**4. Implementation of REs for cancer diagnosis and prognosis**

### **4.1 Structural variations (SVs) associated with REs in cancer**

Identification of somatic mutation hotspots associated with cancer is very important for functional analysis and diagnosis [137]. Several methods have been developed for the identification of somatic RE insertions in cancers (L1-seq, TIPseq, and ERVcaller), and many bioinformatics tools to discover somatic L1 insertions in silico using WGS or WES data have been developed [138, 139]. SVs via L1 insertion associated with cancer have been well investigated in a couple of genes, such as the APC gene that is considered to be a tumor suppressor of colorectal polyposis in colorectal cancer [89]. A potential suppressor of L1, TP53 mutation by L1 insertions, has been observed frequently in tumors. In addition, L1 insertional mutation of MOV10, which is a key L1 suppressor, decreased the expression of the MOV10 in tumors with high L1 insertions [140].

On the other hand, instead of cancer-associated SVs caused by RE insertion, genome variations that might be associated with HERVs or around gene expression in cancer have been identified. Chang et al. identified that four HERVs with mutation hotspots overlapped with exons of four human protein coding genes, which are TNN (HERV-9/LTR12), OR4K15 (HERV-IP10F/LTR10F), ZNF99 (HERV-W/ HERV17/LTR17), and KIR2DL1 (MST/MaLR). They also evaluated the effect of each non-synonymous SNV on the survival of kidney cancer patients. Furthermore, they identified 788 HERVs harboring significantly increased the numbers of somatic single-nucleotide variations (SNVs) [141].

*Methods in Molecular Medicine*

repressive proteins to the p21 promoter. The Alu sequence is crucial to the localization of APTR on the p21 promoter that regulates cell growth and proliferation [109]. Despite the limited contribution of L1 and Alu to lncRNAs, a close association between HERVs and ncRNAs was reported by Kelley and Rinn [110]. Hundreds of ncRNAs originated from HERV-H. For example, the lncRNA ROR known to promote the progression of human cancers is one of the ncRNAs promoted by a HERV-H element [111]. Moreover, the lncRNA produced by HERV-K11 directly binds to polypyrimidine tract-binding protein-associated splicing factor (PSF), of which the function is to repress proto-oncogene transcription, reversing the PSF-mediated repression of proto-oncogene transcription and subsequently driving tumorigenesis [46, 112]. Other HERV-related lncRNAs with tumor-suppressive potential have also been identified in the intronic RNAs arising from ERV-9 [45]. It has been reported that its antisense RNA at 3′-untranslated regions was found to physically bind to key transcription factors for cell proliferation such as NF-Y, p53, and sp1. This means that the HERV-related lncRNAs may have a function as decoy targets or traps for the

transcription factors resulting in the growth retardation of cancer cells [113].

**3.4 RE proteins associated with tumorigenesis**

Another role of RE transcripts related to human disease is to form a complex with the cytoplasmic cDNA of the reactivated RE transcripts to trigger the signal of the inflammatory pathway [23]; for example, RE-derived cytosolic DNA accumulated in Aicardi-Goutières syndrome (AGS) [114]. IFNB1 expression also has an anticorrelation with L1 retrotransposition in cancer cells [115]. Moreover, the study by Ishak et al. showed that mutation of the RB1 gene causes both genome-wide upregulation of L1 expression in somatic cells as well as increased susceptibility to leukemia [116]. Gasche et al. reported that the IL-6 treatment of a cancer cell line induced genome-wide L1 promoter hypomethylation [117]. Altogether, the evidence indicates that REs modify an important aspect of human tumorigenesis.

ORF1 and ORF2 in L1 and GAG, POL, and ENV in HERV are proteins encoded by REs that are essential to complete the replication cycle, whereas Alu's are RNA polymerase III-transcribed sequences without coding potential [118]. Most REs lose their coding potential due to accumulated mutations; however, it is well known that hundreds of L1 are still active to produce two essential proteins, ORF1 (p40, RNA binding protein) and ORF2 (p109, endonuclease and reverse transcriptase activities) [119, 120]. Additionally, although no infectious virus formed by HERVs is reported, multiple protein expressions and their functions have been studied in various HERV families [46]. Most comprehensive studies have reported on envelop proteins (ENV) and their pathogenic properties. The transcripts encoding capsid and protease (GAG) and reverse transcriptase with RNase H domain and integrase (POL) ORFs have been detected in many cells and tissues from both diseased and healthy individuals [121]. Remarkably, HERV-W encodes an ENV protein known as ERVWE1 (Syncytin1), which has been adopted by the human to functionally contribute in placenta biogenesis [122]. Similarly, Syncytin2 encoded by ERVFRD1 is known to have a key role in the implantation of human embryos [123]. Aberrant expression of HERV-W has been known to be associated with various human diseases including cancer [122, 124, 125]. In cancer, an increase in retroviral protein expression was generally detected. Overexpression of L1 ORF1 protein was detected from more than 90% of breast, ovarian, and pancreatic cancers followed by tubular gastrointestinal tract, lung, and prostate cancers (about 50%) [126, 127]. However, the high expression of L1 ORF1p expression is dependent on tumor origin, and it differs case by case even within a similar histological type of cancer. For example, L1 ORF1p is detected in lung adenocarcinoma at greatly varying levels (about 20% are very high, about 30% are

**86**

#### **4.2 Global hypomethylation in cancer and identification of cancer associated RE methylation**

Several studies have shown that global hypomethylation is very common in cancer [142]. The DNA methylation levels of L1 5′-untranslated region (UTR) in cancer have been extensively evaluated for potential use as an epigenomic marker for cancer diagnosis. The level of L1 hypomethylation increases in more advanced cancers; however, other types of REs, such as Alu and HERVs, have been lesser evaluated [143]. Since DNA methylation analysis has some benefits in handling tumor specimens, such as similar efficiency in fresh frozen and formalin-fixed paraffin-embedded tissue, many studies indeed have proposed DNA methylation as a diagnostic marker using fresh tumor biopsies or fixed tissue blocks [144]. Association between L1 hypomethylation and diagnostic and prognostic needs, such as tumor stage group, metastasis, the recurrence rate, and the survival rate, has been studied [145]. Also, L1 hypomethylation has been demonstrated to be a surrogate marker for predicting the response to cancer treatment [146]. Moreover, L1 hypomethylation is observed in very different types of specimen, including blood leukocyte DNA, serum, and oral rinse [147]. Hypomethylation of Alu was reported in several cancers, whereas hypomethylation of HERV-K and HERV-W genomes were found in urothelial cancer and ovarian cancer, respectively [47, 131, 148].

Classically, CpG methylation analyses have been performed in targeted sequence by discriminating between methylated and unmethylated DNA using bisulfite treatment followed by PCR amplification [149]. Although recent nanopore technology can separate between methylated and unmethylated DNA without any treatment, most analyses are usually based on methylation-specific PCR after bisulfite treatment (MSP) [150, 151]. Pyrosequencing-based analysis, specifically methylation-sensitive single-nucleotide-primer extension (MsSNuPE) and Methylight, is a promising method that can be used to reliably measure L1 methylation in paraffinembedded cancer tissues with higher reproducibility [152]. Using this method, L1 hypomethylation has been tested in various human cancer patients, including gastric cancer, colon cancer, colorectal cancer, melanoma, and breast cancer, and its clinical implications have been suggested [153]. Recent studies have addressed that methylated L1 in circulating cell-free DNA (cfDNA) can be used as a potential prognostic and diagnostic target in cancers, and have promoted its potential as a minimally invasive screening technique. Lee et al. showed L1 hypomethylation in cfDNA of both human breast cancer and dog mammary tumor [154, 155].

Unfortunately, there are not many products in the marketplace that capitalize on the association between RE hypomethylation and diverse cancer types and features, even though many studies have provided evidence for it. Representatively, the only clinical test targeting methylation of L1 is used in the detection of bladder cancer in voided urine [156].

#### **4.3 RE transcripts in cancer diagnosis**

First of all, the quantitation of various HERV gene expressions was performed using a real-time PCR. The transcript expression of HERV-H, -K, -P, and -R ENV was significantly increased in the blood of lung cancer patients, and the level was generally much higher in the squamous cell carcinoma (SCC) subtype than the small-cell lung cancer (SCLC) subtype [157]. The level of HERV-K (HML-2) was found to be an independent prognostic factor for the overall survival rate of hepatocellular carcinoma patients [158]. The expression of HERV-H LTR-associating protein 2 (HHLA2) was significantly upregulated in bladder cancer, and it was suggested as a prognostic factor of tumor metastasis and poor survival of bladder

**89**

challenges to measure it in clinic.

**5. RE in cancer therapy**

patients [11].

*Endogenous Retroelements in Cancer: Molecular Roles and Clinical Approach*

cancer patients [159]. The elevated HERV-K (HML-2) was detected in both protein and transcripts level in the blood of breast cancer patients at an early stage and was further increased with developing metastasis. Thus, HERV-K (HML-2) expression will be one of a best candidate for the early detection of an increased risk for breast cancer in women [160]. The expression of HERV-E transcripts is observed in von Hippel-Lindau (VHL)-deficient renal carcinomas. Interestingly, the introduced VHL gene suppressed HERV-E expression in VHL-deficient carcinoma [11]. In addition, high blood levels of the ENV transcripts of various HERV types have been detected in breast cancer patients and that are decreased by treatment of adjuvant chemotherapy which means that alteration of blood HERV transcripts is a very good candidate for diagnosis and is a prognosis marker of breast cancer [132].

A correlation between HERV protein expression and human cancer has been described [11]. HERV proteins, GAG, POL, and ENV, have been identified in cancer tissues, and several factors from environment and hormone response, such as UV radiation, inflammation, estrogen and smoking, have been proposed as a cause of HERV protein expression in various cancer tissues [161]. Remarkably, the envelop protein, ENV, of HERV-K has been identified in melanoma by immunohistochemistry [162]. In melanomas, the expression of HERV-K ENV is higher than that in benign lesions, especially in metastatic tumors. Moreover, it has also been found in other types of cancers, such as breast, ovarian, and bladder cancer. Antibodies targeting HERV-E, HERV-K (HML-2), and ERV3 have also been detected in more than 30% of ovarian cancer patients and are higher in patients with lymph-node-positive breast tumors. In addition, the presence of serum antibodies against HERV-K proteins has been suggested as a prognostic factor for poor survival of melanoma

In L1 proteins, high levels of ORF1 protein was prevalent in certain cancers, including breast and ovarian cancer, whereas no or little expression was detected from other cancers such as renal, liver, and cervical cancer [36]. Rodic et al. and Ardeljan et al. separately detected ORF1 protein via IHC in ~90% of ovarian cancer and in ~90% of the breast cancer samples examined [127, 163]. Chen et al. reported that the ORF1 protein level is very high in ductal carcinoma in situ (DCIS) [164]. Moreover, the ORF1 level was the highest in high-grade ovarian carcinoma, but the expression of ORF1 in prostate cancer has not been fully confirmed [36]. Ardeljan et al. reported ORF1 positivity in ~41% of all prostate cancer tissue samples examined [163]. ORF1 levels could be clinically measured using CT scans on the blood of lung cancer patients. On the other hand, ORF2 has only been limitedly tested as a diagnostic marker for cancer when compared to ORF1 expression. However, since ORF2 encodes a reverse transcriptase that is heavily associated with L1 activity, similar to L1 hypomethylation, it may yet be a better diagnostic marker for L1-associated disease development. High expression of ORF2 in transitional colon mucosa but no expression in normal colon mucosa was detected via IHC. ORF2 was also detected in prostate intraepithelial neoplasia [36]. However, since the ORF2 expression has been reported to be much less than that of ORF1, there are

Aberration of RE activities in various aspects has been suggested as a potential

target for cancer therapy [165]. Several studies have shown that inhibiting RT

*DOI: http://dx.doi.org/10.5772/intechopen.93370*

**4.4 Detection of RE proteins in cancer specimens**

*Endogenous Retroelements in Cancer: Molecular Roles and Clinical Approach DOI: http://dx.doi.org/10.5772/intechopen.93370*

cancer patients [159]. The elevated HERV-K (HML-2) was detected in both protein and transcripts level in the blood of breast cancer patients at an early stage and was further increased with developing metastasis. Thus, HERV-K (HML-2) expression will be one of a best candidate for the early detection of an increased risk for breast cancer in women [160]. The expression of HERV-E transcripts is observed in von Hippel-Lindau (VHL)-deficient renal carcinomas. Interestingly, the introduced VHL gene suppressed HERV-E expression in VHL-deficient carcinoma [11]. In addition, high blood levels of the ENV transcripts of various HERV types have been detected in breast cancer patients and that are decreased by treatment of adjuvant chemotherapy which means that alteration of blood HERV transcripts is a very good candidate for diagnosis and is a prognosis marker of breast cancer [132].

#### **4.4 Detection of RE proteins in cancer specimens**

A correlation between HERV protein expression and human cancer has been described [11]. HERV proteins, GAG, POL, and ENV, have been identified in cancer tissues, and several factors from environment and hormone response, such as UV radiation, inflammation, estrogen and smoking, have been proposed as a cause of HERV protein expression in various cancer tissues [161]. Remarkably, the envelop protein, ENV, of HERV-K has been identified in melanoma by immunohistochemistry [162]. In melanomas, the expression of HERV-K ENV is higher than that in benign lesions, especially in metastatic tumors. Moreover, it has also been found in other types of cancers, such as breast, ovarian, and bladder cancer. Antibodies targeting HERV-E, HERV-K (HML-2), and ERV3 have also been detected in more than 30% of ovarian cancer patients and are higher in patients with lymph-node-positive breast tumors. In addition, the presence of serum antibodies against HERV-K proteins has been suggested as a prognostic factor for poor survival of melanoma patients [11].

In L1 proteins, high levels of ORF1 protein was prevalent in certain cancers, including breast and ovarian cancer, whereas no or little expression was detected from other cancers such as renal, liver, and cervical cancer [36]. Rodic et al. and Ardeljan et al. separately detected ORF1 protein via IHC in ~90% of ovarian cancer and in ~90% of the breast cancer samples examined [127, 163]. Chen et al. reported that the ORF1 protein level is very high in ductal carcinoma in situ (DCIS) [164]. Moreover, the ORF1 level was the highest in high-grade ovarian carcinoma, but the expression of ORF1 in prostate cancer has not been fully confirmed [36]. Ardeljan et al. reported ORF1 positivity in ~41% of all prostate cancer tissue samples examined [163]. ORF1 levels could be clinically measured using CT scans on the blood of lung cancer patients. On the other hand, ORF2 has only been limitedly tested as a diagnostic marker for cancer when compared to ORF1 expression. However, since ORF2 encodes a reverse transcriptase that is heavily associated with L1 activity, similar to L1 hypomethylation, it may yet be a better diagnostic marker for L1-associated disease development. High expression of ORF2 in transitional colon mucosa but no expression in normal colon mucosa was detected via IHC. ORF2 was also detected in prostate intraepithelial neoplasia [36]. However, since the ORF2 expression has been reported to be much less than that of ORF1, there are challenges to measure it in clinic.

#### **5. RE in cancer therapy**

Aberration of RE activities in various aspects has been suggested as a potential target for cancer therapy [165]. Several studies have shown that inhibiting RT

*Methods in Molecular Medicine*

**RE methylation**

**4.2 Global hypomethylation in cancer and identification of cancer associated** 

Several studies have shown that global hypomethylation is very common in cancer [142]. The DNA methylation levels of L1 5′-untranslated region (UTR) in cancer have been extensively evaluated for potential use as an epigenomic marker for cancer diagnosis. The level of L1 hypomethylation increases in more advanced cancers; however, other types of REs, such as Alu and HERVs, have been lesser evaluated [143]. Since DNA methylation analysis has some benefits in handling tumor specimens, such as similar efficiency in fresh frozen and formalin-fixed paraffin-embedded tissue, many studies indeed have proposed DNA methylation as a diagnostic marker using fresh tumor biopsies or fixed tissue blocks [144]. Association between L1 hypomethylation and diagnostic and prognostic needs, such as tumor stage group, metastasis, the recurrence rate, and the survival rate, has been studied [145]. Also, L1 hypomethylation has been demonstrated to be a surrogate marker for predicting the response to cancer treatment [146]. Moreover, L1 hypomethylation is observed in very different types of specimen, including blood leukocyte DNA, serum, and oral rinse [147]. Hypomethylation of Alu was reported in several cancers, whereas hypomethylation of HERV-K and HERV-W genomes were found in urothelial cancer and ovarian cancer, respectively [47, 131, 148].

Classically, CpG methylation analyses have been performed in targeted sequence

by discriminating between methylated and unmethylated DNA using bisulfite treatment followed by PCR amplification [149]. Although recent nanopore technology can separate between methylated and unmethylated DNA without any treatment, most analyses are usually based on methylation-specific PCR after bisulfite treatment (MSP) [150, 151]. Pyrosequencing-based analysis, specifically methylation-sensitive single-nucleotide-primer extension (MsSNuPE) and Methylight, is a promising method that can be used to reliably measure L1 methylation in paraffinembedded cancer tissues with higher reproducibility [152]. Using this method, L1 hypomethylation has been tested in various human cancer patients, including gastric cancer, colon cancer, colorectal cancer, melanoma, and breast cancer, and its clinical implications have been suggested [153]. Recent studies have addressed that methylated L1 in circulating cell-free DNA (cfDNA) can be used as a potential prognostic and diagnostic target in cancers, and have promoted its potential as a minimally invasive screening technique. Lee et al. showed L1 hypomethylation in

cfDNA of both human breast cancer and dog mammary tumor [154, 155].

Unfortunately, there are not many products in the marketplace that capitalize on the association between RE hypomethylation and diverse cancer types and features, even though many studies have provided evidence for it. Representatively, the only clinical test targeting methylation of L1 is used in the detection of bladder cancer in

First of all, the quantitation of various HERV gene expressions was performed using a real-time PCR. The transcript expression of HERV-H, -K, -P, and -R ENV was significantly increased in the blood of lung cancer patients, and the level was generally much higher in the squamous cell carcinoma (SCC) subtype than the small-cell lung cancer (SCLC) subtype [157]. The level of HERV-K (HML-2) was found to be an independent prognostic factor for the overall survival rate of hepatocellular carcinoma patients [158]. The expression of HERV-H LTR-associating protein 2 (HHLA2) was significantly upregulated in bladder cancer, and it was suggested as a prognostic factor of tumor metastasis and poor survival of bladder

**88**

voided urine [156].

**4.3 RE transcripts in cancer diagnosis**

activity is a great therapeutic target for cancer. Sciamanna et al., 2005, uncovered that pharmacologic L1 inhibition by two reverse transcriptase inhibitors slows down the growth of malignant melanoma and prostatic cancer [166]. Carlini et al. similarly demonstrated the efficacy of reverse transcription inhibition of prostate cancer growth [167]. Furthermore, a clinical trial showed that Efavirenz, a nonnucleoside reverse transcriptase inhibitor (NNRTI), provides a therapeutic benefit by increasing the progression free survival in a high-stage castration-resistant prostate cancer cohort [168]. Recently, Efavirenz has been shown to suppress L1 activity and promote morphological differentiation in melanoma cells [169]. On the other hand, another class of RT inhibitor, the nucleoside reverse transcriptase inhibitor (NRTI), has also been shown to suppress L1 activity and induce anticancer activity in prostate cancer cell lines. Importantly, no significant effects were observed in normal cells [167]. Despite these successful findings, it is still unclear regarding the molecular function of RT inhibition in the gene expression regulation.

RNA interference (RNAi)-mediated downregulation of L1 generated identical effects to those observed with RT inhibitory drugs in human melanoma, which indicates that RT activity has a crucial role in L1 activity in human cancer [170]. Recently, a phase II human trial using Efavirenz on a cohort of metastatic patients with prostate cancer showed nonprogression when Efavirenz reached an optimal concentration in the blood [171]. Altogether, preclinical and clinical data provide evidence that RT inhibition is a potentially effective tool in a novel anticancer therapy against diverse human cancers with noncytotoxic effects on non-cancer cells [172].

Another approach regarding REs is an immunotherapy approach to target the pro-oncogenic effects of HERV ENV, which is possibly involved in tumor progression and in downstream metastatic spread, in a number of tissues. HERV ENVs exclusively upregulated in tumor tissues will be suitable targets to direct both passive and active immunotherapy against in cancer cells [130]. The antibodies recognizing the HERV ENVs has been developed, and currently, a monoclonal antibody against HERV-K (HML2) ENV successfully inhibits human breast cancer proliferation, with the activation of apoptosis [173]. On the other hand, various HERV-derived ENVs have been investigated as candidates of anticancer immunotherapy, either as tumor-associated or tumor-specific antigens in cancer cells [130]. ERVs were first used for antitumor immunization in the murine cancer models expressing ERV [9]. Similarly, in humans, protective immunity against the HERV-K MEL antigen in melanoma development has been investigated. This active immunotherapy is considered more advantageous with respect to passive immunization [130]. However, despite the antigenic similarity between HERV-K-MEL and yellow fever virus (YFV), no significant protective effects were shown in the 10 years post-anti-YFV vaccinations in the melanoma cohorts [174, 175]. HERV-H ENV (Xp22.3) is an another antigen significantly upregulated in a subset of gastrointestinal cancers. T cells that was sensitized to HERV-H ENV (Xp22.3) had lytic effects against colorectal cancer expressing the ENV. HERV-E ENV showed similar effects in renal carcinoma [130, 176].

In addition, demethylating drugs are commonly used as anticancer agents and are known to trigger RE reexpression [177]. Interestingly, DNA methyl transferase inhibitors are caused by immune attacks that increase the expression of HERV and thereby increase the viral dsRNA [178]. Accordingly, individual knocking down of MDA5, MAVS, or IRF7 inhibits the ability of DNA methyl transferase inhibitor to target colorectal cancers resulting in significantly reduced the anticancer activity [179]. Altogether, immunotherapy approaches targeting HERV ENV in a broad spectrum of cancers might be valuable for the expansion of target cancers and for use with other cancer therapies.

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*Endogenous Retroelements in Cancer: Molecular Roles and Clinical Approach*

In this chapter, we reviewed and summarized the functions and regulatory mechanisms of retroelements in the development and progression of cancers, and further presented applications in the development of diagnosis and treatment targets using these characteristics (**Table 1**). We looked at the retrovirus as a functional genomic element that forms the genome, not as an ancient infected virus and its useless remnants. Reactivation of retroelements means that it affects various regulation processes of cells beyond not only controlling the functions of surrounding genes but also increasing the protein formed therefrom or its function, or prompting its reinsertion into a new position. Because of this, it is very important to analyze and understand retroelements' functions with regard to various target substances, for example, miRNA, transcription factors, epigenetic modifiers, and

**RE type Cancer type Experimental technique References**

L1 Prostate cancer Southern blot analysis [49]

HERV-K Breast cancer RT-PCR, northern blot, in situ hybridization [51] HERV-K Breast cancer TCGA RNA-seq, RPPA data anaylsis [52] HERV-K Melanoma IHC, immunoblotting [53]

assay HERV-K Kidney cancer RT-PCR, northern blot [54] HERV-K Head and neck cancer Microarray [34] LI Ovarian cancer Southern hybridization, RT-PCR [57] HERV-W Ovarian cancer Southern hybridization, RT-PCR [57]

HERV-K Urothelial carcinoma RT-PCT, bisulfite-pyrosequencing [60] L1 Urothelial carcinoma RT-PCT, bisulfite-pyrosequencing [60]

HERV-K Prostate cancer RT-PCR, bisulfite-pyrosequencing, ChIP [64]

L1 11 types of cancer Whole genome, exome sequencing [94] L1 Lung, brain cancer L1-seq [97]

(RC-seq)

L1 Breast cancer Absolute quantitative assessment of

HERV-K Pancreatic cancer RT-PCR, IHC, IF, ELISA, female

L1 Liver cancer Retrotransposon capture sequencing

L1 Colon cancer Southern blot, isolation of the fragment

LI Colorectal, blood, brain,

prostate, ovarian cancer

Alu Colon cancer Next-generation sequencing of unmethylated Alu

MCA/CpG island microarray

Cell fusion-dependent colony formation

methylated alleles (AQAMA) PCR

Tea (TE analyzer) from paired-end, whole-

immunodeficient nude

genome sequencing

containing the insertion

Bisulfite-pyrosequencing [45]

Bisulfite-pyrosequencing [48]

[50]

[58]

[61]

[96]

[95]

[98]

*DOI: http://dx.doi.org/10.5772/intechopen.93370*

**6. Conclusions**

so on (**Figure 1**).

L1 Colon, breast, lung,

L1 Intrahepatic

ovarian, prostate cancer

cholangiocarcinoma
