RESUMEN
Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a--a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis--as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Imidazoles/farmacología , Ovario/efectos de los fármacos , Piperazinas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/agonistas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Exones , Femenino , Humanos , Concentración 50 Inhibidora , Intrones , Mutación , Ovario/metabolismo , Ovario/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal , Proteína p53 Supresora de Tumor/agonistas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranty.
RESUMEN
OBJECTIVE: To validate the overexpression of insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) in low-grade serous ovarian carcinoma (SOC), and to investigate whether the IGF-1 pathway is a potential therapeutic target for low-grade SOC. METHODS: Gene expression profiling was performed on serous borderline ovarian tumors (SBOTs) and low-grade SOC, and overexpression of IGF-1 in low-grade SOC was validated by RT-PCR and immunohistochemistry. The effect of exogenous IGF-1 on cell proliferation was determined in cell lines by cell proliferation assays, cell migration assays, and Western blot. Signaling pathways downstream of IGF-1 and the effects of the AKT inhibitor MK-2206 were investigated by Western blot analysis and by generating IGF-1R short hairpin RNA stable knockdown cell lines. Low- and high-grade cell lines were treated with the dual IGF-1R- and insulin receptor-directed tyrosine kinase inhibitor OSI-906, and cellular proliferation was measured. RESULTS: mRNA analysis and immunostaining revealed significantly higher IGF-1 expression in low-grade SOCs than in SBOTs or high-grade SOCs. In response to exogenous treatment with IGF-1, low-grade cell lines exhibited more intense upregulation of phosphorylated AKT than did high-grade cell lines, an effect that was diminished with IGF-1R knockdown and MK-2206 treatment. Low-grade SOC cell lines were more sensitive to growth inhibition with OSI-906 than were high-grade cell lines. CONCLUSIONS: IGF-1 is overexpressed in low-grade SOCs compared with SBOTs and high-grade SOCs. Additionally, low-grade SOC cell lines were more responsive to IGF-1 stimulation and IGF-1R inhibition than were high-grade lines. The IGF-1 pathway is therefore a potential therapeutic target in low-grade SOC.
Asunto(s)
Cistadenocarcinoma Seroso/metabolismo , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Neoplasias Ováricas/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Imidazoles/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Terapia Molecular Dirigida/métodos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazinas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
Low-grade (LG) serous ovarian carcinoma is believed to arise from serous borderline ovarian tumors; yet the progression from serous borderline tumors to LG serous ovarian carcinoma remains poorly understood. The purpose of this study was to identify differentially expressed genes between the 2 groups. Expression profiles were generated from 6 human ovarian surface epithelia, 8 serous borderline ovarian tumors (SBOTs), 13 LG serous ovarian carcinomas, and 24 high-grade (HG) serous ovarian carcinomas. The anterior gradient homolog 3 (AGR3) gene was found to be highly upregulated in serous borderline ovarian tumors. This finding was validated by real-time quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry. Anti-AGR3 immunohistochemistry was performed on an additional 56 LG and 103 HG tissues, and the results were correlated with clinical data. Expression profiling determined that 1254 genes were differentially expressed (P<0.005) among SBOT, LG, and HG tumors. SBOTs exhibited robust positive staining for AGR3, with a lower percentage of tumor cells stained in LG and HG. Immunofluorescence staining indicated that AGR3 expression was limited to ciliated cells. Tumor samples with a high percentage (>10%) of AGR3 positively stained tumor cells were associated with improved longer median survival in both the LG (P=0.013) and HG (P=0.008) serous ovarian carcinoma groups. The progression of SBOT to LG serous ovarian carcinoma may involve the dedifferentiation of ciliated cells. AGR3 could serve as a prognostic marker for survival in patients with LG and HG serous ovarian carcinomas.
Asunto(s)
Proteínas Portadoras/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Biomarcadores de Tumor/metabolismo , Western Blotting , Proteínas Portadoras/metabolismo , Recuento de Células , Desdiferenciación Celular , Supervivencia Celular , Cistadenocarcinoma Seroso/metabolismo , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Pronóstico , Texas/epidemiología , Regulación hacia ArribaRESUMEN
Low-grade ovarian serous carcinomas are believed to arise via an adenoma-serous borderline tumor-serous carcinoma sequence. In this study, we found that advanced-stage, low-grade ovarian serous carcinomas both with and without adjacent serous borderline tumor shared similar regions of loss of heterozygosity. We then analyzed 91 ovarian tumor samples for mutations in TP53, BRAF, and KRAS. TP53 mutations were not detected in any serous borderline tumors (n = 30) or low-grade serous carcinomas (n = 43) but were found in 73% of high-grade serous carcinomas (n = 18). BRAF (n = 9) or KRAS (n = 5) mutation was detected in 47% of serous borderline tumors, but among the low-grade serous carcinomas (39 stage III, 2 stage II, and 2 stage I), only one (2%) had a BRAF mutation and eight (19%) had a KRAS mutation. The low frequency of BRAF mutations in advanced-stage, low-grade serous carcinomas, which contrasts with previous findings, suggests that aggressive, low-grade serous carcinomas are more likely derived from serous borderline tumors without BRAF mutation. In addition, advanced-stage, low-grade carcinoma patients with BRAF or KRAS mutation have a better apparent clinical outcome. However, further investigation is needed.
Asunto(s)
Cistadenocarcinoma Seroso/genética , Mutación/genética , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Ovario/metabolismo , Polimorfismo de Nucleótido Simple/genética , Tasa de SupervivenciaRESUMEN
Ovarian tumors of low malignant potential and low-grade ovarian serous carcinomas are thought to represent different stages on a tumorigenic continuum and to develop along pathways distinct from high-grade ovarian serous carcinoma. We performed gene expression profiling on three normal human ovarian surface epithelia samples, and 10 low-grade and 10 high-grade ovarian serous carcinomas. Analysis of gene expression profiles of these samples has identified 80 genes upregulated and 232 genes downregulated in low-grade ovarian serous carcinomas. PAX2 was found to be one of the most upregulated genes in low-grade ovarian serous carcinoma. The upregulation of PAX2 was validated by real-time quantitative RT-PCR, western blot and immunohistochemical analyses. Real-time RT-PCR showed a statistically significant difference in PAX2 mRNA expression (expressed as fold change in comparison to normal human ovarian surface epithelia) among ovarian tumors of low malignant potential (1837.38, N=8), low-grade (183.12, N=17), and high-grade (3.72, N=23) carcinoma samples (P=0.015). Western blot analysis revealed strong PAX2 expression in ovarian tumors of low malignant potential (67%, N=3) and low-grade carcinoma samples (50%, N=10) but no PAX2 protein expression in high-grade carcinomas (0%, N=10). Using immunohistochemistry, tumors of low malignant potential (59%, N=17) and low-grade carcinoma (63%, N=16) samples expressed significantly stronger nuclear staining than high-grade ovarian carcinoma samples (9.1%, N=263). Furthermore, consistent with earlier immunohistochemical findings, PAX2 expression was expressed in the epithelial cells of fallopian tubes but not in normal ovarian surface epithelial cells. Our findings further support the two-tiered hypothesis that tumors of low malignant potential and low-grade ovarian serous carcinoma are on a continuum and are distinct from high-grade ovarian carcinomas. In addition, the absence of PAX2 expression in normal ovarian epithelia but expression in fallopian tube fimbria and ciliated epithelial inclusions would suggest the potential development of tumors of low malignant potential and of low-grade ovarian serous carcinomas from secondary Müllerian structures.