RESUMO
Progression to hormone-independent growth leading to endocrine therapy resistance occurs in a high proportion of patients with estrogen receptor alpha (ERα) and progesterone receptors (PR) positive breast cancer. We and others have previously shown that estrogen- and progestin-induced tumor growth requires ERα and PR interaction at their target genes. Here, we show that fibroblast growth factor 2 (FGF2)-induces cell proliferation and tumor growth through hormone-independent ERα and PR activation and their interaction at the MYC enhancer and proximal promoter. MYC inhibitors, antiestrogens or antiprogestins reverted FGF2-induced effects. LC-MS/MS identified 700 canonical proteins recruited to MYC regulatory sequences after FGF2 stimulation, 397 of which required active ERα (ERα-dependent). We identified ERα-dependent proteins regulating transcription that, after FGF2 treatment, were recruited to the enhancer as well as proteins involved in transcription initiation that were recruited to the proximal promoter. Also, among the ERα-dependent and independent proteins detected at both sites, PR isoforms A and B as well as the novel protein product PRBΔ4 were found. PRBΔ4 lacks the hormone-binding domain and was able to induce reporter gene expression from estrogen-regulated elements and to increase cell proliferation when cells were stimulated with FGF2 but not by progestins. Analysis of the Cancer Genome Atlas data set revealed that PRBΔ4 expression is associated with worse overall survival in luminal breast cancer patients. This discovery provides a new mechanism by which growth factor signaling can engage nonclassical hormone receptor isoforms such as PRBΔ4, which interacts with growth-factor activated ERα and PR to stimulate MYC gene expression and hence progression to endocrine resistance.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptores de Progesterona/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Prognóstico , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Progesterona/genética , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In C4-HD murine mammary carcinomas and in human breast cancer T47D cells, we showed that medroxyprogesterone acetate (MPA) induces a nuclear physical association between estrogen receptor alpha (ERa) and progesterone receptors (PR). The blockade of ERa inhibits cell proliferation mediated by progestins. We hypothesized that this nuclear association between ERa/PR is necessary to trigger progestin-induced cell proliferation and tumor growth. We demonstrated that fulvestrant (FUL, ICI182.780) induced complete regression of C4-HD tumors growing with progestins. MPA treatment induced an early increase in both CCND1 and MYC expression in T47D cells. The blockade of ERa prevented the MPA-dependent transcription of both genes. Specific binding of PR/ERa was observed at the same MPA-sensitive regions at the CCND1 and MYC gene promoters after chromatin immunoprecipitation (ChIP) analysis. ICI inhibited binding of ERa to both gene regulatory sequences while PR binding was unaffected. The nuclear colocalization between both receptors in T47D cells was confirmed by: confocal microscopy, Duolink assays and co-immunoprecipitation assays. In breast cancer samples we also observed a nuclear interaction between both steroid receptors. Our results indicate that the presence of ERa interacting with activated PR at the CCND1 and MYC promoters is required to trigger progestin-induced gene transcription and cell proliferation in breast cancer cells.
Assuntos
Carcinoma/patologia , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/fisiologia , Neoplasias Mamárias Experimentais/patologia , Receptores de Progesterona/fisiologia , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Carcinoma/induzido quimicamente , Carcinoma/tratamento farmacológico , Proliferação de Células , Imunoprecipitação da Cromatina , Ciclina D1/metabolismo , Estradiol/administração & dosagem , Receptor alfa de Estrogênio/efeitos dos fármacos , Feminino , Fulvestranto , Genes myc , Humanos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/tratamento farmacológico , Acetato de Medroxiprogesterona/farmacologia , Murinae , Progestinas/metabolismo , Receptores de Progesterona/efeitos dos fármacos , Transcrição GênicaRESUMO
Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.
Assuntos
Neoplasias da Mama/patologia , Ciclina D1/genética , Receptor alfa de Estrogênio/metabolismo , Genes myc , Neoplasias Mamárias Experimentais/patologia , Receptores de Progesterona/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Processos de Crescimento Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Acetato de Medroxiprogesterona/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Regiões Promotoras Genéticas , Receptores de Progesterona/genéticaRESUMO
More than 60% of all breast neoplasias are ductal carcinomas expressing estrogen (ER) and progesterone receptors (PR). By contrast, most of the spontaneous, chemically or mouse mammary tumor virus induced tumors, as well as tumors arising in genetically modified mice do not express hormone receptors. We developed a model of breast cancer in which the administration of medroxyprogesterone acetate to BALB/c female mice induces mammary ductal carcinomas with a mean latency of 52 weeks and an incidence of about 80%. These tumors are hormone-dependent (HD), metastatic, express both ER and PR, and are maintained by syngeneic transplants. The model has been further refined to include mammary carcinomas that evolve through different stages of hormone dependence, as well as several hormone-responsive cell lines. In this review, we describe the main features of this tumor model, highlighting the role of PR as a trigger of key signaling pathways mediating tumor growth. In addition, we discuss the relevance of this model in comparison with other presently used breast cancer models pointing out its advantages and limitations and how, this model may be suitable to unravel key questions in breast cancer.
Assuntos
Carcinoma Ductal/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Receptores de Progesterona/fisiologia , Animais , Antineoplásicos Hormonais/toxicidade , Feminino , Neoplasias Mamárias Experimentais/patologia , Acetato de Medroxiprogesterona/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Estrogênio/metabolismoRESUMO
To explore mechanisms related to hormone resistance, three resistant variants of the MPA mouse breast cancer tumor model with low levels of progesterone receptor (PR) isoform A (PR-A)/high PR-B expression were developed by prolonged selective pressure with antiprogestins. The resistant phenotype of one tumor line was reversed spontaneously after several consecutive passages in syngeneic BALB/c mice or by 17-beta-estradiol or tamoxifen treatment, and this reversion was significantly associated with an increase in PR-A expression. The responsive parental tumors disclosed low activation of ERK and high activation of AKT; resistant tumors on the other hand, showed the opposite, and this was associated with a higher metastatic potential, that did not revert. This study shows for the first time in vivo a relationship between PR isoform expression and antiprogestin responsiveness, demonstrating that, whereas acquired resistance may be reversed, changes in kinase activation and metastatic potential are unidirectional associated with tumor progression.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Antagonistas de Hormônios/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Mifepristona/farmacologia , Receptores de Progesterona/metabolismo , Animais , Western Blotting , Proliferação de Células , Feminino , Imunofluorescência , Metástase Linfática , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Taxa de SobrevidaRESUMO
We have developed a murine mammary tumor cell line, MC4-L4, and after 15 passages, a spindle-shaped population became evident. The cuboidal cells, MC4-L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti-progestin treatment and stopped growing after 17-beta-estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17-beta-estradiol, and EGF and were inhibited by TGFbeta1. They expressed mutated p53 and estrogen receptors alpha; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle-shaped cells, MC4-L4F, were selected by differential attachment and detachment and proved to be non-epithelial non-tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFbeta1 and EGF, showed a wild-type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors alpha, they were insensitive to 17-beta-estradiol in proliferation assays. Co-cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17-beta-estradiol increased cell proliferation only in co-cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial-stromal interactions in the context of hormone responsiveness in hormone related tumors.
Assuntos
Neoplasias da Mama , Linhagem Celular Tumoral , Glândulas Mamárias Animais/citologia , Células Estromais , Animais , Antineoplásicos Hormonais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/fisiologia , Forma Celular , Técnicas de Cocultura , Fator de Crescimento Epidérmico/metabolismo , Estradiol/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Marcadores Genéticos , Antagonistas de Hormônios/metabolismo , Humanos , Queratinas/metabolismo , Acetato de Medroxiprogesterona/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mifepristona/metabolismo , Mutação , Transplante de Neoplasias , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
INTRODUCTION: Previous data from our laboratory suggested that progesterone receptors (PRs) are involved in progestin-independent growth of mammary carcinomas. To investigate this possibility further, we studied the effects of PR antisense oligodeoxynucleotides (asPR) on in vivo tumor growth. METHOD: BALB/c mice with subcutaneous 25 mm2 mammary carcinomas expressing estrogen receptor-alpha and PR were either injected intraperitoneally with 1 mg asPR every 24 or 12 hours for 5-10 days, or subcutaneously with RU 486 (6.5 mg/kg body weight) every 24 hours. Control mice received vehicle or scPR. RESULTS: Significant inhibition of tumor growth as well as a significant decrease in bromodeoxyuridine uptake was observed in asPR-treated mice, which correlated with histological signs of regression and increased apoptosis. Mice treated with RU 486 experienced almost complete tumor regression. No differences were detected between vehicle-treated and scPR-treated mice. Anti-progestin-treated and asPR-treated mice were in a continuous estrous/meta-estrous state. Decreased phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2 levels and estrogen receptor-alpha expression were observed as late events in RU 486-treated and asPR-treated mice with regressing tumors. CONCLUSION: We demonstrate, for the first time, inhibition of tumor growth in vivo using asPR. Our results provide further evidence for a critical and hierarchical role of the PR pathway in mammary carcinomas.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Receptores de Progesterona/fisiologia , Animais , Neoplasias da Mama/fisiopatologia , Receptor alfa de Estrogênio/biossíntese , Feminino , Humanos , Infusões Parenterais , Injeções Subcutâneas , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/administração & dosagem , Receptores de Progesterona/antagonistas & inibidoresRESUMO
Using medroxyprogesterone acetate (MPA) as a carcinogen, we were able to induce in BALB/c female mice, several progestin-dependent mammary ductal carcinomas that regress completely with estrogen or antiprogestins and are maintained by serial transplantations in syngeneic mice. Progestin-independent variants were subsequently generated or appeared spontaneously. Based on their response to estrogen or antiprogestins, we subdivided them into responsive progestin-independent (R-PI) variants which regress completely and unresponsive progestin-independent (UR-PI) carcinomas which are resistant to both families of compounds. In this study we have investigated progesterone receptor (PR) expression in six responsive progestin-dependent, six R-PI, and three UR-PI tumors. Progestin-dependent and R-PI tumors disclosed a higher expression of the PR(A) isoform as compared with PR(B), as well as an additional band of 78 kDa that was not detected in uterine tissue; all were down-regulated by progestins. UR-PI tumors expressed lower levels of all bands in western blots, but were highly reactive by immunohistochemistry. PR RNA expression was detected in both, UR-PI and R-PI tumors. PR binding was comparable in progestin-dependent and R-PI tumors. In the three UR-PI tumors, only 29/61 (48%) of the samples evaluated showed low binding levels, the rest were negative. This report is the first to describe in an experimental model of breast cancer the expression of PR isoforms and their distribution. Our results suggest the expression of functionally altered isoforms in a subgroup of mammary carcinomas, which may explain their lack of hormone response.
Assuntos
Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/fisiopatologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/fisiopatologia , Acetato de Medroxiprogesterona/efeitos adversos , Congêneres da Progesterona/efeitos adversos , Progestinas/farmacologia , Receptores de Progesterona/biossíntese , Animais , Western Blotting , Regulação para Baixo , Estrogênios/farmacologia , Feminino , Imuno-Histoquímica , Acetato de Medroxiprogesterona/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Congêneres da Progesterona/administração & dosagem , RNA/biossínteseRESUMO
The purpose of this study was to evaluate the effect of the selective estrogen receptor modulators raloxifene and tamoxifen and of the pure antiestrogen fulvestrant on tumor growth and progesterone receptor (PR) expression in an experimental model of breast cancer. The effects of these compounds on cell proliferation were studied in primary cultures of a progestin-dependent mammary carcinoma tumor line, in the presence of medroxyprogesterone acetate (MPA) or 17-beta-estradiol (E2). In in vivo studies the tumor was inoculated subcutaneously in BALB/c female mice treated with 20 mg MPA depot. Raloxifene (12.5 mg/kg) or tamoxifen (5 mg/kg) were administered in daily doses or E2 silastic pellets (5 mg) were implanted. When the tumors reached about 25-50 mm2 MPA was removed in half of the animals. E2 induced complete tumor regressions, tamoxifen inhibited tumor growth in vivo while raloxifene disclosed proliferative effects in animals in which MPA had been removed. In vitro, E2 inhibited cell proliferation at concentrations higher than 10(-14)M. Raloxifene and fulvestrant, but not tamoxifen, partially reverted E2-induced inhibition. Fulvestrant and tamoxifen inhibited MPA-induced cell proliferation while raloxifene had a stimulatory effect. Tamoxifen and E2 increased, raloxifene induced no effect, and fulvestrant significantly decreased PR expression. In this study we provide evidence for differential effects of tamoxifen and raloxifene on experimental mammary tumors. Since raloxifene is under evaluation for use in breast cancer prevention, these results may have important clinical implications.