RESUMEN
In order to identify genes involved in the pathogenesis of clear cell carcinoma of the ovary (CCC), functional screening using a cDNA expression library was performed. We extracted mRNA from a CCC cell line (RMG-1), established a cDNA library using a retroviral vector, transfected that library into mouse NIH3T3 cells and sequenced the resultant foci. The tissue-type specific expression of isolated genes and their transforming activities were evaluated. Seven genes were isolated. Of these genes, the mRNA expression of SEC61B and DVL1 is significantly stronger in CCC than in other histological types (p < .05). Immunohistochemical staining reveals the stronger expression of SEC61B and C1ORF38 than normal ovarian tissues (p < .05). Focus formation is confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells. The present study identified novel genes including SEC61B, C1ORF38, and DVL1, involved in the pathogenesis of CCC. These genes may be additional therapeutic targets for CCC.Impact statementWhat is already known on this subject? Several important genetic abnormalities, including ARID1A and PIK3CA mutations, have been reported in ovarian clear cell carcinoma (CCC).What the results of this study add? SEC61B, C1ORF38, and DVL1 were newly detected as candidate genes involved in ovarian clear cell carcinogenesis.What the implications are of these findings for clinical practice and/or further research? Functional screening using a cDNA expression library may be a useful technique to identify functional genes for pathogenesis. The information obtained using this technique may provide new therapeutic targets of CCC.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Carcinogénesis/genética , Neoplasias Ováricas/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proteínas Dishevelled/metabolismo , Femenino , Biblioteca de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Células 3T3 NIH , Ovario/metabolismo , Canales de Translocación SEC/metabolismoRESUMEN
AIMS: It has been reported that the expression of core 2 ß1,6-N-acetylglucosaminyl transferase 1 (C2GnT1), which synthesizes the core 2 branching structure on O-glycans, may be associated with the biological aggressiveness of tumour cells. Therefore, the aim of this study was to examine the relationship between the expression of C2GnT1 and clinicopathological parameters of patients with endometrial carcinoma. METHODS AND RESULTS: The immunohistochemical expression of C2GnT1 was examined in 84 cases of endometrioid-type endometrial carcinoma, 15 cases of endometrial hyperplasia, and 30 normal endometria. The staining intensity was reported according to a positivity index (PI, full score 100), calculated from the percentage of positive cells. The expression of C2GnT1 was significantly higher in endometrial carcinoma (PI = 8.31 ± 15.29) than in normal endometrium (PI = 0.52 ± 1.24) (P < 0.0005). In carcinomas, the PI was higher in high-grade or advanced-stage tumours, but not significantly. Topologically, C2GnT1 was strongly expressed at sites of deep myometrial invasion. In addition, patients with C2GnT1 overexpression (PI ≥ 10) had significantly shorter survival (P < 0.0005). Multivariable analysis also indicated that C2GnT1 overexpression was an independent prognostic factor (P = 0.017). CONCLUSIONS: C2GnT1 appears to be involved in the biological aggressiveness of endometrial carcinoma. C2GnT1 might become a novel prognostic factor for endometrial carcinoma.
Asunto(s)
Carcinoma Endometrioide/diagnóstico , Neoplasias Endometriales/diagnóstico , N-Acetilglucosaminiltransferasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/mortalidad , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/mortalidad , Femenino , Humanos , Japón/epidemiología , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Tasa de SupervivenciaRESUMEN
AIMS: The aim of this study was to investigate the significance of the expression of Notch-related molecules in endometrial carcinoma. METHODS AND RESULTS: The expression of Notch receptors (Notch1 and 3) and Notch ligands [Jagged (JAG) 1 and Delta-like (DLL) 4] was examined immunohistochemically in 37 normal and 76 malignant endometrial tissue samples. For each section, immunohistochemical staining was scored using a positivity index (PI, full score; 200). The effects of a Notch inhibitor, DAPT, on cell proliferation, invasion and motility were investigated using endometrial carcinoma cell lines. The PIs for Notch1 (mean±SD 90.4±15.3), Notch3 (95.6 ± 20.4), JAG1 (95.5±10.0) and DLL4 (88.2±9.6), were significantly higher in endometrial carcinoma than normal endometrium. The PI for Notch1 was associated significantly with advanced International Federation of Gynecologists & Obstetricians (FIGO) stage. In addition, patients with tumours showing high expression of both Notch1 and JAG1 had a poor prognosis compared with those having double-negative carcinomas (P=0.015). DAPT suppressed invasiveness of cells derived from the endometrial carcinoma cell line KLE. CONCLUSIONS: The Notch1-JAG1 axis may enhance the invasive properties of endometrial carcinomas, which suggests the Notch pathway may be a promising target for the treatment of this malignancy.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Carcinoma Endometrioide/metabolismo , Neoplasias Endometriales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Biomarcadores de Tumor/metabolismo , Carcinoma Endometrioide/tratamiento farmacológico , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dipéptidos/farmacología , Neoplasias Endometriales/tratamiento farmacológico , Femenino , Humanos , Proteína Jagged-1 , Invasividad Neoplásica , Pronóstico , Receptor Notch3 , Proteínas Serrate-JaggedRESUMEN
BACKGROUND: We previously reported the overexpression of lipocalin2 (LCN2), a 25kDa secretory protein involved in iron-transportation, in endometrial carcinoma and its possible contribution to endometrial carcinogenesis. Recently, a specific receptor for LCN2, solute carrier family 22 member 17 (SLC22A17), was identified. The present study was undertaken to investigate the expression of SLC22A17 in endometrial carcinoma. METHODS: The expression of the SLC22A17 and LCN2 proteins was examined immunohistochemically using 69 cases of endometrial carcinoma and adjacent normal endometrial tissues. Immunoreactivity was evaluated according to the percentage of positive cells and described as a positivity index (PI, full score 100). RESULTS: The expression of SLC22A17 was negligible in normal endometria, but positive staining for SLC22A17 (PIâ§1) was observed in 35 cases of endometrial carcinoma. The PI for SLC22A17 was significantly higher in cases with histological grade 3 (P<0.0005), advanced FIGO stage (P=0.002), deep myometrial invasion (P=0.029), positive lymph-vascular space invasion (P=0.029), positive intraperitoneal cytology (P=0.020) and adnexal metastasis (P=0.029). The expression of SLC22A17 and LCN2 was positively correlated with a significant difference (P=0.002), and the patients who overexpressed both SLC22A17 and LCN2 showed poorer survival than those without the expression of SLC22A17 or LCN2 (P=0.002). Moreover, the overexpression of both SLC22A17 and LCN2 was indicated to be an independent prognostic factor by multivariable analysis. CONCLUSIONS: These results suggested that SLC22A17, in cooperation with LCN2, to be involved in the acquisition of aggressive behavior among endometrial carcinoma cells.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Lipocalinas/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Endometriales/diagnóstico , Endometrio/metabolismo , Endometrio/patología , Femenino , Humanos , Inmunohistoquímica , Lipocalina 2 , Persona de Mediana Edad , Pronóstico , Análisis de SupervivenciaRESUMEN
Although overexpression of cyclin A2 is reportedly an indicator of a poor prognosis of various malignancies including endometrial carcinoma, its molecular mechanism remains undetermined. To address this issue, we examined the effect of cyclin A2 on the development of resistance to chemotherapeutic drugs. The expression of cyclin A2 protein was increased in advanced-stage and chemotherapy-refractory stage endometrial carcinomas compared with that in early-stage tumours. The expression levels of cyclin A2 in endometrial carcinoma cell lines correlated positively with the IC(50) for cisplatin. Endometrial carcinoma HHUA cells that overexpressed cyclin A2 showed increased resistance to cisplatin in vitro and in vivo, via the activation of a survival pathway, the inositol-3 phosphate kinase (PI3K) cascade. The use of a cDNA microarray identified an Akt-binding protein, periplakin, as a novel target of cyclin A2. The cyclin A2-induced up-regulation of periplakin was mediated via direct binding of Sp1 to the promoter that was activated by cyclin A2 along with chromatin remodelling involving CBP/p300, and the siRNA-mediated silencing of periplakin suppressed the PI3K pathway. These results indicate cyclin A2 to be involved in the acquisition of aggressive behaviour of tumour cells through the activation of PI3K by cyclin A2-induced periplakin, and to be a promising therapeutic target.
Asunto(s)
Cisplatino/farmacología , Ciclina A2/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Endometriales/patología , Plaquinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/genética , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Plaquinas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/metabolismoRESUMEN
Histone acetylation/deacetylation controls chromatin activity and subsequent gene transcription. Recent studies demonstrated the activation of histone deacetylases (HDACs) in various human malignancies; however, the expression and function of HDACs in ovarian tumors are not fully understood. In this study, we examined the immunohistochemical expression of HDAC1, HDAC2 and HDAC3 using tissues obtained from 115 cases of ovarian tumors and compared it with that of Ki-67 (a growth marker), p21, and E-cadherin and clinicopathological parameters. In addition, we analyzed the effect of specific siRNA for HDAC1, HDAC2 and HDAC3 on the expression of cell cycle-related molecules and E-cadherin to clarify the functional difference among the 3 HDACs. The results indicated that the immunohistochemical expression of nuclear HDAC1, HDAC2 and HDAC3 proteins increased stepwise in benign, borderline and malignant tumors. The expression of HDAC1 and HDAC2 was correlated with Ki-67 expression and that of HDAC3 was inversely correlated with E-cadherin expression. Among the HDACs examined, only HDAC1 was associated with a poor outcome, when overexpressed. Treatment with HDAC inhibitors suppressed the proliferation of ovarian cancer cells in association with apoptosis. A specific siRNA for HDAC1 significantly reduced the proliferation of ovarian carcinoma cells via downregulation of cyclin A expression, but siRNA for HDAC3 reduced the cell migration with elevated E-cadherin expression. Our results suggested that HDAC1 plays an important role in the proliferation of ovarian cancer cells, whereas HDAC3 functions in cell adhesion and migration. Therefore, specific therapeutic approaches should be considered according to the HDAC subtypes.
Asunto(s)
Cadherinas/metabolismo , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Histona Desacetilasa 1/metabolismo , Histona Desacetilasas/metabolismo , Neoplasias Ováricas/enzimología , Western Blotting , Línea Celular Tumoral , Femenino , Histona Desacetilasa 1/genética , Histona Desacetilasas/genética , Humanos , Inmunohistoquímica , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Interferencia de ARN , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cyclin-dependent-kinase (cdk) inhibitor, p27(Kip1) (p27), has been shown to participate in progestin-induced growth suppression of normal endometrial glands. To analyse the molecular mechanisms regulating p27 protein, we examined immunohistochemical expression of the SCF(Skp2) (Skp1-Cullin-F-box protein) complex factors, i.e. Skp1, Cul1 and Skp2, and compared them with that of p27, steroid receptors and Ki-67. In normal endometrial glands, the expression of Skp2 was observed in the proliferative phase, whereas that of p27 was observed in the secretory phase. Cultured normal endometrial glandular cells showed that progesterone induced the down-regulation of Skp2 along with up-regulation of p27. In endometrial carcinomas, the inverse topological correlation between Skp2 and p27 was evident in 39/66 (59%) cases, and the expression of Skp2 showed a strong correlation with Ki-67. These findings suggest that the expression of SCF(Skp2) complex changes during the menstrual cycle in normal endometrium and the SCF(Skp2) ubiquitin-proteasome pathway may also work in endometrial carcinomas.
Asunto(s)
Carcinoma/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Adulto , Anciano , Proteínas Cullin/metabolismo , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Persona de Mediana Edad , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismoRESUMEN
To examine estrogen-induced growth mechanisms of endometrial carcinoma, we investigated the estrogen-induced activation of the mitogen-activated protein kinase (MAPK) pathway and cell cycle regulators. Estradiol (E(2)) treatment at concentrations of 10(-8) M and 10(-6) M to estrogen receptor (ER)-positive endometrial carcinoma Ishikawa cells for 24 h resulted in increased cell proliferation by 20% and 28% respectively. The E(2)-induced proliferation was associated with the activation of extracellular signal-regulated kinase (MAPK)3/1 and up-regulation of cyclin D1 and E, which were suppressed by the addition of an MAP2K inhibitor (U0126) or an ER antagonist (ICI 182 780). Then, our screening for estrogen-inducible growth factors identified that IGF1 was up-regulated remarkably by E(2). Immunoprecipitation using conditioned medium of Ishikawa cells after E(2) treatment confirmed the E(2)-induced secretion of IGF1 protein. Treatment with recombinant IGF1 stimulated cell proliferation in a dose-dependent fashion, in association with MAPK3/1 phosphorylation and up-regulation of cyclin D1 and E. These IGF1-induced responses were suppressed by treatment with MAP2K inhibitor or anti-IGF1 receptor antibody. Immunohistochemical staining confirmed the expression of activated MAPK3/1 in normal proliferative phase endometria and endometrial carcinomas, indicating the involvement of this pathway in actively proliferating endometrial tissues in vivo. These findings suggest that E(2)-induced proliferation of endometrial carcinoma cells is mediated by the MAPK3/1 pathway via autocrine stimulation of IGF1.
Asunto(s)
Ciclina D1/metabolismo , Ciclina E/metabolismo , Neoplasias Endometriales/metabolismo , Estradiol/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Comunicación Autocrina/fisiología , División Celular/fisiología , Línea Celular Tumoral , Ciclina D1/genética , Ciclina E/genética , Relación Dosis-Respuesta a Droga , Neoplasias Endometriales/patología , Endometrio/metabolismo , Endometrio/patología , Estradiol/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND: Although progestins have been used for the treatment of endometrial neoplasias, the mechanisms of progestin-induced growth suppression remain undetermined. MATERIALS AND METHODS: Immunostaining for steroid receptor coactivators (SRC-1, p300/CBP), corepressors (NCoR, SMRT) and Ki-67 in 15 neoplastic endometria before and after the treatment with medroxyprogesterone acetate (MPA) was performed. The effect of progestin on cell proliferation and cofactors expression were examined using T47D cells. RESULTS: Of the 15 cases, 10 showed good histological responses to MPA (Responder) and 5 poor responses (Non-responder). In Responders, MPA treatment resulted in reduced expression of Ki-67 by 78% (p=0.0076) along with increased NCoR expression by 158 % (p=0.0077). Progestin treatment for T47D cells resulted in up-regulation of NCoR mRNA and protein with the suppression of cell proliferation. Immunoprecipitation revealed that NCoR was bound to estrogen receptor alpha, but not to progesterone receptor in T47D cells. CONCLUSION: The up-regulation of NCoR by progestins is associated with the suppression of estrogen-induced growth of neoplastic cells.
Asunto(s)
Hiperplasia Endometrial/tratamiento farmacológico , Neoplasias Endometriales/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Progestinas/farmacología , Proteínas Represoras/metabolismo , Adulto , Western Blotting , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patología , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Histona Acetiltransferasas/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Proteínas Nucleares/genética , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Coactivador 1 de Receptor Nuclear , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba , Adulto Joven , Factores de Transcripción p300-CBP/metabolismoRESUMEN
PURPOSE: Research has revealed abnormal activation of the hedgehog pathway in human malignancies. The present study was undertaken to examine the expression and functional involvement of the hedgehog pathway in endometrial tissues. EXPERIMENTAL DESIGN: The expression of sonic hedgehog (Shh), patched (Ptch), Smoothened (Smo), and Gli1 was examined in various endometrial tissues and endometrial carcinoma cell lines. The effect of hedgehog signaling on the proliferation of endometrial carcinoma cell lines was also examined. RESULTS: The expression of Shh, Ptch, Smo, and Gli1 was very weak in normal endometrium, but was increased in endometrial hyperplasia and carcinoma stepwisely with significant differences. There was no marked difference in the expression of these molecules in carcinomas according to stages and histologic grades. Treatment with cyclopamine, a specific inhibitor of the hedgehog pathway, for endometrial carcinoma Ishikawa and HHUA cells suppressed growth by 56% and 67%, respectively, compared with the control. The addition of recombinant Shh peptide to HHUA cells enhanced their proliferation by 41%. The silencing of Gli1 using small interfering RNA (siGli1) resulted in the growth suppression and down-regulation of Ptch expression. In addition, the cyclopamine/siGli1-induced growth suppression was associated with the down-regulation of cyclins D1 and A and N-myc. No somatic mutations for ptch and smo genes were detected in the endometrial carcinoma cases examined. CONCLUSIONS: The abnormal activation of this pathway is involved in the proliferation of endometrial carcinoma cells possibly in an auto-/paracrine fashion, suggesting the possibility of the hedgehog pathway being a novel candidate for molecular targeting.
Asunto(s)
Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal/fisiología , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Análisis Mutacional de ADN , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Impaired mismatch repair (MMR) is reportedly crucial in the early stages of endometrial carcinogenesis. Although estrogen exposure is considered an important risk factor for endometrial carcinoma, the relationship between estrogen and MMR activity remains undetermined. The present study was undertaken to elucidate the effect of estrogen on MMR activity in normal and malignant endometrial cells. The expression of MMR proteins, hMLH1 and hMSH2, and its correlation with estrogen was examined using immunohistochemical and immunofluorescent techniques. The effect of estradiol (E2) on the expression of hMLH1/hMSH2 protein/mRNA and in vitro MMR activity using two types of heteroduplex (G/T mismatches, 2-base insertion-deletion loops) was examined in cultured normal endometrial glandular cells and estrogen receptor-positive endometrial carcinoma Ishikawa cells. Immunohistochemical expression of hMLH1 and hMSH2 in normal endometrial glands was positively correlated with the serum E2 levels. The expression of hMLH1/hMSH2 protein and mRNA was increased in normal endometrial glandular and Ishikawa cells by E2 treatment. In vitro MMR activity was up-regulated by E2 in both types of cell and heteroduplex. Immunofluorescent analysis demonstrated that E2 enhanced proliferation and hMLH1/hMSH2 expression in both cells; however, proliferating cells without hMLH1/hMSH2 expressions implying high-risk cells were more frequently observed under low E2 concentrations. Collectively, the E2-induced up-regulation of MMR activity in endometrial cells suggests that high estrogen levels act as an intrinsic defense against endometrial carcinogenesis, whereas the imbalance between cell growth and MMR under low E2 environment as seen at postmenopause is vulnerable to carcinogenesis.
Asunto(s)
Disparidad de Par Base/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Estrógenos/farmacología , Proteínas Adaptadoras Transductoras de Señales , Adulto , Western Blotting , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Células Cultivadas , Neoplasias Endometriales/patología , Endometrio/citología , Endometrio/efectos de los fármacos , Estrógenos/sangre , Femenino , Técnica del Anticuerpo Fluorescente Directa , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/biosíntesis , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/efectos de los fármacosRESUMEN
PURPOSE: Although several gene abnormalities have been reported in endometrial carcinoma, the genetic alterations have not fully been elucidated. Recent studies have revealed frequent activating mutations of the gene for BRAF, an effector of Ras protein in the mitogen-activated protein kinase pathway, in several malignancies. However, the prevalence and significance of BRAF mutations in endometrial carcinoma remain unclear. EXPERIMENTAL DESIGN: We examined BRAF mutations in exons 11 and 15 in 97 cases of endometrial carcinoma (endometrioid type, 78; nonendometrioid type, 19), 9 cases of atypical endometrial hyperplasia, and 20 cases of normal endometrium by direct sequencing. In addition, mutations of KRAS and p53 and the immunohistochemical expression of hMLH1 and hMSH2 were also examined. RESULTS: Of the 97 carcinomas and 9 hyperplasias, 20 (21%) and 1 (11%) had BRAF mutations, most of them at previously unreported sites. Twenty samples of normal endometrium and 21 samples of normal endometrium obtained from sites adjacent to neoplastic lesions had no BRAF mutations. There was no apparent difference in the prevalence of BRAF mutation among stages, histologic subtypes, or grades. Mutations of KRAS and p53 were found in 18 (19%) and 22 (23%) cases, and 65 (67%) and 92 (95%) cases showed positive immunostaining for hMLH1 and hMSH2, respectively. BRAF mutation was more frequently found in hMLH1-negative cases (12 of 32, 41%) than in hMLH1-positive cases (7 of 65, 11%; P = 0.008), suggesting that it is associated with an abnormal mismatch repair function. CONCLUSIONS: These findings suggest that mutations of the BRAF gene are partly involved in the malignant transformation of the endometrium.
Asunto(s)
Hiperplasia Endometrial/genética , Neoplasias Endometriales/genética , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Proteínas Portadoras , Análisis Mutacional de ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endometrio/fisiología , Femenino , Genes ras/fisiología , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Aurora kinases such as Aurora A and Aurora B are key regulators of mitosis and have been reported to be overexpressed in various malignancies. However, the expression and localization of Aurora kinases in normal and neoplastic endometrial tissues remain undetermined. In the present study, immunohistochemical expression of Aurora A and B was examined in 40 normal, 30 hyperplastic, and 73 malignant endometria. The data were compared with the expression of Ki-67 and patient survivals. The expression of Aurora A and B at protein and messenger RNA levels was also examined using Western blotting and the reverse transcriptase polymerase chain reaction. The expression of Aurora A in normal endometrium was observed mainly in the proliferative phase and was decreased in the secretory phase. The Aurora A expression was significantly increased in carcinomas compared with normal proliferative endometrium; however, there was no correlation of Aurora A expression with Ki-67 expression or patient survival. The expression of Aurora B in normal endometrium was significantly higher in the proliferative phase than in the secretory phase. In endometrial carcinomas, the expression of Aurora B was correlated with Ki-67 expression and was significantly increased in high-grade tumors. In addition, patients with Aurora B-positive carcinoma showed poor prognosis compared with those with Aurora B-negative carcinoma (P = .0135). Accordingly, the present study indicates the aberrant expression of Aurora A and Aurora B in endometrial carcinomas and the clinical importance of Aurora B expression in relationship to patient prognosis.
Asunto(s)
Adenocarcinoma/enzimología , Carcinoma de Células Escamosas/enzimología , Hiperplasia Endometrial/enzimología , Neoplasias Endometriales/enzimología , Endometrio/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adulto , Anciano , Aurora Quinasa B , Aurora Quinasas , Biomarcadores de Tumor , Western Blotting , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Proliferación Celular , Hiperplasia Endometrial/patología , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Endometrio/anatomía & histología , Endometrio/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Antígeno Ki-67/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Tasa de SupervivenciaRESUMEN
We previously reported the overexpression of cyclins in uterine cervical carcinoma; however, their clinicopathological significance remained undetermined. In the present study, we examined the immunohistochemical expression of cyclins (D1, E, A, B1), p53 and Ki-67 in squamous cell carcinoma (stage Ib+II; 80 cases, stage III+IV; 23 cases). Correlations between the expression of cyclins and clinicopathological parameters and patient survival were statistically evaluated. The results indicated that in the normal squamous epithelium, the expression of cyclins and Ki-67 was sporadically observed in the parabasal layer. Of the 103 cervical carcinomas, overexpression of cyclins D1, E, A, B1 and p53 was observed in 13 (13%), 23 (22%), 25 (24%), 18 (18%) and 23 (22%) cases, respectively, with a slight predominance in advanced stage tumors. The expression of cyclin D1, E, A and p53 significantly correlated with that of Ki-67 (Spearman's rank correlation). Univariate and multivariate analyses revealed that lymph node metastasis and cyclin A overexpression were independent prognostic factors for unfavorable outcomes in stage Ib+II patients. These findings suggest that the overexpression of various cyclins is involved in the acquisition of the vigorous growth potential of cervical carcinoma cells, and that cyclin A is an independent prognosticator of cervical carcinoma in early stages.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/metabolismo , Ciclina A/biosíntesis , Antígeno Ki-67/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Neoplasias del Cuello Uterino/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Análisis de Supervivencia , Neoplasias del Cuello Uterino/mortalidad , Neoplasias del Cuello Uterino/patologíaRESUMEN
To further elucidate the significance of p53 mutation in endometrial carcinoma, we investigated it in endometrioid-type endometrial carcinomas showing intratumoral heterogeneous p53 expression. In addition, we also examined the correlation of p53 mutation and cyclin A expression, because we previously reported a topological correlation between the expression of p53 and cyclin A. The p53 mutation in exons 5-8 in 54 cases of endometrial carcinoma showing immunohistochemical expression of p53 was examined using microdissected tissue DNAs. Of the 54 p53-positive endometrial carcinomas, 23 (43%) had p53 mutation with a tendency in histologically higher grade tumors. Ten of the 54 showed a heterogeneous p53 expression, and in 9 of the 10 cases, p53 mutation was present only in p53-positive sites, which were often found in histologically less differentiated areas with elevated Ki-67 in the same tumor. Cyclin A expression was topologically observed in p53-positive areas; however, it was noted in both tumors with (12/23, 52%) and without (18/31, 58%) p53 mutation. These results suggest that p53 mutation is a late event and plays an important role in the acquisition of malignant potentials in endometrioid-type endometrial adenocarcinomas. Unexpectedly, accumulation of the p53 protein itself may be important in cyclin A overexpression.
Asunto(s)
Carcinoma Endometrioide/metabolismo , Ciclina A/metabolismo , Neoplasias Endometriales/metabolismo , Genes p53 , Antígeno Ki-67/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patología , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Alineación de SecuenciaRESUMEN
Catechol estrogens, such as 4-hydroxyestradiol (4-OHE2), are estrogen metabolites that form DNA adducts and may induce mutations and subsequent cell transformation in mammary cells; however, little is known about their roles in endometrial carcinogenesis. Furthermore, it remains unclear whether 4-OHE2 is able to induce DNA damage on specific genes involved in carcinogenesis or a 'pro'-mutation status such as microsatellite instability (MSI). Therefore, we modified terminal transferase-dependent PCR by the application of a capillary sequencer to detect DNA damage at the single base level. Using this method, we demonstrated that 4-OHE2 directly induced DNA damage on codon 130/131 in exon 5 of PTEN, which is a mutation hot spot for PTEN in endometrial carcinoma. Whereas, both estradiol and 4-OHE2 treatment did not affect MSI status in immortalized endometrial glandular cells. 4-OHE2 might contribute to endometrial carcinogenesis by inducing PTEN mutation on codon 130/131.
Asunto(s)
Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Células Epiteliales/efectos de los fármacos , Estrógenos de Catecol/farmacología , Fosfohidrolasa PTEN/genética , Mutación Puntual , Secuencia de Bases , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Codón , Aductos de ADN/química , Daño del ADN , Análisis Mutacional de ADN , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Endometrio/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Estrógenos de Catecol/química , Exones , Femenino , Expresión Génica , Humanos , Inestabilidad de Microsatélites/efectos de los fármacos , Datos de Secuencia Molecular , Fosfohidrolasa PTEN/metabolismo , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Lobular endocervical glandular hyperplasia (LEGH) is a benign proliferative disease of cervical glands. Although histological resemblance of minimal deviation adenocarcinoma (MDA) to LEGH and frequent association of LEGH with MDA have been reported, it still remains unclear whether LEGH is a precancerous lesion of MDA. The present study was undertaken to examine the pathogenetic relationship between LEGH and MDA using a clonality analysis and mutational analyses of the STK11 gene, of which mutations have been reported in MDA. Of nine cases of LEGH only, four were polyclonal and five were monoclonal in composition. Of six LEGH lesions associated with MDA or adenocarcinoma, two were polyclonal and four were monoclonal. In cases of MDA or adenocarcinoma coexisting with LEGH, the patterns of X chromosome inactivation in malignant lesions were identical to those in coexisting LEGH lesions. A mutation of STK11 was only identified in one MDA, but not in LEGH. These results indicate that a subset of LEGH may be a precursor to malignant tumors including MDA and that a mutation of STK11 may be involved in progression of LEGH to MDA.
Asunto(s)
Adenocarcinoma/patología , Cuello del Útero/patología , Mutación , Proteínas Serina-Treonina Quinasas/genética , Neoplasias del Cuello Uterino/patología , Quinasas de la Proteína-Quinasa Activada por el AMP , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Células Clonales , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Progresión de la Enfermedad , Femenino , Humanos , Hiperplasia , Persona de Mediana Edad , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Displasia del Cuello del ÚteroRESUMEN
Inflammation in the ovary, including ovulation and pelvic inflammatory disease, has been proposed to play a role in the pathogenesis of ovarian cancer. Endometriotic lesions trigger a local inflammatory reaction and have been reported to be associated with an increased risk of epithelial ovarian cancer. However, the precise molecular mechanisms of ovarian cancer arising from endometriosis are still to be elucidated. To clarify the involvement of mismatch repair (MMR) abnormalities in the inflammation-associated malignant transformation of endometriosis, the immunohistochemical expression of mismatch repair proteins (human mutL homolog 1 [hMLH1] and human mutS homolog 2 [hMSH2]) was examined in 27 cases of ovarian endometriosis, 25 cases of ovarian carcinoma accompanied by endometriosis, and 39 cases of solitary ovarian carcinoma. In addition, the relationship between mismatch repair abnormalities including the microsatellite instability, PTEN (phosphatase and tensin homolog) mutation, and clinicopathologic parameters was analyzed. The expression of mismatch repair proteins was stepwisely decreased in endometriosis, ovarian carcinoma accompanied by endometriosis, and ovarian carcinoma. Tumors harboring multiple microsatellite instability (high-frequency microsatellite instability [MSI-H]) were detected in 4 (14.8%) of 27 cases of endometriosis and 7 (30.4%) of 23 cases of ovarian carcinomas. The frequency of PTEN mutations was higher in MSI-H cases than in microsatellite instability-stable (MSI-S) cases. In 2 cases of ovarian carcinoma accompanied by endometriosis, the decreased expression of mismatch repair proteins and MSI-H was observed in both the endometriosis and carcinoma lesions. Clinicopathologically, the MSI-H cases were associated with elevated serum levels of C-reactive protein and higher white blood cell counts. These findings suggest that mismatch repair abnormalities might be involved in the malignant transformation of ovarian endometriosis and that inflammation induces mismatch repair abnormalities during ovarian carcinogenesis arising from endometriosis.
Asunto(s)
Transformación Celular Neoplásica , Cistadenocarcinoma Seroso/patología , Endometriosis/patología , Inestabilidad de Microsatélites , Neoplasias Ováricas/patología , Enfermedad Inflamatoria Pélvica/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias Encefálicas , Neoplasias Colorrectales , Cistadenocarcinoma Seroso/complicaciones , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Progresión de la Enfermedad , Endometriosis/complicaciones , Endometriosis/genética , Endometriosis/metabolismo , Femenino , Humanos , Homólogo 1 de la Proteína MutL , Proteínas MutL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndromes Neoplásicos Hereditarios , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Enfermedad Inflamatoria Pélvica/complicaciones , Enfermedad Inflamatoria Pélvica/genética , Enfermedad Inflamatoria Pélvica/metabolismo , Mutación PuntualRESUMEN
Endometrial carcinoma often arises from normal endometrial glandular cells via a precursor, atypical endometrial hyperplasia. However, the genetic changes involved in this carcinogenetic process are not fully understood. Differentially expressed genes were selected from glandular cells of normal proliferative-phase endometria, atypical endometrial hyperplasia, and endometrial carcinoma using laser-captured microdissection and microarray. The microarray analysis revealed a total of 51 genes to be up-regulated and 23 genes to be down-regulated in neoplastic endometrial epithelia. We focused on lipocalin2 (LCN2), which showed the largest magnitude of up-regulation. Immunostaining for lipocalin2 confirmed a stepwise increase in its expression in endometrial hyperplasia and carcinoma. In addition, elevated expression of lipocalin2 was correlated with the poor outcome of endometrial carcinoma patients. The subcellular distribution of lipocalin2 was both cytoplasmic and nuclear, despite reports that lipocalin2 is a secretory protein. Treatment of endometrial carcinoma cells with 5-azacytidine increased the expression of lipocalin2, suggesting the expression to be controlled by methylation of the promoter. The forced expression of lipocalin2 resulted in the enhanced cell proliferation and invasion in vitro. The expression of lipocalin2 increased with the endometrial carcinogenesis, and accumulation of the protein conferred biological aggressiveness to endometrial carcinoma cells. These results suggest lipocalin2 to be a novel target in the treatment of endometrial carcinoma.
Asunto(s)
Proteínas de Fase Aguda/genética , Neoplasias Endometriales/genética , Endometrio/metabolismo , Lipocalinas/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Hiperplasia Endometrial/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Rayos Láser , Lipocalina 2 , Análisis por Micromatrices , Microdisección , Persona de Mediana EdadRESUMEN
AIMS: The authors previously reported the expression of keratan sulfate (KS), a glycosaminoglycan, in the epithelium of normal and neoplastic endometria. The aim of this study was to evaluate its potential use as a diagnostic marker, and the expression of KS was investigated in other human epithelial tissues. METHODS: Expression was examined immunohistochemically using 102 samples of normal epithelia and 110 samples of carcinomas from the female genital tract (FGT; cervix, endometrium, ovary, fallopian tube), digestive organs (gastrointestinal tract, pancreas, liver), urinary tract, lung, mammary gland, thyroid and mesothelium. RESULTS: In normal tissues, KS was consistently detected in the FGT and ectopic endometrium (25/26), but was not found in the digestive organs (1/42) and urinary tract (0/6), and was only partly detected in the lung (7/10), mammary gland (3/9) and thyroid (4/4). In malignant tissues, KS was consistently observed in carcinomas of the endometrium, ovary and fallopian tube (29/32), and was partly detected in carcinomas of the lung, mammary gland, thyroid, pancreas and mesothelium, but was absent in carcinomas of the gastrointestinal tract (0/17), liver (0/5) and urinary tract (0/11). Among carcinomas of the FGT, digestive organs and urinary tract, KS positivity suggested the possibility of FGT carcinomas, with 79.5% (31/39) sensitivity and 92.9% (39/42) specificity. CONCLUSIONS: KS is a potentially useful marker for the supportive diagnosis of the primary site of metastatic carcinomas or unknown primary carcinomas, especially in the abdominal cavity.