Biomedical subjects

Meiyun Fan

Publications and source records attributed to Meiyun Fan.

10 recordsLinked to original sources

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

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↗

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↗

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↗

PubMed2002

The NEDD8 pathway is required for proteasome-mediated degradation of human estrogen receptor (ER)-alpha and essential for the antiproliferative activity of ICI 182,780 in ERalpha-positive breast cancer cells.

Steroid hormone receptors, including estrogen receptor-alpha (ERalpha), are ligand-activated transcription factors, and hormone binding leads to depletion of receptor levels via preteasome-mediated degradation. NEDD8 (neural precursor cell-expressed developmentally down-regulated) is an ubiquitin-like protein essential for protein processing and cell cycle progression. We recently demonstrated that ubiquitin-activating enzyme (Uba)3, the catalytic subunit of the NEDD8-activating enzyme, inhibits ERalpha transcriptional activity. Here we report that Uba3-mediated inhibition of ERalpha transactivation function is due to increased receptor protein turnover. Coexpression of Uba3 with ERalpha increased receptor degradation by the 26S proteasome. Inhibition of NEDD8 activation and conjugation diminished polyubiquitination of ERalpha and blocked proteasome-mediated degradation of receptor protein. The antiestrogen ICI 182,780 is known to induce ER degradation. In human MCF7 breast cancer cells modified to contain a disrupted NEDD8 pathway, ICI 182,780 degradation of ERalpha was impaired, and the antiestrogen was ineffective at inhibiting cell proliferation. This study provides the first evidence linking nuclear receptor degradation with the NEDD8 pathway and the ubiquitin-proteasome system, suggesting that the two pathways can act together to modulate ERalpha turnover and cellular responses to estrogens. Based on our observation that an intact NEDD8 pathway is essential for the antiproliferation activity of the ICI 182,780 in ERalpha positive breast cancer cells, we propose that disruptions in the NEDD8 pathway provide a mechanism by which breast cancer cells acquire antiestrogen resistance while retaining expression of ERalpha.

Antineoplastic Agents, Hormonal↗

PubMed2002

The activating enzyme of NEDD8 inhibits steroid receptor function.

Coregulator proteins, coactivators and corepressors, have a profound influence on steroid receptor activity and play a role in regulating receptor levels. To identify novel coregulators of nuclear receptors, we used the ligand-binding and hinge region of ERalpha as bait in a yeast two-hybrid screen of a cDNA library derived from rat uterine luminal epithelium. We report the cloning and characterization of a cDNA encoding a protein homologous to yeast and human ubiquitin-activating enzyme 3 (Uba3), the catalytic subunit of the activating enzyme of the ubiquitin-like NEDD8 (neural precursor cellexpressed developmentally down-regulated) conjugation pathway (known as neddylation). Sequence analysis revealed that Uba3 contains multiple nuclear receptor (NR)-interacting motifs (NR boxes), which are known to mediate interactions between coregulatory proteins and ligand-activated NRs. Yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that Uba3 directly interacts with ligand-occupied ERalpha and ERbeta. Transient transfection of Uba3 in mammalian cells inhibited ER-mediated transactivation in a time-dependent fashion; Uba3 had no effect on the initial events of transcriptional activation by liganded ER, but it blocked the progressive increase in target gene expression during continuous stimulation. Uba3 also inhibited transactivation by AR and PR in mammalian cells but had no effect on a steroid receptor-independent transactivation pathway. An enzymatically silent form of Uba3 did not inhibit ER-induced transcription, and a Uba3-binding fragment of amyloid precursor protein-binding protein, the other subunit of the NEDD8-activating enzyme, partially overcame Uba3-mediated inhibition, demonstrating that the neddylation activity of Uba3 is required for its inhibition of steroid receptor transactivation. Thus, Uba3 inhibits transcription induced by steroid hormone receptors through a novel mechanism that involves the neddylation pathway. Understanding the mechanisms controlling hormone responsiveness of target tissues, such as the uterus and mammary gland, may lead to novel insights of therapeutic intervention.

Amino Acid Sequence↗