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INK4 Locus of the Tumor-Resistant Rodent, the Naked Mole Rat, Expresses a Functional P15/P16 Hybrid Isoform

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INK4 Locus of the Tumor-Resistant Rodent, the Naked Mole Rat, Expresses a Functional P15/P16 Hybrid Isoform INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform Xiao Tiana,1, Jorge Azpuruaa,1,2, Zhonghe Kea, Adeline Augereaub, Zhengdong D. Zhangc, Jan Vijgc, Vadim N. Gladyshevb, Vera Gorbunovaa,3, and Andrei Seluanova,3 aDepartment of Biology, University of Rochester, Rochester, NY 14627; bBrigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and cAlbert Einstein College of Medicine, Bronx, NY 10461 Edited* by Eviatar Nevo, Institute of Evolution, Haifa, Israel, and approved December 1, 2014 (received for review September 21, 2014) The naked mole rat (Heterocephalus glaber) is a long-lived and because, in mammals, it encodes three distinct tumor suppressors: tumor-resistant rodent. Tumor resistance in the naked mole rat p16INK4a, p15INK4b, and p19ARF (p14ARF in human) (9). These is mediated by the extracellular matrix component hyaluronan three proteins coordinate a signaling network that depends on of very high molecular weight (HMW-HA). HMW-HA triggers hy- the activities of the retinoblastoma protein (RB) and the p53 persensitivity of naked mole rat cells to contact inhibition, which is tumor suppressor protein. The p16INK4a and p15INK4b pro- associated with induction of the INK4 (inhibitors of cyclin depen- teins are cyclin-dependent kinase inhibitors that directly in- dent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is hibit the binding of cyclins to their target cyclin-dependent among the most frequently mutated in human cancer. This locus kinases (10). p16INK4a is involved in establishing replicative encodes three distinct tumor suppressors: p15INK4b, p16INK4a, and senescence, oncogene-induced senescence, and stress-induced INK4b ARF (alternate reading frame). Although p15 has its own ORF, premature senescence (11, 12), plays a role in stem-cell aging INK4a p16 and ARF share common second and third exons (9, 13), and acts as a barrier to induced pluripotency (14). Many with alternative reading frames. Here, we show that, in the naked genes involved in senescence are also associated with age-related mole rat, the INK4a/b locus encodes an additional product that disease (15, 16). Indeed, evidence has accumulated to support INK4b INK4a consists of p15 exon 1 joined to p16 exons 2 and 3. We the role of the locus in atherosclerosis and other aging-related have named this isoform pALTINK4a/b (for alternative splicing). We – INK4b INK4a INK4a/b pathologies (17 20). Differences between p15 and p16 show that pALT is present in both cultured cells and naked include expression profile and stability, with p15INK4b having a mole rat tissues but is absent in human and mouse cells. Addition- INK4a/b lower overall half-life (21, 22). ARF (alternate reading frame) ally, we demonstrate that pALT expression is induced during protein p19ARF (in mice) also has a tumor-suppressing function early contact inhibition and upon a variety of stresses such as UV, (23): it acts to repress the oncogene MDM2, which in turn binds gamma irradiation-induced senescence, loss of substrate attach- p53, repressing its activation (24). ARF has other functions in ment, and expression of oncogenes. When overexpressed in regulating the cell cycle, independently of p53 (25). naked mole rat or human cells, pALTINK4a/b has stronger ability INK4b INK4a The structure of the INK4a/b locus is complex and has un- to induce cell-cycle arrest than either p15 or p16 . We hy- dergone several modifications during its evolutionary history (26). pothesize that the presence of the fourth product, pALTINK4a/b of the INK4a/b locus in the naked mole rat, contributes to the in- creased resistance to tumorigenesis of this species. Significance naked mole rat | INK4 | p16 | p15 The naked mole rat is a longest lived and cancer-resistant ro- dent. Tumor resistance in the naked mole rat is mediated by signals from the extracellular matrix component hyaluronan he naked mole rat (Heterocephalus glaber) is notable for be- triggering the induction of INK4 (inhibitors of cyclin dependent ing the longest-lived rodent, with an observed maximum T kinase 4) locus expression. The human and mouse INK4 locus lifespan of over 30 y (1). Additionally, despite being kept in encodes three critical tumor-suppressor proteins, p15INK4b, ARF captivity since 1974, there has been no reported incidence of (alternate reading frame), and p16INK4a, which are among the neoplasia in these animals (2, 3). Naked mole rats remain most frequently mutated in cancer. Furthermore, p16INK4a is healthy until almost the end of their lives, showing resistance implicated in aging and senescence. Here, we show that the to multiple age-related diseases (4). Understanding the mecha- naked mole rat INK4 locus encodes an additional product, nisms responsible for tumor resistance of this unusual rodent a hybrid between p15INK4b and p16INK4a. The novel product, may lead to the discovery of novel targets to cancer therapy that named pALTINK4a/b, may contribute to tumor resistance and cannot be found in cancer-prone laboratory species. longevity of the naked mole rat. Understanding the regulation In tissue culture, naked mole rat fibroblasts display hyper- of the INK4 locus is critical for cancer and aging research. sensitivity to contact inhibition, termed early contact inhibition (ECI) (5). ECI is directly linked to the cancer resistance of naked Author contributions: X.T., J.A., V.N.G., V.G., and A.S. designed research; X.T., J.A., Z.K., mole rats. The signal triggering ECI comes from an extracellular and A.A. performed research; Z.D.Z., J.V., and V.N.G. contributed new reagents/analytic matrix component, hyaluronan, which, in the naked mole rat, is tools; X.T., J.A., Z.K., A.A., Z.D.Z., J.V., V.N.G., V.G., and A.S. analyzed data; and X.T., J.A., CELL BIOLOGY characterized by an extremely high molecular weight (6). Re- A.A., V.N.G., V.G., and A.S. wrote the paper. moval of high molecular weight hyaluronan abrogates ECI and The authors declare no conflict of interest. makes naked mole rat cells susceptible to malignant trans- *This Direct Submission article had a prearranged editor. INK4a formation (6). ECI is associated with an increase in p16 1X.T. and J.A. contributed equally to this work. expression, and cell lines that spontaneously lost expression from 2Present address: University of Texas Health Science Center at San Antonio, San Antonio, TX. the INK4a/b (inhibitors of cyclin dependent kinase 4) locus no 3To whom correspondence may be addressed. Email: [email protected] or longer display ECI (5). [email protected]. Deletion or silencing of the INK4a/b locus is the hallmark of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. human cancer (7, 8). This small (<50 kb) locus is remarkable 1073/pnas.1418203112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1418203112 PNAS | January 27, 2015 | vol. 112 | no. 4 | 1053–1058 Downloaded by guest on September 29, 2021 The emergence of separate p16INK4a and p15INK4b genes is the INK4b INK4a result of an ancient duplication event (27). They are structurally very A p15 pARF p16 similar, featuring several ankyrin repeats. The ARF is a completely different protein produced by using an alternative first exon that E1 E2 E1 E1 E2 E3 splices to the second exon of p16INK4b in a different frame (28). p15 p15 E2 p16 E1 p16 E2 The first exon of ARF was acquired before the INK4a/b dupli- cation event (29). The whole-genome sequencing of the naked mole rat revealed that the INK4a/b locus was altered by early NSF2 mut NSF8 NEF9 translation termination events (30), suggesting that it may have B undergone specific evolutionary changes to increase cancer resistance in this species. This genomic finding complemented our earlier results that INK4a/b was required for ECI and p15 p16 pALT p15 p16 pALT p15 p16 pALT cancer resistance. Because of the importance of INK4a/b in cancer resistance, we decided to characterize the expression of its products. Un- INK4 expectedly, we found that, in the naked mole rat, the INK4a/b locus produced a novel hybrid protein isoform from the INK4b promoter that splices p15INK4b exon 1 to p16INK4a exon 2. This Actin isoform, which we named pALTINK4a/b, is unique to the naked mole rat and is not observed in human or mouse. pALTINK4a/b is present in cultured cells and in naked mole rat tissues in vivo. C E1/E2 pALTINK4a/b is activated by a variety of stresses, similar to Splice Junction p15INK4b and p16INK4a, but has higher capacity to induce cell- p15INK4b cycle arrest. We hypothesize that the presence of the additional, pALTINK4a/b INK4a/b more potent, cyclin-dependent kinase inhibitor pALT con- p16INK4a tributes to the cancer resistance of the naked mole rat. Results D NSF -RT Heart Muscle Kidney Identification of the Novel INK4 Isoform in the Naked Mole Rat. Previous work in naked mole rat fibroblasts had indicated that the INK4 locus was an important mediator of the tumor re- p15 p16 pALT sistance of this species (5). To examine the expression of INK4 p15 p16 pALT p15 p16 pALT p15 p16 pALT p15 p16 pALT products, we designed two pairs of primers (Fig. 1A) to amplify INK4b INK4a p15 and p16 cDNAs from three independent lines INK4 of naked mole rat fibroblasts. We observed the expected p16INK4a, p15INK4b products in primary skin fibroblasts (NSF8) and embry- onic fibroblasts (NEF9). The cell line NSF2 mut, which sponta- neously lost the early contact inhibition phenotype, did not express any INK4 products (Fig. 1B), consistent with our previous obser- Liver Lung Brain Spleen Testes vations (5).
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