Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences

Abstract

Tumour cells release an abundance of microvesicles containing a selected set of proteins and RNAs. Here, we show that tumour microvesicles also carry DNA, which reflects the genetic status of the tumour, including amplification of the oncogene c-Myc. We also find amplified c-Myc in serum microvesicles from tumour-bearing mice. Further, we find remarkably high levels of retrotransposon RNA transcripts, especially for some human endogenous retroviruses, such as LINE-1 and Alu retrotransposon elements, in tumour microvesicles and these transposable elements could be transferred to normal cells. These findings expand the nucleic acid content of tumour microvesicles to include: elevated levels of specific coding and non-coding RNA and DNA, mutated and amplified oncogene sequences and transposable elements. Thus, tumour microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer.

Figure 1. Analysis of microvesicle profiles and RNA yields from different human cell lines

Microvesicles were isolated from three medulloblastoma cell lines (a, D384, b, D425 and c, D458), one melanoma (d, Yumel 0106), two GBMs (e, 20/3 and f, 11/5) and two normal fibroblasts (g, HF19 and h, HF27) and measured with Nanoparticle Tracking Analysis (NanoSight). The number of particles per cell per 48 h is shown on the y axis, and the size distribution (particle diameter) on the x axis. The sum refers to the total number of particles released per cell over 48 h and the exoRNA refers to the total microvesicle RNA yield per 1×10⁶ cells per 48 h. The results are presented as the mean ± s.e.m. (n = 3).

Figure 2. Medulloblastomas with amplified c-Myc oncogenes have elevated c-Myc exoRNA and exoDNA in their microvesicles

(a) c-Myc amplification levels were quantified in genomic DNA (gDNA) from one normal human fibroblast line (HF19), one GBM line (11/5), one atypical teratoid rhabdoid tumour (AT/RT) line (NS224) and three medulloblastoma (MB) lines (D425, D458 and D384). ExoRNA and exoDNA were also isolated from their corresponding microvesicles. (b) qRT-PCR and (c) qPCR were carried out on nucleic acid from microvesicles from the same cell lines to measure exoRNA and exoDNA, respectively. c-Myc levels were normalized to GAPDH in the same preparation and expressed as fold increase relative to normal fibroblasts. In all cases, values are expressed as mean ± s.e.m. (n = 3) and analysed by two-tailed t-test comparing MB lines to HF19 (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3. Detection of amplified c-Myc sequences in serum microvesicles from tumour-bearing mice

M edulloblastoma (MBT; D425) and epidermoid carcinoma (ECT; A431) cells were used to generate subcutaneous tumours with and without c-Myc amplification, respectively. (a) c-Myc amplification was evaluated on all tumour samples at the RNA level after tumour resection. Values were normalized to GAPDH, presented as fold change compared with epidermoid carcinoma and shown as mean ± s.e.m. (n = 3). (b) ExoRNA was extracted from serum samples from five MBT and five ECT (MBT 1–5 and ECT 1–5, respectively). c-Myc PCR product was amplified using human specific primers. Amplified DNA was resolved by electrophoresis in a 2% agarose gel and visualized with ethidium bromide staining. c-Myc is shown as an 89 bp fragment (arrow). MW, molecular weight; NTC, no template control.

Figure 4. GBM microvesicles are enriched for retrotransposon elements

The levels of transposon and retrotransposon sequences were compared with the rest of the RNA transcriptome in cells and microvesicles. ExoRNA and cellular RNA were isolated from GBM 20/3 cells and analysed on an Agilent two-color 44k array. (a) Relative levels of all represented RNA sequences (44,000 RNA probes) in cells and microvesicles were evaluated with an MA plot; y axis M = log₂Exo−log₂Cell, x axis A = 0.5× (log₂Exo + log₂Cell). RNA intensities from capture probes aligning to (b) DNA transposons, (c) L1, (d) HERV and (e) Alu sequences were extracted and plotted on separate MA plots. (f) Identification of members of the HERV families enriched more than 16-fold in the microvesicles (M≥4). Some HERV families are represented more than once on the array.

Figure 5. Microvesicles are enriched in retrotransposon elements and can mediate horizontal gene transfer

Retrotransposon elements were quantified in cell RNA and exoRNA from three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. The ratio of RNA abundances for (a) L1, (b) Alu and (c) HERV-K in microvesicles versus cells is shown, with RNA levels normalized to the housekeeping gene GAPDH. The relative ratios are presented as the average ± s.e.m. (n = 3–6). Enrichment of retrotransposons in either cells (y < 0) or microvesicles (y > 0) is expressed as log₂ values. (d) HUVECs were exposed to medulloblastoma microvesicles (D384) and their expression level of HERV-K RNA (normalized to GAPDH) was analysed by qRT-PCR over 72 h following exposure and plotted as fold change compared with non-infected cells (MOCK). P-values were calculated using the two-tailed t-test, comparing levels to MOCK infected cells (*P < 0.05, **P < 0.01).

Figure 6. Retrotransposon DNA sequences in microvesicles

Cellular and microvesicle DNA was isolated from three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. qPCR analysis was carried out for (a) L1, (b) Alu and (c) HERV-K in gDNA and exoDNA. Ct values were normalized to GAPDH levels and are shown as relative enrichment of transposons in cells (y < 0) and microvesicles (y > 0). (d) RT activity was measured in the microvesicles using the EnzChek RT Assay Kit (Invitrogen) and normalized to protein content. Results are expressed as average ± s.e.m. (n = 3). (*P < 0.05, **P < 0.01); NS = not significantly different from HF19).