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Title: Thyroid cell senescence: implications for
carcinogenesis and molecular therapy of thyroid cancer
Authors; T. Zouki, I. Alwine, A. Podtcheko
Abstract:
Senescence is terminal growth arrest that can be induced by a variety of processes, such
as oncogenic signaling, DNA damage, oxidative stress and chemotherapeutic drugs. The
manifestations of senescence are morphological changes, and increased gene expression
that leads to cell cycle arrest and the induction of secretomes to be released by senescent
cells. Senescence is known to limit tumor progression by a variety of pathways with
upregulation of p16 and p53; both which are commonly seen in premalignant lesions and
absent as tumor progresses to anaplastic form. This review will focus to explore the role
of thyroid cancer cell senescence in thyroid carcinogenesis, specifically we will discuss
up to date approaches on how to utilize this knowledge for therapy of advanced types of
thyroid carcinomas such as anaplastic thyroid carcinoma. After senescence is induced in
thyroid carcinomas, what are the important secretomes that may have an effect on the
surrounding microenvironment. Then, finally addressing the therapeutic correlations
between senescence and thyroid carcinomas, what is being done in other tissue cancers
now, which may shed light on the future of treatment in thyroid carcinomas.
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Introduction:
The first description of “cellular senescence” was an inevitable proliferative arrest of
primary human cells in culture (Hayflick, 1965). Hayflick showed that after 55
population doublings the cell would succumb to proliferation arrest that was irreversible.
However, since then cellular senescence has been a topic of interest showing that there
are many other ways to induce senescence, such as activated oncogenes, oxidative stress,
DNA damage, radiation, and chemotherapeutic drugs. Much is known about the
mechanism behind senescence and the pathways that inaugurate and maintain a
senescence state, such as p53 and RB. After induction of cellular senescence program
cancer cells do not just end at terminal growth arrest become passive bystanders but
actively involved in crosstalk with other senescent cells, their microenvironment, and
other neighboring actively proliferating tumor cells through an active secretory program
mediated by paracrine mechanisms or by passing molecules through cell-to-cell contacts
(Kuilman, 2009,). It seems to appear that this senescence-messaging secretome system
has specific physiological roles and pathological consequences. It is well known that
BRAF, RAS point mutations and RET/PTC rearrangement constitute the majority of
mutations found in the two most common types of thyroid cancer, papillary and follicular
carcinoma (Nikiforov, 2011). Therefore taking into account what is known about
senescence, it was found that expression of different thyroid tumor –associated
oncogenes in primary human thyrocytes triggers senescence (Vizioli, 2011). This leads to
several important topics this paper explores; first thyroid oncogenes can induce
senescence of epithelial cells by what mechanism? Second, what factors do these thyroid
senescent epithelial cells secrete and what are their effects on the surrounding
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microenvironment? Third, what is on the forefront in treating thyroid cancer by inducing
senescence, as it was done in other tissue cancers.
Senescence
Replicative Senescence
Replicative senescence described by Hayflick resulted in shortening or damage of
chromosome telomeres that are brought about by repeat DNA replication coupled with
cell division (Campisi and d’Adda di Fagana, 2007). The shortening of telomeres
happens to be the inability of the replication machinery to copy the ends of linear
molecules every round of cell division (Blasco, 2005). Telomeres a critical shorten
length; behave as double-stranded DNA breaks that activate p53 tumor suppressor
proteins resulting in either senescence or apoptosis (de Lange, 2005; Von Zglinicki,
2005). It is known now that in the case of in vitro cultured cells ectopic expression of
telomerase was sufficient enough to prevent the shortening of telomeres and resulted in
immortalization of human fibroblasts, demonstrating how replicative senescence is
induced once telomeres have shorten to the length that induces DNA damage, a critical
factor leading to cellular senescence (Bodnar, 1998). This sort of telomere clock is
important to recognize due to essentially all human cancers have acquired mechanisms
that maintain telomere length through expression of high levels of telomerase (Muntoni
and Reddel, 2005). The idea that telomerase is a very important factor that enables tumor
progression is supported by mice that are deficient in telomerase activity. These animals
are significantly resistant to cancer induced by a variety of genetic defects and
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carcinogenic treatments (Blasco, 2005). Feldser and Greider et al. found that telomereinduced senescence has been shown to act as a tumor suppressor mechanism in telomeredeficient mice expressing the oncogene Myc in B cells. By shortening of the telomeres
the incidence in cancer was reduced in these animals even when apoptosis was blocked
by overexpression of the antiapoptotic protein Bcl2. This observation demonstrates that
telomere-dependent senescence is an efficient mechanism to suppress cancer in vivo
(Felder and Greider, 2007). However in many different cell types senescence can be
induced by non-telomere shortening triggers and actually most primary cells are prone to
senescence through oncogene induced senescence or inactivation of tumor suppressor
genes.
Senescence’s Signaling Pathways
To achieve senescence there must be DNA damage, this signal contributes to the
activation of p53 and pRb tumor suppressor pathways, key regulators of the senescence
process. Activation of p53 happens through Ataxia telangiectasia mutated (ATM), which
is activated by DNA damage stabilizing p53 by blocking MDM2, which is important for
the breakdown of p53. CDKN1a is one of the target genes that p53 up regulates during
senescence; this gene encodes p21CIP1 which then activates pRb tumor suppressor
pathway leading to cell cycle arrest (Adams, 2009). This pathway has been regarded as a
pathway for telomere shortening spoken about previously and oxidative stress it can also
be viewed in (fig. 1). In addition, in human cells pRb can be activated independently of
p53 through up regulation of p16INK4a an inhibitor of cyclin D/cd4-6 kinase also seen in
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(fig. 1), this p16INK4a comes from the INK4a locus, which is responsible for keeping
pRb in its hypophosphorylated state and inhibiting downstream targets such as E2F that
lead to cell cycle arrest (Adams, 2009; Bringold, 2000). This pathway can be triggered by
activated oncogenes which is explained below and by other forms of stress as well, these
are the two important mechanisms disregarding the trigger, that seem to be up regulated
during senescence.
Oncogene-induced senescence (OIS) has been recognized as a formidable barrier against
oncogenic transformation, acting to suppress the increased proliferative action of early
neoplastic cells. In an old study it was shown that Ras signals premature senescence
through the activation of MAPK cascade. Ras effector loop mutants that retain their
ability to bind Raf-1 induce premature senescence and among a series of downstream
components examined, only activated MEK was capable of inducing p53, p16 and other
aspects of senescence (Lin, 1998). However, by using a MEK inhibitor Ras-induced
senescence was prevented in these primary murine fibroblasts. It was then shown that
activated MEK forced uncontrollable proliferation and transformation in primary murine
fibroblasts that were absent of p53 and p16 (Lin, 1998). Leading to believe that onocgene
induced senescence is dependent up on the presences of p53 and p16, but others have
found that high levels of Raf activity can produce immediate cell cycle arrest that occurs
independently of p53 and p16 (Sewing, 1997).
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In more recent studies it has been shown that the ability of Ras to induce senescence
depends on the expression of Ras, with lower expression levels of Ras leading to
mammary gland epithelial hyperplasia, and high levels of Ras inducing senescence and
INK4a up-regulation (Sarkisian, 2007). Other premalignant lesions that is associated
with oncogenes other than Ras is the overexpression of BRAFV600E mutant, results in lung
adenomas and melanocytic nevi with a presents of increased number of senescent cells
(Goel, 2009; Dankort, 2009; Dhomen 2009). The nevi lesions induced by the oncogene
BRAFV600E takes months or years to spontaneously evolve into melanomas but
inactivation of Cdkn2a or p53 accelerates this transformation (Dankort, 2009; Dhomen,
2009). Thus leading us to believe recently in oncogene induced senescence (OIS) is not
only triggered in preneoplastic lesions but also acts as a formidable barrier that needs to
be overcome during tumor progression (Acosta, 2011). Also stating that if there were
some sort of mutation that would disable senescence, OIS could be reversed and give rise
to tumors. However this evidence in vivo is lacking.
Thyroid Senescence
With a good understand of the molecular progression of thyroid carcinogenesis known
from previous studies. It helped lead to the idea that BRAFV600E, RET/PTC, TRK-T3 and
H-RASG12V could be optimal oncogenes that induce senescence. Vizioli et al. displayed
oncogene-induced senescence by taking the oncogenes and tranfecting them into
thyrocytes by different methods and noting changes in morphology, accumulation of
senescent marker SA--Gal, and senescence-associated heterochromatic foci, with
upregulation of cyclin dependent kinase inhibitors p16INK4a and p21CIP1. It was
determined papillary thyroid carcinoma associated oncogenes increase expression of
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ERK1/2 along with continual induction of p16INK4a. However, p53 and p21CIP1 were not
significantly unregulated, exception being RET/PTC-expressing thyrocytes in which p53
increase was detected (Vizioli, 2011). To think about the molecular mechanism
responsible for thyroid oncogene induced senescence noting the above data, it was shown
in previous work that ERK1/2 activate senescence programme through critical
involvement of p16INK4a/pRB and p53/p21CIP1 pathways (Lin, 1998). Also, in many
previous research studies it was displayed that there was an increase in expression of
p16INK4a in adenomas, papillary and follicular tumors. However more importantly there
is an absence of p16INK4a in anaplastic thyroid carcinoma (Ferru, 2006, Ball 2007). In
concordance, p21CIP1 overexpression is noted in papillary micro-thyroid carcinoma but
again accelerated loss of p21CIP1 is seen in advancing papillary thyroid carcinoma.
Yielding an understanding of how senescence needs to be overcome to induce anaplastic
thyroid carcinoma. This collective information is important now in understanding a
small window into the mechanism of thyroid senescence and also how senescence plays a
role in tumor progression. However much is still to be determined about the mechanism
of thyroid senescence.
Senescence Secretome
Senescence is not just terminal growth arrest, but a senescent cell is metabolically active
and has undergone widespread changes in protein expression, secretion of soluble
signaling factors, and insoluble factors as well, to develop the senescence associated
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secretory phenotype (Coppe, 2008). The most recent studies have shown that senescent or
terminally growth arrested liver stellate cells, endothelial cells, and epithelial cells of
retinal pigment, mammary gland, colon, pancreas, and prostrate to secrete biologically
active molecules (Coppe, 2008). Senescent cells are known now to secrete interleukins,
inflammatory cytokines and growth factors that affect surrounding cells. The most
dominant cytokine of the senescence secretome is interleukin-6 (IL-6) a pleiotropic
proinflammatory cytokine associated with DNA damage and OIS in human kertinocytes,
melanocytes, fibroblasts and epithelial cells (Kuilman, 2008; Coppe, Patil 2008; Lu,
2006; Sarkar 2004).
Intriguing about the secretion of IL-6 by senescent cells is that in papillary thyroid
carcinomas certain data states an inverse relationship with IL-6 and epithelial tumor
aggressiveness (Basolo, 1998). Basolo used tissue culture methods and histochemical
techniques to search for possible alterations of cytokine expression in thyroid carcinomas.
As compared to cultures from normal tissue and well-differentiated carcinoma, IL-6 was
strongly down regulated in cultures derived from undifferentiated carcinoma (Basolo,
1998). Leading to perceive that senescent could help keep the levels of IL-6 raised to a
level that may be beneficial in slowing down tumor progression or keeping tumors highly
differentiated.
Senescent epithelial, endothelial, and fibroblast cells have been to known to express high
levels of almost all of the IGF-binding proteins (IGFBPs) and the most important of these
is IGFBP-7. Recently it ha been shown in OIS that activation of BRAF in primary
fibroblasts has lead to the secretion of IGFBP-7, which acts through autocrine/paracrine
and apoptosis in neighboring cells (Wayapeyee, 2008). Furthermore the role of IGFBP-7
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has been shown to mediate senescence in melanocytes, and to suppress melanoma growth
in vivo by inducing apoptosis (Wayapeyee, 2008). In papillary thyroid carcinoma (PTC)
it has been shown that IGFBP-7 is down regulated, and when using PTC-derived NIM1
cell line, in which expression of IGFBP-7 is lacking, exposure to soluble IGFBP-7 by
complementary DNA (cDNA) transfection reduced growth rate, migration and
tumorigenicity of NIM1 cells (Vizoli, Sensi, 2010). These observations lead us to believe
that oncogene or treatment-induced senescence could help decrease tumor progression of
papillary thyroid carcinomas by senescent messaging secretome IGFBP-7. However
there is still much debate on the role of IGFBP-7 and its role in senescence.
In addition to the secreted factors mentioned above there are still many other factors that
could play a strong role in supporting or inducing senescence in the surrounding
microenvironment. Increased production of PAI-1, TGF-1(Kortlever, 2006; Tremain,
2000; Wajapeyee, 2008) are factors that reinforce senescence-associated proliferation
arrest. Senescent cells also express increased CXCR and IL-8, which reinforce the
senescence program, including proliferation arrest (Acosta, 2008). Specifically in colon
carcinoma cells, HCT116 cells, which are p16-defiecient and wild type for p53, when
treated with a chemotherapeutic drug, doxorubicin, induced senescence (Chang, 2002). It
was noted then senescent HTC116 cells overexpress several growth inhibitor factors such
as a serine protease inhibitor Mapsin, which as been shown to inhibit invasion and
angiogenesis of breast and prostate cancer, also TGF- and amphiregulin an epidermal
growth factor that has been shown to inhibit multiple carcinoma cell lines while
increasing proliferation of normal epithelial cells (Chang, 2002). The real question is
what genes are upregulated in thyroid carcinomas, what factors are secreted then in
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response to upregulation and what are the effects in the thyroid surrounding
microenvironment.
There also seems to be a rather unpleasant side to the secretome-associated senescent
phenotype. There are many secreted factors by senescent cells that promote tumor
progression. One of these is the matrix metalloproteinase that is consistently upregulated
in human fibroblasts undergoing senescence, which are stromelysin-1 and -2 (MMP-3
and MMP-10, respectively) and collagenase-1 (Coppe 2009). These matrix
metalloproteinase’s have been shown to increase tumor progression of breast epithelial
cell xenografts in mice, which is believed to allow mitogenic and chemotatic signals
greater access to breast cancer cells (Liu, Hornsby, 2007; Tsai, 2005). An example that
mechanistically could be close to thyroid is the formation of melanoma. Malignant
melanomas express high levels of CXCR-2 receptor (Dhawan, 2002) and can be induced
to grow by the ligand GRO (Balentien, 1991) and interleukin-8 (IL-8) (Schadendorf,
1993) both of these factors are main components of the senescent-associated secretory
phenotype. This stimulation may lead to the proliferation of rare premalignant nevi to
malignant melanoma (Coppe 2009). Therefore, it is very important for future studies of
thyroid senescence to focus on the vast array of factors that are secreted by these cells
and what are the good and bad effects.
Modern Therapy Utilization of Senescence
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It well known that p53, p16 and p21 are frequently inactivated in the majority of cancers.
Therefore, one would venture to assume that with these genes being so vital to inducing
senescence, whether or not trying to induce senescence through chemotherapy treatment
would be beneficial and result in express a senescent phenotype. It has been shown that
chemotherapeutic drugs induce senescence in vitro in p16-deficient tumor cell lines,
HT1080 and HCT116 (Chang, 1999). It was also demonstrated that doxorubicin induced
senescence in p53-deficient Saos-2 cell line, SW480 and U251 cells with a mutant p53
(Chang, 1999). In vivo breast cancer study done by te Poele et al. showed 20% of the SA-gal + tumors displayed high p53 (suggesting p53 mutation) staining and 13% of SA-gal + tumors were absent for p16, suggest wild type p53, and p16 induction is not
necessary for senescence (te Poele, 2002). In consideration of p21, a study was shown to
determine in homozygous knockout of either p21 or p53 strongly reduced but did not
completely abolish radiation or drug induced-senescence which was determined by PKH2
analysis of cell division and SA--gal staining (Chang, Xuan, 1999). Therefore, it can be
drawn that p53, and p21 may act to push forward senescence in tumor cells but are not
required for this response same reasoning goes for p16.
It is important to note the new thoughts of treatment-induced senescence. In senescence
induced by doxorubicin about one third of the genes displayed a delay induction when
colon carcinoma cells, HCT116 were p21 null. Determining p21 plays a role not only in
inhibition but also gene expression in senescent cells (Chang, Swift, 2002). These
findings were also present in HT1080 fibrosarcoma cells and were readily reproduced by
another senescent associated CKD inhibitor p16 (Igor, 2003). A group of p21-induced
genes that were upregulated encode for the secretion of proteins with known mitogenic,
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angiogenic and antiapoptotic activities, most of these secretomes have been mentioned
earlier. Unfortunately the induction of these genes by p21 produces paracrine growthpromoting activities as seen in condition media from p21-arrested HT1080 cells have
mitogenic and antiapoptotic effects (Chang, Wantanabe, 2000). In regards to p16, it has
been determined that although it seems to be a very potent inducer of cell cycle arrest,
p16 does not seem to have an important role of developing senescence associated
phenotype and secrete factors at much lower levels (Coppe, 2009). It is still up to be
determined that these factors have mitogenic, antiapoptotic or angiogenic effects.
However it is believed that p21 and other CDK inhibitors like p16 have undesirable
effects of senescence suggest that treatments inducing senescence in the absence of p21
or p16 would be more beneficial (Igor, 2003). It will be discussed below on what
treatment it was determined to be beneficial in MCF-7 cells that were induced to
senescence with lack of p21. Showing positive effects of tumor senescence with
permanent growth arrest and secretion of tumor-suppressing factors, this in hopes of
separating out disease promoting activities of senescent cells.
Thyroid Carcinogenesis:
It must first be mentioned that thyroid carcinogenesis is a process lacking a definite
general mechanism of progression from the normal thyroid tissue all the way to the
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anaplastic thyroid cancer. In fact, thyroid cancer itself can manifest itself with many
subtypes with each of these having multiple possible tumor progression pathways. The
two most common subtypes being papillary thyroid carcinoma (PTC, >85% of cases) and
follicular thyroid carcinoma (FTC, 5% to 15% of cases) have favorable prognosis but can
both lead to the more aggressive, almost certainly fatal, anaplastic (undifferentiated)
thyroid carcinoma (ATC). It should be pointed out that a variant of papillary carcinoma,
the papillary micr-ocarcinoma (microPTC), represents an earlier stage of disease, which
evolves into PTC (1, Lupi et al. 2007). This will become very interesting when
comparing protein expression in tumor progression as well as the pathway of overcoming
senescence in tumor progression.
Since PTC and FTC both originate from different genetic mutations and hence have a
different pathway of carcinogenesis, it is only fair to discuss these two subtypes
separately:
Papillary thyroid carcinoma:
A common finding in microPTC is BRAF mutation (29, 8). MicroPTC (less than 1cm in
diameter) are common precursors of PTC. Adding these facts together shows clearly that
BRAF mutations can occur at early stages of development of PTC. In fact, about 45% of
PTC shows a point mutation in BRAF (29, 4) the most common of these mutations is a
valine to glutamate substitution at position 600 of the protein (V600E mutation). The
V600E mutation is thought to mimic T599/S602 phosphorylation, rendering BRAF
constitutively active. As discussed earlier, this has the effect to drive uncontrolled
proliferation through MAPK-induced hyperphosphorylation of Rb, which in turn releases
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inhibition of E2F-dependant transcription factors, allowing passage from G1 in to S (29).
Another common mutation found in PTC is the RET/PTC rearrangement, which renders
a receptor tyrosine kinase constitutively active. Although many rearrangement type can
be found among the RET/PTC, all of them seem to involve an intact tyrosine kinase
domain of the RET receptor enabling the RET/PTC chimeric protein to activate the
RAS/RAF/MAPK cascade and initiate thyroid tumorigenesis. This mutation is common
in 10-20% of adult sporadic PTC (30) although its’ incidence is a lot higher in children,
young adults (40-70%) and in patients with a history of radiation exposure (50-80%) (30,
31-34). RAS mutation occurs in 15-20% of PTC (30, 45-50), but this kind of mutation is
more prominent in FTC (discussed below).
One can quickly recognize that whether a mutation occurs at RET/PTC, BRAF or RAS,
rendering these constitutively active, the same mitogen activated protein kinase (MAPK)
pathway activation will be the downstream effect. (figure X). This pathway is usually
transiently activated with cell division being one of its many downstream effects. When
one protein in the pathway is mutated, it can be stuck in the “on” or “off” position, which
is a necessary step in the development of many cancers (50). Thyroid cancer and
melanomas are of particular interest when it comes to the MAPK pathway (29).
Follicular thyroid carcinoma:
Unlike in PTC, a BRAF is not found in follicular carcinoma (30). On the other hand,
RAS point mutation is not restricted to any particular type of thyroid tumor, being present
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in 40-50% of conventional type follicular carcinoma (30, 48, 52-56). Point mutation of
the RAS gene either increases its affinity for GTP (mutations in codons 12 and 13) or
inactivates its autocatalytic GTPase function (mutation in codon 61), resulting in
permanent RAS activation and chronic stimulation of its downstream targets along the
MAPK and PI3K/AKT (figure X) signaling pathways.
PAX8/PPAR rearrangement is also commonly found in FTC (30-40%). Although the
mechanisms of cell transformation induced by this genetic event are yet to be fully
understood, it consists of a translocation between chromosome 2 and 3, t(2;3)(q13;p25),
leading to the fusion between the PAX8 genes coding for the thyroid-specific paired
domain transcription factor, and the PPAR gene.
Road to anaplastic differentiation:
Although these mutations are commonly found in PTC and FTC, they are not sufficient
on their own to cause malignant transformation. In fact, as discussed previously, it has
been shown that activation of RAS/MAPK by oncoproteins is followed by induction of
senescence, which is a barrier for tumor progression (29.10, 39, 40). More precisely,
BRAF V600E induces senescence in association with the activation of the ERK ½
pathway, p16INKa, p21CIP1 and p53. We also mentioned earlier that microPTC
commonly show a BRAF mutations without being malignant. Therefore, it is obvious that
progression of thyroid tissue to malignancy requires bypass of senescence. Unfortunately
this process is not thoroughly studied in thyroid cancer.
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The thyroid oncogenesis is often compared to that of melanomas since evidence has
shown that MAPK activation is the most compelling in both. Some studies have reported
different ways of overcoming senescence in different tissues including melanomas: (1)
Concomitant activation of AKT3, which phosphorylates BRAF V600E and dampens its
kinase activity, thus allowing transformation of human epidermal melanocytes (29,14) (2)
overexpression of C-MYC, which can be overexpressed in PTC (29.17) abrogates the
senescence induced by oncogenic BRAF in normal melanocytes (29.15) (3) Decreased
expression of insulin-like growth factor binding protein 7 (IGFBP7) which can also
inhibit BRAF V600E-induced senescence in human primary foreskin fibroblasts (29.16).
Recall that we have previously mentioned that IGFBP7 was recently shown to have a
possible important counter-act effect, along with p16 and p21, to the proliferative effect
of oncogens. It was shown that there is a progressive loss of IGFBP7 protein during
thyroid tumour progression of both PTC and FTC (1.vizioli et al. 2010)
The factors that are involved in tumor progression are believed to be different than the
ones involved in malignant transformation. In fact, defects in p16 and p53 are rarely if
ever found in PTC. However, they are common in poorly differentiated and anaplastic
thyroid cancers (29.13). This could lead to the belief that the more well-differentiated
thyroid cancers have an epigenomic silencing of p16 and p53, while the less
differentiated forms acquire mutations in them. This would suggest a two-step pathway
leading to anaplastic thyroid carcinoma (ATC) (figure X).
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Thus, the carcinogenesis of thyroid cancer and the journey to anaplastic thyroid cancer is
a complex mechanism by itself. First, the proliferative mechanism could be of different
routes. Possibly a BRAF or RET/PTC mutation leading to PTC and then ATC, or perhaps
a RAS mutation or PAX8/PPAR rearrangement leading to FTC and then ATC.
Whatever the mechanism is, there seems to be a triggering event that leads from the more
differentiated cancer (PTC and FTC) to the more brutal and less differentiated ATC.
Putting all this information together leads us to two important concepts: 1) Anaplastic
thyroid cancer having a mortality rate of almost 100% is one of the most fatal form of
cancers you can have, if not the most fatal. 2) The road to anaplastic cancer shows
progressive loss of senescence. These two concepts highlight the important role of
senescence in cancer and survival. There leaves no doubt that energy, time and resources
should be attributed toward the improvement and discovery of new treatment by using
senescence as a central mediator.
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Senescence in cancer treatment:
Senescence in cancer treatment with other tissues:
It is now known that even tumor cells can be induced to undergo senescence. Since
senescence markers are progressively lost during the progression of thyroid
carcinogenesis, (1, 28) we raise many questions on the possibility of restoring senescence
in thyroid cancer cells as a possible treatment for thyroid cancer. It was discussed earlier
that senescent cells produce secreted proteins with both tumor-suppressing and tumorpromoting activities, the key for treatment using senescence would be to benefit from the
suppressing factors while avoiding the promoting ones. A good example would be that
of retinoid treatment in breast carcinoma, where retinoid induces senescence rather than
differentiation (26.55). Exposure of MCF-7 breast carcinoma to a low noncytotoxic dose
of all-trans RA induced growth arrest and the senescent phenotype without upregulating
any genes encoding secreted factors with tumor-promoting activities, or other disease
promoting activities (26.56). Although retinoids used experimentally to treat thyroid
cancer showed a little or no effect (only a slight re-differentiation in a small minority of
patients) (51), the goal would be to find a therapy that would offer similar benefits. It is
possible that the balance between tumor-promoting and growth-inhibiting secretomes that
will end up deciding the impact that senescence will have on the growth. The effects that
p21 can have depend on which type of cells it is acting on. Different mechanisms are
known to cause senescence in other tissues. DNA-demethylating drugs, ceramide and
inhibitors of histones deacetylase (Holiday 1986, Venable et al. 1995; Orgryzko et al.
1996; Linke et al 1997).
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A wide variety of anti-cancer agents induced senescence-like morphological changes and
SA-ß-gal expression in tumor cells in a study demonstrating the effects of
chemotherapeutic drugs and radiation on cell lines derived from different types of human
solid tumors (26.41) Moderate doses of doxorubicin induced the senescent phenotype in
11 of the 14 cell lines derived form different types of human solid tumors. Similar
evidence of senescence-like phenotype has been demonstrated with different drugs
including
cisplatin
(26.41),
hydroxyurea
(26.42,43)
camptothecin
(26.46),
or
bromodeoxyuridine (26.47,48). Drug induced senescent phenotype in tumor cells was not
associated with telomere shortening and was not prevented by the over-expression of
telomerase (26.45). Additional determinants of drug induced senescence in tumor cells
were identified by gene expression profiling of doxorubicin-induced senescence in
HCT116 colon carcinoma cells, which are p16 deficient and wild-type for p53 (26.49). It
was shown that more than half of the genes that were strongly inhibited in senescent cells
are known to play a role in cell cycle progression, with the largest groups of genes
involved in mitosis and DNA replication. The inhibition of these genes became apparent
1-2 days after treatment with doxorubicin and was likely to contribute to the maintenance
of drug-induced growth arrest.
In another study (26.60), it was demonstrated in a mouse model of B-cell lymphoma that
when the program of apoptosis was inhibited by the transduction with a retrovirus that
expresses the anti-apoptotic gene BCL-2, senescence became the principal tumor
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response to cyclophosphamide, as indicated by apparently complete cessation of DNA
replication and mitosis and by drastic induction of the SA-ß-gal marker. Treatment
induced senescence became undetectable on the knockout of either p53 (which also
inhibited the apoptotic response) or p16 (which had no effect on apoptosis).
Throughout this paper we have discussed about the involvement of p16 and p53
inactivation in cancers. Also, these genes seem to be involved for treatment-induced
senescence. One could assume that since the majority of cancers are deficient in one or
both of these genes, than this could these cancer cells unable to undergo senescence.
However, studies show that chemotherapeutic drugs readily induce senescence in vitro
and in vivo in p16-deficient tumor cell lines (26.40). Also, moderate doses of doxorubicin
induced the senescent phenotype in p53-null Saos-2 cell line, in SW480 and U251 cells
carrying mutant p53, and HeLa and Hep-2 cell lines, in which p53 function has been
inhibited by papillomavirus protein E6. Thus showing the involvement of other regulators
of senescence.
Senescence in thyroid cancer treatment:
Re-expression of ABI3BP in thyroid cells has resulted in decrease in transforming
activity, migration, and invasion and tumor growth in nude mice. ABI3BP re-expression
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appears to trigger cellular senescence through the p21 pathway. Indeed, expression of
ABI3BP inhibited cell growth, induced senescence in undifferentiated thyroid carcinoma
(ARO) that was shown by the elevated senescence markers, decreased ERK
phosphorylation, reduced tumor growth and reduced invasion in well differentiated
thyroid carcinoma (WRO). Therefore, it would be beneficial to indentify the mechanism
by which ABI3BP expression promotes senescence and try to reproduce the mechanism
as an attempt to rescue senescence in thyroid carcinoma.
It was mentioned previously that IGFBP7 expression was frequently lost in FTC and
PTC, its restoration modulates the transformed features of PTC-derived tumour cells
(1.Vizioli et al 2010).
The prognosis of FTC and PTC are relatively good, with a 10-year survival rate of 95%
and 90%, respectively (robbins). On the other hand, Anaplastic carcinoma has a mortality
rate approaching 100%. Radiation therapy has shown poor results due to the bad response
of anaplastic cells to therapy. Different strategies are then brought upon to increase the
sensitivity of anaplastic cells to radiation by using senescence. Podchetko et al. have
demonstrated that a combination of Gleevec ® (Imatinib) and radiation potentiates the
effect of radiation-induced senescence. Gleevec ® induced S- and G2/M-phase cell cycle
arrest: the growth arrest was accompanied by the down-regulation of c-ABL
phosphorylation and of cyclins A and B1 levels, and regulation of CDKN1A (p21).
Whereas radiation alone had shown to significantly increase the levels of senescence
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marker SA-ß-gal-positive in ARO and FRO cell lines, the numbers of SA-ß-gal-positive
we markedly increased when Gleevec ® was combined with radiation compared to
radiation alone (11). According to these facts, we can point out that not only senescence
can slow down or alt tumor progression, but it can also be used as an adjunct to increase
the effects of radiation, which already proves to induce senescence-like terminal growth
arrest in thyroid cancer cells, (19.10) in ARO and FRO.
It has been shown that IR-induced temporal activation of JNK (Jun Nh2-terminal kinase)
is linked to cell survival is one of the manifestations of radiation response in ATC cells
(19.18). Bulgin et alhave reported that JNK inhibitor SP6000125 potentiates radiationinduced terminal growth arrest of ATC-cells. This is another example of senescence-like
phenotype promoting the effects of radiation on ATC. The combination of the drug and
radiation, indeed, greatly enhanced senescence-like phenotype in ATC cell lines
Conclusion:
Senescence whether it be replicative with induction via telomere shortening, oncogeneinduced with the overexpression of Ras, Raf, BRAFV6003, or treatment induced
senescence all seem to lead to a common pathway by upregualting p53, p16, and pRb.
The mechanism of these different ways to induce senescence is known quite well,
however once the cells assume there senescent phenotype is when the true story begins.
In thyroid carcinogenesis it is known that papillary thyroid carcinoma is the most
common and mutations in BRAF600E, RET/PTC, and RAS genes are also frequent.
Though, the mechanism of senescence in thyroid carcinogenesis is not well understood, it
was proven recently that papillary thyroid carcinoma-associated oncogenes trigger
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oncogene-induced senescence in primary thyrocytes and this was based on the presence
of cell morphology and senescent markers (Vizioli, 2011). Once thyrocytes have entered
senescence what factors do these cells secrete and what are their affect on the
microenvironment they secrete it in.
Two things could be apparent; first the cells secrete factors that conducive to the
microenvironment to sustain senescence and induce other cells to enter senescence, such
as mapsin, TGF-, IGF-binding proteins, and amphiregulin. Unfortunately the second is
directly the opposite and these secreted factors could contribute to tumor progression or
help sustain the tumor cell population. Examples being in breast cancer epithelial cells
that are induced into senescence secrete GRO which as been shown to increase
inappropriate epithelial growth in the mammary gland (Tsai, 2005; Sun, 2007). Other
factors that can lead to mitogenic and chemotactic signals are matrix metalloproteinases,
IL-1, IL-6 which as been known to be good and bad for the surrounding
microenvironment. The question that needs to be answered in upcoming research about
thyroid senescence is what factors are being secreted and what are their affects being on
surrounding microenvironment.
Treatment-induced senescence in thyroid carcinoma is not yet in the mainstream
discussion, but seems to be in clinical trials for breast, and prostate cancer. The idea
behind future treatments involving senescence in thyroid carcinomas is upregulating
genes important for senescence but inhibiting the expression of genes that regulate the
factors secreted by senescent cells that promote tumor progression. Genes that seem to
be involved in these unwanted factors are p21 and p16 (Chang and Wanatabe, 2000).
Then the question arises how do we induce senescence without upregulating two very
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important parts of the senescent pathway. This has been spoken about previously with
retinoid-induced senescence of MCF-7 breast carcinoma cells, which also lack increased
expression of p16, and p21 (Chang, Broude, 1999). Fortunately with p21and p16
downregulated the unwanted factors that promote tumor progression were not
upregulated in the breast carcinoma cells.
Future treatments chemotherapeutic drugs such as doxorubicin, UV-irradiation, need to
be geared to inducing senescence in the best way possible that lead to and upregulation of
secretomes that reduce tumor progression, sustain senescence and induce senescence in
the surrounding microenvironment. Thyroid tumors being the most common endocrine
malignancy, really puts an urge in revealing the implications about these secretomes in
the mechanisms of thyroid senescence, and how treatment-induced senescence can affect
tumor progression. These implications are therefore among of the main topics that must
be assessed in thyroid cancer in order to work toward optimal treatment and cures.
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Figure 1.
Figure 1. The mechanism of Senescence:
Senescence begins with initial DNA damage, oxidative stress, and oncogenic
overexpression that will the lead to a cascade of events that activate p53/p21CIP1 and
p16/pRB pathways. The genes will arrest cell cycle in hopes of DNA repair or forcing
the cell into terminal growth arrest and prevention of tumor progression. Or as scene to
the right, if one or both of these mechanisms are mutated and inactivated, tumor
progression will lead to malignant transformation.
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Figure 2.
Figure 2. Senescence Associated Secretory Phenotype:
During terminal growth arrest the cell may be not proliferating, but metabolic activity has
not ceased. Senescence Associated Secretory Phenotype is now known to be quite
relevant in regards to how senescent cells affect the surrounding microenvironment.
There are factors that will advance tumor progression, such as GRO and factors that
maintain senescence and promote tumor regression, TGF-, IGF-binding proteins and
mapsin.
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Figure 3.
Figure 3. MAPK pathway
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1: Ligand binding, 2: Autophosphorylation of RTK, 3: Growth factor receptor bound
protein 2 (GRB2) binds to Phosphate site, 4: SOS binds to GRB2, 5: SOS catalyses the
exchange of GDP to GTP to activate RAS, 6: Activation of B-RAF, 7: B-RAF activates
MEK ½, 8: MEK ½ activates ERK ½ , 9: ERK ½ activates FOS and JUN (among others)
10: FOS and JUN activates Activating protein 1 (AP-1) forming a heterodimer which
regulates gene expression 11: GTP-ase activating protein (GAP) should disactivate RASGTP so that BRAF would unbind and MAPK is terminated.
A mutation in RAS or BRAF would inhibit this disactivation making the pathway
constitutively active.
Figure 4.
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Figure 4.
The activation of RAS could either lead into the MAPK pathway or could take a different
route as seen in FTC, which consists of PIP3/AKT pathway. This pathway leads to cell
proliferation, differentiation and angiogenesis.
Figure 5.
Figure 5.
This figure illustrates the two-hit theory leading to anaplastic thyroid carcinoma. In the
first step, a mutation in BRAF or a RET/PTC mutation leads the normal thyroid cells to
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PTC, or, mutation in RAS or a PAX8/PPAR rearrangement leads to FTC. The second hit
consists of a mutation in p53 and p16 leading both PTC and FTC to ATC.