Telomerase Transcripts Floating in Cancer- Derived Exosomes Reprogram the MicroRNA Transcriptome Profile of the Recipient Cells

Background: Previously, we showed that hTERT transcripts are exported in exosomes to various types of cancer derived cells. These transcripts, formed almost exclusively in neoplastic cells, are subsequently taken up by non-neoplastic cells and alter their phenotype. How neoplastic cells modify recipient cells is currently unknown, but since hTERT has been shown to regulate microRNA expression, we hypothesized that hTERT(⁺) exosomes alter the microRNA profile of recipient cells.

Aims: 1) To verify that hTERT(⁺) exosomes are taken up by fibroblasts.

2) To determine whether exposure to hTERT(⁺) exosomes alter the microRNA profile recipient cells.

3) To determine whether the phenotype observed in recipient cells is driven by reprograming of the microRNA transcriptome.

Methods: We grew cells derived from T-cell leukemia cell line (Jurkat cells) in exosome free medium and isolated the exosomes after 72 hours by serial ultracentrifugation. Subsequently we verified that Jurkat cells shed exosomes at large quantities by NanoSight tracking device. We then stained the exosomes with FM-134, exposed foreskin derived fibroblasts to these exosomes and used flow cytometry to determine the time of maximal exosomal uptake. In addition we verified the uptake of the hTERT transcripts via exosomes by Q-PCR. We then extracted the RNA from recipient cells using RNeasy and performed high throughput analysis of the microRNA transcriptome (Rosetta Genomics, Rehovot, Israel). We transfected the fibroblasts with a microRNA mimic harboring the sequence of microRNA 342 and studied proliferation (by the SRB method) and cells cycle rate (by PI) in the treated cells. In addition we have used ANAT software (Advanced Network Analysis Tool) to detect the protein- protein interactions between the targets of microRNA 342 and telomerase.

Results: As we previously showed, Jurkat-cell derived exosomes carry the hTERT transcripts. These exosomes were taken up by fibroblast in a time and dose dependent manner. By microarray we detected seven microRNAs that were more than 2 folds upregulated and two microRNAs that were more than 2 folds downregulated after exosomal exposure. The overexpression of these microRNAs was verified by Q-PCR. Furthermore, after transfection of naïve fibroblasts with miR342, we observed a marked increase in proliferation rate and a significant decrease in apoptotic rates, recapitulating the phenotype of fibroblasts following exposure to hTERT(⁺) exosomes.

Conclusion: Our study shows that CLL-derived hTERT(⁺) exosomes alter the microRNA transcription profile of recipient fibroblasts and that these changes contribute to the telomerase- dependent aggressive phenotype of the naïve fibroblasts.