A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to miRNA overexpression

Abstract Large‐scale molecular annotation of epithelial ovarian cancer (EOC) indicates remarkable heterogeneity in the etiology of that disease. This diversity presents a significant obstacle against intervention target discovery. However, inactivation of miRNA biogenesis is commonly associated with advanced disease. Thus, restoration of miRNA activity may represent a common vulnerability among diverse EOC oncogenotypes. To test this, we employed genome‐scale, gain‐of‐function, miRNA mimic toxicity screens in a large, diverse spectrum of EOC cell lines. We found that all cell lines responded to at least some miRNA mimics, but that the nature of the miRNA mimics provoking a response was highly selective within the panel. These selective toxicity profiles were leveraged to define modes of action and molecular response indicators for miRNA mimics with tumor‐suppressive characteristics in vivo. A mechanistic principle emerging from this analysis was sensitivity of EOC to miRNA‐mediated release of cell fate specification programs, loss of which may be a prerequisite for development of this disease.

. Distinct miRNA sensitivities in patient-matched cell lines from recurrent disease. A A schematic depicting the derivation of the PEO1 and PEO4 cell lines from a patient with high-grade serous adenocarcinoma of the ovary. B A scatter plot of z-scores corresponding to inhibition of viability in PEO1 versus PEO4 in response to each of 400 miRNA mimics. Values were derived from the mean of triplicate experiments. Mimics with a z-score < 2 were considered to have significantly reduced viability in a cell line. 17 mimics reduced PEO1 cell viability, 15 mimics reduced PEO4 viability, and 2 mimics reduced viability in both cell lines. C The scatter plot indicates the expression value of each miRNA in PEO1 (y-axis) and the effect of each miRNA on cell viability in the same cell line as represented by z-score (x-axis). D The scatter plot indicates the expression value of each miRNA in PEO4 (y-axis) and the effect of each miRNA on cell viability in the same cell line as represented by z-score (x-axis). E The scatter plots indicate the expression values of each miRNA in PEO1 (x-axis) and PEO4 (y-axis) (left panel), and miRNA expression relative to that in HOSE (Normal) cells (right panel). Points are color-coded according to the corresponding miRNA mimic activity in the viability screens. F PEO1-specific hit miR-210 continues to show a robust phenotype at even a 10-fold dilution while increasing the dosage 4-fold does not sensitize PEO4 cells, suggesting that selectivity was not due to overt dosage effects. Each data point represents the mean of 3 independent experiments AE SD.
Source data are available online for this figure.   Figure EV4. Genomic characterization of patient-matched cell lines from recurrent disease. A A summary of the PEO1 and PEO4 genomic characterization is displayed by circos plot. The outer track indicates relative expression of genes (the log 2 of the ratio of (RPKM+1) values of PEO1 versus PEO4). Red peaks correspond to genes with greater than two-fold overexpression in PEO1 cells, and blue peaks correspond to genes with greater than two-fold overexpression in PEO4 cells. The next three tracks represent SNVs unique to PEO1 in red, unique to PEO4 in blue, and common in black. This is followed by an axis indicating chromosome position. The inner four tracks summarize CNV as indicated (variation is defined by a read depth ratio of ≥ 1.5 (gain) or ≤ 0.5 (loss) as compared to the reference cell line). See Datasets EV2, EV3, and EV4 for detailed annotations.   PEO4 C Figure EV5. miR-155 and miR-181b are predicted to target the AKT, MAPK, and TGFb pathways. A miR-155 and miR-181b are predicted to target multiple nodes in the AKT and MAPK signaling pathways. Nodes predicted to be targeted by miR-155 are in yellow, miR-181b in red, those predicted to be targeted by both are in yellow-to-red gradient, and those predicted to be targeted by neither are in gray. Note only miR-155 is predicted to target Rictor, which is part of the complex responsible for phosphorylation of AKT at S473. B Genomic alterations predicted to influence AKT and MAPK signaling pathways in PEO1 and PEO4 cells. Genes in red were overexpressed at least 2-fold in PEO1 relative to PEO4 cells, while genes in blue were overexpressed in PEO4 relative to PEO1. Arrows indicated copy gain or loss with red indicating PEO1 cells and blue indicating PEO4. Asterisks denote genes containing SNVs. C Immunoblots from whole-cell lysates indicate decreased AKT phosphorylation at both T308 and S473 in PEO4 cells relative to PEO1 cells in response to serum stimulation of cells deprived of serum overnight. D Dose-dependent consequences of exposure to the PI3K inhibitor LY294002 or AKT Inhibitor X on cell viability was examined in the indicated cell lines. All values were normalized to DMSO carrier alone. Each point represents the mean of 3 experiments AE SD. No significant differences were detected.
Source data are available online for this figure.  Figure EV6. miR-517a reduces ovarian cancer cell viability, in part, by inhibiting ARCN1 expression. A Correlation of the consequence of USP1 depletion and miR-517a sensitivity in 12 NSCLC cell lines (plot as in Fig 2F). B Schematic summarizing predicted miR-517a relationships with ARCN1 and USP1.
Source data are available online for this figure.
A Expression of miR-124 mimic reduced expression of PTBP1 (PTB) and CTDSP1 (SCP1) but not other members of the REST complex on a microarray. Each bar represents the log 2 of the ratio of the means of three experiments of mir-124 mimic expression to mimic negative control expression of a single probe on the microarray. B miR-124 mimic expression reduced expression of mesenchymal markers ITGB1, VIM, and TWIST2 and increased expression of neuronal markers CDH2, VAMP2, and MAP2 according to the microarray data. Each bar represents the log 2 of the ratio of the means of three experiments of mir-124 mimic expression to mimic negative control expression of a single probe on the microarray. C SIX4, EYA1, EYA2, EYA3, and EYA4 are predicted targets of miR-124 in TargetScan (www.targetscan.org). D SIX4 expression is significantly enriched in human ovarian tumors relative to normal ovarian tissue. P-value from Welch two-sample t-test. N, unmatched normal ovarian tissue; P, primary epithelial ovarian tumors; R, recurrent epithelial ovarian tumors. E Knockdown of SIX4 reduced cell viability similar to expression of a miR-124 mimic in PEO1 cells (mean AE SD of 3 experiments). F Expression of a SIX4 construct that cannot be targeted by miR-124 partially rescues toxicity due to miR-124 over expression in PEO1 cells. Mean AE SD of 6 experiments. Two-way ANOVA analysis, ***P < 0.005. G SIX4 knockdown did not induce activation of caspase-3 and caspase-7. Depletion of SIX4 reduced BrdU incorporation in ES2 cells and resulted in decreased Hoechstpositive nuclei 48 h post-transfection. SIX4 knockdown also resulted in decreased cells in S-phase as seen in PI-stained ES2 cells 48 h post-transfection. All bars represent the mean AE SD of three experiments. H Knockdown of SIX4 reduced expression of multiple cyclin proteins in PEO1 and PEO4 cells on the microarray. Each bar represents the log 2 of the ratio of the means of three experiments of SIX4 siRNA transfection to siRNA negative control transfection of a single probe on the microarray. I SIX4 knockdown induced expression of STRADB in PEO1 and PEO4 cells according to the microarray data. Each bar represents the log 2 of the ratio of the means of three experiments of SIX4 siRNA transfection to siRNA negative control transfection of a single probe on the microarray. J Schematic of predicted interactions of miR-124 with cell cycle and cell differentiation machinery.  Log2(miR-124/miNC)   ITGB1  ITGB1  ITGB1  ITGB1  ITGB1  ITGB1  VIM  VIM  VTN  SNAI1  1NAI2  SNAI2  SNAI3  TWIST1  TWIST2  TWIST2  TWIST2  CDH2  TUBB3  MAP2  MAP2  MAP2  MAP2  VAMP2  NEUROD1  NEUROD2