RESUMEN
BACKGROUND: Breast cancer is the common malignancy with high morbidity and mortality in women. S-phase kinase-associated protein 2 (Skp2) has been characterized to play an oncogenic role in the breast carcinogenesis and progression. Therefore, inactivation of Skp2 in breast cancer might be a novel approach for fighting breast malignancy. A natural compound diosgenin has been reported to exert anticancer activity in a variety of human cancers. However, the underlying mechanism has not been fully determined. METHODS: In this study, we aim to explore whether diosgenin performed antitumor activity via inhibition of Skp2 in breast cancer cells using several methods including MTT, Transwell invasion assay, RT-PCR, western blotting, and transfection. RESULTS: We found that diosgenin inhibited cell viability and stimulated apoptosis. Moreover, we found that diosgenin reduced cell invasion in breast cancer cells. Furthermore, diosgenin inhibited the expression of Skp2 in breast cancer cells. Notably, diosgenin reduced cell viability and motility and induced apoptosis via suppression of Skp2 in breast cancer cells. CONCLUSION: Our findings revealed that diosgenin could be a potential inhibitor of Skp2 for treating breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Diosgenina/farmacología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/genéticaRESUMEN
Down-regulation of cyclin-dependent kinase inhibitor protein p27, due to enhanced degradation, is frequently observed in various cancers. The ubiquitin ligases that mediate this degradation have been identified as S-phase kinase-associated protein-2 (Skp2), Kip1 ubiquitylation-promoting complex (KPC), and p53-inducible protein with RING-H2 domain (Pirh2) as well. We investigated the correlation among expression of these 3 ligases and p27 status in surgical specimens of human lung carcinomas by immunohistochemical analysis. Among 93 cases, expressions of p27, Skp2, KPC, and Pirh2 were found in 89.2%, 59.1%, 59.1%, and 67.7%, respectively. Down-regulation of p27 in cancer cells was frequently observed in adenocarcinoma (AC) and squamous cell carcinoma (SCC), but not in small cell carcinoma (SmCC). Overexpression of ubiquitin ligases was variously observed among histological types: Skp2 was more frequently observed in SCC and SmCC, KPC in SCC and Pirh2 in AC, followed by SCC. Several novel findings were obtained: (i) cytoplasmic p27 was observed in 8.6%, most frequently in SCC (13.3%), and correlated with nodal metastasis (P=.0044), (ii) significant inverse correlation between nuclear p27 and Pirh2 expression was observed by statistical analysis and at the cellular level, and (iii) cytoplasmic Pirh2 and total (cytoplasmic and/or nuclear) Pirh2 were significantly correlated with the nodal status (P=.0225, 0.0314), the pathological stage (P=.0213, 0.0475) and recurrence-free survival (P=.0194, 0.0482, respectively) in AC. Altogether, our data suggests that p27 and its cognate ubiquitin ligases are specifically involved in the clinical profiles, and thus, molecular targeting of these ubiquitin ligases, in particular, Pirh2, may have therapeutic value for human lung carcinomas.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma/enzimología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Neoplasias Pulmonares/enzimología , Proteínas Quinasas Asociadas a Fase-S/análisis , Ubiquitina-Proteína Ligasas/análisis , Carcinoma/mortalidad , Carcinoma/secundario , Carcinoma/cirugía , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Metástasis Linfática , Masculino , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Factores de Riesgo , Fumar/efectos adversos , Factores de Tiempo , Resultado del TratamientoRESUMEN
Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and ß-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Lactonas/farmacología , Neoplasias Pulmonares/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Sesquiterpenos/farmacología , Anciano , Animales , Antineoplásicos/química , Western Blotting , Supervivencia Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoprecipitación , Lactonas/química , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , ARN Interferente Pequeño , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Sesquiterpenos/química , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To construct a eukaryotic expression vector for the human gene S-phase kinase-associated protein 2 (Skp2) with a FLAG tag (pcDNA3-FLAG-Skp2) and detect the effect of Skp2 over-expression on the cell growth and cell cycle in MCF-7 breast cancer cells. METHODS: Skp2 was amplified from MDA-MB-231 breast cancer cells by reverse-transcription polymerase chain reaction (RT-PCR) and then used to construct the eukaryotic expression vector pcDNA3-FLAG-Skp2. Integration of Skp2 into the vector was confirmed via restriction digest and sequencing; The pcDNA3-FLAG-Skp2 was then transfected into MCF-7 breast cancer cells. Expression of Skp2 protein was verified by Western blotting. Cell growth was assessed by Alamar blue proliferation assay, cell cycle analysis was carried out by flow cytometry. RESULTS: The PCR amplified fragment was matched up with the anticipated result and its sequence was the same as the data published on GenBank, indicating that the recombinant plasmid pcDNA3-FLAG-Skp2 was constructed successfully. Western blotting revealed that the expression of Skp2 protein was markedly up-regulated in the pcDNA3-FLAG-Skp2 transfected MCF-7 cells at 48 hours. Furthermore, cell growth was significantly promoted in Skp2 over-expressed MCF-7 cells, and the cell count in S phase were also raised. CONCLUSION: The recombinant eukaryotic expression vector pcDNA3-FLAG-Skp2 has been constructed and expressed in MCF-7 breast cancer cells successfully. Over-expression of Skp2 resulted in the increased cell growth and number of S phase cells in Skp2 transfected MCF-7 cells.
Asunto(s)
Neoplasias de la Mama/patología , Fase S , Proteínas Quinasas Asociadas a Fase-S/fisiología , Proliferación Celular , Femenino , Humanos , Células MCF-7 , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/genética , TransfecciónRESUMEN
OBJECTIVE: The aim of this study was to analyze the immunohistochemical expression of Skp2 protein in 38 oral nevi and 11 primary oral melanomas. STUDY DESIGN: Expression of this ubiquitin protein was evaluated by immunohistochemistry in 49 oral melanocytic lesions, including 38 intramucosal nevi and 11 primary oral melanomas. The labeling index (LI) was assessed considering the percentage of cells expressing nuclear positivity out of the total number of cells, counting 1000 cells per slide. RESULTS: Skp2 protein was rarely expressed in intramucosal nevi, in contrast to oral melanomas, which showed high levels of this protein. CONCLUSION: These results indicate that Skp2 protein may play a role in the development and progression of oral melanomas, and it also could be useful as an immunohistochemical marker for differential diagnosis of oral benign and malignant melanocytic lesions.
Asunto(s)
Melanoma/metabolismo , Neoplasias de la Boca/metabolismo , Nevo/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Adolescente , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Melanoma/química , Persona de Mediana Edad , Neoplasias de la Boca/química , Nevo/química , Proteínas Quinasas Asociadas a Fase-S/análisis , Adulto JovenRESUMEN
PURPOSE: The purpose of this study is to clarify the prognostic significance of expression of Jab1, p16, p21, p62, Ki67 and Skp2 in soft tissue sarcomas (STS). Optimised treatment of STS requires better identification of high risk patients who will benefit from adjuvant therapy. The prognostic significance of Jab1, p16, p21, p62, Ki67 and Skp2 in STS has not been sufficiently investigated. EXPERIMENTAL DESIGN: Tissue microarrays from 193 STS patients were constructed from duplicate cores of viable and representative neoplastic tumor areas. Immunohistochemistry was used to evaluate the expression of Jab1, p16, p21, p62, Ki67 and Skp2. RESULTS: In univariate analyses, high tumor expression of Ki67 (Pâ=â0.007) and Skp2 (Pâ=â0.050) correlated with shorter disease-specific survival (DSS). In subgroup analysis, a correlation between Skp2 and DSS was seen in patients with malignancy grade 1 or 2 (Pâ=â0.027), tumor size >5 cm (Pâ=â0.018), no radiotherapy given (Pâ=â0.029) and no chemotherapy given (Pâ=â0.017). No such relationship was apparent for Jab1, p16, p21 and p62; but p62 showed a positive correlation to malignancy grade (Pâ=â0.019). Ki67 was strongly positively correlated to malignancy grade (Pâ=â0.001). In multivariate analyses, Skp2 was an independent negative prognostic factor for DSS in women (Pâ=â0.009) and in patients without administered chemotherapy or radiotherapy (Pâ=â0.026). CONCLUSIONS: Increased expression of Skp2 in patients with soft tissue sarcomas is an independent negative prognostic factor for disease-specific survival in women and in patients not administered chemotherapy or radiotherapy. Besides, further studies are warranted to explore if adjuvant chemotherapy or radiotherapy improve the poor prognosis of STS with high Skp2 expression.
Asunto(s)
Biomarcadores de Tumor/análisis , Sarcoma/metabolismo , Sarcoma/mortalidad , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Complejo del Señalosoma COP9 , Preescolar , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/análisis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Antígeno Ki-67/análisis , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Pronóstico , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Sarcoma/patología , Sarcoma/terapia , Proteína Sequestosoma-1 , Análisis de Matrices Tisulares , Adulto JovenRESUMEN
The forkhead box proteins (FOXO proteins) comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation, differentiation and longevity. Recently, ubiquitination and proteasome degradation of FOXO3a have been reported. In this study, we investigated the role of FOXO3a and Skp2 in human ovarian cancer. We detected the expression of FOXO3a and Skp2 in ovarian cancer by immunohistochemistry (IHC) and analyzed the relationship of FOXO3a and Skp2 with clinicopathological parameters, including prognosis. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 46 specimens in vivo. We found that the expression of FOXO3a was negatively related to Skp2 expression (r = -0.743; p < 0.05) and FOXO3a expression correlates significantly with disease stage (p = 0.007) and lymph node (p = 0.009) while Skp2 expression correlates significantly with age (p = 0.040), disease stage (p = 0.003) and lymph node (p = 0.019). Kaplan-Meier analysis revealed that survival curves of low versus high expressers of FOXO3a and Skp2 showed a highly significant separation in ovarian cancer (p < 0.01). FOXO3a and Skp2 may be considered to be important prognoses in human ovarian cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Factores de Transcripción Forkhead/biosíntesis , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Femenino , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/análisis , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Ováricas/mortalidad , Pronóstico , Proteínas Quinasas Asociadas a Fase-S/análisisRESUMEN
The mammalian target of rapamycin (mTOR) plays a role in controlling malignant cellular growth. mTOR inhibitors, including rapamycin (sirolimus), are currently being evaluated in cancer trials. However, a significant number of tumors are rapamycin resistant. In this study, we report that the ability of rapamycin to downregulate Skp2, a subunit of the ubiquitin protein ligase complex, identifies tumors that are sensitive to rapamycin. RNA interference (RNAi)-mediated silencing of Skp2 in human tumor cells increased their sensitivity to rapamycin in vitro and inhibited the growth of tumor xenografts in vivo. Our findings suggest that Skp2 levels are a key determinant of antitumor responses to mTOR inhibitors, highlighting a potentially important pharmacogenomic marker to predict sensitivity to rapamycin as well as Skp2 silencing strategies for therapeutic purposes.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Proteínas Quinasas Asociadas a Fase-S/fisiología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Células HeLa , Humanos , Ratones , Fosfohidrolasa PTEN/fisiología , Fosforilación , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The TGFß signalling pathway is a growth inhibitor system that operates in both normal and tumour cells. Alterations to components of this pathway, including SMAD4, result in resistance to growth inhibition and uncontrolled proliferation. The aim of this study was to analyse the relationships between SMAD4, a key protein in the growth-inhibiting TGFß pathway; cell proliferation proteins Ki67, p27 and S-phase kinase-associated protein 2 (SKP2); and mismatch repair (MMR) proteins as well as prognostic indicators in colorectal adenocarcinomas. A series of 230 sporadic colorectal adenocarcinomas were studied using tissue microarrays by immunohistochemistry for SMAD4, Ki67, p27, SKP2 and MMR protein (hMLH1, hMSH2 and hMSH6) expression. Protein expression was analysed with respect to pathological prognostic criteria. Loss of SMAD4 nuclear expression (27/230, 12%) correlated with the presence of lymph node metastases, MMR protein expression and the absence of p27 in tumour cells (p = 0.04, p = 0.08 and p = 0.03, respectively). A high Ki67 index did not correlate with SMAD4 expression; however, it did correlate with moderate or poor histological differentiation, SKP2 expression and aberrant or absent MMR protein expression (p = 0.02, p < 0.01 and p < 0.01, respectively). In conclusion, the results of our study suggest that the loss of SMAD4, occurring in 12% of colorectal adenocarcinomas, correlated with the presence of lymph node metastases and absence of p27 expression but not with high cellular proliferation. However, high proliferation correlated with SKP2 and aberrant MMR protein expression. Although the advantage of immunohistochemistry is high throughput, our results allow only an initial evaluation, and subsequent studies, including genetic analyses, are required.
Asunto(s)
Adenocarcinoma/patología , Neoplasias Colorrectales/patología , Proteína Smad4/fisiología , Proteínas Adaptadoras Transductoras de Señales/análisis , Adenocarcinoma/química , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Neoplasias Colorrectales/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/análisis , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteína Smad4/análisis , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Gallic acid (GA), 3,4,5-trihydroxybenzoic acid, is a natural polyphenolic acid and widely found in gallnuts, tea leaves and various fruits. Previous studies have shown that GA possesses anti-inflammatory, antiallergic and anticarcinogenic activity. In the present study, we aim to investigate the antitumor effects of GA on breast cancer cell. Our results revealed that GA treatment significantly reduced the cell growth of human breast cancer cell MCF-7 in a dose-dependent manner. Further flow cytometric analysis showed that GA induced significant G2/M phase arrest but slightly affected the population of sub-G1MCF-7 cells. Therefore, levels of cyclins, cyclin-dependent kinases (CDKs), and their regulatory proteins involved in S-G2/M transition were investigated. Our findings revealed that levels of cyclin A, CDK2, cyclin B1 and cdc2/CDK1 were diminished; in contrast, levels of the negative regulators p27(Kip1) and p21(Cip1) were increased by GA treatment. Additionally, Skp2, a specific ubiquitin E3 ligase for polyubiquitination of p27(Kip1) was reduced by GA treatment. Further investigation showed that GA attenuated Skp2-p27(Kip1) association and diminished polyubiquitination of p27(Kip1) in MCF-7 cells. Moreover, knockdown of p27(Kip1) but not p21(Cip1) significantly alleviated GA-induced accumulation of G2/M phase. These findings indicate that GA may upregulate p27(Kip1) level via disruption of p27(Kip1)/Skp2 association and the consequent degradation of p27(Kip1) by proteosome, leading to G2/M phase arrest of MCF-7 cell. It is suggested that GA should be beneficial to treatment of breast cancer and p27(Kip1)-deficient carcinomas.
Asunto(s)
Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G2/efectos de los fármacos , Ácido Gálico/farmacología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Quinasas Ciclina-Dependientes/análisis , Ciclinas/análisis , Humanos , Proteínas Quinasas Asociadas a Fase-S/análisisRESUMEN
BACKGROUND: Aberrant activations of Wnt and Notch signaling pathways are individually reported to be associated with the pathogenesis of non-small-cell lung cancer (NSCLC). However, the data about the cross talk between the two signaling pathways are still limited. To elucidate potential Wnt/Notch cross talk within NSCLC, we examined the impact of Notch3 activity on LiCl-induced cell cycle changes. METHODS: The lung cancer cell lines were treated with LiCl, a Wnt activator, in the absence or presence of Notch3-siRNA. Cell cycles and the expression of the regulators of cell cycle, c-MYC, p21 and Skp2 (S phase kinase-associated protein 2) were measured after treatment. RESULTS: The treatment with LiCl increased the percent of cells at S phase and G phase and the expression of c-MYC and Skp2 and decreased the expression of p21. Moreover, the expression of Notch3 and its down-stream genes, HES-1 and HEYL, was up-regulated by LiCl. Notch3-siRNA weakened the effect of LiCl on the cell cycle and resulted in attenuation of the LiCl-induced increment of c-MYC and Skp2 and the LiCl-induced decrement of p21. CONCLUSIONS: These data suggest that Notch3 activation cooperatively takes part in the LiCl-induced cell cycle changes, at least partially, associated with c-MYC, Skp2 and p21.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Cloruro de Litio/farmacología , ARN Interferente Pequeño/genética , Receptores Notch/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/análisis , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas Proto-Oncogénicas c-myc/análisis , Receptor Notch3 , Receptores Notch/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Fase-S/análisisRESUMEN
F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G(0) and during the G(1) phase of the cell cycle, Skp2 is degraded via the APC/C(Cdh1) ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G(0)/G(1) state. APC/C(Cdh1) binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCF(Skp2) ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.
Asunto(s)
Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Serina/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Línea Celular , Proteínas Cullin/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G1 , Humanos , Mutación , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fase de Descanso del Ciclo Celular , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Skp2 is the substrate binding subunit of the SCF(Skp2) ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APC(Cdh1), which targets Skp2 for degradation in early G(1) and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCF(Skp2) ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.
Asunto(s)
Proteínas Quinasas Asociadas a Fase-S/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Fase G1 , Humanos , Fosforilación , Fase S , Proteínas Quinasas Asociadas a Fase-S/análisis , Serina/metabolismoAsunto(s)
Biomarcadores de Tumor/análisis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Linfoma de Células B Grandes Difuso/diagnóstico , Proteínas Quinasas Asociadas a Fase-S/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antineoplásicos/uso terapéutico , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/mortalidad , Masculino , Ratones , Persona de Mediana Edad , Pronóstico , Rituximab , Tasa de Supervivencia , Adulto JovenRESUMEN
S-phase kinase protein 2 (SKP2), an F-box protein, targets cell-cycle regulators including cycle-dependent kinase inhibitor p27KiP1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancer cells and has been implicated in oncogenesis; however, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in a large series (301) of DLBCL patient samples and a panel of DLBCL cell lines. Using immunohistochemistry, SKP2 was detected in 41.6% of DLBCL tumours and was inversely associated with p27Kip1 protein level. The DLBCL subset with high SKP2 and low p27Kip1 showed a strong correlation with the proliferating index marker Ki-67 (p < 0.0001) and also with the germinal centre phenotype (p = 0.0147). Treatment of DLBCL cell lines with bortezomib or expression of SKP2-specific siRNA causes down-regulation of SKP2 and accumulation of p27Kip1, leading to suppression of growth by inducing apoptosis. Furthermore, treatment of DLBCL cells with bortezomib causes apoptosis via involving the mitochondrial pathway and activation of caspases. Finally, treatment of DLBCL cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Altogether, these results suggest that SKP2 and the ubiquitin-proteasome pathway may be a potential target for therapeutic intervention in DLBCL.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Quinasas Asociadas a Fase-S/genética , Apoptosis/efectos de los fármacos , Ácidos Borónicos/uso terapéutico , Bortezomib , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Inhibidores de Proteasas/uso terapéutico , Pirazinas/uso terapéutico , ARN Interferente Pequeño/farmacología , Proteínas Quinasas Asociadas a Fase-S/análisisRESUMEN
BACKGROUND: Low p27 and high Skp2 immunoreactivity are associated with a poor prognosis and other poor prognostic features including resistant phenotypes and antiestrogen drug resistance. We investigated these proteins in two International Breast Cancer Study Group trials studying node-negative early breast cancer. PATIENTS AND METHODS: Trial VIII compared chemotherapy followed by goserelin with either modality alone in premenopausal patients. Trial IX compared chemotherapy followed by tamoxifen with tamoxifen alone in postmenopausal patients. Central Pathology Office assessed p27 and Skp2 expression in the primary tumor by immunohistochemistry among 1631 (60%) trial patients. RESULTS: p27 and Skp2 were inversely related; 13% of tumors expressed low p27 and high Skp2. Low p27 and high Skp2 were associated with unfavorable prognostic factors including larger size and higher grade tumors, absence of estrogen receptor and progesterone receptor, human epidermal growth factor receptor 2 overexpression and high Ki-67 (each P < 0.05). Low p27 and high Skp2 were not associated with disease-free survival (P = 0.42 and P = 0.48, respectively). The relative effects of chemo-endocrine versus endocrine therapy were similar regardless of p27 or Skp2. CONCLUSIONS: We confirm the association of low p27 and high Skp2 with other poor prognostic features, but found no predictive or prognostic value, and therefore do not recommend routine determination of p27 and Skp2 for node-negative breast cancer.
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Antineoplásicos Hormonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Neoplasias de la Mama/tratamiento farmacológico , Antígeno Nuclear de Célula en Proliferación/análisis , Proteínas Quinasas Asociadas a Fase-S/análisis , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/patología , Ciclofosfamida/administración & dosificación , Supervivencia sin Enfermedad , Femenino , Fluorouracilo/administración & dosificación , Goserelina/administración & dosificación , Humanos , Inmunohistoquímica , Metotrexato/administración & dosificación , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Tamoxifeno/administración & dosificación , Resultado del TratamientoRESUMEN
The Cul-1 protein is the scaffold element of SCF complexes that are involved in the proteasomal degradation of numerous proteins regulating cell cycle progression. Owing to this central role in cell growth control, aberrant expression of the components of SCF is thought to play a role during tumourigenesis. Nothing is known about Cul-1 expression in human tumours. In this study, we have analysed its status in a series of 128 human lung carcinomas, comprising 50 non-small cell lung cancers (NSCLCs; 29 squamous cell carcinomas and 21 adenocarcinomas) and 78 neuroendocrine (NE) lung tumours (24 typical and atypical carcinoids, 19 large cell NE carcinomas and 35 small cell lung carcinomas), using immunohistochemistry. We report for the first time an altered pattern of Cul-1 expression in human tumours; indeed, we show that Cul-1 expression is up-regulated in 40% (51/128) of all lung tumours as compared to normal lung tissues, including 34% (17/50), 75% (18/24) and 30% (16/54) of NSCLCs, carcinoids and high grade neuroendocrine lung carcinomas, respectively. Furthermore, we demonstrate that high levels of Cul-1 protein are associated with a low KI67 proliferative index (p = 0.005) and with a decrease in the cyclin E oncoprotein (p = 0.0003), one of the major targets of SCF complexes. These data suggest that up-regulation of Cul-1 could protect cells from hyperproliferative signals through cyclin E down-regulation. Cul-1 is modified by neddylation, a post-translational modification that grafts ubiquitin-like Nedd8/Rub1 residues and controls Cul-1 activity. We also provide evidence that neddylated forms of Cul-1 are specifically expressed in high-grade NE lung tumours and are associated with down-regulation of the Cul-1 inhibitor CAND1 (p = 0.03) and a high level of cyclin E (p = 0.0002). These data support the notion that alterations in the Cul-1 neddylation/deneddylation pathway could contribute to the development of these highly aggressive lung tumours.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Ciclina E/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Adenocarcinoma/química , Adenocarcinoma/metabolismo , Tumor Carcinoide/química , Tumor Carcinoide/metabolismo , Carcinoma Neuroendocrino/química , Carcinoma Neuroendocrino/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ciclo Celular/análisis , Proteínas Cullin/análisis , Ciclina E/análisis , Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Neoplasias Pulmonares/química , Proteína NEDD8 , Estadificación de Neoplasias , Proteínas Oncogénicas/análisis , Procesamiento Proteico-Postraduccional , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Estadísticas no Paramétricas , Factores de Transcripción/análisis , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Loss of expression of the cyclin-dependent kinase inhibitor p27 through enhanced protein degradation frequently occurs in human cancer. Degradation of p27 requires ubiquitination by the S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of Skp1-Cullin-F-box protein ubiquitin ligases. In the present study, we have investigated the role of Skp2 in human thyroid tumours. Immunohistochemistry analysis showed that Skp2 was overexpressed significantly in thyroid carcinomas (26 out of 51) compared with goitres (0 out of 12, P<0.001) or adenomas (1 out of 10, P<0.05), and that high Skp2 expression was detected more often in anaplastic thyroid (ATC; 83%, n=12) than follicular thyroid (FTC; 40%, n=20) or papillary thyroid (PTC; 42%, n=19) carcinomas (P<0.05). Thyroid cancer cell lines and tissues with high levels of Skp2 protein presented high p27 degradation activity and there was an inverse correlation between Skp2 and p27 expression in thyroid cancer tissues (n=68; P<0.05). In most cases, the observed overexpression of Skp2 protein was paralleled by an increase in the levels of Skp2 mRNA, and we observed Skp2 gene amplification at 5p13 in 2 out of 6 cell lines and in 9 out of 23 primary tumours (six out of eight ATCs, two out of nine PTCs and one out of six FTCs) using Q-PCR and/or fluorescence in situ hybridization analysis. Finally, in vitro experiments demonstrated that suppression of Skp2 expression drastically reduced proliferation of thyroid cancer cells and, conversely, forced expression of Skp2 circumvented serum dependency and contact inhibition in Skp2-negative cells by promoting p27 degradation. These findings indicate that Skp2 plays an important role for the development of thyroid cancer.
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Carcinoma/metabolismo , Carcinoma/patología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Carcinoma/química , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Amplificación de Genes , Humanos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas Quinasas Asociadas a Fase-S/análisis , Proteínas Quinasas Asociadas a Fase-S/genética , Neoplasias de la Tiroides/químicaRESUMEN
PURPOSE: Amplified MYCN oncogene defines a subgroup of neuroblastomas with poor outcome. However, a substantial number of MYCN single-copy neuroblastomas exhibits an aggressive phenotype similar to that of MYCN-amplified neuroblastomas even in the absence of high MYCN mRNA and/or protein levels. EXPERIMENTAL DESIGN: To identify shared molecular mechanisms that mediate the aggressive phenotype in MYCN-amplified and single-copy high-risk neuroblastomas, we defined genetic programs evoked by ectopically expressed MYCN in vitro and analyzed them in high-risk versus low-risk neuroblastoma tumors (n = 49) using cDNA microarrays. Candidate gene expression was validated in a separate cohort of 117 patients using quantitative PCR, and protein expression was analyzed in neuroblastoma tumors by immunoblotting and immunohistochemistry. RESULTS: We identified a genetic signature characterized by a subset of MYCN/MYC and E2F targets, including Skp2, encoding the F-box protein of the SCF(Skp2) E3-ligase, to be highly expressed in high-risk neuroblastomas independent of amplified MYCN. We validated the findings for Skp2 and analyzed its expression in relation to MYCN and E2F-1 expression in a separate cohort (n = 117) using quantitative PCR. High Skp2 expression proved to be a highly significant marker of dire prognosis independent of both MYCN status and disease stage, on the basis of multivariate analysis of event-free survival (hazard ratio, 3.54; 95% confidence interval, 1.56-8.00; P = 0.002). Skp2 protein expression was inversely correlated with expression of p27, the primary target of the SCF(Skp2) E3-ligase, in neuroblastoma tumors. CONCLUSION: Skp2 may have a key role in the progression of neuroblastomas and should make an attractive target for therapeutic approaches.