RESUMEN
Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.
Asunto(s)
Leiomioma , Neoplasias Uterinas , Femenino , Humanos , Triptófano , Quinurenina/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Triptófano Oxigenasa/genética , Triptófano Oxigenasa/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Leiomioma/genética , Leiomioma/metabolismo , Leiomioma/patología , Mutación , Complejo Mediador/genética , Complejo Mediador/metabolismoRESUMEN
Neonatal herpes simplex virus (HSV) infections are serious infections that usually occur in the first few weeks of life. Infants generally present with mucocutaneous lesions, central nervous system infection, and/or systemic disease. In this case report, we describe a set of twins that had unexpected presentations of neonatal HSV. Twin A was diagnosed incidentally on routine eye exam, and Twin B was diagnosed only because his twin was found to be infected; both infants were still hospitalized and were beyond 1 month of age. These twins exhibited atypical manifestations that diverge from the 3 main categories of neonatal HSV and expand our understanding of the spectrum of disease.
Asunto(s)
Herpes Simple , Complicaciones Infecciosas del Embarazo , Embarazo , Recién Nacido , Femenino , Humanos , Lactante , Herpes Simple/diagnóstico , Complicaciones Infecciosas del Embarazo/diagnósticoRESUMEN
Uterine leiomyoma is the most common tumor in women and causes severe morbidity in 15 to 30% of reproductive-age women. Epidemiological studies consistently indicate a correlation between leiomyoma development and exposure to endocrine-disrupting chemical phthalates, especially di-(2-ethylhexyl) phthalate (DEHP); however, the underlying mechanisms are unknown. Here, among the most commonly encountered phthalate metabolites, we found the strongest association between the urine levels of mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), the principal DEHP metabolite, and the risk of uterine leiomyoma diagnosis (n = 712 patients). The treatment of primary leiomyoma and smooth muscle cells (n = 29) with various mixtures of phthalate metabolites, at concentrations equivalent to those detected in urine samples, significantly increased cell viability and decreased apoptosis. MEHHP had the strongest effects on both cell viability and apoptosis. MEHHP increased cellular tryptophan and kynurenine levels strikingly and induced the expression of the tryptophan transporters SLC7A5 and SLC7A8, as well as, tryptophan 2,3-dioxygenase (TDO2), the key enzyme catalyzing the conversion of tryptophan to kynurenine that is the endogenous ligand of aryl hydrocarbon receptor (AHR). MEHHP stimulated nuclear localization of AHR and up-regulated the expression of CYP1A1 and CYP1B1, two prototype targets of AHR. siRNA knockdown or pharmacological inhibition of SLC7A5/SLC7A8, TDO2, or AHR abolished MEHHP-mediated effects on leiomyoma cell survival. These findings indicate that MEHHP promotes leiomyoma cell survival by activating the tryptophan-kynurenine-AHR pathway. This study pinpoints MEHHP exposure as a high-risk factor for leiomyoma growth, uncovers a mechanism by which exposure to environmental phthalate impacts leiomyoma pathogenesis, and may lead to the development of novel druggable targets.
Asunto(s)
Dietilhexil Ftalato , Contaminantes Ambientales , Leiomioma , Ácidos Ftálicos , Humanos , Femenino , Dietilhexil Ftalato/toxicidad , Dietilhexil Ftalato/orina , Quinurenina , Triptófano , Supervivencia Celular , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transportador de Aminoácidos Neutros Grandes 1 , Exposición a Riesgos Ambientales/efectos adversos , Leiomioma/inducido químicamente , Leiomioma/orinaRESUMEN
Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.
Asunto(s)
Leiomioma/enzimología , Complejo Mediador/genética , Triptófano Oxigenasa/metabolismo , Adulto , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Mutación , Progestinas/farmacología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs. METHODS: We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74. RESULTS: After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p < 0.001) and decreased LPS-induced IL-8 production (p < 0.01). CONCLUSIONS: Lyophilization and filtration of hAF is feasible with partial loss of TFs but maintains and even improves bioavailability of TFs measured by proliferation and LPS-induced IL-8 production by FHs74.
Asunto(s)
Líquido Amniótico/metabolismo , Enterocolitis Necrotizante/metabolismo , Liofilización , Tracto Gastrointestinal/embriología , Líquido Amniótico/química , Proliferación Celular , Criopreservación , Deglución , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación , Interleucina-8/metabolismo , Embarazo , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
CONTEXT: Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown. OBJECTIVE: To determine the function of PLIN2 in leiomyoma cells. DESIGN: Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Forty-one premenopausal women undergoing surgery for fibroids. MAIN OUTCOME MEASURES: Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation. RESULTS: PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown. CONCLUSIONS: PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Leiomioma/patología , Miometrio/patología , Perilipina-2/metabolismo , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Uterinas/patología , Adulto , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Proliferación Celular , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glucólisis , Humanos , Leiomioma/genética , Leiomioma/metabolismo , Metaboloma , Miometrio/metabolismo , Perilipina-2/antagonistas & inhibidores , Perilipina-2/genética , Pronóstico , ARN Interferente Pequeño , Receptores de Progesterona/genética , Transducción de Señal , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMEN
Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.
Asunto(s)
Proliferación Celular/genética , Metilación de ADN/genética , Leiomioma/genética , Complejo Mediador/genética , Ligando RANK/genética , Receptores de Progesterona/genética , Células Madre/patología , Neoplasias Uterinas/genética , Adulto , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Leiomioma/tratamiento farmacológico , Persona de Mediana Edad , Progesterona/genética , Promegestona/farmacología , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Neoplasias Uterinas/tratamiento farmacológicoRESUMEN
Context: Uterine leiomyomas are the most common type of gynecologic tumor in women. Objective: To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth. Design: Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo. Setting: Research laboratory. Patients: Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy. Interventions: None. Main Outcome Measure: Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc. Results: RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4). Conclusions: Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Leiomioma/patología , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/genética , Proteínas Recombinantes de Fusión/farmacología , Neoplasias Uterinas/patología , Adulto , Estudios de Casos y Controles , Células Cultivadas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leiomioma/genética , Persona de Mediana Edad , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias Uterinas/genéticaRESUMEN
Context: Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective: To define differential gene expression and signaling pathways in leiomyoma cell populations. Design: Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting: Research laboratory. Patients: Eight African American women. Interventions: None. Main Outcomes Measures: Gene expression patterns, cell proliferation, and differentiation. Results: A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions: IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.
Asunto(s)
Antígenos CD/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Factor II del Crecimiento Similar a la Insulina/genética , Leiomioma/genética , Células Madre Neoplásicas/citología , Comunicación Paracrina/genética , Receptor de Insulina/genética , Neoplasias Uterinas/genética , Adulto , Negro o Afroamericano , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Factor II del Crecimiento Similar a la Insulina/metabolismo , Integrina alfa2/metabolismo , Leiomioma/metabolismo , Sistema de Señalización de MAP Quinasas , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Insulina/metabolismo , Análisis de Matrices Tisulares , Neoplasias Uterinas/metabolismoRESUMEN
The endocervix plays an important role in providing appropriate protective mechanisms of the upper female reproductive tract (FRT) while at the same time providing the appropriate milieu for sperm transport. Hormone fluctuations throughout the menstrual cycle contribute to changes in the mucosal environment that render the FRT vulnerable to infectious diseases. The objective of this study was to identify genes in human endocervix tissues that were differentially expressed in the follicular versus the luteal phases of the menstrual cycle using gene expression profiling. A microarray using the IIlumina platform was performed with eight endocervix tissues from follicular and four tissues from luteal phases of the menstrual cycle. Data analysis revealed significant differential expression of 110 genes between the two phases, with a P value <0.05 and a fold change cutoff of 1.5. Categorization of these genes, using Ingenuity Pathway Analysis, MetaCore from Thomson Reuters, and DAVID, revealed genes associated with extracellular matrix remodeling and cell-matrix interactions, amino acid metabolism, and lipid metabolism, as well as immune regulation in the follicular phase tissues. In luteal phase tissues, genes associated with chromatin remodeling, inflammation, angiogenesis, oxidative stress, and immune cell regulation were predominately expressed. Using samples from additional patients' tissues, select genes were confirmed by quantitative real-time PCR; immunohistochemical staining was also done to examine protein levels. This is the first microarray analysis comparing gene expression in endocervix tissues in cycling women. This study identified key genes and molecular pathways that were differentially regulated during the menstrual cycle.
Asunto(s)
Cuello del Útero/metabolismo , Fase Folicular/metabolismo , Perfilación de la Expresión Génica , Fase Luteínica/metabolismo , Adulto , Análisis por Conglomerados , Femenino , Humanos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. DESIGN: Case-control. SETTING: University research setting. PATIENT(S): Premenopausal women. INTERVENTION(S): Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. MAIN OUTCOME MEASURE(S): Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) ß. RESULT(S): Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERß-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERß based on small interfering RNA knockdown and chromatin immunoprecipitation of ERß followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. CONCLUSION(S): ERß leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.
Asunto(s)
Endometriosis/sangre , Endometrio/citología , Endometrio/enzimología , Receptor beta de Estrógeno/fisiología , Proteínas Inmediatas-Precoces/sangre , Proteínas Serina-Treonina Quinasas/sangre , Biomarcadores/sangre , Biomarcadores/metabolismo , Estudios de Casos y Controles , Supervivencia Celular/fisiología , Endometriosis/metabolismo , Endometriosis/patología , Endometrio/patología , Activación Enzimática/fisiología , Femenino , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/ß-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Leiomioma/genética , Células Madre Neoplásicas/metabolismo , Progesterona/metabolismo , Receptores de Progesterona/genética , Neoplasias Uterinas/genética , Vía de Señalización Wnt , Femenino , Humanos , Leiomioma/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Uterinas/metabolismoRESUMEN
CONTEXT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.
Asunto(s)
Antígenos CD34/genética , Transformación Celular Neoplásica , Integrina alfa2/genética , Leiomioma/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Uterinas/patología , Adulto , Antígenos CD34/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Diferenciación Celular/fisiología , Separación Celular/métodos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa2/metabolismo , Factor 4 Similar a Kruppel , Leiomioma/genética , Leiomioma/metabolismo , Persona de Mediana Edad , Células Madre Neoplásicas/fisiología , Células Tumorales Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMEN
CONTEXT: Fetal growth restriction with abnormal umbilical artery Doppler velocimetry (FGRadv), reflective of elevated fetoplacental vascular resistance, is associated with increased risks of fetal morbidity and mortality even in comparison to those of growth-restricted fetuses with normal placental blood flow. One major cause of this abnormally elevated fetoplacental vascular resistance is the aberrantly formed, thin, elongated villous vessels that are seen in FGRadv placentas. OBJECTIVE: The purpose of this study was to determine the role of fetoplacental endothelial cells (ECs) in angiogenesis in normal pregnancies and in those complicated by FGRadv. DESIGN AND PARTICIPANTS: Human placental specimens were obtained from FGRadv and gestational age-matched, appropriately grown control pregnancies for EC isolation/culture and for immunohistochemical studies. Additional mechanistic studies were performed on ECs isolated from subjects with term, uncomplicated pregnancies. MAIN OUTCOME MEASURES: We evaluated tube formation and differential angiogenic gene expression in FGRadv and control ECs, and we used ECs from uncomplicated pregnancies to further elucidate the molecular mechanisms by which angiogenesis is impaired in FGRadv pregnancies. RESULTS: Tube formation assays showed that FGRadv ECs demonstrate fewer branch points and total length compared with those from gestational age-matched controls, and this defect was not rescued by exposure to hypoxia. FGRadv ECs also demonstrated lower aryl hydrocarbon receptor nuclear translocator (ARNT) expression. ARNT knockdown resulted in suppression of key angiogenic genes including vascular endothelial growth factor A expression and led to deficient tube formation. CONCLUSIONS: ARNT expression in the placental vasculature mediates key angiogenic expression and fetoplacental EC angiogenesis, and low ARNT expression in FGRadv ECs appears to be a key factor in deficient angiogenesis. This, in turn, results in malformed thin villous vessels that structurally contribute to the abnormally elevated fetoplacental vascular resistance that is associated with high morbidity and mortality in fetal growth restriction.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Células Endoteliales/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Neovascularización Fisiológica/fisiología , Placenta/metabolismo , Arterias Umbilicales/diagnóstico por imagen , Femenino , Retardo del Crecimiento Fetal/diagnóstico por imagen , Humanos , Placenta/diagnóstico por imagen , Embarazo , Ultrasonografía , Factor A de Crecimiento Endotelial Vascular/metabolismo , Resistencia Vascular/fisiologíaRESUMEN
BACKGROUND: Uterine leiomyoma is the most common benign tumor in women and is thought to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a cellular insult. Leiomyomas cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are the most common indication for hysterectomy in the USA. A slow rate of cell proliferation, combined with the production of copious amounts of extracellular matrix, accounts for tumor expansion. A common salient feature of leiomyomas is their responsiveness to steroid hormones, thus providing an opportunity for intervention. METHODS: A comprehensive search of PUBMED was conducted to identify peer-reviewed literature published since 1980 pertinent to the roles of steroid hormones and somatic stem cells in leiomyoma, including literature on therapeutics that target steroid hormone action in leiomyoma. Reviewed articles were restricted to English language only. Studies in both animals and humans were reviewed for the manuscript. RESULTS: Estrogen stimulates the growth of leiomyomas, which are exposed to this hormone not only through ovarian steroidogenesis, but also through local conversion of androgens by aromatase within the tumors themselves. The primary action of estrogen, together with its receptor estrogen receptor α (ERα), is likely mediated via induction of progesterone receptor (PR) expression, thereby allowing leiomyoma responsiveness to progesterone. Progesterone has been shown to stimulate the growth of leiomyoma through a set of key genes that regulate both apoptosis and proliferation. Given these findings, aromatase inhibitors and antiprogestins have been developed for the treatment of leiomyoma, but neither treatment results in complete regression of leiomyoma, and tumors recur after treatment is stopped. Recently, distinct cell populations were discovered in leiomyomas; a small population showed stem-progenitor cell properties, and was found to be essential for ovarian steroid-dependent growth of leiomyomas. Interestingly, these stem-progenitor cells were deficient in ERα and PR and instead relied on the strikingly higher levels of these receptors in surrounding differentiated cells to mediate estrogen and progesterone action via paracrine signaling. CONCLUSIONS: It has been well established that estrogen and progesterone are involved in the proliferation and maintenance of uterine leiomyoma, and the majority of medical treatments currently available for leiomyoma work by inhibiting steroid hormone production or action. A pitfall of these therapeutics is that they decrease leiomyoma size, but do not completely eradicate them, and tumors tend to regrow once treatment is stopped. The recent discovery of stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma has the potential to provide the missing link between developing therapeutics that temper leiomyoma growth and those that eradicate them.
Asunto(s)
Leiomioma/fisiopatología , Leiomioma/terapia , Neoplasias Uterinas/fisiopatología , Neoplasias Uterinas/terapia , Animales , Apoptosis/fisiología , Inhibidores de la Aromatasa/uso terapéutico , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Receptor alfa de Estrógeno/fisiología , Estrógenos/fisiología , Femenino , Humanos , Miocitos del Músculo Liso/metabolismo , Recurrencia Local de Neoplasia/epidemiología , Recurrencia Local de Neoplasia/prevención & control , Comunicación Paracrina , Progesterona/fisiología , Receptores de Progesterona/fisiología , Células Madre/patologíaRESUMEN
Resistance to progestin treatment is a major hurdle in the treatment of advanced and reoccurring endometrial cancer. Fenretinide is a synthetic retinoid that has been evaluated in clinical trials as a cancer therapeutic and chemo-preventive agent. Fenretinide has been established to be cytotoxic to many kinds of cancer cells. In the present study, we demonstrate that fenretinide decreased cell viability and induced apoptosis in Ishikawa cells, which are an endometrial cancer cell line, in dose dependent manner in-vitro. This effect was found to be independent of retinoic acid nuclear receptor signaling pathway. Further, we have shown that this induction of apoptosis by fenretinide may be caused by increased retinol uptake via STRA6. Silencing of STRA6 was shown to decrease apoptosis which was inhibited by knockdown of STRA6 expression in Ishikawa cells. Results of an in-vivo study demonstrated that intraperitoneal injections of fenretinide in endometrial cancer tumors (created using Ishikawa cells) in mice inhibited tumor growth effectively. Immunohistochemistry of mice tumors showed a decrease in Ki67 expression and an increase in cleaved caspase-3 staining after fenretinide treatment when compared to vehicle treated mice. Collectively, our results are the first to establish the efficacy of fenretinide as an antitumor agent for endometrial cancer both in-vitro and in-vivo, providing a valuable rationale for initiating more preclinical studies and clinical trials using fenretinide for the treatment of endometrial cancer.
Asunto(s)
Neoplasias Endometriales/tratamiento farmacológico , Fenretinida/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Progresión de la Enfermedad , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Fenretinida/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Acetato de Megestrol/farmacología , Proteínas de la Membrana/metabolismo , Ratones Desnudos , Vitamina A/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CONTEXT: Uterine leiomyoma, or fibroids, represent the most common benign tumors of the female reproductive tract. A newly discovered epigenetic modification, 5-hydroxymethylation (5-hmC), and its regulators, the TET (Ten Eleven Translocation) enzymes, were implicated in the pathology of malignant tumors; however, their roles in benign tumors, including uterine fibroids, remain unknown. OBJECTIVE: To determine the role of 5-hmC and TET proteins in the pathogenesis of leiomyoma using human uterine leiomyoma and normal matched myometrial tissues and primary cells. DESIGN: 5-hmC levels were determined by ELISA and immunofluorescent staining in matched myometrial and leiomyoma tissues. TET expression was analyzed by quantitative RT-PCR and immunoblotting. TET1 or TET3 were silenced or inhibited by small interfering RNA or 2-hydroxyglutarate to study their effects on 5-hmC content and cell proliferation. RESULTS: We demonstrated significantly higher 5-hmC levels in the genomic DNA of leiomyoma tissue compared to normal myometrial tissue. The increase in 5-hmC levels was associated with the up-regulation of TET1 or TET3 mRNA and protein expression in leiomyoma tissue. TET1 or TET3 knockdown significantly reduced 5-hmC levels in leiomyoma cells and decreased cell proliferation. Treatment with 2-hydroxyglutarate, a competitive TET enzyme inhibitor, significantly decreased both 5-hmC content and cell proliferation of leiomyoma cells. CONCLUSION: An epigenetic imbalance in the 5-hmC content of leiomyoma tissue, caused by up-regulation of the TET1 and TET3 enzymes, might lead to discovery of new therapeutic targets in leiomyoma.
Asunto(s)
Proliferación Celular/genética , Citosina/análogos & derivados , Epigénesis Genética , Leiomioma/genética , Neoplasias Uterinas/genética , 5-Metilcitosina/análogos & derivados , Adulto , Citosina/metabolismo , Femenino , Humanos , Leiomioma/metabolismo , Leiomioma/patología , Persona de Mediana Edad , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: To reduce sampling error associated with cancer detection in prostate needle biopsies, we explored the possibility of using fluorescence in situ hybridisation (FISH) to detect chromosomal abnormalities in the histologically benign prostate tissue from patients with adenocarcinoma of prostate. METHODS: Tumour specimens from 33 radical prostatectomy (RP) cases, histologically benign tissue from 17 of the 33 RP cases, and 26 benign prostatic hyperplasia (BPH) control cases were evaluated with Locus Specific Identifier (LSI) probes MYC (8q24), LPL (8p21.22), and PTEN (10q23), as well as with centromere enumerator probes CEP8, CEP10, and CEP7. A distribution of FISH signals in the tumour and histologically benign adjacent tissue was compared to that in BPH specimens using receiver operating characteristic curve analysis. RESULTS: The combination of MYC gain, CEP8 Abnormal, PTEN loss or chromosome 7 aneusomy was positive in the tumour area of all of the 33 specimens from patients with adenocarcinomas, and in 88% of adjacent histologically benign regions (15 out of 17) but in only 15% (4 out of 26) of the benign prostatic hyperplasia control specimens. CONCLUSIONS: A panel of FISH markers may allow detection of genomic abnormalities that associate with adenocarcinoma in the field adjacent to and surrounding the tumour, and thus could potentially indicate the presence of cancer in the specimen even if the cancer focus itself was missed by biopsy and histology review.
Asunto(s)
Adenocarcinoma/genética , Aberraciones Cromosómicas , Hibridación Fluorescente in Situ , Neoplasias de la Próstata/genética , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Biomarcadores de Tumor/genética , Biopsia , Humanos , Masculino , Prostatectomía , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Hiperplasia Prostática/cirugía , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Curva ROCRESUMEN
Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/ß-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of ß-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of ß-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/ß-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.