A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

Ilaria Sciamanna,Corrado Spadafora,Emanuele Felice Osimo,Gianni Prosseda,Eleonora Aricò,Cristina Cossetti,Manuela Ferracin,Gianfranco Macchia,Patrizia Vitullo,Tom Misteli,Alberto Gualtieri

doi:10.18632/oncotarget.1403

Abstract

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

Highlights

Evidence that the genome is pervasively transcribed, including in its non-coding component, is an established finding [1] and is changing our views of global mechanisms of regulation of genome function [2]
In past work we found in time-course assays that exposure of cancer cell lines to reverse transcriptase (RT) inhibitors reduced proliferation and promoted differentiation after 4-5 days of treatment [22; 23]; prolonged treatment maintained these features, whereas the cells quickly returned to their original conditions upon discontinuation of the treatment [23]
As newly reverse-transcribed cDNA copies would be virtually undistinguishable from the original DNA templates, we focused on the production of intermediate RNA:DNA hybrid products as a read-out of RT activity: we reasoned that such hybrid molecules might form preferentially in tumor cells, where LINE-1 encoded RT activity is higher compared to normal cells [21 and Fig. 1B]

Summary

Introduction

Evidence that the genome is pervasively transcribed, including in its non-coding component, is an established finding [1] and is changing our views of global mechanisms of regulation of genome function [2]. Full-length LINE-1 elements encode their own reverse transcriptase (RT) enzyme, responsible for retrotranscription and, RNA-dependent mobilization of both LINE-1 elements themselves and of non-autonomous Alus Both families influence the transcriptional output of the genome and growing studies highlight roles of both families in tumorigenesis [6,7,8]. EFV has anti-cancer efficacy in vivo and antagonizes the development of human tumors xenografted in nude mice [23] These results i) identify active LINE-1 retrotransposon families as the major source of functional RT in transformed cells, and ii) indicate that LINE-1-encoded RT might be regarded as a novel target in cancer differentiation therapy. In contrast with the empirical efficacy of RT inhibitors, the molecular role of RT in cancer genomes is still elusive

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