Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription.chromatin ͉ histone ͉ lysine demethylase ͉ methylation

We describe the complete beta-tubulin folding pathway. Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C). The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding. Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation. Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state. Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function.

Purpose: Abnormal DNA CpG island hypermethylation and transcriptionally repressive histone modifications are associated with the aberrant silencing of tumor suppressor genes. Lysine methylation is a dynamic, enzymatically controlled process. Lysinespecific demethylase 1 (LSD1) has recently been identified as a histone lysine demethylase. LSD1 specifically catalyzes demethylation of mono-and dimethyl-lysine 4 of histone 3 (H3K4), key positive chromatin marks associated with transcriptional activation. We hypothesized that a novel class of oligoamine analogues would effectively inhibit LSD1 and thus cause the reexpression of aberrantly silenced genes. Experimental Design: Human colorectal cancer cells were treated with the oligoamines and changes in mono-and dimethyl-H3K4 and other chromatin marks were monitored. In addition, treated cells were evaluated for the reexpression of the aberrantly silenced secreted frizzled-related proteins (SFRP) Wnt signaling pathway antagonist genes. Finally, the effects of the LSD1 inhibitors were evaluated in an in vivo xenograft model. Results: Treatment of HCT116 human colon adenocarcinoma cells in vitro resulted in increased H3K4 methylation and reexpression of silenced SFRP genes. This reexpression is also accompanied by a decrease in H3K9me2 repressive mark. Importantly, cotreatment with low doses of oligoamines and a DNA methyltransferase inhibitor highly induces the reexpression of the aberrantly silenced SFRP2 gene and results in significant inhibition of the growth of established tumors in a human colon tumor model in vivo. Conclusions: The use of LSD1-inhibiting oligoamine analogues in combination with DNA methyltransferase inhibitors represents a highly promising and novel approach for epigenetic therapy of cancer. (Clin Cancer Res 2009;15(23):7217-28) Epigenetics refers to heritable changes in gene expression patterns that are not regulated by changes in the primary DNA sequence. In cancer, epigenetic silencing of gene expression, including of tumor suppressor genes, is a common occurrence (1) that is associated with abnormal DNA methylation patterns and changes in covalent histone modifications (2). The amino-terminal tails of histones are subject to several posttranslational modifications, including acetylation, phosphorylation, and methylation, which are closely tied to transcriptional regulation, DNA replication, and DNA repair (2). As shown by the regulation of histone acetylation by histone acetyltransferases and histone deacetylases (HDAC), the addition and removal of these posttranslational modifications is a dynamic process. A similar dynamic regulation occurs for histone methylation with histone methyltransferases for the addition of methyl groups, and we recently discovered families of enzymes for specific histone demethylation. The first of these demethylating enzymes identified was the lysine-specific demethylase (LSD1/KDM1; ref.3), a flavin adenine dinucleotide (FAD)-dependent amine oxidase, which interacts directly with CoREST and HDAC1/2 pro...

SUMMARY Histone modifications influence higher-order chromatin structures at individual epigenomic states and chromatin environments to regulate gene expression. However, genome-wide higher-order chromatin structures shaped by different histone modifications remain poorly characterized. With stochastic optical reconstruction microscopy (STORM), we characterized the higher-order chromatin structures at their epigenomic states, categorized into three major types in interphase: histone acetylation marks form spatially segregated nanoclusters, active histone methylation marks form spatially dispersed larger nanodomains, and repressive histone methylation marks form condensed large aggregates. These distinct structural characteristics are also observed in mitotic chromosomes. Furthermore, active histone marks coincide with less compact chromatin and exhibit a higher degree of co-localization with other active marks and RNA polymerase II (RNAP II), while repressive marks coincide with densely packed chromatin and spatially distant from repressive marks and active RNAP II. Taken together, super-resolution imaging reveals three distinct chromatin structures at various epigenomic states, which may be spatially coordinated to impact transcription.

Immunotherapy strategies have been emerging as powerful weapons against cancer. Early clinical trials reveal that overall response to immunotherapy is low in breast cancer patients, suggesting that effective strategies to overcome resistance to immunotherapy are urgently needed. In this study, we investigated whether epigenetic reprograming by modulating histone methylation could enhance effector T lymphocyte trafficking and improve therapeutic efficacy of immune checkpoint blockade in breast cancer with focus on triple-negative breast cancer (TNBC) subtype. In silico analysis of The Cancer Genome Atlas (TCGA) data shows that expression of histone lysine-specific demethylase 1 (LSD1) is inversely associated with the levels of cytotoxic T cell-attracting chemokines (C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 9 and 10 (CXCL9, CXCL10)) and programmed death-ligand 1 (PD-L1) in clinical TNBC specimens. Tiling chromatin immunoprecipitation study showed that re-expression of chemokines by LSD1 inhibition is associated with increased H3K4me2 levels at proximal promoter regions. Rescue experiments using concurrent treatment with small interfering RNA or inhibitor of chemokine receptors blocked LSD1 inhibitor-enhanced CD8+ T cell migration, indicating a critical role of key T cell chemokines in LSD1-mediated CD8+ lymphocyte trafficking to the tumor microenvironment. In mice bearing TNBC xenograft tumors, anti-PD-1 antibody alone failed to elicit obvious therapeutic effect. However, combining LSD1 inhibitors with PD-1 antibody significantly suppressed tumor growth and pulmonary metastasis, which was associated with reduced Ki-67 level and augmented CD8+ T cell infiltration in xenograft tumors. Overall, these results suggest that LSD1 inhibition may be an effective adjuvant treatment with immunotherapy as a novel management strategy for poorly immunogenic breast tumors.

The induction of polyamine catabolism and its production of H 2 O 2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N 1 -acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H 2 O 2 . However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N 1 ,N 1 -bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N 1 -acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H 2 O 2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.The natural polyamines, spermine, spermidine, and putrescine, are ubiquitous polycationic alkylamines that are required for normal eukaryotic cell growth and differentiation (1, 2). Neither mammalian cells lacking polyamine biosynthetic enzymes nor cells depleted of polyamines are able to replicate (3). Polyamine metabolism is frequently dysregulated in many types of cancer, including breast, prostate, and lung cancer (1, 4 -6). Consequently, the polyamine metabolic pathway has become an attractive target for the development of antineoplastic agents (5,7,8).Although early work focused on developing drugs that inhibited polyamine biosynthesis, more recent attention has been given to polyamine analogues that, in addition to down-regulating biosynthesis, also upregulate polyamine catabolism (9 -14). Until recently, mammalian intracellular polyamine catabolism was considered to be a consequence of two enzymes, the rate-limiting and inducible cytosolic spermidine/ spermine N 1 -acetyltransferase (SSAT) 3 and a relatively constitutively expressed, peroxisomal N 1 -acetylpolyamine oxidase (PAO) (1, 2). The products of SSAT/PAO activities on spermine and spermidine are the reactive oxygen species, H 2 O 2 , spermidine, and putrescine, re...

Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.

Estrogen receptors (ER) are expressed in f65% of human breast cancer. Cumulative data from clinical trials and retrospective analyses suggest that some chemotherapeutic agents may be less effective in patients with ER-positive (ER+) tumors than those with ER-negative (ERÀ) tumors. Paclitaxel is an active agent used in breast cancer chemotherapy. To investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and its underlying mechanism, we established several isogenic ER+ cell lines by stable transfection of ERA expression vectors into ERÀ breast cancer BCap37 cells. We showed that 17-B estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERA but has no influence on the ERÀ parental cells. Further analyses indicate that expression of ERA in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, without affecting paclitaxel-induced microtubule bundling and mitotic arrest. Moreover, we found that the addition of ICI 182,780 (Fulvestrant), a selective ER down-regulator, could completely reverse the resistance of ER+ BCap37 cells to paclitaxel. These findings showed that ERA-mediated breast tumor cell resistance to paclitaxel was through selective inhibition of paclitaxel-induced tumor cell apoptosis. Additionally, the combination of ICI 182,780 also sensitizes MCF-7 and T47D cell lines to the treatment of paclitaxel, which further confirmed the correlation between ERA and drug resistance in ER+ tumor cells. The results obtained from this study provide useful information for understanding ERmediated resistance to paclitaxel and possibly other antineoplastic agents. [Cancer Res 2007;67(11):5337-44]