Identification of ovarian cancer metastatic miRNAs

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.<br/

miR-146a and miR-150 enhance spheroid formation.

<p>24 hours after transfection with anti-miR LNA inhibitors or pre-miR mimics as indicated, 700/µl SKOV-3 or 600/µl OVCAR-8 cells were seeded in 35 well agarose micromolds with one spheroid forming in each well. <b>A</b>. miR-21 and miR-146a are up-regulated in 4 day SKOV-3 and OVCAR-8 spheroids detected using Taqman qPCR array cards in two replicates. Error bars are s.e.m. <b>B</b>. Inhibition of miR-146a with 10 nM LNA delays spheroid formation and leads to more amorphous and looser formed spheroids in SKOV-3 and OVCAR-8 after 2 days. Red bar is 400 µm. <b>C</b>. Box and whisker plot shows that inhibition of miR-146a with LNAs significantly reduces spheroid size after 4 days in SKOV-3, and modestly in OVCAR-8. Representative expression shown from four replicates. <b>D</b>. Ectopic expression of miR-150 and miR-146a significantly enhances spheroid formation after 4 days. SKOV-3 and OVCAR-8 cells were transfected with 50 nM of pre-miR miR-150 and miR-146a pre-miRs before spheroid formation. SKOV-3 or OVCAR-8 cells were transfected as indicated. Representative spheroids are shown. Red bar is 400 µm. Box and whisker plots of the size distribution of 45 spheroids from a representative experiment. Experiment was reproduced three times. P-values determined by Student's t-test.</p

miRNA expression profile of primary and metastatic ovarian tumors.

<p><b>A</b>. miRNA expression profiling by Taqman qPCR arrays identifies 17 miRNAs differentially expressed between 9 pairs of primary tumors and omental lesions. miRNAs with p<0.05 (paired t-test) were selected. The expression level is presented as the mean +/− standard error of the mean (s.e.m.) of the fold change using the ΔC_t method relative to U6 snRNA. Red, lower expression in metastases, blue, higher expression in metastases. <b>B</b>. Left, unsupervised hierarchical clustering of the fold changes of the analyzed patients. Right, association plot analysis of miRNA expression identifies miRNA clusters indicated in red. Clustering was performed in Gene-E ]50].</p

Validation of miRNA expression.

<p><b>A</b>. <i>In situ</i> hybridization of miR-21. Cancer cells are stained red by Nuclear Red. Higher expression in omental metastases is observed in each case even with both relatively high and low miR-21 expression in the primary tumor. The arrows indicate regions of miR-21 expression co-localizing with Nuclear Red staining. <b>B</b>. Laser Capture Microdissection (LCM) of two cases reveals miRNAs likely expressed in cancer cells. The heat map shows the fold change for the 17 miRNAs identified in the bulk tumor screen. Similar patterns of differential expression are observed for the miRNAs expressed in cancer cells as observed in bulk tumor. Black indicates that the miRNA was not detectable in the LCM isolated cancer cells.</p

Metastatic miRNAs increase surviving cells.

<p><b>A</b>. Treatment of cells with pre-miR-150 mimic modestly increases the cisplatin IC50 in SKOV-3 and IGROV-1, but not OVCAR-8 cells. Wst-1 assays were performed 48 hours after cisplatin treatment. Graph shows average of 3 biological replicates. Error bars represent s.e.m. <b>B</b>. Schematic of cisplatin survival assay. Cells are treated twice with high concentrations of cisplatin leading to survival by approximately 1% of the cells. <b>C</b>. miR-150 and miR-146a are up-regulated in surviving cells after 6 days of 50 µM cisplatin in SKOV-3 and 7 days of 30 µM cisplatin in OVCAR-8 cells compared to untreated, proliferating cells. Data are in duplicate and error bars are s.e.m. The fold change for miR-150 is very large because miR-150 was not detectable in proliferating cells. C_t was set to maximum cycle tested, 40, to estimate the fold change. <b>D</b>. Inhibition of miR-146a with 10 nM LNA inhibitor significantly reduces the number of residual cells in SKOV-3 and modestly reduces the surviving cells in OVCAR-8 after 6 days of 50 µM cisplatin in SKOV-3 and 7 days of 30 µM cisplatin in OVCAR-8 cells. The surviving viable cells were determined by trypan blue exclusion assay. Biological triplicate experiments are shown. Error bars are s.e.m. <b>E</b>. Transfection of 50 nM pre-miR-146a and pre-miR-150 increase long term survival after 6 days of 50 µM cisplatin in SKOV-3 and 7 days of 30 µM cisplatin in OVCAR-8 cells.</p

Specificity of metastatic six gene signature.

<p>P-values are calculated by determining the number of random gene sets with lower p-values than the metastatic gene signature using the log-rank test of Kaplan Meier analysis divided by the number of random sets tested as indicated in parentheses.</p