The development of drug resistance represents a major complication in the effective treatment of breast cancer. Epigenetic therapy, through the use of histone deacetylase inhibitors (HDACi) or demethylation agents, is an emerging area of therapeutic targeting in a number of ontological entities, particularly in the setting of aggressive therapy-resistant disease. Using the well-described HDAC inhibitor trichostatin A (TSA) we demonstrate the suppression of in vitro clonogenicity in the previously described apoptosis-resistant MCF-7TN-R breast carcinoma cell line. Additionally, recent work has demonstrated that these agents can alter the expression profile of microRNA signatures in malignant cells. Using an unbiased microRNA microarray analysis, changes in miRNA expression of MCF-7TN-R cells treated with TSA for 24 h were analyzed. We observed significant up-regulation of 22 miRNAs and down-regulation of 10 miRNAs in response to TSA treatment. Our results demonstrate that the HDACi, TSA, exerts anticancer activity in the apoptosis-resistant MCF-7TN-R breast carcinoma cell line. This activity is correlated with TSA alteration of microRNA expression profiles indicative of a less aggressive phenotype.
microRNA; trichostatin A; histone deacetylase; MCF-7; breast cancer; drug resistance
ovarian cancer; epigenetics; EZH2; histone methyltransferase; 3D culture; H3K27; ECM
In prostate cancer, androgen receptor (AR) binding and androgen-responsive gene expression are defined by hormone-independent binding patterns of the pioneer factors FoxA1 and GATA2. Insufficient evidence of the mechanisms by which GATA2 contributes to this process precludes complete understanding of a key determinant of tissue-specific AR activity. Our observations suggest that GATA2 facilitates androgen-responsive gene expression by three distinct modes of action. By occupying novel binding sites within the AR gene locus, GATA2 positively regulates AR expression before and after androgen stimulation. Additionally, GATA2 engages AR target gene enhancers prior to hormone stimulation, producing an active and accessible chromatin environment via recruitment of the histone acetyltransferase p300. Finally, GATA2 functions in establishing and/or sustaining basal locus looping by recruiting the Mediator subunit MED1 in the absence of androgen. These mechanisms may contribute to the generally positive role of GATA2 in defining AR genome-wide binding patterns that determine androgen-responsive gene expression profiles. We also find that GATA2 and FoxA1 exhibit both independent and codependent co-occupancy of AR target gene enhancers. Identifying these determinants of AR transcriptional activity may provide a foundation for the development of future prostate cancer therapeutics that target pioneer factor function.
Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-Xl/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells. In breast cancer lines, regardless of elevated ERBB1/2 expression, GX15-070 enhanced Lapatinib toxicity within 3–12 h.The promotion of Lapatinib toxicity neither correlated with cleavage of caspase 3 nor was blocked by inhibition caspases; and was not associated with changes in the activities of ERK1/2, JNK1/2 or p38 MAPK but with reduced AKT, mTOR and S6K1 phosphorylation. The promotion of Lapatinib toxicity by GX15-070 correlated with increased cytosolic levels of apoptosis inducing factor (AIF) and expression of ATG8 (LC3), and the formation of large vesicles that intensely stained for a transfected LC3-GFP construct. Knockdown of the autophagy regulatory proteins ATG5 or Beclin1 suppressed the induction of LC3-GFP vesicularization and significantly reduced cell killing, whereas knock down of MCL-1 and BCL-Xl enhanced the induction of LC3-GFP vesicularization and significantly enhanced cell killing. Knockdown of Beclin1 and AIF abolished cell killing. Collectively, our data demonstrate that Obatoclax mediated inhibition of MCL-1 rapidly enhances Lapatinib toxicity in tumor cells via a toxic form of autophagy and via AIF release from the mitochondrion.
lapatinib; obatoclax; autophagy; cell death; resistance
Aberrant DNA methylation of CpG islands, CpG island shores and first exons is known to play a key role in the altered gene expression patterns in all human cancers. To date, a systematic study on the effect of DNA methylation on gene expression using high resolution data has not been reported. In this study, we conducted an integrated analysis of MethylCap-sequencing data and Affymetrix gene expression microarray data for 30 breast cancer cell lines representing different breast tumor phenotypes. As well-developed methods for the integrated analysis do not currently exist, we created a series of four different analysis methods. On the computational side, our goal is to develop methylome data analysis protocols for the integrated analysis of DNA methylation and gene expression data on the genome scale. On the cancer biology side, we present comprehensive genome-wide methylome analysis results for differentially methylated regions and their potential effect on gene expression in 30 breast cancer cell lines representing three molecular phenotypes, luminal, basal A and basal B. Our integrated analysis demonstrates that methylation status of different genomic regions may play a key role in establishing transcriptional patterns in molecular subtypes of human breast cancer.
Preclinical studies have shown that hypomethylating agents reverse platinum resistance in
ovarian cancer. In this phase II clinical trial, based upon the results of our phase I dose defining
study, we tested the clinical and biologic activity of low-dose decitabine administered before
carboplatin in platinum-resistant ovarian cancer patients. Among 17 patients with heavily pretreated
and platinum-resistant ovarian cancer, the regimen induced a 35% objective response rate
(RR) and progression-free survival (PFS) of 10.2 months, with nine patients (53%) free of
progression at 6 months. Global and gene-specific DNA demethylation was achieved in peripheral blood
mononuclear cells and tumors. The number of demethylated genes was greater (P <
0.05) in tumor biopsies from patients with PFS more than 6 versus less than 6 months (311 vs. 244
genes). Pathways enriched at baseline in tumors from patients with PFS more than 6 months included
cytokine–cytokine receptor interactions, drug transporters, and mitogen-activated protein
kinase, toll-like receptor and Jak-STAT signaling pathways, whereas those enriched in demethylated
genes after decitabine treatment included pathways involved in cancer, Wnt signaling, and apoptosis
(P < 0.01). Demethylation of MLH1, RASSF1A, HOXA10, and
HOXA11 in tumors positively correlated with PFS (P < 0.05).
Together, the results of this study suggest that low-dose decitabine altered DNA methylation of
genes and cancer pathways, restoring sensitivity to carboplatin in patients with heavily pretreated
ovarian cancer and resulting in a high RR and prolonged PFS.
Previously, we demonstrated potent antineoplastic activity of a distinctive histone deacetylase inhibitor (HDACI), AR42, against chemoresistant CP70 ovarian cancer cells in vitro and in vivo. Here, in follow-up to that work, we explored AR42 global mechanisms-of-action by examining drug-associated, genome-wide microRNA and mRNA expression profiles, which differed from those of the well-studied HDACI vorinostat. Expression of microRNA genes in negative correlation with their “target” coding gene (mRNA) transcripts, and transcription factor genes with expression positively correlated with coding genes having their cognate binding sites, were identified and subjected to gene ontology analyses. Those evaluations showed AR42 gene expression patterns to negatively correlate with Wnt signaling (> 18-fold induction of SFRP1), the epithelial-to-mesenchymal transition (40% decreased ATF1), and cell cycle progression (33-fold increased 14-3-3σ). By contrast, AR42 transcriptome alterations correlated positively with extrinsic (“death receptor”) apoptosis (> 2.3-fold upregulated DAPK) and favorable ovarian cancer histopathology and prognosis. Inhibition of Wnt signaling was experimentally validated by: (1) > 2.6-fold reduced Wnt reporter activity; and (2) 36% reduction in nuclear, activated β-catenin. Likely AR42 induction of multiple (type I or type II autophagic) cell death cascades was further supported by 57% decreased reliance upon reactive oxygen, increased mitochondrial membrane disruption, and caspase independence, as compared with vorinostat. Taken together, we demonstrate distinct antineoplastic pathway alterations, in aggressive ovarian cancer cells, following treatment with a promising HDACI, AR42. These combined computational and experimental approaches may also represent a straightforward means for mechanistic studies of other promising antineoplastics, and/or the identification of agents that may complement epigenetic therapies.
epithelial-to-mesenchymal transition; histone deacetylase inhibitor; microRNA; ovarian cancer; Wnt signaling
Acquired chemoresistance and epithelial-to-mesenchymal transition (EMT) are hallmarks of cancer progression and of increasing clinical relevance. We investigated the role of miRNA and p38 mitogen-activated protein kinase (MAPK) signaling in the progression of breast cancer to a drug-resistant and mesenchymal phenotype. We demonstrate that acquired death receptor resistance results in increased hormone-independent tumorigenesis compared to hormone-sensitive parental cells. Utilizing global miRNA gene expression profiling, we identified miRNA alterations associated with the development of death receptor resistance and EMT progression. We further investigated the role of p38 MAPK in this process, showing dose-dependent inactivation of p38 by its inhibitor RWJ67657 and decreased downstream ATF and NF-κB signaling. Pharmacological inhibition of p38 also decreased chemoresistant cancer tumor growth in xenograft animal models. Interestingly, inhibition of p38 partially reversed the EMT changes found in this cell system, as illustrated by decreased gene expression of the EMT markers Twist, Snail, Slug and ZEB and protein and mRNA levels of Twist, a known EMT promoter, concomitant with decreased N-cadherin protein. RWJ67657 treatment also altered the expression of several miRNAs known to promote therapeutic resistance, including miR-200, miR-303, miR-302, miR-199 and miR-328. Taken together, our results demonstrate the roles of multiple microRNAs and p38 signaling in the progression of cancer and demonstrate the therapeutic potential of targeting the p38 MAPK pathway for reversing EMT in an advanced tumor phenotype.
p38 mitogen-activated protein kinase; epithelial-tomesenchymal transition; breast cancer; drug discovery
A major goal of the field of systems biology is to translate genome-wide profiling data (e.g., mRNAs, miRNAs) into interpretable functional networks. However, employing a systems biology approach to better understand the complexities underlying drug resistance phenotypes in cancer continues to represent a significant challenge to the field. Previously, we derived two drug-resistant breast cancer sublines (tamoxifen- and fulvestrant-resistant cell lines) from the MCF7 breast cancer cell line and performed genome-wide mRNA and microRNA profiling to identify differential molecular pathways underlying acquired resistance to these important antiestrogens. In the current study, to further define molecular characteristics of acquired antiestrogen resistance we constructed an “integrative network”. We combined joint miRNA-mRNA expression profiles, cancer contexts, miRNA-target mRNA relationships, and miRNA upstream regulators. In particular, to reduce the probability of false positive connections in the network, experimentally validated, rather than prediction-oriented, databases were utilized to obtain connectivity. Also, to improve biological interpretation, cancer contexts were incorporated into the network connectivity.
Based on the integrative network, we extracted “substructures” (network clusters) representing the drug resistant states (tamoxifen- or fulvestrant-resistance cells) compared to drug sensitive state (parental MCF7 cells). We identified un-described network clusters that contribute to antiestrogen resistance consisting of miR-146a, -27a, -145, -21, -155, -15a, -125b, and let-7s, in addition to the previously described miR-221/222.
By integrating miRNA-related network, gene/miRNA expression and text-mining, the current study provides a computational-based systems biology approach for further investigating the molecular mechanism underlying antiestrogen resistance in breast cancer cells. In addition, new miRNA clusters that contribute to antiestrogen resistance were identified, and they warrant further investigation.
Bioinformatics; miRNA; Network; Breast cancer; Antiestrogen resistance
Androgen receptor (AR) is important for prostate cancer development and progression. Genome-wide mapping of AR binding sites in prostate cancer have found that the majority of AR binding sites are located within non-promoter regions. These distal AR binding regions regulate AR target genes (e.g. UBE2C) involved in prostate cancer growth through chromatin looping. In addition to long-distance gene regulation, looping has been shown to induce spatial proximity of two genes otherwise located far away along the genomic sequence and the formation of double strand DNA breaks, resulting in aberrant gene fusions (e.g. TMPRSS2-ERG) that also contribute to prostate tumorigenesis. Elucidating the mechanisms of AR-driven chromatin looping will increase our understanding of prostate carcinogenesis and may lead to the identification of new therapeutic targets.
We determined the efficacy, biological activity, pharmacokinetics and safety of the hypomethylating agent 5-azacitidine (Celgene Corp., Summit, New Jersey) in dogs with naturally occurring invasive urothelial carcinoma.
Materials and Methods
We performed a preclinical phase I trial in dogs with naturally occurring invasive urothelial carcinoma to examine once daily subcutaneous administration of 5-azacitidine in 28-day cycles at doses of 0.10 to 0.30 mg/kg per day according to 2 dose schedules, including days 1 to 5 (28-day cohort) or days 1 to 5 and 15 to 19 (14-day cohort). Clinical efficacy was assessed by serial cystosonography, radiography and cystoscopy. Urinary 5-azacitidine pharmacokinetic analysis was also done. Pretreatment and posttreatment peripheral blood mononuclear cell and invasive urothelial carcinoma DNA, respectively, was analyzed for global and gene specific [CDKN2A (p14ARF)] methylation changes.
Enrolled in the study were 19 dogs with naturally occurring invasive urothelial carcinoma. In the 28-day cohort the maximum tolerated dose was 0.20 mg/kg per day with higher doses resulting in grade 3 or 4 neutropenia in 4 of 6 dogs. In the 14-day cohort the maximum tolerated dose was 0.10 mg/kg per day with grade 3 or 4 neutropenia seen in 2 of 3 dogs treated at higher doses. No grade 3 or 4 nonhematological toxicity was observed during either dosing schedule. Of 18 dogs evaluable for tumor response partial remission, stable disease and progressive disease were observed in 4 (22.2%), 9 (50.0%) and 4 (22.2%), respectively. Consistent 5-azacitidine levels (205 to 857 ng/ml) were detected in urine. Pretreatment and posttreatment methylation analysis revealed no significant correlation with clinical response.
Subcutaneous 5-azacitidine showed promising clinical activity in a canine invasive urothelial carcinoma model, thus meriting further development in humans with urothelial carcinoma.
urinary bladder; urothelium; carcinoma; azacitidine; dogs
Estrogens control multiple functions of hormone-responsive breast cancer cells. They regulate diverse physiological processes in various tissues through genomic and non-genomic mechanisms that result in activation or repression of gene expression. Transcription regulation upon estrogen stimulation is a critical biological process underlying the onset and progress of the majority of breast cancer. ERα requires distinct co-regulator or modulators for efficient transcriptional regulation, and they form a regulatory network. Knowing this regulatory network will enable systematic study of the effect of ERα on breast cancer.
To investigate the regulatory network of ERα and discover novel modulators of ERα functions, we proposed an analytical method based on a linear regression model to identify translational modulators and their network relationships. In the network analysis, a group of specific modulator and target genes were selected according to the functionality of modulator and the ERα binding. Network formed from targets genes with ERα binding was called ERα genomic regulatory network; while network formed from targets genes without ERα binding was called ERα non-genomic regulatory network. Considering the active or repressive function of ERα, active or repressive function of a modulator, and agonist or antagonist effect of a modulator on ERα, the ERα/modulator/target relationships were categorized into 27 classes.
Using the gene expression data and ERα Chip-seq data from the MCF-7 cell line, the ERα genomic/non-genomic regulatory networks were built by merging ERα/ modulator/target triplets (TF, M, T), where TF refers to the ERα, M refers to the modulator, and T refers to the target. Comparing these two networks, ERα non-genomic network has lower FDR than the genomic network. In order to validate these two networks, the same network analysis was performed in the gene expression data from the ZR-75.1 cell. The network overlap analysis between two cancer cells showed 1% overlap for the ERα genomic regulatory network, but 4% overlap for the non-genomic regulatory network.
We proposed a novel approach to infer the ERα/modulator/target relationships, and construct the genomic/non-genomic regulatory networks in two cancer cells. We found that the non-genomic regulatory network is more reliable than the genomic regulatory network.
The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLC) to tyrosine kinase inhibitors (TKIs) has been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression and their dysregulation has been implicated in tumorigenesis. To understand the role of microRNAs in TKI-resistant NSCLC, we examined TK receptor-mediated microRNA changes. Here we report that miR-30b/c and miR-221/222, modulated by both EGF and MET receptors, and miR-103, -203, controlled only by MET, play important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition (EMT) of NSCLC cells, in vitro and in vivo, by inhibiting the expression of Bim, APAF-1, PKC-ε and SRC genes. The finding suggests that modulation of specific microRNAs may provide a therapeutic approach for future treatment of NSCLC.
Background: The organochlorine dichlorodiphenyltrichloroethane (DDT), a known estrogen mimic and endocrine disruptor, has been linked to animal and human disorders. However, the detailed mechanism(s) by which DDT affects cellular physiology remains incompletely defined.
Objectives: We and others have shown that DDT activates cell-signaling cascades, culminating in the activation of estrogen receptor-dependent and -independent gene expression. Here, we identify a mechanism by which DDT alters cellular signaling and gene expression, independent of the estrogen receptor.
Methods: We performed quantitative polymerase chain reaction array analysis of gene expression in MCF-7 breast cancer cells using either estradiol (E2) or o,p´-DDT to identify distinct cellular gene expression responses. To elucidate the mechanisms by which DDT regulates cell signaling, we used molecular and pharmacological techniques.
Results: E2 and DDT treatment both altered the expression of many of the genes assayed, but up-regulation of vascular endothelial growth factor A (VEGFA) was observed only after DDT treatment, and this increase was not affected by the pure estrogen receptor α antagonist ICI 182780. Furthermore, DDT increased activation of the HIF-1 response element (HRE), a known enhancer of the VEGFA gene. This DDT-mediated increase in HRE activity was augmented by the coactivator CBP (CREB-binding protein) and was dependent on the p38 pathway.
Conclusions: DDT up-regulated the expression of several genes in MCF-7 breast cancer cells that were not altered by treatment with E2, including VEGFA. We propose that this DDT-initiated, ER-independent stimulation of gene expression is due to DDT’s ability to initiate crosstalk between MAPK (mitogen-activated protein kinase) signaling pathways and transcriptional coactivators.
breast cancer; CBP; coactivator; crosstalk; DDT; dichlorodiphenyltrichloroethane; endocrine-disrupting chemical; HIF-1α; MAPK; organochlorine; p38 kinase; vascular endothelial growth factor
A number of empirical Bayes models (each with different statistical distribution assumptions) have now been developed to analyze differential DNA methylation using high-density oligonucleotide tiling arrays. However, it remains unclear which model performs best. For example, for analysis of differentially methylated regions for conservative and functional sequence characteristics (e.g., enrichment of transcription factor-binding sites (TFBSs)), the sensitivity of such analyses, using various empirical Bayes models, remains unclear. In this paper, five empirical Bayes models were constructed, based on either a gamma distribution or a log-normal distribution, for the identification of differential methylated loci and their cell division—(1, 3, and 5) and drug-treatment-(cisplatin) dependent methylation patterns. While differential methylation patterns generated by log-normal models were enriched with numerous TFBSs, we observed almost no TFBS-enriched sequences using gamma assumption models. Statistical and biological results suggest log-normal, rather than gamma, empirical Bayes model distribution to be a highly accurate and precise method for differential methylation microarray analysis. In addition, we presented one of the log-normal models for differential methylation analysis and tested its reproducibility by simulation study. We believe this research to be the first extensive comparison of statistical modeling for the analysis of differential DNA methylation, an important biological phenomenon that precisely regulates gene transcription.
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multi-kinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to Class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
Potential epigenetic mechanisms underlying fetal alcohol syndrome (FAS) include alcohol-induced alterations of methyl metabolism, resulting in aberrant patterns of DNA methylation and gene expression during development. Having previously demonstrated an essential role for epigenetics in neural stem cell (NSC) development and that inhibiting DNA methylation prevents NSC differentiation, here we investigated the effect of alcohol exposure on genome-wide DNA methylation patterns and NSC differentiation.
NSCs in culture were treated with or without a 6-hr 88mM (“binge-like”) alcohol exposure and examined at 48 hrs, for migration, growth, and genome-wide DNA methylation. The DNA methylation was examined using DNA-methylation immunoprecipitation (MeDIP) followed by microarray analysis. Further validation was performed using Independent Sequenom analysis.
NSC differentiated in 24 to 48 hrs with migration, neuronal expression, and morphological transformation. Alcohol exposure retarded the migration, neuronal formation, and growth processes of NSC, similar to treatment with the methylation inhibitor 5-aza-cytidine. When NSC departed from the quiescent state, a genome-wide diversification of DNA methylation was observed—that is, many moderately methylated genes altered methylation levels and became hyper- and hypomethylated. Alcohol prevented many genes from such diversification, including genes related to neural development, neuronal receptors, and olfaction, while retarding differentiation. Validation of specific genes by Sequenom analysis demonstrated that alcohol exposure prevented methylation of specific genes associated with neural development [cutl2 (cut-like 2), Igf1 (insulin-like growth factor 1), Efemp1 (epidermal growth factor-containing fibulin-like extracellular matrix protein 1), and Sox 7 (SRY-box containing gene 7)]; eye development, Lim 2 (lens intrinsic membrane protein 2); the epigenetic mark Smarca2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2); and developmental disorder [Dgcr2 (DiGeorge syndrome critical region gene 2)]. Specific sites altered by DNA methylation also correlated with transcription factor binding sites known to be critical for regulating neural development.
The data indicate that alcohol prevents normal DNA methylation programming of key neural stem cell genes and retards NSC differentiation. Thus, the role of DNA methylation in FAS warrants further investigation.
Epigenetics; Epigenomics; MeDIP-Chip; Neural development; Fetal alcohol syndrome
It is now established that, as compared to normal cells, the cancer cell genome has an overall inverse distribution of DNA methylation (“methylome”), i.e., predominant hypomethylation and localized hypermethylation, within “CpG islands” (CGIs). Moreover, although cancer cells have reduced methylation “fidelity” and genomic instability, accurate maintenance of aberrant methylomes that underlie malignant phenotypes remains necessary. However, the mechanism(s) of cancer methylome maintenance remains largely unknown. Here, we assessed CGI methylation patterns propagated over 1, 3, and 5 divisions of A2780 ovarian cancer cells, concurrent with exposure to the DNA cross-linking chemotherapeutic cisplatin, and observed cell generation-successive increases in total hyper- and hypo-methylated CGIs. Empirical Bayesian modeling revealed five distinct modes of methylation propagation: (1) heritable (i.e., unchanged) high- methylation (1186 probe loci in CGI microarray); (2) heritable (i.e., unchanged) low-methylation (286 loci); (3) stochastic hypermethylation (i.e., progressively increased, 243 loci); (4) stochastic hypomethylation (i.e., progressively decreased, 247 loci); and (5) considerable “random” methylation (582 loci). These results support a “stochastic model” of DNA methylation equilibrium deriving from the efficiency of two distinct processes, methylation maintenance and de novo methylation. A role for cis-regulatory elements in methylation fidelity was also demonstrated by highly significant (p<2.2×10−5) enrichment of transcription factor binding sites in CGI probe loci showing heritably high (118 elements) and low (47 elements) methylation, and also in loci demonstrating stochastic hyper-(30 elements) and hypo-(31 elements) methylation. Notably, loci having “random” methylation heritability displayed nearly no enrichment. These results demonstrate an influence of cis-regulatory elements on the nonrandom propagation of both strictly heritable and stochastically heritable CGIs.
The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. This family includes EGFR/ErbB1/HER1, ErbB2/HER2/Neu ErbB3/HER3, and ErbB4/HER4. For many years it was believed that EGFR plays a minor role in the development and progression of breast malignancies. However, recent findings have led investigators to revisit these beliefs. Here we will review these findings and propose roles that EGFR may play in breast malignancies. In particular, we will discuss the potential roles that EGFR may play in triple-negative tumors, resistance to endocrine therapies, maintenance of stem-like tumor cells, and bone metastasis. Thus, we will propose the contexts in which EGFR may be a therapeutic target.
Epidermal growth factor receptor; Resistance to endocrine therapy; Bone metastasis; Triple-negative tumors; Stem-like tumor cells
“Epigenetic plasticity” refers to the capability of mammalian cells to alter their differentiation status via chromatin remodeling-associated alterations in gene expression. While epigenetic plasticity has been best associated with lineage commitment of embryonic stem cells, recent studies have demonstrated chromatin remodeling even in terminally differentiated normal cells and advanced-stage melanoma and breast cancer cells, in context-dependent responses to alterations in their microenvironment. In the current study, we extend this attribute of epigenetic plasticity to aggressive ovarian cancer cells, by using an integrative approach to associate cellular phenotypes with chromatin modifications (“ChIP-chip”) and mRNA and microRNA expression. While we identified numerous gene promoters possessing the well-known “bivalent mark” of H3K27me3/H3K4me2, we also report 14 distinct, lesser known bi-, tri- and tetravalent combinations of activating and repressive chromatin modifications, in platinum-resistant CP 70 ovarian cancer cells. The vast majority (>90%) of all the histone marks studied localized to regions within 2,000 bp of transcription start sites, supporting a role in gene regulation. Upon a simple alteration in the microenvironment, transition from two- to three-dimensional culture, an increase (17–38%) in repressive-only marked promoters was observed, concomitant with a decrease (31–21%) in multivalent (i.e., juxtaposed permissive and repressive histone marked) promoters. Like embryonic/tissue stem and other (non-ovarian) carcinoma cells, ovarian cancer cell epigenetic plasticity reflects an inherent transcriptional flexibility for context-responsive alterations in phenotype. It is possible that this plasticity could be therapeutically exploited for the management of this lethal gynecologic malignancy.
histone modifications; gene expression; chromatin remodeling; ovarian cancer; epigenetic plasticity; tumor microenvironment; bivalent histone mark
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
pemetrexed; sorafenib; autophagy; apoptosis; PDGFR; ZMP; AMP; thymidylate synthase
Aberrant DNA methylation is a hallmark of cancer and DNA methyltransferase inhibitors have demonstrated clinical efficacy in hematologic malignancies. Based on preclinical studies indicating that hypomethylating agents can reverse platinum resistance in ovarian cancer cells, we conducted a phase I trial of low dose decitabine combined with carboplatin, in patients with recurrent, platinum-resistant ovarian cancer.
Decitabine was administered i.v. daily for five days, prior to carboplatin (AUC 5) on day 8 of a 28-day cycle. Using a standard 3+3 dose escalation decitabine was tested at two dose levels: 10 mg/m2 (seven patients) or 20 mg/m2 (three patients). Peripheral blood mononuclear cells (PBMCs) and plasma collected on days 1 (pre-treatment), 5, 8, and 15, were utilized to assess global (LINE-1 repetitive element) and gene-specific DNA methylation.
Dose-limiting toxicity (DLT) at the 20 mg/m2 dose was grade 4 neutropenia (2 patients) and no DLTs were observed at 10 mg/m2. Most common toxicities were nausea, allergic reactions, neutropenia, fatigue, anorexia, vomiting, and abdominal pain, the majority being grades 1-2. One complete response was observed, and three additional patients had stable disease for ≥ six months. LINE-1 hypomethylation on days 8 and 15 was detected in DNA from PBMCs. Of five ovarian cancer-associated methylated genes, HOXA11 and BRCA1 were demethylated in plasma on days 8 and 15.
Repetitive low-dose decitabine is tolerated when combined with carboplatin in ovarian cancer patients, and demonstrates biological (i.e., DNA-hypomethylating) activity justifying further testing for clinical efficacy.
ovarian cancer; decitabine; epigenetic biomarkers; chemosensitization; platinum resistance
Deregulation of the transforming growth factor-β (TGFβ) signaling pathway in epithelial ovarian cancer has been reported, but the precise mechanism underlying disrupted TGFβ signaling in the disease remains unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate genome-wide screening of TGFβ-induced SMAD4 binding in epithelial ovarian cancer. Following TGFβ stimulation of the A2780 epithelial ovarian cancer cell line, we identified 2,362 SMAD4 binding loci and 318 differentially expressed SMAD4 target genes. Comprehensive examination of SMAD4-bound loci, revealed four distinct binding patterns: 1) Basal; 2) Shift; 3) Stimulated Only; 4) Unstimulated Only. TGFβ stimulated SMAD4-bound loci were primarily classified as either Stimulated only (74%) or Shift (25%), indicating that TGFβ-stimulation alters SMAD4 binding patterns in epithelial ovarian cancer cells. Furthermore, based on gene regulatory network analysis, we determined that the TGFβ-induced, SMAD4-dependent regulatory network was strikingly different in ovarian cancer compared to normal cells. Importantly, the TGFβ/SMAD4 target genes identified in the A2780 epithelial ovarian cancer cell line were predictive of patient survival, based on in silico mining of publically available patient data bases. In conclusion, our data highlight the utility of next generation sequencing technology to identify genome-wide SMAD4 target genes in epithelial ovarian cancer and link aberrant TGFβ/SMAD signaling to ovarian tumorigenesis. Furthermore, the identified SMAD4 binding loci, combined with gene expression profiling and in silico data mining of patient cohorts, may provide a powerful approach to determine potential gene signatures with biological and future translational research in ovarian and other cancers.
Several lines of evidence have suggested that estrogen receptor α (ERα)–negative breast tumors, which are highly aggressive and nonresponsive to hormonal therapy, arise from ERα-positive precursors through different molecular pathways. Because microRNAs (miRNAs) modulate gene expression, we hypothesized that they may have a role in ER-negative tumor formation.
Gene expression profiles were used to highlight the global changes induced by miRNA modulation of ERα protein. miRNA transfection and luciferase assays enabled us to identify new targets of miRNA 206 (miR-206) and miRNA cluster 221-222 (miR-221-222). Northern blot, luciferase assays, estradiol treatment, and chromatin immunoprecipitation were performed to identify the miR-221-222 transcription unit and the mechanism implicated in its regulation.
Different global changes in gene expression were induced by overexpression of miR-221-222 and miR-206 in ER-positive cells. miR-221 and -222 increased proliferation of ERα-positive cells, whereas miR-206 had an inhibitory effect (mean absorbance units [AU]: miR-206: 500 AU, 95% confidence interval [CI]) = 480 to 520; miR-221: 850 AU, 95% CI = 810 to 873; miR-222: 879 AU, 95% CI = 850 to 893; P < .05). We identified hepatocyte growth factor receptor and forkhead box O3 as new targets of miR-206 and miR-221-222, respectively. We demonstrated that ERα negatively modulates miR-221 and -222 through the recruitment of transcriptional corepressor partners: nuclear receptor corepressor and silencing mediator of retinoic acid and thyroid hormone receptor.
These findings suggest that the negative regulatory loop involving miR-221-222 and ERα may confer proliferative advantage and migratory activity to breast cancer cells and promote the transition from ER-positive to ER-negative tumors.
Membrane receptors for steroid hormones are currently a subject of considerable debate. One approach to selectively target these putative receptors has been to couple ligands to substances that restrict cell permeability. Using this approach, an analog of the estrogen receptor ligand 4-hydroxytamoxifen was attached to fluorescent dyes with differing degrees of predicted cell permeability. The conjugates bound to estrogen receptor in vitro, but all three conjugates, including one predicted to be cell-impermeable, inhibited estradiol-induced transcriptional activation. Fluorescence microscopy revealed cytoplasmic localization for all three conjugates. We further characterized a 4-hydroxytamoxifen analog conjugated to a BODIPY fluorophore in breast cancer cell lines. Those experiments suggested a similar, but not identical, mode of action to 4-hydroxytamoxifen, as the fluorescent conjugate was equally effective at inhibiting proliferation of both tamoxifen-sensitive and tamoxifen-resistant breast cancer cell lines. While these findings point to significant complicating factors in designing steroid hormone mimics targeted to the plasma membrane, the results also reveal a possible new direction for designing estrogen receptor modulators.