RESUMEN
Loss of tumor-suppressor PTEN is the most common alteration in endometrial carcinoma. However, the relationship between loss of PTEN, growth factors [eg, insulin/insulin-like growth factor (IGF)-1], epidermal growth factor (EGF), and hyperestrogenism in the development of endometrial carcinoma is still controversial. By using three-dimensional (3D) cultures of PTEN(+/+) and PTEN(+/-) endometrial epithelial cells, we investigated the effects of EGF, insulin/IGF, and estradiol in endometrial cell proliferation. We have previously demonstrated that 3D cultures of endometrial cells require EGF and insulin/IGF to proliferate. Herein, we demonstrate that, in the presence of EGF and insulin/IGF, long-term estradiol treatment directly induces proliferation of 3D cultures. Moreover, we show that the mitogenic effects of estradiol require the presence of insulin/IGF and EGF, because withdrawal of such factors completely abolishes estradiol-induced proliferation. In the presence of EGF and insulin/IGF, PTEN(+/-) and PTEN(+/+) spheroids display a similar rate of proliferation. However, the addition of estradiol causes an exaggerated proliferation of PTEN(+/-) cultures, leading to formation of complex structures, such as those observed in endometrial hyperplasia or carcinoma. In summary, we demonstrate that EGF and insulin/IGF prime endometrial epithelial cells to direct the mitogenic effects of estradiol. Furthermore, PTEN deficiency results in enhanced responsiveness to this combination, leading to the development of hyperplasia of endometrial cells in culture.
Asunto(s)
Hiperplasia Endometrial/inducido químicamente , Endometrio/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Estradiol/efectos adversos , Insulina/metabolismo , Mitógenos/efectos adversos , Fosfohidrolasa PTEN/deficiencia , Animales , Biomarcadores/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patología , Endometrio/metabolismo , Endometrio/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Estrógenos/efectos adversos , Femenino , Técnica del Anticuerpo Fluorescente , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides CelularesRESUMEN
We have previously shown that transgenic (tg) mice expressing in B lymphocytes both BCL-2 and a TNFR-associated factor 2 (TRAF2) mutant lacking the really interesting new gene and zinc finger domains (TRAF2DN) develop small lymphocytic lymphoma and chronic lymphocytic leukemia with high incidence (Zapata et al. 2004. Proc. Nat. Acad. Sci. USA 101: 16600-16605). Further analysis of the expression of TRAF2 and TRAF2DN in purified B cells demonstrated that expression of both endogenous TRAF2 and tg TRAF2DN was negligible in Traf2DN-tg B cells compared with wild-type mice. This was the result of proteasome-dependent degradation, and rendered TRAF2DN B cells as bona fide TRAF2-deficient B cells. Similar to B cells with targeted Traf2 deletion, Traf2DN-tg mice show expanded marginal zone B cell population and have constitutive p100 NF-κB2 processing. Also, TRAF3, X-linked inhibitor of apoptosis, and Bcl-X(L) expression levels were increased, whereas cellular inhibitors of apoptosis 1 and 2 levels were drastically reduced compared with those found in wild-type B cells. Moreover, consistent with previous results, we also show that TRAF2 was required for efficient JNK and ERK activation in response to CD40 engagement. However, TRAF2 was deleterious for BCR-mediated activation of these kinases. In contrast, TRAF2 deficiency had no effect on CD40-mediated p38 MAPK activation but significantly reduced BCR-mediated p38 activation. Finally, we further confirm that TRAF2 was required for CD40-mediated proliferation, but its absence relieved B cells of the need for B cell activating factor for survival. Altogether, our results suggest that TRAF2 deficiency cooperates with BCL-2 in promoting chronic lymphocytic leukemia/small lymphocytic lymphoma in mice, possibly by specifically enforcing marginal zone B cell accumulation, increasing X-linked inhibitor of apoptosis expression, and rendering B cells independent of B cell activating factor for survival.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Predisposición Genética a la Enfermedad , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma Folicular/genética , Linfoma Folicular/inmunología , Factor 2 Asociado a Receptor de TNF/deficiencia , Animales , Subgrupos de Linfocitos B/patología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Linfoma Folicular/metabolismo , Ratones , Ratones Transgénicos , Mutación , Estructura Terciaria de Proteína/genética , Factor 2 Asociado a Receptor de TNF/biosíntesis , Factor 2 Asociado a Receptor de TNF/genética , Dedos de Zinc/genéticaRESUMEN
Most glandular tissues comprise polarized epithelial cells organized around a single central lumen. Although there is active research investigating the molecular networks involved in the regulation of lumenogenesis, little is known about the extracellular factors that influence lumen formation and maintenance. Using a three-dimensional culture system of epithelial endometrial cells, we have revealed a new role for pro-inflammatory cytokines such as TNFα and IL1α in the formation and, more importantly, maintenance of a single central lumen. We also studied the mechanism by which glucocorticoids repress TNFα and IL1α expression. Interestingly, regulation of pro-inflammatory cytokine expression and subsequent lumen formation is mediated by estrogen receptor α (ERα) but not by the glucocorticoid receptor. Finally, we investigated the signaling pathways involved in the regulation of lumen formation by pro-inflammatory cytokines. Our results demonstrate that activation of the ERK/MAPK signaling pathway, but not the PI3K/Akt signaling pathway, is important for the formation and maintenance of a single central lumen. In summary, our results suggest a novel role for ERα-regulated pro-inflammatory cytokine expression in lumen formation and maintenance.
Asunto(s)
Regulación hacia Abajo , Endometrio/crecimiento & desarrollo , Endometrio/metabolismo , Receptor alfa de Estrógeno/metabolismo , Glucocorticoides/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-1alfa/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Receptor alfa de Estrógeno/genética , Femenino , Regulación de la Expresión Génica , Interleucina-1alfa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Epithelial to mesenchymal transition is thought to be implicated in tumor invasion and metastasis. To investigate its role in myometrial invasion, samples from 42 stage I (confined to the corpus) endometrioid endometrial carcinomas were analyzed. All E-cadherin repressors (SNAI1, SNAI2 (SLUG), ZEB1, HMGA2, and TWIST1) had a higher expression in endometrioid endometrial carcinomas than in normal endometrium (P < .0001), whereas CDH1 (E-cadherin gene) tended to be lower. In comparison with nonmyoinvasive (stage IA) tumors, those with deep myometrial invasion (stage IC) had increased messenger RNA expression of SLUG, ZEB1, and HMGA2 (P < .001). Furthermore, samples from the myoinvasive front of deeply invasive tumors had higher levels of SLUG, ZEB1, and HMGA2 than the corresponding superficial samples. Immunohistochemical analysis of these cases revealed that the decrease in E-cadherin was concordant with an increase in Snail and Twist protein expression. Trying to induce epithelial to mesenchymal transition in endometrioid endometrial carcinomas, we initially produced persistent activation of this pathway in Ishikawa cells. The cell line was infected with lentiviruses carrying the V600E mutation of BRAF, inducing loss of ß-catenin, E-cadherin, and cytokeratin and increase in vimentin and Snail. These changes were mediated by ERK1/2 phosphorylation, which was also increased at the myoinvasive front. Furthermore, MEK1/2 inhibitor UO126 reversed the mesenchymal phenotype. Our findings suggest that epithelial to mesenchymal transition regulators are implicated in myometrial invasion of endometrioid endometrial carcinoma and may be potential therapeutic targets through the MAPK/ERK pathway.
Asunto(s)
Carcinoma Endometrioide/patología , Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal , Adulto , Anciano , Anciano de 80 o más Años , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/metabolismo , Progresión de la Enfermedad , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Endometrial carcinoma (EC) is a common female cancer, treated mainly by surgery and adjuvant radiotherapy. Relapse following treatment is associated with increased risk of metastases. Hypoxia, a common microenvironment in solid tumors, correlates with malignant progression, rendering tumors resistant to ionizing therapy. Hence, we assessed here the immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α) and members of the NF-κB family in 82 primary EC and 10 post-radiation recurrences of EC. Post-radiation recurrences were highly hypoxic, with a higher expression of HIF-1α and also RelA (p65) and p52 when compared with primary EC. We next investigated the effects of hypoxia on EC cell lines. We found that EC cell lines are highly resistant to hypoxia-induced apoptosis. We thus focused on the molecular mechanisms involved in conferring hypoxic cell death resistance. We show that in addition to the classical NF-κB, hypoxia activates the alternative NF-κB pathway. To characterize the upstream kinases involved in the activation of these pathways, we used lentiviral-mediated knockdown and mouse embryonic fibroblasts lacking IKKα and IKKß kinases. Both IKKα and IKKß kinases are required for RelA (p65) and p100 accumulation, whereas p52 processing under hypoxia is IKKα dependent. Furthermore, Ishikawa endometrial cell line harboring either RelA (p65) or p52 short-hairpin RNA was sensitive to hypoxia-induced cell death, indicating that, in addition to the known prosurvival role of RelA (p65) under hypoxia, alternative NF-κB pathway also enhances hypoxic survival of EC cells. Interestingly, although HIF-1α controlled classical NF-κB activation pathway and survival under hypoxia through RelA (p65) nuclear accumulation, the alternative pathway was HIF-1α independent. These findings have important clinical implications for the improvement of EC prognosis before radiotherapy.
Asunto(s)
Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Neoplasias Endometriales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción ReIA/metabolismo , Animales , Western Blotting , Bromodesoxiuridina , Línea Celular Tumoral , Cartilla de ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunohistoquímica , Lentivirus , Luciferasas , Ratones , Análisis por Micromatrices , Plásmidos/genética , ARN Interferente Pequeño/genética , Transducción de Señal/genética , TransfecciónRESUMEN
The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.
Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Carcinoma/metabolismo , Neoplasias Endometriales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Quinasas/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Línea Celular Tumoral , Proliferación Celular , ADN Complementario/metabolismo , Femenino , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , ARN/metabolismoRESUMEN
Development of three-dimensional (3D) cultures that mimic in vivo tissue organization has a pivotal role in the investigation of the involvement of cell adhesion and polarity genes in the pathogenesis of epithelial cancers. Here we describe a novel 3D culture model with primary mouse endometrial epithelial cells. In this model, isolated endometrial epithelial cells develop single-lumened, polarized glandular structures resembling those observed in endometrial tissue. Our in vitro 3D culture model of endometrial glands requires the use of serum-free defined medium with only epidermal growth factor and insulin as growth supplements and 3% Matrigel as reconstituted extracellular matrix. Under these culture conditions, glands of epithelial cells displaying typical apicobasal polarity and proper positioning of tight and adherent junctions are formed by hollowing as early as 7 to 8 days in culture. Addition of the phosphatidylinositol 3-kinase inhibitor LY294002 completely inhibits bromodeoxyuridine incorporation and cyclinD1 expression, confirming that in vitro growth of endometrial glands depends on phosphatidylinositol 3-kinase/Akt signaling. To prove that our culture method is a good model to study endometrial carcinogenesis, we knocked down E-cadherin or phosphatase and tensin homolog expression by lentivirus-delivered short hairpin RNAs. Down-regulation of E-cadherin resulted in complete loss of epithelial cell polarity and glandular formation, whereas phosphatase and tensin homolog down-regulation resulted in increased proliferation of glandular epithelial cells. These properties indicate that our 3D culture model is suitable to study the effect of growth factors, drugs, and gene alterations in endometrial carcinogenesis and to study normal endometrial biology/physiology.
Asunto(s)
Técnicas de Cultivo de Célula , Polaridad Celular , Neoplasias Endometriales/patología , Endometrio/patología , Células Epiteliales/metabolismo , Animales , Cadherinas/metabolismo , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Neoplasias Endometriales/metabolismo , Endometrio/citología , Células Epiteliales/citología , Femenino , Laminina/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteoglicanos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which mediates apoptosis by the extrinsic pathway. Up-regulation of decoy receptors, DcR1 and DcR2, may result in diminished binding of TRAIL to their functional receptors. DcR1 expression was assessed in normal endometrial tissue (NE) and endometrial carcinoma (EC) samples by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (PCR). IHC was performed in two tissue microarrays; one composed of 80 samples of NE and a second one constructed from paraffin-embedded blocks of 62 EC. For quantitative real-time RT-PCR analysis, RNA was obtained from 19 NE and 28 EC samples using Trizol. mRNA expression of DcR1 was assessed with Taqman-based assays in an Abi-Prism 700 SDS. Results were correlated with stage, histological type, and grade. By IHC, cytoplasmic expression of DcR1 was frequently seen in NE (79.6%) and varied according to the menstrual cycle. Positive DcR1 immunostaining was also detected in EC (98.1% of the cases) without any specific statistical association with histological type, grade, and stage. By quantitative real-time PCR, all NE had similar levels of DcR1expression (0.8-1.7 RQ), which were considered the basal levels of DcR1 expression in NE. Increased DcR1 expression (> or =5-fold higher than the basal levels) was detected in 13 of 28 EC (46.4%). High DcR1 expression levels were found in ECs of different stages: IA, four of 12 (33%); IB, two of four (50%); IC, four of six (66%); and IIA and IIB three of six (50%). Results suggest that DcR1 expression occurs in a subset of EC and may contribute to resistance to TRAIL-induced apoptosis.
Asunto(s)
Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Apoptosis/fisiología , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Proteínas Ligadas a GPI , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Análisis por Micromatrices , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Señuelo del Factor de Necrosis Tumoral/biosíntesis , Receptores Señuelo del Factor de Necrosis Tumoral/genéticaRESUMEN
OBJECTIVE: To review the literature regarding the use of tissue microarray (TMA) technology in understanding the biology, diagnosis and prognosis of endometrial carcinoma (EC). STUDY DESIGN: This review of TMA technology in EC was based on a large number of published articles. We focused on the use of TMA technology as a tool to gain insight in endometrial carcinogenesis and to validate data obtained from DNA microarrays, proteomics and cellular models. RESULTS: We summarized the technical aspects of the 37 articles that were reviewed. The number of EC cases in each series varied from 32-485 (median, 128). The number of cores ranged from 1-4 (median, 2), and the size of the cores ranged from 0.6-2 mm (median, 0.6 mm). Only 3 studies applied fluorescence in situ hybridization technology, while the remaining 34 studies used immunohistochemistry. CONCLUSION: TMA can help to establish new prognostic markers and to define protein biomarkers that help in differential diagnosis.
Asunto(s)
Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/metabolismo , Análisis de Matrices Tisulares/métodos , Biomarcadores/metabolismo , Neoplasias Endometriales/etiología , Neoplasias Endometriales/patología , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica , Análisis de Matrices Tisulares/instrumentaciónRESUMEN
Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.
Asunto(s)
Apoptosis/fisiología , Biomarcadores de Tumor/análisis , Quinasa de la Caseína II/biosíntesis , Proliferación Celular , Neoplasias Endometriales/metabolismo , Fragmentos de Péptidos/biosíntesis , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Tumoral , Neoplasias Endometriales/patología , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Análisis de Matrices Tisulares , beta Catenina/metabolismoRESUMEN
Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) regulates both innate and adaptive immunity by modulating signaling by Toll-like receptors (TLR) and TNF receptors. TRAF3 was recently identified as a tumor suppressor in human multiple myeloma, suggesting a prominent role in plasma cell homeostasis. We have generated transgenic mice expressing human TRAF3 in lymphocytes. These mice are normal at birth, but they develop over time plasmacytosis and hypergammaglobulinemia, as well as systemic inflammation and tertiary lymphoid organ formation. The analysis of the humoral responses of the TRAF3 mice demonstrated increased responses to T-dependent and T-independent antigens with increased production of antigen-specific immunoglobulin Gs (IgGs) compared with wild-type mice. Furthermore, TLR-mediated IgG production is also increased in TRAF3 B cells. In addition, TRAF3 mice develop autoimmunity and are predisposed to cancer, particularly squamous cell carcinomas of the tongue ( approximately 50% incidence) and salivary gland tumors. In summary, TRAF3 renders B cells hyperreactive to antigens and TLR agonists, promoting autoimmunity, inflammation, and cancer, hereby providing a new model for studying de novo carcinogenesis promoted by B cell-initiated chronic inflammation.
Asunto(s)
Autoinmunidad , Linfocitos B/inmunología , Regulación de la Expresión Génica/fisiología , Inflamación/etiología , Neoplasias/etiología , Células Plasmáticas/patología , Factor 3 Asociado a Receptor de TNF/genética , Animales , Proliferación Celular , Femenino , Citometría de Flujo , Humanos , Hipergammaglobulinemia/etiología , Hipergammaglobulinemia/patología , Immunoblotting , Técnicas para Inmunoenzimas , Inmunoglobulina G/sangre , Inflamación/patología , Masculino , Ratones , Ratones Transgénicos , Neoplasias/patología , Transducción de Señal , Receptores Toll-LikeRESUMEN
The article reviews the main molecular pathology alterations of endometrial and ovarian carcinomas and melanoma. Several promising drugs targeting the genes most frequently altered in these tumors are under consideration. The most promising signaling pathways to be targeted for therapies in these tumors are the tyrosine kinase receptor (EGFR, HER2, c-KIT), the RAS/B-RAF/MAPK, the PI3K-mTOR, and apoptosis signaling pathways.
Asunto(s)
Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos/tendencias , Neoplasias de los Genitales Femeninos/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Animales , Ensayos Clínicos como Asunto , Sistemas de Liberación de Medicamentos/métodos , Femenino , Neoplasias de los Genitales Femeninos/genética , Humanos , Melanoma/genéticaRESUMEN
The nuclear factor-kappaB (NF-kappaB) family of transcription factors regulates a wide variety of cellular processes including cell growth, differentiation, and apoptosis. NF-kappaB has been shown to be activated through several signaling pathways that involve growth factor receptors. The aim of the study was to assess the immunohistochemical expression of members of the NF-kappaB family and the putative targets of NF-kappaB in a series of medullary thyroid carcinomas (MTCs), in correlation with RET mutational status. A tissue microarray was constructed from paraffin-embedded blocks of 48 MTCs (13 familial, 35 sporadic) previously evaluated for germ line and somatic RET mutations. Immunohistochemical evaluation included members of the NF-kappaB (p50, p65, p52, c-Rel, RelB) family, as well as putative targets of NF-kappaB such as Flip, Bcl-xL, and cyclin D1. Nuclear immunostaining for members of NF-kappaB was frequent in MTCs (p50, 19%; p65, 68%; p52, 86.6%; c-Rel, 75%; RelB, 36%). MTCs with germ line or somatic RET mutations (29 cases) showed NF-kappaB nuclear translocation (particularly of p65, P = .035) more frequently than MTCs without RET mutations (19 cases). Immunostaining for putative targets of NF-kappaB showed a significant statistical association between p65 and Bcl-xL (P = .024). In addition, Bcl-xL expression was statistically higher in the tumors with exon 16 RET mutation in comparison with those with exon 10 and 11 RET mutations or wild-type RET (P = .002). Moreover, the significance of RETsignaling in NF-kappaB activation was evaluated in the RET-mutated TT cell line. TT cells were infected with lentiviruses carrying short hairpin RNA to knock down RET expression, and NF-kappaB activity was assessed by luciferase reporter assays. Silencing of RET in the TT cell line produced a significant decrease in NF-kappaB activation and reduction in ERK1/2. The results suggest that the NF-kappaB is frequently activated in MTCs. The results also support the hypothesis that RET activation by somatic or germ line mutations may be responsible for NF-kappaB activation in MTCs.
Asunto(s)
Carcinoma Medular/genética , Mutación de Línea Germinal , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Medular/metabolismo , Carcinoma Medular/patología , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Proteínas Proto-Oncogénicas c-ret/metabolismo , Interferencia de ARN , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Análisis de Matrices Tisulares , Factor de Transcripción ReIA/metabolismo , Proteína bcl-X/metabolismoRESUMEN
We have recently demonstrated that proteasome inhibitors can be effective in inducing apoptotic cell death in endometrial carcinoma cell lines and primary culture explants. Increasing evidence suggests that reactive oxygen species are responsible for proteasome inhibitor-induced cell killing. Antioxidants can thus block apoptosis (cell death) triggered by proteasome inhibition. Here, we have evaluated the effects of different antioxidants (edaravone and tiron) on endometrial carcinoma cells treated with aldehyde proteasome inhibitors (MG-132 or ALLN), the boronic acid-based proteasome inhibitor (bortezomib) and the epoxyketone, epoxomicin. We show that tiron specifically inhibited the cytotoxic effects of bortezomib, whereas edaravone inhibited cell death caused by aldehyde-based proteasome inhibitors. We have, however, found that edaravone completely inhibited accumulation of ubiquitin and proteasome activity decrease caused by MG-132 or ALLN, but not by bortezomib. Conversely, tiron inhibited the ubiquitin accumulation and proteasome activity decrease caused by bortezomib. These results suggest that edaravone and tiron rescue cells of proteasome inhibitors from cell death, by inhibiting blockade of proteasome caused by MG-132 and ALLN or bortezomib, respectively. We also tested other antioxidants, and we found that vitamin C inhibited bortezomib-induced cell death. Similar to tiron, vitamin C inhibited cell death by blocking the ability of bortezomib to inhibit the proteasome. Until now, all the antioxidants that blocked proteasome inhibitor-induced cell death also blocked the proteasome inhibitor mechanism of action.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Proteasoma , Antipirina/análogos & derivados , Antipirina/farmacología , Ácido Ascórbico/farmacología , Western Blotting , Ácidos Borónicos/farmacología , Bortezomib , Hidroxianisol Butilado/farmacología , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Inhibidores de Caspasas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cumarinas/farmacología , Relación Dosis-Respuesta a Droga , Edaravona , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Ergotioneína/farmacología , Femenino , Humanos , Leupeptinas/farmacología , Oligopéptidos/farmacología , Pirazinas/farmacología , Ubiquitina/metabolismo , Vitamina E/farmacología , Vitaminas/farmacologíaRESUMEN
Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway are common in endometrial carcinoma. Inactivation of the tumor suppressor gene PTEN leads to a constitutively active PI3K pathway, which plays a role in the early steps of endometrial tumorigenesis. Other alterations in the PI3K/AKT pathway are mutations in the PIK3CA gene, which encode the p110alpha catalytic subunit of PI3K. PIK3CA mutations cluster to the helical (exon 9) and the kinase (exon 20) domains of the gene. In endometrial carcinomas, PIK3CA mutations have been found to coexist frequently with PTEN mutations, but it is not clear whether they occur in cells with monoallelic or biallelic inactivation of PTEN. In the present study we have evaluated PIK3CA mutational status in a series of 33 endometrial carcinomas, previously screened for microsatellite instability and mutations in PTEN, K-RAS, and CTNNB-1. The tumors were also evaluated for loss of heterozygosity on 10q23 and hypermethylation of the promoter region of PTEN/psiPTEN to assess the monoallelic or biallelic inactivation status of PTEN. PIK3CA mutations were detected in 8 (24%) of the 33 cases. Seven mutations were located in exon 20 and 1 in exon 9. PTEN alterations were found in 19 cases (57%). Biallelic inactivation of PTEN was demonstrated in 11 tumors, whereas 8 tumors exhibited alteration in only 1 of the 2 alleles. PIK3CA mutations coexisted with monoallelic alterations of PTEN in 4 cases (2 mutations and 2 allelic imbalances), with biallelic PTEN inactivation in 1 case (mutation and promoter methylation), and 3 tumors showed PIK3CA mutations in association with wild-type PTEN. PIK3CA mutations did not correlate with microsatellite instability or mutations in CTNNB-1. However, PIK3CA and K-RAS mutations (8 cases) were mutually exclusive alterations. In summary, the results confirm that PIK3CA mutations are frequent in endometrial carcinoma and support the hypothesis that PIK3CA mutations may have an additive effect to PTEN monoallelic inactivation in endometrial carcinoma.
Asunto(s)
Neoplasias Endometriales/genética , Genes ras/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Humanos , Inmunohistoquímica , Reacción en Cadena de la PolimerasaRESUMEN
Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-kappaB, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-kappaB, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKK alpha/beta, phosphorylation and degradation of IkappaB alpha, and phosphorylation of the p65 NF-kappaB subunit on serine 536. Proteasome inhibitor-induced NF-kappaB activity can be blocked by a non-degradable form of IkappaB alpha or dominant negative forms of either IKK alpha or IKK beta. Lentiviral delivery of shRNAs to either IKK alpha or IKK beta cause blockade of NF-kappaB transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IkappaB alpha degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-kappaB activation. Accordingly, siRNA knockdown of p65 inhibits proteasome inhibitor-induced NF-kappaB transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-kappaB instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NF-kappaB activation should be carefully examined in particular cell types.
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Neoplasias Endometriales/tratamiento farmacológico , FN-kappa B/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Neoplasias Endometriales/patología , Femenino , Humanos , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Evasion of apoptotic cell death plays a key role in cancer development. Survivin is a member of the inhibitor of apoptosis proteins, which also has a role in the control of cell division. Survivin may be overexpressed in some tumors and has been suggested to be related to PTEN, beta-catenin, p53 [all of them frequently abnormal in endometrial carcinomas (ECs)], and STAT-3. A tissue microarray was constructed from paraffin-embedded blocks of 95 ECs, previously studied for microsatellite instability and for alterations in PTEN, k-RAS, and CTNNB-1. Immunohistochemical evaluation included 1) survivin, 2) markers of cell proliferation and apoptosis (Ki67-MIB1 and M 30-neoepitope cytokeratin 18), and 3) proteins involved in cell signaling pathways (PTEN, phospho-AKT, beta-catenin, p53, and STAT-3). Survivin expression was frequent in ECs (75.95%) but did not show any statistical significant correlation with histological type and grade, stage, overall survival, or mitotic and apoptotic indexes. Survivin expression had a statistical significant correlation with decreased PTEN expression (r = -0.383, p = 0.001), increased phospho-AKT (r = 0.70, p < 0.001), and positive STAT-3 immunostaining (r = 0.6, p < 0.001). Survivin expression did not show statistical correlation with either beta-catenin or p53 alterations. The results suggest that increased survivin expression is frequent in ECs and may be dependent on STAT-3 and PI3 K/AKT activation. Because PTEN abnormalities are very frequent in ECs, the results from this study indicate that PTEN may interfere with the process of apoptosis and cell proliferation by promoting survivin expression.
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Carcinoma/metabolismo , Proteínas de Unión al ADN/biosíntesis , Neoplasias Endometriales/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Monoéster Fosfórico Hidrolasas/biosíntesis , Transactivadores/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Apoptosis/fisiología , Carcinoma/patología , Procesos de Crecimiento Celular/fisiología , Proteínas del Citoesqueleto/biosíntesis , Neoplasias Endometriales/patología , Femenino , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis , Antígeno Ki-67/biosíntesis , Fosfohidrolasa PTEN , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-akt , Factor de Transcripción STAT3 , Transducción de Señal , Survivin , Análisis de Matrices Tisulares , Proteína p53 Supresora de Tumor/biosíntesis , beta CateninaRESUMEN
The FLICE-inhibitory protein (FLIP) plays a key role in the regulation of apoptosis triggered by death ligands. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in some types of tumor but not in others. To assess the possible role of FLIP in apoptosis resistance in endometrial carcinoma, we performed an immunohistochemical study on a tissue microarray composed of 95 endometrial carcinomas. We found positive signals in 43% of the cases, as well as a significant difference in FLIP expression between stage I and II tumors. Moreover, we observed that endometrial carcinoma cell lines Ishikawa and KLE did not undergo apoptosis after TRAIL treatment. Cotreatment of these cells with the inhibitor of transcription actinomycin D resulted in a dramatic decrease in cell viability and induced activation of caspase-8. These events coincided with downregulation of FLIP mRNA and protein. Inhibitors of caspase-8 or overexpression of FLIP completely blocked apoptosis induced by actinomycin D plus TRAIL cotreatment. More importantly, downregulation of endogenous FLIP expression by specific siRNAs sensitized endometrial carcinoma cells to TRAIL-induced apoptosis in the absence of actinomycin D. Taken together, our results suggest for the first time a critical role for FLIP in the regulation apoptosis triggered by TRAIL in endometrial carcinoma cells.
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Adenocarcinoma/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias Endometriales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Línea Celular Tumoral , Dactinomicina/farmacología , Regulación hacia Abajo , Combinación de Medicamentos , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Análisis por Matrices de Proteínas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
The nuclear factor kB (NF-kB) comprises a family of transcription factors involved in the regulation of a wide variety of biological responses. NF-kB plays a well-known function in the regulation of immune responses and inflammation, but growing evidences support a major role in oncogenesis. NF-kB regulates the expression of genes involved in many processes that play a key role in the development and progression of cancer such as proliferation, migration and apoptosis. Aberrant or constitutive NF-kB activation has been detected in many human malignancies. In recent years, numerous studies have focused on elucidating the functional consequences of NF-kB activation as well as its signaling mechanisms. NF-kB has turned out to be an interesting therapeutic target for treatment of cancer.
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FN-kappa B/fisiología , Neoplasias , Apoptosis , División Celular , Resistencia a Antineoplásicos , Humanos , FN-kappa B/antagonistas & inhibidores , Invasividad Neoplásica , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/radioterapia , Transducción de SeñalRESUMEN
The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r=-0.249, P=0.037). The monoclonal antibody 10P03 exhibited an association with phospho-AKT that did not have statistical significance. Both 6.H2.1 and 10P03 antibodies stained PTEN-transfected cells, and were negative in the PTEN-deficient cell line blocks. The polyclonal antibody and the monoclonal antibody 28H6 produced positive staining in PTEN-deficient cell line blocks, suggesting nonspecific staining. The results indicate that monoclonal antibody 6.H2.1 may be a suitable alternative for tumors with inactivation of PTEN.