Accelerated aging in normal breast tissue of women with breast cancer.

BACKGROUND: DNA methylation alterations have similar patterns in normal aging tissue and in cancer. In this study, we investigated breast tissue-specific age-related DNA methylation alterations and used those methylation sites to identify individuals with outlier phenotypes. Outlier phenotype is identified by unsupervised anomaly detection algorithms and is defined by individuals who have normal tissue age-dependent DNA methylation levels that vary dramatically from the population mean. METHODS: We generated whole-genome DNA methylation profiles (GSE160233) on purified epithelial cells and used publicly available Infinium HumanMethylation 450K array datasets (TCGA, GSE88883, GSE69914, GSE101961, and GSE74214) for discovery and validation. RESULTS: We found that hypermethylation in normal breast tissue is the best predictor of hypermethylation in cancer. Using unsupervised anomaly detection approaches, we found that about 10% of the individuals (39/427) were outliers for DNA methylation from 6 DNA methylation datasets. We also found that there were significantly more outlier samples in normal-adjacent to cancer (24/139, 17.3%) than in normal samples (15/228, 5.2%). Additionally, we found significant differences between the predicted ages based on DNA methylation and the chronological ages among outliers and not-outliers. Additionally, we found that accelerated outliers (older predicted age) were more frequent in normal-adjacent to cancer (14/17, 82%) compared to normal samples from individuals without cancer (3/17, 18%). Furthermore, in matched samples, we found that the epigenome of the outliers in the pre-malignant tissue was as severely altered as in cancer. CONCLUSIONS: A subset of patients with breast cancer has severely altered epigenomes which are characterized by accelerated aging in their normal-appearing tissue. In the future, these DNA methylation sites should be studied further such as in cell-free DNA to determine their potential use as biomarkers for early detection of malignant transformation and preventive intervention in breast cancer.

c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution.

Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity.

Structure and dynamic regulation of Abl kinases.

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity.

Enhanced SH3/linker interaction overcomes Abl kinase activation by gatekeeper and myristic acid binding pocket mutations and increases sensitivity to small molecule inhibitors.

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

The Roles of DNA Demethylases in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) are very heterogenous, molecularly diverse, and are characterized by a high propensity to relapse or metastasize. Clinically, TNBC remains a diagnosis of exclusion by the lack of hormone receptors (Estrogen Receptor (ER) and Progesterone Receptor (PR)) as well as the absence of overexpression and/or amplification of HER2. DNA methylation plays an important role in breast cancer carcinogenesis and TNBCs have a distinct DNA methylation profile characterized by marked hypomethylation and lower gains of methylations compared to all other subtypes. DNA methylation is regulated by the balance of DNA methylases (DNMTs) and DNA demethylases (TETs). Here, we review the roles of TETs as context-dependent tumor-suppressor genes and/or oncogenes in solid tumors, and we discuss the current understandings of the oncogenic role of TET1 and its therapeutic implications in TNBCs.

N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor.

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH(2)-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH(2)-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

De novo N-palmitoylsphingosine synthesis is the major biochemical mechanism of ceramide accumulation following p53 up-regulation.

The tumor suppressor protein p53 and the putative lipid tumor suppressor ceramide play pivotal roles in inducing cell cycle arrest or in driving the cell towards apoptosis. Previously we had shown that, in a p53-dependent model of cell death, ceramide accumulated in a p53-dependent manner [Dbaibo GS, Pushkareva MY, Rachid RA, Alter N, Smyth MJ, Obeid LM, Hannun YA. J Clin Invest 1998;102:329-339]. In the current study, we investigated the biochemical pathways by which ceramide accumulated following p53 up-regulation. In both Molt-4 LXSN leukemia cells exposed to gamma-irradiation and in EB-1 colon cancer cells treated with ZnCl(2), p53 up-regulation led to de novo ceramide synthesis with predominance of N-palmitoylsphingosine (C16-ceramide) synthesis. The activation of the de novo pathway was not associated with increased activity of the key enzyme serine palmitoyltransferase (SPT) but rather with the increased activity of ceramide synthase. Furthermore, transcriptional up-regulation of the palmitoyl-specific Lass5 ceramide synthase gene was observed in Molt-4 but not in EB-1 cells. The SPT inhibitor ISP-1 or the ceramide synthase inhibitor fumonisin B1 led to substantial inhibition of ceramide accumulation in response to p53 up-regulation. Other biochemical pathways of ceramide generation such as sphingomyelinase activation were examined and found unlikely to contribute to p53-dependent ceramide formation. These studies indicate that p53 specifically drives de novo ceramide synthesis by activation of a ceramide synthase that favors the synthesis of N-palmitoylsphingosine.

TET1-Mediated Hypomethylation Activates Oncogenic Signaling in Triple-Negative Breast Cancer.

Both gains and losses of DNA methylation are common in cancer, but the factors controlling this balance of methylation remain unclear. Triple-negative breast cancer (TNBC), a subtype that does not overexpress hormone receptors or HER2/NEU, is one of the most hypomethylated cancers observed. Here, we discovered that the TET1 DNA demethylase is specifically overexpressed in about 40% of patients with TNBC, where it is associated with hypomethylation of up to 10% of queried CpG sites and a worse overall survival. Through bioinformatic analyses in both breast and ovarian cancer cell line panels, we uncovered an intricate network connecting TET1 to hypomethylation and activation of cancer-specific oncogenic pathways, including PI3K, EGFR, and PDGF. TET1 expression correlated with sensitivity to drugs targeting the PI3K-mTOR pathway, and CRISPR-mediated deletion of TET1 in two independent TNBC cell lines resulted in reduced expression of PI3K pathway genes, upregulation of immune response genes, and substantially reduced cellular proliferation, suggesting dependence of oncogenic pathways on TET1 overexpression. Our work establishes TET1 as a potential oncogene that contributes to aberrant hypomethylation in cancer and suggests that TET1 could serve as a druggable target for therapeutic intervention.Significance: This study addresses a critical gap in knowledge of how and why methylation is prognostic in breast cancer and shows how this information can be used to stratify patients with TNBC for targeted therapy. Cancer Res; 78(15); 4126-37. ©2018 AACR.

Arsenic trioxide induces accumulation of cytotoxic levels of ceramide in acute promyelocytic leukemia and adult T-cell leukemia/lymphoma cells through de novo ceramide synthesis and inhibition of glucosylceramide synthase activity.

BACKGROUND AND OBJECTIVES: Arsenic trioxide (ATO) is an effective treatment for acute promyelocytic leukemia (APL) and potentially for human T-cell leukemia virus type I (HTLV-I) associated adult T-cell leukemia/lymphoma (ATL). Many cytotoxic drugs induce apoptosis through the generation and accumulation of the sphingolipid breakdown product, ceramide, a coordinator of the cellular response to stress. We, therefore, investigated the contribution of ceramide to the mechanism of action of ATO in APL and ATL. DESIGN AND METHODS: A human APL-derived cell line (NB4), various ATL-derived lines and an HTLV-I-negative malignant T-cell line were cultured and treated with ATO. Growth and apoptosis assays were conducted. Measurements were made of ceramide, diacylglycerol, sphingomyelinase activity, sphingomyelin mass, glucosylceramide synthase activity and the de novo ceramide synthesis. RESULTS: Treatment of APL and ATL-derived cells with a clinically achievable concentration of ATO induced accumulation of cytotoxic levels of ceramide. The effects of ATO on ceramide levels in APL cells were more potent than those of all-trans retinoic acid (ATRA). ATO downregulated neutral sphingomyelinase activity. In contrast to the effect of ATRA, ATO-induced ceramide accumulation was not due to induction of acidic sphingomyelinase, but rather resulted from both de novo ceramide synthesis and inhibition of glucosylceramide synthase activity. Interestingly, the effects of ATO on de novo ceramide synthesis were similar in APL and ATL-derived cells despite the defective pathway in ATL cells. INTERPRETATION AND CONCLUSIONS: These results indicate that ATO-induced ceramide accumulation may represent a general mediator of the effects of ATO, which paves the way for new therapeutic interventions that target the metabolic pathway of this important sphingolipid secondary messenger.

Transcriptional Selectivity of Epigenetic Therapy in Cancer.

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.

Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia.

BACKGROUND: The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1beta and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2). METHODS: Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1beta expression. RESULTS: TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1beta expression was significantly increased at 14 days. CONCLUSION: Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

Ceramide and glutathione define two independently regulated pathways of cell death initiated by p53 in Molt-4 leukaemia cells.

The tumour suppressor p53 induces cell death by launching several pathways that are either dependent on or independent of gene transcription. Accumulation of the sphingolipid ceramide and reactive oxygen species are among these pathways. Crossregulation of these two pathways is possible owing to the demonstrated inhibition of neutral sphingomyelinase by glutathione, the predominant cellular antioxidant, and has been observed in some cytokine-dependent cell-death models. In a model of irradiation-induced cell death of Molt-4 leukaemia cells, it was found that ceramide accumulation and glutathione depletion were dependent on p53 up-regulation. The loss of p53 owing to expression of the papilloma virus E6 protein inhibited both pathways after irradiation. However, in this model, these two pathways appeared to be independently regulated on the basis of the following observations: (1) glutathione supplementation or depletion did not alter irradiation-induced ceramide accumulation, (2) exogenous ceramide treatment did not induce glutathione depletion, (3) glutathione depletion was dependent on new protein synthesis, whereas ceramide accumulation was independent of it and (4) caspase activation was required for ceramide accumulation but not for glutathione depletion. Furthermore, caspase 9 activation, which is dependent on the release of mitochondrial cytochrome c, was not required for ceramide accumulation. This suggested that a caspase, other than caspase 9, was necessary for ceramide accumulation. Interestingly, Bcl-2 expression inhibited these pathways, indicating a possible role for mitochondria in regulating both pathways. These findings indicate that these two pathways exhibit cross-regulation in cytokine-dependent, but not in p53-dependent, cell-death models.

Architecture of epigenetic reprogramming following Twist1-mediated epithelial-mesenchymal transition.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is known to impart metastasis and stemness characteristics in breast cancer. To characterize the epigenetic reprogramming following Twist1-induced EMT, we characterized the epigenetic and transcriptome landscapes using whole-genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. RESULTS: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRα and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the Polycomb repressive complex 2 (PRC2), blocks EMT and stemness properties. CONCLUSIONS: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb and Trithorax complexes leading to increased cellular plasticity. This suggests that inhibiting epigenetic remodeling and thus decrease plasticity will prevent EMT, and the associated breast cancer metastasis.