Seminars in Cancer Biology xxx (xxxx) xxx–xxx

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Seminars in Cancer Biology

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Review

The role of senescence in cancer development

⁎
Eleni Mavrogonatou, Harris Pratsinis, Dimitris Kletsas
Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Centre for Scientific Research “Demokritos”, Athens, Greece

A R T I C LE I N FO A B S T R A C T

Keywords: While research on cancer development is traditionally focusing mainly on the neoplastic cell per se, nowadays
Senescence the role of tumor stroma in this process is indisputable. The stroma - mainly composed of extracellular matrix
Stroma (ECM) - is a source of mediators and signals originating from heterotypic cell-cell and cell-matrix interactions
Extracellular matrix that steer the progression of the disease in a context- and a cancer type-dependent manner. With advancing age
Senescence-associated secretory phenotype
the stroma exhibits alterations, important being the accumulation of senescent cells. Senescence is often trig-
Senolytic
gered by exogenous stresses, including genotoxic anticancer treatment modalities (such as chemotherapy or
radiotherapy) and is manifested as an inhibition of cell proliferation, ascribing to cellular senescence the role of a
potent antitumor barrier. On the other hand, senescent cells, through their specific senescence-associated se-
cretory phenotype (SASP) - comprising cytokines, growth factors, ECM components and ECM-degrading enzymes
- can establish an immunosuppressive, inflammatory and catabolic microenvironment that may stimulate tumor
growth and metastasis. Given that the persistent presence of senescent cells could prove detrimental for tissue
homeostasis, inclusion of a senotherapeutic arm in novel anticancer approaches seems compulsory.

1. Introduction
In oversimplified terms, cancer is the outcome of the unlimited and
uncontrolled cell proliferation and its manifestation is often associated
with increased chronological age. The hallmarks of cancer consist of
distinct and at the same time complementary traits that are required for
the survival, proliferation and dissemination of malignant cells, as well
as for the succeeding progression of the tumor, i.e. incessant pro-
liferative signaling, escape from growth suppressors, resistance to cell
death, induction of angiogenesis, activation of invasion and metastasis,
replicative immortality, genomic instability generating random muta-
tions, tumor-promoting inflammation, metabolism reprogramming to-
wards anaerobic glycolysis to support sustained cell proliferation and
active evasion from the elimination by immune cells [1].
Given that most of human cancers are of epithelial origin [2,3], for
many years carcinogenesis and further tumor growth was believed to be
a one-dimensional process, depending solely on the accumulation of
sequential mutations in the genes carried by epithelial cells [2]. This
reductionist approach is incomplete as it is now well established that
the stroma is an additional key player in cancer development [4–6].
The primary goal of traditional anticancer therapies was merely to
abrogate tumor cells’ unrestrained proliferation by driving them to cell
cycle arrest or apoptosis. Nevertheless, coping with cancer nowadays
has moved ahead to more sophisticated strategies that take into account
several parameters of the multistep process of tumor growth, the bio-
chemical alterations of the stroma (including changes in stromal cells,
such as the induction of senescence), the interconnection of neoplastic
cells with the stroma, the activation of the immune system, as well as
the putative side-effects of the treatment itself on the future progress of
the disease. Even though our knowledge is growing and advance has
been achieved, new therapeutic approaches are needed targeting the
induction, abolishment or disruption of molecular pathways in malig-
nant cells and metabolic components of the stroma. Still, innovative
drugs and therapy schemes should be cautiously designed, since dis-
turbance of the subtle equilibrium between tumor promotion, preven-
tion and cure could prove deleterious for cancer development.
2. The role of the stroma in cancer development
The stroma mainly consists of extracellular matrix (ECM) and covers
the region between the tissue’s basement membrane and the cancer
parenchyma, thus forming a structure that surrounds malignant cells
and can often represent more than 90% of the solid tumor [2,6,7].
Embedded in the ECM, non-cancerous stromal cells include fibroblasts,
endothelial cells, specialized mesenchymal cells and immune cells
[4,8], with fibroblasts constituting the most important cell type, since

⁎
Corresponding author at: Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Centre for Scientific Research
“Demokritos”, 153 10 Athens, Greece.
E-mail address: dkletsas@bio.demokritos.gr (D. Kletsas).

https://doi.org/10.1016/j.semcancer.2019.06.018
Received 20 May 2019; Received in revised form 24 June 2019; Accepted 27 June 2019
1044-579X/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Eleni Mavrogonatou, Harris Pratsinis and Dimitris Kletsas, Seminars in Cancer Biology,
https://doi.org/10.1016/j.semcancer.2019.06.018
E. Mavrogonatou, et al.
they are responsible for the production of the structural components of
the stroma and the basement membrane, as well as for the secretion of
cytokines, growth factors, ECM components and ECM-degrading en-
zymes that can result in local tissue remodeling and alterations in the
architecture of the tumor microenvironment [2,4,9–11]. In the course
of tumor development fibroblasts become activated, lapsing into cancer
associated fibroblasts (CAFs) that promote malignant growth, invasion,
and metastasis [12–15]. Furthermore, the decisive role of the stroma in
tumorigenesis has been evidenced in numerous studies demonstrating
on one hand cancer growth suppression by a normal stroma and on the
other hand promotion of tumor development by an activated stroma
[16–20]. Paradigms of mediators produced by the stroma that de-
termine the fate of cancer progression through heterotypic cell and cell-
matrix interactions are transforming growth factor-β (TGF-β) and in-
terleukin (IL)-6 secreted by CAFs - that can affect proliferative, mi-
gratory, survival and invasive capacities of malignant cells - [21–23] or
matrix metalloproteases (MMPs) and members of the A Disintegrin And
Metalloproteases (ADAM) protein family - that can catabolize ECM
constituents and affect tissue homeostasis, creating a permissive en-
vironment for tumor growth [5,24–31]. Evolving ECM plays a dynamic
role in tumor behavior, as it seems to adapt in every stage of tumor-
igenesis, influencing the progression of the disease. It may abolish
proliferative restriction and increase replicative capacity via telomerase
(the enzyme that secures telomere size) overexpression, inhibit tumor
suppressors, reduce chemotherapeutic responsiveness, favor angiogen-
esis and enhance invasion through actin assembly remodeling or in-
tegrin-dependent and -independent cell adhesion [1,32]. In addition,
cancer-associated ECM alterations may negatively affect immune re-
sponses, subserve the metabolic shift to pathways that feed the in-
creased energy requirements of the highly proliferating malignant cells,
hamper DNA repair machineries and influence the activation state of
innate immune cells [32]. However, predisposition to malignancy in
different contexts and/or types of cancer may stem from opposing ECM
alterations, rendering it difficult to make general assumptions and to
correlate specific ECM modifications to anticipated outcomes in every
cancer type; for instance, fibrosis and collagen cross-linking have been
associated with a greater risk for breast cancer development, but MMPs’
overexpression is a poor prognostic factor in breast cancer patients, as
well [32].
Age-related cancer has been initially ascribed to an age-dependent
raise in genomic instability and to the exponential increase and accu-
mulation of somatic mutations and epigenetic alterations over time
[33–36]. However, given the pronounced implication of the stroma in
cancer progression already described above, the role of the aged stroma
- which undoubtedly differentiates from that of a young individual -
should be added to these factors [4,37]. A major alteration of the aged
stroma concerns its cellular components and stems from the accumu-
lation of senescent cells and especially that of senescent fibroblasts [4].
3. Cellular senescence
Cellular senescence was first described by Hayflick and Moorhead in
normal human embryonic lung fibroblasts, back in 1961, as the per-
manent cessation of cell divisions in vitro [38] and was subsequently
demonstrated in several other cell types [39]. This type of cellular se-
nescence reached after serial subculturing in vitro is called replicative
senescence and has been attributed to the gradual loss of telomere
length after each cell division due to the “end replication problem”, i.e.
the incomplete replication of the end of chromosomes during lagging
strand DNA synthesis [40–42]. Telomere shortening has been proven to
serve as the “mitotic clock” leading normal cells to senescence, since
germ and cancer cells expressing telomerase display an infinite re-
plicative potential, while introduction of the enzyme’s catalytic subunit
in telomerase-deficient cells may extend their lifespan and even render
them immortalized [43,44]. Loss of telomeric length beyond a certain
threshold is perceived by the cells as a DNA damage, which drives DNA
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
Fig. 1. Major signaling pathways leading to cellular senescence. Telomere
shortening or a broad spectrum of other genotoxic stresses is triggering a DNA
damage response, which through the indicated pathways leads to a persistent
growth arrest, the major feature of cellular senescence.
damage response (DDR) pathways, such as the activation of the ATM-
Chk2-p53-p21WAF1-pRb axis leading to an irreversible growth arrest
[45–48] (Fig. 1).
Besides replicative exhaustion, there are additional internal and
external stressors that lead to another type of senescence (usually in-
dependent of telomere erosion), the so-called stress-induced premature
senescence (SIPS) [49,50]. Among the stresses eliciting SIPS lay oxi-
dative stress, exposure to ionizing radiation, treatment with che-
motherapeutic drugs and other genotoxic insults, while oncogene ac-
tivation fuels a special type of SIPS, namely the oncogene-induced
senescence (OIS) [51–55]. OIS was initially described in primary
mammalian cells after the overexpression of ras [51] and was further
shown to be induced by a long list of oncogenes [54,55] and to occur in
vivo, as well [56], strongly supporting the notion that cellular senes-
cence is a potent antitumor barrier. The shared trigger in replicative
senescence and SIPS seems to be the provocation of a persistent DNA
damage and the subsequent launching of a DDR [46,55,57–62] (Fig. 1).
Irrespective of the stimulus evoking senescence, senescent cells
display some common features: 1. an irreversible cell cycle arrest at the
G1 and occasionally at the G2 phase, even in the presence of mitogens
[59,63–66], which also corroborates the original hypothesis that the
evolutionary role of senescence is the avoidance of cellular transfor-
mation and cancer development [42]. This growth arrest has been as-
sociated with the up-regulation of several tumor suppressor genes, i.e.
p16INK4a, p14ARF, pRb, p53, p21WAF1 or p27KIP1, which could be used as
molecular markers of cellular senescence [67–73]; 2. an enlarged
morphology and high granularity due to the presence of an expanded
lysosomal compartment and vacuoles, which is conspicuously distinct
from that of young (early-passage) cells [4,74,75]; 3. an increased
metabolic rate, e.g. higher glucose consumption and lactate production
[76,77]; 4. resistance to apoptosis [78,79]; 5. nuclear and chromatin
alterations (i.e. lamin B1 deficiency [80,81], reduction in hetero-
chromatin, site-specific changes in DNA methylation patterns and his-
tone modification [82], appearance of DNA damage response structures
DNA-SCARS [83] and DNA damage markers, such as γH2A.X and
53BP1 foci, senescence-associated distension of satellites (SADS) and
senescence-associated heterochromatin foci (SAHF) at E2F-regulated
genes [84–86]); 6. a unique bioactive, pro-inflammatory and proteo-
lytic secretome, known as the senescence-associated secretory pheno-
type (SASP) [87]. Given that for a long period of time it was unclear if
senescence does indeed occur in vivo or is an artifact of the culture
E. Mavrogonatou, et al.
conditions in vitro, the necessity for a reliable marker for the identifi-
cation of senescent cells was indispensable. In this direction, the se-
nescence-associated beta galactosidase (SA-β-Gal) histochemical pro-
cedure was developed, which detects senescent cells by their prominent
beta galactosidase activity at pH 6.0, resulting in a characteristic blue
perinuclear staining [88,89]. Even though revolutionary, we are now
aware that SA-β-Gal staining should be cautiously interpreted, as the
methodology is cell density- and stimulus-dependent and may thus lead
to pseudo-positive results [90–92]. The recently discovered staining for
Sudan Black B of lipofuscin (that accumulates in the cytoplasm of se-
nescent cells) overrides most of the limitations of SA-β-Gal staining and
can be performed even in archival samples [93–95]. Based on the fact
that senescence is complicated and multiparametric, it seems that a
combination of morphologic, molecular and functional traits is rather
necessary for the characterization of the senescent phenotype [96].
Since senescent cells have been thus far evidenced to accumulate
with age [97] and to create a permissive environment for cancerous
outgrowth [86,95,98–101], it remains to fully elucidate the mechan-
isms by which senescent cells can synergize with malignant cells and
promote cancer development.
4. The senescence-associated secretory phenotype (SASP)
As already mentioned, cellular senescence is a complex phenotype
elicited by diverse inputs and manifested through multiple effector
programs. Beyond the inability to proliferate, senescent cells are char-
acterized by a specific secretory phenotype, namely the SASP, that
consists of numerous inflammatory mediators, proteolytic enzymes,
cytokines, growth factors and even nucleic acids and proteins enclosed
in exosome-like small extracellular vesicles [87,102–104]. The SASP is
positively regulated by p38 MAPK [105] and nuclear factor κB (NF-κB)
[106], while it is dependent on the DDR proteins ATM, Nbs1 and Chk2,
but not on the cell cycle regulators p53 and pRb [107]. SASP has been
shown to be a conserved senescence-associated feature in several cell
types including cancer cells [103,108,109]. On one hand SASP pro-
motes tissue regeneration through the induction of plasticity and
stemness [110,111] and participates in the clearance of cancer cells by
attracting immune cells [112,113]; on the other hand it can establish an
immunosuppressive, inflammatory and catabolic microenvironment
and stimulate tumor growth and metastasis [95,114–120] (Fig. 2). In
addition, SASP factors have been shown to reinforce senescence via
autocrine and paracrine mechanisms [121], but also to affect adjacent
cells and tissue homeostasis in a paracrine manner, even at great dis-
tance causing a bystander effect [122–125].
5. Senescence-associated changes in the extracellular matrix
(ECM)
It has been stated above that SASP comprises of numerous insoluble
and soluble factors, including ECM components and proteolytic
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
enzymes that cleave ECM, respectively. Up-regulation of MMPs has
been firmly connected with senescence in several cell types like fibro-
blasts [86,103,105,126–131], vascular smooth muscle cells (VSMCs)
[132,133], intervertebral disc (IVD) cells [134–136] and hepatic stel-
late cells (HSCs) [137] regardless of the stimulus inducing senescence
and of the culture conditions [105,130,136,138]. Additionally, tissue
inhibitors of MMPs (TIMPs) have been mainly shown to be down-
regulated during senescence [126,129,134,139]. The aforementioned
increased senescence-associated proteolytic activity is reinforced by the
up-regulation of enzymes belonging to the A Disintegrin And Metallo-
proteinase with Thrombospondin motifs (ADAMTS) protein family
[134,135,140], cathepsins [127,141], urokinase-type plasminogen ac-
tivator (uPA) and tissue plasminogen activator (tPA) [103,127,142]
and plasminogen activator inhibitors (PAI) -1 and -2 [142–144]. Ex-
tracellular PAI-1 has been also shown to be downstream of p53 in the
induction of replicative senescence [145]. Moreover, it was found that
prolonged activation of nuclear factor-erythroid 2-related factor 2
(Nrf2) leads fibroblasts to senescence and that ECM deposited by these
senescent cells further promotes senescence through PAI-1 [125].
In contrast to ECM-degrading molecules that are mainly over-
expressed in senescent cells, ECM constituents have been reported to be
down-regulated in general, thus establishing the catabolic aspect of the
senescent phenotype [95]. In detail, collagen and collagenous proteins
show lower expression in senescent cells, in parallel to the down-reg-
ulation of prolyl 4-hydroxylase that catalyzes collagen biosynthesis
[103,127,128,130,133,137,141,146,147]. Elastin has been demon-
strated to be down-regulated in senescent cells [103,127], as well, in
contrast to fibronectin, tenascin and laminin that have been found to be
induced [103,146,148–152]. Regarding proteoglycans, they have been
mainly shown to be underexpressed and to display modifications con-
cerning their molecular size, binding properties, etc. in senescent fi-
broblasts, chondrocytes, endothelial cells, VSMCs and IVD cells or in
vivo in the skin [132,134,153–158]. On the contrary, the cell surface
heparan sulfate proteoglycan syndecan-1 (SDC1) has been shown to be
up-regulated in prematurely senescent human breast fibroblasts in vitro
and in vivo [99]. Furthermore, hyaluronic acid biosynthesis - whose
overexpression has been linked to the extended lifespan and resistance
to cancer development of the naked mole-rat [159,160] - is reduced in
senescent mesenchymal stem cells due to the down-regulation of hya-
luronic acid synthases [161,162].
Beyond quantitative changes in ECM components and degrading
enzymes, ECM produced by senescent cells also differentiates qualita-
tively and in terms of organization from that produced by young cells
[95,163]. More explicitly, the inflammatory phenotype of senescent
cells accompanied by changes in the levels of ECM components and
ECM-degrading molecules disturb elastin and collagen fiber networks
and basal membrane integrity in the aged tissue. Additionally, it has
been shown that inappropriate collagen cross-linking observed during
ageing renders ECM more rigid, less elastic and mechanically weaker
[95,163].
Fig. 2. Role of senescent cells in tumor progression. Various
factors trigger senescence of normal and cancer cells. Senescence
characterized by an irreversible growth arrest acts as an antitumor
barrier. On the other hand, the soluble and insoluble factors of the
senescence-associated secretory phenotype (SASP) stimulate
tumor growth and metastasis, but also trigger immune recognition
and tumor clearance.
E. Mavrogonatou, et al.
The great importance of the ECM interplay with cells has been va-
lidated by the reversal of the senescent phenotype of fibroblasts when
they were cultured onto ECM isolated from young cells. These cells
displayed signs of a youthful phenotype, i.e. a spindle-like morphology
and an actin structure pattern similar to that of young cells, lower SA-β-
Gal activity, reduced intracellular reactive oxygen species (ROS) levels,
increased novel DNA synthesis, improved mitochondrial function, a
better response to mitogenic signals like epidermal growth factor (EGF),
decreased levels of p53, p21WAF1, p16INK4a and caveolin-1 and even
longer telomeres [164]. When, in their turn, young cells were plated
onto ECM isolated from senescent cells, their proliferation was only
decelerated [164]. In contrast to this finding, young fibroblasts grown
onto ECM produced by Nrf2-induced senescent cells not only showed a
lower proliferation rate, but they also displayed significantly more SA-
β-Gal activity compared to cells grown onto control ECM [125]. Ad-
ditionally, it has been demonstrated that autologous decellularized
ECM protects adipose-derived stem cells against H2O2-induced senes-
cence and stimulates their proliferation in vitro [165].
6. The role of senescent cells in cancer progression
Since cancer has been considered as “a wound that never heals”,
similarities in the implication of senescent cells in wound healing and
cancer development are expected [166]. It has yet been well established
that transient presence of senescent cells is beneficial during normal
tissue repair due to their anti-fibrotic phenotype [137,167–170], but
accumulation of senescent cells can be detrimental to local tissue
homeostasis due to their pro-inflammatory properties [171–174]. In
this vein, even though senescence is a potent tumorigenic barrier when
initially induced [53,55,72,175–177], the persistent presence of se-
nescent cells often allies with malignant cells and supports tumor ex-
pansion [178]. Indeed, accumulating senescent cells through their non-
cell-autonomous activities become accomplice with cancer cells and are
main drivers of cancer initiation and progression [87,121,179], in-
dicating that cellular senescence is a “double-edged sword” in cancer
development [180]. We have mentioned above that among all senes-
cence-associated traits, SASP is mainly culpable for this two-pronged
role of cellular senescence. Although the specific outcome of the SASP is
context-dependent, the net effect of its paracrine action in advanced
cancer is to directly enhance the proliferative and metastatic potential
of neoplastic cells or to indirectly allow them to expand by forming a
permissive milieu, as it may locally result in tissue remodeling
[95,120]. In favor of this, it has been reported that stem cell senescence
and a stem cell-associated SASP drive cell transformation and induction
of pituitary tumors in mice [181,182], while in the case of pediatric
pilocytic astrocytoma (PA), SASP has been shown to be a strong reg-
ulator of PA tumor growth by inducing OIS in proliferating PA cells
through IL-1β [183]. IL-6 secreted from senescent mesenchymal stem
cells promoted proliferation and migration of breast cancer cells [184]
and IL-6 secreted by senescent osteoblasts increased breast cancer co-
lonization of the bone [116]. Senescence-associated alterations in the
production and secretion of matricellular proteins and ECM con-
stituents (hyaluronic acid, collagens, laminins and integrins) of prostate
cells have been shown to create a favorable milieu for tumor develop-
ment [185]. Furthermore, it has been demonstrated that senescent fi-
broblasts promote the development of cancer from ovarian surface
epithelial cells at an early stage of neoplastic transformation [186],
stimulate premalignant and malignant epithelial cells to proliferate in
culture and to form tumors in vivo [98] and enhance the malignant
progression of keratinocytes via MMP-1 and MMP-2 or ROS secretion
[187,188]. This phenomenon has been in some cases attributed to
epithelial-to-mesenchymal transition (EMT) of cancer cells [98,187]. In
addition, premalignant mammary epithelial cells exposed to senescent
human fibroblasts in vivo underwent full malignant transformation and
non-malignant breast epithelial cells co-cultured with senescent fibro-
blasts in vitro showed an MMP-3-mediated disruption in their branching
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
morphogenesis [189]. Finally, co-injection of Nrf2-induced senescent
fibroblasts with weakly malignant human squamous cells into the ear or
the back skin of immunocompromised mice led to the development of
larger and more invasive tumors [125]. These senescent fibroblasts
were found to deposit a senescence-promoting ECM, thus leading to a
perpetuating series of events reinforcing senescence and promoting
tumor growth [125].
Harking back to the tight relationship of the ECM with the mani-
festation of the major features of cancer discussed above, we should
point out that an interrelation of the senescence-associated ECM al-
terations with the onset and progression of cancer could be also pro-
posed. As mentioned earlier, ECM in aged tissues and several age-re-
lated pathologies (where the accumulation of senescent cells has been
identified) is characterized by the loss of tensile strength and by in-
creased stiffness [95,163], which change the mechanical properties of
the matrisome (i.e. all ECM proteins and associated factors) that serves
for epithelial growth and expansion and could thus rigorously interfere
with tumor progression.
7. Therapy-induced senescence (TIS)
We have referred so far to replicative senescence and SIPS provoked
in stromal fibroblasts due to the normal stressors cells routinely face.
What we should also take under consideration when discussing the
implication of senescent cells and SASP in tumor development is the
therapy-induced senescence (TIS), a type of senescence occurring not
only in normal stomal cells, but also in cancer cells during treatment of
the disease. The TIS program is launched as a response to genotoxic
chemotherapy and radiation and has indeed been demonstrated to be
adopted not only by the neighboring to the tumor healthy cells
[86,99,190–192], but also by cancer cells themselves, depending on
their molecular profile [177,193–202]. In detail, regarding che-
motherapeutic compounds, it has been shown that doxorubicin, eto-
poside and cisplatin (which introduce double- or single-strand breaks
into the DNA) can lead normal human fibroblasts to premature senes-
cence [190,191,203] and that busulfan (which can cause DNA-DNA and
DNA-protein cross-links) also results in the senescence of human fi-
broblasts in a ROS-dependent manner [192,204]. On the other hand,
chemotherapeutic drugs like doxorubicin, cisplatin, camphotecin (a
topoisomerase I inhibitor) and dicodermolide (which stabilizes micro-
tubules) have been shown to trigger accelerated senescence in several
types of cancer cells, including colon carcinoma, fibrosarcoma, breast
cancer, nasopharyngeal cancer and non-small cell lung cancer cells,
directly or through a bystander effect [195,197–199,201,205,206], a
phenomenon that has been also verified in vivo [193,200]. Besides
chemotherapy, ionizing radiation used in radiotherapy has been shown
to drive stromal fibroblasts [86,207,208] and cancer cells [202,209] to
premature senescence, as well. It is noteworthy that p53 and p16INK4a
(known to be often mutated in tumors) are not always mandatory for
the manifestation of TIS in cancer cells [193–196,210].
The SASP of stromal and cancer cells becoming senescent in the
course of anticancer therapies has also been demonstrated to affect
tumor progression and the therapeutic outcome. Hence, TIS has been
deservedly now considered a significant side-effect of anticancer
treatments that should be taken into account when designing a parti-
cular therapeutic scheme. Bleomycin-induced senescent human fibro-
blasts enhanced the growth of co-transplanted breast cancer cells in
immunodeficient mice, which was diminished when MMP activity was
inhibited [101]. Additionally, irradiation-induced senescent lung
stromal fibroblasts stimulated cancer cells’ growth both in vitro and in
vivo, partly through MMP up-regulation [86]. In contrast to the soluble
SASP elements (and predominantly MMPs) that have been thoroughly
investigated during carcinogenesis [87,120], to the best of our knowl-
edge the implication of the insoluble segment of the SASP in cancer
progression has been barely studied so far. Focusing on the insoluble
ECM components encompassed in SASP, we have recently shown that
E. Mavrogonatou, et al.
along with its profound catabolic character, the tumor-promoting
phenotype of ionizing radiation-induced senescent human breast
stromal fibroblasts is filled in by the overexpression of SDC1 [99]. SDC1
overexpression in the stroma is known to represent a poor prognostic
factor for the development of several types of cancer [99,211–216].
Increased SDC1 levels in ionizing radiation-induced senescent breast
stromal fibroblasts have been found to be TGF-β-regulated via the
SMAD pathway in collaboration with the transcription factor Sp1 [99].
Regarding therapy-induced senescent cancer cells, the supernatant of
cisplatin-induced senescent A375 melanoma cells promoted the growth
of non-senescent A375 cells and co-transplantation of senescent with
non-senescent A375 cells resulted in accelerated tumor growth into
nude mice, with IL-1α and IL-8 being the key SASP factors regulating
these events [217]. Finally, senescent tumor cells can contribute to
cancer recurrence and increased mortality rates, since they are able to
retrieve their proliferative potential after a period of dormancy [218].
In favor of this, it has been reported that p53- and p16INK4a-deficient
human lung cancer cells can bypass chemotherapy-induced accelerated
senescence and resume cell cycle progression [210]. It has been also
proposed that the SASP of persisting therapy-induced senescent cells in
the microenvironment of the tumor may drive escape from senescence
and re-entry of malignant cells into proliferation, while it provokes
local and systemic inflammation [218–221]. These deleterious out-
comes may often exacerbate the side-effects of anticancer treatments
and even allow cancer relapse, which could justify poor prognosis in
patients expressing senescence markers after treatment with neoadju-
vant chemotherapy [222,223].
8. Clearance of senescent cells in vivo
Having in mind the pro-inflammatory character of SASP, one could
anticipate the elimination of senescent cells through immune system
responses. Indeed, in liver carcinomas of p53-deficient mice, the re-
activation of p53 was observed to activate a premature senescence
program and an up-regulation of inflammatory cytokines, leading to the
triggering of an innate immune response, elimination of senescent cells
and eventually tumor regression [112]. Moreover, in mice with liver
damage, normal hepatic cells are activated, proliferate and overexpress
ECM molecules towards tissue repair. Persistence of this effect may lead
to fibrosis, however the activated cells enter senescence, thereby fa-
cilitating the resolution of fibrosis due to their catabolic phenotype and,
moreover, they attract natural killer (NK) cells effecting senescent cells’
clearance. In contrast, in mice lacking both p53 and p16INK4a (hence
their liver cells are unable to undergo senescence) the same treatment
causes severe fibrosis [137]. Especially in the case of OIS in hepato-
cytes, it has been shown that the latter secrete soluble factors triggering
their immune-mediated clearance, a phenomenon termed “senescence
surveillance”, while if these pre-malignant senescent hepatocytes are
not eliminated, murine hepatocellular carcinomas are being developed
[224]. In this vein, senescent HSCs, beyond their inability to proliferate
per se, promote also an antitumor microenvironment through paracrine
factors that - in a p53-dependent manner - facilitates macrophage po-
larization toward the M1-state, which enables senescent cell elimina-
tion [225]. A mechanism underlying senescence surveillance includes
the overexpression by senescent cells of ligands for an activating NK cell
receptor (NKG2D) as a result of DDR [226]. The importance of the
immune system in limiting senescent cell accumulation in various tis-
sues has been shown in mice lacking perforin, an important mediator of
immune cytotoxicity [227]. These mice in turn develop chronic in-
flammation, a common denominator in most age-related diseases
[228]. Regarding tumor development, it seems that the combination of
cancer cell senescence with immune system recognition can be bene-
ficial, hence cytokine-induced “bystander” senescence has been sug-
gested as a promising novel therapeutic modality [229–231]. In this
direction senescence of the immune system itself, i.e. “im-
munosenescence”, may play an important role for the accumulation of
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
senescent cells in various tissues [232,233], hence future therapies in-
volving the modulation of the immune responses to favor senescent cell
clearance may be envisaged [234].
9. Emerging senotherapeutics
Given that the persistent presence of senescent cells and their me-
tabolic products could impede remedy of cancer and thus prove detri-
mental for rehabilitation, it becomes clear that inclusion of a se-
notherapeutic arm (i.e. a mode to specifically target senescent cells
and/or their metabolic products) should perhaps be considered in the
design of novel anticancer treatments. As mentioned above, in certain
cases clearance of senescent cells is achieved by the immune system,
thus limiting their detrimental effects [137]. Moreover, genetic ap-
proaches have been developed to totally eliminate senescent cells from
the organism of experimental animals, hence preventing tissue dys-
function and even extending lifespan [235,236]. Since, however, such
approaches cannot be applied to humans, in the last five years an in-
tensive search for senolytics has begun, i.e. for natural products or
synthetic compounds capable for selectively destructing senescent cells
without affecting non-senescent ones. The first senolytic compounds
reported were the natural product (flavonoid) quercetin and the tyr-
osine kinase inhibitor dasatinib [237]. The rationale behind their dis-
covery is related to the increased resistance of senescent cells to
apoptosis [78]; indeed quercetin among a plethora of other biological
effects inhibits the PI3K/Akt/mTOR pro-survival signaling pathway,
while dasatinib interferes with the Ephrin B ligands, known to prevent
apoptosis [237,238]. Similarly, other reported senolytics include the
small molecule inhibitors navitoclax (or ABT-263) and ABT-737 that
target the BCL-2/BCL-W/BCL-XL family of anti-apoptotic proteins
[239–241], the relatively selective against BCL-XL inhibitors A1331852
and A1155463, as well as, the flavone fisetin that is related to quercetin
[242], the natural product piperlongumine and its synthetic analogues
that target the cellular oxidative stress sensor oxidation resistance 1
(OXR1) [243–245], the synthetic FOXO4 D-Retro-Inverso peptide that
disrupts the FOXO4-p53 interaction [246], and geldanamycin and its
semi-synthetic derivative 17-dimethylaminoethylamino-17-demethox-
ygeldanamycin (17-DMAG) that inhibit Heat-Shock Protein-90 (HSP-
90) thereby destabilizing Akt and phospho-Akt [247]. Interestingly,
senolytics may also target cancer cells led to senescence due to standard
chemotherapy, thereby augmenting therapy, as shown for the histone
deacetylase inhibitor panobinostat [248]. An important observation
from all these reports was that the effects of all verified senolytics up to
now appear to be cell-type dependent [238,249]. Notably, the dasa-
tinib-quercetin senolytic cocktail was found to be ineffective in an an-
imal model of hepatocellular carcinoma, alone or in synergy with se-
nescence-inducing chemotherapy [250]. Moreover, the in vivo
application of senolytics may be accompanied by toxic side-effects,
especially if they target central anti-apoptotic cellular mechanisms
[251]. As a consequence, the use of senomorphics may be preferable,
i.e. compounds modulating the catabolic and/or inflammatory pheno-
type of senescent cells [252]. In this direction, many substances sup-
pressing the SASP have been identified, most notably interfering with
the NF-κB pathway, like various flavonoids, such as apigenin and
kaempferol [253] and metformin [254], or inhibiting JAK (ruxolitinib
[255]), or generally conveying anti-inflammatory and antioxidant ef-
fects, such as glucocorticoids [256], rapamycin [257] or resveratrol and
its analogues [258]. Notably the latter seem to exert their senomorphic
effects through modulation of splicing factors, in accordance with the
recent finding that knocking down the expression of the alternative
splicing regulator polypyrimidine tract binding protein 1 (PTBP1) in-
hibits the SASP of senescent hepatocytes and, most importantly, its pro-
tumorigenic effects [259]. Of course, SASP suppression may also have
“off-target” effects [260] and could theoretically even impair clearance
of preneoplastic cells by the immune system [259]. In fact, many of the
senolytic and semomorphic compounds are long known and have been
E. Mavrogonatou, et al.
tested in various clinical trials, and some of them were initially iden-
tified as anticancer drugs [238]. Moreover, depleting senescent cells in
pre-clinical studies has been shown to successfully treat chronic con-
ditions, such as frailty, cardiac dysfunction, vascular hyporeactivity and
calcification, diabetes, liver steatosis, osteoporosis, IVD degeneration,
pulmonary fibrosis, and radiation-induced damage [261], while a re-
cent in vivo study in mice, where senescent cells’ transplantation was
shown to cause physical dysfunction and decreased survival, indicated
also that senolytics can alleviate physical dysfunction and increase late-
life survival without extending morbidity [262].
10. Concluding remarks
Since senescent cells - occuring also as a side-effect of genotoxic
anticancer treatments - may create a favorable milieu for tumor ex-
pansion or even assist malignant cells to escape and promote cancer
relapse, it seems crucial that future anticancer therapies will not only
target neoplastic cells, but they will also aim at the elimination of ac-
cumulating senescent cells or the modification of their senescence-as-
sociated secretome. Given the great heterogeneity in various cancer
types, a general therapeutic scheme could not always be applied and
personalized optimization may be necessary towards an effective cancer
remedy.
Acknowledgements
This work was partly supported by the EU Horizon 2020 project
MSCA-RISE-2014, action No. 645756 “GLYCANC - Matrix glycans as
multifunctional pathogenesis factors and therapeutic targets in cancer”
and by the project “Target Identification and Development of Novel
Approaches for Health and Environmental Applications” (MIS
5002514), which is implemented under the Action for the Strategic
Development on the Research and Technological Sectors, funded by the
Operational Programme "Competitiveness, Entrepreneurship and
Innovation" (NSRF 2014-2020) and co-financed by Greece and the
European Union (European Regional Development Fund).
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