Rapamycin, proliferation and geroconversion to senescence

Introduction

Two-step geroconversion model of cellular senescence

In vitro, senescence involves two conflicting events: (i) cell cycle arrest and (ii) growth stimulation [15]. Growth stimulation drives conversion from arrest to senescence (geroconversion) [22,30]. Thus, a senescence program consists of arrest followed by geroconversion (Figure 1), which is driven in part by the growth-promoting mTOR pathway [1,22,25,30]. When the cell cycle is arrested but mTOR is still active, senescence develops [1].

Senescence cannot be completely understood in the realm of cell cycle arrest alone; mTOR-driven geroconversion must also be considered (Figure 2). In a two-dimensional model, markers of cell cycle arrest (p16 and p21) are accompanied by growth markers (phospho-S6 and cyclin D1). This two-dimensional view of senescence (cell cycle arrest plus geroconversion, Figure 2) enables us to not only to reconcile seemingly contradictory published findings, but also to manipulate and suppress cellular senescence. Rapalogs such as rapamycin and everolimus, as well as the pan-mTOR inhibitors, all suppress geroconversion and maintain quiescence [1,2,31–34].

Geroconversion and markers of senescence

During geroconversion, the mTOR and ERK/MAPK pathways are active, while cell cycling is blocked by p16/p21 (Figure 3). In a futile attempt to overcome the p16/p21-induced block, cyclin D1 is hyper-induced. At such high levels, cyclin D1 is a marker of senescence rather than of proliferation [10,35–38]. mTOR-driven geroconversion is associated with cellular hypertrophy [17,24,39] and hyperfunctions such as hypersecretion (or SASP) [40–44], ROS production [45], and lysosomal activation (β-Gal staining) [46–50]. These hyperfunctions in turn provoke compensatory reactions such as growth factor and insulin resistance [51–57], further lysosomal hyperactivation [49], and loss of RPP [10,38].

So-called golden marker of senescence and mTOR inhibitors

When the conflicting model of cellular senescence outlined above was published in 2003 [15], the golden marker of senescence was permanent arrest. We therefore tested whether rapamycin could prevent this “golden marker.” Certainly, rapamycin decreases other markers of cellular senescence such as cellular hypertrophy [2,8,17,33,58,59] and the hypersecretory and hyperinflammatory phenotypes [8,40,60–63]. But these effects were anticipated, as rapamycin is a known antihypertrophic [25] and anti-inflammatory agent [64]. In contrast, the prediction that rapamycin would preserve proliferative potential (RPP) is counterintuitive. After all, rapamycin inhibits proliferation, which makes it crucial to confirm this prediction.

HT-p21 and HT-p16 cells respectively express IPTG-inducible p21 and p16 [1,9,65,66]. Cell cycle arrest can be switched off and on in these cells through the addition and removal of IPTG. If arrest was induced for only 1–2 d, proliferation re-started in most cells after removal of the IPTG. When arrest lasted longer than 3–4 d, however, cell proliferation did not re-start after IPTG removal [9]. When cells were treated with IPTG in the presence of rapamycin, cell proliferation re-started after the IPTG and rapamycin were washed out. Thus, rapamycin preserved RPP in p21/p16-arrested cells. Rapamycin similarly preserved RPP during cell cycle arrest caused by pharmacologic inhibitors of CDK4/6, DNA damaging drugs, HDACi and phorbol ester [29,58,65–67]. Suppression of senescence by rapamycin was further confirmed in vitro and in vivo [26,40,62–81]. In addition to rapamycin, everolimus and ridaforolimus (two rapalogs), pan-mTOR inhibitors, nutlin-3a (a p53-inducer), hypoxia and contact inhibition all inhibit mTOR and thus maintain RPP in arrested cells [2,32–34,58,82–86]. Suppression of senescence by pan-mTOR inhibitors is closely associated with dephosphorylation of 4E-BP1 at both rapamycin-sensitive and -insensitive sites [2,31–34].

Irreversible proliferative arrest due to loss of RPP

Although senescent cell cycle arrest is often said to be irreversible, it is technically reversible, if the correct method is used. It cannot be reversed using serum, nutrients, growth factors or other stimuli. Serum reverses quiescence caused by serum withdrawal, but serum stimulation causes senescence when the cell cycle is blocked by p21 or p16 [1,58]. Similarly, quiescence caused by contact inhibition can be reversed by splitting cell cultures, but splitting senescent cultures only deepens senescence because mTOR is activated in sparse cell cultures [84,87,88]. It has therefore been suggested that the term “irreversible” be narrowed to “irreversible by mitogenic or oncogenic stimuli” [7].

Consider the mTOR-driven model of senescence. In quiescent cells, mTOR is deactivated (by serum/nutrient withdrawal, contact inhibition, hypoxia, etc.) and cyclin D1 is low; cells do not cycle and do not grow. Growth stimuli activate mTOR and induce cyclin D1, causing proliferation. However, strong growth stimuli can cause proliferation that is followed by arrest and geroconversion. For example, oncogenic Ras and Akt activate mTOR and induce cyclinD1, causing proliferation. But they can simultaneously induce p53, p21 and p16, thereby blocking the cell cycle [8,34]. This block cannot be reversed by growth stimulation, which only deepens the block and enhances mTOR-dependent geroconversion, but it can be reversed by inactivating p53, p21 and p16, for instance [3,15,89]. Once the cell cycle is unblocked, senescent cells re-enter the cell cycle but cannot undergo mitosis [9,10]). Moreover, these cells are hypermotile and literally tear themselves apart and eventually die (see micro-video in ref [10].). Thus, while cell cycle arrest is formally reversible, the loss of RPP renders it irreversible in practical terms. However, because rapamycin maintains RPP, cells in culture can regenerate once the cell cycle is unblocked.

Molecular definition of senescence

Although senescence can be defined as arrest that is irreversible by mitogenic or oncogenic (mTOR-activating) stimuli, this definition cannot be easily used in practice. Furthermore, RPP is a “potential” and is therefore difficult to test, especially in vivo. Defining senescence based on β-Gal staining is also problematic. β-Gal-staining is a marker of lysosomal hyperfunction [46–50]. Consequently, serum-starved and contact-inhibited cells are β-Gal-positive too [46,84], although these cells are not senescent (Figure 4). So can we distinguish β-Gal-positive quiescent and senescent cells? In β-Gal-positive quiescent cells, levels of phosphorylated S6, S6K and 4E-BP1 are low or undetectable (Figure 4). In contrast, these proteins are highly phosphorylated in senescent cells (Figure 4). In β-Gal-positive quiescent cells, insulin and other growth factors induce phospho-S6, whereas in senescent and proliferating cells, phospho-S6 is not further induced upon stimulation.

We can define senescence as practically irreversible arrest, a non-proliferative state, associated with proliferation-like mTOR activity (high levels of phospo-S6/S6K/4E-BP1). In addition, high levels of phospho-ERK and cyclin D1 coexist with p21 and/or p16 (Figure 4), and are associated with hypertrophy and hyperfunctions, including SASP, lysosomal hyperfunction (β-Gal staining), lipid synthesis (oil red O staining), ROS and lactate production. We suggest such cells can be identified using double-staining for phospho-S6 plus p16/p21, phospho-S6 plus β-Gal, or p16/p21 plus cyclin D1. A combination of all these markers may be the most valuable (Figure 4).

Cell culture and the organism

Rapamycin inhibits growth and slows geroconversion, which is a continuation of growth. In analogous fashion, organismal aging is a continuation of developmental growth [90–98]. Rapamycin (at high doses) slows cell proliferation within the organism, causing leucopenia, thrombocytopenia and mucositis and also decelerates organismal aging and its manifestations: age-related diseases [92].

In cultured cells, the senescence program consists of two steps: arrest plus geroconversion. Because most cells within organisms are quiescent, senescence consists of slow geroconversion. Why is it so slow? Contact inhibition and high cell density [84], hypoxia [83,99], and serum/nutrient starvation each deactivate mTOR. Within the organism, most cells are confluent or contact inhibited, and oxygen tension (1–3% O₂) as well as levels of nutrients/growth factors are low. These growth-limiting conditions may maintain quiescence for decades during a human lifespan.

In vitro, senescence is induced in sparse cell cultures in the presence of 21% oxygen and high levels of growth factors and nutrients. For example, glucose levels in DMEM are 5-fold higher than in normal blood, corresponding to levels associated with diabetic coma and causing complete insulin-resistance [55]. As a result, geroconversion is a fast event in vitro, especially in cancer cells. In fact, mTOR activity is much higher in cultured cells than in the organism and is inversely related to Akt activity [87].

Geroconversion and disease

mTOR-driven geroconversion is associated with enhanced tissue-specific functions (hyperfunctions), which drive age-related diseases. For example, vascular smooth muscle cell contraction, hypertrophy and hyperplasia all contribute to hypertension and atherosclerosis. Hyperfunction of adipocytes and hepatocytes increase blood cholesterol levels, contributing to atherosclerosis. Atherosclerosis, hypertension and thrombosis (due to platelet hyperfunction) can culminate in stroke and infarction and subsequent loss of organ function. Therefore, initial hyperfunction eventually leads to dysfunction and functional decline [90,100].

It was known by 2006 that rapamycin delays most age-related diseases [90]. As predicted [90], rapamycin prolongs the lifespan of mice [60,75,101–122]. Rapamycin and everolimus have been tested in healthy volunteers [123,124] and in the elderly [125–127]. A combination of rapamycin with several conventional life-extending drugs, known as the Koschei formula, is already being used for the elderly in the Alan Green Clinic in Little Neck, New York (https://rapamycintherapy.com).

“Repetitio est mater studiorum”

Rapamycin and other mTOR inhibitors suppress geroconversion. In the presence of rapamycin, time moves slower for cell growth, cycling and geroconversion [128]. Rapamycin does not reverse cell cycle arrest and does not stimulate proliferation. Like all gerosuppressants, rapamycin slows cell growth and geroconversion in arrested cells. Rapamycin also maintains RPP in quiescent/arrested cells. To observe this effect, the cell cycle block must be relieved – for example, by decreasing p16 and p21.

Two paradoxical effects of rapamycin

Notably, a two-dimensional model of aging predicts contradictory events.

Disclosure statement

No potential conflict of interest was reported by the author.