The Molecular Balancing Act of P16 in Cancer and Aging

Published OnlineFirst October 17, 2013; DOI: 10.1158/1541-7786.MCR-13-0350

Molecular Cancer Review Research

The Molecular Balancing Act of p16INK4a in Cancer and Aging

Kyle M. LaPak1 and Christin E. Burd1,2

Abstract p16INK4a, located on chromosome 9p21.3, is lost among a cluster of neighboring tumor suppressor genes. Although it is classically known for its capacity to inhibit cyclin-dependent kinase (CDK) activity, p16INK4a is not just a one-trick pony. Long-term p16INK4a expression pushes cells to enter senescence, an irreversible cell-cycle arrest that precludes the growth of would-be cancer cells but also contributes to cellular aging. Importantly, loss of p16INK4a is one of the most frequent events in human tumors and allows precancerous lesions to bypass senescence. Therefore, precise regulation of p16INK4a is essential to tissue homeostasis, maintaining a coordinated balance between tumor suppression and aging. This review outlines the molecular pathways critical for proper p16INK4a regulation and emphasizes the indispensable functions of p16INK4a in cancer, aging, and human physiology that make this gene special. Mol Cancer Res; 12(2); 1–17. 2013 AACR.

Introduction frames (ORF) to yield two distinct proteins: p16INK4a and ARF ARF Every day, we depend on our cells to make the right ARF (p14 in humans and p19 in mice). In addition to — CDKN2A,theINK4/ARF locus encodes a third tumor decision to divide or not to divide. Proliferation is essential INK4b for tissue homeostasis, but, when deregulated, it can both suppressor protein, p15 ,justupstreamoftheARF promoter (3). Discovered through homology-based cDNA promote cancer and lead to aging. For this reason, the INK4b INK4a decision to replicate is tightly controlled by a complex library screens, p15 functions analogously to p16 , – directly blocking the interaction of CDK4/6 with D-type network of cell-cycle regulatory proteins. In the early INK4b INK4a 1990s, it was clear that the catalytic activity of cyclin- cyclins (2, 3). In contrast to p15 and p16 ,which dependent kinases (CDKs) was required to drive cellular function to inhibit RB phosphorylation, ARF expression division. Less obvious were the signals that regulate CDK stabilizes and thereby activates another tumor suppressor, activity and how these became altered in neoplastic disease. p53 (5, 6). Like the INK family of inhibitors, p53 functions to In an attempt to address this very question, Beach and block inappropriate proliferation and cellular transformation. colleagues made the observation that CDK4 bound a dis- Through a poorly understood mechanism, likely dependent tinct, 16-kDa protein in cells transduced with a viral onco- upon cell type and transcriptional output, p53 activation can gene (1). Biochemical characterization of this protein, later trigger either apoptosis or cell-cycle arrest (7). A fourth INK4/ named p16INK4a, placed it amongst the INK4-class of cell- ARF transcript, ANRIL (Antisense Noncoding RNA in the cycle inhibitors, which bind directly to CDK4 and CDK6, INK4/ARF Locus), was recently discovered in a familial blocking phosphorylation of the retinoblastoma tumor sup- melanoma kindred with neural system tumors (8). The ANRIL transcript runs antisense to p15INK4b and encodes a pressor (RB) and subsequent traversal of the G1/S cell-cycle checkpoint (Fig. 1A; refs 2, 3). In the presence of various long, noncoding RNA elevated in prostate cancer and leu- stressors (e.g., oncogenic signaling, DNA damage), p16INK4a kemia (9, 10). ANRIL is proposed to function as an epigenetic expression blocks inappropriate cellular division, and pro- regulator of INK4/ARF gene transcription, targeting histone- longed induction of p16INK4a leads to an irreversible cell- modifying enzymes to the locus (see the Discussion section). cycle arrest termed "cellular senescence". In summary, the INK4/ARF locus is a relatively small (110 The gene encoding p16INK4a, CDKN2A,lieswithinthe kb), but complex locus, essential to the proper maintenance of INK4/ARF tumor suppressor locus on human chromosome cell-cycle control and tumor suppression. In this review, we 9p21.3 (Fig. 1B). CDKN2A encodes two transcripts with focus on the founding member of the INK4/ARF locus, p16INK4a, and discuss what is known and unknown about alternative transcriptional start sites (4). Both transcripts share INK4a exons 2 and 3, but are translated in different open reading p16 regulation in cancer and aging. CDK4/6-independent roles of p16INK4a INK4a Authors' Afﬁliations: Departments of 1Molecular Genetics and 2Molecular Several lines of evidence suggest that p16 may and Cellular Biochemistry, The Ohio State University, Columbus, Ohio function both through CDK4/6-dependent and -indepen- – Corresponding Author: Christin E. Burd, Biomedical Research Tower, Rm dent mechanisms to regulate the cell cycle. CYCLIN D 586, The Ohio State University, 460 W. 12th Avenue, Columbus, OH 43210. CDK4/6 complexes are stabilized by interactions with the Phone: 614-688-7569; Fax: 614-292-6356; E-mail: [email protected] CDK2 inhibitors, p21CIP1, p27KIP1, and p57KIP2, and serve doi: 10.1158/1541-7786.MCR-13-0350 to titrate these proteins away from CDK2 (11–14). Subse- INK4a INK4b 2013 American Association for Cancer Research. quent expression of p16 or p15 causes these

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Figure 1. Function, structure, and polymorphisms of the INK4/ARF locus. A, p15INK4b and p16INK4a both function in the RB tumor suppressor pathway through inhibition of CDK4/6 activity. Expression of p14ARF inhibits the E3 ubiquitin ligase activity of MDM2, leading to stabilization of p53. The p53 and RB pathways play integral roles in blocking inappropriate cellular proliferation. B, packed into 35 kb of chromosome 9p21.3 are three well-characterized tumor ARF INK4b INK4a suppressor genes: p14 , p15 , and p16 . GWAS have implicated 9p21.3 SNPs in cancer, heart disease, glaucoma, type 2 diabetes, autism, and endometriosis. The majority of the SNPs lie outside of the coding regions in a recently discovered long, noncoding RNA, ANRIL. Of the identified SNPs, those that have been shown to correlate with CDKN2A expression in at least one study are filled with gray. Other SNPs that have not been correlated with CDKN2A expression in validation studies, or have yet to be examined are filled with black or white, respectively.

complexes to disassociate, releasing sequestered CDK2 inhi- or p19INK4d) develop normally, and are born at expected bitors (15). This process, known as "CDK inhibitor re- mendelian ratios (23–25). In contrast, p18INK4c KOs are shuffling", has been documented in a growing list of cell characterized by organomegaly; yet, the association of lines, and several lines of evidence support the biologic p27KIP1 with CDK2 complexes is unchanged in these relevance of this model. Mice harboring kinase-dead Cdk4 animals (26). Work examining combined loss of p15INK4b or Cyclin D1 alleles that retain p27KIP1-binding capacity and p18INK4c (24) or p27KIP1 and p18INK4c (26) in mice (Cyclin D1K112E, Cdk4D158N) display heightened CDK2 suggests that distinct mechanisms are used by each inhibitor activity (16–18) and fewer developmental defects than to control cellular proliferation. This result is in contrast to Cyclin D1 knockouts (KOs). The same observation holds the CDK inhibitor reshuffling model wherein co-deletion true for a Cyclin D1 knockin mutation incapable of binding would be predicted to concertedly promote CDK2 activity. D RB (Cyclin D1 LxCxE; ref. 19). As such, it is not surprising However, it is important to note that none of these pub- that p27KIP1 deletion can rescue the retinal hypoplasia and lications contest the fact that CDK2 inhibitors bind early mortality phenotypes of Cyclin D1-null mice (20, 21). CYCLIN D–CDK4/6 complexes and are released upon More recently, the biologic relevance of CDK inhibitor p16INK4a expression. Moreover, recent findings suggest that reshuffling has come under scrutiny. Knockin mice harbor- p16INK4a may contribute to cell-cycle regulation through ing p16INK4a-insensitive Cdk4 and Cdk6 alleles still capable additional CDK-independent mechanisms. Specifically, of binding p27KIP1 (Cdk6R31C and Cdk4R21C, respectively) expression of p16INK4a has been reported to stabilize do not display the phenotypes predicted by this model p21CIP1, and may inhibit the AUF1-dependent decay of (18, 22). The decreased p16INK4a-binding capacity of these p21CIP1, cyclin D1, and e2f1 mRNA (27, 28). As a whole, mutants should promote p27KIP1 sequestration and these data provide evidence that the cell-cycle–related func- enhanced CDK2 activity, but neither cells from the liver tions of p16INK4a may extend beyond CDK4/6 inhibition to or testes of Cdk4R21C mice show changes in the composition include the regulation of other CDK-CYCLIN targets. of CDK2–Cyclin complexes, nor do thymocytes harboring the Cdk6R31C allele (18, 22). These data suggest that, in at Transcriptional, Translational, and Epigenetic p16INK4a least a subset of cell types, the kinase activity of CDK4/6 is Regulation of predominantly responsible for proliferative control. KO To maintain tissue homeostasis and prevent cancer, the mice lacking a single CDK4/6 inhibitor (p16INK4a, p15INK4b ability of p16INK4a to inhibit cellular proliferation must be

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tightly controlled. In this section, we discuss the role of to be determined whether recently reported noncanonical chromatin, transcriptional cofactors, and RNAs in main- PRC1 complexes that lack a Pc homolog contain RYBP/ taining proper p16INK4a expression. In addition, we high- YAF2, are recruited to chromatin independent of light the complexity and redundancy of p16INK4a regulatory H3K27me3 (33), and can also function to regulate INK4/ pathways required for proper proliferative control. ARF gene expression. However, regardless of their composition, PRC1 and -2 complexes clearly bind throughout the p16INK4a repression by polycomb group complexes INK4/ARF locus and repress p16INK4a expression in young, Chromatin modifications by the polycomb group com- unstressed cells (31). Maintenance of this repression may be plexes (PcG), PRC1 and PRC2, are critical to the homeo- partially dependent on the ubiquitin-specific protease, static regulation of INK4/ARF gene expression (Fig. 2A). USP11, which was also shown by Maertens and colleagues The PRC2 complex is made up of four core components: to bind and stabilize the PRC1 complex (34). In their work, EZH1/2, EED, SUZ12, and RBAp46/48. EZH1 or -2 depletion of USP11 caused polycomb complexes to disso- serves as the catalytic subunit of PRC2, and functions only ciate from the INK4/ARF locus leading to subsequent de- in the presence of EED and SUZ12 to compact chromatin repression of p16INK4a. through the di- and trimethylation of histone H3 lysine 27 The importance of PRC1 and PRC2 for proper p16INK4a (H3K27me2/3; refs 29, 30). H3K27me3 is recognized by a regulation may be best exemplified by the phenotypes of chromodomain-containing CBX protein family member polycomb KO mice. Deletion of the PRC1 component, associated with PRC1. In this manner, PRC1 is recruited Bmi1, results in homeotic skeletal transformations and to the INK4/ARF locus, where it catalyzes the ubiquitylation lymphoid and neurologic defects (35). Many of these phe- of histone H2A lysine 119 (H2AK119ub), resulting in notypes are attributable to the deregulation of homeobox further chromatin compaction and gene silencing (31). gene expression; however, the lymphoid and neurologic Multiple variants of the PRC1 complex have been identified defects observed in Bmi1 KO mice can be almost completely in vivo, each containing homologs of the Drosophila Poste- rescued by INK4/ARF deletion (36). Here, INK4/ARF loss rior Sex Comb (Psc; NSPC1/PCGF1, MEL-18/PCGF2, reverses the self-renewal defects of Bmi1-null hematopoietic RNF3/PCGF3, BMI1/PCGF4, RNF159/PCGF5, and neuronal progenitors (37, 38). Together, these data RNF134/PCGF6), Polycomb (Pc; CBX2, CBX4, CBX6, show that PRC1 regulation of p16INK4a expression is CBX7, CBX8), Sex Combs Extra (RING1, RING2), and required for proper development, stem cell maintenance, Polyhomeotic proteins (Ph; HPH1, HPH2, HPH3; ref. 32). and homeostasis. In contrast to PRC1-null animals that PRC1 complexes contain a single Psc and Pc homolog; yet, survive gestation, deletion of the PRC2 members, Ezh2 or Maertens and colleagues observed binding of MEL-18, Suz12, is embryonic lethal (39, 40). For this reason, con- BMI-1, CBX7, and CBX8 to the repressed INK4/ARF locus ditional KO alleles are required to assess the biologic func- (32). Further investigation revealed that multiple PRC1 tions of PRC2. By using such alleles to delete Ezh2 in the variants bind to the p16INK4a promoter, working in a brain, pancreas, and embryonic skin, phenotypic outcomes concerted manner to control gene expression (32). It remains have been observed. Specifically, loss of Ezh2 in murine

INK4a INK4a Figure 2. Mechanisms of p16 regulation by chromatin modiﬁcation. A, p16 is negatively regulated by the histone-modifying complexes, PRC1 and PRC2, which combine to lay down repressive marks (e.g., H3K27me3, H2AK119ub) throughout the INK4/ARF locus. Several associated proteins (AP), INK4a including RNA-binding proteins (RBP), are reported to facilitate PRC1/2 interaction with the p16 promoter. APs and RBPs discussed in the text are shown. Elevated expression of the PRC2 component, EZH2, is also reported to enhance INK4/ARF gene silencing. Proteins reported to transactivate EZH2, leading to INK4a INK4a PRC-mediated silencing of p16 are depicted. B, activation of p16 is associated with decreases in PRC1/2 levels and the removal of repressive INK4a histone marks. JMJD3 demethylates H3K27me3 and subsequent chromatin decondensation promotes access by transcription factors and p16 transcription whereas JDP2 binds H3K27 and prevents further methylation. CTCF maintains chromosomal boundaries and three-dimensional structure INK4a of the chromatin surrounding p16 .

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pancreatic islets causes a diabetic phenotype associated with binds ANRIL to facilitate PRC1 interaction with the INK4/ increased b-cell expression of both p16INK4a and ARF (41). ARF locus has yet to be determined. However, together, In contrast, Ezh2 deletion in the brain and skin resulted in these data make a strong case for the role of ANRIL in only mild differentiation defects (42, 43). The recent obser- epigenetic silencing of p16INK4a. vation that EZH1 is expressed in many adult tissues and also ANRIL-independent mechanisms are also suggested to catalyzes histone H3 methylation led to the hypothesis that recruit PcG complexes to the p16INK4a locus. Recently, EZH1 may compensate for EZH2 loss in some settings. H2.0-like homeobox 1 (HLX1), a homeobox (HOX) protein, Indeed, deletion of both Ezh1 and -2 caused severe defects in was shown by Martin and colleagues to facilitate PRC2 murine skin morphogenesis associated with a >70-fold recruitment to the p16INK4a promoter (Fig. 2A; ref. 52). increase in p16INK4a/ARF expression (43). Although the mechanism has yet to be defined, six other Supporting a role for EZH2 in maintaining proliferative homeobox-containing proteins (HOXA9, DLX3, HOXB13, homeostasis, human germline mutations in EZH2 give rise HOXC13, HOXD3, and HOXD8) were similarly reported to Weaver syndrome, a congenital disorder characterized by to participate in p16INK4a silencing (52). Given the role of uncontrolled and rapid growth (44). Although this pheno- HOX genes in developmental patterning, it is interesting to type could be attributed to p16INK4a silencing, few reports speculate that proteins like HLX1 and HOXA9 initiate tissue- have attempted to functionally characterize the EZH2 mis- specificsilencingofp16INK4a. Like HOX proteins, both sense mutations commonly associated with Weaver syn- TWIST1, a basic helix–loop–helix (bHLH) transcription drome (44). Instead, work has focused on recurring point factor, and KDM2B, a histone demethylase, may also facil- mutations reported in B-cell lymphoma. These mutations itate polycomb-mediated silencing of the INK4/ARF locus. localize to tyrosine 641 of the EZH2 SET domain, resulting Ectopic expression of TWIST1 and KDM2B can cause in the production of a neomorphic protein with enhanced cellular levels of EZH2 to rise, resulting in an increase in H3K27 di- and trimethylation activity (45). Of importance, PRC2 activity (53). KDM2B was further reported to function not all cancer-associated EZH2 mutants are gain-of-function in demethylating H3K36me2/3, a common marker for DNA alleles. In myleloid neoplasms, missense, nonsense, and polymerase II transcription (53). Furthermore, TWIST1 frameshift mutations in EZH2 have been described that appears to increase BMI1 expression (54), and subsequent lack a catalytic SET domain (46). In addition, the expression recruitment of BMI1 to the p16INK4a promoter has been of wild-type EZH2 has been reported to cause p16INK4a linked to interactions with phosphorylated RB (pRB) and zinc silencing in SNF5-deficient malignant rhabdoid tumors finger domain-containing protein 277 (ZFP277; refs 55, 54). (MRT; ref. 47). Deletion or pharmaceutical inhibition of In particular, the link between pRB and p16INK4a silencing is EZH2 activity in cells from these tumors increases p16INK4a intriguing, as it suggests the presence of a feedback loop expression, resulting in cell-cycle inhibition (47, 48). wherein cells entering the S-phase repress p16INK4a expression Together, these observations suggest that EZH2 activity (55). ZFP277-mediated recruitment of BMI1 to the p16INK4a must be tightly controlled in order to maintain proliferative promoter may also be linked to the cell cycle. A study by homeostasis and proper p16INK4a regulation. Negishi and colleagues showed that reductions in ZFP277 expression caused by oxidative stress could lead to PRC1 Recruiting polycomb to CDKN2A dissociation from the p16INK4a promoter and subsequent cell- In Drosophila melanogaster, polycomb group proteins are cycle arrest (54). How these mechanisms of PRC recruitment recruited to defined DNA-binding sites termed, Polycomb interplay with ANRIL remains to be established, but, cer- repressive elements (PRE; reviewed in ref. 49). In contrast, tainly, the complexity of p16INK4a silencing is indicative of the few mammalian PREs have been identified to date, leading importance of this gene in maintaining tissue homeostasis. many to speculate that other DNA-binding proteins or RNAs must guide polycomb complexes to target genes like Reversing polycomb silencing of the INK4/ARF locus – the INK4/ARF locus. Recent work suggests that long, non- In order for normal cells to traverse the G1 S checkpoint, coding RNAs (lncRNA) can serve as scaffolds for polycomb p16INK4a must be maintained in a repressed state. At the recruitment and epigenetic gene silencing. The discovery of same time, induction of p16INK4a expression in the presence disease-linked polymorphisms within ANRIL prompted of stress signals is required to prevent inappropriate cell-cycle investigation into whether this lncRNA could function in progression. Linking proliferative control to epigenetic reg- a similar manner to regulate INK4/ARF gene transcription. ulation of the p16INK4a promoter, Bracken and colleagues fi Through RNA binding assays, a study by Yap and colleagues rst demonstrated that RB phosphorylation during the G1 to showed that CBX7 interacts with both ANRIL and S-phase transition releases E2F1 to transactivate EZH2 and H3K27me3 to promote INK4/ARF gene silencing (Fig. EED (56). Later, these data were confirmed by an indepen- 2A; ref. 10). Subsequently, ANRIL binding to SUZ12, a dent group who showed that p53 activation represses EZH2 component of the PRC2 complex, was reported to promote expression through RB-mediated inhibition of E2F1 activity silencing of p15INK4b, but not p16INK4a (50). Together with (57). Although these results explain how the activity of these data, a report showing that MOV10—a putative RNA PRC2 might be curbed in the presence of stress, they do helicase and PRC1 binding partner—is required for not explain how epigenetic silencing of the p16INK4a pro- p16INK4a repression, supports a role for ANRIL in polycomb moter is reversed. One mechanism of removing repressive recruitment to the INK4/ARF locus (51). Whether MOV10 histone marks is via the activity of histone demethylases. In

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response to oncogenic stressors, levels of the H3K27me3 Chromatin regulation of p16INK4a by nonpolycomb demethylase, Jmjd3, increase, removing repressive histone proteins marks from the p16INK4a promoter (Fig. 2B; refs 58, 59). Epigenetic modification of the INK4/ARF locus extends Following histone demethylation, researchers at the beyond polycomb-mediated silencing. The well-conserved Yokoyama laboratory showed that Jun dimerization protein genomic insulator, CCCTC-motif binding factor (CTCF), 2 (JDP2) may help maintain p16INK4a in an active state by binds throughout the INK4/ARF locus (65, 66) and func- binding and sequestering H3K27 away from the actions of tions to regulate both chromatin compaction and gene PRC2 (60). These findings suggest that the activity of JDP2 expression (Fig. 2B). Witcher and colleagues first reported and JMJD3 establishes a permissive state for p16INK4a interaction of CTCF with a chromosomal boundary approx- expression. Furthermore, unmethylated H3K27 is no longer imately 2 kb upstream of the p16INK4a promoter (65). In recognized by PRC1, and chromatin surrounding the their studies of cancer cell lines, knockdown of CTCF p16INK4a promoter is decondensed. Based upon this activity, resulted in the spread of heterochromatin DNA into the it is not surprising that JMJD3, like p16INK4a, serves as a INK4/ARF locus, leading to epigenetic silencing of p16INK4a barrier to induced pluripotency (See the section "p16INK4a as (65). In contrast to this model, Hirosue and colleagues a Barrier to Pluripotency"; refs 61, 62). Clearly, interplay recently reported that decreases in CTCF expression asso- among PRC1, PRC2, and histone demethylases is required ciated with oncogene-induced senescence and promotes for homeostatic regulation of the p16INK4a promoter, yet, decondensation of the INK4/ARF locus leading to height- how this crucial balance is maintained is still in question. ened levels of p16INK4a mRNA (66). Reconciliation of these Moreover, it is possible that other histone demethylases, two results is possible as rapid p16INK4a induction following such as lysine-specific demethylase 6A (KDM6A/UTX), CTCF knockdown would place enormous pressure on may also play a role in p16INK4a regulation. would-be cancer cells to epigenetically silence the INK4/ In Drosophila, the SWI–SNF chromatin remodeling com- ARF locus. In fact, the observation that epigenetic silencing plex serves as a trithorax group activator, opposing the actions of p16INK4a in breast cancers is accompanied by CTCF of polycomb-mediated silencing. Similarly, SWI–SNF func- disassociation from the locus (65) is consistent with a role for tions to counteract PcG silencing of p16INK4a in mammals. CTCF in proper epigenetic regulation of INK4/ARF. Cancer cell lines deficient in the SWI–SNF component, SNF5, induce high levels of p16INK4a upon restoration of Transcriptional activators and repressors of p16INK4a SNF5 expression (63). In untransformed cell lines, SNF5 Opposing transcriptional regulators. The presence of a binds and directly inhibits the transcription of EZH2,result- permissive chromatin state alone is insufficient for p16INK4a ing in decreased polycomb occupancy at the p16INK4a pro- expression. Binding of activation factors and the subsequent moter (47). Tumor cells lacking SNF5 overexpress EZH2 and recruitment of RNA polymerase is required to initiate are dependent upon the activity of PRC2 to silence p16INK4a p16INK4a transcription (Fig. 3). Similar to the interplay expression and drive proliferation (47, 64). between PRC2 and JMJD3, transcriptional regulation of the p16INK4a promoter is tightly controlled by antagonistic

INK4a INK4a Figure 3. Transcriptional regulation of p16 . Expression of p16 requires the action of transcription factors (green) that recruit and/or facilitate RNA INK4a polymerase association with the promoter. Opposing this action are transcriptional repressors (red). Direct interactions with the p16 promoter are depicted by a solid line and indirect interactions with a dotted line. The numbers below each binding site indicate the position of protein interaction relative to INK4a the p16 transcriptional start site. All locations correspond to the human genome unless designated by an "(m)," which signiﬁes the mouse genome. Proteins predicted to share a common binding site are depicted on top of one another.

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pathways. Serving as a classic example, induction of p16INK4a of this, developmental defects are not observed in p16INK4a by the E-box binding transcription factors E-26 transfor- KO mice or melanoma-prone kindreds harboring germline mation-specific 1 (ETS1), E-26 transformation-specific2 p16INK4a deficiencies (25, 82). Whether compensatory (ETS2) and E47, is directly opposed by Inhibitor of DNA mechanisms are required to combat the functional loss of Binding 1 (ID1; ref. 67). In response to oncogenic and p16INK4a during development is still to be determined. senescent signaling, ETS1, ETS2, and/or E47 bind to E-box Role of acetyltransferases and deacetylases in p16INK4a motifs (CANNTG) within the p16INK4a promoter to stim- regulation. Histone acetylation facilitates chromatin ulate gene expression (67, 68). This action is directly decondensation and subsequent gene transactivation. As antagonized by ID1, which prevents the interaction of such, transcriptional coactivators often harbor or recruit ETS1, ETS2, and E47 with the p16INK4a promoter (67, histone acetyltransferase (HAT) activity to target gene pro- 68). As such, it is not surprising that Id1-null MEFs undergo moters. In a pair of recent publications, Wang and colleagues premature senescence in culture (69) and that age-related describe how the transcription factors, SP1 and HMG box- increases in p16INK4a expression correlate directly with ets-1 containing protein 1 (HBP1), recruit p300, a well-known levels in mice and rats (70). HAT, to the p16INK4a promoter (83, 84). In their studies, Similar to ID1, TWIST1 is reported to oppose transcrip- acetylation of local chromatin as well as HBP1 promoted tional activation of p16INK4a. The relationship between ID1 decondensation of the p16INK4a promoter and subsequent and TWIST1 was initially suggested in studies examining gene transactivation. However, numerous targets of p300 the progression of benign human nevi to melanoma. In acetylation have been identified to date, including B-MYB, a general, nevi are nonproliferative and express elevated levels putative repressor of p16INK4a transcription (83, 85, 86). of p16INK4a; yet, upon progression to melanoma, these Therefore, the p300–p16INK4a relationship is likely complex lesions frequently silence p16INK4a (71). Analysis of and may be dependent upon the available pool of transcrip- TWIST1 and p16INK4a expression in nevi and melanomas tional cofactors within a given cell type. revealed an inverse correlation between these two proteins, HAT activity is opposed by histone deacetylases (HDAC), putting forth the hypothesis that they function in antago- which promote transcriptional silencing. In human cell lines, nistic pathways (72). Preliminary work suggested that this HDACs 1 to 4 have all been reported to bind and repress antagonism might be mediated through direct interaction of transcription of the p16INK4a promoter (52, 87–89). Most of TWIST1 with ETS2 (72), however, in a recent publication these interactions have been linked to bridging transcription by Cakouros and colleagues, TWIST1 was reported to factors such as Lymphoid Specific Helicase (LSH), HLX1, inhibit p16INK4a transcription by decreasing E47 expression and ZBP-89 (87, 89), albeit one report suggested that (73). Clearly, further work is required to fully understand the HDAC2 may directly bind the p16INK4a promoter (88). physiological relationship between p16INK4a and TWIST1. Although loss of Hdac1, 2, 3,or4 causes developmental In addition, it will be of interest to assess the potential role of abnormalities and lethality in mice, none of these pheno- p16INK4a in classical TWIST1 pathways including epithelial types have been attributed to defects in p16INK4a regulation to mesenchymal transition, stem cell maintenance, and (90). tumor metastasis. Age-related signaling pathways influence p16INK4a In line with the relationship among TWIST, ID1, and E- expression. The observation that p16INK4a levels are high box binding transcription factors, antagonistic interplay has in senescent cells has prompted several investigations into also been described between members of the Activator the connection between prosenescent signaling and Protein-1 (AP-1) family of transcription factors, c-JUN and p16INK4a regulation. For example, age-related metabolic JUNB. Although JUNB serves to activate p16INK4a tran- pathologies have long been associated with activation of the scription by binding to three identified AP1-like sites within peroxisome proliferator-activated receptors (PPARs). It is the p16INK4a promoter (74), c-JUN limits p16INK4a expres- now known that these nuclear receptors directly bind and sion (75). Certainly, a delicate balance between inhibitory activate the p16INK4a promoter leading to subsequent cell- (ID1, TWIST, c-JUN), and stimulatory (JUNB, ETS, E47) cycle arrest (91, 92). Similarly, alterations in TGF-b signal- pathways is required for proper regulation of p16INK4a. ing have been linked to a variety of age-related diseases Upsetting this balance through the overexpression of inhi- including cancer, osteoarthritis, cardiovascular disease, and bitors or repression of p16INK4a activators promotes the Alzheimer’s (93). Work by researchers at the Conboy lab- bypass of senescence and can lead to cancer (72, 73, 76–78). oratory has demonstrated that elevated TGF-b signaling The HOX family of proteins could also be viewed as reduces the capacity of muscle stem cells to regenerate (94). antagonistic p16INK4a regulators. Earlier, we discussed the Here, phospho-SMAD3 has been shown to directly bind the potential role of HLX1, HOXA9, DLX3, HOXB13, p16INK4a promoter, stimulating gene transcription and cell- HOXC13, HOXD3, and HOXD8 in polycomb-mediated cycle arrest (95). Due to cross-talk between the TGF-b and repression of the INK4/ARF locus. In contrast, the HOX b-catenin/Wnt signaling pathways, it is not surprising that proteins, VENTX, MEOX1, and MEOX2 have each been aberrant Wnt signaling is also associated with age-related reported to bind the p16INK4a promoter and activate gene disease (95). b-catenin can directly bind and activate the transcription (79–81). This proposed interplay between p16INK4a promoter in both human and murine cells (96– HOX genes and p16INK4a regulation is suggestive of a role 98), and recent evidence links the induction of p16INK4a by for CDK4/6 inhibition in embryonic development. In spite reactive oxygen species (ROS) to b-catenin/Wnt signaling

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(99). Together, these findings suggest a strong association (108). Although let-7b did not bind p16INK4a mRNA between age-promoting, "gerontogenic" signals and directly, overexpression of let-7b reduced the expression of p16INK4a expression. High-Mobility Group AT-Hook 2 (HMGA2), causing p16INK4a levels to increase (108). In addition to hmga2,a Cellular structure and p16INK4a expression growing number of RNAs involved in growth and prolifer- An emerging theme in p16INK4a regulation is the ative control have been identified as let-7b targets, and, potential for cytoskeletal rearrangements to influence therefore, it is not surprising that the levels of most let-7 INK4/ARF gene transcription. In an siRNA screen of family members decrease during tumor progression (109). >20,000 genes, Bishop and colleagues recently identified In addition to miRNAs, RNA-binding proteins can also GLI2, a member of the Hedgehog signaling pathway, as regulate p16INK4a translation. Interaction of the Hu RNA- an activator of p16INK4a expression (100). Interestingly, binding protein, HuR, with the p16INK4a 30UTR have been GLI2 partially localizes to a nonmotile cytoskeletal pro- reported to destabilize the transcript in an miRNA-inde- trusion called the primary cilium, and cultured human pendent fashion (110). Work by Zhang et al. suggests that mammary epithelial cells with a primary cilium expressed this action is opposed by the tRNA methyltransferase, lowerlevelsofp16INK4a than those without (100). Upon NSUN2, which methylates the 30-untranslated region ablation of p16INK4a, the number of cells with a primary (UTR) of p16INK4a to prevent HuR binding and subsequent cilium increased, suggesting a link between cellular struc- mRNA degradation (111). In this way, interplay between ture and p16INK4a expression (100). Supporting this HuR and NSUN2 may tightly control p16INK4a translation. observation, the ACTIN-nucleating enzymes ARP2 and For example, in the presence of oxidative stress, NSUN2 3 have been implicated in a second connection between levels appear to increase, tipping the balance toward mRNA cytoskeletal structure and Ink4/Arf gene transcription. stability, p16INK4a expression, and subsequent cell-cycle Here, the generation of stable ARP2/3 knockdown cells arrest (111). required concomitant Ink4/Arf deletion (101). These data fi INK4a suggested that lamellipodial dysfunction triggers growth- The Function and Signi cance of p16 inhibitory Ink4/Arf gene transcription; however, the spe- Expression in Cancer cificroleofp16INK4a in this process is yet to be examined. In the absence of p16INK4a, both mice and humans are predisposed to cancer (25, 82). Loss of p16INK4a function The role of miRNAs and RNA-binding proteins in can occur through gene deletion, methylation, or mutation, p16INK4a regulation and, therefore, comprehensive genetic analyses are required Although literature defining transcriptional and epigenetic to determine the frequency of p16INK4a silencing in cancer. regulators of p16INK4a is extensive, far fewer studies have Adding to the challenge of assessing p16INK4a status in examined posttranscriptional mechanisms of p16INK4a regu- human tumors, few studies distinguish between the two lation. Discovered in an unbiased search for miRNAs silenced CDKN2A transcripts: p16INK4a and ARF. Here, we com- during senescence (102), two independent groups have piled data from The Cancer Genome Atlas (TCGA) to reveal reported that miR-24 binds and inhibits p16INK4a translation that functional inactivation of CDKN2A is a frequent event (102, 103). Consistent with this observation, antagonists of in most tumor types (Fig. 4; refs 112–118). We probed 16 miR-24 cause a moderate decrease in the proliferation of tumor types to determine the percentage of cases wherein normal human keratinocytes (103). In a similar manner, mutational and/or copy-number changes disrupted genes knockdown of miR-31 has been proposed to regulate cell- critical to the p16INK4a tumor suppressor pathway (i.e., RB1, cycle progression in murine embryo fibroblasts with altered CDKN2A, CCND1 (CYCLIN D1), CCND2 (CYCLIN nuclear structure (104). Here, loss of Lamin B1 was associated D2), CCND3 (CYCLIN D3), CCNE1 (CYCLIN E1), and with increased miR-31 expression and p16INK4a instability CCNE2 (CYCLIN E2)). Tumor types displaying a high (104). This work suggests another possible connection percentage of p16INK4a tumor suppressor pathway altera- between cellular structure and p16INK4a regulation, linking tions included urothelial carcinoma (BLUCA; 77%), glio- changes in nuclear integrity to p16INK4a expression. Together, blastoma multiforme (GBM; 77%), head and neck squa- the associations among miR-31, miR-24, and p16INK4a mous cell carcinoma (HNSC; 63%), lung squamous cell suggest that miRNAs function to control proliferation via carcinoma (LUSC; 58%), and cutaneous melanoma interactions with p16INK4a mRNA; however, it is important to (SKCM; 58%; Fig. 4A). Examination of individual tumor note that other cell-cycle regulators have been identified as profiles revealed infrequent overlap between CDKN2A and miR-24 targets (e.g., cdk4, cyclin A2, cyclin B1, myc, e2f2, RB1 deletion, implying that these are often mutually exclu- p14ARF,andp27KIP1; refs 103, 105, 106) and miR-31 (e.g., sive tumorigenic events (Fig. 4B). In support of these data, ets1, cdk1; ref. 107). Therefore, cell-cycle defects caused by an inverse correlation between p16INK4a and RB inactivation perturbations in miRNA expression likely reflect the outcome was previously described in lung cancer cell lines (119) and of both p16INK4a-dependent and -independent pathways. human tumors (120). In contrast to RB, we frequently noted The let-7 family of miRNAs has also been implicated in alterations within other components of the p16INK4a tumor p16INK4a regulation, proliferative control, and stem cell suppressor pathway (i.e., Cyclin amplification) in CDKN2A aging. The Morrison group first reported that murine let- mutant tumors from the TCGA dataset (Fig. 4B). However, 7b expression increased with age in neuronal stem cells in cases where the p16INK4a tumor suppressor pathway

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INK4a Figure 4. Alterations in the p16 tumor suppressor pathway are frequent in human cancer. A, data from TCGA was obtained and analyzed using cBioPortal (112, 113). The tumors analyzed are as follows: bladder urothelial carcinoma (BLUCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), skin cutaneous melanoma (SKCM), ovarian serous cystadenocarcinoma (OV), lung adenocarcinoma (LUAD), stomach adenocarcinoma (STAD), breast invasive cancer (BRCA), uterine corpus endometrial carcinoma (UCEC), brain lower grade glioma (LGG), colon and rectum adenocarcinoma (COAD/READ), prostate adenocarcinoma (PRAD), kidney renal papillary cell carcinoma (KIRP), kidney renal clear cell carcinoma (KIRC), acute myeloid leukemia (AML). The percentage of tumors with mutations or copy-number changes in the p16INK4a tumor suppressor pathway are shown. For the purposes of this analysis, the p16INK4a tumor suppressor pathway was deﬁned to contain: CDKN2A (p16INK4a), RB1 (RB), CCND1 (CYCLIN D1), CCND2 (CYCLIN D2), CCND3 (CYCLIN D3), CCNE1 (CYCLIN E1), and CCNE2 (CYCLIN E2). B, OncoPrints from cBioPortal show aberrations in individual tumors across the x-axis. C, methylation of CDKN2A (black bars) and RB1 (gray bars) was quantiﬁed using HM27 or HM450 TCGA data. Genes were considered methylated if b-values exceeded 0.2.

appeared genetically intact, high levels of RB1 or CDKN2A tumors will require thorough experimental and bioinfor- methylation were observed [e.g., kidney cancers (KIRP, matic analyses. KIRC), colorectal adenocarcinomas (COAD/READ), low-grade glioma (LGG), and acute myeloid leukemia p16INK4a expression in tumors (AML); Fig. 4C]. Combining these genetic and epigenetic In most tumor types, p16INK4a inactivation occurs early data suggests that functional inactivation of the p16INK4a– in tumorigenesis. For example, pancreatic intraepithelial RB axis approaches 100% in many cancer types. A prior neoplasias frequently inactivate p16INK4a upon progres- study by Schutte and colleagues supports this claim, dem- sion to invasive disease (122). Pressure to silence p16INK4a onstrating inactivation of the p16INK4a–RB-axis in 49 of 50 presumably stems from oncogenic engagement of the RB pancreatic carcinomas (121). However, further comprehen- tumor suppressor pathway. Therefore, tumors with early sive assessment of p16INK4a functionality in a wide variety of defects in RB signaling continue to express p16INK4a

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independent of progression (reviewed in ref. 123). Intense methods. This incredible sensitivity, along with the obser- activation of the p16INK4a promoter is believed to repre- vation that tumors maintained luciferase activity during sent a futile attempt by the cell to curb oncogenic progression, led to the hypothesis that p16INK4a transcrip- – proliferation in the absence of a functional G1 Scheck- tion is activated in the surrounding tumor stroma. Indeed, point. In the clinic, immunohisotchemical p16INK4a stain- this was the case as syngeneic transplantation of six p16LUC- ing is used to identify cervical and head and neck tumors negative tumor cell lines harboring a wide variety of onco- driven by oncogenic human papilloma virus (HPV) infec- genic drivers caused stromal induction of p16LUC (Fig. 5A tion. Here, the HPV viral oncoprotein, E7, functionally and (130)). Although the specific stromal cell types respon- inactivates RB leading to increases in p16INK4a expression sible for this observation are yet to be identified, bone (124). Although it is believed that RB inactivation is marrow transplantation studies suggest that the p16LUC requisite for the elevation of p16INK4a expression in signal originates in part from bone marrow–derived cells. cancer, aberrations in the RB pathway are not obvious Therefore, it appears that alterations in the milieu surround- in every tumor. The RB checkpoint is deregulated by ing a growing neoplasm can promote the expression of multiple mechanisms independent of RB1 mutation, p16INK4a in a cell-nonautonomous fashion (Fig. 5B). deletion, or methylation. Viral oncogene expression and Mounting evidence supports a role for stromal p16INK4a cyclin gene amplification represent just two possible forms expression in tumor initiation and progression. Fibroblasts of alternative RB inactivation. Unfortunately, there is a paucity of studies that comprehensively examine the status of the RB pathway in multiple human tumor types. Therefore, the critical role of this p16INK4a-induced checkpoint may be underestimated. Limited analyses of the RB pathway in human tumors have revealed that p16INK4a expression can be predictive of both tumor subtype and therapeutic response. Elevated p16INK4a levels discern small-cell lung cancer from lung adenocarcinoma (119, 125) and characterize the basal-like breast cancer subtype (126). Given that tumor subtypes often show distinct therapeutic response profiles, it is not surprising that p16INK4a expression can predict therapeutic efficacy. For example, elevated p16INK4a levels predict a higher initial response to radiation therapy in prostatic adenocarcinoma (127). However, these same p16INK4a positive tumors are the most likely to fail androgen-depri- vation therapy (128). In oropharyngeal cancer, p16INK4a serves as a marker of oncogenic HPV infection and is predictive of improved therapeutic response and patient survival (129). Although p16INK4a expression clearly serves as a relevant clinical marker, combined analysis of multiple RB pathway members may be of additional value. Likewise, recent data suggests that the localization of p16INK4a staining within a tumor may provide further clinical insight (see further sections).

The significance of stromal p16INK4a expression Although a myriad of signals are linked to p16INK4a regulation, most of these are triggered by intrinsic, cell- autonomous events. Employing a recently developed luciferase reporter mouse, p16LUC, we have observed a second, cell-nonautonomous mechanism of p16INK4a activation (130). The p16LUC allele expresses firefly luciferase from the endogenous p16INK4a promoter, allowing investigators INK4a INK4a to dynamically track p16 transcription in live animals. Figure 5. Extrinsic versus intrinsic activation of p16 . A, injection of LUC When the p16 allele was crossed with genetically engi- Lkb1-null endometrial cancer cells into a syngenic mouse heterozygous LUC neered mouse models (GEMM) of human cancer, a lumi- for the p16 reporter causes stromal luciferase expression upon tumor growth. Injection of Matrigel vehicle on the opposite flank does not alter nescent signal appeared only in the location of future tumor INK4a p16 expression. (See also ref. 130.) B, intrinsic signals induces INK4a formation (130). These luminescent foci were visible weeks p16 expression in damaged, senescent, or transformed cells. before tumors could be visualized or palpated, providing a Alterations in surrounding the cellular milieu can trigger the induction of INK4a significant detection advantage over traditional monitoring p16 in nearby undamaged cells through an unknown pathway.

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ectopically expressing p16INK4a have altered cellular metab- MK-7965/dinaciclib (Merck) and PD-0332991/palboci- olism and overproduce high-energy mitochondrial fuels such clib (Pfizer); ref. 137). INK4a as L-lactate (131). In xenograft studies, co-injection of these p16 selectively binds CDK4 and -6 in vitro (2); fibroblasts with MDA-MB-231 breast cancer cells increased however, achieving such therapeutic specificity is more tumor size 2 fold, suggesting that altered stromal metabolism challenging. In fact, Merck attributes the success of dinaci- caused by p16INK4a expression promotes tumor growth clib in chronic lymphocytic leukemia to inhibition of (131). In support of this observation, elevated p16INK4a CDK9, not CDK4/6 (137). Moreover, the observation that levels in the stroma of human mammary ductal carcinoma in p16INK4a may contribute to CDK4/6-independent cell- situ (DCIS) lesions are predictive of disease recurrence cycle regulation (i.e., through CDK inhibitor reshuffling) independent of other histopathologic markers such as ER suggests that pharmaceutical CDK4/6 inhibitors may not positivity (132). It remains to be determined whether the fully recapitulate the potency of p16INK4a. Although adverse promotion of tumorigenesis by p16INK4a-positive stromal effects associated with CDK4/6-selective inhibitors are mild cells is solely a reflection of altered local metabolism. Studies (i.e., limited bone marrow suppression; ref. 136), concerns from the p16LUC mouse model suggest that p16INK4a induc- about the long-term efficacy of such therapeutics is increas- tion in tumor-infiltrating immune cells may also promote ing. In particular, mechanisms to bypass the requirement for cancer progression by dampening the antitumor response. CDK4/6 are already known, including RB loss and increased Regardless of the mechanism, these data make it clear that CDK2 activity. How quickly tumors will exploit these p16INK4a expression in the tumor stroma should not be pathways to subvert therapeutic treatment is unknown. ignored. Initial trials of PD-0332991 in mantle cell lymphomas speak to this concern. Although 89% of study participants showed Therapeutic mimicry of p16INK4a reduced phospho-RB staining at 3 weeks, only 18% of Pharmaceutical efforts to mimic the function of tumors responded to therapy, suggesting that resistance was p16INK4a arose after the antitumor effects of flavopiridol rapidly acquired (138). In addition to concerns about were linked to CDK inhibition (reviewed in ref. 133). resistance, the role of p16INK4a expression in the tumor Flavopiridol (alvocidib) was originally touted as an inhib- stroma remains undefined, and inhibition of the local itor of EGF receptor (EGFR), but later was shown to immune response or altered cellular metabolism may func- inhibit the growth of a wide range of cancer cells at an tion to promote growth and metastasis. In fact, fibroblasts IC50 much lower than that required to block EGFR exposed to PD-0332991 adapt a tumor-promoting meta- activation. Broad inhibition of CDKs, including CDK4 bolic phenotype similar to that of fibroblasts overexpressing < fi INK4a (IC50 120 nmol/L), was later identi ed as the mechanism p16 (131). Therefore, therapeutic mimetics could have of flavopiridol action (134). After several promising phase I similar, detrimental consequences on the tumor microen- trials, flavopiridol failed in phase II, showing significant vironment. A final concern is the long-term effects of activity only in cases of relapsed chronic lymphocytic CDK4/6 inhibition on cellular senescence. Prolonged leukemia (135). Based upon these disappointing results, expression of p16INK4a promotes senescence and decreases Sanofi-Aventis halted production of flavopiridol in 2010. regenerative capacity (see following sections). In a similar For other nonselective CDK inhibitors, multiple toxicities manner, CKD4/6 inhibitors may influence tissue aging. and poor solubility prevented successful transition to the However, under normal physiologic conditions, stem cell clinic. However, recent efforts to generate potent and populations are, by and large, quiescent and, thus, unlikely selective CDK inhibitors appear more fruitful. to be altered by CDK4/6 inhibitors. It may be that stress Of particular interest to the pharmaceutical industry, and/or mitogenic signaling, which often accompany selective CDK4/6 inhibitors have shown promise in early p16INK4a induction in vitro, is required to elicit senescence, clinical trials (reviewed in ref. 136). Knowledge of p16INK4a and, therefore, the effects of CDK4/6 inhibition on aging regulation and function has bolstered these efforts by pro- will be minimal. As more than half of all cancers are viding biomarkers of therapeutic efficacy and identifying diagnosed in people 65 years and older, determining whether patient populations wherein response is likely. Often, clin- CDK4/6 therapeutics exacerbate age-related disease will be ical trials of CDK4/6 inhibitors now exclude RB-null of significant interest. tumors, which are naturally resistant to the effects of ectopic INK4a p16INK4a expression. In addition, the observation that The Role of p16 in Senescence and Aging p16INK4a is markedly upregulated in response to RB-inacti- p16INK4a marks biologic senescence vating viral oncoproteins (e.g., E7, T-Antigen) has prompted As noted first by Sherr and colleagues (139), p16INK4a the exclusion of tumors expressing high levels of p16INK4a. levels increase with aging. In fact, detailed quantification has Finally, studies of CDK KO mice are assisting these demonstrated a direct increase in p16INK4a expression with efforts, identifying cell and tumor types that are more chronologic age in all mammalian species tested to date (140). reliant upon the activity of CDK4/6 to drive proliferation Such increases in p16INK4a are exponential, increasing than that of CDK2. Employing this knowledge, CDK4/6 approximately 16-fold during the average human lifespan inhibitors are showing efficacy, and a number of drugs and making p16INK4a one of the most robust aging biomar- have now entered phase II and III clinical trials [phase II: kers characterized to date (141). The induction of senescence LEE011(Novartis) and Ly2835219(Eli Lilly); phase III: and p16INK4a expression is traditionally associated with a wide

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variety of intrinsic cellular stressors including: DNA damage, mechanisms of curbing age-related p16INK4a induction have telomere erosion, ROS, and stalled replication forks (reviewed been reported in mice. For example, activation of platelet- in ref. 142). However, use of the p16LUC reporter mouse has derived growth factor receptor (PDGFR) signaling in elderly provided evidence that undefined, extrinsic signals can also murine pancreatic b-cells increased the regenerative capacity trigger p16INK4a transcription in a cell nonautonomous of these cells via repression of p16INK4a expression (158). fashion (130). Using this same reporter to compare the Likewise, administration of fibroblast growth factor 7 dynamics of p16INK4a expression in mice with that observed (FGF7) reduced p16INK4a levels and increased the number in humans showed a direct correlation between the rate of of early T-cell progenitors in 15- to 18-month-old mice, p16INK4a accumulation and lifespan (130). Furthermore, data thereby partially rescuing the known decline in thymopoiesis from progeroid and calorically restricted rodents suggest that with age (159). Apart from these studies, analyses of p16INK4a serves as a marker of biologic, rather than chrono- p16INK4a-mediated regenerative decline are limited to a logic aging (70, 143, 144). Supporting this observation in small number of tissues; therefore, the potential outcome humans, smoking and chemotherapy are associated with of p16INK4a-directed therapies remains uncertain. Clearly, elevated p16INK4a expression in the human population further characterization of the relationship among p16INK4a (141, 145). Moreover, skin biopsies from long-lived nona- expression, senescence, and regenerative capacity in vivo genarian cohorts have fewer p16INK4a-positive cells than their would have broad implications for the therapeutic treatment age-matched partners (146). Clinically, use of p16INK4a as a of age-related disease. marker of biologic aging could provide quantitative measures of patient fitness including immune function and chemo- The role of p16INK4a in intrinsic versus extrinsic aging therapeutic tolerance (145, 147). Assessment of p16INK4a Senescence is typically viewed as a response to detrimental, levels prior to organ transplantation may also aid in the intrinsic cellular events; however, recent evidence suggests identification of biologically "younger" donor tissues with that extrinsic signals also contribute to tissue aging. Earlier, increased potential for success (148–150). we discussed the potential for neoplastic transformation to initiate cell nonautonomous p16INK4a expression. Although A causal role for p16INK4a in aging the extracellular mediators of stromal p16INK4a induction are Several lines of evidence suggest that p16INK4a is not only a undefined, several signaling pathways have been linked to biomarker of aging, but also causes aging in many cell types. the expression of p16INK4a in aging tissues. In muscle Using p16INK4a transgenic and KO mice, the cell-autono- satellite cells, age-associated increases in local TGF-b mous role of p16INK4a in aging has been investigated. In production activate SMAD3, which in turn binds to the murine hematopoietic stem cells, T cells, pancreatic b-cells, p16INK4a promoter to initiate gene transcription (94). and neural progenitors of the subventricular zone, age- NOTCH signaling antagonizes TGF-b,and,indoingso, related p16INK4a expression causes a decline in regenerative can alleviate age-related declines in satellite cell function capacity (151–154). A caveat to these initial studies was the (94). Similar to TGF-b signaling, changes in thymic and use of germline KO mice; however, strategies employing bone marrow structure are reported to promote aging in conditional p16INK4a loss or siRNAs have since confirmed local progenitor cell populations (142). Together, these these findings in T cells and pancreatic b-cells (154, 155). findings put forth the model of "niche aging," wherein Supporting the idea that tissues expressing less p16INK4a are stromal changes influence the regenerative capacity of more biologically fit, kidney transplant success is higher in local progenitors. However, parsing the role of the senes- p16INK4a-low donor organs (148–150). These data put forth cent cell versus the niche in aging biology becomes the hypothesis that p16INK4a expression drives cellular somewhat of a chicken and egg question. After all, senes- senescence, resulting in decreased regenerative potential. As cent cells themselves secrete a large number of proinflam- a proof of principal, work from the Van Deursen laboratory matory cytokines associated with age-related disease showed that deletion of p16INK4a-expressing cells from a (reviewed in ref. 160). Future studies aimed at identifying BubR1-deficient, progeroid mouse model reduced many cell nonautonomous p16INK4a activation signals are clearly aging phenotypes (e.g., sarcopenia, cataracts, loss of adipos- needed to better understand the induction of senescence ity; ref. 144). Further work by this group showed that both during physiologic aging. muscle and adipocyte progenitors from these mice express high levels of p16INK4a, suggesting that even the deletion of p16INK4a as a barrier to pluripotency senescent stem cells can promote improved regenerative Work with induced pluripotent stem cells (iPS) also capacity (156). Unfortunately, these animals did not live implicates p16INK4a as a modulator of regenerative capacity. longer, owing to the development of cardiac pathologies, and During iPS generation, senescence induced by the four- a similar experiment has yet to be reported in wild-type mice factor cocktail of OCT4, SOX2, KLF4, and c-MYC serves as (144). One explanation for why these animals were not long- a barrier to efficient reprogramming (161). In cells where lived is that the gerontogenic effects of p16INK4a expression reprogramming is effective, silencing of INK4/ARF gene are tissue specific. Indeed, p16INK4a levels do not seem to transcription is observed concomitant with the induction influence the regenerative capacity of murine melanocyte of molecular markers indicative of stem cell phenotypes (61). stem cells or neuronal progenitors of the dentate gyrus Therefore, it is not surprising that iPS production efficiency (unpublished observations and refs 153, 157). Other is increased by cellular immortalization (162), or short

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hairpin RNA (shRNA) knockdown of INK4/ARF transcripts clearly extends beyond cancer and aging. Dynamic induc- (61, 161). Currently, efficient generation of iPS from older tion of p16INK4a is observed during mammary involution, patients represents a major hurdle for regenerative medicine. wound healing, nerve regeneration, and infection (unpub- Therefore, novel reprogramming approaches aimed at curb- lished data and refs 130, 172–174). It has been proposed that ing the senescent phenotype may improve iPS technology for the induction of p16INK4a during these highly proliferative the future (163). events is critical to the maintenance of proper tissue homeostasis. Whether the same signals that trigger p16INK4a Genome-Wide Association Studies linking p16INK4a to expression under physiologic conditions play a role in age-related disease tumorigenesis or aging is still unclear. However, p16INK4a Meta-analysis of genome-wide association studies expression is only transient during processes like mammary (GWAS) suggests that we have only begun to scratch the involution and wound healing (130, 172, 173). Are surface in understanding the role of p16INK4a in age-related p16INK4a-positive cells cleared by the immune system or disease. Single-nucleotide polymorphisms (SNP) in chro- can they revert to a phenotype conducive to proliferation? mosome 9p21.3 have been linked to cancer, atherosclerosis, Understanding the role of p16INK4a in normal physiology diabetes, frailty, cataracts. and late-onset Alzheimer’s disease will be critical to the development of "senolytic" therapies, (Fig. 1B; ref. 164). In many cases, the expression of p16INK4a which aim to lengthen our healthspan by eliminating senes- correlates directly with SNP genotype, suggesting a causal cent cells in the body. role for p16INK4a in diverse, age-associated diseases (164). p16INK4a is different from other INK4/ARF family Several mechanistic models have been suggested to explain members. The dynamics of p16INK4a expression during how SNPs, some of which are >100 kb from the gene senescence make it a robust biomarker of mammalian promoter, influence p16INK4a expression. One model pro- aging. Human tumors silence p16INK4a with greater fre- poses that the activity of distal enhancer elements is modified quency than ARF or p15INK4b (140), suggesting that the by SNP genotype (165). Another provides evidence that tumor suppressor function of p16INK4a is somehow more ANRIL expression and splicing is directly influenced by critical than that of the other INK4/ARF family members. 9p21 SNPs (166). We believe that these models are not As such, the therapeutic restoration of p16INK4a activity mutually exclusive. appears to be a promising avenue for antineoplastic Although mechanisms by which 9p21.3 SNPs influence development. Ironically, although drug development p16NK4a transcription have been proposed, the link teams in the field of oncology work fervidly to move between p16INK4a expression and age-related diseases is CKD4/6 inhibitors into the clinic, aging biologists aim to not always apparent. For example, atherosclerosis is fre- block the accumulation of p16INK4a-positive cells. Oddly, quently associated with lipid metabolism; yet, 9p21.3 the key to longevity likely lies in the hands of both groups, SNPs have emerged as robust indicators of atherosclerotic as a careful balance of p16INK4a expression is required to risk in some of the most widely replicated GWASs con- stave off cancer and prevent aging. ducted to date (167). Evidence from animal models sug- geststhat9p21.3SNPsmayreduceINK4/ARF gene tran- Disclosure of Potential Conflicts of Interest scription, leading to altered cellular proliferation and apo- No potential conflicts of interest were disclosed. ptosis, which exacerbate disease progression (168–170). However, transgenic mice carrying multiple copies of the Acknowledgments The authors thank M. Waqas, J. Gillahan, S.T. Nguyen, and Drs. D. Beach INK4/ARF locus are equally susceptible to atherosclerosis (Bartholomews Hospital, United Kingdom), C.J. Burd (Ohio State University), and (171). Therefore, the mechanisms by which 9p21.3 SNPs N. Sharpless (University of North Carolina at Chapel Hill) for critical reading of the fl manuscript. Dr. K. Hoadley (University of North Carolina at Chapel Hill) provided in uence a large number of age-related human disease advice and guidance regarding the analysis of TCGA data. remain a subject of ongoing investigation. Grant Support Conclusions This work was supported by NIH R00AG036817 (C.E. Burd) and American Since the discovery of p16INK4a >20 years ago, numerous Cancer Society IRG-67-003-50 (C.E. Burd). advances have led to an increasingly complex view of Received July 1, 2013; revised September 23, 2013; accepted September 24, 2013; INK4a INK4a p16 regulation and function. The role of p16 published OnlineFirst October 17, 2013.

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p16INK4a in Cancer and Aging

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www.aacrjournals.org Mol Cancer Res; 12(2) February 2014 OF17

The Molecular Balancing Act of p16INK4a in Cancer and Aging

Kyle M. LaPak and Christin E. Burd

Mol Cancer Res Published OnlineFirst October 17, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-13-0350

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