Paradoxical down-regulation of p16INK4a mRNA with
advancing age in Acute Myeloid Leukemia
Abstract
Aging
is generally considered to be the consequence of stem cell attrition caused
by the activity of tumor suppressor pathways that censor potentially
malignant clones by eliciting apoptosis or senescence. An important
effector of aging is the cyclindependent kinase inhibitor p16INK4a,
which is also a known suppressor of cancer. The expression of p16INK4a
is very low or absent in young organisms but increases with advancing age.
We recently showed that, unlike healthy cells, acute myeloid leukemia (AML)
derived blasts show a down-regulation of p16INK4a mRNA with
increasing age. Based on this observation we hypothesize that suppression
of defense mechanisms which protect older cells against cellular and DNA
damage might facilitate oncogenesis in older individuals.
Aging
In human biology aging is accompanied by a diminished
capacity to adequately maintain tissue homeostasis or to repair tissues after
injury. When homeostatic control diminishes to a point at which tissue/organ
integrity and function are no longer sufficiently maintained, physiologic
decline ensues, and aging is manifested. Consistent with this, many of the
pathophysiological conditions afflicting the elderly, such as anemia,
sarcopenia and osteoporosis, suggest an imbalance between cell loss and
renewal.
Numerous theories have been put forth to
explain the decline of stem cell function with advancing age. The free-radical
theory of aging proposes that reactive oxygen species (ROS), which are
by-products of normal metabolism, are responsible for damage to many cellular components, including DNA [1]. It is clear, however,
that in addition to ROS, a much broader range of
extrinsic and intrinsic sources, such as UV irradiation, alkylating agents,
telomere attrition, and DNA replication errors, can also infringe upon genomic
integrity [2]. In response
to this damage, tumor suppressor pathways are activated, including those
mediated by the tumor suppressor proteins p16 and p53, to ensure that
potentially dangerous lesions do not lead to malignancy [2,3]. So, when aging
advances and damage accumulates the activity of these tumor suppressors
increases and consequently has the potential to negatively modulate stem cell
function through the induction of apoptosis or senescence.
p16INK4a and aging
The cyclin-dependent kinase inhibitor p16INK4a
has emerged as an important player in aging and age-related disease. p16INK4a
has the ability to bind and inhibit the cyclin-D-dependent kinases CDK4 and
CDK6. These kinases are known to have oncogenic potential and phosphorylate the
retinoblastoma (Rb) family of tumor suppressors. Hence, expression of p16INK4a
maintains Rb-family proteins in hypophosphorylated state, which promotes
binding of E2F to effect a G1 cell cycle arrest [3,4]. Several
studies have shown that p16INK4a expression increases markedly with
advancing age in a variety of tissues [5-8].
Consequently, the increased expression of p16INK4a induces an age-dependent decrease in the proliferative capacity of
certain tissue-specific stem- and progenitor-cells [5-8].
Senescence characterized by p16INK4a upregulation as well as
telomere shortening has been observed in human and mouse cardiomyocytes and may
contribute to myocardial aging [9,10]. Because
p16INK4a expression can be upregulated by a wide variety of stresses
[4,11] it may be
involved in many, maybe all, forms of senescence, and has thus recently
received attention as a promising biomarker [12,13].
Also in the hematopoietic system it has been shown
that the induction of p16INK4a correlated with the in vivo
senescence of hematopoietic stem cells [14-16]. Indeed,
mice lacking BMI1, a repressor of p16INK4a, displayed a striking
loss of hematopoietic cells which correlated with increased expression of p16INK4a
expression and replicative failure of stem cells [16-18].
Conversely, an increase in regenerative capacity was found in the bone marrow
of p16INK4a deficient mice [19]. These
findings reinforce the notion that the age-associated upregulation of p16INK4a
restricts self-renewal and unbalances tissue homeostasis. We have confirmed
increased p16INK4a expression in healthy human CD34+ hematopoietic
cells upon aging [20].
Aging and Acute Myeloid Leukemia
Aging not only affects normal
hematopoietic development (e.g. increased incidence of anemia), but also
impacts on the clinical biology of AML. In particular, the incidence of AML
increases with increasing age [21,22].
Moreover, older AML patients have a markedly reduced long-term survival due to
the combination of poor chemotherapeutic tolerance and inherent chemotherapy
resistance compared to younger AML patients [21-25]. AML in
older patients shows also a lower frequency of favorable core-binding
chromosomal abnormalities and a higher incidence of complex aberrant karyotypes
[26,27]. We
wondered whether these differences in clinical and cellular behavior of AML in
older patients were reflected by differences in gene expression profiles.
Therefore, a cohort of 525 adult AML patients was studied to compare gene
expression profiles of the one-third of youngest cases (median age 31 years)
with the one third of oldest cases (median age 59 years) [20]. Biological
processes (represented by GO-ontologies) associated with aging in AML were compared
with a published list of significantly differentially expressed GO-ontologies
between young and old purified murine long-term hematopoietic stem cells. This
analysis revealed that in both sets the NF-κB cascade was up-regulated and
that maintenance of chromatin architecture, chromatin modification and
organelle organization were down-regulated [20, 28].
Subsequently, comparison of gene expression profiles of AML samples of the 175
youngest with the 175 oldest AML patients revealed that 477 probe sets were
up-regulated and 492 probe sets were down-regulated with increasing age at the
significance level of P <1.0x10-5. Additionally, two in- dependent AML gene array
cohorts were used for validation. The final list of validated genes which were differently
expressed depending on age in three independent AML cohorts yielded a number of
interesting genes, including p16INK4a. The level of expression of
p16INK4a in AML samples during aging was reciprocal to the usual
trend of an increased expression at higher age; i.e. the expression of p16INK4a
declined significantly with increasing age. Of note, this was only noticed in
the intermediate- and unfavorable-risk group and not in the favorable-risk
group and the molecularly defined subset ‘NPM1 mutant without FLT3-ITD'.
Multivariate analysis revealed p16INK4a, besides cytogenetic
risk-groups, as an independent prognostic parameter for overall survival (OS)
in older (and not in younger) AML patients. Lower p16INK4a
expression in AML samples of patients of older age predicts for reduced OS.
Further studies unraveling the regulation and molecular mechanisms responsible
for the down-regulation of p16INK4a during aging in AML are awaited.
Striking in this perspective is our observation that BMI1, a potent repressor
of p16INK4a, was significantly
inversely correlated with p16INK4a in older (P = <.001, rho = -.274, n = 175) and not in
younger (P = .322, rho = -.075, n = 175) AML patients.
Is the downregulation of p16INK4a a broader phenomenon?
We wondered whether the paradoxical down-regulation of
p16INK4a with advancing age in AML might be a more general
phenomenon, i.e. is p16INK4a expression also down-regulated with
advancing age in other malignancies? An extensive search in the Gene Expression
Omnibus (GEO) repository revealed only three publically available Affymetrix
gene array datasets of malignancies (i.e. lymphoma, glioblastoma and breast
cancer) which also presented the patient characteristic age (GSE4475, GSE7696
and GSE3494) [29-31]. The median age at diagnosis is
given in Table 1 for all three cohorts. Interestingly, the expression of p16INK4a
declined significantly with advancing age in lymphoma patient samples (P =
<.001, rho = -.252 n = 219, Table 1) and in glioblastoma patient samples (P
= .005, rho = -.310, n= 80, Table 1). In breast cancer patient samples the
continuous variables age and p16INK4a did not correlate (P = .407,
rho = -.053, n = 251, Table 1).
Table 1. Correlation between the expression of p16 INK4a with age with age
at diagnosis in three malignancies.
The relation between the expression of p16INK4a mRNA level and age at
diagnosis in three malignancies (publicly available micro arrays, i.e. GSE4475, GSE7696
and GSE3494)[29-31]. Spearman rank correlation
coefficients between the continuous variables age and the averaged p16INK4a
probe sets (n=3) were calculated. The characteristic age is given as median (range).
Cancer type | age | n | P | Rho |
lymphoma
|
61 (2-61)
|
219
| <.001 |
-.252
|
glioblastoma
|
52 (26-70)
|
80
| .005 |
-.310
|
breast cancer
|
64
(28-93)
|
251
|
.407
|
-.053
|
Conflicts of Interest
The authors of this
manuscript have no conflict of interest to declare.
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