RESUMO
BACKGROUND: The Bcl-3 protein is an atypical member of the inhibitor of -κB family that has dual roles as a transcriptional repressor and a coactivator for dimers of NF-κB p50 and p52. Bcl-3 is expressed in mammary adenocarcinomas and can promote tumorigenesis and survival signaling and has a key role in tumor metastasis. In this study, we have investigated the role of Bcl-3 in the normal mammary gland and impact on tumor pathology. METHODS: We utilized bcl-3-/- mice to study mammary gland structure in virgins and during gestation, lactation and early involution. Expression of involution-associated genes and proteins and putative Bcl-3 target genes was examined by qRT-PCR and immunoblot analysis. Cell autonomous branching morphogenesis and collagen I invasion properties of bcl-3-/- organoids were tested in 3D hydrogel cultures. The role of Bcl-3 in tumorigenesis and tumor pathology was also assessed using a stochastic carcinogen-induced mammary tumor model. RESULTS: Bcl-3-/- mammary glands demonstrated reduced branching complexity in virgin and pregnant mice. This defect was recapitulated in vitro where significant defects in bud formation were observed in bcl-3-/- mammary organoid cultures. Bcl-3-/- organoids showed a striking defect in protrusive collective fibrillary collagen I invasion associated with reduced expression of Fzd1 and Twist2. Virgin and pregnant bcl-3-/- glands showed increased apoptosis and rapid increases in lysosomal cell death and apoptosis after forced weaning compared to WT mice. Bcl-2 and Id3 are strongly induced in WT but not bcl-3-/- glands in early involution. Tumors in WT mice were predominately adenocarcinomas with NF-κB activation, while bcl-3-/- lesions were largely squamous lacking NF-κB and with low Bcl-2 expression. CONCLUSIONS: Collectively, our results demonstrate that Bcl-3 has a key function in mammary gland branching morphogenesis, in part by regulation of genes involved in extracellular matrix invasion. Markedly reduced levels of pro-survival proteins expression in bcl-3 null compared to WT glands 24 h post-weaning indicate that Bcl-3 has a role in moderating the rate of early phase involution. Lastly, a reduced incidence of bcl-3-/- mammary adenocarcinomas versus squamous lesions indicates that Bcl-3 supports the progression of epithelial but not metaplastic cancers.
Assuntos
Adenocarcinoma , Proteína 3 do Linfoma de Células B , Neoplasias da Mama , Carcinoma de Células Escamosas , Glândulas Mamárias Animais , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose/genética , Proteína 3 do Linfoma de Células B/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Carcinoma de Células Escamosas/patologia , Colágeno/metabolismo , Células Epiteliais/metabolismo , Feminino , Lactação , Glândulas Mamárias Animais/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
BACKGROUND: The alternative NF-κB pathway is activated by the NF-κB-inducing kinase (NIK) mediated phosphorylation of the inhibitor of κ-B kinase α (IKKα). IKKα then phosphorylates p100/NFKB2 to result in its processing to the active p52 subunit. Evidence suggests that basal breast cancers originate within a subpopulation of luminal progenitor cells which is expanded by signaling to IKKα. OBJECTIVE: To determine the role of IKKα in the development of basal tumors. METHODS: Kinase dead IkkαAA/AA mice were crossed with the C3(1)-TAg mouse model of basal mammary cancer. Tumor growth and tumor numbers in WT and IkkαAA/AA mice were assessed and immunopathology, p52 expression and stem/progenitor 3D colony forming assays were performed. Nik-/- mammary glands were isolated and mammary colonies were characterized. RESULTS: While tumor growth was slower than in WT mice, IkkαAA/AA tumor numbers and pathology were indistinguishable from WT tumors. Both WT and IkkαAA/AA tumors expressed p52 except those IkkαAA/AA tumors where NIK, IKKαAA/AA and ErbB2 were undetectable. Colonies formed by WT and IkkαAA/AA mammary cells were nearly all luminal/acinar however, colony numbers and sizes derived from IkkαAA/AA cells were reduced. In contrast to IkkαAA/AA mice, virgin Nik-/- mammary glands were poorly developed and colonies were primarily derived from undifferentiated bipotent progenitor cells. CONCLUSIONS: C3(1)-TAg induced mammary tumors express p100/p52 even without functional IKKα. Therefore the development of basal-like mammary cancer does not strictly rely on IKKα activation. Signal-induced stabilization of NIK may be sufficient to mediate processing of p100NFKB2 which can then support basal-like mammary tumor formation. Lastly, in contrast to the pregnancy specific role of IKKα in lobuloalveogenesis, NIK is obligatory for normal mammary gland development.
Assuntos
Quinase I-kappa B , Neoplasias Mamárias Animais , Animais , Feminino , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Neoplasias Mamárias Animais/genética , Camundongos , NF-kappa B/metabolismo , Fosforilação , Gravidez , Transdução de SinaisRESUMO
Cellular inhibitor of apoptosis proteins-1 and -2 (cIAP1/2) are integral to regulation of apoptosis and signaling by the tumor necrosis factor (TNF) and related family of receptors. The expression of cIAP2 in tissues is typically low and considered functionally redundant with cIAP1, however cIAP2 can be activated by a variety of cellular stresses. Members of the TNFR family and their ligands have essential roles in mammary gland biology. We have found that cIAP2-/- virgin mammary glands have reduced ductal branching and delayed lobuloalveogenesis in early pregnancy. Post-lactational involution involves two phases where the first phase is reversible and is mediated, in part, by TNFR family ligands. In cIAP2-/- mice mammary glands appeared engorged at mid-lactation accompanied by enhanced autophagic flux and decreased cIAP1 protein expression. Severely stretched myoepithelium was associated with BIM-EL expression and other indicators of anoikis. Within 24 h after forced or natural weaning, cIAP2-/- glands had nearly completed involution. The TNF-related weak inducer of apoptosis (Tweak) which results in degradation of cIAP1 through its receptor, Fn14, began to increase in late lactation and was significantly increased in cIAP2-/- relative to WT mice by 12 h post weaning accompanied by decreased cIAP1 protein expression. Carcinogen/progesterone-induced mammary tumorigenesis was significantly delayed in cIAP2-/- mice and tumors contained high numbers of apoptotic cells. We conclude that cIAP2 has a critical role in the mammary gland wherein it prevents rapid involution induced by milk stasis-induced stress associated with Tweak activation and contributes to the survival of mammary tumor cells.
Assuntos
Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Carcinogênese/metabolismo , Lactação/metabolismo , Glândulas Mamárias Animais/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , DesmameRESUMO
Estrogen receptor (ER)-positive breast cancer cells have low levels of constitutive NF-kappaB activity while ER negative (-) cells and hormone-independent cells have relatively high constitutive levels of NF-kappaB activity. In this study, we have examined the aspects of mutual repression between the ERalpha and NF-kappaB proteins in ER+ and ER- hormone-independent cells. Ectopic expression of the ERalpha reduced cell numbers in ER+ and ER- breast cancer cell lines while NF-kappaB-binding activity and the expression of several NF-kappaB-regulated proteins were reduced in ER- cells. ER overexpression in ER+/E2-independent LCC1 cells only weakly inhibited the predominant p50 NF-kappaB. GST-ERalpha fusion protein pull downs and in vivo co-immunoprecipitations of NF-kappaB:ERalpha complexes showed that the ERalpha interacts with p50 and p65 in vitro and in vivo. Inhibition of NF-kappaB increased the expression of diverse E2-regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF-7 cells by supershift analysis while p65 antibody reduced ERalpha:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF-kappaB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF-kappaB undergo mutual repression, which may explain, in part, why expression of the ERalpha in ER- cells does not confer growth signaling. Secondly, the acquisition of E2-independence in ER+ cells is associated with predominantly p50:p50 NF-kappaB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , NF-kappa B/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade p50 de NF-kappa B/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Estradiol and related estrogens have been widely used as supplements to relieve menopausal symptoms, but they lead to an increased risk of breast and endometrial cancer. Here we report the synthesis of a new family of compounds where we have removed the B-ring from the steroid ABCD structure, and functionalized the A-ring. These A-CD compounds show a preferential affinity for the estrogen receptor subtype ERbeta. Some show binding affinities which are greater than estradiol. The presence of electron-withdrawing substituents on the A-ring should reduce the tendency of these compounds to form carcinogenic metabolites, so they might lead to a safer approach to hormone replacement therapy.
Assuntos
Estradiol , Receptores de Estradiol/agonistas , Estradiol/agonistas , Estradiol/análogos & derivados , Estradiol/síntese química , Estradiol/farmacologia , Feminino , Terapia de Reposição Hormonal/métodos , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
SRC-3/AIB1 (Amplified in Breast Cancer-1) is a nuclear receptor coactivator for the estrogen receptor in breast cancer cells. It is also an intrinsically disordered protein when not engaged with transcriptional binding partners and degraded upon transcriptional coactivation. Given the amplified expression of SRC-3 in breast cancers, the objective of this study was to determine how increasing SRC-3 protein levels are regulated in MCF-7 breast cancer cells. We found that endogenous SRC-3 was expelled from the nucleus in vesicle-like spheres under normal growth conditions suggesting that this form of nuclear exclusion of SRC-3 is a homeostatic mechanism for regulating nuclear SRC-3 protein. Only SRC-3 not associated with CREB-binding protein (CBP) was extruded from the nucleus. We found that overexpression in MCF-7 cells results in aneuploid senescence and cell death with frequent formation of nuclear aggregates which were consistently juxtaposed to perinuclear microtubules. Transfected SRC-3 was SUMOylated and caused redistribution of nuclear promyelocytic leukemia (PML) bodies and perturbation of the nuclear membrane lamin B1, hallmarks of nucleophagy. Increased SRC-3 protein-induced autophagy and resulted in SUMO-1 localization to the nuclear membrane and formation of protrusions variously containing SRC-3 and chromatin. Aspects of SRC-3 overexpression and toxicity were recapitulated following treatment with clinically relevant agents that stabilize SRC-3 in breast cancer cells. We conclude that amplified SRC-3 levels have major impacts on nuclear protein quality control pathways and may mark cancer cells for sensitivity to protein stabilizing therapeutics.
Assuntos
Proteína de Ligação a CREB/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Apoptose/genética , Apoptose/fisiologia , Autofagia/genética , Autofagia/fisiologia , Proteína de Ligação a CREB/genética , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Senescência Celular/fisiologia , Ciclina E/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Células MCF-7 , Microscopia de Fluorescência , Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , Mutagênese Sítio-Dirigida , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Coativador 3 de Receptor Nuclear/genética , Fosforilação , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Tamoxifeno/farmacologiaRESUMO
Recent studies have shown that progesterone receptor (PR)-expressing cells respond to progesterone in part through the induction of the receptor activator of NF-κB ligand (RANKL), which acts in a paracrine manner to induce expansion of a RANK-expressing luminal progenitor cell population. The RANK+ population in human breast tissue from carriers of BRCA1 mutations (BRCA1mut/+) as well as the luminal progenitor population in Brca1-deficient mouse mammary glands is abnormally amplified. Remarkably, mouse Brca1+/- and human BRCA1mut/+ progenitor cells are able to form colonies in vitro in the absence of progesterone, demonstrating a hormone-independent proliferative capacity. Our research has demonstrated that proliferation in BRCA1-deficient cells results in a DNA damage response (DDR) that activates a persistent NF-κB signal, which supplants progesterone/RANKL signaling for an extended time period. Thus, the transcriptional targets normally activated by RANKL that promote a proliferative response in luminal progenitors can contribute to the susceptibility of mammary epithelial cells to BRCA1-mutated breast cancers as a consequence of DDR-induced NF-κB. Together, these latest findings mark substantial progress in uncovering the mechanisms driving high rates of breast tumorigenesis in BRCA1 mutation carriers and ultimately reveal possibilities for nonsurgical prevention strategies. Cancer Prev Res; 11(2); 69-80. ©2017 AACR.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/patologia , Mama/patologia , Transformação Celular Neoplásica/patologia , Mutação , NF-kappa B/metabolismo , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Humanos , Transdução de SinaisRESUMO
Pluripotential embryonal carcinoma cells such as those of the P19 line differentiate when exposed to retinoic acid (RA). The RAC65 cell line is a mutant clone of P19 cells selected to be RA nonresponsive. RAC65 cells carry a rearrangement affecting one of the genes encoding a nuclear retinoic acid receptor (RAR alpha). The mutant gene encodes a protein, RAR alpha', that has lost its 70 C-terminal amino acids, thus truncating the RA-binding domain. The RAR alpha' was found to be a dominant repressor of transcription from an RA-responsive target gene; however, expression of RAR alpha' was insufficient to confer RA nonresponsiveness, suggesting that RAC65 cells carry an additional mutation(s) affecting RA-induced genes.
Assuntos
Proteínas de Transporte/genética , Genes Dominantes , Mutação , Teratoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Diferenciação Celular , Linhagem Celular , DNA de Neoplasias/genética , Resistência a Medicamentos , Biblioteca Gênica , Genes , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores do Ácido Retinoico , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
About one-third of breast cancers express a functional estrogen (beta-estradiol [E2]) receptor (ER) and are initially dependent on E2 for growth and survival but eventually progress to hormone independence. We show here that ER(+), E2-independent MCF-7/LCC1 cells derived from E2-dependent MCF-7 cells contain elevated basal NF-kappaB activity and elevated expression of the transcriptional coactivator Bcl-3 compared with the parental MCF-7 line. LCC1 NF-kappaB activity consists primarily of p50 dimers, although low levels of a p65/p50 complex are also present. The ER(-) breast cancer cell lines harbor abundant levels of both NF-kappaB complexes. In contrast, nuclear extracts from MCF-7 cells contain a significantly lower level of p50 and p65 than do LCC1 cells. Estrogen withdrawal increases both NF-kappaB DNA binding activity and expression of Bcl-3 in MCF-7 and LCC1 cells in vitro and in vivo. Tumors derived from MCF-7 cells ectopically expressing Bcl-3 remain E2 dependent but display a markedly higher tumor establishment and growth rate compared to controls. Expression of a stable form of IkappaBalpha in LCC1 cells severely reduced nuclear expression of p65 and the p65/p50 DNA binding heterodimer. Whereas LCC1 tumors in nude mice were stable or grew, LCC1(IkappaBalpha) tumors regressed after E2 withdrawal. Thus, both p50/Bcl-3- and p65/p50-associated NF-kappaB activities are activated early in progression and serve differential roles in growth and hormone independence, respectively. We propose that E2 withdrawal may initiate selection for hormone independence in breast cancer cells by activation of NF-kappaB and Bcl-3, which could then supplant E2 by providing both survival and growth signals.
Assuntos
Neoplasias da Mama/genética , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteína 3 do Linfoma de Células B , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Núcleo Celular/química , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Feminino , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/química , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Oncogenes , Ovariectomia , Receptores de Estrogênio/metabolismo , Fatores de Transcrição , Ativação TranscricionalRESUMO
Recent studies have shown that the Bcl-2 protein suppresses programmed cell death or apoptosis induced by a variety of stimuli including chemotherapeutic drugs. Because estrogen promotes the survival of estrogen-dependent breast cancer cells in vivo, we investigated whether estrogen might regulate levels of Bcl-2 gene expression in an estrogen-responsive human breast cancer cell line. Estrogen receptor-positive MCF-7 human breast cancer cells cultured in the presence of estrogen express the 8.5-kb Bcl-2 mRNA transcript. Depletion of estrogen from the medium results in loss of expression of the mRNA, whereas reexposure to estrogen markedly induces the Bcl-2 transcript. The changes in Bcl-2 mRNA are paralleled by changes in Bcl-2 protein levels. Estrogen-induced increases in Bcl-2 are significantly inhibited by inclusion of the pure antiestrogen ICI 164,384 in the medium. The Bax protein that heterodimerizes with Bcl-2 and promotes cell death is expressed in MCF-7 cells grown in the presence of estrogen and is unaffected by culture in estrogen-free medium. Estrogen depletion doubles the sensitivity of MCF-7 cells to the cytotoxic effects of Adriamycin compared with cells cultured in medium supplemented with estrogen, consistent with a decrease in the Bcl-2 levels. MCF-7 cells treated simultaneously with estrogen and ICI 164,384 exhibit markedly lower resistance to Adriamycin compared with cells treated with estrogen alone. In the absence of estrogen, MCF-7 cells transfected with Bcl-2 expression plasmids display a marked increase in resistance to Adriamycin. In the presence of estrogen, MCF-7 cells expressing Bcl-2 antisense transcripts are rendered twice as sensitive to acute Adriamycin cytotoxicity as a control clone. We conclude that estrogen can promote resistance of estrogen receptor bearing human breast cancer cells to chemotherapeutic drugs through a mechanism that involves regulation of the Bcl-2 proto-oncogene.
Assuntos
Neoplasias da Mama/metabolismo , Estrogênios/farmacologia , Neoplasias Hormônio-Dependentes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Apoptose , Neoplasias da Mama/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Doxorrubicina/farmacologia , Resistência a Medicamentos , Estradiol/análogos & derivados , Estradiol/farmacologia , Humanos , Neoplasias Hormônio-Dependentes/química , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/genética , Alcamidas Poli-Insaturadas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Human BRCA1 mutation carriers and BRCA1-deficient mouse mammary glands contain an abnormal population of mammary luminal progenitors that can form 3D colonies in a hormone-independent manner. The intrinsic cellular regulatory defect in these presumptive breast cancer precursors is not known. We have discovered that nuclear factor kappaB (NF-κB) (p52/RelB) is persistently activated in a subset of BRCA1-deficient mammary luminal progenitors. Hormone-independent luminal progenitor colony formation required NF-κB, ataxia telangiectasia-mutated (ATM), and the inhibitor of kappaB kinase, IKKα. Progesterone (P4)-stimulated proliferation resulted in a marked enhancement of DNA damage foci in Brca1(-/-) mouse mammary. In vivo, NF-κB inhibition prevented recovery of Brca1(-/-) hormone-independent colony-forming cells. The majority of human BRCA1(mut/+) mammary glands showed marked lobular expression of nuclear NF-κB. We conclude that the aberrant proliferative capacity of Brca1(-/-) luminal progenitor cells is linked to the replication-associated DNA damage response, where proliferation of mammary progenitors is perpetuated by damage-induced, autologous NF-κB signaling.
Assuntos
Proteína BRCA1/deficiência , Mama/patologia , Dano ao DNA , Glândulas Mamárias Animais/patologia , NF-kappa B/metabolismo , Células-Tronco/patologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Subunidade p52 de NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Progesterona/farmacologia , Ligação Proteica/efeitos dos fármacos , Sesquiterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Ensaio Tumoral de Célula-TroncoRESUMO
All-trans retinoic acid inhibits growth associated with downregulation of cyclin D1 and can cause low level apoptosis in estrogen receptor positive breast cancer cell lines. The cyclin D1 gene is amplified and/or the protein overexpressed in about one-third of breast cancers. Constitutive expression of cyclin D1 in estrogen receptor positive MCF-7 and ZR-75 breast cancer cells (MCF-7(cycD1) and ZR-75(cycD1)) Increased the fraction of cells in S phase and reduced the G1 accumulation following retinoic acid treatment compared with control cells. However, culture of MCF-7(cycD1) with 1 microM all-trans retinoic acid resulted in about threefold greater growth inhibition compared with vector-transfected cells. Hoechst staining of DNA and in situ DNA end-labeling analysis indicated that MCF-7(cycD1) and ZR-75(cycD1) cultures contained 4-6-fold more retinoic acid-induced apoptotic nuclei as vector-transfected cells. Retinoic acid treatment of vector-transfected clones resulted in Bax protein activation as assessed by exposure of the NH(2)-terminus of Bax but the proportion of cells containing activated Bax was increased in cyclin D-expressing cells treated with retinoic acid. The latter cells also displayed both immunocytochemical and biochemical evidence of translocation of cytochrome c into the cytosol following RA-treatment. Retinoic acid markedly decreased the Bcl-2 levels in MCF-7 and ZR-75 cells. Accordingly, coexpression of Bcl-2 and cyclin D1 rendered the cells resistant to retinoic acid-induced apoptosis. We conclude that constitutive expression of cyclin D1 sensitizes ER-positive breast cancer cells to a retinoic acid-induced mitochondrial death pathway involving Bax activation, cytochrome c release and caspase-9 cleavage.
Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Ciclina D1/fisiologia , Mitocôndrias/fisiologia , Tretinoína/farmacologia , Apoptose/efeitos dos fármacos , Caspase 9 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular , Núcleo Celular/efeitos dos fármacos , Ciclina D1/genética , Grupo dos Citocromos c/análise , Feminino , Fase G1/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/metabolismo , Fase S/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Retinoic acid (RA) inhibition of breast cancer cell growth is associated with an accumulation of cells in G1 phase of the cell cycle. We have investigated the effects of RA on the expression and activity of cell cycle-regulatory proteins in MCF-7 human breast cancer cells. Flow cytometry analysis of MCF-7 cells treated with RA revealed a decrease in the percentage of cells in S phase by 48 h, which was maximal by 72 h. Phosphorylation of the retinoblastoma protein (pRb) was partially reduced in RA-treated cells accompanied by a decrease in the level of retinoblastoma protein. Expression of the cyclin D1 transcript was reduced by 48 h and cyclin-dependent kinase 2 (cdk2) mRNA levels declined within 8 h posttreatment followed by a decrease in cyclin D1 and cdk2 protein levels. Message and protein levels of cdk4 and cdc2 were not affected by RA. While cdk4 activity was similar in control and RA-treated cells, cdk2 activity began to decrease within 48 h of exposure to RA and was profoundly reduced after 72 h. This reduced activity was associated with decreased phosphorylation of cdk2. The decrease in cdk2 activity occurred in the absence of RA-mediated increases in the levels of the cdk inhibitors p21 and p27. However, assays of cdk2 from pooled lysates from RA-treated and control cells showed that RA-treated cells contain a cdk2-inhibitory activity. Our results show that RA inhibits cell cycle progression of MCF-7 cells by inhibiting cdk2 mRNA and protein production and by decreasing cdk2 activity.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas , Tretinoína/farmacologia , Proteínas Supressoras de Tumor , Neoplasias da Mama/genética , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ciclina D1 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/efeitos dos fármacos , Ciclinas/genética , Ciclinas/metabolismo , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Oncogênicas/efeitos dos fármacos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Proteína do Retinoblastoma/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Células Tumorais Cultivadas , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
We recently showed that estrogen induces expression of the anti-apoptotic protein, Bcl-2 in MCF-7 human breast cancer cells. Since estrogen-dependent breast tumours can regress following estrogen withdrawal, we hypothesized that stable Bcl-2 expression would prevent estrogen-withdrawal induced regression of MCF-7 tumours. We therefore established tumours in ovariectomized female nude mice implanted with an estrogen-release pellet using untransfected MCF-7 cells or MCF-7 cells stably transfected with a Bcl-2 cDNA sense or antisense expression vector. All tumours grew at similar rates indicating that Bcl-2 levels have no effect on tumour formation. After removal of the estrogen pellet, Bcl-2 antisense tumours and untransfected MCF-7 tumours regressed means of 49% and 52%, respectively, after estrogen pellet removal whereas Bcl-2 sense tumours were significantly stabilized. Regressing tumours displayed characteristics of apoptotic cells. These results show that Bcl-2 can prevent hormone-dependent breast tumour regression and are consistent with the notion that decreased Bcl-2 levels following estrogen withdrawal renders hormone-dependent breast tumour cells sensitive to apoptotic regression.
Assuntos
Estrogênios/fisiologia , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Animais , Apoptose , Estrogênios/administração & dosagem , Feminino , Humanos , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Síndrome de Abstinência a Substâncias , Células Tumorais CultivadasRESUMO
Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1-1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300-500 muU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Carcinógenos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Iodeto Peroxidase/metabolismo , Iodetos/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/metabolismo , Animais , Northern Blotting , Eletroforese em Gel de Poliacrilamida , RNA Mensageiro/análise , OvinosRESUMO
In this study we report the spontaneous production of autoimmune anti-thyroglobulin antibodies (Tg Ab) by peripheral blood lymphocytes from patients with autoimmune thyroid disease and the use of these cells to study the regulation of this production in vitro. Using a mitogen free microculture system we have found a significant correlation between an individuals' serum titre and the amount of Tg Ab produced in vitro. At low B:T cell ratios Tg Ab production decreased. Specific depletion of suppressor T cells and helper T cells was accomplished using the monoclonal antibodies OKT 8 and OKT 4 respectively. Suppressor cell depletions resulted in increases in Tg Ab production while depletion of helper cells had no consistent effect. The addition of pokeweed mitogen had no stimulatory effect on spontaneous thyroglobulin antibody production. In contrast, when T cell conditioned media was added to the cells, Tg Ab production increased substantially in all patient cultures tested but not in normal control cultures. The results of this study are consistent with the existence of functional thyroid-antigen specific suppressor cells in the peripheral blood which are actively involved in the regulation of autoantibody producing B cells. The results obtained with the cultures maintained in conditioned media show that autoimmune lymphocytes are extremely sensitive to one or more stimulatory factors present in the media and suggests an important role for the production of soluble lymphocyte factors and the expression of receptors for these factors in the aetiology of autoimmune thyroid disease.
Assuntos
Autoanticorpos/biossíntese , Linfócitos T/imunologia , Tireoglobulina/imunologia , Linfócitos B/imunologia , Humanos , Técnicas In Vitro , Cooperação Linfocítica/imunologia , Linfocinas/imunologia , Solubilidade , Linfócitos T Reguladores/imunologia , Tireoidite Autoimune/imunologiaRESUMO
Vasomotor thermo-dysregulation (hot flashes) are an often debilitating symptom of menopause. Effective treatment is achieved primarily through activation of the estrogen receptor (ER)α with estrogens but is also associated with increased risk for breast and uterine cancer. In this study, we have tested novel compounds lacking the B ring of 17-hydroxy-ß-estradiol (E2) (A-CD compounds) with differing ratios of ERα:ERß binding affinities for the ability to reduce diurnal/nocturnal tail-skin temperatures (TSTs) in the ovariectomized female rat menopausal hot flash model. Normal mammary tissue expresses the predominantly antiproliferative ERß. Therefore, we hypothesized that a preferential ERß agonist with fractional ERα activity would safely reduce TSTs. The A-CD compound, L17, is a preferential ERß agonist that has a ratio of ERß:ERα binding affinity relative to E2 of 9.3 (where ERß:ERα for E2, 1.0). In the ovariectomized rat, daily administration of low doses (1 mg/kg) of the A-CD compound TD81 (ERα:ERß relative affinity, 15.2) was ineffective in temperature regulation, whereas L17 showed a trend toward TST reduction. Both E2 and the A-CD compound, TD3 (ERß:ERα relative affinity, 5.0), also reduced TSTs but had marked proliferative effects on mammary and uterine tissues. At 2 mg/kg, L17 strongly reduced TSTs even more effectively than E2 but, importantly, had only minimal effect on uterine weight and mammary tissues. Both E2- and L17-treated rats showed similar weight reduction over the treatment period. E2 is rapidly metabolized to highly reactive quinones, and we show that L17 has 2-fold greater metabolic stability than E2. Finally, L17 and E2 similarly mediated induction of c-fos expression in neurons within the rat thermoregulatory hypothalamic median preoptic nucleus. Thus, the A-CD compound, L17, may represent a safe and effective approach to the treatment of menopausal hot flashes.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Menopausa , Modelos Animais , Sistema Vasomotor/fisiologia , Adiposidade/efeitos dos fármacos , Animais , Ligação Competitiva , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Estradiol/química , Estradiol/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Estrogênios/química , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Fogachos/metabolismo , Fogachos/fisiopatologia , Humanos , Imuno-Histoquímica , Ligantes , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Estrutura Molecular , Ovariectomia , Área Pré-Óptica/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimento , Útero/metabolismo , Sistema Vasomotor/efeitos dos fármacos , Aumento de Peso/efeitos dos fármacosRESUMO
Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER(+)) breast cancers. There are two ER subtypes, ERα and ERß, which promote and inhibit breast cancer cell proliferation, respectively. Although ER(+) breast cancers typically express a high ratio of ERα to ERß, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERß. On some gene enhancers, ERß has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERß agonists, WAY-20070 and a novel "A-CD" estrogen called L17, produce a marked reduction in G(2)-M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERß agonists recruited both the ERα and ERß to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERß-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERß. Exposure to the ERß ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERß agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERß agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Oxazóis/farmacologia , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Ligantes , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The cold inducible RNA binding protein (CIRBP) responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC), at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6) encoded proteins important in inflammation with IL-1ß apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS) was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1ß expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.
Assuntos
Interleucina-1beta/genética , NF-kappa B/genética , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Temperatura Baixa , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/imunologia , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Lipopolissacarídeos/farmacologia , Inibidor de NF-kappaB alfa , NF-kappa B/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Raios UltravioletaRESUMO
Cellular inhibitors of apoptosis proteins 1 and 2 (cIAP1/2) are members of the inhibitor of apoptosis protein (IAP) family that has been implicated in the pathology of human cancers due to their overexpression and function as blockers of cell death in various cancers. As a result, small molecule IAP antagonists have been developed and are currently under clinical evaluation for potential therapeutic use. In contrast, recent evidence has indicated a tumour-suppressing role for the cIAPs. Mutations in or loss of cIAPs have been identified as molecular lesions that contribute to constitutive activation of NF-κB in hematopoietic malignancies. These studies reveal a context-dependent role for the cIAPs wherein both their overexpression and loss may contribute to tumourigenesis.