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Uterine leiomyomas or fibroids are the most common tumors of the female reproductive tract. Estrogen (E2), a steroid-derived hormone, and its receptors (ERs), particularly ER-α, are important drivers for the development and growth of leiomyomas. We previously demonstrated that simvastatin, a drug used for hyperlipidemia, also possesses anti-leiomyoma properties. The aim of this work is to investigate the impact of simvastatin on ER-α signaling in leiomyoma cells, including its expression, downstream signaling, transcriptional activity, post-translational modification, trafficking and degradation. Primary and immortalized human uterine leiomyoma (HuLM) cells were used for in vitro experiments. Immunodeficient mice xenografted with human leiomyoma tissue explants were used for in vivo studies. Leiomyoma samples were obtained from patients enrolled in an ongoing double-blinded, phase II, randomized controlled trial. Here, we found that simvastatin significantly reduced E2-induced proliferation and PCNA expression. In addition, simvastatin reduced total ER-α expression in leiomyoma cells and altered its subcellular localization by inhibiting its trafficking to the plasma membrane and nucleus. Simvastatin also inhibited E2 downstream signaling, including ERK and AKT pathways, E2/ER transcriptional activity and E2-responsive genes. To explain simvastatin effects on ER-α level and trafficking, we examined its effects on ER-α post-translational processing. We noticed that simvastatin reduced ER-α palmitoylation; a required modification for its stability, trafficking to plasma membrane, and signaling. We also observed an increase in ubiquitin-mediated ER-α degradation. Importantly, we found that the effects of simvastatin on ER-α expression were recapitulated in the xenograft leiomyoma mouse model and human tissues. Thus, our data suggest that simvastatin modulates several E2/ER signaling targets with potential implications in leiomyoma therapy and beyond.
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Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Leiomioma/metabolismo , Simvastatina/farmacología , Neoplasias Uterinas/metabolismo , Adolescente , Adulto , Animales , Línea Celular Tumoral , Supervivencia Celular , Método Doble Ciego , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Leiomioma/tratamiento farmacológico , Lipoilación , Ratones , Persona de Mediana Edad , Transporte de Proteínas , Proteolisis , Transducción de Señal/efectos de los fármacos , Simvastatina/uso terapéutico , Neoplasias Uterinas/tratamiento farmacológico , Adulto JovenRESUMEN
BACKGROUND: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress ß1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. OBJECTIVE: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 µM, or control) for 48 hours. Protein and mRNA levels of ß1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. RESULTS: We found that simvastatin significantly reduced the protein expression of ß1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in ß1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. CONCLUSION: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Leiomioma/genética , Mecanotransducción Celular/efectos de los fármacos , Simvastatina/farmacología , Neoplasias Uterinas/genética , Proteínas de Anclaje a la Quinasa A/efectos de los fármacos , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Colágeno Tipo I/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ciclina D1/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Integrina beta1/efectos de los fármacos , Integrina beta1/genética , Integrina beta1/metabolismo , Leiomioma/metabolismo , Mecanotransducción Celular/genética , Antígenos de Histocompatibilidad Menor/efectos de los fármacos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Quinasa de Cadena Ligera de Miosina/efectos de los fármacos , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Neoplasias Uterinas/metabolismo , Quinasas Asociadas a rho/efectos de los fármacos , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/efectos de los fármacos , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
OBJECTIVE: To evaluate the impact of coenzyme Q-10 (CoQ-10) on the dysregulated synthesis of extracellular matrix proteins mediated by transforming growth factor beta 3 (TGF-ß3) in uterine leiomyomas. DESIGN: Laboratory study. SETTING: University. PATIENTS: None. INTERVENTIONS: Treatment of immortalized uterine myometrial and leiomyoma cells to TGF-ß3 and CoQ-10. MAIN OUTCOME MEASURES: The protein concentrations of collagen 1A1 (COL1A1), collagen 3A1 (COL3A1), collagen 11A1 (COL11A1), and fibronectin (FN1) were assessed through western blot analysis after treatment of immortalized uterine myometrial and leiomyoma cells with both transforming growth factor beta (TGF-ß) 3 and concentrations of CoQ-10 at 10, 50, and 100 µM concurrently for 24 hours. RESULTS: Immortalized uterine leiomyoma and myometrial cells exposed to TGF-ß3 for 24 hours demonstrated a significant up-regulation of COL1A1, COL3A1, COL11A1, and FN1 compared with untreated cells. In leiomyoma cells, concurrent treatment with CoQ-10 over the same timeframe revealed a dose-dependent decrease in these protein concentrations compared with those in cells treated with TGF-ß3 alone. At the highest concentration of 100 µM of CoQ-10, significant decreases in the amounts of COL1A1 (0.59 ± 0.10-fold), COL3A1 (0.46 ± 0.09-fold), COL11A1 (0.53 ± 0.09-fold), and FN1 (0.56 ± 0.09-fold) were observed. Similarly, myometrial cells exposed to both TGF-ß3 and CoQ-10 demonstrated a dose-responsive decline in the amount of extracellular matrix protein compared with cells exposed to TGF-ß3 alone. Significant reductions in the amounts of COL1A1 (0.75 ± 0.03-fold), COL3A1 (0.48 ± 0.06-fold), COL11A1 (0.38 ± 0.06), and FN1 (0.69 ± 0.04-fold) were appreciated at 100-µM CoQ-10. CONCLUSION: Coenzyme Q-10 mitigated the aberrant production of key biomarkers of the extracellular matrix mediated by TGF-ß3 in uterine leiomyomas. Our findings highlight a promising nonhormonal compound that can counteract the fibroproliferative process inherent to leiomyomas.
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OBJECTIVES: To assess the effect of simvastatin on uterine leiomyoma growth and extracellular matrix (ECM) deposition. DESIGN: Laboratory analysis of human leiomyoma cell culture, xenograft in a mouse model, and patient tissue from a clinical trial. SETTING: Academic research center. PATIENT(S): Tissue culture from human leiomyoma tissue and surgical leiomyoma tissue sections from a placebo-controlled randomized clinical trial. INTERVENTION(S): Simvastatin treatment. MAIN OUTCOME MEASURE(S): Serum concentrations, xenograft volumes, and protein expression. RESULTS: Mice xenografted with 3-dimensional human leiomyoma cultures were divided as follows: 7 untreated controls; 12 treated with activated simvastatin at 10 mg/kg body weight; and 15 at 20 mg/kg body weight. Simvastatin was detected in the serum of mice injected at the highest dose. Xenograft volumes were significantly smaller (mean 53% smaller at the highest concentration). There was dissolution of compact ECM, decreased ECM formation, and lower collagen protein expression in xenografts. Membrane type 1 matrix metalloproteinase was increased in vitro and in vivo. Matrix metalloproteinase 2 and low-density lipoprotein receptor-related protein 1 were increased in vitro. CONCLUSIONS: Simvastatin exhibited antitumoral activity with ECM degradation and decreased leiomyoma tumor volume in vivo. Activation of the matrix metalloproteinase 2, membrane type 1 matrix metalloproteinase, and low-density lipoprotein receptor-related protein 1 pathway may explain these findings.
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Leiomioma , Neoplasias Uterinas , Femenino , Humanos , Ratones , Animales , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/farmacología , Simvastatina/farmacología , Simvastatina/metabolismo , Simvastatina/uso terapéutico , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/farmacología , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patología , Leiomioma/tratamiento farmacológico , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Peso Corporal , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Lipoproteínas LDL/uso terapéuticoRESUMEN
OBJECTIVE: To determine whether a curcumin-supplemented diet would prevent and/or treat uterine leiomyoma growth in our mouse xenograft model. DESIGN: Animal study. SETTING: Laboratory study. PATIENT(S): N/A. INTERVENTION(S): Curcumin-supplemented diet. MAIN OUTCOME MEASURE(S): Dietary intake, blood concentrations, tumor size, extracellular matrix protein concentrations, apoptosis markers. RESULT(S): We found that curcumin was well tolerated as a dietary supplement, free curcumin and its metabolites were detected in the serum, and exposure resulted in approximately 60% less leiomyoma xenograft growth as well as dissolution of the peripheral extracellular matrix architecture of the xenografts. The production of matrix proteins, including collagens, decreased, whereas the number of apoptotic cells in the xenografts increased. Additionally, when xenografts were placed in a uterine intramural location, we found a significantly increased apoptotic response to curcumin in the diet. CONCLUSION(S): Mice on a diet supplemented with curcumin could achieve serum concentrations sufficient to regulate human leiomyoma xenograft growth, and curcumin could play both preventive and curative roles in the treatment of uterine leiomyoma as an oral nutritional supplement.
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Curcumina , Leiomioma , Neoplasias Uterinas , Femenino , Humanos , Animales , Ratones , Curcumina/farmacología , Curcumina/uso terapéutico , Curcumina/metabolismo , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patología , Xenoinjertos , Solubilidad , Leiomioma/tratamiento farmacológico , Matriz Extracelular/metabolismo , Matriz Extracelular/patologíaRESUMEN
OBJECTIVE: To determine the effect relugolix and elagolix have on the production of extracellular matrix (ECM) proteins in human leiomyoma cells. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S) OR ANIMALS: None. January 5, 2022 Cell culture, protein analysis, immunohistochemistry. MAIN OUTCOME MEASURE(S): Production of GnRHR, COL1A1, FN1, VCAN, p-ERK, & ERK in treated/untreated leiomyoma cells. RESULTS: 100 nM relugolix resulted in decreased production of COL1A1 at 24 (1.78 0.06-fold; P < .05) and 48 hours (1.92 0.14-fold; P < .05). Elagolix treatment resulted in a decrease in COL1A1 production at 24 but not 48 hours. In 2D and 3D, 100 nM relugolix resulted in decreased production of FN1 at 24 (1.7 ± 0.07-fold; P < .05) and 48 hours (1.8 ± 0.07-fold; P < .05); 100 nM elagolix resulted in decreased production of FN1 at 24 (1.7 ± 0.14-fold; P < .05) and 48 hours (2.0 ± 0.09-fold; P < .05). For cells treated with relugolix 100 nM resulted in decreased VCAN production by 48 hours (0.66 ± 0.07-fold; P < .05). Contrary to our 3D data, 2D elagolix-treated cells demonstrated a decrease in VCAN production that was identified only at 24 hours. For GnRHR, no significant difference between the drugs was seen at 24 hours; at 48 hours production was only significantly decreased for relugolix (P < .05). Comparing both drugs, there was a significant difference in the concentration of p-ERK to ERK at 24 hours (P < .05); there was no difference by 48 hours. CONCLUSIONS: Our findings demonstrated that treatment with either drug can 1) decrease ECM protein production and 2) inhibit the MAPK pathway.
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Leiomioma , Neoplasias Uterinas , Técnicas de Cultivo Tridimensional de Células , Matriz Extracelular , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Hidrocarburos Fluorados , Leiomioma/tratamiento farmacológico , Compuestos de Fenilurea , Pirimidinas , Pirimidinonas , Neoplasias Uterinas/tratamiento farmacológicoRESUMEN
A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Corazón Fetal/embriología , Corazón Fetal/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Corazón Fetal/anomalías , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Hibridación in Situ , Factores de Transcripción MEF2 , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Antígenos de Histocompatibilidad Menor , Modelos Cardiovasculares , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Embarazo , ARN Interferente Pequeño/genética , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
OBJECTIVE: To characterize the method by which angiogenesis occurred in three-dimensional (3D) leiomyoma xenografts, and to assess the impact of hypoxia on two-dimensional (2D) and 3D myometrial and leiomyoma cells and leiomyoma xenografts in vivo. DESIGN: Laboratory study. SETTING: Academic research. PATIENT(S): Cell cultures from patient-matched myometrial and leiomyoma tissues. INTERVENTION(S): In vivo 3D leiomyoma xenografts from ovariectomized mice treated with gonadal hormones; myometrial and leiomyoma cells in 2D and 3D growth formats exposed to 1% oxygen. MAIN OUTCOME MEASURE(S): Protein expression. RESULT(S): Blood vessels in the xenograft estradiol group are identified with anti-mouse/anti-rat CD31/PECAM-1 antibody. Hormone-stimulated 3D leiomyoma xenografts stain positively for adrenomedullin (ADM). Myometrial cells exposed to 1% oxygen demonstrated an increase in hypoxia-inducible factor (HIF)-1α at 6 hours and a marked increase at 24 hours. Under normoxic conditions, leiomyoma cells at 6 hours show increased expression of HIF-1α, which is further increased at 24 hours. Leiomyoma cells under hypoxia demonstrated a 1.14-fold decrease in HIF-1α expression at 6 hours and no change at 24 hours. Hypoxic myometrium decreased the proangiogenic protein ADM expression at 6 hours and showed a >1.5-fold increase at 24 hours. Normoxic leiomyoma decrease ADM at 24 hours and showed a >1.5-fold increase at 24 hours of hypoxia. CONCLUSION(S): Hypoxia-induced HIF-1α expression facilitates angiogenesis in 3D xenografts in vivo by increasing the expression of the proangiogenic protein ADM. Angiogenesis contributes to the viability and extended survival of these xenografts. Furthermore, 2D myometrial and leiomyoma cells increase HIF-1α and ADM expression in vitro under hypoxic conditions.
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Leiomioma , Animales , Femenino , Xenoinjertos , Humanos , Hipoxia , Leiomioma/metabolismo , Ratones , Neovascularización Patológica , Oxígeno , RatasRESUMEN
OBJECTIVE: To assess and characterize the role of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the development of uterine leiomyoma. DESIGN: Laboratory study. SETTING: Academic research center. PATIENT(S): Not applicable. INTERVENTION(S): Laboratory investigation. In vitro assessment of human leiomyoma and myometrial tissue specimens as well as immortalized leiomyoma and myometrial cell lines. MAIN OUTCOME MEASURE(S): Western blotting and immunohistochemical analyses were performed to assess differences in CEACAM1 content between leiomyoma and myometrial samples. Small interfering RNA silencing experiments and transient transfection experiments were performed to characterize the regulatory role of CEACAM1 on downstream signaling cascades. RESULT(S): Analysis of RNA sequencing data revealed decreased CEACAM1 expression in human uterine leiomyoma specimens compared with that in myometrial samples. This translated to a significant down-regulation in CEACAM1 protein content in human leiomyoma compared with patient-matched myometrial tissue samples (0.236 ± 0.05-fold). A similar decrease in CEACAM1 protein content was observed in matched immortalized leiomyoma cell (ILC) and immortalized myometrial cell lines (0.21 ± 0.07). Immunohistochemical analysis revealed decreased staining intensity in leiomyoma surgical specimens compared with the matched myometrium of placebo patients. Lower CEACAM1 levels in leiomyoma were associated with increased activation of both the mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase/protein kinase B pathways compared with that in myometrial cells. This is significant because activation of these pathways plays an important role in leiomyoma growth. Treatment of myometrial cells with CEACAM1 small interfering RNA resulted in a significant down-regulation of CEACAM1 at the protein level (0.272 ± 0.06-fold) and was associated with increased activation of the MAPK (1.62 ± 0.21-fold) and phosphoinositide 3-kinase/protein kinase B (1.79 ± 0.35-fold) pathways, as well as increased collagen production (2.1 ± 0.49-fold). Rescue of CEACAM1 expression in leiomyoma cells by transient transfection restored regulatory control and resulted in lower activation of the MAPK pathway (0.58 ± 0.37-fold). CONCLUSION(S): CEACAM1 is an important protein involved in regulating many signal transduction pathways. Decreased CEACAM1 expression in leiomyoma allows permissive uncontrolled overactivation and up-regulation of downstream pathways that may contribute to leiomyoma growth.
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Leiomioma , Neoplasias Uterinas , Antígenos CD , Antígeno Carcinoembrionario/uso terapéutico , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular , Femenino , Humanos , Leiomioma/genética , Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt , ARN Interferente Pequeño/uso terapéutico , Neoplasias Uterinas/genéticaRESUMEN
Multiple in vivo animal models for uterine leiomyoma do not adequately represent human disease based on etiology, molecular phenotype, or limited fixed life span. Our objective was to develop a xenograft model with sustained growth, by transplanting a well-established actively growing three-dimensional (3D) cell culture of human leiomyoma and myometrium in NOD/SCID ovariectomized female mice. We demonstrated continued growth to at least 12 weeks and the overexpression of extracellular matrix (ECM). Further, we confirmed maintenance of hormonal response that is comparable to human disease in situ. Leiomyoma xenografts under hormonal treatment demonstrated 8 to12-fold increase of volume over the xenografts not treated with hormones. Estradiol-treated xenografts were more cellular as compared to progesterone or combination milieu, at the end of 8-week time frame. There was also a non-statistically significant 2-4 mm3 increase in volume between 8-week and 12-week xenografts with higher matrix to cell ratio in 12-week xenografts compared to the 8-week and placebo xenografts. Increased expression of ECM proteins, fibronectin, versican, and collagens, indicated an actively growing cell matrix formation in the xenografts. In conclusion, we have developed and validated a xenograft in vivo model for uterine leiomyoma that shares the genomic and proteomic characteristics with the human surgical specimens of origin and recapitulates the most important features of the human tumors, the aberrant ECM expression that defines the leiomyoma phenotype and gonadal hormone regulation. Using this model, we demonstrated that combination of estradiol and progesterone resulted in increased cellularity and ECM production leading to growth of the xenograft tumors.
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Estradiol/administración & dosificación , Leiomioma/patología , Miometrio/patología , Progesterona/administración & dosificación , Neoplasias Uterinas/patología , Animales , Femenino , Ratones , Ratones Endogámicos NOD , Miometrio/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To characterize the potential interaction between interleukin-6 (IL6), Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-3 (JAK/STAT3) pathway, and Transforming growth factor beta (TGFß)-3 , and to determine whether such cross-talk was a contributing factor in the dysregulation of type I collagen production in leiomyomas. DESIGN: Laboratory study. SETTING: University research laboratory. PATIENTS: None. INTERVENTIONS: Exposure of leiomyoma and myometrial cell lines to IL6 and STAT3 activators/inhibitors. Western immunoblot analysis and immunohistochemistry. MAIN OUTCOME MEASURES: Expression of STAT3, pSTAT3, SOCS3, COL1A1, and TGFb3. RESULTS: We observed that IL6 increased pSTAT3 as well as collagen1A1 in uterine leiomyoma cells. Direct activation of the JAK/STAT3 pathway increased collagen1A1 production in leiomyoma cells, whereas inhibition of the pathway significantly decreased collagen1A1 production. We further observed that modulation of the JAK/STAT3 pathway also increased the expression of TGFß3 protein. Leiomyoma cells exposed to TGFß3 demonstrated a significant decrease in pSTAT3 protein. Myometrial cells demonstrated a less sensitive response to STAT3 modulation and collagen production. CONCLUSION: Cross-talk between the TGFß pathway and JAK/STAT3 pathway contributes to the fibrotic nature of uterine leiomyomas.
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OBJECTIVE: To characterize the role Activator Protein 1 (AP 1) family members play in mediating extracellular matrix deposition in uterine leiomyoma. DESIGN: Laboratory study. SETTING: University research laboratory. INTERVENTION(S): Exposure of leiomyoma and myometrial cell lines to either an AP 1 inhibitor alone, AP 1 inhibitor plus transforming growth factor (TGF)ß3, or TGFß3 alone. MAIN OUTCOME MEASURE(S): Western immunoblot analysis was performed to assess for changes in AP 1 family member protein expression. RESULT(S): In patient-matched myometrial and leiomyoma cell lines, the only AP 1 member found to be elevated significantly in leiomyoma compared with myometrium was FOSB (3.47 ± 0.12-fold), whereas others were decreased significantly: FRA1 (0.67 ± 0.02-fold), FRA2 (0.45 ± 0.01-fold), c FOS (0.37 ± 0.01-fold), Phos c FOS (0.19 ± 0.02-fold), Phos c JUN (0.75 ± 0.02-fold), JUNB (0.81 ± 0.04-fold), and JUND (0.65 ± 0.03-fold). c JUN (0.93 ± 0.03-fold) concentration was reduced but at nonsignificant levels. Following stimulation with TGF ß 3, fibronectin (2.16 ± 0.14-fold) and versican (4.71 ± 0.15-fold) protein concentrations were increased at 24 hours. Collagen 1A demonstrated a time-dependent significant increased concentration beginning at 6 hours (1.32 ± 0.01-fold) and increased to (6.49 ± 0.02-fold) at 24 hours. Following treatment with AP 1 inhibitor (SR11302), there were significant reductions in Collagen 1A concentration at 4 hours (0.59 ± 0.03-fold) and 6 hours (0.42 ± 0.05-fold). Activator Protein 1 inhibition did not reduce significantly versican concentration until 6 hours of treatment (0.84 ± 0.04-fold). SR11302 also decreased significantly fibronectin concentration (0.68 ± 0.05-fold) at 8 hours of treatment. CONCLUSION(S): Activator Protein 1 signaling is well described in fibrotic diseases, and, herein, we demonstrated that signaling via AP 1 family members promotes extracellular matrix deposition in leiomyoma.
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NAV 3 is a tumor suppressor of unknown function in leiomyomas. The objective of this study is to assess NAV3 expression and its potential role in human uterine leiomyomas. NAV3 protein expression was examined in patient leiomyoma and patient-matched myometrial tissue samples by Western blot and immunohistochemistry. NAV3 mRNA and protein expression was assessed in leuprolide acetate- and cetrorelix-treated cell line leiomyoma samples. RNAseq analysis of placebo-treated leiomyoma compared with myometrium demonstrated the presence of transcripts encoding for several neuronal proteins. For NAV3, RNA sequence analysis demonstrated decreased expression in leiomyoma as compared with myometrium (0.86 ± 0.03 fold). Presence of NAV3 mRNA was also decreased in leiomyoma surgical samples (0.43 fold ± 0.05, p = 0.026) compared with patient-matched myometrium. Confirmatory qRT-PCR results on immortalized leiomyoma and myometrial cell lines similarly demonstrated a decrease in expression of NAV3 in leiomyomas (0.28 ± 0.02, p = 0.00075). Immunohistochemical analysis demonstrated a significant decrease in NAV 3 protein in leiomyomas (H-score 154.7 ± 6.2) as compared with myometrium (H-score; 312.5 ± 14.7, p < 0.0001). Leuprolide acetate-treated leiomyoma cells demonstrated an increase in NAV 3 mRNA expression (1.53 ± 0.13, p < 0.0001). Similarly, Western blot analysis on leuprolide-treated leiomyoma cells showed a non-significant increase in NAV 3 protein expression (1.26 ± 0.09, p = 0.063). NAV 3, a tumor suppressor in numerous cancers, is decreased in leiomyoma cells and tissue compared with myometrium, and increased by GnRH analog treatment, suggesting that NAV3 may mediate steroid hormone-independent leiomyoma regulation by GnRH analogs.
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Genes Supresores de Tumor , Leiomioma/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Cultivadas , Femenino , Humanos , Leiomioma/genética , Leiomioma/patología , Proteínas de la Membrana/genética , Miometrio/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. DESIGN: Laboratory study of placebo and ulipristal acetate (UPA)-treated patient tissue. In vitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. SETTING: Academic research center. PATIENT(S): Not applicable. INTERVENTIONS(S): Exposure of leiomyoma cell lines to 17ß-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. MAIN OUTCOME MEASURE(S): Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. RESULT(S): Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57-fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17ß-E2 yielded a 1.98 ± 0.11-fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97-fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). In vitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. CONCLUSION(S): NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and in vitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. CLINICAL TRIAL REGISTRATION NUMBER: NCT00290251.
Asunto(s)
Anticonceptivos Femeninos/farmacología , Hormonas Esteroides Gonadales/farmacología , Antagonistas de Hormonas/farmacología , Leiomioma/metabolismo , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Biomarcadores/metabolismo , Línea Celular Tumoral , Anticonceptivos Femeninos/uso terapéutico , Método Doble Ciego , Estradiol/farmacología , Estradiol/uso terapéutico , Femenino , Proteínas Ligadas a GPI/agonistas , Proteínas Ligadas a GPI/biosíntesis , Hormonas Esteroides Gonadales/uso terapéutico , Antagonistas de Hormonas/uso terapéutico , Humanos , Leiomioma/tratamiento farmacológico , Leiomioma/patología , Moléculas de Adhesión de Célula Nerviosa/agonistas , Norpregnadienos/farmacología , Norpregnadienos/uso terapéuticoRESUMEN
OBJECTIVE: To characterize the effect of ulipristal acetate (UPA) treatment on transforming growth factor (TGF) canonical and noncanonical signaling pathways in uterine leiomyoma tissue and cells. UPA decreased extracellular matrix in surgical specimens; we characterize the mechanism in this study. DESIGN: Laboratory study. SETTING: University. INTERVENTION(S): Exposure of leiomyoma cell lines to UPA. MAIN OUTCOME MEASURE(S): RNAseq was performed on matched myometrium and leiomyoma surgical specimens of placebo- and UPA-treated patients. Changes in gene expression and protein were measured using quantitative polymerase chain reaction and western immunoblot analysis, respectively. RESULT(S): In surgical specimen, mRNA for TGF-ß3 was elevated 3.75-fold and TGFR2 was decreased 0.50-fold in placebo leiomyomas compared with myometrium. Analysis of leiomyomas from UPA-treated women by western blot revealed significant reductions of active TGF-ß3 (0.64 ± 0.12-fold), p-TGFR2 (0.56 ± 0.23-fold), pSmad 2 (0.54 ± 0.04-fold), and pSmad 3 (0.65 ± 0.09-fold) compared with untreated leiomyomas. UPA treatment demonstrated statistically significant reduction in collagen 1, fibronectin, and versican proteins. Notably, there was a statistically significant increase of the extracellular matrix protein fibrillin in leiomyoma treated with UPA (1.48 ± 0.41-fold). Data from in vitro assays with physiologic concentrations of UPA supported the in vivo findings. CONCLUSION(S): TGF-ß pathway is highly up-regulated in leiomyoma and is directly responsible for development of the fibrotic phenotype. UPA attenuates this pathway by reducing TGF-ß3 message and protein expression, resulting in a reduction in TGF-ß canonical signaling. In addition, UPA significantly increased fibrillin protein expression, which can serve to bind inactive TGF-ß complexes. Therefore, UPA inhibits leiomyoma fibrosis by decreasing active TGF-ß3 and diminishing signaling through the canonical pathway. CLINICAL TRIAL REGISTRATION NUMBER: NCT00290251.
Asunto(s)
Leiomioma/genética , Norpregnadienos/farmacología , Factor de Crecimiento Transformador beta3/genética , Neoplasias Uterinas/genética , Adulto , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leiomioma/metabolismo , Leiomioma/patología , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Crecimiento Transformador beta3/metabolismo , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
Nuclear factor of activated T-cells (NFAT5) is a tissue specific, osmoadaptive transcription factor essential for the control of hydration homeostasis in mammalian cells. Nuclear factor of activated T-cells regulates osmolyte transporters aldo-keto reductase family 1 member B1 (AKR1B1) and solute carrier family 5 member 3 (SLC5A3) to maintain fluid equilibrium in cells. The osmotic potential of the extracellular matrix of leiomyomas is attributed to the role of proteoglycans. In leiomyoma cells, NFAT5 is overexpressed compared to myometrial cells. The selective progesterone receptor modulator, ulipristal acetate, has been reported to decrease the size of leiomyomas in clinical trials. When treated with ulipristal acetate, both patient leiomyoma tissue and leiomyoma cells grown in 3-dimensional cultures show a decrease in the expression of NFAT5 protein, solute transporters AKR1B1 and SLC5A3, and results in an associated decline in the expression of proteoglycans, versican, aggrecan, and brevican. In summary, ulipristal acetate induces changes in leiomyoma cell osmoregulation which result in a decrease in proteoglycan expression.
Asunto(s)
Leiomioma/metabolismo , Factores de Transcripción NFATC/metabolismo , Norpregnadienos/farmacología , Proteoglicanos/metabolismo , Neoplasias Uterinas/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Leiomioma/tratamiento farmacológico , Norpregnadienos/uso terapéutico , Neoplasias Uterinas/tratamiento farmacológicoRESUMEN
Context: Uterine leiomyomata (fibroids) are prevalent sex hormoneâdependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood. Objective: Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids. Methods and Results: Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferaseâbinding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus. Conclusion: These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Leiomioma/patología , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Uterinas/patología , Proteínas de Anclaje a la Quinasa A/genética , Adulto , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Leiomioma/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor/genética , Norpregnadienos/farmacología , Norpregnadienos/uso terapéutico , Progesterona/metabolismo , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/metabolismo , Receptores de Progesterona/antagonistas & inhibidores , Neoplasias Uterinas/tratamiento farmacológico , Útero/efectos de los fármacos , Útero/patologíaRESUMEN
BACKGROUND: Uterine leiomyomas are clinically significant tumours that may develop due to an altered differentiation pathway. We have previously identified a dysregulated retinoic acid (RA) pathway that reduced retinoic exposure in human leiomyoma surgical specimens, and have shown that the leiomyoma phenotype was characterized by excessive and disorganized extracellular matrix (ECM). OBJECTIVE: The goal of this study was to determine the impact of RA exposure on the disrupted ECM phenotype of leiomyomas. DESIGN AND METHODS: Study of immortalized and molecularly confirmed cells generated from surgical specimens of spontaneous uterine leiomyoma and matched myometrium. RESULTS: Immortalized leiomyoma and myometrial cells retained the molecular characteristics of their progenitor tissue. Proliferation of leiomyoma cells was inhibited by all-trans retinoic acid (ATRA). Furthermore, there was a dose-dependent decrease in soluble extracellular collagen protein in ATRA-treated leiomyoma cells. Exposure of leiomyoma cells to ATRA resulted in a dose-dependent inhibition of templates for specific ECM protein production including collagen 1, collagen 4, fibronectin and versican. Notably, expression levels in treated leiomyoma cells approached those found in myometrial cells. These mRNA alterations translated into altered protein. Down-regulation was also observed among the RA pathway genes such as CYP26A1 with exposure to ATRA. Finally, ATRA down-regulated TGF-beta3 mRNA expression and the TGF-beta regulated genes in leiomyoma cells. CONCLUSION: Exposure of leiomyomas to ATRA down-regulated cell proliferation, ECM formation, RA metabolism and TGF-beta regulation, suggesting that RA exposure can alter the leiomyoma phenotype to one that more closely approximates normal myometrium.
Asunto(s)
Leiomioma/patología , Miometrio/citología , Tretinoina/farmacología , Neoplasias Uterinas/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Matriz Extracelular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leiomioma/genética , Leiomioma/metabolismo , Miometrio/efectos de los fármacos , Fenotipo , Transducción de Señal/efectos de los fármacos , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMEN
OBJECTIVE: Uterine leiomyoma produce an extracellular matrix (ECM) that is abnormal in its volume, content, and structure. Alterations in ECM can modify mechanical stress on cells and lead to activation of Rho-dependent signaling and cell growth. Here we sought to determine whether the altered ECM that is produced by leiomyoma was accompanied by an altered state of mechanical homeostasis. STUDY DESIGN: We measured the mechanical response of paired leiomyoma and myometrial samples and performed immunogold, confocal microscopy, and immunohistochemical analyses. RESULTS: Leiomyoma were significantly stiffer than matched myometrium. The increased stiffness was accompanied by alteration of the ECM, cell shape, and cytoskeleton in leiomyoma, compared with myometrial samples from the same uterus. Levels of AKAP13, a protein that is known to activate Rho, were increased in leiomyoma compared to myometrium. AKAP13 was associated with cytoskeletal filaments of immortalized leiomyoma cells. CONCLUSION: Leiomyoma cells are exposed to increased mechanical loading and show structural and biochemical features that are consistent with the activation of solid-state signaling.
Asunto(s)
Procesos de Crecimiento Celular , Matriz Extracelular/metabolismo , Leiomioma/metabolismo , Miometrio/fisiología , Proteínas de Anclaje a la Quinasa A/biosíntesis , Adulto , Femenino , Homeostasis , Humanos , Inmunohistoquímica , Leiomioma/fisiopatología , Microscopía Confocal , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Proteínas Proto-Oncogénicas/biosíntesis , Transducción de Señal , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/fisiopatología , Proteína de Unión al GTP rhoA/biosíntesisRESUMEN
In a prior randomized controlled study, patients treated with ulipristal acetate (UPA) or placebo for 3 months had a decrease in leiomyoma size. A total of 10 patients' tissue samples (5 placebo and 5 treated with 10 mg/d UPA) that underwent hysterectomy and tissue preservation were identified from this study. Quantitative real-time reverse transcriptase polymerase chain reaction and Western blotting were used to assess fold gene and protein expression of extracellular membrane (ECM) proteins: collagen 1A (COL1A), fibronectin (FN1), and versican (VCAN) of the samples. Confirmatory immunohistochemical analysis was performed. Changes in total matrix collagen were examined using Masson trichrome staining. Multiplex measurement of the matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases was performed. Compared to placebo-treated surgical specimens, 80% of the treated specimens showed decrease in VCAN protein, 60% showed decrease in FN1, but no consistent alteration in COL1A. This effect was also supported by immunohistochemistry where leiomyoma surgical specimens demonstrated decreased amount of FN1 and VCAN on UPA treatment. Increased MMP2 and decreased MMP9 in treated patient leiomyomas indicate both degradation of the matrix and inhibition of the pathway involved in matrix production. Treatment with UPA decreased fibroid volume in placebo-controlled, randomized trials. Treatment with UPA decreased gene expression and protein production in leiomyoma tissue, suggesting both an impact on water content and ECM protein concentration as a mechanism of ulipristal-mediated decrease in leiomyoma size.