Chromatin In Cancer Cells Research Articles

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

Mechanism of curaxin-dependent nucleosome unfolding by FACT.

Human FACT (FACT) is a multifunctional histone chaperone involved in transcription, replication and DNA repair. Curaxins are anticancer compounds that induce FACT-dependent nucleosome unfolding and trapping of FACT in the chromatin of cancer cells (c-trapping) through an unknown molecular mechanism. Here, we analyzed the effects of curaxin CBL0137 on nucleosome unfolding by FACT using spFRET and electron microscopy. By itself, FACT adopted multiple conformations, including a novel, compact, four-domain state in which the previously unresolved NTD of the SPT16 subunit of FACT was localized, apparently stabilizing a compact configuration. Multiple, primarily open conformations of FACT-nucleosome complexes were observed during curaxin-supported nucleosome unfolding. The obtained models of intermediates suggest "decision points" in the unfolding/folding pathway where FACT can either promote disassembly or assembly of nucleosomes, with the outcome possibly being influenced by additional factors. The data suggest novel mechanisms of nucleosome unfolding by FACT and c-trapping by curaxins.

The Combination of Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Delays KMT2A-Rearranged Leukemia Progression.

Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.

SSX2 promotes the formation of a novel type of intranuclear lamin bodies.

SSX proteins are normally restricted to spermatogenic cells, but ectopic expression can be observed in many types of human cancer. We recently demonstrated that SSX family members may contribute to tumorigenesis by modifying chromatin structure and, in specific settings, compromise chromatin stability. Here, we used normal and tumorigenic breast epithelial cell line models to further study the effect of ectopic expression of SSX2 on nuclear organization. We show that SSX2 induces the formation of a novel type of nucleoplasmic lamin bodies. Ectopic expression of SSX2 in various breast epithelial cell lines led to the formation of a previously undescribed type of intranuclear bodies containing both A and B type lamins but no other components of the nuclear lamina. SSX2-expressing cells contained a highly variable number of lamin bodies distributed throughout the nuclear space. SSX2-mediated establishment of intranuclear lamin bodies could not be linked to previous molecular interactions of SSX proteins, including polycomb proteins and the Mediator complex, but was, however, dependent on S-phase progression. These results reveal a novel interaction between SSX2 and lamins in the nucleoplasmic space. They further suggest that SSX2 promotes the formation of chromatin neighborhoods supporting the organization of lamins into nuclear bodies. We speculate that this may have implications for the organization and functional regulation of chromatin in cancer cells. Our study contributes to the further understanding of the biology of SSX proteins in tumorigenesis.

Targeting Histone Modifications in Breast Cancer: A Precise Weapon on the Way.

Histone modifications (HMs) contribute to maintaining genomic stability, transcription, DNA repair, and modulating chromatin in cancer cells. Furthermore, HMs are dynamic and reversible processes that involve interactions between numerous enzymes and molecular components. Aberrant HMs are strongly associated with tumorigenesis and progression of breast cancer (BC), although the specific mechanisms are not completely understood. Moreover, there is no comprehensive overview of abnormal HMs in BC, and BC therapies that target HMs are still in their infancy. Therefore, this review summarizes the existing evidence regarding HMs that are involved in BC and the potential mechanisms that are related to aberrant HMs. Moreover, this review examines the currently available agents and approved drugs that have been tested in pre-clinical and clinical studies to evaluate their effects on HMs. Finally, this review covers the barriers to the clinical application of therapies that target HMs, and possible strategies that could help overcome these barriers and accelerate the use of these therapies to cure patients.

Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication and repair; which can reversibly unfold nucleosomes in presence of ATP. FACT is necessary for the viability and growth of breast tumor cells meanwhile in normal cells it can be knocked out without loss of vitality. Human FACT (hFACT) is a target for promising anticancer drug curaxins, which causes FACT trapping in chromatin of cancer cells and destabilizes the nucleosome. The nucleosome-unfolding activity is an important function of hFACT in vivo; however, the mechanism of FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained hFACT structure using single particle electron microscopy using JEOL 2100 TEM. Micrographs were captured with 25k magnification, and 4.1 Å pixel size. EM images pre-processing and single particles collection were performed in EMAN2.3, followed by 2D-particles analysis in RELION2.0. Final 2D-classes included ~70 000 single particles images. Results: Based on 2D-classess data analysis we evaluated several states of hFACT reflecting its conformational flexibility: the “closed” complex is characterized by four domains localized close to each other and forming a compact structure; “intermediate” state represented by classes with identified three domains having compact structure and more disordered fourth domain, and the “open” complex, represented by three domains forming almost linear structure. The “closed” and “open” states are present in comparable amounts and significantly outnumber the “intermediate” state. It has been shown that hFACT domains are connected through flexible linkers and SPT16 and SSRP1 dimerization domains (DDs) form the “joint”-like connection between the two subunits. In the “сlosed” conformation, the DNA-binding surface of FACT is covered by its two C-terminal and middle domains (MDs). The N-terminal domain (NTD) of SPT16 was not resolved previously, but it is the best candidate for the forth domain that is clearly visible only in the “closed” conformation of hFACT, based on its dimensions and the longest linker length. Conclusion: We propose that during conversion to the “open” complexes SPT16 NTD is moving away from the other subunits leading to formation of the first intermediate state with the NTD domain poorly resolved or not resolved, while less mobile DDs and MDs maintain more compact structure and the DNA-binding site is still protected by the CTDs. In the “open” state SPT16/SSRP1 visible MDs and DDs form almost linear structure, unmasking the DNA-binding sites and making them accessible for the interaction with a nucleosome.

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach.

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

Cyclin E2 Promotes Whole Genome Doubling in Breast Cancer

Genome doubling is an underlying cause of cancer cell aneuploidy and genomic instability, but few drivers have been identified for this process. Due to their physiological roles in the genome reduplication of normal cells, we hypothesised that the oncogenes cyclins E1 and E2 may be drivers of genome doubling in cancer. We show that both cyclin E1 (CCNE1) and cyclin E2 (CCNE2) mRNA are significantly associated with high genome ploidy in breast cancers. By live cell imaging and flow cytometry, we show that cyclin E2 overexpression promotes aberrant mitosis without causing mitotic slippage, and it increases ploidy with negative feedback on the replication licensing protein, Cdt1. We demonstrate that cyclin E2 localises with core preRC (pre-replication complex) proteins (MCM2, MCM7) on the chromatin of cancer cells. Low CCNE2 is associated with improved overall survival in breast cancers, and we demonstrate that low cyclin E2 protects from excess genome rereplication. This occurs regardless of p53 status, consistent with the association of high cyclin E2 with genome doubling in both p53 null/mutant and p53 wildtype cancers. In contrast, while cyclin E1 can localise to the preRC, its downregulation does not prevent rereplication, and overexpression promotes polyploidy via mitotic slippage. Thus, in breast cancer, cyclin E2 has a strong association with genome doubling, and likely contributes to highly proliferative and genomically unstable breast cancers.

Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins.

Human FACT (facilitates chromatin transcription) is a multifunctional protein complex that has histone chaperone activity and facilitates nucleosome survival and transcription through chromatin. Anticancer drugs curaxins induce FACT trapping on chromatin of cancer cells (c-trapping), but the mechanism of c-trapping is not fully understood. Here, we show that in cancer cells, FACT is highly enriched within the bodies of actively transcribed genes. Curaxin-dependent c-trapping results in redistribution of FACT from the transcribed chromatin regions to other genomic loci. Using a combination of biochemical and biophysical approaches, we have demonstrated that FACT is bound to and unfolds nucleosomes in the presence of curaxins. This tight binding to the nucleosome results in inhibition of FACT-dependent transcription in vitro in the presence of both curaxins and competitor chromatin, suggesting a mechanism of FACT trapping on bulk nucleosomes (n-trapping).

Abstract 4320: p53 binding sites in normal and cancer cells are characterized by distinct chromatin context

Abstract The tumor suppressor protein p53 interacts with DNA in a sequence-dependent manner. Thousands of p53 binding sites have been mapped genome-wide in normal and cancer cells. However, the way p53 selectively binds its cognate sites in different types of cells is not fully understood. Here, we performed a comprehensive analysis of 25 published p53 cistromes and identified 3,551 and 6,039 "high-confidence" binding sites in normal and cancer cells, respectively. Our analysis revealed two distinct epigenetic features underlying p53-DNA interactions in vivo. First, p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. Intriguingly, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is nonpermissive to most DNA-binding transcription factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin. Citation Format: Feng Cui. p53 binding sites in normal and cancer cells are characterized by distinct chromatin context [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 4320.

Metabolic perturbation of epigenome by inhibiting S-adenosylhomocysteine hydrolase elicits senescence through DNA damage response in hepatoma cells.

Cellular senescence is a key physiological barrier against tumor and represents an option for therapeutic intervention. One pivotal intracellular stimulus causing senescence is DNA damage response, while the senescence-associated heterochromatin in cancer limits the strength of the DNA damage response to endogenous genotoxic stress or DNA-damaging agents. Therefore, targeting the maintenance of compacted chromatin in cancer cells represents an optional intervention to improve the therapeutic efficacy in cancer treatment. Given a crosstalk between methionine cycle and histone methylation, we hypothesize that pharmacologically disrupting methylation potential, defined as the ratio of cellular S-adenosylmethionine to S-adenosylhomocysteine, could affect the chromatin structures in cancer cells and thus enhance their sensitivity to DNA damage response signaling. Our results showed that 3-deazaneplanocin A, a chemical inhibitor of S-adenosylhomocysteine hydrolase, elicited a typical cellular senescence in hepatoma cells. Therapy-induced senescence by 3-deazaneplanocin A was mediated through p53-p21 pathway and triggered by enhanced ataxia-telangiectasia mutated activation related to chromatin changes. In conclusion, our study demonstrated that metabolic perturbation of chromatin status in oncogene-activated cancers could be an optional intervention to sensitize DNA damage response signaling.

PARP1 and CBP lose their footing in cancer.

The human histone macroH2A.1.1 recruits activated PARP1 enzyme to chromatin through its poly(ADP-ribose)-binding macrodomain. New work shows that PARP1 and CBP can be displaced from chromatin in cancer cells that have lost macroH2A.1.1, thus leading to changes in histone H2B acetylation at cancer-relevant genes.

STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain

Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.

In vitro pro-oxidant action of Methotrexate in presence of white light

Methotrexate (MTX) an anti-cancer drug as well as a photosensitizer is able to generate reactive oxygen species (ROS). Cu (II) is present associated with chromatin in cancer cells and has been shown to be capable of mediating the action of several anti-cancer drugs through production of ROS. The objective of the present study is to determine Cu (II) mediated anti-cancer mechanism of MTX under photoilluminated condition as well as alone, using alkaline single cell gel electrophoresis (comet assay). We have shown that cellular DNA breakage was enhanced when Cu (II) is used with MTX as compared to MTX alone. It is also shown that MTX alone as well as in combination with Cu (II) is able to generate oxidative stress in lymphocyte which is inhibited by scavengers of ROS but the pattern of inhibition was differential as was also demonstrated by plasmid nicking assay. Thus, we can say that MTX exhibit pro-oxidant action in presence of white light which gets elevated in presence of Cu (II). Hence, we propose that the mobilization of endogenous copper is possibly involved in killing of cancer cells by MTX during chemo-radio therapy besides acting as antifolate.

A Ruthenium Antimetastasis Agent Forms Specific Histone Protein Adducts in the Nucleosome Core

His-tones are not ruth-less: A ruthenium anticancer agent, RAPTA-C, was found to associate with chromatin in cancer cells and to form stable, well-defined adducts in the nucleosome core at specific histone protein sites (see picture). This illuminates the macromolecular binding properties of this antimetastasis agent and indicates potential for developing metallodrugs for site-selective targeting of chromatin.

Targeting Tyrosine Phosphorylation of PCNA Inhibits Prostate Cancer Growth

The proliferation cell nuclear antigen (PCNA) is a critical protein required for DNA replication in proliferating cells including cancer cells. However, direct inhibition of PCNA in cancer cells has been difficult due to the lack of targetable sites. We previously reported that phosphorylation of tyrosine 211 (Y211) on PCNA is important for the proliferative function of PCNA when this protein is associated with the chromatin in cancer cells. Here, we show that the Y211 phosphorylation of PCNA is a frequent event in advanced prostate cancer. To explore the potential of this signaling event in inhibition of cancer cell growth, we used a synthetic peptide, the Y211F peptide, which when present inhibits phosphorylation of Y211 on endogenous PCNA. Treatment with this peptide, but not a scrambled control peptide, resulted in S-phase arrest, inhibition of DNA synthesis, and enhanced cell death in a panel of human prostate cancer cell lines. In addition, treatment with the Y211F peptide led to decreased tumor growth in PC3-derived xenograft tumors in vivo in nude mice. Our study shows for the first time that PCNA phosphorylation at Y211 is a frequent and biologically important signaling event in prostate cancer. This study also shows a proof of concept that Y211 phosphorylation of PCNA may be used as a therapeutic target in prostate cancer cells, including cells of advanced cancers that are refractory to standard hormonal therapies.

New doors to open...and so many!

New doors to open...and so many!

Organization of chromatin in cancer cells: role of signalling pathways

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.Key words: histone acetylation, histone phosphorylation, nuclear matrix, cytoskeleton, histone deacetylase, cancer.

Organization of chromatin in cancer cells: role of signalling pathways

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.