Rachel Varughese

University of Oxford Medical School

Essay for consideration for 2011 BGS Amulree Essay Prize:

Unlocking ageing and age-related carcinogenesis: does senescence

hold the key?

Word Count: 5090

Man’s obsession with ageing is as old as man itself. The pure inevitability of it is ultimately

frustrating to the human nature, which endeavours to constantly solve and overcome the many

challenges of life. As medical intelligence has grown, this preoccupation has grown to include age-

related disease as a key obstacle to unlocking extended life. A review by Kirkwood et al. in 2000,

gave a dishearteningly accurate definition of ageing as ‘the progressive loss of function accompanied

by decreasing fertility and increasing mortality with advancing age’1. As a medical student, it was

easy during pre-clinical studies to be lulled into a false sense of security, when hearing about dramatic

life-expectancy increases, that the elderly are becoming less and less afflicted with age-related

morbidity. Taking a step back, from a simplistic black and white point of view, living longer is

ultimately desirable and death is bad. However, only when entering the world of clinical medicine did

I discover what I believe to be the two key facets of delaying ageing. Firstly, I began to differentiate

between lifespan and wellspan. This is what society really aspires to, although they may not know it

to articulate; a longer life, associated with no extra morbidity. In addition, from my time on the wards,

emerged another inevitable feature of ageing: if given enough time, cancer will be the primary factor

increasing mortality with advancing age.

The strangely intimate relationship between cancer and age is a consequence of the relentless

accumulation of non-proliferative senescent cells. Since the first cell culture in the early 1900s it was

a common belief that given the correct conditions, cultured cells would replicate indefinitely. In 1965

Leonard Hayflick first formally described the existence of a cellular replicative ‘Hayflick’ limit2. This

is known as cellular senescence and is defined as the cell-autonomous program where previously

mitotically competent cells irreversibly lose proliferative potential. Two conflicting theories connect

cellular senescence to age-related pathologies. Firstly, senescence is potentially a robust tumour

suppression mechanism, limiting excessive or aberrant proliferation. On the other hand, mounting

evidence suggests that senescence might contribute to the physical hallmarks of age-related

pathologies, including carcinogenesis3, through the adoption of a senescence-associated secretory

phenotype (SASP). In this essay I will address the question of whether senescence deserves its title as

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University of Oxford Medical School

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a tumour suppressor mechanism or whether it may paradoxically promote environments suitable for

age-related carcinogenesis. Furthermore, I will discuss the potential of harnessing the beneficial

effects of senescence, in order to explore possibilities for creating a treatment for cancer and a

deterrent for ageing.

The inevitability of cancer

Perhaps most puzzling, is that neither ageing, nor the associated pathologies can be explained to exist

for an evolutionary advantage. There are several theories as to why genes are selected that lead to age-

related carcinogenesis. The most popular are antagonistic pleiotropy and the mutation accumulation

theory. Antagonistic pleiotropy is defined as the accumulation of late-acting deleterious genes,

selected for their beneficial effects early in life, regardless of unintentional detrimental late life

effects. Senescence follows this pattern, where protection against cancer sustains the population in

early life, after which, in a natural environment extrinsic hazards would restrict lifespan before

senescence-related cancer developed. The mutation accumulation theory states that genes leading to

negative consequences after reproductive age are not accounted for by natural selection, as their

effects manifest after the genes have been passed to the next generation. A combination of the two

theories may be the most plausible answer. Understanding these evolutionary theories are an

important part in understanding the molecular and cellular basis of ageing, and thus an insight into

how to target age-related pathologies.

In mammals, as in many other species, age is the largest single risk factor for developing cancer.

Cancer is a major killer in Western countries, accounting for 1 in 4 deaths4. There is an exponential

rise in cancer (Fig.1) that occurs with age after 405. The intimate relationship between cancer and age

is a consequence of the effects of non-proliferative senescent cells. On one hand, senescence halts

uncontrolled proliferation in early life, acting as a barrier that cells must overcome to progress to

Figure 1: The rise in cancer with age. Lifetime risk for males is 1:2, females 1:3.3,9

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University of Oxford Medical School

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malignancy. However, advancing age is an inescapable carcinogen that causes the sudden mid-life

increase in cancer risk, due to amassing senescent cells, culminating in a near inevitable affliction of

cancer6-8

. Tumorigenesis in humans is a multistep process, where several genetic alterations

collaborate to transform normal cells to highly proliferative and invasive derivatives9. Progression to

malignancy involves the acquisition of various features, such as invasion, metastasis and insensitivity

to anti-growth signals. The majority of malignant cells are immortalised; meaning they have limitless

replicative proliferation through circumvention of senescence.

4,10

Molecular themes underpinning senescence

Two main pathways establish and maintain senescence (Fig.2). Both involve gateway tumour

suppressor proteins acting as transcriptional regulators: p53 and p16INK4a

, which positively regulates

pRB. pRB was the first tumour suppressor to be discovered, named after its initial finding in

retinoblastoma. p53 was serendipitously discovered during the investigation of Simian Virus-40

driven transformation of hamster fibroblasts. The p53 gene is evolutionarily ancient as a stress and

damage response coordinator11

. However, its role as a tumour suppressor is thought to be a relatively

recent adaptation, arising as organisms have gained sufficient regenerative capacity as to need

protection from accumulating cellular damage. Its abundance in tumours initially suggested oncogenic

function, however, it was later confirmed that these were p53 mutations12

.

Figure 2: p53 and p16(INK4a)-pRB pathways controlling senescence.2,14

ARF: P14 Alternative Reading Frame product, HDM2: Human Double Minute, CDKs: Cyclin Dependent Kinases, E2F: E2

Transcription Factor.

ARF inhibits HDM2/4 (MDM2 in mice), a negative regulator of p53. p53 transcriptionally activates p21. This inhibits

CDK2, and thus cell division. p16INK4a inhibits the functional impairment of pRB by CDK phosphorylation.

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University of Oxford Medical School

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p53 and pRB are the two most commonly mutated tumour suppressors in malignant tumours3, and

their experimental inactivation at the germ-line leads to senescence-resistant cells6. This is mirrored in

Li Fraumeni syndrome, where germline p53 mutations lead to risks of developing multiple cancers.

p53 and pRB interact to achieve irreversible tumour suppression13

, although they are able to act

independently, albeit to a less permanent degree. Beausejour et al., refuted previous hypotheses

claiming that senescence is always irreversible14

. They found p53 inactivation alone could not reverse

senescence, however, upon removal or lowering of p16INK4a

, p53 inactivation reinstated vigorous

growth. Consequently, it appears that senescence is reversible when maintained primarily by p53,

however additional p16INK4a

provides an irreversible outcome.

3,15

Activation of senescence

Despite evidence implicating senescence as a cause of age-related carcinogenesis, its protective

effects must not be undervalued. So why are genes that ultimately lead to cancer, selected and

conserved over generations? A prominent theory is ‘antagonistic pleiotropy’, defined as accumulation

of late-acting deleterious genes, selected for early beneficial effects, regardless of unintentional

detrimental late-life effects. The benefits of cellular senescence are demonstrated by its response as a

protective measure against adverse stimuli. Although these stimuli are diverse, they all trigger

senescence via DNA damage response (DDR) pathways and converge on p53 and pRB. In addition to

the replicative exhaustion demonstrated by Hayflick, cellular senescence can also be an outcome of

DNA damage, chromatin disruption, shortened or dysfunctional telomeres16

, or activated oncogenes17

,

all of which are risk factors for malignancy (Fig.3).

d’Adda di Fagagna asserts that ‘senescence, triggered by different stimuli, is the outcome of a

protracted DDR’18

. This is an evolutionarily conserved pathway designed to sense DNA damage, and

relay this via a signal amplification cascade to induce one of three outcomes: repair, apoptosis or

senescence. Moreover, the signalling pathway continues to actively contribute to stable cell arrest,

long after induction of senescence19

. Ataxia-telangiectasia Rad3-related (ATR) and ataxia-

telangiectasia mutated (ATM) protein kinases control responses to single-strand DNA exposure and

double-strand breaks (DSBs), respectively20

. Recruitment of these kinases facilitates formation of

DNA damage foci containing the activated histone H2AX, which amplifies ATM/ATR activity in a

positive feedback loop. Once local concentration of ATM/ATR has increased past a threshold,

checkpoint kinases, CHK-1/2, are activated, which converge on p53, resulting in cell-cycle arrest.

DNA damage has been repeatedly observed to be constitutively active in premalignant lesions through

staining for activated ATM, γH2Ax, CHK-2 and p5321

. However, DDR markers peak early and wane

progressively as cancer progresses, consistent with DDR activation and senescence being an initial

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University of Oxford Medical School

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barrier to tumorigenesis22

. Thus, as an outcome to DNA discontinuities, senescence is a necessary

cancer defence, as demonstrated when this response is abrogated: mutated ATM in inherited ataxia-

telangiectasia, causes lymphoreticular malignancies and ATM-/-

mice have a strong predisposition to

lymphoid tumours23

. In addition, DSB repair defects in Bloom syndrome24

, cause a powerful cancer

predisposition25

.

The chromatin state describes the balance of euchromatin and heterochromatin. Histone deacetylase

inhibition (HDAi), promotes euchromatin formation and establishes and maintains cell-cycle arrest26

.

Meanwhile, excessive heterochromatin formation also induces senescence27

, therefore it is likely that

the precipitating factor is simply the imbalance of chromatin. HDAis are a potential source of cancer

treatment, with phase II clinical trials well underway28

. Progressive telomere shortening in every cell

cycle, eventually causes chromosome ends to be recognised as DNA breaks, thus somatic cells are

ultimately destined to undergo telomere-driven senescence. Alternatively, oncogene driven

hyperproliferation activates ARF, inhibiting H/MDM2 (Fig.2). The importance of ARF in mice is

highlighted by their increased propensity to widely distributed tumours in ARF-/-

mice29

. However,

human ARF may not share this importance: although p16INK4a

overlaps reading frames with ARF,

mutations appear to affect ARF much less than p16INK4a

. This suggests that p16INK4a

may have a

greater impact on tumour suppression in humans. Such results emphasise how effects of genes in mice

cannot be directly translated to homologous genes in humans. Ideally, all experiments must be tested

with cells such as human fibroblasts before drawing reliable conclusions.

How senescence supports age-related carcinogenesis

Senescence causes inevitable depletion of renewable proliferative progenitor and stem cells30

.

Senescent cells in mammalian tissues increase in number with age, with some aged tissues such as

fibroblasts containing as many as 15%31

. The accumulation of senescent cells with age was first

demonstrated in vivo by Jeyapalan et al.31

using baboon skin fibroblasts, who showed senescent

markers accumulating in mitotic tissue with age. Since baboons demonstrate similar cellular

senescence to humans, with cells arresting after 56 population doublings, these results are deemed

applicable to humans. The ability of renewable tissues to regenerate may therefore be compromised,

implicating senescence in the undesirable aspects of ageing and age-related pathologies. However, the

full irony of the negative aspects of senescence was only realised when it was implicated as

encouraging an environment suitable for age-related cancer: the very disease it has evolved to protect

us from.

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University of Oxford Medical School

5

The protumorigenic effects of senescence were neatly demonstrated by Krtolica et al.6, by comparison

of co-cultured senescent and presenescent cells. Senescent cells caused hyperproliferation of

preneoplastic and accelerated tumorigenesis of neoplastic epithelial cells, while presenescent cells

encouraged far less proliferation. The additional growth stimulated by senescent cells suggests their

acquisition of an aberrant phenotype. In this study, Rhodanile blue first preferentially stained

epithelial colonies, before quantification with fluorescence using DAPI (4,6-diamidino-2-

phenylindole) stain and EGFP (enhanced-green-fluorescent-protein) expression. Thus, Krtolica et al.

validated their results using multiple methods for evaluating growth in order to obtain trustworthy

results.

This aberrant phenotype may be explained by marked changes in gene expression of senescent cells,

which create a number of distinct physical characteristics. These include actin stress fibres,

accumulation of protein aggregates consequent to failure of autophagy, increased cell size and

decreased proliferative capacity. In addition, human senescent cells secrete myriad matrix

metalloproteinases and inflammatory cytokines, a phenotype known as the senescence-associated

secretory phenotype (SASP), which takes around 7-10 days to develop under DDR32

(Fig.3). It

recruits immune cells to tumour sites allowing clearance of senescent masses and promoting tumour

Figure 3: The main interactions of senescence. (SASP: Senescence-associated secretory phenotype)

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University of Oxford Medical School

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regression, via macrophage engulfment and death receptor mediated apoptosis such as Fas-FasL

cytotoxicity33

.

However, while a normal tissue environment supports cell function, an aberrant tissue environment

can promote tumorigenesis32

. Secretions of the SASP enzymatically target the extracellular matrix and

change tissue microenvironments, promoting ageing and cancerous phenotypes34

(Fig.4). Differences

across various cell strains imply that the SASP is not an invariant phenotype, but the result of a

“conserved core secretory program”32

triggered by senescent cells. As the SASP intensity increases

with the strength of the mutagenic stimuli, so does the aggression of the resulting proliferation.

Cancers of the breast6,35

, skin36,37

, pancreas38

, prostate31,37

and oropharyngeal mucosa39

respond to the

SASP, which promotes hyperproliferation and migration of epithelial cells. Thus, most often,

senescent cells do not precipitate carcinogenesis by themselves changing, but by distorting the

surrounding microenvironment via secretions.

Furthermore, mitochondrial dysfunction triggered by the DDR leads to activation of long-lived

reactive oxygen species (ROS), such as H2O2. Passos et al.40

recently elucidated the previously

unknown cause-effect relationship between ROS and DDR by confirming the existence of a positive

feedback loop, whereby ROS activation further exacerbates senescence through direct DNA damage

and acceleration of telomere shortening. Experiments were conducted using proliferation competent

human MCR5 fibroblasts, comparing data to the normal proliferative limit, in order to produce

Figure 4: The SASP effect. (Campisi, 2003, with permission).6

4a) Stroma maintains the basement membrane. Tissue environment suppresses neoplasia of the ‘initiated’ cell,

containing an oncogenic mutation.

4b) Stromal fibroblasts senesce with age, secreting degradative factors, disrupting tissue environments, promoting

neoplastic growth of the ‘initiated’ cell.

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University of Oxford Medical School

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thorough and reliable findings, relevant to humans. In response to both telomere-dependent and

independent DNA damage, ROS were found to be necessary and sufficient in maintaining growth

arrest in establishment of an irreversible senescent phenotype, through their stochastic contribution to

the long-term maintenance of DNA damage foci. Passos et al. speculate that mitochondrial

dysfunction and consequent ROS production may contribute to the ‘bystander effect’ of the SASP,

whereby neighbouring tissues are stressed. They cause a retrograde response, resulting in major

reprogramming of nuclear gene expression patterns. Moreover, while interruption of the DDR is not

sufficient to rescue cells in established senescence19

, Passos et al.40

highlight the possibility of

interventions against individual components of the SASP, e.g. ROS, in order to ameliorate the

secretory damage to the surrounding microenvironment.

SASPs arise both in cells in culture and in epithelial tumour cells in vivo, only in response to

potentially oncogenic events, such as oncogenic RAS or the loss of p53 function. The most

predominant factors noticed in the SASP are the high levels of inflammatory cytokines: interleukin

(IL)-6, -7 and -8, and immune modulators: MCP-2 and MIP-3a, growth factors such as GRO, HGF

and IGFBPs and shed surface molecules such as ICAMs, uPAR and TNF receptors. IL-6 and IL-8

stimulate epithelial-mesenchyme transitions (EMT), underscoring the change of cancer from an

isolated carcinoma in situ to an invasive cancer. Immune modulation is facilitated by cytokine

receptors shed by senescent cells, which subsequently act as decoys meaning nearby preneoplastic or

neoplastic cells avoid immune surveillance. In vivo studies, induced an SASP using the clinically

approved DNA-damaging chemotherapy, topoisomerase 2β inhibitor, mitoxantrone, used to treat

prostate cancer32

. These findings suggest a potential mechanism for side effects of chemotherapy,

whereby secretions by chemotherapy-induced senescent cells create a perfect environment for the

later development of secondary cancers. A prominent finding was the amplification of the SASP with

the loss of p53 function and oncogenic RAS expression; further solidifying the link of senescence to

cancer.

Modulating senescence to influence age-related cancerous phenotypes: The future of age-related

carcinogenesis

The elusive cure for cancer is modern medicine’s biggest quandary yet, it may appear that

intervention in aspects of senescence could hold an answer. Research using mouse models with

modified p53 sequences highlight the delicate balance between tumour suppression and promotion of

cancerous phenotypes. Total abrogation of senescence is not a solution as p53 deficient mice are

strongly predisposed to spontaneous transformation and tumour development41

, further enhanced by

administration of carcinogenic stimuli such as radiation42

. Moreover, there seems to be a fine line

between whether overexpression of p53 shortens or lengthens lifespan30

. Maier et al.43

bred transgenic

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University of Oxford Medical School

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mice overexpressing p44 (P44+/+

), a natural short p53 isoform lacking the main transactivation

domain. This resulted in wild-type p53 hyperactivity, and a low incidence of cancer was counteracted

by early ageing phenotypes, attributed to p53-induced premature senescence. However, Garcia-Cao et

al.44

challenged the developing view that extra p53 was harmful by using super-p53 mice (transgenic

mice carrying one or two extra wild-type p53 genes). No accelerated ageing was observed, with

normal basal p53 levels and activity in unstressed conditions. Nevertheless, they retained a

remarkable cancer resistance, probably due to a decreased likelihood of p53 inactivation from

stochastic mutations. Then again, when exposed to DNA-damaging radiation or carcinogens, p53

activity was markedly elevated and ageing rapidly ensued.

There are several explanations for the discrepancy between the P44+/+

and super-p53 results. Chronic

p53 hyperactivity in the P44+/+

mouse could explain the accelerated ageing, accounting for absence

thereof in the super-p53 mouse. Assuming this is a factor, unless it is chronically stressed, the super-

p53 mouse is able to confer tumour suppressive properties without concurrently accelerating ageing.

It is possible that by manipulating p53 in this way, cancer resistance can be conferred without any

undesirable ageing side-effects. Also, the P44+/+

mouse overexpressed an N-terminally truncated form

of p53, which perhaps caused detrimental qualitative imbalance in the p53 activity. Maier et al. failed

to appreciate the potential ramifications of using different p53 isoforms, and the importance of the

p53 gene context in reliably representing the physiological situation. Garcia-Cao et al., however,

ensured p53 retained most of its natural expression properties by performing transgenesis with large

DNA segments to keep the p53 gene in context. Therefore, prospects for direct comparison between

these two results could be tenuous.

Clinical links between senescence and age-related carcinogenesis lie within the cancer-prone

progeroid Werner syndrome, described as the best clinical model of human ageing45

. The effects of

cellular senescence are reflected in tissue cultures, where cells senesce extraordinarily rapidly with

vigorous SASPs, providing an in vivo link between cancer susceptibility and senescence. Werner

syndrome arises from an autosomal recessive germline mutation in the DNA helicase WRN,

highlighting the profound relationship between genome maintenance and increased incidence of

cancer.

Further possibilities for solving the problem of inescapable cancer with longevity could lie in animals

such as the naked mole-rat, the first reported mammal to show negligible senescence over the

majority of their lifespan46

. Not only do these animals live to 2847

, over ten times longer than the

average lifespan of normal mice, they appear to not develop any spontaneous neoplasms at all, a

remarkable feat considering Lipman et al. diagnose cause of death in over 80% of inbred laboratory

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University of Oxford Medical School

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mice to be cancer48

. Buffenstein et al.’s initial comparison of lifespan between these two creatures

was inadequate proof of inherent genetic predispositions to longevity in the mole-rat. Lifestyle

matters, such as the mole-rats’ natural caloric restriction and exercise, in contrast to the lonely,

restricted life of a captive lab-mouse could be influencing factors. Nonetheless, Buffenstein has since

qualified this, by comparing mole-rats and mice kept in similar captivity and of a similar size46

.

Activated oncogenic RAS fails to induce transformation in mole-rat cells as it does in mouse cells.

Mole-rat cells appear to need more perturbation than even human cells to induce cancer, which as yet

seems to be elusive to them47

. Uncovering the genetic foundation to this cancer resistance may reveal

a novel cancer treatment. Both the mole-rat and humans employ anticancer mechanisms known as

contact-inhibition, where cell division is arrested to prevent uncontrollable proliferation of cells on

top of each other47

. Cultured human fibroblasts arrest proliferation once the cells have created a

monolayer, while fibroblasts of mole-rats senesce after just a few cell-cell contacts, making a loose

network. The mole-rats exhibit hypersensitive early inhibition using p16INK4a

as well as regular

inhibition using p27Kip1

. Humans only use p27Kip1

in contact-inhibition, lacking the protection

conferred from a two-tiered system49

(Fig.5). Furthermore, despite the close association between

telomerase activity and cancer in humans, mole-rats exhibit such active contact-inhibition that they

can afford to continuously express telomerase, replicating prolifically throughout life without cells

entering replicative senescence49

.

Unsurprisingly, as in all fields of science, astoundingly radical ideas are being sought after from

‘extremist’ ends of the spectrum. While recent trends indicate that average lifespan is approaching

Figure 5: A model comparing the contact inhibition between naked mole-rat and mouse/human. The two-tiered

protection present in naked mole-rats may be the foundation to their cancer resistance. Figure adapted from Seluanov

(2009)

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University of Oxford Medical School

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100, these radical ideas claim that some babies born today will live until 100050

. Dr Aubrey de-Grey

claims to be developing ways to restore humans on a molecular level, returning them to a biological

age decades younger than their chronological one. They seek to accomplish the ambitious feat of

eliminating not only age-related cancer, but ageing itself by removing the telomerase enzyme gene

from all existing cells. However, a major flaw in this theory is that telomerase is not present in

effective quantities across the majority of somatic cells. As a scientist, it therefore appears that this

may be just another unrealistic claim to unlocking eternal life.

A cure for cancer; a victory against ageing; a downfall for society?

Finally, despite all of the potential possibilities outlined in this essay, the unanswered ‘elephant in the

room’ question is whether, if we did succeed in overcoming ageing, would we really want to? Where

can we draw the line between promoting longevity and artificially creating life? As previously

asserted in this paper, age-related carcinogenesis is the primary factor causing mortality in age.

Therefore if this were preventable, the lifespan of the population would increase by years

immediately.

As doctors, we strive to overcome disease to extend life, and so instinctively, targeting senescence to

do just this seems like a perfect solution. However, already with increasing life-expectancy, society is

beginning to suffer under the weight of the dependent population. In both harsh economic and social

terms, there just isn’t scope for an ever increasing dependent elderly cohort ‘sapping’ the resources.

The answer to this aspect lies in the lessons I gleaned from entering the clinical wards. Society aspires

to a longer wellspan. Targeting senescence to overcome age-related carcinogenesis and the ageing

process itself would decrease age-associated morbidity and therefore increase wellspan. In this case,

the societal effects may be less, as people would experience a loss of function at a much older age,

and so could continue to work, thus creating a larger pool of population to depend on. Nevertheless,

there are many other concerns that would need to be considered such as job opportunities, spatial

requirements for such an expanded population, food resources and energy supplies.

In conclusion, while senescence has many protective effects in preventing early-life cancer, its

deleterious phenotype is a potential condemnation to death by cancer in advancing age. Modulation of

senescence to include only its tumour suppressive effects, while preventing its destruction of the

stromal support, is the foundation of what should be a focus to achieving a treatment for cancer.

Senescence cannot altogether be abrogated, as this may significantly increase cancer risk by removing

the beneficial effects for which it was selected. However, anti-SASP interventions, for example

intervening at ROS, have the potential to delay ageing. A ‘cure for cancer’, with all of its

complications and facets, may be viewed as more of a catchphrase than an actual possibility. On the

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University of Oxford Medical School

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other hand, maybe finding a cure for cancer is a modern-day version of man walking on the moon, an

unfathomable feat that was in the end achievable. If used responsibly, to target disease and not to

create long-living humans at whim, then there is a potential for hope within senescence. Moreover,

with such interventions, it may be able to earn back its title as a functioning tumour suppressor.

Acknowledgments

I would like to thank Dr. Lynne Cox at the University of Oxford for her invaluable insight into the

intricate components of ageing and senescence, and the paradoxical relationship that exists between

them both. I would also like to thank Dr. Paul Mathew, Tufts Medical Centre, for giving me the

inspiration to research this area of geriatric medicine in the first place.

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University of Oxford Medical School

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