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The Molecular Balancing Act of P16 in Cancer and Aging

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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); 167–83. Ó2013 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 Ó2013 American Association for Cancer Research. quent expression of p16 or p15 causes these www.aacrjournals.org 167 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst October 17, 2013; DOI: 10.1158/1541-7786.MCR-13-0350 LaPak and Burd 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.Ofthe 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 168 Mol Cancer Res; 12(2) February 2014 Molecular Cancer Research
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