RESUMO
BACKGROUND: While estrogen receptor (ER) positive breast tumors generally respond well to endocrine therapy (ET), up to 40% of patients will experience relapse, either while on endocrine therapy or after ET is completed. We previously demonstrated that the selective pressure of tamoxifen activates the NFκB pathway in ER + patient tumors, breast cancer cell lines, and breast cancer xenograft tumors, and that this activation allows for survival of a subpopulation of NFκB + cells that contribute to cell regrowth and tumor relapse after ET withdrawal. However, the mechanisms contributing to the expansion of an NFκB + cell population on ET are unknown. METHODS: Here, we utilized single-cell RNA sequencing and bioinformatics approaches to characterize the NFκB + cell population and its clinical relevance. Follow-up studies were conducted to validate our findings and assess the function of the integrated stress response pathway in breast cancer cell lines and patient-derived models. RESULTS: We found that the NFκB + population that arises in response to ET is a preexisting population is enriched under the selective pressure of ET. Based on the preexisting NFκB + cell population, we developed a gene signature and found that it is predictive of tumor relapse when expressed in primary ER + tumors and is retained in metastatic cell populations. Moreover, we identified that the integrated stress response (ISR), as indicated by increased phosphorylation of eIF2α, occurs in response to ET and contributes to clonogenic growth under the selective pressure of ET. CONCLUSIONS: Taken together, our findings suggest that a cell population with active NFκB and ISR signaling can survive and expand under the selective pressure of ET and that targeting this population may be a viable therapeutic strategy to improve patient outcome by eliminating cells that survive ET. Understanding the mechanisms by which breast cancer cells survive the selective pressure of ET may improve relapse rates and overall outcome for patients with ER + breast tumors.
Assuntos
Neoplasias da Mama , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Transdução de Sinais , Tamoxifeno/uso terapêuticoRESUMO
Aromatase inhibitors are the preferred treatment for certain women with estrogen receptor (ER)-positive breast cancer, but evidence suggests that women with obesity experience aromatase inhibitor resistance at higher rates. To compare how stromal cells derived from women who are lean or obese influence response to the aromatase inhibitor (anastrazole), we incorporated patient-derived stroma in a previously characterized MCF7-derived in vitro duct model. Coculture with adipose stromal cells enabled the metabolism of testosterone (T) to E2, which induced estrogen response element activity, epithelial proliferation, and hyperplasia in MCF7 cells. The effects of T were inhibited by the ER antagonist tamoxifen and aromatase inhibitor anastrazole and were increased by the aromatase inducer dexamethasone. Primary mammary adipose stromal cells derived from women with obesity displayed increased aromatase mRNA compared with lean controls. MCF7-derived ducts cocultured with obese stromal cells exhibited higher maximal aromatization-induced ER transactivation and reduced anastrazole sensitivity, a difference not seen in 2-dimensional coculture. Finally, tamoxifen was more effective than anastrazole at reducing aromatization-induced ER transactivation and proliferation. These findings suggest that patient-specific responses to hormone therapies can be modeled and studied organotypically in vitro and add to evidence advocating obesity as a parameter to consider when identifying treatments for patients with ER-positive breast cancer.-Morgan, M. M., Arendt, L. M., Alarid, E. T., Beebe, D. J., Johnson, B. P. Mammary adipose stromal cells derived from obese women reduce sensitivity to the aromatase inhibitor anastrazole in an organotypic breast model.
Assuntos
Adipócitos/efeitos dos fármacos , Anastrozol/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Obesidade/metabolismo , Células Estromais/efeitos dos fármacos , Adipócitos/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura/métodos , Estrogênios/metabolismo , Feminino , Humanos , Células MCF-7 , Receptores de Estrogênio/metabolismo , Células Estromais/metabolismo , Tamoxifeno/farmacologiaRESUMO
The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)(118)-Pro(119) in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function.
Assuntos
Neoplasias da Mama/metabolismo , DNA/química , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptidilprolil Isomerase/metabolismo , Sítio Alostérico , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células MCF-7 , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Fosforilação , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Receptor levels are a key mechanism by which cells regulate their response to stimuli. The levels of estrogen receptor-α (ERα) impact breast cancer cell proliferation and are used to predict prognosis and sensitivity to endocrine therapy. Despite the clinical application of this information, it remains unclear how different cellular processes interact as a system to control ERα levels. To address this question, experimental results from the ERα-positive human breast cancer cell line (MCF-7) treated with 17-ß-estradiol or vehicle control were used to develop a mass-action kinetic model of ERα regulation. Model analysis determined that RNA dynamics could be captured through phosphorylated ERα (pERα)-dependent feedback on transcription. Experimental analysis confirmed that pERα-S118 binds to the estrogen receptor-1 (ESR1) promoter, suggesting that pERα can feedback on ESR1 transcription. Protein dynamics required a separate mechanism in which the degradation rate for pERα was 8.3-fold higher than nonphosphorylated ERα. Using a model with both mechanisms, the root mean square error was 0.078. Sensitivity analysis of this combined model determined that while multiple mechanisms regulate ERα levels, pERα-dependent feedback elicited the strongest effect. Combined, our computational and experimental results identify phosphorylation of ERα as a critical decision point that coordinates the cellular circuitry to regulate ERα levels.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Modelos Teóricos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Imunoprecipitação da Cromatina , Estrogênios/farmacologia , Retroalimentação Fisiológica , Feminino , Humanos , Cinética , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.
Assuntos
Neoplasias da Mama/genética , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica , Células Estromais/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Análise de Componente Principal , Microambiente TumoralRESUMO
Estrogen receptor (ER)-positive breast cancer is characterized by late recurrences following initial treatment. The epithelial cell fate transcription factor Grainyhead-like protein 2 (GRHL2) is overexpressed in ER-positive breast cancers and is linked to poorer prognosis as compared to ER-negative breast cancers. To understand how GRHL2 contributes to progression, GRHL2 was overexpressed in ER-positive cells. We demonstrated that elevated GRHL2 imparts plasticity with stem cell- and dormancy-associated traits. RNA sequencing and immunocytochemistry revealed that high GRHL2 not only strengthens the epithelial identity but supports a hybrid epithelial to mesenchymal transition (EMT). Proliferation and tumor studies exhibited a decrease in growth and an upregulation of dormancy markers, such as NR2F1 and CDKN1B. Mammosphere assays and flow cytometry revealed enrichment of stem cell markers CD44 and ALDH1, and increased self-renewal capacity. Cistrome analyses revealed a change in transcription factor motifs near GRHL2 sites from developmental factors to those associated with disease progression. Together, these data support the idea that the plasticity and properties induced by elevated GRHL2 may provide a selective advantage to explain the association between GRHL2 and breast cancer progression.
RESUMO
Context: Positron emission tomography imaging with 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) is used clinically for initial staging, restaging, and assessing therapy response in breast cancer. Tumor FDG uptake in steroid hormone receptor-positive breast cancer and physiologic FDG uptake in normal breast tissue can be affected by hormonal factors such as menstrual cycle phase, menopausal status, and hormone replacement therapy. Objective: The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells. Methods and Results: PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake. GLUT1 and PFKFB3 mRNA expression and the enzymatic activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were increased after progestin treatment of T47D cells. Conclusion: Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.
RESUMO
Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant posttranslational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group revealed enrichment of the GRHL2 motif near pS118-ER binding sites. In this study, we used cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300, compared to sites where each factor binds independently. Transcriptomic analysis yielded four subsets of ER/GRHL2-coregulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis indicated that coregulated genes are involved in cell migration. Accordingly, knockdown of GRHL2, combined with estrogen treatment, resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to selected enhancers and facilitates pS118-ER recruitment to chromatin, which then results in differential activation and repression of genes that control estrogen-regulated ER-positive breast cancer cell migration.
Assuntos
Receptor alfa de Estrogênio , Receptores de Estrogênio , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Ativação Transcricional/genética , Cromatina , Ligantes , Estrogênios/metabolismo , Serina/metabolismo , Linhagem Celular TumoralRESUMO
The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as "background", that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.
Assuntos
Separação Celular/métodos , Células Epiteliais , Filtração/instrumentação , Microfluídica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Desenho de Equipamento , Filtração/métodos , Humanos , Modelos Teóricos , Tensão SuperficialRESUMO
The fibril orientation of type I collagen has been shown to contribute to tumor invasion and metabolic changes. Yet, there is limited information about its impact on tumor cells' behavior in a restrictive growth environment. Restrictive growth environments are generated by the inhibition of a proliferation stimulus during therapy or as an inflammatory response to suppress tumor expansion. In this study, the impact of a type I collagen matrix orientation and fibrous architecture on cell proliferation and response to estrogen receptor (ER) therapy were examined using estrogen-dependent breast tumor cells (MCF-7 and T-47D) cultured in a hormone-restricted environment. The use of hormone-free culture media, as well as pharmacological inhibitors of ER, Tamoxifen, and Fulvestrant, were investigated as hormone restrictive conditions. Examination of cultures at 72 h showed that tumor cell proliferation was significantly stimulated (1.8-fold) in the absence of hormones on collagen fibrous substrates, but not on polycaprolactone fibrous substrates of equivalent orientation. ER inhibitors did not suppress cell proliferation on collagen fibrous substrates. The examination of reporter cells for ER signaling showed a lack of activity, thus confirming a shift toward an ER-independent proliferation mechanism. Examination of two selective inhibitors of α2ß1 and α1ß1 integrins showed that cell proliferation is suppressed in the presence of the α2ß1 integrin inhibitor only, thereby indicating that the observed changes in tumor cell behavior are caused by a combination of integrin signaling and/or an intrinsic structural motif that is uniquely present in the collagen fibrils. Adjacent coculture studies on collagen substrates showed that tumor cells on collagen can stimulate the proliferation of cells on tissue culture plastic through soluble factors. The magnitude of this effect correlated with the increased surface anisotropy of the substrate. This sensing in fibril orientation was further supported by a differential expression pattern of secreted proteins that were identified on random and aligned orientation substrates. Overall, this study shows a new role for electrospun collagen I fibrous substrates by supporting a shift toward an ER-independent tumor cell proliferation mechanism in ER+ breast tumor cells.
Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Linhagem Celular Tumoral , Proliferação de Células , Colágeno Tipo I , Feminino , Fulvestranto/farmacologia , Humanos , Receptores de Estrogênio/genética , Microambiente TumoralRESUMO
BACKGROUND: Sin3A is an evolutionarily conserved transcriptional repressor which regulates gene expression as part of the multi-protein Sin3 repressive complex. It functions as a scaffold upon which proteins with enzymatic activity dock, including chromatin modifying histone deacetylases. Although regulation of transcription by Sin3A has been studied in detail, little is understood about the function of Sin3A in cancer cells. We previously showed that Sin3A is expressed in breast cancer cells and is a repressor of estrogen receptor-alpha (ERα, ESR1) gene expression. Here, we expand our previous studies to elucidate the function of Sin3A in the control of gene expression and growth of breast cancer cells. RESULTS: Analysis of gene expression following knockdown of Sin3A revealed changes in both basal and regulated gene transcription. Genes of known importance in breast cancer and estrogen signaling, including ERBB2, PGR, MYC, CLU, and NCOA2, were among those identified as Sin3A-responsive. The mechanism of Sin3A action varied among genes and was found to be mediated through both HDAC1/2 -dependent and -independent activities. Loss of Sin3A inhibited breast cancer cell growth by increasing apoptosis without affecting cell cycle progression. Analysis of both ERα-positive and ERα-negative cell lines revealed that the effects of Sin3A on growth were cell-type specific, as Sin3A expression promoted maximum growth of only the ERα-positive cells, and, notably, Sin3A protein itself was increased by estrogen. Further gene expression experiments revealed that Sin3A repressed expression of key apoptotic genes, including TRAIL, TRAILR1, CASP10, and APAF1, in ERα-positive, but not ERα-negative, cell lines, which could provide a mechanistic explanation for cell-type differences in growth. CONCLUSIONS: This study identifies Sin3A as a regulator of gene expression, survival, and growth in ERα-positive breast cancer cells. Sin3A regulates the transcription of genes involved in breast cancer and apoptosis and acts through multiple mechanisms not limited to histone deacetylase function. These findings reveal previously undescribed functions of Sin3A in breast cancer and provide evidence for an important role of this transcriptional repressor in ERα-positive tumor cell growth.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas Repressoras/metabolismo , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Neoplasias da Mama/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Transcriptional regulation of ESR1, the gene that encodes for estrogen receptor α (ER), is critical for regulating the downstream effects of the estrogen signaling pathway in breast cancer such as cell growth. ESR1 is a large and complex gene that is regulated by multiple regulatory elements, which has complicated our understanding of how ESR1 expression is controlled in the context of breast cancer. Early studies characterized the genomic structure of ESR1 with subsequent studies focused on identifying intrinsic (chromatin environment, transcription factors, signaling pathways) and extrinsic (tumor microenvironment, secreted factors) mechanisms that impact ESR1 gene expression. Currently, the introduction of genomic sequencing platforms and additional genome-wide technologies has provided additional insight on how chromatin structures may coordinate with these intrinsic and extrinsic mechanisms to regulate ESR1 expression. Understanding these interactions will allow us to have a clearer understanding of how ESR1 expression is regulated and eventually provide clues on how to influence its regulation with potential treatments. In this review, we highlight key studies concerning the genomic structure of ESR1, mechanisms that affect the dynamics of ESR1 expression, and considerations towards affecting ESR1 expression and hormone responsiveness in breast cancer.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Proliferação de Células , Feminino , Humanos , MasculinoRESUMO
Accumulating evidence suggests that our ability to predict chemical effects on breast cancer is limited by a lack of physiologically relevant in vitro models; the typical in vitro breast cancer model consists of the cancer cell and excludes the mammary microenvironment. As the effects of the microenvironment on cancer cell behavior becomes more understood, researchers have called for the integration of the microenvironment into in vitro chemical testing systems. However, given the complexity of the microenvironment and the variety of platforms to choose from, identifying the essential parameters to include in a chemical testing platform is challenging. This review discusses the need for more complex in vitro breast cancer models and outlines different approaches used to model breast cancer in vitro. We provide examples of the microenvironment modulating breast cancer cell responses to chemicals and discuss strategies to help pinpoint what components should be included in a model.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Carcinoma Intraductal não Infiltrante/diagnóstico , Linhagem Celular Tumoral , Progressão da Doença , Ensaios de Seleção de Medicamentos Antitumorais , Epitélio/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Inflamação , Modelos Estatísticos , Neoplasias , Fenótipo , Engenharia TecidualRESUMO
The purpose of this study was to identify critical pathways promoting survival of tamoxifen-tolerant, estrogen receptor α positive (ER+) breast cancer cells, which contribute to therapy resistance and disease recurrence. Gene expression profiling and pathway analysis were performed in ER+ breast tumors of patients before and after neoadjuvant tamoxifen treatment and demonstrated activation of the NF-κB pathway and an enrichment of epithelial-to mesenchymal transition (EMT)/stemness features. Exposure of ER+ breast cancer cell lines to tamoxifen, in vitro and in vivo, gives rise to a tamoxifen-tolerant population with similar NF-κB activity and EMT/stemness characteristics. Small-molecule inhibitors and CRISPR/Cas9 knockout were used to assess the role of the NF-κB pathway and demonstrated that survival of tamoxifen-tolerant cells requires NF-κB activity. Moreover, this pathway was essential for tumor recurrence following tamoxifen withdrawal. These findings establish that elevated NF-κB activity is observed in breast cancer cell lines under selective pressure with tamoxifen in vitro and in vivo, as well as in patient tumors treated with neoadjuvant tamoxifen therapy. This pathway is essential for survival and regrowth of tamoxifen-tolerant cells, and, as such, NF-κB inhibition offers a promising approach to prevent recurrence of ER+ tumors following tamoxifen exposure. IMPLICATIONS: Understanding initial changes that enable survival of tamoxifen-tolerant cells, as mediated by NF-κB pathway, may translate into therapeutic interventions to prevent resistance and relapse, which remain major causes of breast cancer lethality.
Assuntos
Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Recidiva Local de Neoplasia/patologia , Tamoxifeno/administração & dosagem , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , NF-kappa B/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Transplante de Neoplasias , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.
Assuntos
Membrana Celular/efeitos dos fármacos , Dimetilpolisiloxanos/farmacologia , Técnicas Analíticas Microfluídicas/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Linhagem Celular , Membrana Celular/metabolismo , Meios de Cultura/química , Dimetilpolisiloxanos/análise , Dimetilpolisiloxanos/química , Ensaio de Imunoadsorção Enzimática , Estrogênios/química , Humanos , Luciferases/genética , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/normas , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
In mammals, the grainyhead-like transcription factor (GRHL) family is composed of three nuclear proteins that are responsible for driving epithelial cell fate: GRHL1, GRHL2, and GRHL3. GRHL2 is important in maintaining proper tubulogenesis during development and in suppressing the epithelial-to-mesenchymal transition. Within the last decade, evidence indicates both tumor-suppressive and oncogenic roles for GRHL2 in various types of cancers. Recent studies suggest that GRHL2 may be especially important in hormone-dependent cancers, as correlative relationships exist between GRHL2 and various steroid receptors, such as the androgen and estrogen receptors. Acting as a pioneer factor and coactivator, GRHL2 may directly affect steroid receptor transcriptional activity. This review will highlight recent discoveries of GRHL2 activity in cancer and in maintaining the epithelial state, while also exploring recent literature on the role of GRHL2 in hormone-dependent cancers and epigenetics.
Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Estrogen receptor α (ER) is the target of endocrine therapies in ER-positive breast cancer (BC), but their therapeutic effectiveness diminishes with disease progression. Most metastatic BCs retain an ER-positive status, but ER expression levels are reduced. We asked how the bone tumor microenvironment (TME) regulates ER expression. We observed ESR1 mRNA and ER protein downregulation in BC cells treated with conditioned media (CM) from patient-derived, cancer-activated bone marrow stromal cells (BMSCs) and the BMSC cell line HS5. Decreases in ESR1 mRNA were attributed to decreases in nascent transcripts as well as decreased RNA polymerase II occupancy and H3K27Ac levels on the ESR1 promoter and/or distal enhancer (ENH1). Repression extended to neighboring genes of ESR1, including ARMT1 and SYNE1. Although ERK/MAPK signaling pathway can repress ER expression by other TME cell types, MAPK inhibition did not reverse decreases in ER expression by BMSC-CM. ESR1 mRNA and ER protein half-lives in MCF7 cells were unchanged by BMSC-CM treatment. Whereas ER phosphorylation was induced, ER activity was repressed by BMSC-CM as neither ER occupancy at known binding sites nor estrogen response element-luciferase activity was detected. BMSC-CM also repressed expression of ER target genes. In cells expressing the Y537S and D538G ESR1 mutations, BMSC-CM reduced ESR1, but expression of target genes PGR and TFF1 remained significantly elevated compared with that of control wild-type cells. These studies demonstrate that BMSCs can transcriptionally corepress ESR1 with neighboring genes and inhibit receptor activity, but the functional consequences of the BMSC TME can be limited by metastasis-associated ESR1 mutations.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptor alfa de Estrogênio/genética , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.
Assuntos
DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Estrogênios/metabolismo , Feminino , Humanos , Células MCF-7 , Fosforilação , Ligação Proteica , Transdução de SinaisRESUMO
The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor alpha (ERalpha) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERalpha proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERalpha and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERalpha. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERalpha are mechanistically separable functions of ERalpha. We find that proteolysis of ERalpha correlates with the ability of ERalpha mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERalpha proteolysis and transcription.
Assuntos
Estradiol/análogos & derivados , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica , Transcrição Gênica , Sequência de Aminoácidos , Linhagem Celular , Imunoprecipitação da Cromatina , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Etanol/farmacologia , Fulvestranto , Humanos , Hidrólise , Rim/citologia , Rim/embriologia , Ligantes , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Deleção de Sequência , Serina/metabolismo , Ativação Transcricional , Ubiquitina/análise , Ubiquitina/metabolismoRESUMO
Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17ß-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer.