Forced expression of hsa-miR-302b increased cisplatin sensitivity in IGROV-1 cells without affecting cell proliferation.

<p>(A) Percent cell death of hsa-miR-302b- and scrambled-transfected cells after cisplatin treatment. IGROV-1 cells were transfected with 50 nmol/l hsa-miR-302b precursor molecule or scrambled control, and 72 h later, exposed to cisplatin (50 µM) for 1 h. Cell viability was assessed 24 h after cisplatin treatment by propidium iodide staining and flow cytometry. Data represent mean ± SEM of 6 independent experiments. ***p<0.0001 by paired t-test. (B) Evaluation of cell proliferation by SRB assay. Transfected cells were seeded in a 96-well plate at a density of 10³, 1.5×10³, and 2×10³ cells/well. Cell growth was assessed by optical density (OD) determination 72 h after transfection. Data represent mean ± SEM of 3 independent experiments.</p

Computational integration of miRNA and gene expression profiles of tumor samples from CpG-ODN- and saline-treated mice.

<p>Network between 15 of 20 differentially expressed miRNAs and their anti-correlated target genes. The top 250 interactions were used to generate the network using the MAGIA tool.</p

Increased Sensitivity to Chemotherapy Induced by CpG-ODN Treatment Is Mediated by microRNA Modulation

<div><p>We recently reported that peritumoral CpG-ODN treatment, activating TLR-9 expressing cells in tumor microenvironment, induces modulation of genes involved in DNA repair and sensitizes cancer cells to DNA-damaging cisplatin treatment. Here, we investigated whether this treatment induces modulation of miRNAs in tumor cells and their relevance to chemotherapy response. Array analysis identified 20 differentially expressed miRNAs in human IGROV-1 ovarian tumor cells from CpG-ODN-treated mice versus controls (16 down- and 4 up-regulated). Evaluation of the role of the 3 most differentially expressed miRNAs on sensitivity to cisplatin of IGROV-1 cells revealed significantly increased cisplatin cytotoxicity upon ectopic expression of hsa-miR-302b (up-modulated in our array), but no increased effect upon reduced expression of hsa-miR-424 or hsa-miR-340 (down-modulated in our array). Accordingly, hsa-miR-302b expression was significantly associated with time to relapse or overall survival in two data sets of platinum-treated ovarian cancer patients. Use of bio-informatics tools identified 19 mRNAs potentially targeted by hsa-miR-302b, including HDAC4 gene, which has been reported to mediate cisplatin sensitivity in ovarian cancer. Both HDAC4 mRNA and protein levels were significantly reduced in IGROV-1 cells overexpressing hsa-miR-302b. Altogether, these findings indicate that hsa-miR-302b acts as a “chemosensitizer” in human ovarian carcinoma cells and may represent a biomarker able to predict response to cisplatin treatment. Moreover, the identification of miRNAs that improve sensitivity to chemotherapy provides the experimental underpinning for their possible future clinical use.</p> </div

Targeting of HDAC4 in IGROV-1 cells by hsa-miR-302b.

<p>IGROV-1 cells were transfected with 50 nmol/l hsa-miR-302b or a scrambled oligonucleotide and RNA and proteins were collected after 72 h. HDAC4 mRNA levels were evaluated by RT-qPCR (A) and protein expression was evaluated by Western blot (B). GAPDH was used to normalize protein loading per lane. Data are representative of 6 independent experiments with superimposable results. (C) Schematic representation of the interaction between hsa-miR-302b and the binding site on the wild-type HDAC4-3′UTR and the mutated control. (D) Relative luciferase activity in IGROV-1 cells for HDAC4-3′UTR-wt co-transfected with reporter vector and with hsa-miR-302b precursor molecule or negative scrambled control for 48 h. (E) Relative luciferase activity in IGROV-1 cells for HDAC4-3′UTR-mut co-transfected with reporter vector and with hsa-miR-302b precursor molecule or negative scrambled control for 48 h.</p

Independent biological validation of CpG-ODN miRNA profile.

<p>miRNA expression was assessed by RT-qPCR on IGROV-1 xenografts collected from a replica of a previous experiment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058849#pone.0058849-Sommariva1" target="_blank">[3]</a>. RT-qPCR data are plotted as −ΔCt. P-values were calculated using two-tailed Student’s t-test.</p

miRNA expression profiling in IGROV-1 ovarian tumors from CpG-ODN-treated athymic mice.

<p>Heat-map of 23 modulated miRNAs with FDR <0.1 and fold change >1.8 in CpG-ODN- versus saline-treated mice. Among the 20 miRNAs belonging to miRBase12.0, 16 were down- and 4 up-modulated in CpG-ODN-treated mice (red: up-regulated miRNAs; green: down-modulated miRNAs). Columns and rows represent samples and miRNAs, respectively.</p

Image_7_A Bispecific Antibody to Link a TRAIL-Based Antitumor Approach to Immunotherapy.TIF

T-cell-based immunotherapy strategies have profoundly improved the clinical management of several solid tumors and hematological malignancies. A recently developed and promising immunotherapy approach is to redirect polyclonal MHC-unrestricted T lymphocytes toward cancer cells by bispecific antibodies (bsAbs) that engage the CD3 complex and a tumor-associated antigen (TAA). The TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) is an attractive immunotherapy target, frequently expressed by neoplastic cells, that we decided to exploit as a TAA. We found that a TRAIL-R2xCD3 bsAb efficiently activates T cells and specifically redirect their cytotoxicity against cancer cells of different origins in vitro, thereby demonstrating its potential as a pan-carcinoma reagent. Moreover, to mimic in vivo conditions, we assessed its ability to retarget T-cell activity in an ex vivo model of ovarian cancer patients' ascitic fluids containing both effector and target cells—albeit with a suboptimal effector-to-target ratio—with remarkable results.</p

In silico evaluation of ovarian cancer patients’ clinical course according to hsa-miR-302b and hsa-miR-340 expression levels.

<p>Kaplan-Meier survival curves of patients stratified according to hsa-miR-302b expression (A) and has-miR-340 expression (B) on GSE25204 and referred to TTR. (C) Kaplan-Meier survival curves for hsa-miR-302b expression on GSE27290 and referred to OS. Patients were dichotomized using median expression as threshold.</p

Supplementary Figure S2 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S2 - PDF file 2513K, A and B: Analysis of the correlation between IL-18 and IL-18BP protein levels in EOC sera (n. 47) (A) and ascites (n. 17) (B). No significant correlation (P=ns) was found by Pearson's test. C: Analysis of the correlation between IL18 and IL-18BP protein levels and IFN-? in the ascites of 15 EOC patients. IFN-? levels are low to undetectable and show no correlation with IL-18 or IL-18BP levels (P=ns). Pearson's correlation coefficients are shown (r). Lines represent the best-fit linear regression analysis with the 95% Confidence Interval</p

Supplementary Figure S6 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S6 - PDF file 185K, A: Analysis of the correlation between IL18BP and EBI3 mRNA levels in high grade (Type II) tumors in two microarray datasets of EOC. A significant correlation was found between EBI3 and IL18BP in both datasets, suggesting a relationship between the expression of EBI3 and IL18BP mRNA in EOC cell primary tumors. Pearson's correlation coefficients are shown (r). Lines represent the best fit linear regression analysis with the 95% Confidence Interval. B: Association between different EBI3 mRNA expression levels and Progression Free Survival (PFS) in Type II EOC of the Tothill microarray dataset. High EBI3 mRNA levels are associated to a shorter PFS time. Median PFS was 13 months for EBI3 levels higher than third quartile versus 21 months for EBI3 levels lower than first quartile (P=0.016). Solid line: cases with EBI3 levels lower than first quartile (n.52). Dashed line: cases with EBI3 levels higher than third quartile (n.49). P values were determined using log-rank test.</p