Too big not to fail: emerging evidence for size‐induced senescence

Cellular senescence refers to a permanent and stable state of cell cycle exit. This process plays an important role in many cellular functions, including tumor suppression. It was first noted that senescence is associated with increased cell size in the early 1960s; however, how this contributes to permanent cell cycle exit was poorly understood until recently. In this review, we discuss new findings that identify increased cell size as not only a consequence but also a cause of permanent cell cycle exit. We highlight recent insights into how increased cell size alters normal cellular physiology and creates homeostatic imbalances that contribute to senescence induction. Finally, we focus on the potential clinical implications of these findings in the context of cell cycle arrest‐causing cancer therapeutics and speculate on how tumor cell size changes may impact outcomes in patients treated with these drugs.


Introduction
Eukaryotic cells exist in a wide range of sizes and morphologies, even within the same organism.Human cells span approximately five orders of magnitude in size, with the smallest cells (sperm) measuring ~25 fL in volume [1] and the largest (oocytes) measuring 3.5 nL [2].Despite the wide size distribution across healthy cells, it has long been observed that the distribution of sizes within a single cell type is typically narrow [3].Such a uniform distribution is a strong indicator that retaining a particular cell size is important for normal cellular function.Indeed, increased cell size heterogeneity has been observed in cancers [3,4], and this heterogeneity has been predicted to increase tumor invasiveness [5].Despite this, cells can drastically change size under some biological circumstances (e.g., differentiation) without detrimental consequences [6][7][8].These observations suggest that changes in cell size are well-tolerated in some cases but not others, but what drives this discrepancy is not yet clear.
A well-studied circumstance under which cells depart from their typical size range but lose normal cellular function is cellular senescence.This was first observed in the 1960s in seminal work by Hayflick and Moorhead, who identified a permanent state of cell cycle arrest in serially cultivated fibroblasts [9] that later became known as replicative senescence [10].Since then, many other causes for senescence have been established, including oncogene activation, mitochondrial dysfunction, and others.Despite diverse causes for senescence induction, senescent cells share several common traits, which are extensively detailed in other reviews [11][12][13][14].Among these traits is the common tendency to become enlarged, which occurs because senescent cells continue to accumulate biomass but do not divide.Thus, increased cell size has long been a biomarker for senescent cells in vitro [9,11,[15][16][17] and in vivo [18].
Recent work from our laboratory and others has demonstrated that excess cell size is not only a consequence but also a cause of senescence [19][20][21][22][23][24][25][26].Excess cell size compromises a number of critical cellular functions, and the contributions of these defects to permanent cell cycle exit have recently come into focus.In this review, we examine recent insights into how excess cell size drives permanent cell cycle exit.We further describe homeostatic defects observed in enlarged cells that may contribute to persistent proliferative failure.Finally, we discuss the implications of these findings in the context of clinical drugs that cause persistent cell cycle arrest and expose the need for careful controls before translating the in vitro mechanisms of these drugs into a clinical setting.

Replicative senescence
Replicative senescence refers to the permanent cell cycle withdrawal that occurs in non-transformed somatic cells that have reached their division limit [10].Although it is not yet clear how cells "count" how many divisions they have experienced, the current model of replicative senescence implicates the progressive shortening of telomeres in causing a loss of DNA [27].Cells register this loss as DNA double-stranded breaks (DSBs), and induction of the DNA damage response causes permanent cell cycle exit [28].Indeed, overexpression of telomerase-the enzyme that protects chromosome ends by synthesizing telomeric repeats [29]-is sufficient to extend cells' replicative lifespans [30].

Oncogene-induced senescence (OIS)
Oncogene-induced senescence refers to senescence that is prematurely induced following either hyperactivation of an oncogene or silencing of a tumor suppressor-both of which can result in unscheduled proliferation.Examples of this include the hyperactivation of RAS [31], BRAF [32], CCNE1, or MYC [33], or the repression of PTEN [34] or NF1 [35].By blocking cell cycle progression in response to proproliferative signaling, OIS is a tumor-suppressive mechanism that prevents oncogenic transformation [36].Consistent with this role, OIS is the form of senescence that is most often identified in precancerous tissues [37].Similar to replicative senescence and or senescence induced by DSBs, OIS can activate the DNA damage response.This is a consequence of unscheduled replication causing replication stress and subsequent DNA damage [38,39].

Mitochondrial dysfunction-associated senescence
Mitochondrial dysfunction is defined by a decrease in mitochondrial membrane potential and decreased respiratory capacity.This can be caused by imbalances in the NAD + /NADH ratio, defects in mitochondrial turnover, changes in calcium or nutrient signaling, mitochondrial DNA mutations [40], and others.Low mitochondrial membrane potential is correlated with an increase in reactive oxygen species (ROS) production [40][41][42], which has been implicated in cell cycle exit and aging since the 1950s [43].Although mitochondrial dysfunction has long been observed in senescent cells [44,45], more recent work has demonstrated that compromised mitochondrial function is itself sufficient to trigger senescence.Mechanistically, this is thought to be a consequence of a decreased NAD + / NADH ratio activating AMPK signaling, which subsequently results in a p53-dependent cell cycle arrest [46].

Size-induced senescence
Based on recent studies discussed in this review, we propose that-in addition to being a consequence of prolonged cell cycle arrest-excess cell size is itself an independent cause of senescence.A prolonged cell cycle arrest that enables continuous cell growth causes permanent cell cycle exit upon release, whereas restricting growth during the arrest is sufficient to preserve proliferative potential [19][20][21][22][23][24] (Fig. 1A).An elegant study from Lanz and colleagues also shows thatwhen cycling cells are stratified by size-populations of larger cells carry proteomic signatures characteristic of senescence and tend to senesce earlier than small cells [26].Such size-dependent proteome changes are also observed on a single-cell basis [47].Importantly, size-dependent senescence induction is rescued when cellular ploidy is increased [19,26], suggesting that excess cell size drives senescence due to limiting DNA abundance.Although it is still not clear why a low DNA:cytoplasm ratio drives cells into senescence, one potential explanation is that the failure to scale macromolecule production in relation to cell volume causes cytoplasmic dilution, which compromises normal cell physiology [19].Consistent with this notion, enlarged yeast fail to scale both inducible and general transcription [19,48], resulting in the global subscaling of RNA levels.
Because nuclear size increases with cell size [49][50][51], defects associated with cellular enlargement could also be a consequence of altered nuclear organization, given that DNA content remains constant.
Importantly, because other forms of senescence cause excess cell growth, increased cell size likely reinforces senescence initially triggered by other causes.This hypothesis is supported by observations that senescence arising from genotoxic stress exposure or from persistent cell cycle arrest signaling also requires continuous growth [52,53].

Cell cycle arrest signaling in enlarged cells
Senescence induction typically requires at least one of two signaling axes: the p16/Rb pathway or the p53/ p21 pathway.In its classical interpretation, the p16/Rb pathway blocks the G 1 /S transition by interfering with the feedback loop that drives the expression of genes required for cell cycle progression.Mechanistically, this is a consequence of the Cdk inhibitory protein p16 blocking Cdk4/6 activity.A critical target of Cdk4/6 is Rb, which is an inhibitor of the E2F family of transcription factors.In its hypophosphorylated state, Rb is a potent inhibitor of E2F-mediated transcription.Upon Cdk4/6-mediated phosphorylation, Rb is released from E2F target gene promoters, and E2F inhibition is relieved.Thus, p16 blocks E2F target gene expression and restrains the G 1 /S transition [54].In cycling cells, Rb protein levels are also important for linking cell growth to cell division: Rb is diluted as cells grow during G 1 , which promotes cell cycle entry in response to increasing cell size [55,56].It is not yet known what role Rb dilution plays under conditions of excess cell growth that lead to senescence.The p53/p21 pathway is activated by diverse cellular stresses, including DNA damage, hypoxia, oxidative stress, aneuploidy, ribosomal stress, and others [57].In unperturbed conditions, p53 is constitutively turned over by the ubiquitin ligase MDM2.Disruption of this turnover results in p53 stabilization and facilitates its transcriptional activity [58].One of p53's main transcriptional targets is p21, which-like p16-is a Cdk inhibitor.Unlike p16, p21 can inactivate Cdk1/2 in addition to Cdk4/6, leading to cell cycle arrest in G 1 or G 2 [59][60][61][62].Depending upon the stimulus, p21 expression can lead to temporary cell cycle arrest (quiescence) [63], DNA damage repair, apoptosis, or permanent cell cycle arrest (senescence) [64].Nonetheless, p21 is frequently considered a marker for senescent cells [12].P53 activation also triggers the formation of the DREAM complex, which represses the expression of genes involved in cell cycle progression [65].
Triggers of replicative senescence and genotoxic stress-induced senescence activate DNA-damage sensing pathways that converge upon p53/p21 activation to drive cell cycle arrest [28,36,66,67].In contrast, mitochondrial dysfunction-associated senescence can induce both p16 as well as p53 [46,68,69].OIS is also regulated by either p53/p21 or p16/Rb depending on context: OIS can enforce unscheduled replication, resulting in the activation of DNA damage sensing pathways [38,39] that converge upon p53/p21 [67].However, in some cases (e.g., RAS hyperactivation), oncogene activation also triggers p16 expression [31].Thus, despite diverse triggers, nearly all forms of senescence rely on either (or often both) the p53/p21 pathway or the p16/Rb pathway.Given the emerging evidence for cell growth-induced senescence, an important question is how cells sense that they have accumulated excess biomass and how this in turn activates cell cycle arrest signaling.
Although it has previously been observed that cell cycle withdrawal following a prolonged, Cdk4/6 inhibitor-mediated cell cycle arrest depends on the p53/p21 pathway [70,71], recent studies demonstrate that this is a consequence of increased cell size.Enlarged primary fibroblasts, hTERT-RPE1, MCF7, and MCF10A cells upregulate p21 [21,22,26,71] in response to a prolonged G 1 arrest that sustains continuous cell growth.Because p21 upregulation is blocked when cell growth is restricted during a prolonged G 1 arrest [21,22], this is a response to excess cell size.Conversely, unbiased approaches have also revealed that upregulation of p53/p21 is associated with enlarged phenotypes [72].
P21 expression in enlarged pre-senescent cells occurs prior to S-phase entry and therefore is not a consequence of replication.Moreover, enlarged G 1 cells harbor no signs of basal DNA damage, and p21 levels cannot be lowered by blocking DNA damage signaling pathways [21].Thus, the upregulation of p21 observed in enlarged cells is not a consequence of DNA damage.Still, it is unclear how cell size sensing machinery converges upon the p53/p21 pathway.
A recent study [22] suggests that excess cell size triggers an osmotic stress response that drives p53/p21 signaling through p38 MAPK.p38 has been implicated in p53-mediated p21 induction in varied circumstances, including loss of centrosome integrity [73,74], downregulation of PLK4 [75], DNA double-stranded breaks [76], Aurora B inhibition [77], and others.Moreover, p38 activity directly modulates p21 expression [77,78].In enlarged cells, p38 inhibition reduces p21 levels.Consistent with this, p38 inhibition accelerates cell cycle entry upon G 1 arrest release.Still, the requirement for p38 in p21 induction is not limited to G 1 : p38 inhibition also prevents p21 expression after enlarged cells have passed through the S-phase, where enlarged cells accumulate high levels of replicationacquired DNA damage [21].Thus, whether the signaling that activates p38 in different cell cycle stages is the same or whether these results reflect a more general requirement for p38 to induce p21 expression is unclear.
Finally, excess cell size is associated with p16 upregulation in some cell lines, but not others.Recent studies show that primary human lung fibroblasts accumulate p16 as they become larger [26], but hTERT-RPE1 cells do not [21,26,79].Nonetheless, proteomic measurements reveal that p16 superscales with respect to size across a cancer cell line panel [79].Thus, p16 dynamics in response to excess cell size may vary based on cell type and context.

Mitochondrial dysfunction in enlarged cells
Mitochondrial content increases with respect to cell size [80][81][82].Indeed, mitochondrial DNA content has been shown to scale with cell volume rather than nuclear genome content in yeast [83,84], demonstrating an intrinsic relationship between cell size and mitochondrial abundance.Still, the scaling of mitochondrial content does not guarantee mitochondrial function.Indeed, mitochondrial membrane potential and oxidative phosphorylation become compromised both in too-small and too-large cells [80].Based on this observation, excess cell size is predicted to severely compromise mitochondrial function.Consistent with this prediction, prolonged Cdk4/6 inhibition increases mitochondrial mass [81,85,86] but also causes an accumulation of mitochondria-derived ROS [85,87,88], suggesting mitochondrial dysfunction.Moreover, excessively large cells upregulate SOD2-which neutralizes superoxide species-suggesting an increased need to neutralize ROS [21,71].Because ROS can activate p53/p21 signaling [89,90], an intriguing hypothesis is that mitochondrial dysfunction contributes to senescence induction in excessively large cells.Still, recent work from Crozier et al. [22] demonstrates that increased antioxidant capacity does not lower p21 levels in enlarged cells.This finding makes it unlikely that high levels of ROS alone account for the p21 upregulation observed in enlarged cells but does not rule out the possibility that other consequences of mitochondrial dysfunction may drive senescence induction in enlarged cells (e.g., imbalances in the NAD + / NADH ratio or altered ATP concentrations).

Dysregulated ribosome biogenesis in enlarged cells
Although ribosome components scale with cell size within a normal physiological range, they are diluted at excessive cell sizes in yeast [19].Recent evidence suggests that this is not a consequence of active stress response signaling [91], and thus it is possible that the DNA template encoding ribosomal RNA becomes limiting when cells are too large.Similarly, proteomic analysis of enlarged mammalian cells reveals a failure to scale protein biosynthetic machinery [26].Consistent with this, enlarged human cells have decreased cytoplasmic density, suggesting decreased ribosome concentrations [19,92].Increased cell size is also associated with decreased nucleolar size in C. elegans embryos [93].Since the nucleolus is the site of ribosome biogenesis, this could also suggest changes in ribosome synthesis and assembly.Because nucleolar stress is a conserved hallmark of senescence between yeast and humans [94][95][96], an intriguing possibility is that sizeassociated alterations in ribosome biogenesis trigger a stress response that contributes to size-associated cellular senescence.
Disruption of ribosome biogenesis activates p53 signaling and can trigger senescence [97].This is thought to occur when unincorporated ribosomal proteins (r-proteins) titrate the interaction between p53 and Mdm2.In this manner, high levels of unincorporated ribosomes stabilize p53 and lead to subsequent p21 transcription.At least 13 ribosomal proteins and several other accessory factors have been implicated in activating p53 [98].Some senescence-associated ribosome biogenesis defects have also been reported to trigger cell cycle arrest through the Rb pathway and not p53 [99], which may explain how ribosome biogenesis stress causes senescence induction in p53-null cells.One hypothesis is that p53-dependent cell cycle arrest signaling in enlarged cells is a downstream consequence of dysregulated ribosome biogenesis.This could occur if rDNA becomes limiting for rRNA transcription, leading to the accumulation of unassembled r-proteins.Further work is required to understand whether excess cell size compromises ribosome biogenesis by this or other mechanisms.
Still, large hematopoietic stem cells (HSCs) show no evidence of ribosome biogenesis defects but nonetheless lose fitness as a consequence of increased size [25].Because the downregulation of ribosome synthesis is a broad cellular response to stress [100,101], it is also possible that disrupted ribosome biogenesis is instead a consequence of another yet-to-be-identified senescence-inducing stimulus in some cells (e.g., fibroblasts [26]) but not others (e.g., HSCs [25]).Thus, the question of how alterations in ribosome biogenesis affect the long-term fitness of enlarged cells is an important topic for future research.

Cell cycle progression defects in enlarged cells
Although earlier work has shown that enlarged cells undergo long-term proliferative failure [9, 19,53], the mechanism by which this occurs has not become clear until recently.Several recent publications demonstrate that there are three routes by which enlarged cells become senescent following a prolonged G 1 cell cycle arrest: (1) failing to re-enter the cell cycle at all, (2) re-entering the cell cycle but undergoing "G 2 slippage" (i.e., exiting G 2 into G 1 without undergoing mitosis), and (3) re-entering the cell cycle but undergoing mitotic failure (Fig. 1B) [21,22,24,71].The fraction of enlarged cells that fail to re-enter the cell cycle is eliminated when p53-dependent p21 expression is blocked [21,22,71], demonstrating that the cell cycle entry defects observed in enlarged cells arise from activation of the p53/p21 pathway.In contrast, cells that are maintained at physiological size during the G 1 arrest re-enter the cell cycle and proliferate normally regardless of p53 status [21].
The G 2 slippage and mitotic failures that are observed in enlarged cells are characteristic phenotypes of cells that acquired DNA damage from replication and have failed to repair it during G 2 [21,71,102].Consistent with this, cells that become large during a prolonged G 1 arrest accumulate replication-acquired DNA damage whereas cells that are maintained at physiological size do not.Previous work has suggested that replication stress following a prolonged, Cdk4/6 inhibitor-mediated arrest is a consequence of origin under-licensing since proteomic data revealed that G 1 arrested cells downregulate replisome components over time [71].Still, a more recent study of DNA replication dynamics using DNA fiber analysis did not reveal evidence of decreased origin firing in enlarged cells [21].In contrast, cells released from a prolonged G 1 arrest have reduced replication fork speed, which is typically associated with increased origin firing [103,104].Although physiologically-sized cells released from a G 1 arrest still encounter replication stress, this phenotype is more pronounced in enlarged cells [21,22].Interestingly, supplementing enlarged cells with additional nucleosides rescues fork progression, suggesting that size-associated replication stress may be due to an underlying metabolic problem [22].
It is not clear whether the observed differences in replication fork progression can account for the discrepancy in outcomes between enlarged and physiologically-sized cells released from G 1 , the former of which go on to obtain high levels of damage and undergo concomitant cell division failure [21].Thus, the cause of replication stress following a prolonged G 1 arrest and why it only leads to subsequent cell cycle progression defects in enlarged cells are still open questions.
The observation that enlarged cells acquire damage from replication stress whereas physiologically-sized cells are largely unscathed suggests that enlarged cells have additional defects that sensitize them to replication stress.Enlarged cells possess defects in DNA damage signaling pathways [21] (discussed in detail below), which likely contribute to this.Still, there may be other contributing factors that drive the high levels of damage observed in enlarged cells after replication.An interesting possibility is that enlarged cells-which upregulate gene expression to account for their increased size-are subject to more replicationtranscription conflicts, which may result in higher levels of DNA damage.This and other potential contributors to enlarged cells' propensity for replicationacquired damage are important topics for future investigation.

DNA damage signaling defects in enlarged cells
In addition to being highly sensitive to replication stress, enlarged cells are also hypersensitive to exogenous DNA damage.Recent work demonstrates that enlarged G 1 cells acquire more damage from genotoxins compared to physiologically-sized cells [21,26] and fail to clear damage to the same extent [21].Moreover, recent proteomic analyses of size-stratified cells revealed that enlarged cells are depleted in DNA repair factors relative to smaller cells [26,72].Together, these observations suggest that enlarged cells bear defects in DNA damage-responsive pathways.
Non-homologous end joining (NHEJ) is a major DSB repair pathway in mammalian cells, and it is strongly favored for DSB repair during G 1 [105].The initiation of NHEJ involves the formation of 53BP1 foci at DNA damage sites, which serve as adaptors for downstream signaling and suppress alternative repair pathways [106].Recent studies in enlarged G 1 cells revealed that excess cell size prevents the efficient formation of 53BP1 foci at DNA damage sites induced by doxorubicin.This correlates with a failure to recruit downstream factors, indicating a significant defect in the initiation of NHEJ [21].
53BP1 foci are phase-separated entities [107,108], and their formation is highly dependent on the biophysical properties of the nucleoplasm [108,109], which are generally robust to perturbation [110].Because excess cell size causes nuclear dilution [19,111,112], an interesting possibility is that excess cell size precludes the biophysical requirements for 53BP1 foci formation.Thus, the impact of cell size on other phase-separated compartments-including others involved in DNA damage repair [113,114]-should be examined further.
Enlarged G 1 cells also fail to efficiently stabilize p53 in response to DNA damage to the same extent as G 1 cells that were maintained close to physiological size.This correlates with a failure to upregulate p21 in response to DNA damage, indicating a defect in the expression of one of p53's main transcriptional targets.Enlarged and physiologically-sized G 1 cells stabilize p53 to the same extent upon nutlin3a treatment (which induces p53 expression without causing DNA damage), suggesting that the p53 induction in response to DNA damage is compromised in enlarged cells [21].Because 53BP1 foci formation is a requirement for p53 induction, the 53BP1 foci formation defects observed in enlarged G 1 cells may explain large cells' failure to robustly upregulate p53 in response to DNA damage [108,109,115], but this hypothesis has yet to be tested further.The failure to activate p53 in response to replication stress may also lead to nucleotide exhaustion and reduced replication fork speed [116], providing a potential explanation for replication stress associated with increased cell size [21,22].
The recent finding that enlarged cells fail to robustly signal through DNA damage-responsive pathways may explain the long-standing observation that stable cellular senescence is associated with signs of persistent DNA damage [117][118][119][120]. Indeed, cells that have entered replicative senescence have been reported to maintain cH2AX foci for months to years following senescence induction [117].Similarly, aged HSCs (which are larger than their younger counterparts [25]) have been reported to show signs of persistent DNA damage [121,122] and restricted DNA damage repair [122].Thus, the observation that excess cell size hampers the DNA damage response may unify these observations and provide an explanation for why aged and senescent cells fail to efficiently clear DNA damage.
Finally, the discovery that excessive cell size impedes DNA damage signaling raises new questions about what other DNA-dependent signaling pathways may be impacted by increased cell volume.Given that DNA does not scale with increased size during G 1 [19], an interesting possibility is that excess cell volume dilutes signals that originate from the genome itself.Indeed, this has also been observed in the context of the spindle assembly checkpoint (SAC)-which generates signals from DNA-bound kinetochores-during embryogenesis in C. elegans: the strength of the SAC increases with decreasing cell volume [123].Together, these results suggest that a defined DNA:cytoplasm ratio may play an important role in maintaining diverse DNA-dependent signaling pathways.

Clinical use of drugs that cause cell cycle arrest while sustaining cell growth
Many cell cycle arrest-causing drugs that are either currently used for cancer therapies or are in clinical trials (e.g., doxorubicin, Cdk4/6 inhibitors, Cdk7 inhibitors, etoposide) have been reported to drastically increase cell size in vitro [19,20,[22][23][24]81,124].Still, this is not true in all cultured cell lines: many blood cancers are reported to maintain their size in response to the Cdk4/6 inhibitor palbociclib [21,81].A list of human cell lines where size has been evaluated upon multi-day Cdk4/6 inhibition is included in Table 1.Some cells' failure to gain excess biomass following Cdk4/6 inhibition may be due to cell-type specific crosstalk between Cdk4/6 and mTOR: Cdk4/6 inhibitors can block mTORC1 activity via TSC2 in some cell lines but not others [125].Consistent with the hypothesis that increased cell size is the determinant of senescence induction following prolonged Cdk4/6 inhibition, at least one blood cancer cell line (NALM6) that fails to grow large during palbociclib treatment resumes normal proliferation following drug removal [21].Together, these findings raise an important question about cell cycle arrest-causing drugs: do changes in cell size contribute to their efficacy in vivo?
Recent studies demonstrate that the suppression of biomass accumulation (e.g., by mTOR inhibition, contact inhibition, or serum starvation [19,21,22,26,81,126]) in combination with drugs that cause cell cycle arrest rescues long-term proliferation in vitro.This has been demonstrated in the context of palbociclib [19,21,22,24,25], the MDM2 inhibitor nutlin3a [21,127], etoposide [52], and doxorubicin [52].An essential prerequisite for understanding the in vivo mechanisms of these drugs is to identify whether treated cancer cells are able to undergo unchecked biomass accumulation in the tumor microenvironment, or whether signaling cues (either in cis or in trans) limit their growth.Although growth in a tissue context is often regulated by contact inhibition [128], mutations that hyperactivate growth pathways are frequently found in tumors [23,24], and it has long been known that loss of contact inhibition drives metastasis [129][130][131].On the other hand, mutations that overcome contact inhibition could sensitize cells to cell cycle arrest-causing drugs.Such mutations would allow senescence-driving overgrowth in tumor cells, whereas surrounding healthy cells could restrict their growth and preserve their proliferative potential (Fig. 2).Consistent with this, recent studies demonstrate that oncogenic mutations that promote excess cell growth render cells more sensitive to palbociclib and the Cdk7 inhibitor ICEC0942 [23,24].Moreover, constitutive activation of mTOR also sensitizes ER + breast cancer cells to palbociclib-induced senescence [132].Still, it is not yet clear whether other factors limit biomass accumulation in cancer cells within an organism.Thus, extending the in vitro cytotoxic mechanisms of cell cycle arrest-causing drugs to an in vivo setting requires a thorough understanding of how cell size dynamics change within a tumor upon drug treatment.
To complicate matters further, some studies report that the combination of drugs that cause persistent cell cycle arrest with drugs that restrict cell growth can be more effective than the former alone in some cancer contexts.Indeed, recent work demonstrates the synergy between PI3K/AK5/mTOR inhibitors and doxorubicin in eliminating leiomyosarcoma [133], hepatocellular and renal cell carcinomas [134,135], and others [136].Similarly, using Cdk4/6 inhibitors in combination with mTOR inhibition reduced cell proliferation in xenograft models of anaplastic thyroid cancer [137] and ER + breast cancer [138].Thus, the effect of increased cell size on cell cycle withdrawal in dysregulated cancer environments requires further investigation and may be highly context-dependent.

Outlook and future considerations
In this review, we discuss several recent publications that implicate cellular enlargement as a cause of permanent cell cycle exit.Based on the evidence presented in these works, we propose that cellular enlargement is a well-defined cause (in addition to consequence) of cellular senescence.This revelation provides insight into a long-standing, fundamental question in cell biology: why is senescence permanent?The recent findings described in this review suggest that senescence may arise when a temporary insult causes G 1 arrest, but it is able to persist due to the accumulation of excess biomass during the arrest.
Although recent research has uncovered several essential cellular processes that become compromised as a function of excess size, there are still many mechanistic questions remaining regarding how and if these defects drive permanent cell cycle exit.For example, though it has recently been established that enlarged G 1 cells possess NHEJ signaling defects, it is not yet clear if these defects persist later in the cell cycle and contribute to enlarged cells' failure to repair replication stress-acquired damage.Moreover, though the cell cycle arrest signaling pathways that become activated in response to excess cell size have been identified, it is still not known what upstream stimuli trigger these pathways.Thus, the question of how cells sense their size and how this in turn translates to cell cycle arrest signaling remains an exciting topic for ongoing research.
Despite recent evidence of size-induced senescence in some cell types, it is an open question why others can grow to considerable sizes in a physiological context without compromising cellular fitness.In agreement with the hypothesis that maintaining a particular DNA:cytoplasm ratio is essential for cellular fitness, some naturally large cell types (e.g.hepatocytes), are polyploid.Because increased ploidy rescues biosynthetic capacity when cells become enlarged, this likely serves as a mechanism for preserving cellular fitness in naturally large cell types [139].Another strategy employed by oocytes-which are also naturally largeinvolves obtaining organelles and cytoplasm from surrounding cells [140].Nonetheless, how other naturally enlarged cell types (e.g., neurons) retain cellular function remains an exciting topic for future research.
The findings summarized here also raise an important consideration for researchers studying cell cycle withdrawal in other contexts: whether treatments that cause cell cycle exit do so as an indirect consequence of altering cell size.Indeed, Cdk4/6 inhibitors have long been known to induce cellular senescence; however, it has only recently become clear that this is a consequence of their effect on cell volume.Restricting cell growth rescues proliferative failures in the context of DNA damaging agents [52,124], Cdk7 inhibitors [23], and nutlin-3a [21,127] suggesting that excess size accumulation may be a broadly applicable mechanism for driving permanent cell cycle arrest.Thus, cell size is an essential parameter for researchers in diverse areas to consider when diagnosing cell cycle exit mechanisms.

Fig. 1 .
Fig. 1. (A) Model for how long-term G 1 cell cycle arrests may lead to senescence or quiescence as a function of biomass accumulation during the arrest.(B) Depiction of cell cycle progression in cells that were maintained at physiological size (top) or allowed to grow large (bottom) during a prolonged G 1 cell cycle arrest.

Fig. 2 .
Fig. 2. Illustration for how Cdk4/6 inhibitors may lead to tumor cell senescence as a product of increased cell size during treatment.

Table 1 .
Effect of Cdk4/6 inhibition on cell size in established human cell lines.Collection of commonly used cell lines whose size has been assessed following Cdk4/6 inhibition.