吳 華 席 Hua-Hsi Wu, MD OB/GYN, VGH-TPE Aug 12, 2008 10 Minutes Talk 吳 華 席 Hua-Hsi Wu, MD OB/GYN, VGH-TPE Aug 12, 2008

Ovarian cancer, MicroRNA, Exosome

Introduction Ovarian cancer Worldwide, 6th most common in female & 125,000 deaths annually 75% diagnosed as extra-ovarian dz (EM ca 73%, Breast ca 55% & Cx ca 50% in stage 1) 5-yr survival: stage 1 > 90%, advanced stage 21% How to early Dx?

Introduction MicroRNA Small (21-25 nucleotides in length) non-coding RNAs Suppress the translation of target mRNAs by binding to their 3” untranslateed region Post-transcriptional silencing Cleavage of homologus mRNA Specific inhibition of protein synthesis Critical regulator of cellular processes Proliferation, differentiation, development & cell death

Introduction MicroRNA Distinct MicroRNAs signature, classify human cancers Ovarian cancers: miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205 & miR-214 Up-modulation of specific microRNA could be the amplification of its gene. Is microRNAsignature better than mRNA signature, in cnacer Dx, staging, progression, and response to treatment

Biogenesis of microRNAs Fig. 1  Biogenesis of microRNAs(miRNAs). miRNA genes are transcribed by RNA polymerase II and the resulting primary transcripts (pri-miRNAs) are poly-adenylated and the 3' end and capped at the 5' end. Pri-miRNA molecules contain local stem-loop structures (hairpins; see Fig. 3 for details) that are recognized by the Drosha-DGCR8 complex. The hairpin structures are trimmed by the complex in the nucleus and the precursor-miRNA (pre-miRNA) is transported to the cytoplasm by Exportin-5. In the cytoplasm Dicer processes the pre-miRNAs and one miRNA duplex is released from each pre-miRNA. The two strands of the duplex are separated from each other by the Dicer-TRBP complex and one of the strands is incorporated into the RNA induced silencing complex (RISC). This strand is the mature miRNA (orange) that will target mRNAs (see Fig. 2). The strand, which is not incorporated into RISC is called the miRNA* strand (red) and it is degraded

Detailed structures of pri-, pre- and mature miRNAs Fig. 2  Detailed structures of pri-, pre- and mature miRNAs. Hairpins on pri-miRNAs consist of a stem and a loop. Most nucleotides within the stem region are paired between the two arms of the stem but there are nucleotides that are not paired and form bulges. Nucleotides in the terminal loop are not paired to each other. Pri-miRNAs contain the flanking nucleotides outside of the hairpin and pre-miRNAs only consist of the hairpin structure. Dicer releases the miRNA duplex (orange and red) from the pre-miRNA that have 2 nucleotide 3' overhangs at both sides of the duplex. Both the orange (mature miRNA) and the red (miRNA*) strands are usually 21 nucleotide long and 16–18 nucleotides from each strands form base pairs with the other strand [3–5 nucleotides are in bulge

Introduction Exosome small (50-100nm) membrane vesicles of endocytic origin, in the peripheral circulation (of ovarian cancer patients was found initially in 1979) Source Except cancers, including reticulocytes, dendritic cells, B cells, T cells, mast cells, epithelial cells, and embryonic cells. All exosomes share certain common characteristics (structures, size, density & general protein composition)

Introduction Exosome Cell-cell communication Directly by surface expressed ligands By transferring molecules between cells Exosomes contains both cellular mRNA and miRNAs While tumor-derived exosomes exhibit some common, shared proteins, they also express an array of tumor antigens that reflect the originating tumor cells.

Material and Methods Serum & biopsied tissue Serous papillary adenocarcinoma Stage I n=10, stage II n=10, stage III n=20, stage IV n=10 Ovarian adenoma n=10 NED of ovarian diseases n=10 Isolation of circulating exosomes MACS (EpCAM, anti-epithelial cell adhesion molecules) Isolation & profiling of miRNAs mirVana microRNA isolation kit

Results

Panel A: The levels of circulating tumor-derived exosomes compared to stage of ovarian cancer Panel B: Electron micrograph of circulating exosomes isolated by magnetic beads. Fig. 1. Panel A: The levels of circulating tumor-derived exosomes compared to stage of ovarian cancer. Exosomes were isolated from sera obtained from age-matched female controls (n = 10), age-matched women with benign ovarian disease (n = 10), and women diagnosed with ovarian cancer (n = 10 for each stage). Levels of exosomes are presented as protein concentrations. Panel B: Electron micrograph of circulating exosomes isolated by magnetic beads. Ultrathin sections (65 nm) were cut and stained with uranyl acetate and Reynold's lead citrate. The sections were examined in a Jeol 1210 transmission electron microscope

 Presence of small RNA associated with circulating EpCAM-positive exosomes from ovarian cancer patients Fig. 2. Presence of small RNA associated with circulating EpCAM-positive exosomes from ovarian cancer patients. Panel A: Representative analysis of the RNA isolated from tumor exosomes using Agilent 2100 Bioanalyzer. Panel B: Agarose gel (1%) separation of total RNA from circulating exosomes and corresponding tumors. This total RNA was used as the starting material for microRNA profiling.

Fig. 3. Intensities for specific microRNAs derived from the advanced-staged ovarian tumors (□) and from EpCAM-positive exosomes (■) isolated from the sera of these patients. miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, and miR-214 have previously been demonstrated to be upregulated markers for ovarian cancer. Each bar presents the average intensities of duplicate samples with the results of four representative patients presented. **Intensities for specific microRNAs derived from the advanced-staged ovarian tumors (□) and from EpCAM-positive exosomes (■) isolated from the sera of these patients. **miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, and miR-214 have previously been demonstrated to be upregulated markers for ovarian cancer

Comparison of miRNA with peripheral blood-derived exosomes and their corresponding tumors

miRNAs in exosomes compared by stages Fig. 4. Intensities for specific microRNAs derived from EpCAM-positive exosomes isolated from the peripheral blood (2.5 ml) of the patients with benign ovarian disease and patients with ovarian cancer. Patients with ovarian cancer were separated between Stages I, II, and III. The bars represent the mean ± standard deviation of the normalized intensities of each group of patients (n = 10 for each group).

Comparison of specific exosomal miRNAs immediately after blood draw or 24, 48, & 96 hr later Fig. 5. Comparison of specific exosomal microRNAs derived from the serum of an ovarian cancer patient, immediately after blood draw or 24, 48, and 96 h later with sera stored at 4 °C (panel A) or after 7 to 28 days, stored at − 70°C (panel B). Tumor exosomes were isolated by MACS using anti-EpCAM

Appendix A

Results MicroRNA from ovarian tumor cells and exosomes from the same patients were positive for 218 of 467 mature microRNAs analyzed. The levels of the 8 specific microRNAs were similar between cellular and exosomal microRNAs (exhibiting correlations from 0.71 to 0.90). While EpCAM-positive exosomes were detectable in both patients with benign ovarian disease and ovarian cancer, exosomal microRNA from ovarian cancer patients exhibited similar profiles, which were significantly distinct from profiles observed in benign disease. Exosomal microRNA could not be detected in normal controls.

Conclusions These results suggest that microRNA profiling of circulating tumor exosomes could potentially be used as surrogate diagnostic markers for biopsy profiling, extending its utility to screening asymptomatic populations.