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International Journal of

Molecular Sciences

Review
The Role of Telomerase in Breast Cancer’s Response to Therapy
Eliza Judasz 1 , Natalia Lisiak 1 , Przemysław Kopczyński 2 , Magdalena Taube 1 and Błażej Rubiś 1, *

1 Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences,
60-806 Poznan, Poland
2 Centre for Orthodontic Mini-Implants at the Department and Clinic of Maxillofacial Orthopedics and
Orthodontics, Poznan University of Medical Sciences, 60-812 Poznan, Poland
* Correspondence: blazejr@ump.edu.pl; Tel.: +48-616418304

Abstract: Currently, breast cancer appears to be the most widespread cancer in the world and the
most common cause of cancer deaths. This specific type of cancer affects women in both developed
and developing countries. Prevention and early diagnosis are very important factors for good
prognosis. A characteristic feature of cancer cells is the ability of unlimited cell division, which makes
them immortal. Telomeres, which are shortened with each cell division in normal cells, are rebuilt
in cancer cells by the enzyme telomerase, which is expressed in more than 85% of cancers (up to
100% of adenocarcinomas, including breast cancer). Telomerase may have different functions that are
related to telomeres or unrelated. It has been shown that high activity of the enzyme in cancer cells
is associated with poor cell sensitivity to therapies. Therefore, telomerase has become a potential
target for cancer therapies. The low efficacy of therapies has resulted in the search for new combined
and more effective therapeutic methods, including the involvement of telomerase inhibitors and
telomerase-targeted immunotherapy.

Keywords: telomerase; breast cancer; therapy; resistance

Citation: Judasz, E.; Lisiak, N.;


Kopczyński, P.; Taube, M.; Rubiś, B. 1. Introduction
The Role of Telomerase in Breast
Cancer is still one of the most dangerous diseases of the 21st century, and the most
Cancer’s Response to Therapy. Int. J.
common type in women worldwide is breast cancer [1,2]. The progress in the field of
Mol. Sci. 2022, 23, 12844.
molecular biology in recent decades enabled understanding the molecular basis of cancer
https://doi.org/10.3390/
and provided significant progress in cancer diagnostics and therapy. However, due to
ijms232112844
increasing environmental risk factors and the aging of societies, the incidence numbers
Academic Editor: Erica Salvati continue to rise. Over the past three decades, scientific interest in the field of cancer cell
immortality related to telomerase and telomeres has increased significantly. In 2009, the
Received: 8 September 2022
Accepted: 24 October 2022
Nobel Prize in Physiology and Medicine was awarded to three scientists, E. Blackburn, J.
Published: 25 October 2022
Szostak, and C. Greider, for “the discovery of how the ends of chromosomes are protected
by structures called telomeres and the enzyme telomerase” [3]. This knowledge has
Publisher’s Note: MDPI stays neutral allowed us to understand some of the mechanisms involved in cancer cells and to plan
with regard to jurisdictional claims in
promising new treatments. Importantly, the idea of how telomerase contributes to cancer
published maps and institutional affil-
cell metabolism has significantly extended since that time, suggesting important roles of
iations.
this enzyme in telomere-unrelated mechanisms. Progressive studies have shown that the
role of telomerase extends beyond mechanisms associated with telomere restoration, e.g.,
resistance to therapy, adhesion, migration, etc. [4]. We hope that proceedings in this area
Copyright: © 2022 by the authors.
will provide the development of novel and effective oncological therapy strategies. A
Licensee MDPI, Basel, Switzerland. telomerase-based therapeutic approach may be particularly efficient in breast cancer cells
This article is an open access article that, similar to other adenocarcinomas, abundantly express this enzyme [4].
distributed under the terms and
conditions of the Creative Commons
2. Breast Cancer—A Challenge
Attribution (CC BY) license (https:// Breast cancer originates from the epithelial tissue of the ducts or lobules of the mammary
creativecommons.org/licenses/by/ gland and primarily develops locally in the breast [5]. Cancer cells are capable of translo-
4.0/). cating due to a very primitive but effective amoeboid mechanism [6]. Breast cancer cells

Int. J. Mol. Sci. 2022, 23, 12844. https://doi.org/10.3390/ijms232112844 https://www.mdpi.com/journal/ijms


Int. J. Mol. Sci. 2022, 23, 12844 2 of 13

acquire a very dynamic characteristic that is associated with tumor cells’ ability to induce
the epithelial–mesenchymal transition (EMT) and, conversely, the mesenchymal–epithelial
transition (MET) [7]. Consequently, by changing their phenotype, cells are able to escape the
bulk, enter the cardiovascular system, and metastasize to lymph nodes and distant organs
such as bones, the liver, or the brain [7,8]. The ability of breast cancer cells to metastasize
is the main cause for treatment failure and patient deaths. However, the mechanisms that
contribute to this phenomenon are not yet fully understood. Different metabolic phenotypes
of breast cancer cells are modulated by both intrinsic (such as MYC amplification, PIK3CA,
and TP53 mutations) and extrinsic factors (such as hypoxia, oxidative stress, and acidosis) [9].
The pathways involved in the modulation of breast cancer cell mobility are associated with
the metalloproteinases MMP1 and ADAMTS1, which drive metastasis to bones. In turn, when
metastasis to the lungs is reported, the combinatorial effects of COX2, EREG (cyclooxygenase-2
and epiregulin, respectively), and MMP1 and 2 are raised [10]. Importantly, the pharmacologi-
cal inhibition of pathways mediated by these factors was shown to diminish the aggressiveness
of the metastatic phenotypes [11]. Thus, as the EMT accounts for a broad spectrum of tran-
sitional stages between the epithelial and mesenchymal phenotypes, it is critical to find a
common feature of all these stages. Apart from the modulation of the mentioned pathways,
some specific genes and proteins are directly involved in adhesion and cell-to-cell interactions,
including α1 and β1 integrins, cadherins, selectins, focal adhesion kinase (FAK), Akt, and
other cell adhesion molecules as well as epigenetic factors, including miRNA [12]. Importantly,
one of the most characteristic features of the vast majority of adenocarcinomas, including
breast cancer, is the restoration of the telomerase expression (specifically the key catalytic
telomerase subunit hTERT) and activity that provide tumor cell immortality. Telomerase
is also postulated to modulate cancer cells’ responses to drugs as well as their metastatic
potential via affecting adhesion pathways [13]. For these reasons, telomerase seems to be a
good therapeutic target in breast cancer.

3. Obstacles in Therapeutic Strategies


Breast cancer can be treated locally or systemically. The choice of treatment method
should be made by a multidisciplinary team of specialists in consultation with the patient
and should be based on clinical and pathomorphological assessments, including the grade
of malignancy, the histological type, ER/PgR, Ki67, and HER2 expression, the presence and
location of metastases, etc. Treatment may include surgical treatment (sometimes including
mastectomy), radiotherapy, chemotherapy with alkylating drugs (anthracyclines and tax-
oids), hormone therapy, and subsequent rehabilitation [14]. The increasing effectiveness of
cancer treatment is due to better and earlier diagnostics supported by molecular markers
and the screening of therapeutic targets. In cancer cells, many mechanisms are altered,
including cell death inhibition (apoptosis suppression), changed drug metabolism, epige-
netic modulation, changes in drug targets, enhanced DNA repair, and gene amplification
that may all lead to resistance to therapy [15]. Some of the key players in drug resistance
development are ATP-binding cassette (ABC) family proteins. However, as DNA stability is
also critical for the process, telomeres and telomerase (specifically the catalytic telomerase
subunit hTERT) are also suggested to contribute to the drug-resistant phenotype of cancer
and the therapy response [16].

4. Immortality of Cancer Cells


Cancer cells share a common set of characteristics that distinguish them from normal
cells. They allow tumor formation and provide the possibility of infiltration and metastasis
to other organs. Although cancer cells retain the structural and functional features of the
cells from which they were formed, during the process of tumor formation they acquire,
among others, the following features: unlimited proliferation potential, independence
from external growth signals, self-sufficiency in growth factors, resistance to proapoptotic
signaling, the ability to generate angiogenesis, infiltration, and metastasis [17–19].
Int. J. Mol. Sci. 2022, 23, 12844 3 of 13

One of the most important features of cancer cells is their ability to undergo an
unlimited number of cell divisions, which provides them with immortality. This is due to
the autocrine secretion of growth factors, which results from the cancer cells’ independence
from external factors. In addition, cancer cells can interact with the surrounding normal
cells, creating a specific microenvironment as well as stimulating nearby normal cells to
produce growth factors [20]. Cancer resistance to apoptosis results from various defense
mechanisms, but the most common cause is mutations leading to the loss of p53 protein
function [18]. It is also characteristic for cancer cells to increase the expression of telomerase,
whose role is, among others, to restore telomeres, which allows cells to divide in an
unlimited way and avoid replicative senescence [18].

5. Telomerase—Key Factor in Immortality


Telomerase is expressed in more than 85% of cancers [21,22]. Human telomerase is a
ribonucleic complex consisting of several subunits, but two of them are considered to be
critical and are required for full activity in vitro: hTERT and hTERC (human telomerase
reverse transcriptase and the human telomerase RNA component). The molecular weight
of the holoenzyme exceeds 500 kDa [3]. The catalytic protein subunit hTERT is encoded by
the hTERT gene located in locus 5p15.33. The hTERC unit is an RNA template for DNA
synthesis and is encoded by a gene located in locus 3q26 [23]. It forms a structure called
the “glove structure”, which allows telomerase to wrap around the end of the chromosome
and synthesize new telomeric repeats. While the hTERC gene is expressed in most tissue
types, the hTERT gene is limited to certain groups of tissues, including stem cells, activated
lymphocytes, embryos, cancer, and cancer stem cells [21]. In vivo, the entire complex
contains additional proteins such as NOP10, NHP2, GAR, and dyskerins that bind to
hTERC and stabilize the entire complex [24].
The mechanism of telomerase action can be divided into three basic steps: primer
recognition and binding, the elongation of the DNA strand, and enzyme translocation or
dissociation. In the first step, the sequence of the telomeric DNA strand located at the 3’
end of the chromosome and the sequence of the matrix RNA of the enzyme are matched
complementarily. In the next step, the hTERT subunit elongates the DNA strand, using the
RNA fragment as a template. Then, the telomerase moves towards the 3’ end of the newly
synthesized chromosome to add further repeats or dissociates from the telomere [3].

6. Canonical and Noncanonical Functions of Telomerase


The main function of telomerase is to provide immortality to cells by maintaining the
integrity of the chromosome ends. This RNA-dependent DNA polymerase synthesizes
telomeres by reverse transcription and eliminates the end replication problem as well as
prevents chromosomes from fusing. Consequently, telomerase has been recognized to
have a huge role in cancer metabolism, aging, and degenerative diseases. The function of
telomerase associated with the altering telomere metabolism is called the canonical function
of the enzyme [3,21].
However, the role of telomerase (or its subunits) is not limited to telomeric functions.
It also contributes to the regulation of key metabolic mechanisms in cells, such as gene
expression, mitochondrial metabolism, signal transduction pathways, and stress responses
(Figure 1). These nontelomere functions are called noncanonical telomerase functions [21].
The hTERT subunit is associated with key signaling pathways that control proliferation,
migration, and cell differentiation during embryonic development or carcinogenesis, among
others [21]. One of these is the Wnt/β-catenin pathway, in which hTERT acts as a cofactor
in the β-catenin transcription complex by binding to BRG1. Thus, hTERT is involved in
the regulation of gene expression, including cyclin D1, MYC, and AXIN2 [25,26]. Recent
studies indicate that the role of hTERT is also to stabilize MYC protein and regulate the
binding of MYC to target promoters. Thus, telomerase contributes to the activation or
repression of MYC family genes [26,27].
Int. J. Mol. Sci. 2022, 23, 12844 4 of 13

Telomerase is also involved in the NFκB signaling pathway by regulating inflamma-


tory signals in cancer cells. NFκB can be regulated by various factors, including phos-
phatases, kinases, cytokines, and lncRNAs, that modulate the expression of proinflamma-
tory genes [28–31]. Telomerase (hTERT) can mediate these processes by interacting with
transcription factors or chromatin-modifying factors [21]. Together with the p65 subunit
of NFκB, hTERT can bind to gene promoters in the presence of an inflammatory stimulus.
Studies suggests that the p65 subunit is the main regulator of proliferation, while hTERT
enhances its function [32]. In addition, the inhibition of telomerase activity reduces the
binding of p65 to the promoter sequence. Therefore, it is suggested that the expression
of p65-dependent genes in the NFκB signaling pathway is linked to the expression of
hTERT [28,32].
It is known that telomerase can protect cells from oxidative stress, which is associated
with a reduced level of reactive oxygen species (ROS). However, due to conflicting findings,
these relationships are still not fully understood [33–35]. The result of oxidative stress is
the presence of 10–20% of the cellular hTERT pool in mitochondria. The oxidative-stress-
induced overexpression of hTERT in mitochondria has been shown to limit UV-induced
mtDNA damage and mutagenic agents (e.g., ethidium bromide) and to protect mitochon-
drial DNA by reducing the cellular production of superoxides and ROS [21,35,36]. Other
studies reported impaired mitochondrial function due to the reduced expression of hTERT,
which manifested in increased or decreased membrane potentials, changes in the mtDNA
copy number, and altered mitochondrial mass and ATP levels [37–39]. An association
Int. J. Mol. Sci. 2022, 23, 12844 5 of 13
between the mitochondrial localization of hTERT and the drug resistance of cancer cells
was also observed [38,40,41]. All these results, although still not fully understood, point to
a complex relationship between mitochondria, telomerase, and ROS [21,35].

Figure 1. Association between hTERT, key signaling pathways, and senescence (acc. [21,22,39],
Figure 1. Association
modified). Thebetween hTERT,ofkey
hTERT subunit signalingcan
telomerase pathways, andinsenescence
be involved (acc. [21,22,39],
various metabolic pathways (e.g.,
modified).
NFκB, Wnt/β-catenin, and adhesion as well as senescence) and affect thepathways
The hTERT subunit of telomerase can be involved in various metabolic (e.g.,
expression of various
NFκB, Wnt/β‐catenin, and adhesion as well as senescence) and affect the expression of various
genes. Under the influence of stress, hTERT is translocated to mitochondria, where hTERT protects
genes. Under the influence of stress, hTERT is translocated to mitochondria, where hTERT protects
mitochondrial metabolism by lowering the level of ROS and binding to mtDNA.
mitochondrial metabolism by lowering the level of ROS and binding to mtDNA.
It was shown that hTERT is not the only subunit that exhibits extratelomeric functions.
7. Therapeutic Potential
The hTERC of Telomerase
subunit Subunits
is also able to control the expression of other genes, as was demon-
7.1. Telomerase
strated as A Target
during for Cancer
studies Therapymelanoma cell line. In this study, the suppression of
on a mouse
mTERC (mouse TERC) resulted in the inhibition
In most somatic cells, telomerase activity is below ofthethe expression
detection limitof[3,45].
more than 100 genes.
The ex‐
Some of them were involved in the glycolytic pathway, indicating a possible
ceptions are embryonic stem cells, marrow stem cells, intestinal crypts, hair capsules, tes‐ relationship
between
ticular cells, and hTERC
ovarian and
cellscancer
[46–48].metabolism [42]. Studies
Most fetal tissues, in human
apart from cell lines
brain cells, showsuggest
high that
the upregulation
telomerase of hTERC
activity at 16–20 weeks ofcould lead to the
development inhibition
[49]. However,of it
angiogenesis and the
was not detected in down-
cells from a 2‐month‐old baby, with the exception of reproductive cells [49,50]. High te‐
lomerase activity is characteristic of most cancers, especially advanced and metastatic can‐
cers [45]. Normal breast tissue has been shown to lack telomerase activity [48]. In the case
of noninvasive ductal carcinoma, telomerase activity was demonstrated in 75% of exam‐
Int. J. Mol. Sci. 2022, 23, 12844 5 of 13

regulation of metastasis-related gene expression [43]. It has also been shown that hTERC
is involved in the transcription of genes mediated by the telomerase-dependent NFκB
signaling pathway [32,44].
Telomerase subunits may have many different nontelomeric functions. Many discrep-
ancies can be seen in the results of studies on their noncanonical functions. This is probably
a result of the diversity of research models and the lack of appropriate experimental tools
to gain a thorough understanding of the mechanisms involved in cells. Moreover, in cancer
cells hTERT is a weakly expressed protein, and the existence of alternative splice variants
of hTERT complicates the interpretation of the results [35].

7. Therapeutic Potential of Telomerase Subunits


7.1. Telomerase as a Target for Cancer Therapy
In most somatic cells, telomerase activity is below the detection limit [3,45]. The
exceptions are embryonic stem cells, marrow stem cells, intestinal crypts, hair capsules,
testicular cells, and ovarian cells [46–48]. Most fetal tissues, apart from brain cells, show
high telomerase activity at 16–20 weeks of development [49]. However, it was not detected
in cells from a 2-month-old baby, with the exception of reproductive cells [49,50]. High
telomerase activity is characteristic of most cancers, especially advanced and metastatic
cancers [45]. Normal breast tissue has been shown to lack telomerase activity [48]. In the
case of noninvasive ductal carcinoma, telomerase activity was demonstrated in 75% of
examined samples, and for ductal or lobular carcinoma this value was 88% [48]. Due to
the high expression of telomerase in tumor cells, it has become an important biomarker of
cancer and a promising therapeutic target [45].
The idea of telomerase-targeted therapy is to selectively induce the apoptosis of cancer
cells with minimal side effects of the therapy on normal cells. To achieve this goal, the
following therapeutic strategies are being pursued: the direct inhibition of telomerase,
the inhibition of hTERT or hTERC promoters, strategies based on telomeres, and the
development of vaccines based on the telomerase protein complex elements/antigens.
Several strategies directed against telomerase, e.g., imetelstat, Vx-001, and GRNVAC1, are
currently at various stages of clinical trials, which suggests that in the coming years a drug
targeting telomerase might be included in the pharmacopoeia [4,51–54].
One of the biggest problems in modern oncology is the lack of drug sensitivity of
cancer cells, mainly due to acquired resistance to anticancer agents. Studies indicate a link
between telomerase inhibition/repression and the increased sensitivity of cancer cells to
certain drugs, e.g., doxorubicin, 5-fluorouracil, and cisplatin [55–57]. The mechanisms of
telomerase resistance vary depending on the type of cancer and the type of drug used,
which means that they are still not fully understood. Nevertheless, telomerase-targeted
therapy shows great promise and is being tested in a wide range of cancer models [22].
One of these approaches is based on the direct inhibition of telomerase’s enzymatic
activity. There are currently several compounds that cause direct telomerase inhibition.
The first and probably the best-studied is GRN163L, also known as imetelstat. It is a 13-
mer oligonucleotide that inhibits telomerase by antagonistically binding to the telomerase
RNA template. Preclinical studies have demonstrated high efficacy of GRN163L [4,45,58].
For example, the treatment of breast cancer cells with this drug resulted in the inhibition
of telomerase activity and progressive telomere shortening, leading to a reduced risk of
tumor formation and cell invasiveness [58,59]. Imetelstat showed an inhibitory effect on
telomerase activity in CSC (cancer stem cell) populations of HER2+ breast cancer cells.
Moreover, imetelstat, both alone and in combination with trastuzumab, reduced the CSC
fraction and inhibited CSC functional ability, as shown by decreased mammosphere counts
and invasive potential. These results strongly suggest that the addition of imetelstat to
trastuzumab may enhance the effects of HER2 inhibition therapy, especially in the CSC
population [60]. Unfortunately, in clinical trials, imetelstat did not achieve the expected
efficacy and showed strong toxic effects, mainly neutropenia and thrombocytopenia [4].
Despite this, there are still high hopes for this drug, and more clinical trials are underway.
Int. J. Mol. Sci. 2022, 23, 12844 6 of 13

Telomerase inhibitors have shown limited improvement in patient prognosis. This is


related to the fact that, despite telomerase inhibition, cancer cells are still viable, and some
of them acquire resistance to inhibitors through ALT (alternative lengthening of telomeres)
induction. It is suggested that a much more effective strategy would be to use the hTERT
or hTERC promoters to express cytotoxic factors in a cancer-tissue-specific manner or to
generate mutations in hTERT promoters to reduce its expression [4,45].
Simultaneously, gene editing technology based on CRISPR interference and pro-
grammable base editing seems to be a very useful approach and might validate activating
TERT promoter mutations as a cancer-specific therapy. Li et al. exploited CRISPR to
downregulate TERT expression in glioblastoma cells. Introduced modifications blocked the
binding of E26 transcription factor family members to the TERT promoter, reduced TERT
transcription as well as TERT protein expression, and induced cancer cell senescence and
proliferative arrest [61].

7.2. Targeting Telomeres


Much attention has been given to strategies in which telomeres are destabilized
while telomerase activity is not affected. This approach has the advantage of therapy
efficacy, even in cancers that maintain telomeres through an alternative telomere (ALT)
elongation mechanism. Three main categories of potential drugs affecting telomeres can be
distinguished: G-quadruplex stabilizers, tankyrase inhibitors, and T-oligo [4].
One of the functions of the G-quadruplexes is to protect the ends of the chromosomes
from the action of exonucleases [62]. To enable telomere elongation, they are partially un-
wound by telomerase [63]. The G-quadruplex stabilizers prevent the access of the enzyme
to the substrate and thus prevent telomere restoration [45,64]. Importantly, this may be
efficient in ALT-positive cancer cells as well by conferring antiproliferative properties to the
cells [64,65]. However, due to the diversity of G-quadruplex structures, there are difficulties
in determining the conformations of the structures under physiological conditions, which
makes drug design much more complicated [65]. Currently, several formulations are under
investigation, including TMPyP4, BRACO-19, and telomestatin, but they have not yet
entered the clinical trial phase, unlike CX-5461, which is currently in phase I clinical trials,
with promising results [4,66–68].
Another category of drugs affecting telomeres is tankyrase inhibitors, which prevent
telomerase from binding to telomeres, thereby inhibiting their elongation. In addition,
they also show nontelomeric functions such as affecting the β-catenin-pathway-dependent
transcription pathway [4,69,70]. Tankyrase inhibitors were shown to cause more rapid
telomere shortening, which could lead to cell death [70,71]. These include, e.g., IWR1,
XAV939, and JPI-547, which are currently in clinical trials [4,72].
Another compound that is still under investigation is T-oligo. It is an 11-mer oligonu-
cleotide that is homologous to the 30 end of the telomere, which, when introduced into cancer
cells, increases p53 activity and induces autophagy and apoptosis, contributing to the elimina-
tion of cancer cells [4,70,73]. This therapeutic strategy induces DNA damage responses (DDR)
in several cancers, e.g., melanoma, breast carcinoma, and lung cancer [74–77]. Interestingly,
T-oligo has little or no effect on most normal cells [70,76,78]. Initial studies suggested that
T-oligo did not affect telomerase activity [78,79]. However, recent studies indicate that the
action of T-oligo may include a reduction in telomerase expression [73,75].

7.3. Genetic Profiling in Different Cancers


In 2013, Huang et al. [80] and Horn et al. [81] reported high frequencies of hTERT pro-
moter mutations in melanoma. Following their discoveries, some further reports showed
varying distributions of these mutations across cancers and indicated hTERT promoter mu-
tations to be the most frequent noncoding mutations in cancers. [82,83]. As demonstrated,
somatic mutations in the regulatory regions of target genes (transcription modulation)
also affected telomere length [84–87]. Consequently, hTERT mutations were indicated
to be important factors in oncogenic activation, cancer stemness, and proliferation [86].
Int. J. Mol. Sci. 2022, 23, 12844 7 of 13

Importantly, breast cancer has a very low frequency of TERT promoter mutation, which
implies epigenetic regulation in this type of cancer and possibly a wider systemic effect,
especially in HER2-positive breast cancer [88].

7.4. Immunotherapy
A novel approach is to use telomerase as a target for the development of immunother-
apy, and two main strategies have been adopted. The first is based on direct in vivo immune
activation with a vaccine directed against hTERT, which sensitizes cells of the immune sys-
tem to tumor cells [4,51]. Telomerase is degraded by proteasomes, leading to the formation
and presentation of hTERT-derived peptides as antigens on the cell surface [4,51,89]. This
induces an antitumor response of CD8+ or CD4+ cytotoxic T lymphocytes [51]. Promising
vaccines include GV1001 and Vx-001 (Geron, Foster City, CA, USA), both of which effec-
tively stimulate the immune system while inducing minimal side effects, as demonstrated
in clinical trials [4,53,90,91]. Another strategy is the ex vivo activation and expansion
of immune cells using the GRNVAC1 vaccine. The patient’s isolated dendritic cells are
transfected ex vivo and then administered to the patient via intradermal injection. This
induces a strong immune response with only grade 1 toxicity [4,51,91]. Importantly, these
studies have already progressed to in vivo experiments, which makes this strategy less
elusive and more realistic [92–94].
Another peptide-based cancer vaccine consisted of two human leukocyte antigens.
The subcutaneous injection of the TERT (572Y) peptide followed by the subcutaneous
administration of the TERT (572) peptide was aimed to elicit a specific and possibly optimal
cytotoxic T-cell (CTL) response against hTERT-expressing tumor cells [95]. The new trend in
cancer immunology is based on nucleic acids and an adjuvant approach. As shown in 2007,
chemokine adjuvant strategies might enhance the tumor-antigen-specific (hTERT-targeted)
immunity induced by vaccines. The hTERT DNA vaccine consisted of a plasmid containing
the carboxyl terminal end of the TERT (cTERT) gene encapsulated in multilayered liposomes
with a hemagglutinating virus of Japan coating. The study demonstrated that CCL21
administration before cTERT DNA vaccination significantly augmented tumor-antigen-
specific immunity against breast cancer [96].
Other studies on hTERT-based immunotherapy (three phase I/IIa clinical trials covering
malignant melanoma, non-small-cell lung cancer, and prostate cancer) showed that UV1, a
second-generation telomerase-targeting therapeutic cancer vaccine, triggered highly dynamic
and persistent telomerase-peptide-specific immune responses that lasted up to 7.5 years after
the initial vaccination. The superior immune response kinetics were observed in the melanoma
study [97].

7.5. New Generation Vaccines


Recent studies showed an optimized DNA plasmid encoding an inactivated form of
hTERT, named INVAC-1, capable of triggering cellular immunity against tumors. The intra-
dermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse
models elicited broad hTERT-specific cellular immune responses, including in highly CD4+
Th1 effector and memory CD8+ T cells. Furthermore, therapeutic INVAC 1 immunization
in an HLA-A2 spontaneous and aggressive mouse sarcoma model slowed tumor growth
and increased the survival rate of 50% of tumor-bearing mice. INVAC-1 vaccination was
safe, well-tolerated, and immunogenic when administered intradermally at the three tested
doses in patients with relapsed or refractory cancers. Disease stabilization was observed
for the majority of patients (58%) during the treatment period and beyond [98].
Altogether, there are nine ongoing hTERT-immunotherapy-based clinical trials at
different stages, out of which two are being carried out in solid tumors, including breast
cancers [99]. These are: NCT02960594—hTERT Immunotherapy Alone or in Combination
With IL-12 DNA and NCT02301754—INVAC-1 Anti-Cancer hTERT DNA Immunotherapy.
As hTERT is reported to affect the tumor metastatic potential, there is a need for an
appropriate model to study this phenomenon. One of the models that reflects the in vivo
Int. J. Mol. Sci. 2022, 23, 12844 8 of 13

conditions in humans is a mouse breast tumor model based on 4T1 tumor cells. The 4T1
mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic, invasive,
and, unlike most tumor models, can spontaneously metastasize from the primary tumor
in the mammary gland to multiple distant sites, including the lymph nodes, blood, liver,
lung, brain, and bone. Moreover, the progressive spread of 4T1 metastases to the draining
lymph nodes and other organs is very similar to that of human mammary cancer. Another
advantage of 4T1 is its resistance to 6-thioguanine, which enables the precise quantitation
of metastatic cells [100].
Despite being considered a promising antitumor strategy, to date, cancer vaccination
has not been shown to be a clinically useful approach in patients with severe disease [101].
This overall negative result is at least in part due to the poor effectiveness of gene transfer
with the currently employed strategies and, particularly, the frequent onset of a neu-
tralizing antibody response against the viral vectors, which limits the use of repeated
vaccinations [102,103]. Therefore, new immunization regimens that are able to reduce the
neutralizing antibody response and maintain an active immune response over time are
needed to assess the actual potential of cancer vaccination in clinical practice. The results
of a recent study suggested the safety and feasibility of V934/V935 hTERT vaccination (an
adenoviral type 6 vector vaccine expressing a modified version of hTERT, administered
alone or in combination with V934, a DNA plasmid that also expresses the same antigen) in
cancer patients with solid tumors. Further investigation is needed for a proper evaluation
of the efficacy of this strategy and its potential impact on clinical practice [103].

8. Summary
Intensive research on the regulation of telomerase in cancer cells in recent years has
allowed for the understanding of the mechanisms accompanying tumor proliferation,
telomere elongation, and noncanonical enzyme functions. All of these make it possible
to develop new, effective anticancer drugs. Unfortunately, there are still many unsolved
questions concerning telomeres and telomerase, such as whether telomerase expression has
oncogenic properties [51]. It is also important to note that it has still not been possible to
develop an effective telomerase-based drug that would be approved for clinical use. This
shows the need for further research. However, it is undeniable that the study of telomerase
has contributed to a deeper understanding of cancer biology and has opened up promising
new therapeutic avenues [4,51,104]. Research conducted in recent years has led to the
development of many structurally diverse compounds with different mechanisms of action.
Therapies based on targeting telomerase and telomeres or combination therapies may
prove to have high efficacy against telomerase-positive tumors while sparing neighboring
normal cells [45,105]. In addition, the use of molecularly targeted therapy makes it possible
to bypass the side effects characteristic of conventional therapies [45,73]. Hopefully, the
knowledge accumulated so far will soon allow researchers to improve the therapeutic
efficacy and eliminate the side effects of previously known drugs targeting telomeres and
telomerase or will allow the development of new and effective drugs. However, further
research is needed to make this possible [105].

Author Contributions: Conceptualization, E.J. and B.R.; writing—original draft preparation, E.J. and
B.R.; writing—review and editing, N.L. and P.K.; visualization, E.J.; supervision, B.R.; immunotherapy
supplementation, M.T. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by Poznan University of Medical Sciences.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This work was supported by Poznan University of Medical Sciences.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2022, 23, 12844 9 of 13

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