BRAF and KRAS mutations in ovarian serous borderline tumors (OSBTs) and ovarian low-grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumor samples from 23 recurrent LGSC patients with known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for 5 patients, and either OSBT or LGSC were available for another 18 patients. Tumor cells from paraffin-embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumors that appeared to have wild-type KRAS by conventional PCR–Sanger sequencing were further analyzed by full COLD (coamplification at lower denaturation temperature)-PCR and deep sequencing. Full COLD-PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in 10 patients. Full COLD-PCR deep sequencing detected low-abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in 7 OSBT samples and 6 LGSC samples. To our surprise, patients with the KRAS G12V mutation appeared to have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumor cells with or without detectable KRAS mutations.
Objective 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 (HGOSC) 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 HGOSC 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 HGOSCs 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 Patients with wild-type TP53 high grade ovarian serous carcinomas 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.
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 warranted.
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.
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.
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.
Ovarian cancer treatment consists of surgery followed by a chemotherapy combination entailing a platinum agent (cisplatin/carboplatin) and a taxane (paclitaxel). While 70-80% of patients initially respond to the standard treatment, the majority will relapse, and of these patients, 70-90% die because of drug resistance. Therefore, identifying markers that can predict therapeutic response and/or therapeutic targets is of great interest. To identify genes involved in chemoresistance, a microarray study of an ovarian cancer cell line (A2780) treated with varying doses of cisplatin and at varying time points was conducted. This study showed GDF15 had the highest fold-induction by cisplatin treatment. GDF15 induction by cisplatin was further validated by RT-PCR and Western blot analysis in the A2780 and 12 other cell lines with varying sensitivity/resistance to cisplatin. To further test whether GDF15 plays a role in cisplatin resistance, we knocked down its expression by esiRNA. GDF15 knockdown revealed an increase in the cisplatin IC50, suggesting that GDF15 sensitizes ovarian cancer cells to cisplatin. To determine whether GDF15 was also induced in vivo we injected mice intraperitoneally with either A2780 (cisplatin sensitive) or RMG1 (cisplatin resistant) cells. Once the tumors developed, the mice were treated with either 2.5 mg/kg or 5 mg/kg cisplatin and both the tumors and sera collected 24 and 48 hours after treatment. ELISA results on the serum showed an increase in GDF15 in mice after cisplatin treatment. Although there was no correlation between the two cisplatin doses used and GDF15 there was a correlation between GDF15 and tumor weight. Thus, our results suggest that GDF15 is induced by cisplatin both in vitro and in vivo and has a potential role as a serum biomarker to predict chemotherapeutic response. Citation Format: Daisy I. Izaguirre, Suet Yan Kwan, Zhifei Zu, Yvonne T. Tsang-Lee, Kwong-Kwok Wong. GDF15 as a serum marker of cisplatin response in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3474. doi:10.1158/1538-7445.AM2013-3474
Introduction: Ovarian Clear Cell Carcinoma (OCCC) is generally aggressive and chemoresistant, with five year survival rate of approximately 40%. Recent mutation analysis has indicated a high frequency of PIK3CA mutations, the gene encoding for the p110α subunit in PI3K, in OCCC. Aberrant signaling of the PI3K/AKT pathway might be linked to the aggressiveness of OCCC. In this study, we investigated the sensitivity of ovarian clear cell carcinoma cells with different PIK3CA mutations to inhibitors targeting the PI3K signaling pathway. Experimental design: To elucidate the functional roles of PIK3CA mutations in OCCC, 12 OCCC cell lines carrying PIK3CA wildtype or different PIK3CA mutations were used. By performing WST-1 assay, the IC50 values for cisplatin of the cell lines were compared. Eight stable OCCC cell lines expressing a luciferase reporter fused with a FOXO3a promoter were constructed. FOXO3a is a transcription factor that is exported out of the nucleus by AKT. The difference in the PI3K/AKT pathway activation in these cell lines was then correlated with luciferase activity. PI3K inhibitor LY294002 and dual PI3K/mTOR inhibitor NVP-BEZ235 were used to elucidate the dependency of OCCC cell lines on the PI3K/AKT pathway. Results: When compared with PIK3CA wildtype cell lines, cell lines with PIK3CA mutations K111N, E545K and H1047R had higher IC50 values for cisplatin and PI3K inhibitor LY294002. These 3 cell lines also have lower luciferase activity, indicating increased activation of the PI3K/AKT pathway. The IC50 values for NVP-BEZ235 are similar in PIK3CA wildtype or mutated cell lines. Conclusion: The constitutive activation of the PI3K/AKT pathway in ovarian clear cell carcinoma appears to lead to increased resistance to chemotherapy, possibly by promoting cell survival and inhibiting apoptosis. Inhibition of PI3K/AKT pathway may overcome this resistance. NVP-BEZ235 was shown to be a potent drug in OCCC, it was effective in the nano molar range (IC90 value was reached in all cells lines at drug concentration of 100nM) and was active against PIK3CA mutants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5037. doi:10.1158/1538-7445.AM2011-5037
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