentiation, over 1100 publications related to bone biology and pathologies can be found, as well as many recent comprehensive reviews summarizing miRNA in skeletal cells. Delineating the activities and targets of specific miRNAs regulating differentiation of osteogenic and resorptive bone cells, coupled with in vivo gain- and loss-of-function studies, discovered unique mechanisms that support bone development and bone homeostasis in adults. We present here "guiding principles" for addressing biological control of bone tissue formation by ncRNAs. This review emphasizes recent advances in understanding regulation of the process of miRNA biogenesis that impact on osteogenic lineage commitment, transcription factors and signaling pathways. Also discussed are the approaches to be pursued for an understanding of the role of lncRNAs in bone and the challenges in addressing their multiple and complex functions. Based on new knowledge of epigenetic control of gene expression to be gained for ncRNA regulation of the skeleton, new directions for translating the miRNAs and lncRNAs into therapeutic targets for skeletal disorders are possible. This article is part of a Special Issue entitled Epigenetics and Bone.
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