Research progress of LINE-1 in the diagnosis, prognosis, and treatment of gynecologic tumors.

The LINES family includes LINE-1, LINE-2, and LINE-3, of which the most abundant and functionally rich is LINE-1 with approximately 500,000 copies, accounting for 17% of the human genome ( 8 ). About 100 of these are still complete and functioning, while the majority have lost retrotransposition owing to deletion, mutation, or truncation ( 4 , 9 ). The full-length LINE-1 sequence is 6000-7000 base pairs (bp) and contains a 5’ untranslated region (UTR), two open reading frames (ORF) ORF1 and ORF2, and a 3’ UTR containing a polyadenylation signal (polyA) ( 10 ). LINE-1 activity is driven by RNA polymerase II binding to the 5’UTR internal promoter, and ORF1 and ORF2 encode two proteins required for reverse transcription, ORF1p, and ORF2p, respectively, ORF1p is an RNA-binding protein with nucleic acid chaperone activity ( 11 ); ORF2p contains nucleic acid endonuclease (EN) and reverse transcriptase (RT) structural domains ( 12 ). Among them, the endonuclease cleaves genomic DNA by specifically recognizing the 5’-TTTT/AA-3’ sequence, and its induced single-stranded DNA break triggers the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) at the LINE-1 integration site to promote reverse transcriptional translocation ( 13 ). Reverse transcriptase activity can use LINE-1mRNA as a template to produce cDNA ( 14 ). ORF1p and ORF2p are translated in the cytoplasm and first bind to LINE-1 mRNA to form the LINE-1RNA/ORF1p/ORF2p ribonucleoprotein (RNP) complex particle ( 15 ), RNP is then transported into the nucleus using the membrane-associated nuclear endosome sorting complex (ESCRT) required for transport ( 16 ), Generation of a new copy of LINE-1 and its insertion into host DNA through a unique mechanism of target-primed reverse transcription (TPRT) ( 17 ) ( Figure 1 ). Additionally, LINE-1 can assist in transposing components without an independent reverse transcription function, such as SINE and ALU ( 18 ). The transposition process of LINE-1. (A) Transcription: The LINE-1 mRNA is produced. (B) Translation: Proteins ORF1p and ORF2p are produced. (C) LINE-1 RNP formation: ORF1p and ORF2p bind to LINE-1 RNA to generate LINE-1 RNP. (D) TPRT: The nucleic acid endonuclease activity and reverse transcriptase activity of ORF2p insert LINE-1 into the new locus through the TPRT process. LINE-1 played an essential role in the evolution of the species, however, in normal somatic cells, the host cell has tight control over LINE-1 translocation to maintain genomic stability ( Table 1 ). The 5’UTR promoter region of LINE-1 is rich in guanine and cytosine enriched regions (cytosine phosphate-guanosine, CpG) ( 28 ), which are often repressed by the organism in normal tissues, while the absence or deficiency of methylation, i.e. CPG hypomethylation, enhances its reverse transcription activity and promotes retrotranscription transposition ( 29 ). Sanchez-Luque et al. reported a conserved Yin-Yang 1 (YY1) transcription factor binding site that mediates LINE-1 promoter methylation in pluripotent and differentiated cells, thereby inhibiting LINE-1 reverse transcriptional translocation ( 19 ). N6-methyladenosine (m6A), the most prevalent methylation modification in eukaryotic mRNA, promotes LINE-1 RNA expression and reverse transcriptional translocation, enhances translation efficiency, and promotes RNP formation ( 20 , 21 ). In addition, oncogenes and tumor suppressor genes are also involved in the regulation of LINE-1. Myc oncoprotein may be a major regulator of LINE-1 transcription during cancer development, Sun et al. found that Myc expression levels significantly and inversely correlated with LINE-1 expression in breast and ovarian tumors ( 22 ). Tumor suppressor P53, which inhibits LINE-1 in cancer tissues derived from human cell cultures by acting on the 5’UTR promoter of LINE-1 and stimulating local deposition of inhibitory histone marks, limits autonomous replication of mobile elements in cells ( 23 ). Mita et al. revealed that the tumor suppressor BRCA1, which is involved in DNA homologous recombination repair (HR), has a strong inhibitory effect on LINE-1 retrotransposition, and in the cytoplasm, BRCA1 inhibits ORF2p translation by binding to its mRNA ( 24 ). Therefore, LINE-1 expression and its activity in an environment of HR dysregulation may increase genomic instability and accelerate tumor development or the formation of drug-resistant clones after treatment. In addition, endogenous proteins also regulate the expression of LINE-1. Liang et al. showed that The apolipoprotein B mRNA-editing catalytic polypeptide 3 APOBEC3 (A3) family, APOBEC3 (A3DE) inhibited LINE-1 reverse transcription translocation by interacting with ORF1p and affecting LINE-1 reverse transcriptase activity ( 25 ). Dual specificity protein phosphatase 1 (DUSP1), is involved in the negative regulation of cell proliferation and suppression of inflammatory responses, and its activity downregulates LINE-1 in cancer cells ( 26 ). The human silencing center (Hush) complex, a newly discovered epigenetic complex, represses LINE-1 expression in the organism, selectively binds evolutionarily young full-length LINE-1 and promotes the deposition of histone H3Lys9 trimethylation (H3K9me3) and the formation of heterochromatin for transcriptional silencing ( 27 , 30 – 32 ). Regulation of LINE-1.

DNA methylation and histone modifications are the main forms of epigenetic modifications, and studies have shown that genome-wide LINE-1 hypomethylation is associated with immune escape features in aneuploid tumors, promotes high mutations and high chromosomal copy number changes, and is an important manifestation of epigenetic abnormalities in a variety of tumors ( 80 ). Studies related to ovarian cancer have shown that LINE-1 hypomethylation directly activates the ATM-MRN-SMC S-phase checkpoint pathway by promoting mRNA expression, genomic DNA rearrangements, and chromatin mutations, leading to double-strand breaks (DSBs) and replication stress, which ultimately cause cancer development ( 67 ). In addition, LINE-1 hypomethylation promotes IL20 expression in cervical cancer, induces altered signaling pathways, and increases cell invasiveness, which is an important epigenetic alteration in several cancers, including gynecologic tumors ( 65 ). Therefore, inhibition of LINE-1 hypomethylation is an effective therapeutic strategy. In a clinical trial, LINE-1 hypomethylation was found to be associated with the progression of oral precancerous lesions to head and neck squamous cell carcinoma (HNSCC), and short-term soy isoflavone intake resulted in a significant increase in tissue-specific overall methylation in patients, suggesting that patients’ LINE-1 hypomethylation levels could be modulated by soy isoflavone supplementation. Also, the association of LINE-1 hypomethylation with genetic instability, tumorigenesis, and prognosis suggests that soy isoflavones may be used as potential cancer-preventive agents ( 81 ). Szabo et al. also found that olive oil (EVOO) improved the hypomethylation of LINE-1 reverse transcription transposon DNA in an environmental carcinogen model compared to controls, resulting in reduced tumor incidence ( 82 ). Therefore, it can be considered that the treatment of gynecological tumors by targeting and regulating LINE-1 hypomethylation. In addition, DNA methyltransferase (DNMT), an important epigenetic molecule that causes DNA methylation, plays an important role in the occurrence and development of various tumors. Zhang et al. showed that in breast cancer cell lines treated with paclitaxel, DNMT3a positively regulated LINE-1 mRNA expression levels by increasing methylation within the LINE-1 gene, while promoting the development of a drug-resistant phenotype in breast cancer ( 83 ). Therefore, DNA methyltransferase inhibitors (DNMTis) can be used to downregulate DNMT3a levels and thus inhibit LINE-1 expression. Thus, DNA methylation alterations also suggest epigenetics in precision immunotherapy. Targeted histone modifications provide another important epigenetic treatment modality. The inhibitory-modified histones (H3K9me3 and H3K9me2) are highly enriched in LINE-1 and silence LINE-1 expression at different stages ( 84 , 85 ). It has been shown that the histone demethylase KDM4B, a novel regulator of LINE-1, activates the reverse transcriptional activity of LINE-1 by catalyzing the demethylation reaction of the repressive histone H3K9me3 leading to DNA damage, and exhibits increased LINE-1 expression and copy number in breast cancers expressing high levels of KDM4B ( 86 ). In addition, hypoxia-induced KDM4B is expressed in approximately 60% of epithelial ovarian cancer (EOC) and positively correlates with the tumor hypoxia marker CA-IX, which is strongly induced in EOC cell lines under hypoxic conditions, and inhibition of KDM4B expression can effectively control ovarian cancer cell invasion and migration in vitro ( 87 ). Therefore, LINE-1 expression in tumors can be inhibited by using histone demethylase inhibitors. Later, Fukuda et al. identified the H3K9 methyltransferase SETDB1 as a key repressor of retrotransposable elements in mouse embryonic stem cells (mESCs), i.e., histone methylation mediates transcriptional silencing of LINE-1 ( 88 ). Taken together, targeting histone demethylation is a major focus of epigenetic therapy for tumors. In addition, the small RNA-mediated pathway governs gene expression in a sequence-dependent manner, inhibiting LINE-1 expression and exerting anti-tumor activity. P-element-induced weak testis protein (PIWI) interacting RNA (piRNA) PIWI-piRNA complex enters the nucleus and targets nascent TE transcripts, recruiting epigenetic silencers, including histone methyltransferases, and even ab initio DNA methylation to maintain transcriptional silencing in the long term ( 89 , 90 ). RNA interference induced the downregulation of LINE-1 expression in mouse models, leading to a significant reduction in tumorigenicity of cancer cells, and this RNA interference also led to post-transcriptional silencing of LINE-1 elements in human melanoma cells and prostate cancer cells ( 91 ). Therefore, RNA interference technology can be considered to reduce the expression level of reverse transcriptase, thus slowing down the proliferation of tumor tissue. The reverse transcriptase (RT) machinery is mainly present within ORF2p, and preclinical studies have shown that RT inhibitors (nevirapine and efavirenz) promote cancer cell differentiation, reduce cell proliferation and antagonize tumor progression in mouse models. Phase II trial species in patients with prostate tumors confirmed the anticancer efficacy of RT inhibitors ( 91 ). Furthermore, clinical studies on lung adenocarcinoma have shown that LINE-1 forms a chimeric transcript LCT (L1-FGGY) with the tumor suppressor gene FGGY via reverse transcriptional translocation, and analysis of its function suggests that LCT deletion activates the arachidonic acid (AA) metabolic pathway and promotes tumor growth, which can be effectively targeted by the combination of anti-HIV drugs (NVR) and metabolic inhibitors (ML355) ( 79 ). These studies suggest that endogenous RT can be considered as an epigenetic regulator of cell differentiation and proliferation and can be a new target for cancer therapy, and RT inhibitors are effective tools for novel anti-cancer, non-cytotoxic therapy. The chaperone ORF1p also causes loss of retrotransposon activity. Recently, Kou et al. provided new insights into the potential of ORF1p expression inhibitors in antitumor therapy by suggesting that ORF1p may have an independent role in driving tumor development and is considered as a cancer marker ( 92 ). They screened LINE-1 ORF1p expression inhibitors in lung cancer cell lines, non-small cell lung adenocarcinoma cell lines, and breast cancer cell lines by efficient high-throughput ICW assays and showed that inhibition of LINE-1 ORF1p expression can affect reverse transcriptional translocation of LINE-1 and inhibit the proliferation of cancer cell lines. These studies point us to the RT and ORF1p encoded by LINE-1 as potential targets in future cancer therapy.

Effective treatment strategies and reliable biomarkers are essential to improve cancer treatment. Currently, among all gynecologic tumors, endometrial and cervical cancers are more often diagnosed at early stages due to different symptoms and screening tools, while ovarian cancer still faces great challenges in its diagnosis and treatment due to the lack of obvious early symptoms. Therefore, the development of effective early diagnostic markers is of great importance for the early detection of cancer. In recent years, The expression of LINE-1 in tumors has aroused particular interest in the scientific community, and numerous studies have shown that LINE-1, as the only autonomously mobile transposon in the human genome, plays an important role in tumorigenesis and development due to its reverse transcriptional activity leading to genomic instability. LINE-1 hypomethylation is an early stage in gynecological tumors such as ovarian, cervical, and endometrial cancers events, and can be used as a molecular marker for early diagnosis and prognosis determination. In addition, ORF1p generated by LINE-1 retrotransposition is highly expressed in ovarian, endometrial, and differentiated vulvar epithelial cancers, and it has been noted that ORF1p is expressed in 90% of HGSOC and 90% of STIC, but is undetectable in 99% of controls; therefore, plasma ORF1p appears to be a highly specific cancer biomarker in gynecologic cancers as well as in high-risk precursor lesions and has important value for tumor prognosis ( 93 ). In summary, exploring the function and mechanism of LINE-1 in gynecologic tumors will provide a novel direction for targeted cancer therapy. Its expression in gynecologic tumor tissues such as ovarian cancer, cervical cancer, and endometrial cancer suggests that it is expected to be a novel molecular marker for early diagnosis and prognosis of gynecologic tumors and a potential therapeutic target. However, the mechanism of LINE-1 in the development of gynecologic tumors has not been fully understood, and few clinical trials have been reported on LINE-1 as a therapeutic target in gynecologic tumors. Therefore, in-depth studies on the mechanism of LINE-1 in gynecologic tumors and related clinical studies are valuable for the early diagnosis of tumors and also help us to find more effective treatments. In conclusion, the use of LINE-1 as a biological agent in gynecologic tumors is of great value. In conclusion, the use of LINE-1 as a biomarker has generated considerable excitement in the scientific community, and it is reasonable to expect that in the coming years, LINE-1 may become a useful biomarker for early diagnosis and prognosis of gynecologic tumors, and research on LINE-1 is expected to become a new strategy for effective treatment of gynecologic tumors.

JF, TQ, CL, JZ, YD, and MZ performed literature searches. JF, and TQ wrote the first draft of the manuscript. FC, QY, XL, and JZ wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.