Cancer Relevance of Circulating Antibodies Against LINE-1 Antigens in Humans.

Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant family of autonomous retrotransposons occupying over 17% of human DNA, is epigenetically silenced in normal tissues by the mechanisms involving p53 but is frequently derepressed in cancer, suggesting that L1-encoded proteins may act as tumor-associated antigens recognized by the immune system. In this study, we established an immunoassay to detect circulating autoantibodies against L1 proteins in human blood. Using this assay in >2,800 individuals with or without cancer, we observed significantly higher IgG titers against L1-encoded ORF1p and ORF2p in patients with lung, pancreatic, ovarian, esophageal, and liver cancers than in healthy individuals. Remarkably, elevated levels of anti-ORF1p-reactive IgG were observed in patients with cancer with disease stages 1 and 2, indicating that the immune response to L1 antigens can occur in the early phases of carcinogenesis. We concluded that the antibody response against L1 antigens could contribute to the diagnosis and determination of immunoreactivity of tumors among cancer types that frequently escape early detection. SIGNIFICANCE: The discovery of autoantibodies against antigens encoded by L1 retrotransposons in patients with five poorly curable cancer types has potential implications for the detection of an ongoing carcinogenic process and tumor immunoreactivity.

Inflammatory response to retrotransposons drives tumor drug resistance that can be prevented by reverse transcriptase inhibitors.

Activation of endogenous retrotransposons frequently occurs in cancer cells and contributes to tumor genomic instability. To test whether inhibition of retrotranspositions has an anticancer effect, we used treatment with the nucleoside reverse transcriptase inhibitor (NRTI) stavudine (STV) in mouse cancer models, MMTV-HER2/Neu and Th-MYCN, that spontaneously develop breast cancer and neuroblastoma, respectively. In both cases, STV in drinking water did not affect tumor incidence nor demonstrate direct antitumor effects. However, STV dramatically extended progression-free survival in both models following an initial complete response to chemotherapy. To approach the mechanism underlying this phenomenon, we analyzed the effect of NRTI on the selection of treatment-resistant variants in tumor cells in culture. Cultivation of mouse breast carcinoma 4T1 in the presence of STV dramatically reduced the frequency of cells capable of surviving treatment with anticancer drugs. Global transcriptome analysis demonstrated that the acquisition of drug resistance by 4T1 cells was accompanied by an increase in the constitutive activity of interferon type I and NF-κB pathways and an elevated expression of LINE-1 elements, which are known to induce inflammatory responses via their products of reverse transcription. Treatment with NRTI reduced NF-κB activity and reverted drug resistance. Furthermore, the inducible expression of LINE-1 stimulated inflammatory response and increased the frequency of drug-resistant variants in a tumor cell population. These results indicate a mechanism by which retrotransposon desilencing can stimulate tumor cell survival during treatment and suggest reverse transcriptase inhibition as a potential therapeutic approach for targeting the development of drug-resistant cancers.

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.