Oral Oncology 161 (2025) 107169

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Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Review

From infection to immortality: The role of HPV and telomerase in head and
neck cancer☆
Silvia Giunco a,b, Annarosa Del Mistro b , Marzia Morello b, Jacopo Lidonnici a, Helena Frayle b,
Silvia Gori b , Anita De Rossi a,* , Paolo Boscolo-Rizzo c
a
Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padova, 35128 Padova, Italy
b
Immunology and Diagnostic Molecular Oncology Unit, Veneto Institute of Oncology IOV – IRCCS, 35128 Padova, Italy
c
Department of Medical, Surgical and Health Sciences, University of Trieste, 34149 Trieste, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of malignancies with
HPV multifactorial aetiologies. High-risk human papillomavirus (hrHPV) infections, particularly HPV16, and the
Telomerase dysregulation of telomerase activity, specifically through its catalytic subunit, telomerase reverse transcriptase
TERT
(TERT) are among the key contributors to HNSCC development and progression. HPV promotes oncogenesis via
HNSCC
Carcinogenesis
the E6 and E7 oncoproteins, which inactivate tumour suppressors TP53 and RB1, leading to unchecked cellular
Tumour progression proliferation. Concurrently, telomerase activation plays a critical role in HNSCC by maintaining telomere length,
Therapeutic targets thus enabling cellular immortality, and facilitating tumour development and progression. The interplay between
HPV and telomerase is significant; HPV oncoprotein E6 enhances telomerase activity through multiple regulatory
mechanisms, including upregulating TERT expression. Beyond telomere maintenance, TERT influences signalling
pathways, cellular metabolism, and the tumour microenvironment, contributing to aggressive tumour behaviour
and poor prognosis. This review integrates the roles of HPV and telomerase in HNSCC, focusing on their mo­
lecular mechanisms and interactions that drive carcinogenesis and influence disease progression. Understanding
the synergistic effects of HPV and TERT in HNSCC may be crucial for risk stratification, prognostic assessment,
and the development of novel therapeutic strategies targeting these specific molecular pathways.

HPV and telomerase: Key players in HNSCC have been observed in the incidence of oropharyngeal squamous cell
carcinoma (OPSCC), primarily due to the rise in HPV-associated carci­
Globally, in 2022 head and neck cancer represented 4 % of all can­ nomas in many countries [6]. HPV-associated OPSCC constitutes a
cers, ranking as the 7th most prevalent cancers worldwide [1]. Partic­ distinct entity with a different clinical presentation, a unique genetic
ularly, head and neck squamous cell carcinomas (HNSCCs) encompass a profile [7], a distinct tumour immune environment [8], and increased
heterogeneous group of malignancies (i.e. cancer of the oral cavity, sensitivity to chemotherapy and radiotherapy [9]. HPV-associated
oropharynx, larynx and hypopharynx) with diverse drivers contributing OPSCC is characterized by a significantly better prognosis than HPV-
to its development and progression [2]. Among these, high risk human independent counterpart [10,11] with HPV status being the single
papillomavirus (hrHPV) infection and dysregulated telomerase activity most important determinant of outcome [10,11].
have emerged as key players [3,4]. In parallel, the reactivation of telomerase, an enzymatic complex
HPV infection, particularly by the oncogenic type 16, promotes consisting of the catalytic subunit telomerase reverse transcriptase
neoplastic transformation through the action of the viral oncoproteins (TERT) and the telomerase RNA component (TERC), enables immor­
E6 and E7, which inactivate the major tumour suppressor proteins, talization of cancer cells by maintaining telomere length [12]. Telo­
tumour protein p53 (TP53) and RB transcriptional corepressor 1 (RB1), merase is upregulated in most HNSCCs through increased expression of
respectively [5]. In the last decades, significant time-trend variations TERT enzyme and the RNA component TERC. The interplay between

This article is part of a special issue entitled: ‘HPV and Head and Neck Cancers’ published in Oral Oncology.
☆

* Corresponding author at: Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padova, Via Gattamelata
64, 35128 Padova, Italy.
E-mail address: anita.derossi@unipd.it (A. De Rossi).

https://doi.org/10.1016/j.oraloncology.2024.107169
Received 14 August 2024; Received in revised form 19 December 2024; Accepted 25 December 2024
Available online 3 January 2025
1368-8375/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Giunco et al. Oral Oncology 161 (2025) 107169

HPV and TERT is significant in HNSCC. HPV oncoprotein E6 enhances secondary genetic alterations that confer a more aggressive phenotype
telomerase activity through various regulatory mechanisms, aiding in [24].
cell immortalization and cancer development and progression [13]. HPV-associated OPSCC typically develops from the reticulated
Besides telomere maintenance, TERT influences signalling pathways, epithelium covering the crypts of the palatine tonsils and the base of the
cellular metabolism, and the tumour immune microenvironment tongue, which represent immunologically privileged sites for HPV-
[14,15], contributing to tumour progression, metastasis, and poor transforming infections [25]. Consequently, only 3 % of OPSCCs
prognosis [16,17]. arising from non-tonsillar sites harbour hrHPV [26]. Due to its function
Understanding the roles of HPV and TERT in HNSCC and their in capturing inhaled and ingested antigens and transporting them into
interaction is crucial for risk stratification, prognosis prediction, and the subepithelial spaces, the reticulated epithelium features a porous
development of novel therapeutic strategies tailored to target specific basal membrane, consisting of a network of specific collagen types
molecular pathways implicated in HNSCC, such as inhibiting HPV E6- which facilitate close interaction between the stationary epithelial cells
mediated TERT upregulation and telomerase activity in tumours. This and the migratory lymphoid cell population [27]. Transformed cells can
review aims to provide an in-depth analysis of the roles played by HPV thus rapidly and easily spread through efferent lymphatic vessels to
and TERT in HNSCC, as well as to explore the potential interactions regional cervical lymph nodes. Furthermore, HPV16 E6 and E7 onco­
between these two factors in driving carcinogenesis and influencing gene expression can modify the phenotype of infected tissue stem cells,
disease progression. thereby increasing the number of migratory cancer stem cells [28].
These metastases often exhibit cystic degeneration and may undergo
From infection to malignancy rapid changes in size, attributed to the neoplastic cells attempting to
recapitulate the morphology of the originating crypts within the lymph
Molecular mechanisms of HPV-associated carcinogenesis nodes [29]. Clinically, this implies that carcinoma in situ is somewhat
elusive in these cases [30], and the presentation of a relatively early-
Many different HPV types have been characterized so far; all infect stage tumour with a large cystic lateral cervical lymph node package
the epithelial cells, and distinct types are involved in mucosal and is highly indicative of a HPV-associated OPSCC [31], and in many cases
cutaneous infections [18]. Among the mucosal HPV types, only a subset the primary tumour remains occult [32,33]. Compared to the cervix,
is associated with cancer, and constitute the hrHPV group, that includes specific features of the oropharynx include a much higher frequency of
types HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, whereas other HPV16, the low prevalence of productive HPV infections, and the
HPV types are associated with asymptomatic productive infections and absence of dysplastic lesions preceding the invasive phase [34].
benign lesions [19]. The HPV genome is a circular double-stranded DNA
of approximately 8000 base pairs, containing a regulatory region (LCR, Identifying HPV-associated carcinomas
long control region) and open reading frames (ORF). HPV ORFs are
between 7–9 depending on the genotype and are divided in Early and Understanding the good prognosis and better response to chemo-
Late regions. The early (E) region encodes proteins involved in virus radiotherapy of the HNSCC causally related to HPV, compared to the
replication (E1, E2) and cellular transformation (E6, E7, E5), while the HPV-independent carcinomas, poses new challenges to the diagnostic
late (L) proteins constitute the viral capsid. The cellular transformation process. It is necessary to accurately identify HPV-associated tumours to
process represents a state of abortive infection, where the pathway of provide appropriate patient counselling and management [35]. Several
viral gene expression leading to virus particles production is interrupted, methodologies have been employed to detect HPV (DNA or RNA) se­
resulting in abnormal and deregulated expression of the E6 and E7 on­ quences and the expression of the p16INK4A protein in the neoplastic
cogenes [20]. tissue. These assays differ in sensitivity and specificity; HPV-DNA PCR
Knowledge on the natural history of HPV infection and the HPV and/or p16INK4A overexpression by immunohistochemistry (IHC) are the
neoplastic transformation process primarily derives from studies per­ most widely utilized diagnostic tools and there is a consensus on
formed in the uterine cervix, and the HPV carcinogenetic pathways in defining as HPV-associated HNSCCs only those positive for both HPV-
the other body sites in which HPV plays a carcinogenetic role are DNA and p16INK4A protein [36,37].
inferred from these studies. Common features across all body sites More recently, besides tissues, other less invasive sample types for
include persistent infection by a hrHPV, the involvement of cells highly investigating HPV involvement in HNSCCs have been used. These
susceptible to transformation (such as those present in the squamo- include cytological samples obtained by fine needle aspiration or
columnar junction of the cervix, the dentate line of the anal canal, and mucosal brushing/rinsing specimens, and body fluids (mainly blood)
the tonsillar crypts of the oropharynx), and the overexpression of the [36,38]. Cytological samples have been primarily employed as means to
viral E6 and E7 oncoproteins. These proteins interact with key regula­ investigate neck squamous cell carcinomas of unknown primary
tors of the cell cycle and apoptosis, leading to up-regulation of the (NSCCUP), a sizable proportion of which are HPV-associated [33,39].
cellular protein cyclin dependent kinase inhibitor 2A (CDKN2A, also On the other hand, the use of blood as a source of circulating tumour
known as p16INK4A) and degradation of the tumour-suppressor proteins HPV DNA (ctHPVDNA) has been mostly investigated in patients with
TP53 and RB1. Additionally, E6 and E7 interfere with the immune HPV-associated OPSCC for monitoring the response to therapy and/or
response by neutralizing key transcription factors involved in the innate for detecting tumour recurrence [40], particularly in case of indeter­
immunity and by delaying the activation of the adaptive immune minate imaging findings or as a stand-alone tool.
response [21]. The E6 protein also promotes TERT expression [22].
Overexpression of p16INK4A and TERT proteins can be considered useful Telomere and telomerase in carcinogenesis
biomarkers for diagnosis and/or prognosis of HPV-associated carci­
nomas [4,17,23]. Overall, persistent infection with hrHPV types estab­ Guardians of the genome: Understanding telomeres
lishes a replication-competent and immune-compromised environment
that can eventually lead to cancer. Telomeres are specialized DNA structures located at the ends of
In the HPV-associated carcinomas, HPV sequences are often inte­ chromosomes, crucial for maintaining genomic integrity [41]. They
grated into the cellular genome [20]. Viral genome integration has been consist of variable tandem repeats of a short double-stranded DNA
reported to occur in 65–75 % of HPV16-positive HNSCCs, and in seven sequence (5′-TTAGGG-3′) associated with a set of specialized telomeric
out of eight cell lines derived from tissues of HPV-associated primary DNA binding proteins, i.e. the shelterin complex, which has specific
HNSCCs; the cellular integration site varies widely, and it has been capping functions and protects telomere end from being misrecognized
postulated that HPV integration into cancer-related genes may induce as DNA damage [42].

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In normal somatic cells, telomeres progressively shorten with each protein level, thus supporting MYC-driven oncogenesis [64–66].
cell division. When they reach a critical length, they lose their protective Notably, MYC may be overexpressed in HNSCC and is associated with a
shelterin complex and its function, becoming perceived as double-strand poor prognosis [67].
breaks (DSBs) which activate the DNA damage response. This leads to As illustrated in Fig. 1, accumulating evidence suggests the existence
cellular replicative senescence, mediated by checkpoint signalling of feed-forward regulatory loops between TERT and various transcrip­
pathways involving proteins such as TP53 and p16INK4A [42], and cells tional factors, including WNT/β-catenin, NF-κB, and MYC, whereby the
cease to proliferate and undergo senescence or apoptosis, depending on transcriptional factors regulate TERT transcription (see below) and
the cell type [43]. TERT, in turn, regulates their transcriptional levels and/or their cellular
Further erosion of telomeres, which may occur in checkpoint- compartmentalization and stability. These regulatory loops, once acti­
compromised cells, results in genomic instability, a key event in carci­ vated, can contribute to tumour progression through the regulation of
nogenesis initiation [44]. The activation of telomere maintenance multiple hallmarks of cancer [55]. Specifically, TERT’s non-canonical
mechanisms allows cells harbouring tumour-promoting mutations and functions within these loops may significantly influence relevant path­
genomic instability to evade this crisis, conferring unlimited prolifera­ ways in HNSCC, promoting persistent high TERT expression, autono­
tive potential. In the majority of tumours (85–90 %), including HNSCC, mous cancer cell proliferation, and tumour progression.
telomere maintenance is achieved through the activation of telomerase Given telomerase’s extensive involvement in the vast majority of
enzyme [45,46], while a minority (10–15 %) utilize a recombinant- tumours, and the requirement of telomere length maintenance for un­
based mechanism, i.e. the Alternative Lengthening of Telomeres (ALT) limited cell proliferation, TERT and telomere length have been proposed
[47]. as prognostic biomarkers [68]. Indeed, high TERT expression in cancer
cells have been associated with disease aggressiveness, advanced clin­
The enzyme of immortality: Telomerase and its roles ical stage, and poor overall survival (OS) and disease-free survival (DFS)
in several cancer types, including HNSCC [4,16,17,68–70]. As above
Telomerase is a ribonucleoprotein complex containing a catalytic mentioned, it is conceivable that this aggressive behaviour may be
protein with telomere-specific reverse transcriptase activity, TERT, attributable to telomerase’s non-canonical functions.
which synthesizes telomeric sequences de novo using an internal RNA as It has been suggested that telomere length may also have a clinical
a template, TERC [48]. While TERC has a broad tissue distribution, significance, as telomere shortening is a critical early event in carcino­
TERT is the rate-limiting component of the complex. TERT is physio­ genesis in many tumour types [68]. Several studies demonstrated that
logically expressed during embryogenesis to support the high rate of approximately half [16,17] of patients with HNSCC have short telo­
proliferation, but its expression is inhibited in most somatic adult cells meres in the normal tissue adjacent to the cancer. Short telomeres in
[49]. When telomerase is downregulated, telomeric sequences lost at these surrounding tissues are strongly and independently associated
each cell division are not restored, leading to progressive telomere with mucosal failure, supporting the concept that short telomeres may
erosion and subsequent cellular replicative senescence, a pivotal event favour genetic instability, thus increasing the potential for trans­
in the aging process. TERT (re)activation that inappropriately occurs in formation. Consequently, telomere shortening can serve as a marker for
the vast majority of cancers [45,46], prevents cellular replicative field cancerization, aiding in identifying patients at risk for local re­
senescence and leads to unlimited cellular proliferative potential, currences [16,17].
thereby promoting tumour formation and progression [50].
Two main strategies are currently employed to estimate telomerase Mechanisms of TERT activation in tumours
levels from fresh or frozen tissues: quantification of TERT mRNA levels
using PCR-based techniques, and measurement of telomerase activity, In cancers, TERT reactivation includes transcriptional, post-
via the TRAP (Telomeric Repeat Amplification Protocol) assay [51]. transcriptional and epigenetic regulation, as well as multiple changes
Additionally, in clinical settings, immunohistochemistry with specific at the TERT promoter, including chromosomal rearrangements and
anti-TERT antibodies offers a practical method for directly assessing mutations [71]. The transcription of the TERT gene is the key determi­
TERT expression in tumour tissues. [52,53]. nant to regulating telomerase activity. The TERT promoter contains
binding sites for numerous transcriptional activators, such as SP1, MYC,
Beyond telomeres: Non-canonical functions of telomerase HIF-1α, transcription factor AP-2α, β-catenin, NF-κB, E-twenty-six (ETS)
family members, as well as transcriptional repressors, including Wilms’
Although telomere maintenance is the primary canonical function of tumour, TP53, NFX1, MXD1 (also known as MAD) and CTCF [72].
telomerase, several studies indicate that its expression is also associated The wild-type TERT promoter is often silenced by the trimethylation
with many telomere-length independent functions that contribute to of histone H3 Lys27 (H3K27me3) [73]. TERT promoter also contains a
tumour development, including enhancement of proliferation, resis­ cluster of CpG sites that contributes to transcriptional regulation
tance to apoptosis, inflammation, invasion and metastasis through DNA methylation [71]. Additionally, somatic mutations in the
[14,15,54,55]. TERT significantly influences several key signalling TERT core promoter can create de novo ETS binding sites, thus
pathways crucial for cancer progression. TERT binds to promoters enhancing TERT transcription [74,75]. In particular, TERT promoter
responsive to WNT signalling to regulate WNT target genes [56]. This mutations mainly occur at nucleotides 1,295,228 (-124C > T) and
regulation affects cell survival, proliferation, cell polarity, differentia­ 1,295,250 (-146C > T) within the TERT core promoter and are among
tion during embryonic development, and carcinogenesis [56–59]. the most common non-coding mutations in solid tumours, with a broad
Notably, overexpression of TERT in primary human oral epithelial cells spectrum of prevalence [76–79].
induces epithelial-mesenchymal transition (EMT) by activating the In HNSCC, the overall prevalence of -124C > T and -146C > T TERT
WNT/β-catenin pathway, enhancing epithelial cells invasiveness. promoter mutations is 21 %, with the -124C > T mutation being more
Conversely, TERT silencing inhibits WNT/β-catenin signalling and sup­ common than the -146C > T mutation. These mutations are more
presses EMT in oral cancer [60]. TERT also contributes to tumour- prevalent in OCSCCs compared to other head and neck regions; indeed,
promoting processes by inducing the transcription of the NF-κB target nearly 50 % of OCSCC harboured TERT promoter mutations, whereas
genes, including interleukin 6 (IL6), interleukin 8 (IL8), matrix metal­ only 1 % of SCCs from the oropharynx and 12 % from the larynx/hy­
lopeptidase 9 (MMP9), and tumour necrosis factor (TNFα), commonly popharynx exhibited these mutations [80].
over expressed in HNSCC [61–63]. Moreover, TERT interacts with the
MYC pathway by regulating MYC transcription through binding MYC
transcription factor NME2 or by directly interacting with MYC at the

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S. Giunco et al. Oral Oncology 161 (2025) 107169

Fig. 1. HPV and TERT interplay in HNSCC: In both HPV-associated and HPV-independent HNSCC, telomerase activation is an essential process for maintaining
cellular replicative capacity through the maintenance of telomere length. The HPV E6 protein is the main driver of TERT/telomerase activation in infected cells. In
addition to providing cells with unlimited proliferation potential, telomerase interacts with other cancer-related signalling pathways, such as WNT/β-catenin, NF-kB,
and MYC. These non-canonical roles of telomerase can impact cancer progression by activating cellular programs that enhance tumour cell proliferation, motility,
migration, invasion, and epithelial-to-mesenchymal transition within the framework of feed-forward loops. In addition, telomerase non-canonical roles may influence
viral DNA maintenance and HPV persistence. HPV: human papilloma virus; TERT: telomerase reverse transcriptase; TERC: telomerase RNA component; TP53:
tumour protein p53; RB1: RB transcriptional corepressor 1; WNT: WNT/β-catenin signalling; NF-κB: NF-κB signalling; MYC: MYC pathway. Created in BioR
ender.com.

Possible mechanisms of interaction between HPV and TERT [86].

NFX1 isoform 3 (NFX1-91) is a repressor of TERT transcription,
A large number of in vitro studies conducted on primary human constitutively bound to the downstream X-box of the TERT promoter
foreskin keratinocytes have demonstrated that HPV promotes cellular [87,88]. E6 targets NFX1-91 for proteasomal degradation through
immortalization through the activation of telomerase, primarily driven polyubiquitination [87] leading to the dissociation of the SIN3A/histone
by the E6 protein [81]. E6 regulates TERT expression and telomerase deacetylase corepressor complex from the TERT promoter. This results
activity at multiple levels: transcriptional, epigenetic, and post- in increased acetylation of histones H3 and H4, demethylation of H3K4,
transcriptional [22,81,82]. At the transcriptional level, E6 targets the and enhanced TERT transcription [88]. Additionally, E6 collaborates
core promoter region of TERT, collaborating with MYC to stabilize MYC/ with NFX1-123, a splice variant of NFX1-91, and cytoplasmic poly(A)
MYC associated factor X (MAX) heterodimers, displacing the MAD/MAX binding proteins to stabilize TERT mRNA post-transcriptionally [81,89].
and upstream transcription factor 1 (USF1) and USF2 repressors from E- At the post-transcriptional level, E6 enhances telomerase activity by
box sequences, thereby enhancing TERT transcription [82–84]. E6 also directly interacting with the TERT protein at telomeric DNA, thereby
forms complexes with MYC to directly activate the TERT promoter [85]. promoting telomerase activity [22].
Further studies in HPV18-positive keratinocytes and HeLa cells have The observation that E6 mediates telomerase activation via multiple
demonstrated that E6 enhances telomerase expression also by sup­ pathways underscores telomerase as a critical target for HPV-induced
pressing lysine acetyltransferase 5 (KAT5, also known as TIP60)- cell immortalization and oncogenesis. Besides increasing the canonical
mediated acetylation of SP1, thus leading to the accumulation of SP1 function of TERT to maintain the telomere length, it is possible that E6
on the TERT promoter and consequently increasing TERT expression increases TERT non-canonical activities, such as apoptosis blockade,

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gene transcription, cell proliferation, which could have important roles differential telomerase dynamics between HPV-associated and HPV-
in tumour progression as well as in the viral life cycle. In this regard, it independent cases. What has emerged from several investigations is
has been demonstrated that TERT can promote the induction of the EMT that HPV-associated HNSCCs express higher levels of TERT [17,117]
phenotype in E6/E7-expressing keratinocytes [90]. Thus, it is likely that and that TERT promoter mutations are very rare in HPV-associated
high levels of TERT expression induced by HPV could also enhance the oropharyngeal cancer [69,109]. This is likely because there is no se­
non-telomeric functions of telomerase, contributing to the metastatic lective pressure for clones to acquire promoter mutations, as the viral
behaviour observed for some of the HPV-associated malignancies infection itself is sufficient to maintain TERT levels necessary for sus­
[33,91]. taining the unlimited tumour cell replication. However, HPV-associated
Miller et al. demonstrated that during the immortalization of kera­ HNSCC have a markedly better prognosis compared to their HPV-
tinocytes expressing the HPV protein E7, TERT induces the expression of independent counterparts [10]. Considering the non-canonical func­
BMI1, a key component of the polycomb group complex 1 involved in tions of TERT that are potentially associated with enhanced cancer
chromatin remodeling and implicated in tumorigenesis and cancer aggressiveness [55], this observation may present a paradox given also
progression, including HNSCC [92]. BMI1 is crucial in cancer stem cell the capability of E6 to activate TERT. A possible explanation for this
biology and is expressed in head and neck cancer stem cells, regardless contradiction lies in the immune microenvironment linked to TERT
of HPV-association, potentially contributing to chemoresistance, recur­ expression. This microenvironment is characterized by robust B-
rence, and metastasis [93,94]. Its expression may arise early in head and lymphocyte infiltration, and cases with high TERT expression and sig­
neck cancer development [95], underscoring the potential role of TERT nificant B-cell infiltration are associated with improved progression-free
in BMI1 regulation during HPV-associated oncogenesis. survival [117]. Despite the beneficial impact of high TERT levels in the
It has been suggested that the primary intent of HPV in increasing presence of B-cell infiltration occurred regardless of HPV status [117],
TERT protein and telomerase activity is unlikely to be cellular immor­ conditions resulting in very high TERT levels, which can trigger an
talization or oncogenic transformation, given that tumorigenic cells are immune response and a “hot” immunological tumour microenvironment
not permissive to HPV replication. Instead, because TERT expression is are significantly more common in HPV-associated cancers compared to
characteristic of stem cells, E6 might induce keratinocytes to acquire a HPV-independent ones [8,17]. In HPV-independent cancers, elevated
stem-like phenotype, thereby facilitating HPV persistence or latency in TERT levels in a “cold” immunological context are more likely to facil­
the squamous epithelium [22]. On this ground, it is significant that itate the non-canonical functions of telomerase, thereby driving tumour
TERT can transcriptionally activate cellular gene sets of WNT signalling progression. Furthermore, genomic instability, a hallmark of cancer
pathway [22,56], tuning a stem-like state potentially promoting HPV progression [119], emerges as another critical factor that may influence
persistence (Fig. 1). the dual nature of TERT’s role in these tumours. HPV-associated tu­
Additionally, TERT induction and the consequent increase in telo­ mours are indeed characterized by a lower mutational burden, whereas
merase activity might play a role in viral DNA replication, as TERT ac­ HPV-independent HNSCC tend to have higher mutational load, which
tivates the HPV LCR [96]. The LCR is a noncoding segment constituting often correlate with more aggressive behaviour. Interestingly, in
about 10 % of the HPV genome and contains elements critical for viral recurrent and metastatic HPV-associated tumours, genetic alterations
transcription and replication. This finding highlights an interaction be­ such as TP53 mutations (15 %), 3p deletions (55 %), and whole-genome
tween HPV and telomerase in host cells, suggesting that TERT may duplications (25 %) become more frequent, aligning their molecular
regulate HPV-DNA maintenance or replication independently of its profiles more closely with those of HPV-independent HNSCC. These
telomerase activity. changes might reflect tumour adaptation during progression or treat­
ment, distinguishing recurrent and metastatic HPV-associated HNSCCs
Clinical and therapeutic implications of TERT in HNSCC from their primary counterparts [120]. Taken together, these observa­
tions highlight the complex and dynamic role of TERT in HNSCC. The
TERT plays a pivotal role in HNSCC, with potential implications for prognostic impact of TERT expression appears to be modulated not only
clinical practice and treatment strategies [97]. A comprehensive sum­ by its levels but also by the characteristics of the tumour microenvi­
mary of TERT-related studies, including HPV data, is provided in ronment and genomic instability, underscoring the importance of inte­
Table 1. grating molecular and immune landscape analyses to better understand
The high levels of telomerase expression detected in the vast ma­ its role in HNSCC outcomes.
jority of HNSCC cases strongly correlate with aggressive tumour
behaviour and poor patient outcomes [4]. The impact of TERT in HNSCC TERT/telomerase inhibition strategies
extends beyond its canonical role in telomere maintenance, contributing
to various cancer hallmarks, including cell proliferation, angiogenesis, The requirement for TERT overexpression to sustain the immortal
invasion, and metastasis [14,96]. TERT’s interaction with key signalling phenotype, a hallmark of cancer, highlights its potential as a therapeutic
pathways such as WNT/β-catenin and NF-κB amplifies its influence on target in both HPV-associated and HPV-independent HNSCC [97]. Tar­
tumour progression, creating a complex network of pro-oncogenic ef­ geting telomerase in HNSCC could offer significant benefits due to its
fects [56,61,63]. selective upregulation in cancer cells, while being inactive in most
The clinical significance of TERT in HNSCC is evident in its associ­ normal somatic cells. This specificity reduces the likelihood of off-target
ation with several adverse prognostic factors. Increased TERT expres­ effects, a significant advantage over many current chemotherapeutics
sion and telomerase activity are linked to advanced tumour stage, poor which affect both cancerous and normal cells. Inhibiting telomerase
differentiation, lymph node involvement, and extracapsular extension could effectively limit the proliferation of cancer cells, leading to their
[17]. These factors contribute to higher rates of regional and distant eventual senescence and apoptosis.
metastases, reduced treatment response, and ultimately, poor OS [118]. Although telomerase has many attractive qualities as a target for
Notably, telomerase activity has emerged as an independent predictor of cancer treatment, creating effective clinical therapies has been difficult
poor survival in HNSCC patients, even when accounting for other clin­ due to several challenges. These include the limitations of preclinical
ical and pathological factors [16,113]. This underscores its potential as a models, the absence of detailed high-resolution structures of human
valuable prognostic marker in clinical practice. telomerase, and issues with adaptive drug resistance [121]. Telomerase
Despite the well-established role of the HPV oncoprotein E6 in inhibitors work by gradually shortening telomeres, which can take
inducing TERT expression, it is surprising that very few studies have several cell divisions, potentially allowing cancer cells to develop
evaluated TERT in relation to HPV status (Table 1). While data specific resistance mechanisms before the treatment becomes effective, making
to HPV-associated HNSCC are limited, some evidence suggests them less suitable as first-line treatments [121]. Additionally, cancer

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Table 1
Key studies on TERT/telomerase in HNSCC, including data addressing HPV-associated OPSCC.
Authors [Ref] Year Number of cases OPSCC HPV- Detection of TERT/ Main findings
associated telomerase (assay)
cases

Ogawa Y et al. 1998 25 patients with oral and not available TA (TRAP) Lower levels of TA correlated with better response to radiation
[98] oropharyngeal SCC therapy (P = 0.025).
Patel MM et al 2002 110 HNSCC + matched not available TA (TRAP) TA in adjacent mucosa was associated with poor 2-year DFS (P
[99] adjacent mucosa < 0.05).

Koscielny S 2004 80 HNSCC + matched adjacent not available TA (TRAP) No correlation between TA and local and regional recurrences
et al. [100] mucosa and OS.

Liao CT et al. 2004 217 HNSCC + matched not available TA (TRAP) No correlation between TA and local and regional recurrences
[101] adjacent mucosa and OS.

El Samny T 2005 35 patients with laryngeal SCC not applicable TERT mRNA (RT-PCR) TERT levels in tumour edges significantly correlated with OS (P
et al. [102] = 0.04).
Luzar B et al. 2005 40 laryngeal and 16 not applicable TERT mRNA (relative No correlation between level of TERT mRNA and OS.
[103] hypopharyngeal SCC quantification by PCR based
kit)

Freier K et al. 2007 352 HNSCC not available TERT (FISH); TERT (IHC) No differences in OS and DFS in HNSCC with increased TERT
[104] expression.

Pannone G 2007 42 oral SCC + matched not available TERT mRNA (RT-PCR) TERT Stage I patients with higher TERT expression had a lower OS (P
et al. [105] adjacent mucosa (IHC) = 0.04).

Chen HH et al. 2007 82 oral SCC + 116 OED + 21 not available TERT (IHC) Increased expression of TERT is an early event in oral
[106] normal oral mucosa carcinogenesis. Oral SCC patients with nuclear TERT labelling
scores > 100 % had significantly shorter OS than those with
nuclear TERT labelling scores < 100 % (P = 0.011).
Fabricius EM 2009 40 HNSCC + 38 tumor-free not available TA (TRAP) TERT (IHC) No significative correlation was found between high TERT
et al. [107] surgical margins + 18 tumor- expression and OS.
free distant from tumor

Qu Y et al. 2014 235 laryngeal SCC not applicable TERT promoter mutations TERT promoter mutation -146C > T was associated with worse
[108] (pyrosequencing) OS of laryngeal cancer patients (P = 0.01).

Boscolo-Rizzo 2015 139 HNSCC + matched 9/30 cases (30 TERT mRNA (RT-PCR) Higher TERT levels in cancer tissues significantly correlated
P et al. [17] adjacent mucosa %) with higher risk of regional failure (P = 0.045), distant failure
(P = 0.067), and worse DSF (P = 0.037). HPV-associated
tumours had higher TERT levels than HPV-independent (P =
0.020).
Annunziata C 2018 24 HNSCC 5/9 cases (55.6 TERT promoter mutations TERT promoter mutations were frequent in oral SCC (60 %) but
et al. [109] %) (Sanger sequencing) absent in oropharyngeal SCC (P = 0.007).
Mundi N et al. 2019 135 patients with SCC of the not applicable TERT promoter mutations TERT promoter was mutated in 0.4 % of the samples. No
[110] oral cavity (Illumina Ampliseq platform) association with OS (HR: 0.592, P = 0.131).

Boscolo-Rizzo 2019 101 HNSCC + matched 10/22 cases TERT mRNA (RT-PCR) Higher TERT levels in cancer tissues significantly correlated
P et al. [16] adjacent mucosa + plasma (45.5 %) with higher risk of regional failure (P = 0.032), progression (P
= 0.049), and death (P = 0.005).
Arantes LMRB 2020 88 HNSCC not available TERT promoter mutations 27 % of cases harboured TERT promoter mutations (92 % in
et al. [111] (pyrosequencing) oral cavity site). Patients with -124C > T TERT promoter
mutation showed a significant decrease in DFS (20.0 vs. 63.0
%, P = 0.017) and in OS (16.7 vs. 45.1 %, P = 0.017).

Yilmaz I et al. 2020 189 HNSCC not available TERT promoter mutations TERT promoter mutations were detected in the oral cavity (75
[112] (PCR-based direct %); larynx (8.4 %), hypopharynx (16.6 %), and none in
sequencing) oropharynx. No significant association between TERT promoter
mutations and OS was found.
Boscolo-Rizzo 2020 101 HNSCC + matched 11/23 cases (48 TERT promoter mutations TERT promoter harboured mutations in 12 tumours (11.9 %;
P et al. [69] adjacent mucosa %) (Sanger sequencing) 37 % in oral cavity site). No significant association between
TERT promoter status and OS. No TERT-promoter mutations
were detected in HPV-associated tumours.

Giunco S et al. 2021 144 oral SCC and 57 normal not available TERT promoter mutations 31 % of cases harboured TERT promoter mutations. Patients
[113] adjacent mucosal (Sanger sequencing) with -124C > T TERT promoter mutation showed higher risk of
local recurrence (HR: 2.75, P = 0.0143), disease progression
(HR: 2.71, P = 0.0024), and death (HR: 2.71, P = 0.0079).
Kim H et al. 2021 80 tonsillar carcinomas 64/80 cases (80 TERT promoter mutations 7.5 % of cases harboured TERT promoter mutations. TERT
[114] %) (NAClamp™ TERT mutation promoter mutations statistically associated with shorter DFS (P
detection kit) = 0.007).

Yu Y et al. 2021 117 HPV-negative HNSCC ​ NGS panel TERT promoter mutations were associated with a higher
[115] cumulative incidence of locoregional failure (P < 0.001).

(continued on next page)

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S. Giunco et al. Oral Oncology 161 (2025) 107169

Table 1 (continued )
Authors [Ref] Year Number of cases OPSCC HPV- Detection of TERT/ Main findings
associated telomerase (assay)
cases

Haraguchi K 2022 53 oral SCC not available TERT (IHC) Patients with high TERT expression showed higher risk of
et al. [116] disease progression (HR: 5.763, P = 0.008) and death (HR:
10.075, P = 0.034).
Xian S et al. 2023 156 HNSCC 46/156 (49 %) Transcriptomic data from Significant positive correlation was found between TERT
[117] The Cancer Genome Atlas expression and HPV status (P < 0.001). High TERT expression,
(TGCA) combined with elevated B-cell infiltration in tumours, was
associated with improved PFS (P = 0.0048), irrespective of HPV
status.

TERT, telomerase reverse transcriptase; Ref, reference; OPSCC, oropharyngeal squamous cell carcinoma; SCC, squamous cell carcinoma; HNSCC head and neck
squamous cell carcinoma; TA, telomerase activity; TRAP, telomeric repeat amplification protocol; DFS, disease-free survival; PFS, progression-free survival; RT-PCR,
reverse transcription polymerase chain reaction; OS, overall survival; FISH, Fluorescence in situ hybridization; IHC, immunohistochemistry; OED, oral epithelial
dysplasia; HR, hazard ratio; NGS, next generation sequencing.

cells may activate ALT mechanism to maintain telomere length in the As stated above, studies have consistently shown that the presence of
absence of telomerase activity [47,122]. The ALT, though less common, HPV-DNA in tumour cells is associated with a significant reduction in
could become more prevalent as a resistance strategy, necessitating the risk of death compared to HPV-independent tumours [10] with these
combination therapies to effectively target both telomerase and ALT neoplasm being more sensitive to radiation therapy and chemothera­
pathways [123]. peutic agents [134]. The enhanced treatment responsiveness has led to
While telomerase is inactive in most somatic cells, it is active in discussions about de-escalating therapy in HPV-associated patients to
certain stem cell populations [124]. Inhibiting telomerase could reduce treatment-related morbidity without compromising survival
adversely affect these cells, leading to side effects such as bone marrow outcomes [135,136]. However, it is increasingly evident that HPV-
suppression and impaired tissue regeneration. Therefore, careful associated tumours represent a heterogeneous group, with some tu­
consideration and monitoring of side effects are essential in the clinical mours exhibiting unexpectedly aggressive behaviour [137,138]. This
application of telomerase inhibitors. heterogeneity challenges the current paradigm of uniform treatment de-
Despite numerous challenges, several strategies to target telomerase escalation for all HPV-associated cases [139]. Among the most impli­
function have been developed, some of which have entered clinical trials cated factors in modulating prognosis in patients with HPV-associated
[121]. Imetelstat, a direct telomerase inhibitor, shortens telomeres and OPSCC are cigarette smoking [10] and extranodal extension (ENE)
shows efficacy in hematologic malignancies, though it has dose-limiting [140], as well as molecular biomarkers [141,142]. Though still debated
toxicities in solid tumour trials [125,126]. Indirect inhibitors, like G- [29,143], some recent studies suggest that the extent of ENE may
quadruplex stabilizers (e.g., CX-5461), disrupt telomerase function by significantly influence prognosis [140]. This has led some authors to
blocking the resolution of telomeric G-quadruplex DNA inducing DNA reconsider the role of ENE in staging, suggesting that every radiological
damage and cell death [127]. Additionally, suicide gene therapies like ENE + should be reclassified into the cN3b category [144].
Telomelysin utilize a replication-competent oncolytic adenovirus engi­ In this scenario, TERT expression emerges as a promising quantita­
neered with a TERT promoter, enabling tumour-specific replication and tive biomarker to address this clinical challenge. High TERT expression,
showing promising antitumor effects [128,129]. Telomelysin has been coupled with significant B-cell tumour infiltration, may suggest
proposed for a Phase II clinical trial to assess its efficacy in patients with immune-mediated tumour control [117], identifying patients who could
inoperable, recurrent, or progressive HNSCC (NCT04685499). be ideal candidates for de-escalation therapies. Conversely, high TERT
Immunotherapies, including peptide vaccines (such as GV1001, expression without substantial immune infiltration, even in HPV-
GX301, UV1, Vx-001, and UCPVax) and the DNA vaccine INVAC-1, offer associated tumours, may indicate a more aggressive disease, poten­
immunogenic TERT epitopes that activate immune responses against tially identifying a subset of HPV-associated HNSCCs that behave more
cancer cells expressing telomerase. Immunotherapies against TERT like their HPV-independent counterparts, particularly in cases with
display potential when combined with immune checkpoint inhibitors altered genetic and molecular profiles [120]. This insight is crucial for
[130–132]. The ongoing VolATIL study, a Phase II trial, evaluates the patient stratification: HPV-associated tumours with high TERT expres­
combination of atezolizumab (an immune checkpoint inhibitor) and sion but poor immune infiltration might require more aggressive treat­
UCPVax, a therapeutic vaccine based on telomerase-derived peptides, in ment approaches and should be excluded from de-intensification trials
HPV-associated cancers, including head and neck cancer to avoid undertreatment. On the other hand, patients with low TERT
(NCT03946358) [133]. The recent findings that high TERT expression, expression might be ideal candidates for de-escalation therapies. Thus,
combined with elevated B-cell infiltration in tumours, is associated with by integrating TERT expression analysis into clinical assessment, on­
improved progression-free survival, irrespective of HPV status [117], cologists could more accurately predict tumour behaviour, optimize
position TERT as a key antigen in mediating local antitumor immunity in treatment strategies, and improve patient outcomes. This approach
HNSCC. Collectively, these data strongly suggest that innovative aligns with the growing trend towards precision medicine in oncology,
immunotherapeutic strategies targeting TERT could be effective in both offering a more nuanced and personalized approach to managing HPV-
HPV-associated and independent HNSCC, potentially expanding treat­ associated HNSCC [145].
ment options for these challenging malignancies. Understanding how HPV E6 activates TERT opens avenues for novel
therapeutic strategies, potentially leading to drugs that target this spe­
Potential clinical implication of the interaction between HPV cific interaction in HPV-associated HNSCCs while sparing normal cells
and telomerase [84]. Moreover, this relationship might elucidate mechanisms of treat­
ment resistance, such as how HPV’s ability to maintain high TERT
The interaction between TERT and HPV in HNSCC carries potential expression could contribute to radiotherapy or chemotherapy resistance
significant clinical implications, spanning diagnosis, prognosis, and [146].
treatment strategies. Understanding this relationship may be crucial for The potential role of TERT in HPV-associated HNSCC may extend
advancing patient care and outcomes. beyond initial diagnosis and treatment planning to post-treatment

7
S. Giunco et al. Oral Oncology 161 (2025) 107169

surveillance. However, while tumours with a high prevalence of TERT Visualization, Investigation, Conceptualization.
promoter mutations, such as hepatocellular carcinoma, demonstrate
promising potential for utilizing cell-free DNA quantification in treat­ Funding
ment and post-treatment surveillance [147], this approach may have
limited utility in HPV-associated OPSCCs. In these cases, the prevalence This research did not receive any specific grant from funding
of TERT promoter mutations is marginal [79,113], suggesting that agencies in the public, commercial, or not-for-profit sectors.
alternative strategies may be necessary to provide reliable predictive
insights for treatment responses in this specific patient population.
Overall, the integration of TERT expression analysis in the clinical Declaration of competing interest
assessment of HPV-associated HNSCC presents a promising avenue for
more precise risk stratification and personalized treatment strategies. The authors declare that they have no known competing financial
Further research into the HPV-TERT interaction may not only refine our interests or personal relationships that could have appeared to influence
approach to managing these tumours but also pave the way for novel the work reported in this paper.
targeted therapies, potentially transforming the treatment landscape for
HNSCC patients. References

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