Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway.

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.

BRG1 is a prognostic indicator and a potential therapeutic target for prostate cancer.

Brahma-related gene 1 (BRG1) is one of two mutually exclusive ATPases that function as the catalytic subunit of human SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling enzymes. BRG1 has been identified as a tumor suppressor in some cancer types but has been shown to be expressed at elevated levels, relative to normal tissue, in other cancers. Using TCGA (The Cancer Genome Atlas) prostate cancer database, we determined that BRG1 mRNA and protein expression is elevated in prostate tumors relative to normal prostate tissue. Only 3 of 491 (0.6%) sequenced tumors showed amplification of the locus or mutation in the protein coding sequence, arguing against the idea that elevated expression due to amplification or expression of a mutant BRG1 protein is associated with prostate cancer. Kaplan-Meier survival curves showed that BRG1 expression in prostate tumors inversely correlated with survival. However, BRG1 expression did not correlate with Gleason score/International Society of Urological Pathology (ISUP) Grade Group, indicating it is an independent predictor of tumor progression/patient outcome. To experimentally assess BRG1 as a possible therapeutic target, we treated prostate cancer cells with a biologic inhibitor called ADAADi (active DNA-dependent ATPase A Domain inhibitor) that targets the activity of the SNF2 family of ATPases in biochemical assays but showed specificity for BRG1 in prior tissue culture experiments. The inhibitor decreased prostate cancer cell proliferation and induced apoptosis. When directly injected into xenografts established by injection of prostate cancer cells in mouse flanks, the inhibitor decreased tumor growth and increased survival. These results indicate the efficacy of pursuing BRG1 as both an indicator of patient outcome and as a therapeutic target.

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.

Epithelial-to-mesenchymal transition and cancer stem cells contribute to breast cancer heterogeneity.

Breast cancer is the most common cancer in women, and accounts for ~30% of new cancer cases and 15% of cancer-related deaths. Tumor relapse and metastasis are primary factors contributing to breast cancer-related deaths. Therefore, the challenge for breast cancer treatment is to sustain remission. A driving force behind tumor relapse is breast cancer heterogeneity (both intertumor, between different patients, and intratumor, within the same tumor). Understanding breast cancer heterogeneity is necessary to develop preventive interventions and targeted therapies. A recently emerging concept is that intratumor heterogeneity is driven by cancer stem cells (CSCs) that are capable of giving rise to a multitude of different cells within a tumor. Studies have highlighted linkage of CSC formation with epithelial-to-mesenchymal transition (EMT). In this review, we summarize the current understanding of breast cancer heterogeneity, links between EMT and CSCs, regulation of EMT by Runx transcription factors, and potential therapeutic strategies targeting these processes.

Mitotic Gene Bookmarking: An Epigenetic Program to Maintain Normal and Cancer Phenotypes.

Reconfiguration of nuclear structure and function during mitosis presents a significant challenge to resume the next cell cycle in the progeny cells without compromising structural and functional identity of the cells. Equally important is the requirement for cancer cells to retain the transformed phenotype, that is, unrestricted proliferative potential, suppression of cell phenotype, and activation of oncogenic pathways. Mitotic gene bookmarking retention of key regulatory proteins that include sequence-specific transcription factors, chromatin-modifying factors, and components of RNA Pol (RNAP) I and II regulatory machineries at gene loci on mitotic chromosomes plays key roles in coordinate control of cell phenotype, growth, and proliferation postmitotically. There is growing recognition that three distinct protein types, mechanistically, play obligatory roles in mitotic gene bookmarking: (i) Retention of phenotypic transcription factors on mitotic chromosomes is essential to sustain lineage commitment; (ii) Select chromatin modifiers and posttranslational histone modifications/variants retain competency of mitotic chromatin for gene reactivation as cells exit mitosis; and (iii) Functional components of RNAP I and II transcription complexes (e.g., UBF and TBP, respectively) are retained on genes poised for reactivation immediately following mitosis. Importantly, recent findings have identified oncogenes that are associated with target genes on mitotic chromosomes in cancer cells. The current review proposes that mitotic gene bookmarking is an extensively utilized epigenetic mechanism for stringent control of proliferation and identity in normal cells and hypothesizes that bookmarking plays a pivotal role in maintenance of tumor phenotypes, that is, unrestricted proliferation and compromised control of differentiation. Mol Cancer Res; 16(11); 1617-24. ©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.

The BRG1 ATPase of human SWI/SNF chromatin remodeling enzymes as a driver of cancer.

Mammalian SWI/SNF enzymes are ATP-dependent remodelers of chromatin structure. These multisubunit enzymes are heterogeneous in composition; there are two catalytic ATPase subunits, BRM and BRG1, that are mutually exclusive, and additional subunits are incorporated in a combinatorial manner. Recent findings indicate that approximately 20% of human cancers contain mutations in SWI/SNF enzyme subunits, leading to the conclusion that the enzyme subunits are critical tumor suppressors. However, overexpression of specific subunits without apparent mutation is emerging as an alternative mechanism by which cellular transformation may occur. Here we highlight recent evidence linking elevated expression of the BRG1 ATPase to tissue-specific cancers and work suggesting that inhibiting BRG1 may be an effective therapeutic strategy.

Mammalian SWI/SNF Enzymes and the Epigenetics of Tumor Cell Metabolic Reprogramming.

Tumor cells reprogram their metabolism to survive and grow in a challenging microenvironment. Some of this reprogramming is performed by epigenetic mechanisms. Epigenetics is in turn affected by metabolism; chromatin modifying enzymes are dependent on substrates that are also key metabolic intermediates. We have shown that the chromatin remodeling enzyme Brahma-related gene 1 (BRG1), an epigenetic regulator, is necessary for rapid breast cancer cell proliferation. The mechanism for this requirement is the BRG1-dependent transcription of key lipogenic enzymes and regulators. Reduction in lipid synthesis lowers proliferation rates, which can be restored by palmitate supplementation. This work has established BRG1 as an attractive target for breast cancer therapy. Unlike genetic alterations, epigenetic mechanisms are reversible, promising gentler therapies without permanent off-target effects at distant sites.

Identifying Nuclear Matrix-Attached DNA Across the Genome.

Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix-attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix-associated genome is highly cell-context dependent. J. Cell. Physiol. 232: 1295-1305, 2017. © 2016 Wiley Periodicals, Inc.

SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells.

The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation.

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer.

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells.

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer.

Coordinated Dynamics of RNA Splicing Speckles in the Nucleus.

Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.

Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell.

How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons--far greater than typical intracellular motor forces--was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces.

The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation.

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response.

Cancer cells exhibit modifications in nuclear architecture and transcriptional control. Tumor growth and metastasis are supported by RUNX family transcriptional scaffolding proteins, which mediate the assembly of nuclear-matrix-associated gene-regulatory hubs. We used proteomic analysis to identify RUNX2-dependent protein-protein interactions associated with the nuclear matrix in bone, breast and prostate tumor cell types and found that RUNX2 interacts with three distinct proteins that respond to DNA damage - RUVBL2, INTS3 and BAZ1B. Subnuclear foci containing these proteins change in intensity or number following UV irradiation. Furthermore, RUNX2, INTS3 and BAZ1B form UV-responsive complexes with the serine-139-phosphorylated isoform of H2AX (γH2AX). UV irradiation increases the interaction of BAZ1B with γH2AX and decreases histone H3 lysine 9 acetylation levels, which mark accessible chromatin. RUNX2 depletion prevents the BAZ1B-γH2AX interaction and attenuates loss of H3K9 and H3K56 acetylation. Our data are consistent with a model in which RUNX2 forms functional complexes with BAZ1B, RUVBL2 and INTS3 to mount an integrated response to DNA damage. This proposed cytoprotective function for RUNX2 in cancer cells might clarify its expression in chemotherapy-resistant and/or metastatic tumors.

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics.

BACKGROUND: Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress. RESULTS: We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved. CONCLUSIONS: Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.

Nuclear shape changes are induced by knockdown of the SWI/SNF ATPase BRG1 and are independent of cytoskeletal connections.

Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.

Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway.

UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5' end of a nuclear mRNA but are also reported to associate with the exon junction complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly, we used fluorescence recovery after photobleaching to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the phosphatidylinositide 3-kinase (PI3 kinase)/AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intronless histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the endoplasmic reticulum or to mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and promote the nuclear retention of some mRNAs.

Live cell imaging of the cancer-related transcription factor RUNX2 during mitotic progression.

The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I-dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching to measure the relative binding kinetics of enhanced green fluorescent protein (EGFP)-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction." RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased binding affinity of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells.