Keywords

Introduction

Since the discovery of A-type and B-type lamins as components of the nuclear lamina [1], they have been the subject of intense scrutiny regarding possible roles in a range of nuclear functions. The finding that they are targets for mutation in degenerative and progeroid diseases has further driven research in the area [2]. Partially overlapping research threads for nearly three decades have implicated lamins in the control of cell proliferation and differentiation, leading to speculation that lamins may have roles in cancer progression. One obvious connection between lamin function and cell cycle progression comes from the fact that the nuclear envelope breaks down during mitosis in mammalian cells, leading to a dissociation of the lamin intermediate filament structure that exists between the chromatin and the envelope [3]. Upon reformation of the nucleus after mitosis, the lamina also reassembles, and numerous studies have been performed to define a role for A- and B-type lamins in this process.

A full description of A- and B-type lamins is provided in other reviews. Here we provide basic facts relevant to lamin roles in cell cycle regulation and aging. All A-type lamins (lamins A and C in most settings) are encoded by the LMNA gene, which is targeted for mutation in a wide range of pathologies [2]. Among these, forms of dilated cardiomyopathy and muscular dystrophy are generally associated with reduced A-type lamin function and can be phenocopied by knockout of the LMNA locus in the mouse [4]. In contrast, dominant gain-of-function or neomorphic mutations in LMNA can lead to progeroid syndromes [5–7]. The most common of these is Hutchinson–Gilford Progeria syndrome (HGPS), which is most often associated with the LMNA G608G mutation, which is silent with respect to coding sequence but activates a cryptic splice site leading to the production of progerin, a variant of lamin A that lacks 50 amino acids in the C-terminus [6, 7]. Whether progerias are mechanistically linked to normal aging has been an ongoing debate in the aging research field for decades with no consensus yet emerging [8, 9]. Interestingly, however, alternative splicing of LMNA can occur in normal cells leading to low-level production of progerin, and recent studies have demonstrated progerin accumulation with organismal age or with increasing passage in cell culture [10–13]. These findings at least raise the possibility that progerin may in part promote the normal aging process.

B-type lamins, encoded by LMNB1 and LMNB2, are also linked to disease states and have been implicated in cell cycle progression. Interestingly, recent studies indicate that lamin B expression may be altered as cells approach senescence [14], a topic discussed in Sects. 3 and 4. One major difference between A- and B-type lamins is their expression patterns. While B-type lamins are expressed in all cell types, A-type lamins are regulated during development and differentiation. In a mouse, for instance, A-type lamin expression is not evident until mid-gestation, when it can be detected in cells committing to different lineages [15]. This finding has led to the possibility that A-type lamins are cell commitment factors, being expressed when cells adopt certain fates and perhaps ensuring gene expression programs that define those fates. A-type lamins are also not expressed in stem cells (or at least at very low levels), a fact made particularly evident in studies aimed to generate induced pluripotent stem cells from fibroblasts of HGPS patients [16–18]. These fibroblasts have proliferation defects and altered nuclear shape but can be induced to become stem cells. Upon this transition, the cells lose A-type lamin expression (including that of progerin) and no longer display proliferation or nuclear shape abnormalities. When induced to differentiate again, they resume progerin expression and regain abnormal behavior.

A deeper role for lamins in cell cycle regulation was proposed when the discovery was made that lamin tethering of many cell cycle regulators, including c-Myc and retinoblastoma protein (RB), was important for their function [19–23]. This has stimulated investigation by many laboratories into the role of lamins in coordinating the transit through the G1 phase of the cell cycle. Lamins have also been linked to the control of DNA replication (below) and checkpoint pathways, including those involved in repair of DNA lesions [24].

If lamins control cell cycle progression and exit, then their function might be impaired during cancer progression, either through direct mutation or through other events during cancer expression that affect their activity. This theory is augmented by findings in several tumors that nuclear shape and organization are commonly altered in cancer cell lines [25, 26]. Many investigators have examined the possibility of lamin impairment in cancer progression, and, particularly with regard to A-type lamins, expression patterns often change in cancer although few mutations have been identified. This finding has led to the possibility that lamins may be used as biomarkers for cancer progression, and in many different tumor types the expression of A-type lamins or lamin B1 changes during different stages of tumor development. However, there are no clear generalizations to be made, with expression pattern changes often complex and specific to tumor type [27].

In this review, we focus on the role of A-type lamins in cell cycle progression and exit, discussing how these specific functions may relate to both cancer and aging. The answers to these questions remain unresolved, but a number of tantalizing findings have been reported in the last few years that may finally lead to the primary functions of lamins in the nucleus, as well as how they relate to disease progression, both those of a dystrophic and hyper-proliferative nature.

Cell Proliferation

The G1-to-S phase transition of the mammalian cell cycle is tightly regulated in normal cells and a primary target for dysregulation in cancer. Many of the regulatory factors have been associated with the nuclear matrix. Rather than cover a compendium of these factors, which in many cases interact with nuclear lamins, the focus of this section will be to define the phenotypic roles of nuclear lamins in cell proliferation and, wherever possible, to connect these roles to regulation of proliferative factors. In general, proliferative effects of LMNA mutations will be separated into those associated with reduced A-type lamin function and those associated with the expression of progeria alleles, which are either hypermorphs or neomorphs.

Lmna −/− mice develop skeletal muscle dystrophy and dilated cardiomyopathy [4], succumbing between 6 and 8 weeks of age likely due to cardiac conduction defects [28]. To clarify, recent findings suggest that Lmna −/− mice may not be true nulls for the LMNA locus, as a truncated allele of lamin A appears to be expressed in these mice [29]. The most likely scenario is that this mouse is actually a hypomorph, a theory supported by the phenotype of another Lmna disruption in a mouse that results in lethality before weaning [30] and the one known case of a homozygous nonsense mutation of LMNA identified in human patient who died shortly after birth [31]. For purposes of clarity and consistency, the term Lmna −/− will still be applied to the original mouse generated by Sullivan et al. [4].

While speculation about reduced A-type lamin expression and cancer has a long history, none of the mouse models or human patients with LMNA mutations linked to striated and cardiac muscle have been associated with oncogenesis. However, there is strong evidence that A-type lamins regulate key factors involved in controlling the G1-to-S transition, and the most evidence exists for lamin A effects on the RB [32]. The connection between A-type lamins and RB has been examined in a variety of settings, and, while there may be differences, the general consensus is that A-type lamins are required for normal RB function.

One of the first tumor suppressors identified, loss of both copies of the RB gene leads to a range of different cancers including retinoblastomas and osteosarcomas [33]. More broadly, RB activity is deregulated in a wide range of tumors, generally through unchecked activity of cyclin-dependent kinases (CDKs) [34]. RB has myriad binding partners and has been ascribed to a number of functions in the nucleus [35]. Most notably, RB acts as a repressor of the transcriptional factor E2F, which controls a range of genes important for entry into S phase of the cell cycle. When hypophosphorylated and active, RB binds to E2F complexes and acts as a repressor of S-phase genes, retaining cells in G1. CDK-dependent phosphorylation promotes release of RB from E2F and cell cycle progression. Along with p53 (discussed below) and telomere regulation, the RB pathway is a major determinant of cell senescence and also plays a role in cell differentiation in multiple lineages. Finally, control of G1-dependent gene expression is but one function ascribed to RB, which is also linked to DNA replication, mitosis, and checkpoint pathways, including those initiated by DNA damage where its roles are still being fully defined [34].

Both A-type lamins and their binding partner, lamina-associated polypeptide 2α (LAP2α), have been reported to interact with RB [21, 36], repress E2F-dependent transcription, and promote cell cycle arrest [37]. Consistently, loss of LAP2α or lamin A/C impairs normal cell cycle regulation leading to inappropriate S-phase entry. Mouse cells lacking A-type lamins or LAP2α have altered cell cycle profiles with premature S-phase entry and in some contexts enhanced proliferation [38–40]. In contrast, one study in human primary fibroblasts indicated that reduced lamin A/C or LAP2α expression led to cell cycle arrest [41]. The reason(s) for these different observations remains unknown.

In addition to promoting RB-dependent transcriptional repression of E2F target genes, A-type lamins regulate RB by at least three other mechanisms by coordination of RB phosphorylation, localization, and protein stability [39, 42]. Some aspects of control of RB protein stability are beginning to be understood. For instance, in cells lacking lamin A/C, enhanced levels of RB degradation occur through a proteasome-dependent mechanism [39]. Reduced RB levels make Lmna −/− fibroblasts insensitive to p16INK4A-mediated cell cycle arrest [38]. However, the E3 ligase responsible for RB degradation remains to be identified and appears to be independent of the MDM2 and gankyrin pathways that have been linked to RB turnover in other contexts [43]. A number of other proteins are destabilized by loss of RB, including the RB-related protein p107 [39], emerin [44], and ATR kinase [45]. These findings raise the possibility that A-type lamins might coordinate nuclear proteasome function, and altered activity of ubiquitin ligase components has been detected in cells expressing mutant forms of lamin A [45].

Other A-type lamin functions may promote G1 maintenance. Serum stimulation of G1 arrested cells promotes ERK1/2 mitogen-activated protein kinase-dependent phosphorylation of c-Fos, leading to its association with c-Jun- and AP-1-dependent transcription [46]. Prior to stimulation, c-Fos and ERK1/2 were found to be in a complex with lamin A/C at the nuclear periphery preventing premature activation of AP-1, which occurred in Lmna −/− fibroblasts [47, 48]. Independent studies have reported enhanced ERK1/2 activity in cells with reduced A-type lamin expression, and this has been linked to cardiac pathology in dilated cardiomyopathy type 1A (DCM1A) laminopathy patients [49]. Interestingly, a more recent study indicates that ERK1/2–lamin A/C and RB–lamin A/C complexes are mutually exclusive and finds that ERK1/2-dependent lamin A/C binding upon serum stimulation displaces RB, thereby promoting cell cycle progression [50]. If ERK1/2 levels are elevated in cycling cells, this may lead to RB dysregulation and underlie some of the altered cell cycle parameters evident in Lmna −/− cells.

A number of reports have implicated lamins in regulation of DNA replication. For instance, early studies showed that disruption of the lamin structure impaired initiation of DNA synthesis [51–53]. In immortalized cells, lamin B was localized to intranuclear sites of late S-phase replication [54], whereas in primary fibroblasts, intranuclear A-type lamins associate with initial sites of DNA synthesis upon S-phase entry [55]. The impact of lamins on S-phase progression in mammalian cells is less clear. S phase is elongated in fibroblasts lacking A-type lamins, although this could be an indirect effect of premature S-phase entry due to defective RB function [39]. A recent study has shed light on a different aspect of replication by comparing the response of Lmna −/− cells to forms of DNA damage-induced cell cycle arrest, finding that lamin A/C was required for restart of stalled replication forks and genome maintenance after hydroxyurea-induced replication stress [56]. While the roles of A-type and B-type lamins in DNA replication remain to be fully elaborated, this is clearly an important area for further studies.

Mitotic defects have not been reported for cells with reduced A-type lamin function. However, a recently identified novel allele of LMNA, Lmna DHE, was identified as a spontaneous mouse mutation with a subset of progeroid phenotypes [57]. Fibroblasts heterozygous for this Lmna allele exhibit, in addition to reduced levels of hypophosphorylated RB, a reduction in a mitosis-specific centromere condensing subunit that depends on RB activity [58]. These alterations result in a range of chromosome segregation defects. It will be of interest to determine whether other Lmna disease-associated alleles lead to similar defects.

Cell Senescence: A-Type Lamins

Both A-type and B-type lamins have been linked to cell senescence, the process by which primary cells withdraw from the cell cycle in response to extended passaging, irreparable damage, or unbalanced proliferative signals. Cell senescence has typically been viewed as an impedance to cancer progression and not a driving force in aging, but recent findings paint a more complex picture [59]. Senescent cells do accumulate with age, and while they generally never reach a large percentage of the population of a tissue, recent findings indicate that they adopt an altered secretory profile, the senescence-associated secretory phenotype (SASP), that leads to paracrine release of a number of inflammatory cytokines. These cytokines may promote tissue aging and stimulate tumor development in neighboring cells. In this section, we cover links between lamins and senescence, discussing the still tenuous connections between lamins and normal aging process.

Several studies have implicated A-type lamins in cell senescence, although the phenotype is most clearly associated with the expression of progeria-associated LMNA alleles such as progerin [9]. Progerin expression, in addition to delaying cell cycle progression, brings about premature senescence in a variety of contexts [60, 61]. Lamin A is normally farnesylated at its C-terminus but only for a short time because the last 18 amino acid residues are removed in two cleavage steps. The protease that removes the farnesyl group is Zmpste24 in mice (FACE-1 in humans). In HGPS the deleted exon also removes this cleavage site so that the progerin form of lamin A is permanently farnesylated. Zmpste24 −/− cells with defective lamin A processing also exhibit enhanced levels of senescence [62, 63]. The mechanisms behind these effects remain unclear. As cells approach senescence the p16INK4A/RB and p53 pathways both become engaged, leading cells to stop proliferation and enter a permanently arrested state [59]. In addition, telomere attrition during passaging in culture drives senescence, particularly as telomere ends shorten beyond critical thresholds, invoking DNA damage checkpoint response pathways. All three of these networks (p16INK4A/RB, p53, and telomeres) are interrelated, and progerin has been linked functionally to each.

The connection between progerin expression and RB pathway is not entirely understood presently. In one study, where LMNA–progerin alleles were expressed in a lamin A/C-deficient background, it was found that progerin restored RB stability [38]. Moreover, inactivation of the RB pathway by expression of HPV E7 failed to suppress the proliferation defects of human fibroblasts stably expressing progerin [64]. However, an analysis of global gene expression profiles in fibroblasts from HGPS patients identified the RB-E2F pathway as dysregulated [65] due in part to RB gene expression. The mechanisms underlying this effect were unknown. Interestingly, exposure of cells to farnesyl transferase inhibitors mostly restored the normal gene expression profile.

It is generally thought that LMNA mutations are not associated with tumors; however, two instances have been reported in progeria models. In one case, an osteosarcoma was identified in an HGPS patient [66, 67]. This is intriguing since osteosarcomas are commonly associated with RB mutations [68]. Interestingly, this patient expressed a smaller 35 amino acid C-terminal deletion in the C-terminus of LMNA and not progerin [66, 67]. It would be interesting to determine the levels of RB and activity of the RB pathway in this context. One issue possibly limiting cancer progression in progeria patients is the early progression of the disease leading to mortality for patients usually in their teens. A recent study has identified a novel late-onset progeria syndrome, LMNA-associated cardiocutaneous progeria, that is associated with possible cancer susceptibility [69]. This syndrome is associated with a heterozygous novel mutation in the lamin A/C coiled-coil domain that is largely uncharacterized.

The RB pathway is not the only cell cycle regulatory network that is influenced by A-type lamins. Whereas inactivation of RB did not rescue proliferation defects and premature senescence in human fibroblasts stably expressing progerin, either inactivation of p53 (by HPV E7) or expression of telomerase did [64]. Several studies have connected LMNA mutation to p53 engagement due to enhanced DNA damage [24], but a recent study has elaborated this connection further. In this case, depletion of lamin A/C in primary human fibroblasts led to dramatic destabilization of RB as expected; however, the cells also had proliferation defects and a senescent phenotype instead of the expected short G1 phase due to enhanced specific activation of the p53–p21 axis [70]. p53 did not display enhanced levels or activating phosphorylation, and many targets were not upregulated. Instead, a subset of targets including p21 were upregulated leading to repression of E2F targets even in the absence of normal levels of RB. Cross talk between the RB and p53 pathways is not unprecedented. Moreover, these findings suggest that the A-type lamins interact with both pathways in a nuanced manner and whether LMNA mutations lead to altered proliferation with early G1 cell cycle exit or reduced proliferation leading to senescence may depend on the specific nature of the LMNA mutation.

Another interesting recent finding has connected lamins to the p53 pathway in a different manner. In renal carcinoma cells, where genetic inactivation of the von Hippel–Lindau (VHL) gene E3 ligase is a frequent event, progerin expression may play a role in controlling the p53 pathway [71]. p53 is not generally mutated in these tumors but is inactivated functionally. Jung et al. found that progerin was a target of VHL-mediated proteasomal degradation. Loss of VHL led to stabilization of progerin, which was otherwise bound to p14/ARF, sequestering it from MDM2 and leading in turn to p53 degradation. Thus, progerin was required for p53 inactivation in the absence of VHL. If progerin has this activity in a wide array of cell types, this finding may provide a potential link between aging and cancer:

Accumulation of progerin with aging would lead to inactivation of the p53 network, impairing its tumor suppressive and checkpoint activities. More research is needed to test this intriguing hypothesis.

Recent studies have also pointed to a role for lamins in the maintenance of telomere metabolism, another activity that could be closely linked to cell senescence [72]. For instance, HGPS fibroblasts are reported to have faster rates of telomere attrition [73]. This finding does not on its own suggest a direct role for A-type lamins at telomeres; however, a number of studies have reported that telomeres associate with the nuclear matrix and more specifically with A-type lamins [74–77]. Lmna −/− fibroblasts have shorter telomeres but no differences in telomerase activity [78]. Instead, the answer may be related to altered chromatin structure at telomeres and trace back to the reduced function of RB and its related proteins p107 and p130 [72]. Cells lacking A-type lamins exhibit a decrease in histone H4K20me3 [78], a known feature of cells lacking RB family members [79, 80]. However, the latter cells have increased telomere length, leading the authors of the lamin study to suggest that A-type lamins might be required for telomere elongation in the absence of RB family members [81, 82]. A relocalization of telomeres from peripheral to central regions of the nucleus has also been reported in Lmna −/− cells, through at present unknown mechanisms [83].

HGPS fibroblasts have altered telomere chromatin as well, although the changes are distinct from those in Lmna −/− cells [83, 84]. In this case, decreased H4K20me3 and increased H4K20me were found. This finding suggests that, not unexpectedly, progerin influences telomere metabolism in a manner distinct from hypomorphic mutation of LMNA. Recently, it was reported that progerin-induced DNA damage is localized specifically to telomeres [85]. Expression of telomerase resolves this DNA damage, and, once repaired, HGPS fibroblasts regain full potential to proliferate. It will be intriguing to see how DNA damage is restricted in the genome by progerin.

Finally, the proliferative defects leading to senescence with expression of progerin and/or unprocessed lamin A appear to extend to adult stem cell populations. Studies in mesenchymal stem cells have indicated that progerin expression leads to elevated Notch signaling, causing perturbations in stem cell differentiation and maintenance of stem cell identity [13]. In addition, adult bone marrow-derived stem cells from Zmpste24 −/− mice have decreased SIRT1 function due to its dissociation from the nuclear matrix and leading to reduced proliferation and premature senescence [86]. This phenotype may be relevant for aging as restoration of SIRT1 function was associated with improved stem cell function and enhanced survival. Studies in fibroblasts and other cell culture models have provided important insights into lamin function; however, more emphasis needs to be placed on the role of lamins in adult stem cell populations, which could be central to a subset of the pathologies associated with laminopathies.

Cell Senescence: B-Type Lamins

Lamin B1 has also been tightly associated with cell senescence, with altered expression in either direction possibly having deleterious consequences [87]. Initial suggestions of altered lamin B expression came from studies of HGPS cells, where lamin B1 was found to be reduced [87]. Two virtually contemporaneous recent reports have shown that loss of lamin B1 expression is a marker for cell senescence induced by a variety of causes, including replicative exhaustion. Loss of lamin B1 expression was not dependent on many molecular inducers of senescence but was driven by activation of either the p53 or the RB pathway [88]. Changes in lamin B1 could be traced back to reduced mRNA stability, although reduced protein stability could be detected as well in mouse liver induced to senescence by irradiation. In the second study, Shimi et al. also reported loss of lamin B1 as a much needed biomarker of cell senescence [14]. Shimi et al. also examined the consequences of RNAi-mediated knockdown of lamin B1 expression, finding that this was sufficient to both slow proliferation and reduce senescence. The proliferative delay was dependent on p53, and the senescent phenotype was dependent on both p53 and RB. Yet a third very recent study has confirmed and extended the observed reduction in lamin B1 expression to senescent keratinocytes and to chronologically aged human skin tissue [89]. However, in this study enforced reduction in lamin B1 expression failed to lead to senescence. The disparities between the two studies are not known [14, 89]. Together these findings (1) indicate that loss of lamin B1 may serve as an effective biomarker of in vivo senescence and (2) suggest the existence of a complex regulatory loop connecting B-type lamins to the RB and p53 pathways.

Whereas mutations affecting the LMNB1 coding sequence have not been reported, overexpression is linked to at least two diseases, suggesting that too much lamin B1 may be as deleterious as too little. Duplication of the LMNB1 locus results in adult-onset autosomal dominant leukodystrophy (ADLD) [90], and lamin B1 overexpression has been detected in lymphoblasts and fibroblasts from ataxia telangiectasia patients [91]. Interestingly, lamin B1 overexpression also drives cell senescence, a phenomenon also observed in ataxia telangiectasia cells, which are rescued by restoration of normal lamin B1 expression. Induction of senescence by overexpression of lamin B1 has been repeated in a second study [89]. In this case, the senescent phenotype could be rescued by expression of telomerase or inactivation of p53, paralleling observations for progerin-induced senescence [64]. Finally, senescence induced by overexpression of lamins is not restricted to B1. Increased levels of lamin A also reduce the replicative life-span of primary human fibroblasts [92].

Several studies indicate a connection between A-type and B-type lamins in the formation of intermediate filament networks, and there appears to be an interplay between the two nuclear intermediate filament families with respect to cell senescence. For instance, reduced expression of A-type lamins exacerbates the senescent phenotype of cells overexpressing lamin B1 [89]. In the context of senescence in normal cells, one thing that needs to be resolved is whether increased progerin levels and reduced lamin B1 expression are related. Does one family of nuclear intermediate filaments regulate the other during aging and senescence?

Whether reactive oxygen species (ROS) drive aspects of the aging process remains highly debated [93]. Both A- and B-type lamins have been linked to reactive oxygen production and sensing in recent years, and while the details remain murky, these findings represent another promising set of leads as the relationship between lamins and aging is elucidated [87]. There may be a direct connection as conserved cysteines in the C-terminal tail of lamin A have been found to be oxidized in senescent cells [94]. This led to the formation of intra- and intermolecular disulfide bonds and perturbation of the lamina. These cysteine residues may serve as a reservoir or a sensor for oxidation, as mutating the cysteines to alanine led to oxidative stress sensitivity and premature senescence. In the case of B-type lamins, a number of conflicting results have been reported. In some contexts, increased ROS has been reported to lead to elevated and reduced lamin B1 levels [14, 88, 91]. Similarly, both higher and lower levels of lamin B1 lead to reduced ROS levels [14]. Further studies with ROS and lamins will likely clarify this complex and potentially mechanistically rewarding relationship.

Conclusions

While nuclear lamins have been speculated to control cell cycle progression for decades, this area of research has exploded in recent years and many labs have investigated the effect of laminopathy-associated mutations in LMNA and diseases associated with LMNB1 overexpression. These studies have linked nuclear lamins to virtually every major aspect of cell cycle progression and, more recently, cell senescence. This latter connection may be particularly interesting given that LMNA mutations are associated with progeria as well as the findings that both progerin and lamin B1 have altered expression with normal aging.

However, several big questions remain to be resolved: Does altered lamin expression promote tumor progression? Are lamins important regulators of the aging process? With respect to cancer, an increasing number of studies have linked altered expression of both A-type and B-type to different tumors, but the relationships are complex and causal links are generally lacking. It is critical to resolve these issues in more detail to determine for which tumors lamins might be effective biomarkers and perhaps more importantly to understand why changes in lamin expression may promote tumorigenesis.

With respect to aging, the findings are certainly becoming more intriguing. That LMNA mutations cause HGPS is not sufficient to ascribe A-type lamins a role in normal aging. That progerin is expressed in aging and/or senescent cells is also not sufficient, but together the data certainly justify further analysis of lamin roles in the normal aging process. To provide convincing evidence, it will be necessary to manipulate A-type (or B-type) lamins in a manner that leads to enhanced organismal longevity. For instance, it would be informative to apply technologies developed to reduce progerin expression in HGPS models to wild-type mice to determine whether suppression of progerin in this context leads to longer life-span. Experiments such as these will begin to answer the critical questions surrounding aging and lamins.

Progress in understanding disease-relevant functions of lamins has escalated dramatically in recent years, and the next few years will without a doubt provide exciting new findings relevant to cancer and aging. Perhaps the most exciting aspect of the field is that researchers are beginning to understand the roles of lamins at the mechanistic level. Further progress on this front will likely yield effective therapeutic approaches for treatment of laminopathies and, importantly, an increasingly elegant understanding of the organization of the mammalian nucleus.