RESUMEN
SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular , Colágeno/metabolismo , Glándulas Mamarias Humanas/crecimiento & desarrollo , Animales , Matriz Extracelular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones NoqueadosRESUMEN
In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations, including gland-reconstituting mammary stem cells, with largely undefined functions. Here, we have studied how vimentin deficiency affects mouse mammary gland development. We find that, in adult vimentin knockout mice (Vim-/- ), mammary ductal outgrowth is delayed. The adult Vim-/- glands display dilated ducts and a reduced basal-to-luminal mouse mammary epithelial cell (MMEC) ratio indicative of altered progenitor cell activity. Accordingly, isolated Vim-/- MMECs form fewer mammospheres and basal-like organoids in vitro than their wild-type counterparts. Importantly, reduced basal MMEC number translates into defects in Vim-/- mammary gland regeneration in vivo Global gene expression profiling of basal MMECs reveals that lack of vimentin alters multiple pathways, including adhesion, cancer and Wnt signalling. Furthermore, vimentin contributes to stem-like cell properties in MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumoursphere formation and attenuates expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells.
Asunto(s)
Células Epiteliales/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Vimentina/metabolismo , Animales , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/citología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Glándulas Mamarias Animales/citología , Ratones Noqueados , Organoides/metabolismo , Regeneración , Esferoides Celulares/patología , Células Madre/citología , Células Madre/metabolismo , Células del Estroma/metabolismo , Vimentina/deficienciaRESUMEN
To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2(-/-) female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2(-/-) mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2(-/-)mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal ß-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2(-/-) mice. The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.
Asunto(s)
Metabolismo de los Lípidos , Glándulas Mamarias Animales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Peroxisomas/metabolismo , Tejido Adiposo/metabolismo , Animales , Ácidos y Sales Biliares/química , Células Epiteliales/citología , Ácidos Grasos/química , Femenino , Homeostasis , Lactancia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Poliaminas/química , Fracciones Subcelulares , Factores de TiempoRESUMEN
Approximately 70% of all newly diagnosed breast cancers express estrogen receptor (ER)-α. Although inhibiting ER action using targeted therapies such as fulvestrant (ICI) is often effective, later emergence of antiestrogen resistance limits clinical use. We used antiestrogen-sensitive and -resistant cells to determine the effect of antiestrogens/ERα on regulating autophagy and unfolded protein response (UPR) signaling. Knockdown of ERα significantly increased the sensitivity of LCC1 cells (sensitive) and also resensitized LCC9 cells (resistant) to antiestrogen drugs. Interestingly, ERα knockdown, but not ICI, reduced nuclear factor (erythroid-derived 2)-like (NRF)-2 (UPR-induced antioxidant protein) and increased cytosolic kelch-like ECH-associated protein (KEAP)-1 (NRF2 inhibitor), consistent with the observed increase in ROS production. Furthermore, autophagy induction by antiestrogens was prosurvival but did not prevent ERα knockdown-mediated death. We built a novel mathematical model to elucidate the interactions among UPR, autophagy, ER signaling, and ROS regulation of breast cancer cell survival. The experimentally validated mathematical model explains the counterintuitive result that knocking down the main target of ICI (ERα) increased the effectiveness of ICI. Specifically, the model indicated that ERα is no longer present in excess and that the effect on proliferation from further reductions in its level by ICI cannot be compensated for by increased autophagy. The stimulation of signaling that can confer resistance suggests that combining autophagy or UPR inhibitors with antiestrogens would reduce the development of resistance in some breast cancers.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/metabolismo , Proliferación Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/farmacología , Femenino , Citometría de Flujo , Fulvestrant , Humanos , Ratones , Ratones Desnudos , Microscopía Confocal , Modelos Teóricos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: About 70% of all breast cancers are estrogen receptor alpha positive (ER+) and are treated with antiestrogens. However, 50% of ER + tumors develop resistance to these drugs (endocrine resistance). In endocrine resistant cells, an adaptive pathway called the unfolded protein response (UPR) is elevated that allows cells to tolerate stress more efficiently than in sensitive cells. While the precise mechanism remains unclear, the UPR can trigger both pro-survival and pro-death outcomes that depend on the nature and magnitude of the stress. In this study, we identified MYC, an oncoprotein that is upregulated in endocrine resistant breast cancer, as a regulator of the UPR in glucose-deprived conditions. METHODS: ER+ human breast cancer cell lines (LCC1, LCC1, LY2 and LCC9) and rat mammary tumors were used to confirm upregulation of MYC in endocrine resistance. To evaluate functional relevance of proteins, siRNA-mediated inhibition or small molecule inhibitors were used. Cell density/number was evaluated with crystal violet assay; cell cycle and apoptosis were measured by flow cytometry. Relative quantification of glutamine metabolites were determined by mass spectrometry. Signaling molecules of the UPR, apoptosis or autophagy pathways were investigated by western blotting. RESULTS: Increased MYC function in resistant cells correlated with increased dependency on glutamine and glucose for survival. Inhibition of MYC reduced cell growth and uptake of both glucose and glutamine in resistant cells. Interestingly, in glucose-deprived conditions, glutamine induced apoptosis and necrosis, arrested autophagy, and triggered the unfolded protein response (UPR) though GRP78-IRE1α with two possible outcomes: (i) inhibition of cell growth by JNK activation in most cells and, (ii) promotion of cell growth by spliced XBP1 in the minority of cells. These disparate effects are regulated, at different signaling junctions, by MYC more robustly in resistant cells. CONCLUSIONS: Endocrine resistant cells overexpress MYC and are better adapted to withstand periods of glucose deprivation and can use glutamine in the short term to maintain adequate metabolism to support cell survival. Our findings reveal a unique role for MYC in regulating cell fate through the UPR, and suggest that targeting glutamine metabolism may be a novel strategy in endocrine resistant breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Estrógenos/farmacología , Glucosa/metabolismo , Glutamina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Moduladores de los Receptores de Estrógeno/farmacología , Femenino , Humanos , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Women are using estrogens for many purposes, such as to prevent pregnancy or miscarriage, or to treat menopausal symptoms. Estrogens also have been used to treat breast cancer which seems puzzling, since there is convincing evidence to support a link between high lifetime estrogen exposure and increased breast cancer risk. In this review, we discuss the findings that maternal exposure to the synthetic estrogen diethylstilbestrol during pregnancy increases breast cancer risk in both exposed mothers and their daughters. In addition, we review data regarding the use of estrogens in oral contraceptives and as postmenopausal hormone therapy and discuss the opposing effects on breast cancer risk based upon timing of exposure. We place particular emphasis on studies investigating how maternal estrogenic exposures during pregnancy increase breast cancer risk among daughters. New data suggest that these exposures induce epigenetic modifications in the mammary gland and germ cells, thereby causing an inheritable increase in breast cancer risk for multiple generations.
Asunto(s)
Envejecimiento , Neoplasias de la Mama/inducido químicamente , Disruptores Endocrinos/toxicidad , Congéneres del Estradiol/efectos adversos , Glándulas Mamarias Humanas/efectos de los fármacos , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/prevención & control , Carcinógenos/toxicidad , Anticonceptivos Hormonales Orales/efectos adversos , Dietilestilbestrol/efectos adversos , Exposición a Riesgos Ambientales , Epigénesis Genética/efectos de los fármacos , Congéneres del Estradiol/uso terapéutico , Terapia de Reemplazo de Estrógeno/efectos adversos , Estrógenos no Esteroides/efectos adversos , Femenino , Desarrollo Fetal/efectos de los fármacos , Humanos , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Exposición Materna/efectos adversos , Embarazo , RiesgoRESUMEN
We previously showed that environmentally-induced epigenetic inheritance of cancer occurs in rodent models. For instance, we reported that paternal consumption of an obesity-inducing diet (OID) increased breast cancer susceptibility in the offspring (F1). Nevertheless, it is still unclear whether programming of breast cancer in daughters is due to systemic alterations or mammary epithelium-specific factors and whether the breast cancer predisposition in F1 progeny can be transmitted to subsequent generations. In this study, we show that mammary glands from F1 control (CO) female offspring exhibit enhanced growth when transplanted into OID females compared to CO mammary glands transplanted into CO females. Similarly, carcinogen-induced mammary tumors from F1 CO female offspring transplanted into OID females has a higher proliferation/apoptosis rate. Further, we show that granddaughters (F2) from the OID grand-paternal germline have accelerated tumor growth compared to CO granddaughters. This between-generation transmission of cancer predisposition is associated with changes in sperm tRNA fragments in OID males. Our findings indicate that systemic and mammary stromal alterations are significant contributors to programming of mammary development and likely cancer predisposition in OID daughters. Our data also show that breast cancer predisposition is transmitted to subsequent generations and may explain some familial cancers, if confirmed in humans.
Asunto(s)
Epigénesis Genética , Padre , Predisposición Genética a la Enfermedad , Neoplasias Mamarias Animales/genética , Obesidad/fisiopatología , Animales , Apoptosis , Área Bajo la Curva , Peso Corporal , Proliferación Celular , Modelos Animales de Enfermedad , Epigenoma , Epigenómica , Salud de la Familia , Femenino , Prueba de Tolerancia a la Glucosa , Masculino , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , ARN de Transferencia/metabolismo , RNA-Seq , Espermatozoides/metabolismoRESUMEN
Lariciresinol is a dietary lignan that accounts for a significant portion of the total phytoestrogen intake from Western foods. Recent epidemiological studies suggest that high dietary intake of lignans and lariciresinol is associated with reduced breast cancer risk. However, no causal relationship between lariciresinol intake and breast cancer development has been established. In this study, we investigated for the first time the effects and possible mechanisms of action of lariciresinol on hormone responsive mammary cancer in vivo in dimethylbenz[a]anthracene induced mammary cancer in rats, and in human MCF-7 breast cancer xenografts in athymic mice. For tumor bearing rats, lariciresinol (3 or 15 mg/kg of body weight) or vehicle was administered p.o. daily for 9 weeks. For E2-maintained ovariectomized athymic mice bearing orthotopic MCF-7 tumors, control diet (AIN-93G) or lariciresinol containing diet (AIN-93G supplemented with 20 or 100 mg of lariciresinol/kg of diet) was administered for 5 weeks. In both models, lariciresinol administration inhibited the tumor growth and tumor angiogenesis. In MCF-7 cells, enterolactone significantly inhibited the E2-stimulated VEGF secretion. Moreover, in MCF-7 xenografts, lariciresinol administration enhanced tumor cell apoptosis and increased estrogen receptor beta expression. Lariciresinol and its further metabolites secoisolariciresinol, enterodiol and enterolactone were found in serum of both rats and athymic mice confirming a similar lignan metabolism pattern as in humans. These findings indicate conceivable importance of dietary lignan lariciresinol in inhibition of breast cancer development.
Asunto(s)
Antineoplásicos/farmacología , Suplementos Dietéticos , Furanos/farmacología , Lignanos/sangre , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/sangre , 9,10-Dimetil-1,2-benzantraceno , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Butileno Glicoles/sangre , Carcinógenos , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Furanos/sangre , Furanos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lignanos/farmacología , Lignanos/uso terapéutico , Neoplasias Mamarias Experimentales/sangre , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/inducido químicamente , Ratones , Ratones Desnudos , Ovariectomía , Fitoestrógenos/sangre , Ratas , Receptores de Progesterona/metabolismo , Trasplante HeterólogoRESUMEN
The postnatal mammary gland undergoes repeated cycles of proliferation and cell death, most notably when the fully differentiated (lactating) gland dedifferentiates to a prelactation state. Accumulation of milk proteins in the secretory epithelium creates the stress signal that triggers this process (involution). How this stress is perceived, and the cellular processes that are subsequently activated, remain unclear. We now report that Unfolded Protein Response (UPR), autophagy, and apoptosis related genes cluster separately during lactation and involution in the mouse mammary gland. Time-course experiments in rodents show that autophagy and UPR signaling are tightly co-regulated at the transition from reversible to irreversible involution. Inhibition of autophagy by chloroquine or genetic deletion of one ATG7 allele enhanced progression of mammary involution into the irreversible phase, as characterized by an early/precocious induction of apoptosis. These are the first preclinical in vivo data in support of a clinical trial testing an autophagy inhibitor for prevention of intraductal breast malignancy progression to invasive breast cancer. In marked contrast, stimulation of autophagy by low dose tunicamycin treatment reduced apoptosis and extended the reversible phase of involution by sustaining the secretory epithelium. Autophagy stimulators could be used short-term to promote lactation in women experiencing difficulties or irregularities in nursing. Taken together, these data indicate that UPR and autophagy play a key role in regulating the balance between cell survival and apoptosis during normal mammary gland regression.
RESUMEN
Lignans are a large group of fiber-associated phenolic compounds widely distributed in edible plants. Some of the ingested plant lignans are converted by intestinal microbiota to enterolignans, enterodiol (END) and enterolactone (ENL), the latter of which has been thought to be the major biologically active lignan, and suggested to be associated with low risk of breast cancer. In line with this, administration of plant lignans which are further metabolized to ENL, or ENL as such, have been shown to inhibit or delay the growth of experimental mammary cancer. The mechanism of anticarcinogenic action of ENL is not yet fully understood, but there is intriguing evidence for ENL as a modulator of estrogen signaling. These findings have generated interest in the use of lignans as components of breast cancer risk reducing functional foods. Identification of target groups, who would benefit most, is of pivotal importance. Therefore, further identification and validation of relevant biomarkers, which can be used as indicators of lignan or ENL action and breast cancer risk reduction at different stages of the disease, are of importance.
Asunto(s)
Anticarcinógenos/administración & dosificación , Neoplasias de la Mama/prevención & control , Dieta , Lignanos/administración & dosificación , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , 4-Butirolactona/farmacología , Animales , Bacterias/metabolismo , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Femenino , Humanos , Intestinos/microbiología , Lignanos/metabolismo , Lignanos/farmacología , Neoplasias Mamarias Animales/prevención & control , Plantas Comestibles/química , Factores de RiesgoRESUMEN
Breast cancer continues to be a major challenge for public health, since it is the most common cancer of women in the Western world, and its prevalence is still increasing. In order to achieve better results in the prevention and treatment of breast cancer it is crucial to identify the mechanisms behind its initiation, i.e. the changes and deviations that have occurred in the mammary gland growth. It has long been known that a woman's reproductive history is the strongest breast cancer risk factor if genetic background and age are excluded. The reproductive hormones, and the timing of events leading to changes in these hormones, and consequently, in the mammary gland, are the most important players. However, it has become obvious that dietary components may also contribute to breast cancer risk through their effects on the mammary gland. The past few years have added important information to our knowledge of the mechanisms behind breast cancer initiation at the level of target cells (mammary stem cells) and gene expression (genetic 'fingerprint' associated with persistent pregnancy-induced protection against breast cancer), as well as of the effects of certain dietary factors (steroid action modulators). These results and their links to breast cancer initiation and progression will be discussed.
Asunto(s)
Neoplasias de la Mama/dietoterapia , Neoplasias de la Mama/prevención & control , Dieta , Glándulas Mamarias Humanas/crecimiento & desarrollo , Animales , Neoplasias de la Mama/etiología , Células Madre de Carcinoma Embrionario , Femenino , Hormonas Esteroides Gonadales/fisiología , Humanos , Isoflavonas/uso terapéutico , Lignanos/uso terapéutico , Glándulas Mamarias Humanas/citología , Modelos Biológicos , Células Madre Neoplásicas/fisiología , Fitoestrógenos/uso terapéutico , Posmenopausia/fisiología , Embarazo/fisiología , Pubertad/fisiología , Factores de Riesgo , Células Madre/fisiologíaRESUMEN
PURPOSE: Whether it is safe for estrogen receptor-positive (ER+) patients with breast cancer to consume soy isoflavone genistein remains controversial. We compared the effects of genistein intake mimicking either Asian (lifetime) or Caucasian (adulthood) intake patterns to that of starting its intake during tamoxifen therapy using a preclinical model. EXPERIMENTAL DESIGN: Female Sprague-Dawley rats were fed an AIN93G diet supplemented with 0 (control diet) or 500 ppm genistein from postnatal day 15 onward (lifetime genistein). Mammary tumors were induced with 7,12-dimethylbenz(a)anthracene (DMBA), after which a group of control diet-fed rats were switched to genistein diet (adult genistein). When the first tumor in a rat reached 1.4 cm in diameter, tamoxifen was added to the diet and a subset of previously only control diet-fed rats also started genistein intake (post-diagnosis genistein). RESULTS: Lifetime genistein intake reduced de novo resistance to tamoxifen, compared with post-diagnosis genistein groups. Risk of recurrence was lower both in the lifetime and in the adult genistein groups than in the post-diagnosis genistein group. We observed downregulation of unfolded protein response (UPR) and autophagy-related genes (GRP78, IRE1α, ATF4, and Beclin-1) and genes linked to immunosuppression (TGFß and Foxp3) and upregulation of cytotoxic T-cell marker CD8a in the tumors of the lifetime genistein group, compared with controls, post-diagnosis, and/or adult genistein groups. CONCLUSIONS: Genistein intake mimicking Asian consumption patterns improved response of mammary tumors to tamoxifen therapy, and this effect was linked to reduced activity of UPR and prosurvival autophagy signaling and increased antitumor immunity. Clin Cancer Res; 23(3); 814-24. ©2017 AACR.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Moduladores de los Receptores de Estrógeno/uso terapéutico , Genisteína/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Fitoestrógenos/farmacología , Alimentos de Soja , Tamoxifeno/uso terapéutico , 9,10-Dimetil-1,2-benzantraceno , Animales , Antineoplásicos Hormonales/farmacología , Autofagia/efectos de los fármacos , Autofagia/genética , Citocinas/sangre , Dieta , Chaperón BiP del Retículo Endoplásmico , Moduladores de los Receptores de Estrógeno/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genisteína/administración & dosificación , Genisteína/sangre , Isoflavonas/sangre , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/patología , Fitoestrógenos/administración & dosificación , Fitoestrógenos/sangre , Ratas , Ratas Sprague-Dawley , Recurrencia , Alimentos de Soja/efectos adversos , Tamoxifeno/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
Background: Responses to endocrine therapies vary among patients with estrogen receptor (ER+) breast cancer. We studied whether in utero exposure to endocrine-disrupting compounds might explain these variations. Methods: We describe a novel ER+ breast cancer model to study de novo and acquired tamoxifen (TAM) resistance. Pregnant Sprague Dawley rats were exposed to 0 or 0.1 ppm ethinyl estradiol (EE2), and the response of 9,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors to 15 mg/kg TAM, with (n = 17 tumors in the controls and n = 20 tumors in EE2 offspring) or without 1.2 g/kg valproic acid and 5 mg/kg hydralazine (n = 24 tumors in the controls and n = 32 tumors in EE2 offspring) in the female offspring, was assessed. One-sided Chi2 tests were used to calculate P values. Comparisons of differentially expressed genes between mammary tumors in in utero EE2-exposed and control rats, and between anti-estrogen-resistant LCC9 and -sensitive LCC1 human breast cancer cells, were also performed. Results: In our preclinical model, 54.2% of mammary tumors in the control rats exhibited a complete response to TAM, of which 23.1% acquired resistance with continued anti-estrogen treatment and recurred. Mammary tumors in the EE2 offspring were statistically significantly less likely to respond to TAM (P = .047) and recur (P = .007). In the EE2 offspring, but not in controls, adding valproic acid and hydralazine to TAM prevented recurrence (P < .001). Three downregulated and hypermethylated genes (KLF4, LGALS3, MICB) and one upregulated gene (ETV4) were identified in EE2 tumors and LCC9 breast cancer cells, and valproic acid and hydralazine normalized the altered expression of all four genes. Conclusions: Resistance to TAM may be preprogrammed by in utero exposure to high estrogen levels and mediated through reversible epigenetic alterations in genes associated with epithelial-mesenchymal transition and tumor immune responses.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Etinilestradiol/efectos adversos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Tamoxifeno/uso terapéutico , 9,10-Dimetil-1,2-benzantraceno , Proteínas E1A de Adenovirus/genética , Animales , Línea Celular Tumoral , Metilación de ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Galectina 3/genética , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Hidralazina/administración & dosificación , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/química , Recurrencia Local de Neoplasia/prevención & control , Embarazo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/análisis , Tamoxifeno/administración & dosificación , Transactivadores/genética , Ácido Valproico/administración & dosificaciónRESUMEN
The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657-70. ©2016 AACR.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Proteínas de Choque Térmico/fisiología , Metabolismo de los Lípidos , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Antígeno CD47/fisiología , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Ácidos Grasos/metabolismo , Femenino , Humanos , Inmunidad Innata , Metabolómica , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The inverse association between a high enterolactone (ENL) concentration in both urine and serum, and the risk of breast cancer found in epidemiological studies suggests a chemopreventive action for ENL. However, no causal relationship has been established in clinical studies or in experimental models for breast cancer. In the present study, the potential chemopreventive action of p.o. administered ENL (1 or 10 mg/kg of body weight) was tested in 7,12-dimethylbenz(a)anthracene-induced mammary cancers of the rat. Rats were maintained on a standard open-formula chow diet. Daily p.o. administration of ENL at a dose of 10 mg/kg of body weight for 7 weeks significantly inhibited tumor growth. The growth-inhibitory effect of ENL was more pronounced on the new tumors, which developed during the treatment period, but ENL also inhibited the growth of those tumors established before the start of the lignan administration. The rat serum concentration of ENL, which illustrated a permanent positive effect on breast cancer growth, was 0.4 microM, which is >10-fold as compared with the serum concentrations found in the general human population. The effect of ENL was not restricted to any specific histological tumor type. ENL was demonstrated to act as a weak aromatase inhibitor in vitro and to reduce the relative uterine weight of the 7,12-dimethylbenz(a)anthracene-treated nonovariectomized rats. However, in a short-term assay ENL had no effect on the uterine growth of the intact or androstenedione-treated immature rats. Thus, the mechanism of the ENL action and its minimum or optimal daily dose remains to be clarified.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacocinética , 9,10-Dimetil-1,2-benzantraceno , Isoflavonas , Lignanos/farmacocinética , Neoplasias Mamarias Animales/tratamiento farmacológico , 4-Butirolactona/sangre , 4-Butirolactona/orina , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Inhibidores de la Aromatasa , Carcinógenos , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estrógenos/farmacología , Estrógenos no Esteroides/sangre , Estrógenos no Esteroides/orina , Femenino , Lignanos/sangre , Lignanos/metabolismo , Lignanos/orina , Modelos Químicos , Tamaño de los Órganos , Fitoestrógenos , Preparaciones de Plantas , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Útero/efectos de los fármacosRESUMEN
Antiestrogen therapy induces the unfolded protein response (UPR) in estrogen receptor-positive (ER(+)) breast cancer. X-box binding protein 1 (XBP1), which exists in the transcriptionally inactive unspliced form [XBP1(U)] and the spliced active form [XBP1(S)], is a key UPR component mediating antiestrogen resistance. We now show a direct link between the XBP1 and NF-κB survival pathways in driving the cell fate decisions in response to antiestrogens in ER(+) breast cancer cells, both in vitro and in a xenograft mouse model. Using novel spliced and nonspliceable forms of XBP1, we show that XBP1(U) functions beyond being a dominant negative of XBP1(S). Both isoforms regulate NF-κB activity via ERα; XBP1(S) is more potent because it also directly regulates p65/RelA expression. These findings provide new insights into the fundamental signaling activities of spliced and unspliced XBP1 in breast cancer, establish NF-κB to be a mediator of these activities, and identify XBP1 and its splicing to be novel therapeutic targets.
Asunto(s)
Neoplasias de la Mama/metabolismo , Linaje de la Célula/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , FN-kappa B/efectos de los fármacos , Tamoxifeno/farmacología , Factores de Transcripción/efectos de los fármacos , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Ratones , FN-kappa B/metabolismo , Empalme del ARN/genética , Receptores de Estrógenos/metabolismo , Factores de Transcripción del Factor Regulador X , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Interferon regulatory factor-1 (IRF1) is a tumor suppressor that regulates cell fate in several cell types. Here, we report an inverse correlation in expression of nuclear IRF1 and the autophagy regulator ATG7 in human breast cancer cells that directly affects their cell fate. In mice harboring mutant Atg7, nuclear IRF1 was increased in mammary tumors, spleen, and kidney. Mechanistic investigations identified ATG7 and the cell death modulator beclin-1 (BECN1) as negative regulators of IRF1. Silencing ATG7 or BECN1 caused estrogen receptor-α to exit the nucleus at the time when IRF1 nuclear localization occurred. Conversely, silencing IRF1 promoted autophagy by increasing BECN1 and blunting IGF1 receptor and mTOR survival signaling. Loss of IRF1 promoted resistance to antiestrogens, whereas combined silencing of ATG7 and IRF1 restored sensitivity to these agents. Using a mathematical model to prompt signaling hypotheses, we developed evidence that ATG7 silencing could resensitize IRF1-attenuated cells to apoptosis through mechanisms that involve other estrogen-regulated genes. Overall, our work shows how inhibiting the autophagy proteins ATG7 and BECN1 can regulate IRF1-dependent and -independent signaling pathways in ways that engender a new therapeutic strategy to attack breast cancer.
Asunto(s)
Apoptosis , Autofagia , Neoplasias de la Mama/patología , Factor 1 Regulador del Interferón/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/fisiología , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Linaje de la Célula , Femenino , Humanos , Proteínas de la Membrana/fisiología , Ratones , Modelos Teóricos , Enzimas Activadoras de Ubiquitina/fisiologíaRESUMEN
We recently generated a transgenic mouse strain that expresses the human aromatase gene under the ubiquitin C promoter (AROM+). We have previously shown that in these mice the serum estradiol concentration is highly elevated, whereas the testosterone concentration is decreased. In the present study we examined mammary gland development in AROM+ male mice at different ages and found that the mammary glands of AROM+ males undergo ductal and alveolar development morphologically resembling that of terminally differentiated female mammary glands, expressing mRNA for a milk protein gene (beta-casein). The male mammary glands also express multiple hormone receptors typical for female mammary gland: estrogen receptor alpha and beta, progesterone receptor, and PRL receptor. Furthermore, data showed activation of the Stat5 (signal transducer and activator of transcription 5) signaling pathway in the AROM+ male mammary gland. Interestingly, the phenotype observed is in part reversible. Treatment with finrozole, a specific aromatase inhibitor, caused an involution of the differentiated phenotype of the mammary gland, marked by the disappearance of alveolar structures and the majority of the tertiary side branches of the ducts. The present animal model is a valuable tool for better understanding the cellular and molecular mechanisms involved in the development of gynecomastia.
Asunto(s)
Aromatasa/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Animales , Aromatasa/genética , Inhibidores de la Aromatasa , Biomarcadores , Inhibidores Enzimáticos/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Humanos , Masculino , Glándulas Mamarias Animales/anatomía & histología , Ratones , Ratones Transgénicos/genética , Nitrilos/farmacología , Hipófisis/citología , Hipófisis/metabolismo , Prolactina/fisiología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Triazoles/farmacologíaRESUMEN
PURPOSE: Estrogen receptor-α (ERα)-targeted therapies including tamoxifen (TAM) or Faslodex (ICI) are used to treat ER(+) breast cancers. Up to 50% of tumors will acquire resistance to these interventions. Autophagy has been implicated as a major driver of antiestrogen resistance. We have explored the ability of hydroxychloroquine (HCQ), which inhibits autophagy, to affect antiestrogen responsiveness. EXPERIMENTAL DESIGN: TAM-resistant MCF7-RR and ICI-resistant/TAM cross-resistant LCC9 ER(+) breast cancer cells were injected into mammary fat pads of female athymic mice and treated with TAM and/or ICI in combination with oral low-dose HCQ. RESULTS: We show that HCQ can increase antiestrogen responsiveness in MCF7-RR and LCC9 cells and tumors, likely through the inhibition of autophagy. However, the combination of ICI+HCQ was less effective than HCQ alone in vivo, unlike the TAM+HCQ combination. Antiestrogen treatment stimulated angiogenesis in tumors but did not prevent HCQ effectiveness. The lower efficacy of ICI+HCQ was associated with ICI effects on cell-mediated immunity within the tumor microenvironment. The mouse chemokine KC (CXCL1) and IFNγ were differentially regulated by both TAM and ICI treatments, suggesting a possible effect on macrophage development/activity. Consistent with these observations, TAM+HCQ treatment increased tumor CD68(+) cells infiltration, whereas ICI and ICI+HCQ reduced peripheral tumor macrophage content. Moreover, macrophage elimination of breast cancer target cells in vitro was reduced following exposure to ICI. CONCLUSION: HCQ restores antiestrogen sensitivity to resistant tumors. Moreover, the beneficial combination of TAM+HCQ suggests a positive outcome for ongoing neoadjuvant clinical trials using this combination for the treatment of ER(+) ductal carcinoma in situ lesions.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/farmacología , Hidroxicloroquina/farmacología , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Femenino , Fulvestrant , Humanos , Técnicas para Inmunoenzimas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In estrogen receptor-positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX15-070 has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here, we show that GX15-070 is more effective in reducing the cell density of antiestrogen-resistant breast cancer cells versus sensitive cells and that this increased sensitivity of resistant cells to GX15-070 correlates with an accumulation of autophagic vacuoles. Formation of autophagosomes in GX15-070-treated cells was verified by changes in expression of the lipidation of microtubule-associated protein-1 light chain-3 and both confocal and transmission electron microscopy. While GX15-070 treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulate. Moreover, GX15-070 exposure leads to a reduction in cathepsin D (CTSD) and L (CTSL1) protein expression that would otherwise digest autolysosome cargo. Thus, GX15-070 has dual roles in promoting cell death: (i) directly inhibiting antiapoptotic BCL2 family members, thereby inducing apoptosis; and (ii) inhibiting downstream CTSD and CTSL1 protein expression to limit the ability of cells to use degraded material to fuel cellular metabolism and restore homeostasis. Our data highlight a new mechanism of GX15-070-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer.