LINE-1 Cargo and Reverse Transcriptase Activity Profiles in Extracellular Vesicles from Lung Cancer Cells and Human Plasma

Abstract

Long Interspersed Element-1 (LINE-1) is an oncogenic human retrotransposon that ‘copies and pastes’ DNA into new locations via reverse transcription. Given that enzymatically active LINE-1 can be exported in extracellular vesicles (EVs), and that LINE-1 mRNA and its two encoded proteins, ORF1p and ORF2p, are required for retrotransposition, the present study examined LINE-1 EV loading patterns relative to reverse transcriptase (RT) activity in vivo and in vitro. Density gradient ultracentrifugation identified conserved patterns of LINE-1 mRNA and protein distribution in EVs, with RT activity readily detected in EV fractions containing both LINE-1 mRNA and protein. Unlike whole cell and tissue lysates, the ORF1p in EVs was detected as a dimer. EVs from ostensibly healthy plasma donors showed variable but consistent ORF1p profiles, with residual levels of LINE-1 mRNA measured in some but not all samples. EVs from cancer cell lines had elevated mean LINE-1 levels and 5–85 times greater RT activity than EVs from normal cells or healthy plasma. EV RT activity was associated with EV LINE-1 mRNA content and was highest in cell lines that also expressed an elevated expression of ORF1p and ORF2p. Given that LINE-1 activation is a hallmark of many cancer types, our findings suggest that an EV LINE-1 ‘liquid biopsy’ may be developed to monitor LINE-1 activity during the course of malignant progression.

Long Interspersed Nuclear Element-1 Analytes in Extracellular Vesicles as Tools for Molecular Diagnostics of Non-Small Cell Lung Cancer

“…To characterize EVs released by H520 lung cancer cells, EVs were isolated by PEG precipitation of conditioned media for 48 h. H520 cells have high constitutive expression of LINE-1 [24]. EV isolates ranged in diameter from 50 to 225 nm, with most particles concentrating at ~80 or 110 nm (Figure 1B), a range consistent with EVs of endocytic origin, mainly exosomes.…”

“…EVs are released by nearly all cell types and can be collected from saliva, blood, urine, and cerebrospinal fluid [21]. While the presence of LINE-1 products in EVs has been documented by us and others [22][23][24], detailed analyses to determine if LINE-1 products in EVs can be used for cancer detection and future development of point-of-care diagnostics have not been systematically completed.…”

Reactivated endogenous retroviruses promote protein aggregate spreading

“…Still, also HERVs have been shown to produce viral-like particles in cell culture, cancer, and autoimmune diseases 58 , 59 . For example, RNA and protein transcribed from LINE-1 retroelements are packaged into EVs 60 . HERV-W Syncytin-1 is present on EVs derived from placenta 61 .…”

Endogenous retroviruses promote prion-like spreading of proteopathic seeds

“…Still, also HERVs have been shown to produce viral-like particles in cell culture, cancer and autoimmune diseases (67)(68)(69). For example, RNA and protein transcribed from LINE-1 retroelements are packaged into EVs (70). HERV-W Env Syncytin-1 is present on EVs derived from cutaneous T cell lymphoma, BeWo cells and placenta (71,72).…”