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EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer.

ABSTRACT: The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 messenger RNA and EDD transfection enhanced murine Mcl-1 promoter-driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were 4- to 21-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery [1,2-dioleoyl-sn-glycero-3-phophatidylcholine (DOPC)] and cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, P = 0.004; A2780ip2, 75.9% reduction, P = 0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, P = 0.035), with a trend in A2780ip2 (60.3% reduction, P = 0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest that EDD is a therapeutic target for ovarian cancer.

SUBMITTER: Bradley A

PROVIDER: S-EPMC4004201 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer.

Bradley Amber A Zheng Hui H Ziebarth Angela A Sakati Wayne W Branham-O'Connor Melissa M Blumer Joe B JB Liu Yuying Y Kistner-Griffin Emily E Rodriguez-Aguayo Cristian C Lopez-Berestein Gabriel G Sood Anil K AK Landen Charles N CN Eblen Scott T ST

Carcinogenesis 20131230 5

The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780i ...[more]

PMID: 24379240

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Project description:BackgroundMost patients with ovarian cancer (OC) treated with platinum-based chemotherapy have a dismal prognosis owing to drug resistance. However, the regulatory mechanisms of circular RNA (circRNA) and p53 ubiquitination are unknown in platinum-resistant OC. We aimed to identify circRNAs associated with platinum-resistant OC to develop a novel treatment strategy.MethodsPlatinum-resistant circRNAs were screened through circRNA sequencing and validated using quantitative reverse-transcription PCR in OC cells and tissues. The characteristics of circNUP50 were analysed using Sanger sequencing, oligo (dT) primers, ribonuclease R and fluorescence in situ hybridisation assays. Functional experimental studies were performed in vitro and in vivo. The mechanism underlying circNUP50-mediated P53 ubiquitination was investigated through circRNA pull-down analysis and mass spectrometry, luciferase reporters, RNA binding protein immunoprecipitation, immunofluorescence assays, cycloheximide chase assays, and ubiquitination experiments. Finally, a platinum and si-circNUP50 co-delivery nanosystem (Psc@DPP) was constructed to treat platinum-resistant OC in an orthotopic animal model.ResultsWe found that circNUP50 contributes to platinum-resistant conditions in OC by promoting cell proliferation, affecting the cell cycle, and reducing apoptosis. The si-circNUP50 mRNA sequencing and circRNA pull-down analysis showed that circNUP50 mediates platinum resistance in OC by binding p53 and UBE2T, accelerating p53 ubiquitination. By contrast, miRNA sequencing and circRNA pull-down experiments indicated that circNUP50 could serve as a sponge for miR-197-3p, thereby upregulating G3BP1 to mediate p53 ubiquitination, promoting OC platinum resistance. Psc@DPP effectively overcame platinum resistance in an OC tumour model and provided a novel idea for treating platinum-resistant OC using si-circNUP50.ConclusionsThis study reveals a novel molecular mechanism by which circNUP50 mediates platinum resistance in OC by modulating p53 ubiquitination and provides new insights for developing effective therapeutic strategies for platinum resistance in OC.

Project description:BackgroundOvarian cancer is the most common gynecological malignancy and is difficult to manage due to the emergence of resistance to various chemotherapeutic drugs. New efforts are urgently awaited. Aspirin, which is traditionally considered a nonsteroidal anti-inflammatory drug (NSAID), has been reported to exert potential chemopreventive effects. Therefore, we aimed to investigate the anticancer effect and explore the underlying molecular mechanisms of aspirin on epithelial ovarian cancer (EOC) cells.MethodsWe conducted wound healing, transwell migration, EdU cell proliferation, colony formation and apoptosis detection assays to observe the effects of aspirin on the migration, proliferation and apoptosis of EOC cells (A2870, Caov-3, and SK-OV-3). EOC cells were treated with a combination of aspirin and cisplatin (CDDP) to observe the effect of aspirin on enhancing CDDP sensitivity. Orthotopic xenograft models of ovarian cancer established with A2780-Luciferase-GFP cells were applied to compare tumor growth inhibition in the control, CDDP and CDDP plus aspirin groups through in vivo imaging, which can be used to continuously monitor tumor growth. The expression and acetylation levels of p53 in EOC cells treated with aspirin were determined using western blotting, and p53 acetylation levels were examined in tumors harvested from the transplanted mice. Quantitative real-time PCR was used to assess the mRNA expression of p53 target genes.ResultsAspirin inhibited migration and proliferation and induced apoptosis in EOC cell lines in a concentration-dependent manner. In vitro, aspirin enhanced the sensitivity of EOC cells to CDDP by increasing its inhibitory effect on proliferation and its effect on inducing apoptosis. In vivo, the differences in the tumor growth inhibition rates among the different CDDP experimental groups were statistically significant (p < 0.05). Aspirin did not affect p53 protein expression but increased the p53 acetylation level in a concentration-dependent manner. In addition, the mRNA levels of CDKN1A, BAX, FOXF1, PUMA, and RRAD in EOC cells were significantly increased by the aspirin treatment.ConclusionsAspirin inhibits tumor progression and enhances the CDDP sensitivity of EOC cells. These antitumor effects of aspirin might be mediated by p53 acetylation and subsequent activation of p53 target genes.

Project description:Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. Despite initial responses to intervention, up to 80% of patient tumors recur and require additional treatment. Retrospective clinical analysis of patients with ovarian cancer indicates antibiotic use during chemotherapy treatment is associated with poor overall survival. Here, we assessed whether antibiotic (ABX) treatment would impact growth of EOC and sensitivity to cisplatin. Immunocompetent or immunocompromised mice were given untreated control or ABX-containing (metronidazole, ampicillin, vancomycin, and neomycin) water prior to intraperitoneal injection with EOC cells, and cisplatin therapy was administered biweekly until endpoint. Tumor-bearing ABX-treated mice exhibited accelerated tumor growth and resistance to cisplatin therapy compared with control treatment. ABX treatment led to reduced apoptosis, increased DNA damage repair, and enhanced angiogenesis in cisplatin-treated tumors, and tumors from ABX-treated mice contained a higher frequency of cisplatin-augmented cancer stem cells than control mice. Stool analysis indicated nonresistant gut microbial species were disrupted by ABX treatment. Cecal transplants of microbiota derived from control-treated mice was sufficient to ameliorate chemoresistance and prolong survival of ABX-treated mice, indicative of a gut-derived tumor suppressor. Metabolomics analyses identified circulating gut-derived metabolites that were altered by ABX treatment and restored by recolonization, providing candidate metabolites that mediate the cross-talk between the gut microbiome and ovarian cancer. Collectively, these findings indicate that an intact microbiome functions as a tumor suppressor in EOC, and perturbation of the gut microbiota with ABX treatment promotes tumor growth and suppresses cisplatin sensitivity.SignificanceRestoration of the gut microbiome, which is disrupted following antibiotic treatment, may help overcome platinum resistance in patients with epithelial ovarian cancer. See related commentary by Hawkins and Nephew, p. 4511.

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EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer.

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EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer.

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