RESUMO
Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which no effective targeted therapies are available. Growing evidence suggests that chemotherapy-resistant cancer cells with stem-like properties (CSC) may repopulate the tumor. The androgen receptor (AR) is expressed in up to 50% of TNBCs, and AR inhibition decreases CSC and tumor initiation. Runt-related transcription factor 1 (RUNX1) correlates with poor prognosis in TNBC and is regulated by the AR in prostate cancer. Our group has shown that RUNX1 promotes TNBC cell migration and regulates tumor gene expression. We hypothesized that RUNX1 is regulated by the AR and that both may work together in TNBC CSC to promote disease recurrence following chemotherapy. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments in MDA-MB-453 revealed AR binding to RUNX1 regulatory regions. RUNX1 expression is upregulated by dihydrotestosterone (DHT) in MDA-MB-453 and in an AR+-TNBC HCI-009 patient-derived xenograft (PDX) tumors (p < 0.05). RUNX1 is increased in a CSC-like experimental model in MDA-MB-453 and SUM-159PT cells (p < 0.05). Inhibition of RUNX1 transcriptional activity reduced the expression of CSC markers. Interestingly, RUNX1 inhibition reduced cell viability and enhanced paclitaxel and enzalutamide sensitivity. Targeting RUNX1 may be an attractive strategy to potentiate the anti-tumor effects of AR inhibition, specifically in the slow-growing CSC-like populations that resist chemotherapy which lead to metastatic disease.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Recidiva Local de Neoplasia , Receptores Androgênicos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , FemininoRESUMO
Background: The high COVID-19 dissemination rate demands active surveillance to identify asymptomatic, presymptomatic, and oligosymptomatic (APO) SARS-CoV-2-infected individuals. This is of special importance in communities inhabiting closed or semi-closed institutions such as residential care homes, prisons, neuropsychiatric hospitals, etc., where risk people are in close contact. Thus, a pooling approach-where samples are mixed and tested as single pools-is an attractive strategy to rapidly detect APO-infected in these epidemiological scenarios. Materials and Methods: This study was done at different pandemic periods between May 28 and August 31 2020 in 153 closed or semi-closed institutions in the Province of Buenos Aires (Argentina). We setup pooling strategy in two stages: first a pool-testing followed by selective individual-testing according to pool results. Samples included in negative pools were presumed as negative, while samples from positive pools were re-tested individually for positives identification. Results: Sensitivity in 5-sample or 10-sample pools was adequate since only 2 Ct values were increased with regard to single tests on average. Concordance between 5-sample or 10-sample pools and individual-testing was 100% in the Ct ≤ 36. We tested 4,936 APO clinical samples in 822 pools, requiring 86-50% fewer tests in low-to-moderate prevalence settings compared to individual testing. Conclusions: By this strategy we detected three COVID-19 outbreaks at early stages in these institutions, helping to their containment and increasing the likelihood of saving lives in such places where risk groups are concentrated.
RESUMO
Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Conexina 43/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Trombospondinas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia de Fluorescência , Regiões Promotoras Genéticas/genética , Células Tumorais Cultivadas , Cicatrização , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3'-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes.
Assuntos
RNA Polimerase II/metabolismo , Receptores de Progesterona/metabolismo , Elongação da Transcrição Genética , Proteína bcl-X/genética , Processamento Alternativo , Sítios de Ligação , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Humanos , Fator B de Elongação Transcricional Positiva/metabolismo , Promegestona/farmacologia , Proteína bcl-X/biossíntese , Proteína bcl-X/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lactação/efeitos dos fármacos , Lactação/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Physiological cell turnover is under the control of a sharp and dynamic balance of different homeostatic mechanisms such as the equilibrium between cell proliferation and cell death. These mechanisms play an important role in maintaining normal tissue function and architecture. It is well known that apoptosis is the prevalent mode of physiological cell loss in most tissues. Steroid hormones like glucocorticoids have been identified as key signals controlling cell turnover by modulating programmed cell death in a tissue- and cell-specific manner. In this sense, several reports have demonstrated that glucocorticoids are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes. In contrast, they protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, in glandular cells such as the mammary gland epithelia, endometrium, hepatocytes, ovarian follicular cells, and fibroblasts. Although several studies have provided significant information on hormone-dependent apoptosis in an specific tissue, a clearly defined pathway that mediates cell death in response to glucocorticoids in different cell types is still misunderstood. The scope of this review is held to those mechanisms by which glucocorticoids control apoptosis, emphasizing tissue-specific expression of genes that are involved in the apoptotic pathway.
Assuntos
Apoptose , Glucocorticoides/metabolismo , Animais , Biomarcadores , Humanos , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-bcl-2/classificação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de SinaisRESUMO
The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-X(L) (antiapoptotic)/bcl-X(S) (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X(L)/bcl-X(S) favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4.
Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Linfócitos/metabolismo , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT5/metabolismo , Timo/metabolismo , Proteína bcl-X/metabolismo , Acetilação , Animais , Apoptose , Células COS , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Antagonistas de Hormônios/farmacologia , Linfócitos/citologia , Masculino , Camundongos , Mifepristona/farmacologia , Coativador 1 de Receptor Nuclear , Plasmídeos/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/citologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genéticaRESUMO
The antiapoptotic effect of melatonin has been described in several systems. In this study, the antagonistic effect of the methoxyindole on dexamethasone-induced apoptosis in mouse thymocytes was examined. Melatonin decreased both DNA fragmentation, and the number of annexin V-positive cells incubated in the presence of dexamethasone. Analysis of the expression of the members of the Bcl-2 family indicated that the synthetic glucocorticoid increased Bax protein levels without affecting the levels of Bcl-2, Bcl-XL, Bcl-XS, or Bak. This effect correlated with an increase in thymocytes bax mRNA levels. Dexamethasone also increased the release of cytochrome C from mitochondria. All of these effects were reduced in the presence of melatonin, which was ineffective per se on these parameters. In addition, the involvement of cAMP on glucocorticoid/melatonin antagonism was examined. Both melatonin and dexamethasone decreased the levels of this nucleotide in mouse thymocytes, indicating that the antagonistic action between both hormones involves a cAMP-independent pathway. In summary, the present results suggest that the antiapoptotic effect of melatonin on glucocorticoid-treated thymocytes would be a consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bax protein levels.