Biomedical subjects

Kenneth P Nephew

Publications and source records attributed to Kenneth P Nephew.

At least 19 recordsLinked to original sources

PubMed2024

Ovarian tumor cells gain competitive advantage by actively reducing the cellular fitness of microenvironment cells.

Cell competition and fitness comparison between cancer and tumor microenvironment (TME) cells determine oncogenic fate. Our previous study established a role for human Flower isoforms as fitness fingerprints, where the expression of Flower Win isoforms in tumor cells leads to growth advantage over TME cells expressing Lose isoforms. Here we demonstrate that the expression of Flower Lose and reduced microenvironment fitness is not a pre-existing condition but, rather, a cancer-induced phenomenon. Cancer cells actively reduce TME fitness by the exosome-mediated release of a cancer-specific long non-coding RNA, Tu-Stroma, which controls the splicing of the Flower gene in the TME cells and expression of Flower Lose isoform, which leads to reduced fitness status. This mechanism controls cancer growth, metastasis and host survival in ovarian cancer. Targeting Flower protein with humanized monoclonal antibody (mAb) in mice significantly reduces cancer growth and metastasis and improves survival. Pre-treatment with Flower mAb protects intraperitoneal organs from developing lesions despite the presence of aggressive tumor cells.

Female↗

PubMed2006

Diverse gene expression and DNA methylation profiles correlate with differential adaptation of breast cancer cells to the antiestrogens tamoxifen and fulvestrant.

The development of targeted therapies for antiestrogen-resistant breast cancer requires a detailed understanding of its molecular characteristics. To further elucidate the molecular events underlying acquired resistance to the antiestrogens tamoxifen and fulvestrant, we established drug-resistant sublines from a single colony of hormone-dependent breast cancer MCF7 cells. These model systems allowed us to examine the cellular and molecular changes induced by antiestrogens in the context of a uniform clonal background. Global changes in both basal and estrogen-induced gene expression profiles were determined in hormone-sensitive and hormonal-resistant sublines using Affymetrix Human Genome U133 Plus 2.0 Arrays. Changes in DNA methylation were assessed by differential methylation hybridization, a high-throughput promoter CpG island microarray analysis. By comparative studies, we found distinct gene expression and promoter DNA methylation profiles associated with acquired resistance to fulvestrant versus tamoxifen. Fulvestrant resistance was characterized by pronounced up-regulation of multiple growth-stimulatory pathways, resulting in estrogen receptor alpha (ERalpha)-independent, autocrine-regulated proliferation. Conversely, acquired resistance to tamoxifen correlated with maintenance of the ERalpha-positive phenotype, although receptor-mediated gene regulation was altered. Activation of growth-promoting genes, due to promoter hypomethylation, was more frequently observed in antiestrogen-resistant cells compared with gene inactivation by promoter hypermethylation, revealing an unexpected insight into the molecular changes associated with endocrine resistance. In summary, this study provides an in-depth understanding of the molecular changes specific to acquired resistance to clinically important antiestrogens. Such knowledge of resistance-associated mechanisms could allow for identification of therapy targets and strategies for resensitization to these well-established antihormonal agents.

Breast Neoplasms↗

PubMed2006

Characterization of molecular and structural determinants of selective estrogen receptor downregulators.

Antiestrogens used for breast cancer therapy can be categorized into two classes that differ in their effect on estrogen receptor (ER) alpha stability. The selective estrogen receptor modulators (SERMs) stabilize ER alpha and the selective estrogen receptor downregulators (SERDs) cause a decrease in cellular ER alpha levels. A clinically relevant antiestrogen, GW7604, appears to work through a SERD-like mechanism, despite sharing the same molecular scaffold as 4-hydroxytamoxifen, a SERM. In order to investigate potential structural features of GW7604 responsible for SERD activity, GW7604 and two analogs were synthesized using a new, improved synthetic route and tested for their effects on ER alpha function and cell proliferation. The two analogs, which have an acrylamide or a methyl vinyl ketone replacing the acrylic acid group of GW7604, display lower binding affinity for ER alpha than GW7604, but show similar antagonism of estradiol-induced activation of ER alpha-mediated transcription as GW7604 and inhibit estradiol-induced proliferation of the MCF-7 cell line with a similar potency as GW7604. Unlike GW7604, neither analog has a significant effect on cellular ER alpha levels, suggesting that the carboxylate is a key determinant in GW7604 action and, for the first time, showing that this group is responsible for inducing ER alpha degradation in breast cancer cells.

Breast Neoplasms↗

PubMed2006

A mixture model-based discriminate analysis for identifying ordered transcription factor binding site pairs in gene promoters directly regulated by estrogen receptor-alpha.

MOTIVATION: To detect and select patterns of transcription factor binding sites (TFBSs) which distinguish genes directly regulated by estrogen receptor-alpha (ERalpha), we developed an innovative mixture model-based discriminate analysis for identifying ordered TFBS pairs. RESULTS: Biologically, our proposed new algorithm clearly suggests that TFBSs are not randomly distributed within ERalpha target promoters (P-value < 0.001). The up-regulated targets significantly (P-value < 0.01) possess TFBS pairs, (DBP, MYC), (DBP, MYC/MAX heterodimer), (DBP, USF2) and (DBP, MYOGENIN); and down-regulated ERalpha target genes significantly (P-value < 0.01) possess TFBS pairs, such as (DBP, c-ETS1-68), (DBP, USF2) and (DBP, MYOGENIN). Statistically, our proposed mixture model-based discriminate analysis can simultaneously perform TFBS pattern recognition, TFBS pattern selection, and target class prediction; such integrative power cannot be achieved by current methods. AVAILABILITY: The software is available on request from the authors. CONTACT: lali@iupui.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗

PubMed2006

Dominant-negative histone H3 lysine 27 mutant derepresses silenced tumor suppressor genes and reverses the drug-resistant phenotype in cancer cells.

Histone modifications and DNA methylation are epigenetic phenomena that play a critical role in many neoplastic processes, including silencing of tumor suppressor genes. One such histone modification, particularly at H3 and H4, is methylation at specific lysine (K) residues. Whereas histone methylation of H3-K9 has been linked to DNA methylation and aberrant gene silencing in cancer cells, no such studies of H3-K27 have been reported. Here, we generated a stable cell line overexpressing a dominant-negative point mutant, H3-K27R, to examine the role of that specific lysine in ovarian cancer. Expression of this construct resulted in loss of methylation at H3-K27, global reduction of DNA methylation, and increased expression of tumor suppressor genes. One of the affected genes, RASSF1, was shown to be a direct target of H3-K27 methylation-mediated silencing. By increasing DNA-platinum adduct formation, indicating increased access of the drug to target DNA sequences, removal of H3-K27 methylation resensitized drug-resistant ovarian cancer cells to the chemotherapeutic agent cisplatin. This increased platinum-DNA access was likely due to relaxation of condensed chromatin. Our results show that overexpression of mutant H3-K27 in mammalian cells represents a novel tool for studying epigenetic mechanisms and the Histone Code Hypothesis in human cancer. Such findings show the significance of H3-K27 methylation as a promising target for epigenetic-based cancer therapies.

Antineoplastic Agents↗

PubMed2006

Prognostic DNA methylation biomarkers in ovarian cancer.

PURPOSE: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. EXPERIMENTAL DESIGN: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. CONCLUSION: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease.

Adenocarcinoma↗

PubMed2006

Combinatorial analysis of transcription factor partners reveals recruitment of c-MYC to estrogen receptor-alpha responsive promoters.

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

Animals↗

PubMed2006

Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha.

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.

Breast Neoplasms↗

PubMed2005

Hypermethylation of 18S and 28S ribosomal DNAs predicts progression-free survival in patients with ovarian cancer.

PURPOSE: Repetitive ribosomal DNA (rDNA) genes are GC-rich clusters in the human genome. The aim of the study was to determine the methylation status of two rDNA subunits, the 18S and 28S genes, in ovarian tumors and to correlate methylation levels with clinicopathologic features in a cohort of ovarian cancer patients. EXPERIMENTAL DESIGN: 18S and 28S rDNA methylation was examined by quantitative methylation-specific PCR in 74 late-stage ovarian cancers, 9 histologically uninvolved, and 11 normal ovarian surface epithelial samples. In addition, methylation and gene expression levels of 18S and 28S rDNAs in two ovarian cancer cell lines were examined by reverse transcription-PCR before and after treatment with the demethylating drug 5'-aza-2'-deoxycytidine. RESULTS: The methylation level (amount of methylated rDNA/beta-actin) of 18S and 28S rDNAs was significantly higher (P < 0.05) in tumors than in normal ovarian surface epithelial samples. Methylation of 18S and 28S rDNA was highly correlated (R2= 0.842). Multivariate analysis by Cox regression found that rDNA hypermethylation [hazard ratio (HR), 0.25; P < 0.01], but not age (HR, 1.29; P = 0.291) and stage (HR, 1.09; P = 0.709), was independently associated with longer progression-free survival. In ovarian cancer cell lines, methylation levels of rDNA correlated with gene down-regulation and 5'-aza-2'-deoxycytidine treatment resulted in a moderate increase in 18S and 28S rDNA gene expressions. CONCLUSION: This is the first report of rDNA hypermethylation in ovarian tumors. Furthermore, rDNA methylation levels were higher in patients with long progression-free survival versus patients with short survival. Thus, rDNA methylation as a prognostic marker in ovarian cancer warrants further investigation.

Adult↗

PubMed2005

CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha.

In estrogen target cells, estrogen receptor-alpha (ERalpha) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.

Benzoquinones↗

PubMed2005

New anti-cancer strategies: epigenetic therapies and biomarkers.

Epigenetics is the study of chromatin modifications that affect gene expression without altering DNA nucleotide sequences. This review highlights a prominent role for epigenetic therapies, particularly those that reverse aberrant DNA methylation and histone acetylation, in the potential treatment of cancer. Administration of such therapies to reverse epigenetic "silencing" of tumor suppressors, including genes involved in chemotherapy responses, could prove useful in the management of cancer patients. In this review, we summarize recent advances in the use of methyltransferase and histone deacetylase inhibitors and possible synergistic combinations of these to achieve maximal tumor suppressor gene re-expression. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize drug-resistant tumors to established treatments. As specific, aberrant epigenetic modifications are frequently associated with distinct cancer types, and likely occur early in tumorigenesis, these have potential utility as biomarkers. Finally, future directions are addressed, including alternative epigenetic targets, gene-specific modifications, and the use of bioinformatics.

Acetylation↗

PubMed2005

Epigenetic hypothesis tests for methylation and acetylation in a triple microarray system.

To fully elucidate the functional relationship between DNA methylation and histone hypoacetylation in gene silencing, we have developed an integrated "triple" microarray system that allows us to begin to decipher the influence of epigenetic hierarchies on the regulation of gene expression in cancer cells. Our hypothesis is that in the promoter region of a silenced gene, reversal of two epigenetic factors (i.e., DNA demethylation and/or histone hyperacetylation) is highly correlated with gene reexpression after treatment of the human epithelial ovarian cancer cell line CP70 with the drug combination 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and trichostatin A (TSA), an inhibitor of histone deacetylases. To estimate the posterior probabilities for genes with altered expression, DNA methylation and histone acetylation status measured with a triple-microarray system, we have employed an established empirical Bayes model. Two methods have been proposed to test our hypothesis that DNA demethylation and histone hyperacetylation are highly correlated among those up-regulated genes. One method follows a weighted least squares regression, while the other is derived from a chi-square statistic. The data derived by these approaches, which have been further verified through bootstrap analyses, support the proposed epigenetic correlation (p-values are less than 0.001). Further simulations suggest that even if the constant variance and normality assumptions do not hold, the power of those two tests is robust.

Acetylation↗

PubMed2005

Multiple signaling pathways converge on beta-catenin in thyroid cancer.

The beta-catenin pathway has been conclusively demonstrated to regulate differentiation and patterning in multiple model systems. In thyroid cancer, alterations are often seen in proteins that regulate beta-catenin, including those of the RAS, PI3K/AKT, and peroxisome proliferation activated receptor-gamma (PPARgamma) pathways, and evidence from the literature suggests that beta-catenin may play a direct role in the dedifferentiation commonly observed in late-stage disease. RET/PTC rearrangements are frequent in thyroid cancer and appear to be exclusive from mutational events in RAS and BRAF. Activation of AKT by phosphatidylinositide-3 kinase (PI3K), a RAS effector, results in GSK3beta phosphorylation and deactivation and subsequent beta-catenin upregulation in thyroid cancer. Activating mutations in beta-catenin, which have been demonstrated in late-stage thyroid tumors, correlate with beta-catenin nuclear localization and poor prognosis. We hypothesize that activation of the RAS, PI3K/AKT, and PPARgamma pathways ultimately impinges upon beta-catenin. We further propose that if mutations in BRAF, RAS, and RET/PTC rearrangements are mutually exclusive in certain thyroid tumors or tumor types, as has already been shown for papillary thyroid cancer, then these interconnected pathways may cooperate in the initiation and promotion of the disease. We believe that clinical benefit for thyroid cancer patients could be derived from disrupting the middle or distal pathway effectors of these pathways, such as AKT or beta-catenin.

Genetic Therapy↗

PubMed2005

Dose-dependent effects of 4-hydroxytamoxifen, the active metabolite of tamoxifen, on estrogen receptor-alpha expression in the rat uterus.

Tamoxifen, a selective estrogen receptor modulator, has agonist or antagonist activity, depending on the target tissue. The estrogen-like agonist effects of tamoxifen in the uterus are mediated primarily by 4-hydroxytamoxifen (4OH), the major active metabolite. Tamoxifen, 4OH and estradiol-17beta (E2) all bind to estrogen receptors (ERalpha and ERbeta), but with different affinities, suggesting that these ligands are capable of producing differential in vivo effects on the uterus. However, differences in short-term effects of tamoxifen, 4OH and E2 on the uterus have not been compared in the rat in vivo. Thus, we treated adult, ovariectomized rats (225-250 g) with vehicle (sesame oil), tamoxifen (1 mg/kg body weight), 4OH (0.01, 0.1 or 1.0 mg/kg body weight), E2 (40 microg/kg body weight), estradiol valerate (a long-lasting estrogen; 40 microg/kg body weight) or ICI 182,780 (a pure anti-estrogen; 1 mg/kg body weight). Animals were sacrificed at 0, 3, 6, 12 or 24 h post-injection, and protein and mRNA levels for ERalpha and two estrogen-regulated early response genes, c-fos and jun-B, were examined. Administration of E2 and 4OH (1 mg/kg body weight dose) resulted in down-regulation of uterine ERalpha protein in the uterine luminal and glandular epithelium by 6 h post-treatment. In contrast, no change in ERalpha level was observed after treatment with tamoxifen. Rapid (by 3 h) and transient increases in c-fos and jun-B mRNA levels were observed after E2 treatment; however, c-fos and jun-B induction by 4OH was highly dose dependent, and higher 4OH doses induced rapid but persistent proto-oncogene expression in vivo. Our results demonstrate that tamoxifen and its major metabolite have differential effects on uterine gene expression, and 4OH is highly estrogenic in the rat uterus.

Animals↗

PubMed2005

Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer.

Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the development and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemosensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5-aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebularine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1. RASSF1A gene reexpression was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU, although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5- to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as determined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drug-resistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regimens, for the therapy of drug-resistant ovarian cancer.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2004

Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray.

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Animals↗

PubMed2004

Loss of estrogen receptor signaling triggers epigenetic silencing of downstream targets in breast cancer.

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) alpha signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERalpha target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERalpha signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERalpha targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERalpha target genes, providing new direction for research in this classical signaling pathway.

Base Sequence↗

PubMed2004

Inhibiting proteasomal proteolysis sustains estrogen receptor-alpha activation.

Estrogen receptor-alpha (ER alpha) is a ligand-dependent transcription factor that mediates physiological responses to 17 beta-estradiol (E2). Ligand binding rapidly down-regulates ER alpha levels through proteasomal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin-proteasome pathway on ER alpha-mediated transcriptional responses. In HeLa cells transfected with ER alpha, blocking either ubiquitination or proteasomal degradation markedly increased E2-induced expression of an ER-responsive reporter. Time course studies further demonstrated that blocking ligand-induced degradation of ER alpha resulted in prolonged stimulation of ER-responsive gene transcription. In breast cancer MCF7 cells containing endogenous ER alpha, proteasome inhibition enhanced E2-induced expression of endogenous pS2 and cathepsin D. However, inhibiting the proteasome decreased expression of progesterone receptor (PR), presumably due to the heterogeneity of the PR promoter, which contains multiple regulatory elements. In addition, in endometrial cancer Ishikawa cells overexpressing steroid receptor coactivator 1, 4-hydroxytamoxifen displayed full agonist activity and stimulated ER alpha-mediated transcription without inducing receptor degradation. Collectively, these results demonstrate that proteasomal degradation is not essential for ER alpha transcriptional activity and functions to limit E2-induced transcriptional output. The results further indicate that promoter context must be considered when evaluating the relationship between ER alpha transcription and proteasome inhibition. We suggest that the transcription of a gene driven predominantly by an estrogen-responsive element, such as pS2, is a more reliable indicator of ER alpha transcription activity than a gene like PR, which contains a complex promoter requiring cooperation between ER alpha and other transcription factors.

Cathepsin D↗