Dicer, Drosha, and outcomes in patients with ovarian cancer

Abstract

Background: We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer.

Methods: We measured messenger RNA (mRNA) levels of Dicer and Drosha in specimens of invasive epithelial ovarian cancer from 111 patients, using a quantitative reverse-transcriptase-polymerase-chain-reaction assay, and compared the results with clinical outcomes. Validation was performed with the use of published microarray data from cohorts of patients with ovarian, breast, and lung cancer. Mutational analyses of genomic DNA from the Dicer and Drosha genes were performed in a subgroup of ovarian-cancer specimens. Dicer-dependent functional assays were performed by means of in vitro transfection with small interfering RNA (siRNA) and short hairpin RNA (shRNA).

Results: Levels of Dicer and Drosha mRNA correlated with the levels of expression of the corresponding protein and were decreased in 60% and 51% of ovarian-cancer specimens, respectively. Low Dicer expression was significantly associated with advanced tumor stage (P=0.007), and low Drosha expression with suboptimal surgical cytoreduction (P=0.02). Cancer specimens with both high Dicer expression and high Drosha expression were associated with increased median survival (>11 years, vs. 2.66 years for other subgroups; P<0.001). We found three independent predictors of reduced disease-specific survival in multivariate analyses: low Dicer expression (hazard ratio, 2.10; P=0.02), high-grade histologic features (hazard ratio, 2.46; P=0.03), and poor response to chemotherapy (hazard ratio, 3.95; P<0.001). Poor clinical outcomes among patients with low Dicer expression were validated in additional cohorts of patients. Rare missense mutations were found in the Dicer and Drosha genes, but their presence or absence did not correlate with the level of expression. Functional assays indicated that gene silencing with shRNA, but not siRNA, may be impaired in cells with low Dicer expression.

Conclusions: Our findings indicate that levels of Dicer and Drosha mRNA in ovarian-cancer cells have associations with outcomes in patients with ovarian cancer.

Figure 1. The RNA-Interference Cascade in Humans

Long precursor microRNA (miRNA) segments, called pri-miRNA, are first cleaved in the nucleus by Drosha, an RNase III endonuclease, into segments of approximately 70 nucleotides each (called pre-miRNA). Transportation into the cytoplasm by means of exportin 5 leads to cleavage by Dicer, another RNase III endonuclease, which produces mature miRNA segments. Host degradation of messenger RNA (mRNA) and translational repression occurs after miRNA binds to the RNA-induced silencing complex (RISC). Cytoplasmic long double-stranded RNA (dsRNA) is cleaved by Dicer into small interfering RNA (siRNA), which is incorporated into RISC, resulting in the cleavage and degradation of specific target mRNA.

Figure 2. Kaplan–Meier Survival Curves for Patients According to Tumor Expression of Dicer and Drosha

For patients with invasive epithelial ovarian cancer, curves are shown for Dicer and Drosha expression (Panel A), with curves for Dicer and Drosha combined shown for comparison in Fig. 3 in the Supplementary Appendix. Curves from validation analyses are also shown for the expression of Dicer and Drosha in independent cohorts of patients with ovarian cancer (Panel B) and lung cancer (Panel C), as well as Dicer expression in two cohorts of patients with breast cancer (Panels D and E).

Figure 3. Transfection of Small Interfering RNA (siRNA) and Short Hairpin RNA (shRNA) Targeting Galectin-3 in Ovarian-Cancer Cell Lines with Low Dicer Expression and Those with High Dicer Expression

Western blot densitometry analysis was performed for the cell lines transfected with siRNA or shRNA or control (nontargeting) sequences. Actin was used for purposes of normalization.