RESUMO
Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKß establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKß signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , Células MCF-7 , Simulação de Dinâmica Molecular , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Fosforilação , Conformação Proteica , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Tamoxifeno/farmacologia , Transcrição Gênica , Transfecção , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Alcohol consumption is a risk factor for breast cancer, contributing to up to nearly 23,000 new cases each year. Mechanistic studies show that alcohol increases tumor aggressiveness and metastatic potential, promotes angiogenesis, induces chronic inflammation, and dysregulates RNA polymerase III-related genes. Alcohol has also been shown to affect estrogen signaling in breast cancer, including in our study of the transcriptomic effects of alcohol in breast cancer cells. METHODS: To elucidate mechanisms of action of alcohol in breast cancer, we carried out secondary analyses of our alcohol-responsive transcriptome data using gene ontology and pathway databases and analysis tools and cistromic data analysis of candidate transcription factors which may mediate the transcriptomic alterations. Predicted alcohol-responsive pathways and mechanisms were perturbed and examined experimentally in breast cancer cells. The clinical relevance of identified genes was determined by expression profiles in patient samples and correlation with disease outcomes and alcohol consumption in previously published study cohorts. RESULTS: Gene ontology analysis showed that alcohol alters the expression of many metabolism-related genes, and cistromic data of differentially expressed genes revealed the potential involvement of nuclear factor of activated T cells 3 (NFATC3) in mediating the transcriptomic effects of alcohol. Pathway analysis also predicted regulation of calcium signaling by alcohol in breast cancer cells. Chemical perturbation of this pathway reversed the effect of alcohol on breast cancer cell growth and reduced the elevated cytosolic Ca2+ levels induced by alcohol. Expression levels of alcohol-responsive genes in tumor samples from breast cancer patients are associated with poor disease outcomes. Moreover, expression of some of these genes was altered in breast cancer patients who consumed alcohol previously as compared to those who did not drink. CONCLUSION: Alcohol alters expression of genes that regulate intracellular calcium levels and downstream signaling pathways which drive breast cancer cell proliferation and disease progression.
Assuntos
Neoplasias da Mama/induzido quimicamente , Sinalização do Cálcio/genética , Carcinoma/induzido quimicamente , Etanol/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/mortalidade , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Fatores de Transcrição NFATC/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer with a high mortality rate due to the lack of early detection and effective treatment options for advanced diseases. Metabolic reprogramming, a common hallmark of malignant transformation in pancreatic cancer, is critical for the growth and survival of cancer cells and a potential target mechanism for the treatment of pancreatic cancer. PDAC cells have upregulated glutamine metabolism to meet their biosynthetic and oxidative demands. Liver X receptors (LXRs) are ligand-dependent transcription factors involved in maintaining metabolic homeostasis. LXRs regulate critical cancer-related processes and pathways, including cholesterol, glucose and lipid metabolism, and inflammatory and immune responses. Analysis of transcriptomic data from PDAC clinical samples reveals overexpression of LXRs and their target genes in tumors as compared to normal tissue controls. Targeting LXRs with the novel LXR inverse agonist and degrader GAC0001E5 inhibited PDAC cell proliferation. Using a metabolomics approach, we discovered that 1E5 inhibits glutamine anaplerosis and induces oxidative stress, which are detrimental to PDAC cells. These findings highlight a novel role for LXR in regulating cancer metabolism and the potential application of LXR modulators in targeting cancer metabolism in pancreatic cancer and other malignancies.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Glutamina/metabolismo , Receptores X do Fígado/agonistas , Estresse Oxidativo/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/agonistas , Benzoatos/farmacologia , Benzilaminas/farmacologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumor-derived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+ islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.
Assuntos
Transformação Celular Neoplásica/metabolismo , Hidroxicolesteróis/metabolismo , Tumores Neuroendócrinos/etiologia , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo , Animais , Transformação Celular Neoplásica/genética , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Colesterol 24-Hidroxilase , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Feminino , Imunofluorescência , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development.
Assuntos
Desenvolvimento Embrionário/fisiologia , Receptores de Estrogênio/fisiologia , Animais , Aves/embriologia , Aves/genética , Criança , Desenvolvimento Embrionário/genética , Feminino , Peixes/embriologia , Peixes/genética , Hormônios/biossíntese , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais/genéticaRESUMO
The estrogens are female sex hormones that are involved in a variety of physiological processes, including reproductive development and function, wound healing, and bone growth. They are mainly known for their roles in reproductive tissues--specifically, 17ß-estradiol (E2), the primary estrogen, which is secreted by the ovaries and induces cellular proliferation and growth of the uterus and mammary glands. In addition to the role of estrogens in promoting tissue growth and development during normal physiological states, they have a well-established role in determining susceptibility to disease, particularly cancer, in reproductive tissues. The responsiveness of various tissues to estrogen is genetically controlled, with marked quantitative variation observed across multiple species, including humans. This variation presents both researchers and clinicians with a veritable physiological puzzle, the pieces of which--many of them unknown--are complex and difficult to fit together. Although genetics is known to play a major role in determining sensitivity to estrogens, there are other factors, including parent of origin and the maternal environment, that are intimately linked to heritable phenotypes but do not represent genotype, per se. The objectives of this review article were to summarize the current knowledge of the role of genotype, and uterine and neonatal environments, in phenotypic variation in the response to estrogens; to discuss recent findings and the potential mechanisms involved; and to highlight exciting research opportunities for the future.
Assuntos
Estrogênios/fisiologia , Animais , Feminino , Humanos , Glândulas Mamárias Humanas/fisiologia , Útero/fisiologia , Vagina/fisiologiaRESUMO
The uterotropic response of the uterus to 17ß-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high [C57BL/6J (B6)] or low [C3H/HeJ (C3H)] responders to E2 led to the identification of quantitative trait (QT) loci associated with phenotypic variation in uterine growth and leukocyte infiltration. The mechanisms underlying differential responsiveness to E2, and the genes involved, are unknown. Therefore, we used a microarray approach to show association of distinct E2-regulated transcriptional signatures with genetically controlled high and low responses to E2 and their segregation in (C57BL/6J×C3H/HeJ) F1 hybrids. Among the 6664 E2-regulated transcripts, analysis of cellular functions of those that were strain specific indicated C3H-selective enrichment of apoptosis, consistent with a 7-fold increase in the apoptosis indicator CASP3, and a 2.4-fold decrease in the apoptosis inhibitor Naip1 (Birc1a) in C3H vs. B6 following treatment with E2. In addition, several differentially expressed transcripts reside within our previously identified QT loci, including the ERα-tethering factor Runx1, demonstrated to enhance E2-mediated transcript regulation. The level of RUNX1 in uterine epithelial cells was shown to be 3.5-fold greater in B6 compared to C3H. Our novel insights into the mechanisms underlying the genetic control of tissue sensitivity to estrogen have great potential to advance understanding of individualized effects in physiological and disease states.
Assuntos
Caspase 3/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Proteína Inibidora de Apoptose Neuronal/genética , Transcrição Gênica/genética , Útero/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Células Epiteliais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Peroxidase/genética , Análise Serial de Proteínas , Locos de Características Quantitativas/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcriptoma , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimentoRESUMO
The HER2-positive subtype accounts for approximately one-fifth of all breast cancers. Insensitivity and development of acquired resistance to targeted therapies in some patients contribute to their poor prognosis. HER2 overexpression is associated with metabolic reprogramming, facilitating cancer cell growth and survival. Novel liver X receptor (LXR) ligand GAC0001E5 (1E5) has been shown to inhibit cancer cell proliferation by disrupting glutaminolysis and inducing oxidative stress. In this study, HER2-positive breast cancer cells were treated with 1E5 to determine their potential inhibitory effects and mechanisms of action in HER2-positive breast cancers. Similar to previous observations in other cancer types, 1E5 treatments inhibited LXR activity, expression, and cancer cell proliferation. Expression of fatty acid synthesis genes, including fatty acid synthase (FASN), was downregulated following 1E5 treatment, and results from co-treatment experiments with an FASN inhibitor suggest that the same pathway is targeted by 1E5. Treatments with 1E5 disrupted glutaminolysis and resulted in increased oxidative stress. Strikingly, HER2 transcript and protein levels were both significantly downregulated by 1E5. Taken together, these findings indicate the therapeutic potential of targeting HER2 overexpression and associated metabolic reprogramming via the modulation of LXR in HER2-positive breast cancers.
RESUMO
Estrogen receptor alpha (ERα) is a cellular receptor for the female sex hormone estrogen and other natural and synthetic ligands and play critical roles in normal development and physiology and in the etiology and treatment of endocrine-related diseases. ERα is a member of the nuclear receptor superfamily of transcription factors and regulates target gene expression in a ligand-dependent manner. It has also been shown to interact with G-protein coupled receptors and associated signaling molecules in the cytoplasm. Transcriptionally, ERα either binds DNA directly through conserved estrogen response element sequence motifs or indirectly by tethering to other interacting transcription factors and nucleate transcriptional regulatory complexes which include an array of co-regulator proteins. Genome-scale studies of ERα transcriptional activity and localization have revealed mechanistic complexity and insights including novel interactions with several transcription factors, including FOXA1, AP-2g, GATA3, and RUNX1, which function as pioneering, collaborative, or tethering factors. The major challenge and exciting prospect moving forward is the comprehensive definition and integration of ERα complexes and mechanisms and their tissue-specific roles in normal physiology and in human diseases.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Fator de Transcrição AP-2/metabolismoRESUMO
INTRODUCTION: Liver × receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors and have established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment. METHODS: To further determine the effects of LXR ligands and identify their potential mechanisms of action in breast cancer cells, we carried out microarray analysis of gene expression in four breast cancer cell lines following treatments with the synthetic LXR ligand GW3965. Differentially expressed genes were further subjected to gene ontology and pathway analyses, and their expression profiles and associations with disease parameters and outcomes were examined in clinical samples. Response of E2F target genes were validated by real-time PCR, and the posited role of E2F2 in breast cancer cell proliferation was tested by RNA interference experiments. RESULTS: We observed cell line-specific transcriptional responses as well as a set of common responsive genes. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes. Hierarchical clustering of breast cancer patients based on the expression profiles of the commonly downregulated LXR ligand-responsive genes showed a strong association of these genes with patient survival. CONCLUSIONS: Taken together, these results indicate that LXR ligands target gene networks, including those regulated by E2F family members, are critical for tumor biology and disease progression and merit further consideration as potential agents in the prevention and treatment of breast cancers.
Assuntos
Benzoatos/metabolismo , Benzilaminas/metabolismo , Neoplasias da Mama/genética , Fator de Transcrição E2F2/biossíntese , Receptores Nucleares Órfãos/metabolismo , Benzoatos/administração & dosagem , Benzilaminas/administração & dosagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição E2F2/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Receptores X do Fígado , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacosRESUMO
Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors which regulate the expression of lipid and cholesterol metabolism genes. Moreover, LXRs and their ligands have been shown to inhibit tumor growth in a variety of cancers. We have previously identified the small molecule compound GAC0001E5 (1E5) as an LXR inverse agonist and a potent inhibitor of pancreatic cancer cells. Transcriptomic and metabolomic studies showed that 1E5 disrupts glutamine metabolism, an essential metabolic pathway commonly reprogrammed during malignant transformation, including in breast cancers. To determine the role of LXRs and potential application of 1E5 in breast cancer, we examined LXR expression in publicly available clinical samples, and found that LXR expression is elevated in breast tumors as compared to normal tissues. In luminal A, endocrine therapy-resistant, and triple-negative breast cancer cells, 1E5 exhibited LXR inverse agonist and "degrader" activity and strongly inhibited cell proliferation and colony formation. Treatments with 1E5 downregulated the transcription of key glutaminolysis genes, and, correspondingly, biochemical assays indicated that 1E5 lowered intracellular glutamate and glutathione levels and increased reactive oxygen species. These results indicate that novel LXR ligand 1E5 is an inhibitor of glutamine metabolism and redox homeostasis in breast cancers and suggest that modulating LXR activity and expression in tumor cells is a promising strategy for targeting metabolic reprogramming in breast cancer therapeutics.
Assuntos
Neoplasias da Mama , Receptores Nucleares Órfãos , Humanos , Feminino , Receptores X do Fígado/metabolismo , Receptores Nucleares Órfãos/metabolismo , Ligantes , Agonismo Inverso de Drogas , Glutamina/metabolismo , Homeostase , OxirreduçãoRESUMO
MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.
Assuntos
Neoplasias da Mama/genética , Diferenciação Celular/fisiologia , Redes Reguladoras de Genes , Glândulas Mamárias Humanas/metabolismo , MicroRNAs/fisiologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/citologia , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The central dogma of molecular biology relegates RNAs to the role of "messengers" of genetic information, with proteins as the end products that perform key roles as regulators and effectors of biological processes. Notable exceptions include non-protein-coding RNAs, which function as adaptors (tRNAs) and ribosomal components (rRNAs) during translation, as well as in splicing (snRNAs) and RNA maturation including editing (snoRNAs). Genome and transcriptome projects have revealed, however, a significant number, rivaling the protein-coding transcripts, of non-protein-coding RNAs not related to these previously characterized transcript classes. Non-protein-coding RNA research has primarily focused on microRNAs, a small subclass of non-protein-coding RNAs, and their regulatory roles in gene expression, and these findings have been reviewed extensively. Here, we turn our attention to the larger, in number and size, long non-coding RNAs (lncRNAs), and review their evolutionary complexity and the growing evidence for their diverse mechanisms of action and functional roles in basic molecular and cellular biology and in human disease. In contrast to the focus on in-silico and expression studies in existing lncRNA literature, we emphasize direct evidence for lncRNA function, presenting experimental approaches and strategies for systematic characterization of lncRNA activities, with applications to known gene regulatory networks and diseases.
Assuntos
Pesquisa Biomédica/métodos , Evolução Molecular , MicroRNAs/genética , RNA não Traduzido/fisiologia , RNA/fisiologia , Animais , Pesquisa Biomédica/tendências , Regulação da Expressão Gênica/fisiologia , Redes Reguladoras de Genes/fisiologia , Humanos , MicroRNAs/metabolismo , Modelos Biológicos , RNA/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV+ than HPV- cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV+ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.
Assuntos
Proteínas de Transporte/genética , Proliferação de Células/genética , Papillomavirus Humano 16/patogenicidade , Proteínas de Membrana/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Hormônios Tireóideos/genética , Neoplasias do Colo do Útero/virologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Expressão Gênica , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Fosforilação , Hormônios Tireóideos/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
Tumor-suppressor genes (TSG) are often deleted or transcriptionally suppressed in cancer. PGR codes for progesterone receptor (PR), a transcription factor whose function depends on its ligand. Although PR expression is often undetectable in cervical cancer, its relevance to the endocrine-related etiology of this prevalent gynecological disease remains unclear. In this study, we show that the deletion of one Pgr allele in cervical epithelium promoted spontaneous cervical cancer in human papilloma viral oncogene-expressing transgenic mice as efficiently as the ablation of both Pgr alleles. We also show that tumors arising in the transgenic mice with one or both Pgr alleles did not express PR or expressed at the reduced levels compared with the normal epithelium. PR status correlated with estrogen receptor α (ERα) status in the mouse model and the Cancer Genome Atlas (TCGA) dataset. TCGA data analyses revealed that PGR expression significantly decreased in cervical cancer and that the biallelic deletion of PGR was rare. Furthermore, low PGR expression was associated with poor prognosis in young patients with cervical cancer. These discoveries point to PGR as a haploinsufficient TSG in the uterine cervix. They also raise the possibility that the restoration of PGR expression may improve the survival rate. IMPLICATIONS: The decreased expression of PR may increase the risk of cervical cancer in human papillomavirus-infected women. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/42/F1.large.jpg.
Assuntos
Genes Supressores de Tumor/fisiologia , Receptores de Progesterona/metabolismo , Neoplasias do Colo do Útero/genética , Animais , Feminino , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.
Assuntos
Bocavirus/fisiologia , Vesículas Revestidas por Clatrina/virologia , Endocitose , Internalização do Vírus , Animais , Bocavirus/ultraestrutura , Bovinos , Células Cultivadas , Vesículas Revestidas por Clatrina/ultraestrutura , Células Epiteliais , Microscopia Eletrônica de TransmissãoRESUMO
We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/biossíntese , Fatores de Transcrição SOX9/metabolismo , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Imunoprecipitação , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/efeitos dos fármacos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição HES-1 , Transfecção , Tretinoína/farmacologiaRESUMO
Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation.
Assuntos
DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Genoma Humano , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Regulação da Expressão Gênica/fisiologia , HumanosRESUMO
Estrogen receptor ß (ERß) was first identified in the rodent prostate and is abundantly expressed in human and rodent prostate epithelium, stroma, immune cells and endothelium of the blood vessels. In the prostates of mice with inactivated ERß, mutant phenotypes include epithelial hyperplasia and increased expression of androgen receptor (AR)-regulated genes, most of which are also upregulated in prostate cancer (PCa). ERß is expressed in both basal and luminal cells in the prostate while AR is expressed in luminal but not in the basal cell layer which harbors the prostate stem cells. To investigate the mechanisms of action of ERß and its potential cross-talk with AR, we used RNA-seq to study the effects of estradiol or the synthetic ligand, LY3201, in AR-positive LNCaP PCa cells which had been engineered to express ERß. Transcriptomic analysis indicated relatively few changes in gene expression with ERß overexpression, but robust responses following ligand treatments. There is significant overlap of responsive genes between the two ligands, estradiol and LY3201 as well as ligand-specific alterations. Gene set analysis of down-regulated genes identified an enrichment of androgen-responsive genes, such as FKBP5, CAMKK2, and TBC1D4. Consistently, AR transcript, protein levels, and transcriptional activity were down-regulated following ERß activation. In agreement with this, we find that the phosphorylation of the CAMKK2 target, AMPK, was repressed by ligand-activated ERß. These findings suggest that ERß-mediated signaling pathways are involved in the negative regulation of AR expression and activity, thus supporting a tumor suppressive role for ERß in PCa.
Assuntos
Receptor beta de Estrogênio/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Benzopiranos/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Receptor beta de Estrogênio/agonistas , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Masculino , Receptores Androgênicos/genética , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer. PDACs harbor oncogenic mutations in the KRAS gene, and ongoing efforts to directly target its mutant protein product to inhibit tumor growth are a priority not only in pancreatic cancer but in other malignancies such as lung and colorectal cancers where KRAS is also commonly mutated. An alternative strategy to directly targeting KRAS is to identify and target druggable receptors involved in dysregulated cancer hallmarks downstream of KRAS dysregulation. Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-modulated transcription factors and are involved in the regulation of genes which function in key cancer-related processes, including cholesterol transport, lipid and glucose metabolism, and inflammatory and immune responses. Modulation of LXRs via small molecule ligands has emerged as a promising approach for directly targeting tumor cells or the stromal and immune cells within the tumor microenvironment. We have previously shown that only one of the two LXR subtypes (LXRß) is expressed in pancreatic cancer cells, and targeting LXR with available synthetic ligands blocked the proliferation of PDAC cells and tumor formation. In a screen of a focused library of drug-like small molecules predicted to dock in the ligand-binding pocket of LXRß, we identified two novel LXR ligands with more potent antitumor activity than current LXR agonists used in our published studies. Characterization of the two lead compounds (GAC0001E5 and GAC0003A4) indicates that they function as LXR inverse agonists which inhibit their transcriptional activity. Prolonged treatments with novel ligands further revealed their function as LXR "degraders" which significantly reduced LXR protein levels in all three PDAC cell lines tested. These findings support the utility of these novel inhibitors in basic research on ligand design, allosteric mechanisms, and LXR functions and their potential application as treatments for advanced pancreatic cancer and other recalcitrant malignancies.