RESUMEN
BACKGROUND: The standard treatment of ovarian cancer is surgery followed by a chemotherapeutic combination consisting of a platinum agent, such as cisplatin and a taxane-like paclitaxel. We previously observed that patients with ovarian cancer wild-type for p53 had a poorer survival rate than did those with p53 mutations. Thus, a better understanding of the molecular changes of epithelial ovarian cancer cells with wild-type p53 in response to treatment with cisplatin could reveal novel mechanisms of chemoresistance. METHODS: Gene expression profiling was performed on an ovarian cancer cell line A2780 with wild-type p53 treated with cisplatin. A gene encoding a secretory protein growth differentiation factor 15 (GDF15) was identified to be highly induced by cisplatin treatment in vitro. This was further validated in a panel of wild-type and mutant p53 ovarian cancer cell lines, as well as in mouse orthotopic models. The mouse tumor tissues were further analyzed by histology and RNA-seq. RESULTS: GDF15 was identified as one of the highly induced genes by cisplatin or carboplatin in ovarian cancer cell lines with wild-type p53. The wild-type p53-induced expression of GDF15 and GDF15-confered chemotherapy resistance was further demonstrated in vitro and in vivo. This study also discovered that GDF15-knockdown (GDF15-KD) tumors had less stromal component and had different repertoires of activated and inhibited canonical pathways in the stromal cell and cancer cell components from that of the control tumors after cisplatin treatment. CONCLUSIONS: GDF15 expression from the wild-type p53 cancer cells can modulate the canonical pathways in the tumor microenvironment in response to cisplatin, which is a possible mechanism of chemoresistance.
RESUMEN
Inactivating mutations in ARID1A are found in a broad spectrum of cancer types, with the highest frequency in gynecologic cancers. However, therapeutic strategies targeting ARID1A-mutant cancer cells remain limited. In this study, we aimed to identify drugs sensitivities in ARID1A-mutant cancer cell lines. By analyzing the Genomics of Drug Sensitivity in Cancer database, we found that ARID1A-mutant cancer cell lines were more sensitive to treatment with the reactive oxygen species (ROS)-inducing agent elesclomol. In a panel of 14 gynecologic cancer cell lines, treatment with elesclomol inhibited growth and induced apoptosis more potently in ARID1A-mutant cells. Knockdown of ARID1A in RMG1 and OVCA432 ovarian cancer cells resulted in increased sensitivity to elesclomol, whereas restoration of ARID1A expression in TOV21G ovarian cancer cells resulted in increased resistance to elesclomol. Furthermore, we found that knockdown of ARID1A expression resulted in increased intracellular ROS levels. In ovarian clear cell carcinoma patient samples, low expression of ARID1A correlated with high expression of 8-hydroxyguanosine, a marker for oxidative stress. In summary, we demonstrate for the first time that loss of ARID1A leads to accumulation of ROS and suggest that elesclomol may be used to target ARID1A-mutant gynecologic cancer cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Hidrazinas/farmacología , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/genética , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN , Femenino , Genómica , Humanos , Concentración 50 Inhibidora , Mutación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Estrés Oxidativo , ARN Interferente Pequeño/metabolismoRESUMEN
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.
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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
OBJECTIVE: The objective of this study is to investigate whether wild-type TP53 status in high-grade serous ovarian carcinoma is associated with poorer survival. METHODS: Clinical and genomic data of 316 sequenced samples from The Cancer Genome Atlas (TCGA) ovarian high-grade serous carcinoma study were downloaded from TCGA data portal. Association between wild-type TP53 and survival was analyzed with Kaplan Meier method and Cox regression. The diagnosis of high-grade serous carcinomas was evaluated by reviewing pathological reports and high-resolution hematoxylin and eosin (H&E) images from frozen sections. The authenticity of wild-type TP53 in these tumor samples was assessed by analyzing SNP array data with ASCAT algorithm, reverse phase protein array (RPPA) data and RNAseq data. RESULTS: Fifteen patients with high grade serous ovarian carcinomas were identified to have wild-type TP53, which had significantly shorter survival and higher chemoresistance than those with mutated TP53. The authenticity of wild-type TP53 status in these fifteen patients was supported by SNP array, RPPA, and RNAseq data. Except four cases with mixed histology, the classification as high grade serous carcinomas was supported by pathological reports and H&E images. Using RNAseq data, it was found that EDA2R gene, a direct target of wild-type TP53, was highly up-regulated in samples with wild-type TP53 in comparison to samples with either nonsense or missense TP53 mutations. CONCLUSION: Although patients with wild-type TP53 ovarian cancer were rare in the TCGA high grade ovarian serous carcinomas cohort, these patients appeared to have a poorer survival and were more chemoresistant than those with mutated TP53. Differentially expressed genes in these TP53 wild-type tumors may provide insight in the molecular mechanism in chemotherapy resistance.
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Cistadenocarcinoma Seroso/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes p53/genética , Neoplasias Ováricas/genética , Receptor Xedar/genética , Cistadenocarcinoma Seroso/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Ováricas/patología , Polimorfismo de Nucleótido Simple , Modelos de Riesgos Proporcionales , Análisis por Matrices de Proteínas , Análisis de Secuencia de ARN , Regulación hacia ArribaRESUMEN
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.
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Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line data sets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined polypyrimidine tract-binding protein 1 (PTBP1) levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each data set. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5' splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.