Latest p16(Ink4a) senescence-reversing research – an important but not game-changing contribution

By Vince Giuliano

The press has recently picked up heavily on work by Mayo Clinic researchers related to reversal of cell-senescence in-vivo via inhibition of expression of the protein p16(Ink4a).  The research has been heralded by such headlines as Cell Study Finds a Way to Slow Ravages of Age (Wall Street Journal), Clearing Away Old Cells Delays Aging in Mice (TIME) and Removing Deadbeat Cells Slows Aging in Mice and May Spare Humans (Business Week).  Ah, if these and similar headlines were only the simple truth!   Sadly, the actual research delivers far less and is incremental to previous research as I will detail here.  However, the finding is important and worth noting.  Here, I will describe the new research, say why it falls short of the promises of the headlines,  and characterize the stream of research in which the latest finding is an incremental contribution.

The latest finding relating P16(Ink4a) to cell senescence and animal health and longevity

The title of the much-publicized 2 November online  publication is Clearance of p16^Ink4a-positive senescent cells delays ageing-associated disorders.  “Advanced age is the main risk factor for most chronic diseases and functional deficits in humans, but the fundamental mechanisms that drive ageing remain largely unknown, impeding the development of interventions that might delay or prevent age-related disorders and maximize healthy lifespan. Cellular senescence, which halts the proliferation of damaged or dysfunctional cells, is an important mechanism to constrain the malignant progression of tumour cells^{1, 2}. Senescent cells accumulate in various tissues and organs with ageing³ and have been hypothesized to disrupt tissue structure and function because of the components they secrete^{4, 5}. However, whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has remained unknown. To address these fundamental questions, we made use of a biomarker for senescence, p16^Ink4a, to design a novel transgene, INK-ATTAC, for inducible elimination of p16^Ink4a-positive senescent cells upon administration of a drug. Here we show that in the BubR1 progeroid mouse background, INK-ATTAC removes p16^Ink4a-positive senescent cells upon drug treatment. In tissues—such as adipose tissue, skeletal muscle and eye—in which p16^Ink4a contributes to the acquisition of age-related pathologies, life-long removal of p16^Ink4a-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. These data indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.”

Speaking to this latest research, the news item in Gen Clearing Tissues of Senescent Cells Found to Delay Onset of Age-Related Disordersreports “Clearing tissues of senescent cells could represent a new therapeutic approach to delaying the onset or progression of age-related disorders and prolonging healthy human lifespan, researchers claim. Mayo Clinic College of Medicine investigators developed a transgenic mouse model in which cells displaying a specific marker of senescence can be removed on the administration of a drug. They then crossed these mice with a strain of animal that displays early age-related deficits and evaluated the effects of clearing tissues of senescent cells. — The results, reported in Nature, showed that drug-induced clearance of senescent cells from an early age delayed the onset of tell-tale age-related problems such as muscle wastage, specifically in those tissues in which the senescent cells normally accumulate. Moreover, claim Jan M. van Deursen, M.D., and colleagues, starting treatment later in life helped slow the progression of already-established age-related disorders. — The team says its findings indicate that acquisition of the senescence-associated secretory phenotype (SASP), which enables cells to secrete a variety of growth factors, cytokines, and proteases, contributes to age-related tissue dysfunction.”

Why the latest research is incremental

Well and good.  However, despite much publicity:

• The research does not establish that removal of senescent cells can prevent or delay tissue dysfunction in normal (wild-type) mice, let alone humans.    This is because the experiments were done on BubR1 progeroid mice, a laboratory strain that is very seriously compromised to start with.  These mice “express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing(ref).”  Reducing markers of senescence in these mice may or may not say a lot with respect to normal mice or other animals.  Although the inference has been drawn that removal of senescent cells can prevent or delay tissue dysfunction in normal (wild-type) mice, this proposition is yet to be established by experiment and may or may not be correct.  As of now there is no direct experimental evidence that healthspan or lifespan of normal animals can be enhanced by removal of senescent cells.
• There is a stream of antecedent research relating to P16(Ink4a) and cell senescence that makes the latest observed result highly likely.  I will discuss some of this research later in this blog posting.  Directly applicable to this latest research the 2008 paper Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency reported “Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.”

Background on cell senescence and P16(Ink4a)

P16(Ink4a) has long been known to be associated with cell senescence and aging.

In an early draft of my treatise, ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY, some three years ago I wrote “There appear to be several other clocks related to aging in addition to telomere length. One is the accumulation of INK4a/P16 as well as NF-kappaB in cells with age. And this directly relates to decline in stem cell differentiation as a function of age ? –“Buildup of levels of Ink4a/P16 associated with aging slows down the rate of differentiation of adult stem cells. “Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of theInk4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis.” – “ These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16Ink4a is necessary(ref).“ –“ A new line of research(ref) focuses on four genes known to be implicated in both cancer and stem cell activation: Ink4a, Arf, Hmga2 and let-7b. P16/Ink4a in particular, a tumor suppressor gene, appears to become increasingly active with age in mice, humans and other mammals. It is a known mediator of cell senescence and biomarker of aging as well as a possible promoter of mammalian aging. P16/Ink4a works together with the three other genes to articulate a process of simultaneously protecting against cancers and shutting down adult stem cell function and regenerative capacity in aging tissues. Expression of Ink4a and Arf in the absence of a protein Bmi1 results in loss of self-renewing stem cells. The four genes involved, appear to switch on and off in a coordinated fashion that depends on age. Older stem cells don’t wear out or die from damage according to this line of reasoning; they are shut down. Increasing cancer protection is paid for by accelerated aging. This research is based on neural tissues in mice and the extent to which it can be generalized to other human cell types is still to be discovered.”

I have also discussed the relationships among P16(Ink4a) and cell senescence in several blog entries. In the June 2009 blog entry Linking up the theories of aging, I wrote:” Senescent cells tend to strongly express the anti-cancer genes P16(INK4a) and P19(Arf). So, these genes offer senescent cells an alternative to becoming malignant. But senescent cells are likely to become bad neighbors sending out signals that can lead to organ dysfunction or degeneration. Further, in discussing the Programmed epigenomic changes theory, I mentioned how p16(INK4a) tends to be increasingly expressed with age and how it tends to inhibit the differentiation of adult stem and progenitor cells. Thus, P16(INK 4a) plays a central role in the Decline in adult stem cell differentiation theory. Also, it “induces an age-dependent decline in islet regenerative potential(ref).” Increasing expression of P16(INK4a) with age therefore tends to compromise organ repair and regeneration. P16(INK4a) provides a central defense against cancer in the case of senescent cells and is therefore important in theSusceptibility to cancers theory of aging.  — There is another side to cell senescence, however: “Senescent cells, particularly senescent stromal fibroblasts, secrete factors that can disrupt tissue architecture and/or stimulate neighboring cells to proliferate. We suggest that senescent cells can create a tissue environment that synergizes with oncogenic mutations to promote the progression of age-related cancers(ref).” I have mentioned the paradoxical role of P16(INK4a) in the Blog post Dr. Jekyll and-Mister Hyde Proteins. The new research, reported in a publication entitled Polycomb Mediated Epigenetic Silencing and Replication Timing at the INK4a/ARF Locus during Senescence provides a new link between the theories and hints at anti-aging intervention that can address all of these theories.  — Basically, in young cells, Polycomb group proteins act on the INK4/ARF gene regulatory domain so as to the keep the expression of P16(INK4a) turned off, the gene is silenced. In senescent cells, however, there are epigenetic modifications (DNA and histone methylation changes) which block the inhibitory actions of the polycomb group proteins, so the P16(INK4a) and Arf genes are activated. So, cell senescence leads to another pro-aging effect, the activation of the P16(INK4a) and Arf genes. Earlier, in the Anti-Aging Firewalls treatise I identified the increasing expression of P16(INK4a) with aging as a biomarker of aging and possible cause of age-related changes. In fact, I identified this as possibly one of the major aging mechanisms according to the Programmed epigenomic changes theory.

Recent literature relating to P16(Ink4a), cell senescence and aging.

While senescent cells are often extremely bad neighbors secreting inflammatory cytokines and other disease-promoting chemicals, cells with senescence induced by P16(Ink4a) do not excrete such inflammatory or disease-promoting chemicals.  Thus, secretion of such cytokines and other chemicals is a cell damage response separate from simple cell senescence.

An October 21 2011 publication Tumor Suppressor and Aging Biomarker p16INK4a Induces Cellular Senescence without the Associated Inflammatory Secretory Phenotypereports:  “Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16(INK4a), a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16(INK4a) and another cyclin-dependent kinase inhibitor, p21(CIP1/WAF1), induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest.  Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16(INK4a). Cells induced to senesce by ectopic p16(INK4a) expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16(INK4a) by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16(INK4a) in suppressing the SASP. These findings suggest that p16(INK4a)-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16(INK4a) activation or senescence per se, but rather is a damage response that is separable from the growth arrest.”

While elevated levels of P16(Ink4a) and cell senescence is taken as biomarkers of aging in many cells, other cells, particularly hematopoietic stem cells, age via other mechanisms and do not express P16(Ink4a) or become senescent

The 2009 publication Hematopoietic stem cell ageing is uncoupled from p16 INK4A-mediated senescence reports “Somatic stem cells are ultimately responsible for mediating appropriate organ homeostasis and have therefore been proposed to represent a cellular origin of the ageing process-a state often characterized by inappropriate homeostasis. Specifically, it has been suggested that ageing stem cells might succumb to replicative senescence by a mechanism involving the cyclin-dependent kinase inhibitor p16(INK4A). Here, we tested multiple functional and molecular parameters indicative of p16(INK4A) activity in primary aged murine hematopoietic stem cells (HSCs). We found no evidence that replicative senescence accompanies stem cell ageing in vivo, and in line with p16(INK4A) being a critical determinant of such processes, most aged HSCs (>99%) failed to express p16(INK4A) at the mRNA level. Moreover, whereas loss of epigenetically guided repression of the INK4A/ARF locus accompanied replicative senescent murine embryonic fibroblasts, such repression was maintained in aged stem cells. Taken together, these studies indicate that increased senescence as mediated by the p16(INK4A) tumor suppressor has only a minor function as an intrinsic regulator of steady-state HSC ageing in vivo.”

So, hematopoietic stem cells do not become senescent like other cells do when they age.  Instead, as pointed out above and previously, with aging they slow down and eventually stop differentiating.   P16(Ink4a) is again implicated in slowing down differentiation but acts in a different way than in the case of cell senescence in other cell lines such as endothelial progenitor cells..

A mathematical model has been built relating factors that bear on P16(Ink4a) accumulation, like smoking, exercise and certain genetic polymorphisms, to lifespans.

The 2009 publication A quantitative model for age-dependent expression of the p16INK4a tumor suppressor reports “Recent work has shown that expression of the p16(INK4a) tumor suppressor increases with chronological age. Expression is accelerated by gerontogenic behaviors such as tobacco use and physical inactivity, and is also influenced by allelic genotype of a polymorphic single nucleotide polymorphism (SNP) rs10757278 that is physically linked with the p16(INK4a) ORF. To understand the relationship between p16(INK4a) expression, chronologic age, subject characteristics and host genetics, we sought to develop a mathematical model that links p16(INK4a) expression with aging. Using an annotated dataset of 170 healthy adults for whom p16(INK4a) expression and subject genotypes were known, we developed two alternative stochastic models that relate p16(INK4a) expression to age, smoking, exercise and rs10757278 genotype. Levels of p16(INK4a) increased exponentially and then saturated at later chronologic ages. The model, which best fit the data, suggests saturation occurs because of p16(INK4a)-dependent attrition of subjects at older chronologic ages, presumably due to death or chronic illness. An important feature of our model is that factors that contribute to death in a non p16(INK4a)-dependent manner do not affect our analysis. Interestingly, tobacco-related increases in p16(INK4a) expression are predicted to arise from a decrease in the rate of p16(INK4a)-dependent death. This analysis is most consistent with the model that p16(INK4a) expression monotonically increases with age, and higher expression is associated with increased subject attrition.”

It is speculated that, in certain cell lines, endothelial progenitor cells in particular,  the presence of p16(INK4a)-and telomere length attrition may interact to co-modulate cell senescence.

The 2008 publication Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescencereports “Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.”

P16(INK4a) cell senescence is not necessarily accompanied by shortening of telomere lengths.

The 2010 e-publication Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-alpha via the p38 mitogen-activated protein kinase pathway also relates to senescence of endothelial progenitor cells.  It reports:  “Senescence of endothelial cells increases with systemic aging and is thought to contribute to the development of atherosclerosis. Cell therapy with highly proliferative endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote endothelial regeneration, but little is known about their senescence and their vulnerability to inflammatory stressors. We therefore studied the senescence of proliferative human EPCs and investigated the effects of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on their senescence. Human EPCs had a significantly lower rate of senescence at baseline, compared with that of mature endothelial cells. However, EPCs up-regulated the expression of the senescence-associated cell cycle arrest protein p16(INK4a) and markedly increased measured senescence levels when exposed to chronic TNF-alpha treatment. Analysis of telomere length showed that the increases in senescence were not related to changes in telomere length. Inhibition of the p38 mitogen-activated protein kinase pathway blocked the induction of p16(INK4a) and cellular senescence. In conclusion, highly proliferative EPCs have a low rate of intrinsic senescence but are vulnerable to premature senescence induction by chronic proinflammatory stimulation. These findings will lead to a better understanding of physiological endothelial regeneration as well as to targeted therapies with the aim of promoting endothelial regeneration through endothelial progenitor cells.”

Several articles were published in 2010 and 2010 related to epigenetic modifications of the P16(Ink4a) gene as related to disease susceptibilities.  I mention only two of these here, ones dealing with how hypermethylation of P15(Ink4a) may increase cancer susceptibilities..

The 2011 publication P16 gene hypermethylation and hepatocellular carcinoma: a systematic review and meta-analysis reports “AIM: To quantitatively investigate the effect of p16 hypermethylation on hepatocellular carcinoma (HCC) and hepatocirrhosis using a meta-analysis of available case-control studies. METHODS: Previous studies have primarily evaluated the incidence of p16 hypermethylation in HCC and corresponding control groups, and compared the incidence of p16 hypermethylation in tumor tissues, pericancer liver tissues, normal liver tissues and non-tumor liver tissues with that in other diseases. Data regarding publication information, study characteristics, and incidence of p16 hypermethylation in both groups were collected from these studies and summarized. RESULTS: Fifteen studies, including 744 cases of HCC and 645 non-tumor cases, were identified for meta-analysis. Statistically significant odds ratios (ORs) of p16 hypermethylation were obtained from tumor tissues and non-tumorous liver tissues of HCC patients (OR 7.04, 95% CI: 3.87%-12.78%, P < 0.0001), tumor tissues of HCC patients and healthy liver tissues of patients with other diseases (OR 12.17, 95% CI: 6.64%-22.31%, P < 0.0001), tumor tissues of HCC patients and liver tissues of patients with non-tumorous liver diseases (OR 6.82, 95% CI: 4.31%-10.79%, P < 0.0001), and cirrhotic liver tissues and non-cirrhotic liver tissues (OR 4.96, 95% CI: 1.45%-16.96%, P = 0.01). The pooled analysis showed significantly increased ORs of p16 hypermethylation (OR 6.98, 95% CI: 4.64%-10.49%, P < 0.001) from HCC tissues and cirrhotic tissues. CONCLUSION: P16 hypermethylation induces the inactivation of p16 gene, plays an important role in hepatocarcinogenesis, and is associated with an increased risk of HCC and liver cirrhosis.”

Hypermethylation of P16(Ink4a) could be an important biomarker for susceptibility to lung cancers due to exposure to. polycyclic aromatic hydrocarbons.

The October 2011 publication CpG Site-specific Hypermethylation of p16INK4α in Peripheral Blood Lymphocytes of PAHs Exposed Workers reports: BACKGROUND: Sufficient epidemiologic evidence demonstrates an etiologic link between polycyclic aromatic hydrocarbons (PAHs) exposure and lung cancer risk. While the genetic modifications have been found in PAHs-exposed population, it is unclear whether gene-specific methylation involves in the process of PAHs-associated biological consequence.  METHODS:Sixty-Nine PAHs exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16(INK4α) promoter in peripheral blood lymphocytes (PBLs) from PAHs exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16(INK4α) methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. RESULTS: Compared to the control group, PAHs-exposed workers exhibited higher levels of urinary 1-OHP (10.62 μg/L v.s. 2.52 μg/L), p16(INK4α) methylation (7.95% v.s. 1.14% for 22 “hot” CpG sites) and CBMN (7.28‰ v.s. 2.92‰) in PBLs. p16(INK4α) hypermethylation in PAHs-exposed workers exhibited CpG site-specificity. Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. CONCLUSIONS: PAHs exposure induced CpG site-specific hypermethylation of p16(INK4α) gene. The degree of p16(INK4α) methylation was associated with the levels of DNA damage and internal exposure.Impact: p16(INK4α) hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer.”

Sorry that the latest research cannot deliver on the promises of the news headlines.  However the subject remains an important one and I expect we will be hearing more and more about epigenetic regulation of P16(Ink4a) and implications of such for disease processes and aging.