LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Abstract

Background: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results:We report mass spectrometry based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically-acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions:Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth. References

Proteogenomics analysis of non-coding region encoded peptides in normal tissues and five cancer types

“…In comparison, ORF2p peptides were rarely detected. It corroborates findings from an antibody-based study which concluded that LINE-1 ORF2p expression is hardly detectable in human cancers 19 . Surprisingly, we also detected peptides of LINE-1 ORF1 in healthy tissues, including lung, ovary and prostate (supplementary table S5).…”

Measuring and interpreting transposable element expression

“…This proteomic approach has high potential to identify biologically active proteins derived from TEs and to provide an overview of the transposition activity in a given condition or sample (reviewed in 193 ). Similarly, mass spectrometry approaches and mining mass-spectrometry databases has permitted to validate the presence of predicted chimeric TE-derived peptides in tumours or primate embryonic stem cells 153 or to confirm L1 expression in human cancers 194 .…”

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker

“…We demonstrate that ORF1p is an early indicator of chemotherapeutic response in gastric and esophageal cancers at timepoints where other parameters are often ambiguous, opening possibilities for monitoring minimal residual disease or relapse. Importantly, ORF1p appears to provide a level of specificity for cancers not achieved by other protein biomarkers, likely due to the unique biology of the retrotransposon, with repression of L1 in normal somatic tissue (9,13,14). ORF1p is therefore attractive as a putative "binary" cancer biomarker, in which a positive signal is highly specific for disease, with diagnostic utility both in tissue and plasma.…”

“…L1 expression is repressed in normal somatic tissues, resulting in either very low or undetectable levels of L1 RNA and protein that appear to originate from epithelium (9,14). Epigenetic dysregulation of L1 and L1 ORF1p overexpression begin early in carcinogenesis, and histologic precursors of ovarian, esophageal, colorectal, and pancreatic cancers studied all express ORF1p at varying levels (8,15).…”