[Show abstract][Hide abstract] ABSTRACT: The mitotic checkpoint protein CHFR has emerged as a major mediator of taxane resistance in cancer. Here we show that CHFR's PAR-binding zinc finger domain (PBZ) mediates a protein interaction with poly-ADP ribosylated PARP1 leading to stabilization of CHFR. Disruption of the CHFR-PARP1 interaction through either PARP1 shRNA-mediated knockdown or overexpression of a PBZ domain peptide induces loss of CHFR protein expression. In an attempt to exploit this observation therapeutically, and to develop compounds with synthetic lethality in combination with taxanes, we performed a high-throughput computational screen of 5,256,508 chemical structures against the published crystal structure of the CHFR PBZ domain to identify candidate small molecule CHFR protein-protein interaction inhibitors. The 10 compounds with the best docking scores (< -9.7) were used for further in vitro testing. One lead compound in particular, termed 'A3', completely disrupted the protein-protein interaction between CHFR and PARP1, resulting in the inhibition of mitotic checkpoint function, and led to therapeutic synergy with docetaxel in cell viability and colony formation assays. In mouse xenografts, i.p. administration of 'A3' led to a significant reduction in nuclear CHFR protein expression with a maximal effect 4 hours after administration, confirming relevant pharmacodynamics following the peak of 'A3' plasma concentration in vivo. Furthermore, combination of A3 and taxane led to significant reduction of implanted tumor size without increase in hematological, hepatic or renal toxicity. These findings provide a proof-of-principle that small molecule inhibition of CHFR PBZ domain interaction is a novel potential therapeutic approach to increase the efficacy of taxane-based chemotherapy in cancer.
[Show abstract][Hide abstract] ABSTRACT: CpG islands (CGI) are associated with over half of human gene promoters and are characterized by a unique chromatin environment and high levels of bidirectional transcriptional activity relative to surrounding genomic regions, suggesting that RNA polymerase (Pol II) progression past the CGI boundaries is restricted. Here we describe a novel transcriptional regulatory step wherein Pol II encounters an additional barrier to elongation distinct from the promoter-proximal pause and occurring at the downstream boundary of the CGI domain. For most CGI associated promoters, Pol II exhibits a dominant pause at either the promoter-proximal or this distal site that correlates, both in position and in intensity, with local regions of high GC skew, a sequence feature known to form unique secondary structures. Upon signal-induced gene activation, long-range enhancer contacts at the dominant pause site are selectively enhanced, suggesting a new role for enhancers at the downstream pause. These data point to an additional level of control over transcriptional output at a subset of CGI associated genes that is linked to DNA sequence and the integrity of the CGI domain.
Published by Cold Spring Harbor Laboratory Press.
Genome Research 08/2015; 25(11). DOI:10.1101/gr.189068.114 · 14.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.Oncogene advance online publication, 20 April 2015; doi:10.1038/onc.2015.103.
[Show abstract][Hide abstract] ABSTRACT: Chronic liver inflammation precedes the majority of hepatocellular carcinomas (HCC). Here, we explore the connection between chronic inflammation and DNA methylation in the liver at the late precancerous stages of HCC development in Mdr2-/- (Mdr2/Abcb4-knockout) mice, a model of inflammation-mediated HCC. Using methylated DNA immunoprecipitation followed by hybridization with "CpG islands" (CGIs) microarrays, we found specific CGIs in 76 genes which were hypermethylated in the Mdr2-/- liver compared to age-matched healthy controls. The observed hypermethylation resulted mainly from an age-dependent decrease of methylation of the specific CGIs in control livers with no decrease in mutant mice. Chronic inflammation did not change global levels of DNA methylation in Mdr2-/- liver, but caused a 2-fold decrease of the global 5-hydroxymethylcytosine level in mutants compared to controls. Liver cell fractionation revealed, that the relative hypermethylation of specific CGIs in Mdr2-/- livers affected either hepatocyte, or non-hepatocyte, or both fractions without a correlation between changes of gene methylation and expression. Our findings demonstrate that chronic liver inflammation causes hypermethylation of specific CGIs, which may affect both hepatocytes and non-hepatocyte liver cells. These changes may serve as useful markers of an increased regenerative activity and of a late precancerous stage in the chronically inflamed liver.
[Show abstract][Hide abstract] ABSTRACT: We present a pipeline to perform integrative analysis of mate-pair (MP) and paired-end (PE) genomic DNA sequencing data. Our pipeline detects structural variations (SVs) by taking aligned sequencing read pairs as input and classifying these reads into properly paired and discordantly paired categories based on their orientation and inferred insert sizes. Recurrent SV was identified from the discordant read pairs. Our pipeline takes into account genomic annotation and genome repetitive element information to increase detection specificity. Application of our pipeline to whole-genome MP and PE sequencing data from three multiple myeloma cell lines (KMS11, MM.1S, and RPMI8226) recovered known SVs, such as heterozygous TRAF3 deletion, as well as a novel experimentally validated SPI1 - ZNF287 inter-chromosomal rearrangement in the RPMI8226 cell line.
Cancer informatics 09/2014; 13(Suppl 2):49-53. DOI:10.4137/CIN.S13783
[Show abstract][Hide abstract] ABSTRACT: Purpose
Aberrant promoter DNA methylation can serve as a predictive biomarker for improved clinical responses to certain chemotherapeutics. One of the major advantages of methylation biomarkers is the ease of detection and clinical application. In order to identify methylation biomarkers predictive of a response to a taxane-platinum based chemotherapy regimen in advanced NSCLC we performed an unbiased methylation analysis of 1,536 CpG dinucleotides in cancer-associated gene loci and correlated results with clinical outcomes.
We studied a cohort of 49 patients (median age 62 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. Methylation analysis was done on the Illumina GoldenGate Cancer panel 1 methylation microarray platform. Methylation data were correlated with clinical response and adjusted for false discovery rates.
Cav1 methylation emerged as a powerful predictor for achieving disease stabilization following platinum taxane based chemotherapy (p = 1.21E-05, FDR significance = 0.018176). In Cox regression analysis after multivariate adjustment for age, performance status, gender, histology and the use of bevacizumab, CAV1 methylation was significantly associated with improved overall survival (HR 0.18 (95%CI: 0.03–0.94)). Silencing of CAV1 expression in lung cancer cell lines(A549, EKVX)by shRNA led to alterations in taxane retention.
CAV1 methylation is a predictor of disease stabilization and improved overall survival following chemotherapy with a taxane-platinum combination regimen in advanced NSCLC. CAV1 methylation may predict improved outcomes for other chemotherapeutic agents which are subject to cellular clearance mediated by caveolae.
PLoS ONE 09/2014; 9(9):e107124. DOI:10.1371/journal.pone.0107124 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary
to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing
mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage
in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain
insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking
can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were
used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further
investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring
a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive
off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing
potential off-target cleavage.
Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku402 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammation has been associated with fatigue during and after various types of breast cancer treatments. We examined whether prior chemotherapy was associated with DNA methylation patterns that could explain persisting inflammation and/or fatigue in women treated for breast cancer. Prior to breast radiation therapy, DNA was extracted from peripheral blood mononuclear cells (PBMCs) of 61 Stage 0-IIIA breast cancer patients who had received partial mastectomy with or without chemotherapy. DNA methylation was assessed at >485,000 CpG sites across the genome along with fatigue and plasma inflammatory markers previously associated with fatigue. Compared to non-chemotherapy-treated women, women who had received chemotherapy exhibited significantly decreased methylation at eight CpG sites (p<1.03x10-7) including four in exon 11 of transmembrane protein 49 (TMEM49), which demonstrated the largest decreases in methylation. Lower methylation at each identified CpG site was associated with increased plasma soluble tumor necrosis factor receptor 2 (sTNFR2) and interleukin (IL)-6 and mediated the relationship between chemotherapy and increases in these inflammatory biomarkers adjusting for multiple clinical and treatment characteristics. sTNFR2, but not CpG methylation status, was correlated with fatigue. Six months after breast radiation therapy, DNA methylation, inflammatory biomarkers and fatigue assessments were repeated in a subset of subjects (N=39). Reduced methylation in 4 of the 8 identified CpG sites was still observed in chemotherapy versus non-chemotherapy-treated patients, albeit with some decay indicating the dynamic and potentially reversible nature of the changes. Reduced methylation in these 4 CpG sites also continued to correlate with either increased sTNFR2 or IL-6, but not fatigue. In conclusion, prior chemotherapy treatment was associated with decreased methylation of CpG sites in DNA from PBMCs of breast cancer patients, which correlated with increased inflammatory markers prior to and 6 months after radiation therapy. Persisting epigenetic changes secondary to chemotherapy may be one factor that contributes to inflammation and its consequences including cancer-related fatigue in vulnerable breast cancer patients.
[Show abstract][Hide abstract] ABSTRACT: RNA polymerase II (Pol II) promoter-proximal pausing plays a critical role in postinitiation transcriptional regulation at
many metazoan genes. We showed recently that histone H4 lysine 16 acetylation (H4K16Ac), mediated by the MSL complex, facilitates
the release of paused Pol II. In contrast, H4 lysine 20 trimethylation (H4K20me3), mediated by SUV420H2, enforces Pol II pausing
by inhibiting MSL recruitment. However, how the balance between H4K16Ac and H4K20me3 is locally regulated remains unclear.
Here, we demonstrate that PR-SET7/SETD8, which monomethylates histone H4 lysine 20 (H4K20me1), controls both H4K16Ac and H4K20me3
and in doing so, regulates Pol II pausing dynamics. We find that PR-SET7-mediated H4K20me1 is necessary for the recruitment
of the MSL complex, subsequent H4K16Ac, and release of Pol II into active elongation. Although dispensable for SUV420H2 recruitment,
PR-SET7-mediated H4K20me1 is required for H4K20me3. Although depletion of SUV420H2 is sufficient to deplete H4K20me3 and relieve
an H4K20me3-induced pause, pausing is maintained in the absence of PR-SET7 despite H4K20me3 depletion because of an inability
to recruit the MSL complex in the absence of H4K20me1. These findings highlight the requirement for PR-SET7 and H4K20me1 in
establishing both the H4K16Ac and H4K20me3 marks and point to a dual role in the local regulation of Pol II pausing.
Journal of Biological Chemistry 01/2014; DOI:10.1074/jbc.M113.520783 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDACs) 1-3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell cycle independent increased DNMT1 stability and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter and de-repression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability.
Cancer Prevention Research 01/2014; 7(3). DOI:10.1158/1940-6207.CAPR-13-0254 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer cells. Withaniasomnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A, inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin inhibitory activity. To determine if sWRE inhibited EMT, TGF-β was used to induce EMT in MCF10A human mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.
PLoS ONE 09/2013; 8(9):e75069. DOI:10.1371/journal.pone.0075069 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian target of Rapamycin (mTOR) is aberrant activated in many cancer types, and two Rapamycin derivatives are currently approved by FDA for the treatment of renal cell carcinoma. Mechanistically, mTOR is hyperactivated in human cancers either due to the genetic activation of its upstream activating signaling pathways or the genetic inactivation of its negative regulators. The tumor suppressor liver kinase B1 (LKB1; also known as serine/threonine kinase 11, STK11) involves in cell polarity, cell detachment and adhesion, tumor metastasis, and energetic stress response. One of LKB1's key roles is to negatively regulate mTOR complex 1 (mTORC1) activity. This review summarizes the molecular basis of this negative interaction, and recent research progress in this area.
Ai zheng = Aizheng = Chinese journal of cancer 05/2013; 32(8). DOI:10.5732/cjc.013.10086 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2,010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors, as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon pre-existing DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes downregulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.
Genome Research 08/2012; 22(12). DOI:10.1101/gr.132738.111 · 14.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this issue of Molecular Cell, Ginno et al. (2012) describe unusual sequence features at promoter CpG islands that can lead to formation of persistent RNA-DNA hybrids (R loops), which are proposed to prevent genomic DNA methylation.
[Show abstract][Hide abstract] ABSTRACT: Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.
Nucleic Acids Research 02/2012; 40(11):4841-9. DOI:10.1093/nar/gks155 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition and reduced patient survival. As vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data showed that WFA had weak cytotoxic and apoptotic activity at concentrations less than or equal to 500 nM, but retained potent anti-invasive activity at these low doses. Imaging of breast cancer cell lines revealed that WFA induces perinuclear vimentin accumulation followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin-binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti-invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2 μM in plasma with a half-life of 1.36 hr following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose-dependent inhibition of metastatic lung nodules and induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti-metastatic agent and the anti-metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.
International Journal of Cancer 12/2011; 129(11):2744-55. DOI:10.1002/ijc.25938 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brain angiogenesis inhibitor 1 (BAI1) is a putative G protein-coupled receptor with potent antiangiogenic and antitumorigenic properties that is mutated in certain cancers. BAI1 is expressed in normal human brain, but it is frequently silenced in glioblastoma multiforme. In this study, we show that this silencing event is regulated by overexpression of methyl-CpG-binding domain protein 2 (MBD2), a key mediator of epigenetic gene regulation, which binds to the hypermethylated BAI1 gene promoter. In glioma cells, treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-Aza-dC) was sufficient to reactivate BAI1 expression. Chromatin immunoprecipitation showed that MBD2 was enriched at the promoter of silenced BAI1 in glioma cells and that MBD2 binding was released by 5-Aza-dC treatment. RNA interference-mediated knockdown of MBD2 expression led to reactivation of BAI1 gene expression and restoration of BAI1 functional activity, as indicated by increased antiangiogenic activity in vitro and in vivo. Taken together, our results suggest that MBD2 overexpression during gliomagenesis may drive tumor growth by suppressing the antiangiogenic activity of a key tumor suppressor. These findings have therapeutic implications because inhibiting MBD2 could offer a strategy to reactivate BAI1 and suppress glioma pathobiology.
Cancer Research 07/2011; 71(17):5859-70. DOI:10.1158/0008-5472.CAN-11-1157 · 9.33 Impact Factor