Noncanonical functions of telomerase and telomeres in viruses‐associated cancer

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase (TERT) enzyme and telomerase RNA component (TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein–Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA‐binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.


| INTRODUCTION
Cancer is a complex disease characterized by a cascade of genetic mutations and epigenetic modifications, resulting in uncontrolled cellular proliferation.Even though cancer diagnosis and treatment have developed significantly over the years, it still stands as the leading cause of death globally.Because of this reason, there is a pressing need to find new drug targets for developing therapies that can improve the patient survival rate.The most efficient method to cure cancer would be through the usage of procedures that specifically exterminate malignant cells without having any adverse effect on normal ones. 1 Telomeres, repetitive TTAGGG sequences capping chromosome ends, play a pivotal role in both aging and cancer genesis.These protective caps are maintained by a protein complex known as shelterin, ensuring genomic stability. 2,3The telomerase enzyme, consisting of a catalytic subunit called telomerase reverse transcriptase (TERT) and an RNA component known as telomerase RNA component (TERC), controls the length of telomeres. 4Normally, there is a low level of telomerase activity in germline and normal cells where with each round of cell division, telomeres shorten resulting in replicative senescence or apoptosis. 5However, telomerase activity is often upregulated in cancer cells, stem progenitor cells, lymphocytes, and other highly proliferative cells, leading to limitless cell proliferation and tumor growth. 6Furthermore, more than 85% of human cancers have been reported to exhibit detectable telomerase activity 7 along with high hTERT protein levels in different human cancer cases. 8obally, human papillomavirus (HPV) has been identified as a virus infection that is responsible for cancers and several diseases.
HPV is a nonenveloped virus with circular double-stranded DNA strands. 9Based on epidemiological classification, the oncogenic potential of HPVs can be classified into different groups.This classification includes HPVs with low, intermediate, and high-risk levels, depending on their capacity to induce malignant changes in the growth of infected cells. 9,10Based on the information available so far, it is assumed that HPVs most likely select keratinocytes or cells that can differentiate into keratinocytes as primary target cells in the skin and mucous membranes.The way HPV infections take place is postulated to be similar across different tissue types.In particular, for instance, HPV infects the basal layer of the cervix and causes basement membrane exposure.Similarly, HPV enters the basal layer of tonsillar epithelium, causing crypt cell infection and exposure. 11,12man herpesvirus 4, also referred to as EBV, is a virus that is found in many people worldwide and belongs to the Herpesviridae family.It contains a genome composed of linear double-stranded DNA that measures about 184 kb, which means it can cause infection in both epithelial cells and B cells.Although the infection with EBV may lead to Burkitt's lymphoma and nasopharyngeal carcinoma, these diseases are not very common.Other human malignancies affected by this virus are Hodgkin's disease, gastric cancer, and T-cell lymphoma.
Targeting TERT, an essential part of telomerase, 13 has been a promising cancer therapeutic option.Apart from its canonical role in telomere maintenance, TERT was found to have additional noncanonical roles in cancer, such as control of gene expression and DNA repair. 14Moreover, oncogenic viruses such as HPV and EBV have been shown to exploit TERT to enhance viral replication and persistence. 1Investigating the intricate roles of TERT in cancer formation as well as virus life cycles is crucial to pave the way for advanced targeted therapeutic strategies.This review paper shall address the noncanonical functions of TERT and TERC in cancer cells, and the role of TERT in cancers caused by oncogenic viruses, including whether it could have an important role during viral replication.

| Noncanonical functions of TERT and TERC
In addition to their well-established role in telomere maintenance, TERT, and TERC have been shown to have noncanonical functions in cancer that contribute to tumor development and progression (Table 1). 15Previously, it has been shown that telomerase also participates in noncanonical regulation of the Wnt β-catenin pathway, which involves the induction of stem cells, hair regeneration, and the control of transcriptional programs that are similar to those managed by Wnt and Myc. 16Telomerase's cooperation with the Wnt β-catenin pathway is not related to its catalytic role at telomeres, but it plays an important part in maintaining the tumor-initiating cell phenotype in human cancer cells. 17Mice engineered to conditionally express catalytically inactive telomerase in keratinocytes displayed induction of anagen, a proliferative phase of hair follicles that requires activation of stem cells in the hair follicle bulge region, suggesting that telomerase can activate hair follicle stem cells and trigger hair growth independently of its reverse transcriptase T A B L E 1 Noncanonical functions of TERT and TERC in cancer.

Function
Mechanism and impact TERT in Wnt/β-catenin pathway -Activation of stem cells.

TERC interaction with microRNAs
-Alters gene expression and cellular function in cancer.

TERT regulation of VEGF expression
-Activation of VEGF transcription.
-Stimulation of angiogenesis.activity. 16Further studies have shown that telomerase acts as a cofactor in the β-catenin transcriptional complex through its interaction with Brg1, an SWI/SNF-related chromatin remodeling factor. 18Telomerase or its inactive form, in conjunction with βcatenin, promoted anterior-posterior axis duplication in Xenopus embryos, while mice deficient in telomerase displayed homeotic transformations in their vertebrae. 18In addition, telomerase's synergistic role with the Wnt/β-catenin pathway is independent of its catalytic function at telomeres and is essential for maintaining the tumor-initiating cell phenotype in human cancer cells through its interaction with Brg1. 19other example of the factors whose expression is regulated by TERT is vascular endothelial growth factor (VEGF) as another example of a factor whose expression is regulated by TERT.TERT has been found to activate VEGF transcription in embryonic lung cells and HeLa cells. 20Studies have shown that in lung carcinoma tumor xenograft assays in mice, TERT deficiency reduced tumor development and VEGF expression. 21Mechanistically, this activation may occur through hTERT binding to the transcription factor Sp1 at the VEGF promoter to stimulate angiogenesis. 21Our previous studies showed that hTERT-induced gene expression in keratinocytes independent of telomerase activity. 22,23udies have shown that TERC is involved in cell protective mechanisms and can modulate cellular responses to DNA damage.
ATR and DNA-PK are two kinases that are related to TERC, and their activities are modulated by TERC. 24,25Specifically, TERC inhibits ATR kinase and activates DNA-PK, which is required for the repair of double-strand breaks via the nonhomologous end-joining pathway. 26ng and his colleagues showed that HuR regulates telomerase activity via TERC methylation.HuR is a protein that controls telomerase activity by influencing the methylation of TERC.The study identified specific methylation sites in TERC, including C106.
Reducing HuR levels or mutating HuR-recognizing sequences in TERC decreased C106 methylation.The study showed that HuR's interaction with TERC enhanced C106 methylation, and methylated TERC had a stronger interaction with hTERT.Maintaining HuR-regulated TERC methylation was crucial for telomerase activity and telomere length. 27RC, on the other hand, has been shown to regulate gene expression through its interaction with RNA-binding proteins.For example, TERC can interact with the RNA-binding protein hnRNP A1, leading to changes in alternative splicing and promoting tumor growth. 24Moreover, reducing hTERC expression in cells results in an increase in the amount of p53, the tumor suppressor, and the cell cycle regulator CHK1. 28hnRNP A1, a protein involved in the regulation of alternative splicing, was identified as a telomereassociated protein, and suggested to be a regulator of telomerase recruitment to chromosome ends.In the presence of hTERC, DNA-PK phosphorylates hnRNP A1 protein. 29The study proposes that TERC is involved in the regulation of the DNA damage response by activating DNA-PK.However, it is possible that telomerase RNA may be involved in the structural organization of telomeres, which could influence the DNA damage response. 30,31e recent study has provided new insights into how TERT and TERC can interact with RNA-binding proteins as well as microRNAs, which might be essential components in the growth and spread of cancer.RNA-binding proteins take part in the regulation of RNA metabolism through a variety of mechanisms that include the stability of mRNA, translation, and alternative splicing.Conversely, micro-RNAs refer to small RNA molecules controlling gene expression by adhering to their target mRNAs' 3′-UTRs. 32Moreover, TERT has been reported to interact with diverse RNA-binding proteins and microRNAs; hence leading to changes in gene expression and cellular activities.Furthermore, for instance, TERT is capable of interacting with PTBP1, an RNA-binding protein, thus resulting in changes in alternative splicing among others and promoting tumor growth. 33ey performed both transient and stable knockdown experiments targeting PTBP1 in these cell lines and measured the effect on telomerase activity and hTERT splicing.The results suggest that PTBP1 promotes splicing of hTERT transcripts, and that knockdown of PTBP1 leads to a decrease in telomerase activity and a decrease in the percentage of hTERT splicing isoforms. 5In addition, TERT can regulate the expression of miRNAs, which are involved in tumor proliferation and metastasis.TERT gene silencing led to decreased levels of miR-21, miRNA-208a, and miRNA-29a. 34TERC, on the other hand, has also been shown to interact with microRNAs, leading to changes in gene expression and cellular function.For example, TERC can interact with miR-138, leading to changes in telomere length and promoting cancer cell survival. 35e study found that the long noncoding RNA (lncRNA) LINC00909 induces EMT and contributes to osteosarcoma tumorigenesis and metastasis.The study revealed that LINC00909 promotes the migration and invasion of osteosarcoma cells by upregulating the expression of TERT and TERC, which subsequently activates the Wnt/β-catenin signaling pathway. 36Furthermore, LINC00909 was found to interact with RNA-binding protein HuR to stabilize TERT and TERC mRNA, which contributes to the sustained expression of TERT and TERC in osteosarcoma cells. 27sides the canonical processing of TERC, terc-sRNA has been identified that is upregulated in different tumors and can increase telomerase activity. 37A 45nt-long noncoding RNA, T3p, also arises from the 3′ end of TERC and acts as a negative regulator of miRNA function, promoting metastasis in breast cancer. 38The nucleolytic biogenesis of T3p is controlled by DROSHA and TARBP2, two factors involved in miRNA processing, and T3p might be a terc-sRNA precursor, but more experimental evidence is required to link T3p to terc-sRNA biogenesis. 39u and colleagues demonstrated that increasing TERT expression leads to the promotion of epithelial-mesenchymal transition (EMT) while inhibiting TERT reduces EMT.This effect is mediated by TERT's involvement in Wnt/β-catenin signaling, which subsequently upregulates the expression of Snail-1 and vimentin, both of which are crucial markers of EMT.The study highlights that TERT directly binds to β-catenin and localizes to vimentin gene promoters, emphasizing its role as a transcriptional coactivator. 40Another study showed that an hTERT/ZEB1 complex directly regulates E-cadherin to promote EMT in colorectal cancer.The study revealed that the overexpression of TERT in colorectal cancer cells promotes EMT by forming a complex with the transcription factor ZEB1, which directly represses the expression of E-cadherin, a key protein involved in maintaining cell-cell adhesion. 41 is known that many cancers show changes in TERT splicing patterns, leading to the expression of splice variants that are usually silenced in normal cells.While the splicing of TERT plays a significant role in regulating telomerase activity, there is evidence to suggest that these splice variants may have other nonconventional activities.
Studies using TERT mutants transduced into human mammary epithelial cells (HMECs) indicate that TERT has biological actions in cell proliferation and DNA damage signaling that can be genetically separated and functionally independent, supporting the possibility of functions of alternatively spliced TERT isoforms beyond telomerase activity. 42e interaction of TERT and TERC with RNA-binding proteins and microRNAs is an important mechanism underlying their roles in cancer development and progression.Understanding these molecular mechanisms is crucial for developing targeted cancer therapies based on telomerase biology.Therefore, we can conclude that telomerase RNA ectopically expressed in telomerase-negative cells may play a role in interacting with telomeres, disturbing their structure, and compromising their function within the DNA damage response.On another note, TERC seems to be involved in the regulation of important cellular processes and has been hypothesized to contribute to the pathogenesis of diseases such as cancer.

| Modulation of cellular metabolism and immune response
It has been shown that TERT participates in the process of mitochondrial respiration as well as intracellular redox state. 43erexpression of hTERT leads to increased basal COX activity, which is related with enhanced mitochondrial efficiency and decreased ROS production. 43Besides, there is a change in glutathione levels due to hTERT activation leading to an increased GSH/ GSSG ratio and improved cellular antioxidant capacity. 44Consequently, continuous expression of GCLC observed in hTERTexpressing cells counteracts fast decrease in GSH under oxidative stress conditions. 43Conversely, the hyperoxidized forms of Prx are more quickly consumed by hTERT-overexpressing cells implying that these cells have a larger pool of active Prx for targeting cellular peroxides within them. 43,45,46Above all, the results support the idea that cancer cells might be protected from undergoing apoptosis by elevating their capacity to handle intracellular oxidative stress through variation in both antioxidant status and mitochondrial function induced by hTERT. 43In other words, this ROS-mediated mechanism indicates that high levels of hTERT in cancer cells may confer resistance to chemotherapy drugs acting through ROS pathways for cell death induction mechanisms. 43 addition, TERT can modulate the immune response by promoting the expansion of regulatory T cells and inhibiting the function of effector T cells. 47Overexpression of TERT in mouse thymocytes (T cells) also leads to T cell lymphomas without affecting telomere length, supporting a telomere-independent role for telomerase in mouse tumorigenesis across multiple tissue types. 47The NF-κB family consists of transcription factors that can be activated in response to certain stimuli and play important roles in the advancement of cancer. 48It has been found that increasing NF-κB activity can functionally compensate for reduced transformation-related activity of telomerase. 49To test for the functional overlap between telomerase and NF-κB signaling pathways, an experiment was conducted in which hTERC and hTERT were artificially expressed in cells.Additionally, NF-κB activity was suppressed during this experiment. 48,49e results imply a complex association between telomerase and NF-κB, which plays a role in the regulation of cell growth, viability, and perhaps other cellular processes.This research establishes that while telomerase can contribute to these effects, its action is only revealed in the presence of functional NF-κB signaling. 49

| hTERT and telomerase activation by oncogenic viruses
Several oncogenic viruses have been associated with the development and progression of various human cancers, including HPV and Epstein-Barr virus (EBV).These viruses have been shown to upregulate TERT expression, contributing to the pathogenesis and progression of the associated cancers.
EBV is a herpesvirus that infects B lymphocytes and is associated with various lymphoid and epithelial malignancies, including Burkitt lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinoma. 50,51e human gamma herpesvirus 4 belongs to the Herpesviridae family and is a common virus found everywhere.It has a genome size of 184 kb linear double-strand DNA and is capable of infecting both B cells and epithelial cells, 52 infecting B lymphocytes, and establishes a latent infection that can manifest as either asymptomatic or infectious mononucleosis.In immunocompromised individuals, it can lead to a diverse spectrum of lymphoma. 53Within tumor cells, EBV produces multiple groups of proteins associated with latency, which possess transforming properties.These include EBV nuclear antigen (EBNA)-1 and latent membrane proteins (LMP) such as LMP-1, LMP-2A, and LMP-2B.Specifically, LMP-1 activates the hTERT promoter, resulting in increased telomerase activity in B lymphocytes. 50A few amounts of EBV-infected B cells will gain to the immortalization by showing telomere dysfunction and potentially triggering the alternative lengthening of telomeres (ALT) mechanism, 36 for example, just lymphoblastoid cell lines (LCLs) with strong telomerase activity successfully navigate through cellular crises and will be immortalized. 16,54Thus, tumor-associated viruses can contribute to carcinogenesis by transcriptionally activating cellular genes with oncogenic potentials, such as the hTERT gene. 55PVs are nonenveloped viruses with double-stranded circular DNA. 9 The papillomavirus genome is composed of three parts, which are the early, late, and genomic regions.The early region, comprising E1, E2, and E4-E8, accounts for half of the HPV genome.These early fragments have various roles, with E1 and E2 participating in DNA replication regulation, E2 in transcription, and E5, E6, and E7 in cell transformation. 52HPV is the main cause of cervical cancer, as well as other cancers of the anogenital region and head and neck. 56The E6 and E7 viral oncoproteins are responsible for HPV-induced oncogenesis and have been shown to activate TERT expression through various mechanisms. 57The E6 and E7 proteins inhibit the p53 and pRb tumor suppressor pathways in host cells, respectively. 58E6 can interact with the cellular ubiquitin ligase E6AP, which results in the degradation of p53 and activation of TERT expression. 59,60The high-risk HPV E6 oncoproteins promote telomerase activity through various mechanisms that regulate transcriptional, epigenetic, and posttranscriptional processes. 57,61For example, E6 upregulates the hTERT promoter activity in normal human keratinocytes, while E2 can directly repress hTERT transcription through Sp1 binding sites within the promoter. 58,62In past years, we established that hTERT promoter induction depends upon E6 binding to the E6AP E3 ubiquitin which prevents DNA damage and genomic instability. 75However, this protective function may also benefit viruses by protecting their genome from degradation and immune recognition. 56rious herpesviruses, such as EBV 76,77 and Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), 75 have demonstrated the ability to specifically integrate their genetic material into the telomeres of latently infected cells.This integration process also leads to the upregulation of the hTERT promoter activity. 78,79rtain types of cancer are appropriately linked with EBV and KSHV.The balance between EBV latent and lytic cycles is influenced significantly by hTERT.By repressing TERT, lymphoma cells infected with EBV become more sensitive to antiviral drugs due to the activation of lytic replication.Therefore, evaluating the potential of using a combination of TERT suppressors and antivirals as polytherapy for treating EBV-associated malignancies shows promise. 80rly studies showed that the expression of HPV16 E6 in human keratinocytes induced high levels of telomerase. 81E6 protein does not affect the apparent stability of hTERT protein, both in vitro and in vivo.the well-studied roles of the E6 protein, one of them is the degradation of the p53 tumor suppressor protein using the E6/E6AP ubiquitin ligase and proteasome pathway. 82,83Furthermore, TERT levels and telomerase activity were even higher in cells expressing both HPV E6 and E7 oncogenes compared to cells expressing HPV E6 alone. 56 protein increases telomerase activity independently of transcription regulation.The researchers conducted telomerase assays in the presence and absence of E6, using a cell line where the hTERT gene was unresponsive to E6.They found that E6 was unable to induce the hTERT gene or change hTERT mRNA levels.
However, when E6 was expressed in the cells, telomerase activity significantly increased.This indicates that E6 enhances telomerase activity through a posttranscriptional mechanism, likely resulting from direct interactions between E6 and hTERT proteins. 615][86][87][88] Thus, HPV E6 increases telomerase activity through three different mechanisms: hTERT activation, stabilizing hTERT mRNA, and direction with hTERT protein.
Liu and colleagues have demonstrated that E6 has the ability to stimulate the transcription of hTERT by binding to Myc sites located in the promoter region of hTERT. 66,89MYC can activate TERT expression by forming MYC/MAX heterodimers that displace MAD1/ MAX repressors on the E-boxes. 59,60The E6/E6AP complex cooperates with MYC in stabilizing MYC/MAX heterodimers, which causes the shift of MAD1/MAX repressor and enhances TERT transcription and telomerase activity. 60,61The TERT promoter activation mechanism involving a cooperative action of transcription factors binding simultaneously to MYC and SP1 cis elements is complex. 56Nonetheless, the E6 protein does not cause an upregulation of Myc expression, nor does it enhance Myc binding to the hTERT promoter. 73,89,90The presence or absence of E6 protein does not affect the ability of the Myc protein to bind to the hTERT promoter in keratinocytes. 74,91e E7 gene is responsible for producing the E7 protein.Upon expression, the HPV E7 protein interacts with a range of cellular proteins, such as p107 and p130, which are part of the retinoblastoma (Rb) tumor suppressor pathway.This interaction occurs through a conserved LXCXE sequence found within the CR2 sequences. 92is interaction disrupts normal cell cycle regulation and promotes uncontrolled cell proliferation. 93The E7 protein also interferes with other cellular processes involved in DNA repair, apoptosis, and cell differentiation. 94The destabilization of pocket proteins by E7 is crucial for transforming cells.Apart from degrading pRb and related pocket proteins (p107, p130) by binding to them, E7 also targets other cellular factors involved in cellular transformation.HPV E7 has the ability to override the inhibitory effects of cyclin-dependent kinase inhibitors, such as p21CIP1 95,96 and p27KIP1, 97 which regulate cell growth.Various methods have identified additional proteins that interact with E7, including transcription factors p300, CBP, and pCAF [98][99][100] cell cycle regulators cyclin A, cyclin E, p21, p27, and metabolic enzymes.Many of these interactors appear to associate with the carboxyl-terminal sequences of E7. 101 Liu and their colleagues suggest that E6 and E7 oncoproteins are necessary for the immortalization of human foreskin keratinocytes (HFKs) and the maintenance of a cancerous state in cells infected with HPV.
Telomerase activity is also important for this process.The researchers used siRNA constructs to selectively decrease the expression of E6 or E6/ E7 in cervical cancer cells (SiHa cells) and immortalized HFKs.As a result, telomerase activity was reduced by 50-70%, indicating that the continued expression of viral oncoproteins and Myc is crucial for the activity of this enzyme. 66Other viral proteins that can trans-activate hTERT transcription include the KSHV LANA protein and an adenovirus type 12 E1A mutant. 63,64The hepatitis B virus (HBV) genome can also integrate into the hTERT promoter region in hepatocellular carcinoma (HCC) cells, where the HBV enhancer sequence, located ~1.6 kb upstream of the hTERT transcription start site, is responsible for the activation of hTERT transcription in cis. 65Research has shown that hTERT gene expression does not immortalize human mammary epithelial cells (HMECs) unless the expression of the p16 tumor suppressor gene is stopped, despite telomerase activation being a critical step in cell immortalization and oncogenesis. 102Additionally, Shibashish Giri et al. were able to immortalize human fetal hepatocyte cells by introducing the hTERT gene and HPV gene (E7) through ectopic expression. 103 summarize interplay of TERT/telomerase and oncogenic viruses in Table 2.

| TERT and TERC in cervical cancer
Cervical cancer is the most common gynecologic cancer and the leading cause of cancer-related deaths of women worldwide.
Approximately 527 000 women are diagnosed with cervical cancer and >260 000 women die of this disease each year, equal to almost one-tenth of global cancer deaths in women.The National Cancer Institute estimates that there are ~290 000 women living with cervical cancer in the United States. 104While the implementation of HPV vaccination has had a major impact on reducing persistent genital HPV infections, a large proportion of women (approximately 40%) in the United States remain unvaccinated.Worldwide the HPV vaccination rate varies but in developing regions such as Africa and Asia, approximately 99% of the girls between the ages of 10-20 years remain unvaccinated. 105Compounded with the inability of these countries to afford the cost of widespread vaccination programs, it is clear that alternative approaches to treat cervical cancer and precancer will be needed for the foreseeable future.This is especially important since the survival rates of recurrent cervical cancer and advanced-stage cancer are very low and are considered to be incurable.Effective therapeutic and early intervention options for cervical cancer are not available because a detailed mechanism underlying carcinogenic progression is unclear.High-risk HPV DNA, which encodes the E6 and E7 oncogenes, is present in nearly all cervical cancers and HPV is thereby considered a necessary agent for cancer initiation and development. 106Although epidemiological studies show that the estimated lifetime risk of HPV infection in women is more than 75%, the majority of infected women never develop cancer.Therefore, HPV alone is insufficient for cancer development.Unknown factors unique to individual hosts appear to contribute to dysplastic transformation and progression.In addition, the genetic analysis of cervical cancer indicates that there are no commonly occurring mutations that give insight into initiation or progression of the malignancy. 107However, it has been shown that telomerase activity increases with advancing cervical dysplasia and that TERC, one of the components of the telomerase complex, is overexpressed (mainly by amplification) in nearly all human cervical cancers (>90%). 108,109Two studies demonstrate that TERC is amplified in 20%-21% of mild dysplasia of the cervix (CIN I), 50%-68% of moderate dysplasia (CIN II), 81%-82% of severe dysplasia (CIN III) and 95%-100% in invasive cancer. 110,111TERT, the second component of the telomerase complex, is also increasingly expressed in the cascade of cervical dysplasia. 112though we and others have shown increased telomerase activity in cells immortalized by the high-risk E6/E7 oncogenes. 22Recently, we demonstrated that further overexpression of TERT and TERT induced an increased cell growth rate, anchorage-independent growth of HPV E6E7 immortalized cells in soft agar, and the formation of tumors in immunodeficient mice.This represents a distinct progressive step in the conversation of normal to malignant cells.In the TERT/TERC overexpressing cells, the molecular interplay between HPV oncogenes and telomerase can be potentially very important in tumorigenesis. 113These data suggest that TERT and TERC may play critical roles in multisteps of the development of human cervical cancer using telomerasedependent and independent pathways (Figure 2).
T A B L E 2 Interplay of TERT and telomeres and oncogenic viruses.The mechanistic studies of EBV-induced telomerase activation disclose that the LMP-1 EBV oncoprotein have a critical role in increasing telomerase activity in both epithelial cells and B lymphocytes. 33,34The (LMP1) has been shown to activate TERT expression by upregulating c-Myc, which can bind to the TERT promoter and activate its transcription. 50,51Reducing the expression of LMP-1 leads to a decrease in telomerase activity and the transactivation of c-myc.Mutating the Myc-responsive Ebox elements within the essential core of the hTERT promoter can effectively suppress the hTERT expression induced by LMP-1.
inhibiting the transactivation of c-Myc significantly reduces LMP-1-mediated hTERT expression. 53Conversely, the expression of LMP-1 in B lymphocytes has been shown to independently activate TERT through the NF-κB and MAPK/ ERK1/2 pathways, regardless of MYC expression. 50,51This distinction is significant, particularly in the case of EBVassociated tumors such as immunoblastic lymphomas in AIDS patients, where MYC exists in a germ-line configuration.In such cancers, LMP-1 plays a crucial role in activating TERT. 54tably, the inhibition of TERT in Burkitt's lymphomas and LCLs that are EBV-positive increased the effectiveness of antiviral therapy in inducing apoptosis.This suggests potential for new therapeutic approaches, such as using TERT inhibitors, in the treatment of EBV-related cancers. 6,114Given the important role of TERT in the pathogenesis and progression of viralassociated cancers, targeting TERT has been proposed as a potential therapeutic strategy for these cancers. 115Several studies have shown that inhibition of TERT expression or activity can reduce cell proliferation and induce apoptosis in HPVassociated cervical cancer cells 116 and EBV-associated nasopharyngeal carcinoma cells. 53Increasing the telomerase activity in infected cells by retroviruses can be a potentially helpful predictive marker for tumor progress and a good therapeutic target. 117,118In lymphomagenesis that is driven by EBV, TERT makes EBV latency, preventing the lytic cycle and contributing to transformed states. 18,19The NOTCH2/BATF pathway is recognized as the mechanism through which TERT inhibits the viral lytic cycle, suppressing BZLF1 expression. 45Besides, the high telomerase activity of epithelial cells causes an increase in latent EBV gene expression, which confirms TERT's role in promoting the latency program in both B lymphocytes and epithelial cells in EBV-driven malignancies. 9dogenous retroviruses (ERVs) are viral sequences that have become integrated into the genome of a host organism.In general, ERVs are kept silent in the host genome through DNA methylation. 119However, in cancer cells, reduced DNA methylation that can cause the de-repression of these ERVs. 120searchers reported that SP1 can bind to ERVs and activate them by protecting them from methylation. 121Researchers previously showed that TERT could interact with SP1 and activate the expression of VEGF. 21Further analysis using ChIP-seq data in U2OS cells expressing TERT and in HeLa cells confirmed that TERT and Sp1 share common binding sites on transcriptionally active ERVs. 122These findings highlight the crucial role of TERT in activating ERV expression through its interaction with Sp1.
Although HPV E6-mediated telomerase activation is critical for host cell immortalization and transformation, it is unlikely that the primary intent of activating hTERT transcription and increasing telomerase activity is to immortalize cells and facilitate oncogenic conversion, since tumorigenic cells are not permissive for productive HPV life cycle.Since hTERT is expressed in stem or stem-like cells, it is likely that HPV E6 induces the conversion of human keratinocytes into a stem-like phenotype such that it may facilitate HPV persistent or latent stages in squamous epithelium.It is also possible that hTERT protein and/or telomerase activity may participate in HPV DNA maintenance or replication.For example, telomeric proteins regulate episomal maintenance of the EBV origin of plasmid replication. 123Our latest results indicated that hTERT proteins including enzymatic defective hTERT mutant induced HPV LCR (long control regions) activity (Figure 3).This data indicates a novel interplay between HPV and telomerase in host cells and hTERT protein may regulate HPV DNA maintenance or replication independent of telomerase activity.
In normal liver cells compared to hepatoma cells, telomerase activity is inhibited and telomeres are shortened with each cell cycle. 124Xiaodong Zhang and colleagues found that hTERT expression was observed at rates of 67.6% in tumor samples, 73.5% in paratumor samples, and 100% in cirrhosis samples.No hTERT positivity was detected in samples from healthy livers. 125udies also indicate that the HCV RNA-dependent RNA polymerase, along with the NS5B protein, forms a complex with the retinoblastoma tumor suppressor protein (pRb).This complex formation necessitates the involvement of E6-associated protein (E6AP) ubiquitin ligase to facilitate degradation, thereby activating E2F-responsive promoters and promoting cellular proliferation. 126Some research has demonstrated that telomerase activity and hTERT expression are reduced in both the nuclear and cytoplasmic components of CD4(+) T lymphocytes in patients with HIV-1. 127,128Also, telomerase activity in the peripheral blood lymphocytes (PBLs) of HIV-1 patients is significantly lower compared to that in healthy donors; in addition, they indicated that telomerase activity decreases when PBLs are stimulated by HIV-1 under in vitro conditions. 129Moreover, it has been demonstrated that the Tat protein, a transcriptional factor from HIV-1, reduces both telomerase activity and the expression of hTERT in the nucleus of uninfected CD4+T lymphocytes. 130In another study, researchers observed that there was a decrease in both the overall levels of hTERT and its phosphorylated form in HIV-infected cells compared to controls, along with changes in the levels and activation states of proteins like Akt and PP2A, suggesting that HIV-1 might interfere with the pathways that regulate telomerase activity. 128

| CONCLUSION AND FUTURE DIRECTIONS
In conclusion, it has been ascertained that telomerase key F I G U R E 3 hTERT protein induced HPV (highrisk and low-risk types) activity of LCR (long control regions) independent of telomerase activity.The constructs expressing either wildtype hTERT or enzymatic defective hTERT mutant (D868A) and HPV LCR report plasmids were transfected to human keratinocytes and relative luciferase activity was normalized the activity with control plasmids (set to 1).HPV, human papillomavirus; hTERT, human telomerase reverse transcriptase; TERC, telomerase RNA component.
ligase and Myc protein (Figure1).All three proteins (E6, E6AP, and Myc) reside on the activated hTERT promoter.Interestingly, however, we found that Myc (and its associated Max protein) was bound to the hTERT promoter even in the quiescent state, indicating that Myc engagement is not the critical determinant of hTERT transcription.The minimal, E6responsive promoter consists of a 295 bp region which contains binding sites for several transcription factors, including those for Sp1, E2F, Ets, Myc, NF-кB, and smad3.55,[63][64][65][66][67][68]E2F sites are specific DNA sequences recognized and bound by E2F transcription factors, which are critical in controlling the cell cycle.66A simplified diagram of the E6-responsive hTERT promoter (Figure1) includes the critical E box (Myc binding site) and the proteins that we hypothesize regulate this promoter.The hTERT core promoter region contains two canonical consensus sequences for Myc/Max binding (-CACGTG-)64,65,69 and Myc protein transactivates the hTERT promoter via direct binding to these sequences.69Mad overexpression represses hTERT transcription,70,71 apparently by forming Mad/Max complexes which compete with Myc/Max for binding to these E-box sites.Of the two E boxes located in this promoter construct, the proximal (+22 position) E box is the most important.Mutation of the proximal E box abrogates both Myc-and E6-dependent transactivation.[72][73][74]Recent studies have suggested that telomeres may also play a role in the life cycle of oncogenic viruses.Telomeres have been shown to form a protective cap at the ends of chromosomes,F I G U R E 1The proposed model for regulation of the hTERT promoter by HPV E6. Results from our previous studies and others are summarized to illustrate the possible mechanisms for regulation of the hTERT promoter by the HPV E6. 1.The E6/E6AP complex interacts with c-Myc an hTERT expression regulator, and helps it to displace the hTERT repressor complex.2. Myc and RNA polymerase II are pre-existing on the "silent" hTERT promoter.E6 (E6AP) induces epigenetic changes may "push" the "paused Pol II complex" on the promoter, leading an active transcription.3. E6 (E6AP) removes tumor repressor NFX1-91 from the hTERT promoter.3. E6 (E6AP) induces Myc phosphorylation at the promoter.4. E6 (E6AP) increases HAT (histone acetyltransferase) and acetylation of histones, and decreases histone deacetylase (HDAC) activity at the promoter.HPV, human papillomavirus; hTERT, human telomerase reverse transcriptase.(This was adapted from Oncotarget 2017 and modified, this applies the Creative Commons Attribution 4.0 License [CC BY 4.0].)

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Potential role of TERT/telomeres in the life cycle of oncogenic virusesIn addition to their canonical and noncanonical functions in cancer, TERT and telomeres have also been implicated in the life cycle of oncogenic viruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV).57 components TERT and TERC have noncanonical roles in cancer that favor tumor formation and progression.Some of these noncanonical functions include regulation of gene expression and signal transduction pathways, involvement in RNA binding proteins, microRNA interactions, modulation of cellular metabolism and immune response as well as association with oncogenic viruses.These discoveries unravel different aspects of cancer biology related to the function of TERT and TERC, which lead to potential targets for therapy.Based on this information, a new study approach could be focused on investigating whether these noncanonical activities can offer therapeutic options against cancers.It could explore in depth the underlying mechanisms through which these unusual functions cause tumorigenesis.Furthermore, it could also seek out novel therapies aimed at selective targeting those noncanonical functions while minimizing effects on telomere maintenance.Additionally, research examining how TERT or TERC is involved in virus-related tumors might provide possible methods through which interferences between viruses and telomerases can be broken up.Such research could lead to the discovery of innovative approaches for cancer therapy that exploit the multifaceted roles of TERT and TERC in cancer cells.