P16 Controls P53 Protein Expression Through Mir-Dependent Destabilization of MDM2 Huda H

Home , CDKN2A, P16, Tumor suppressor gene

Published OnlineFirst May 8, 2018; DOI: 10.1158/1541-7786.MCR-18-0017

Oncogenes and Tumor Suppressors Molecular Cancer Research p16 Controls p53 Protein Expression Through miR-dependent Destabilization of MDM2 Huda H. Al-Khalaf1,2 and Abdelilah Aboussekhra2

Abstract

p16INK4A and p53 are two major tumor suppressor proteins that ubiquitination. This effect occurs through p16-related promotion are both upregulated in response to various cellular stresses and of the MDM2 mRNA turnover via the p16INK4A downstream during senescence and aging. p53 is a well-characterized tran- effectors miR-141 and miR-146b-5p, which bind specific sites at scription factor, while p16INK4A a cyclin-dependent kinase inhib- the 30 untranslated region of the MDM2 mRNA. itor encoded by the CDKN2A gene, and controls the expression of INK4A several genes through protein–protein interactions and also via Implications: The current findings show p16 -dependent miRNAs. This report demonstrates a p16INK4A-dependent positive stabilization of p53 through miR-141/miR-146b-5p–related regulation of p53 expression, at the protein level, in various posttranscriptional repression of MDM2, thus providing new INK4A human cells as well as in mouse embryonic fibroblasts. p16 insights into the complex functional link between p16 and suppresses p53 turnover through inhibition of its MDM2-related p53. Mol Cancer Res; 1–10. 2018 AACR.

Introduction preventing their activation by D-type cyclins (20, 21). In addition, p16 controls the expression of different miRNAs and genes p16INK4A (p16) and p53 belong to two different, yet over- involved in various cellular processes (22–24). p16 is also a major lapping tumor suppressor pathways p16INK4A/pRB/E2F1 and player in the cellular response to DNA damage, which leads to cell- ARF/p53/p21, which are deregulated in most, if not all, human cycle arrest (25), senescence (26, 27), and/or apoptosis (28, 29). tumors (1–3). Both tumor suppressor proteins p16 and p53 are The sequence-specific transcription factor p53 promotes the upregulated during aging, senescence, as well as in response to expression of genes involved in cell-cycle control, DNA repair, different cytotoxic agents (4–8). Furthermore, we have recently apoptosis, and cellular responses to various stresses (30–32). One shown that the levels of p16 and p53 as well as their common of the major targets of p53 is the MDM2 oncoprotein, which in target p21 are reduced in breast cancer-associated fibroblasts as turn represses p53 functions through promoting its proteasome- compared with their adjacent counterparts from histologically mediated degradation (33–36). The MDM2 protein is overex- normal tissues (9, 10). Further investigations showed that p16 pressed in many human malignancies, and high MDM2 levels are and p53 have non-cell–autonomous tumor suppressor functions associated with poor prognosis (33). in stromal fibroblasts (9, 11, 12). In another recent study, it has In this study, we have shown that p16 controls p53 expression been reported that p16 and p53 are both downregulated in at the protein level through miR-141 and miR-146b-5p–depen- breast stromal fibroblasts when exposed to the cytokine IL6 dent negative regulation of MDM2. (13). In addition, it has also been reported that the levels of the p53 and p16 proteins are positively correlated in triple-negative Materials and Methods breast cancers (14, 15). In mouse models, deletion of CDKN2A or TP53 abrogated senescence and enabled tumor formation (16). Cell lines, cell culture, and chemicals fi þ/þ / These results and others suggest the presence of a direct link Mouse embryonic broblasts (MEF; p16 and p16 ) were between p16 and p53; however, the mechanistic basis of this a generous gift from Dr. R.A. DePinho (Harvard Medical School link remains unclear. and the Dana-Farber Cancer Institute, Boston, MA) and HFSN1 fi p16 is a cyclin-dependent kinase inhibitor (CDKI) encoded by (primary normal human skin broblasts) were routinely cultured the CDKN2A gene (17–19). In response to various stresses, p16 in DMEM-F12 medium supplemented with 10% FCS. Normal breast luminal cells (NBL-10) were obtained and cultured as causes cell-cycle arrest in the G1 phase, by binding to CDK4/6 and described previously (37). The p16-defective osteosarcoma U2OS cell line was a generous gift from Dr. G. Peters (Cancer Research 1The National Center for Genomics Research, King Abdulaziz City for Science and UK, London, United Kingdom). MCF10A cells were obtained Technology, Riyadh, Saudi Arabia. 2Department of Molecular Oncology, Cancer from ATCC and were cultured according to the manufacturer's Biology and Experimental Therapeutics Section, King Faisal Specialist Hospital instructions. All supplements were purchased from Gibco. Cells and Research Centre, Riyadh, Saudi Arabia. were maintained at 5% CO2 ina37 C humidified incubator. All Corresponding Author: Abdelilah Aboussekhra, Department of Molecular cells were split at ratio 1:2 and the population doublings (PD) Oncology, Cancer Biology and Experimental Therapeutics Section, King Faisal were determined accordingly. Cycloheximide was purchased Specialist Hospital and Research Centre, MBC # 03, PO Box 3354, Riyadh 11211, from Sigma-Aldrich. Saudi Arabia. Phone: 9661-1464-7272, ext. 32840; Fax: 9661-1442-7858; E-mail: [email protected] Transfection doi: 10.1158/1541-7786.MCR-18-0017 CDKN2A-shRNA, specifically targeting the CDKN2A-1a exon 2018 American Association for Cancer Research. (38, 39) expressed in pRNAT-U6/Neo vector (GenScript

www.aacrjournals.org OF1

Al-Khalaf and Aboussekhra

Corporation), and the corresponding control plasmid, Immunoprecipitation were used at 1 mg/mL for transfection using human dermal Cell lysates were prepared from confluent cells, and then fibroblast nucleofector kit (Amaxa Biosystems) according to the centrifuged at 14,000 rpm at 4C. Subsequently, 300 mg of protein manufacturer's protocol. pGFP-C-shLenti-MDM2-shRNA extracts were incubated with 2 mg Ub (P4D1) antibody (Santa (specific downregulation of MDM2; Origene), pIRES-Puro2 Cruz Biotechnology; mouse IgG1 was used as control) and mixed encoding FLAG-tagged human p16, and their corresponding at 4C for 4 hours. Equal volume of protein A/G agarose was control plasmids were used at 1 mg/mL for transfection added per immunoprecipitation and mixed overnight at 4C. using Lipofectamine 2000 (Invitrogen) according to the man- After centrifugation, the pellet was washed 5 times with lysis ufacturer's instructions. pLKO.1-miRZip146b-5p (inhibitor of buffer and placed in boiling water for 10 minutes in the presence miR-146b-5p), pLKO.1-miRZip141 (Inhibitor of miR-141), of SDS gel sample buffer (0.5 mol/L Tris pH 6.8, 10% glycerol, pCDH-miR-141 (expressing pre-miR-141), pCDH-miR- 10% SDS, 5% 2-mercaptoethanol, and 1% bromophenol) and 146b-5p (expressing pre-miR-146b-5p; System Biosciences), electrophoresed for 2 hours at 125 V. and the corresponding control plasmids were used at 1 mg/mL each for the transfection of 293FT cells. Lentiviral supernatants RNA purification, PCR, and quantitative RT-PCR were collected 48 hours posttransfection. Culture media were Total RNA was purified using the RNeasy Mini Kit (Qiagen) removed from the target cells and replaced with the lentiviral according to the manufacturer's instructions and was treated with supernatant and incubated for 24 hours in the presence of RNase-free DNase before cDNA synthesis using Advantage RT for 1 mg/mL polybrene (Sigma-Aldrich). Transduced cells were PCR kit (Clontech Laboratories). Quantitative RT-PCR was per- selected after 48 hours with puromycin or G418. formed using FastStart Essential DNA Green qPCR Mastermix (Roche) and the amplifications were performed utilizing the miRNA target prediction LightCycler 96 Real-time PCR detection system (Roche) using miRNA targets were predicted using the algorithms, including the following cycle conditions: 95C for 10 minutes (1 cycle); miRanda Human miRNA targets, miRDB, RNA22, and miROrg. 95C for 10 seconds, 59C for 20 seconds, 72C for 30 seconds To identify the genes commonly predicted by these different (45 cycles). The melting-curve data were collected to check PCR algorithms, results of predicted targets were intersected using specificity, and the amount of PCR products was measured by miRWalk. threshold cycle (Ct) values and the relative ratio of specific genes to GAPDH for each sample was then calculated for normalization. Cellular lysate preparation The obtained values were plotted as mean SD. Three indepen- Cells were washed and scraped in the lysis buffer (50 mmol/L dent experiments were performed for each reaction. The respective Tris.Cl pH 7.5 containing 150 mmol/L NaCl, 1% NP40, and primers were: protease inhibitor cocktail). Lysates were clarified by centrifuga- MDM2: 50-TGTTTGGCGTGCCAAGCTTCTC-30 (forward) and tion at 14,000 rpm for 30 minutes. The supernatant was removed, 50-CACAGATGTACCTGAGTCCGATG-30 (reverse), GAPDH:50- aliquoted, and stored at 80C. GAGTCCACTGGCGTCTTC-30 (forward) and 50-GGGGTGCTAA- GCAGTTGGT-30 (reverse), mature miR-141: UAACACUGUCUG- Immunoblotting GUAAAGAUGG and mature miR-146b-5p: UGAGAACUGAAUU- SDS-PAGE was performed using 12% separating minigels. CCAUAGGCU. Equal amounts of protein extract (30 mg) from different samples were placed in boiling water for 5 minutes in the presence Analysis of mRNA stability of SDS gel sample buffer (0.5 mol/L Tris pH 6.8, 10% glycerol, Exponentially growing cells were challenged with actinomy- 10% SDS, 5% 2-mercaptoethanol, 1% bromophenol) and cin D (5 mg/mL) for various periods of time (0–6hours),and electrophoresed for 2 hours at 125 V. After transfer onto then total RNA was purified and the level of the MDM2 mRNA polyvinylidene difluoride membrane, the membrane was incu- was assessed using qRT-PCR. Nonlinear regression analysis bated overnight with the appropriate antibodies. Visualization (Prism, GraphPad Software) was used to assess the mRNA of the secondary antibody was performed using the enhanced decay kinetics, considering the values at time 0 as 100%. The chemiluminescence detection system (Amersham Biosciences). time corresponding to 50% remaining mRNA was considered The antibodies directed against b-actin (C-11), GAPDH as mRNA half-life. (FL-335), p53 (DO-1), p53 (R-19), p21 (F5), and p19ARF (G-19) were purchased from Santa Cruz Biotechnology, p16 Dual luciferase reporter assay from BD Biosciences, MDM2 (Ab-2) from Calbiochem, MDM2 Cells were plated at 1 105 cells/well on 6-well plates and (3G9) from Merck Millipore, and p14 (4C6/4) from Cell transfected with 3 mg of the luciferase/Renilla reporter vector Signaling Technology. containing either human wild-type of the miR-141 or miR- 146b-5p seed sequence in the MDM2 30UTR, mutated sequences, Quantification of protein expression level or a control sequence containing no-ARE sequence of MDM2 Protein signal intensity of each band was determined using 30UTR (GeneCopoeia). Transfection was carried out using ImageQuant TL Software (GE Healthcare). Next, dividing the Lipofectamine 2000 as recommended by the manufacturer obtained value of each band by the values of the correspond- (Invitrogen). At 24 hours posttransfection, cells were seeded in ing internal control allowed a correction of the loading differ- 96-well plate and Firefly and Renilla luciferase activities were ences. The fold of induction/reduction in the protein levels consecutively measured using the Dual luciferase assay as recom- was determined by dividing the corrected values that corre- mended by the manufacturer (GeneCopoeia). The Firefly sponded to the treated samples by that of the nontreated luciferase signal was normalized to the Renilla luciferase signal one (time 0). for each individual analysis. The mean and SEM were calculated

OF2 Mol Cancer Res; 2018 Molecular Cancer Research

p16 Stabilizes p53 via MDM2 Destabilization

from three wells for each 30-UTR activity and presented as fold Together, these data indicate that p16 positively controls the change over the nonstimulated control. expression of p53.

Statistical analysis p16 stabilizes the p53 protein Statistical analysis was performed using Student t test and To elucidate the molecular basis of p16-dependent regulation P values 0.05 were considered significant. of p53 expression, we sought to investigate the possible implication of p16 in the stabilization of the p53 protein. To this end, HFSN1C and HFSN1p16sh cells were treated with cycloheximide Results (20 mg/mL) for different periods of time, and the level of the p53 p16 positively controls the expression of the p53 protein in protein was assessed by immunoblotting using GAPDH as inter- human and mouse cells nal control. Figure 2A shows faster decrease in the level of p53 in We have recently shown that p16 modulates the expression of the p16-deficient cells as compared with their controls. Indeed, several p53 target genes (40). This prompted us to examine the while p53 half-life was 75 minutes in control cells, it was only 35 potential role of p16 in controlling the expression of the tran- minutes in HFSN1p16sh cells (Fig. 2B). This suggests that p16 scription factor p53. As the level of both proteins increases during stabilizes the p53 protein. cellular aging, whole-cell lysates were prepared from serially passaged human skin fibroblast HFSN1 cells, and were utilized p16 suppresses the expression of MDM2 to assess the level of the tumor suppressor genes p16, p53, and its To explore the molecular mechanism underlying the target p21 by immunoblotting. b-Actin was used as internal p16-dependent stabilization of the p53 protein, we investigated control. Figure 1A shows population doubling (PD)-dependent the effect of p16 on the expression of MDM2, which targets increase in the levels of the three important aging markers p16, p53 for ubiquitin-dependent degradation (35, 44, 45). Therefore, p53, and p21. Similar results were obtained in serially passaged the level of the MDM2 protein was assessed in serially passaged þ þ wild-type MEFs (Fig. 1C). Interestingly, when p16 was down- HFSN1 as well as p16 / MEF cells by immunoblotting. Figure 1A regulated by shRNA, which specifically targets the CDKN2A-1a and C show that while the level of the MDM2 protein decreased in exon, in HFSN1 cells (HFSN1p16sh; a scrambled sequence was a PD-dependent manner, its level was strongly upregulated upon used as control HFSN1C), the levels of p53 and p21 were also downregulation of p16 in HFSN1p16sh cells (Fig. 2C). Similar strongly reduced, while p14 (a well-known regulator of p53; effects were obtained when p16 was knocked-out in MEF cells þ þ refs. 41, 42) was not affected (Fig. 1B). Similar effects were (p16 / ) as compared with WT cells (p16 / ; Fig. 1C). obtained when p16 was knocked-out in MEF cells (p16 / )as Next, the level of the MDM2 mRNA was assessed by quanti- þ þ compared with WT cells (p16 / ; Fig. 1C). In these p16-defective tative RT-PCR (qRT-PCR). Figure 2C (bottom) shows remarkable cells, the decrease in the level of p21 and p53 was enhanced with increase in the level of the MDM2 mRNA in HFSN1p16sh as cellular aging. However, there was an age-dependent increase in compared with HFSN1C cells. To further confirm this, the levels of þ þ the level of p19 in both p16 / and p16 / (Fig. 1C). the MDM2 protein and mRNA were assessed in U2OS and MCF10 The levels of the p53 and p21 proteins were also assessed cells expressing p16ORF or control plasmid by immunoblotting in normal primary human luminal NBL-10 cells expressing and qRT-PCR, respectively. Figure 2D shows that upon expression either CDKN2A-shRNA (NBL-p16sh) or a scrambled sequence of p16, the levels of the MDM2 protein and mRNA were signi- (NBL-Ctl) by immunoblotting. Figure 1D shows a strong decrease ficantly decreased as compared with the respective control cells. in the level of both p53 and p21 in p16-deficient NBL-10 cells as These results indicate that p16 negatively controls the MDM2 compared with their control counterpart cells. However, the level expression at the mRNA level. Thereby, we investigated whether of p14 was only marginally affected. Together, these data suggest p16 has a role in the stability of the MDM2 mRNA. To do this, an important role of p16 in controlling the expression of the p53 HFSN1p16sh and HFSN1C cells were treated with the transcrip- and p21 proteins in a p14-independent manner. To further show tion inhibitor actinomycin D, and then were reincubated for this, p16 was ectopically expressed in the p16-defective osteosar- different periods of time (0–6 hours). Total RNA was prepared coma U2OS cells and breast MCF10A cells (p16ORF) using empty and the mRNA level of MDM2 was assessed by qRT-PCR. Figure 2E vector as control (control), and the levels of the p53 and p21 shows that the MDM2 mRNA is more stable in HFSN1p16sh cells proteins were assessed in these cells by immunoblotting. Figure than in HFSN1C cells. Indeed, while the MDM2 mRNA half-life 1E shows a strong increase in the level of both p53 and p21 in in HFSN1C was 2 hours and 45 minutes, it increased to 6 hours p16ORF cells as compared with their control counterpart cells. in p16-deficient cells (Fig. 2E). This shows that p16 plays a This increase was cell-cycle–independent as there were no major major role in the turnover of the MDM2 mRNA in normal changes in the proportion of cells at G1 phase as well as the level of human fibroblasts. the proliferation marker Ki-67 (Fig. 1F). We have next assessed the senescence status of cells, as p16ORF can trigger senescence, which miR-141 and miR-146b-5p mediate the p16-negative regulation upregulates p53 (31, 43). Interestingly, p16-dependent increase of MDM2 in the level of p21 and p53 was also senescence-independent as To investigate the possible implication of miR-141 and p16ORF did not change cellular shape and did not increase miR-146b-5p, two major p16 downstream effector miRNAs b-galactosidase staining in MCF10A cells (Fig. 1F). Furthermore, (23), in the p16-dependent negative regulation of MDM2, the the level of the other important senescence marker Lamin B1 was levels of both mature miRNAs were assessed in U2OS and not affected in cells wherein p16 was ectopically expressed as MCF10A expressing either p16ORF or control plasmid by compared with their respective controls (Fig. 1E). These results qRT-PCR. While ectopic expression of p16 strongly decreased the show that ectopic expression of p16 enhances the level of p53 and level of the MDM2 mRNA (Fig. 2D), it significantly increased the p21 proteins in a cell-cycle/senescence-independent manner. expression of the mature forms of miR-141 and miR-146b-5p

www.aacrjournals.org Mol Cancer Res; 2018 OF3

Al-Khalaf and Aboussekhra

A BCHFSN1p16sh PD: 6 9 15 18 6 9 15 18 PD: 11 21 33 40 p16 HFSN1C p16 1.1 3.2 3.1 5.6 0.08 1.3 2.7 3.5 p53 1.0 1.4 2.6 3.1 1.6 1.1 0.7 0.5 p53 40 40 p21 0.9 1.8 2.5 2.7 p16 3.1 4.1 5.6 6.2 2.3 0.03 3.4 2.8 1.2 0.05 p21 β-Actin p53 0.1 1.6 2.2 3.7 0.54 0.2 MDM2 β-Actin p21 1.0 0.6 0.3 0.07 0.3 0.8 1.2 1.5 6.4 3.1 p14 p19 GAPDH 0.1 0.8 1.3 3.2 0.4 0.6 1.6 1.6 1.0 1.1 1.2 1.2 GAPDH MDM2 p14 MEF p16+/+ MEF p16-/- 2.1 1.0 0.6 0.1 12 10 GAPDH GAPDH HFSN1

DE F Control p16ORF G0–G1 60.33 G0–G1 54.70 S 18.79 S 26.31 NBL-p16sh NBL-Ctl Control p16ORF Control p16ORF

p16 p16 0 200 400 600 800 1,000 0 200 400 600 800 1,000 2.2 0.8 0.08 4.8 0.01 2.7 p53 p53 1.9 0.3 0.9 2.1 1.1 3.7 Ki-67 p21 p21 2.7 0.7 GAPDH 0.5 1.8 2.5 4.7 Lamin B1 p14 β 2.1 2.2 2.7 2.6 SA- -Gal 0.9 0.7 GAPDH GAPDH NBL-10 MCF10A U2OS MCF10A

Figure 1. p16 positively controls the p53 protein level in human and mouse cells. A–E, Whole-cell lysates were prepared from the indicated cells, and were utilized for immunoblotting using speciﬁc antibodies for the indicated proteins, and b-actin and GAPDH were used as internal controls. The numbers below the bands indicate the corresponding protein expression levels. PD represents population doublings. F, MCF10A cells were transfected with plasmids bearing either p16ORF or control plasmid. After 72 hours, cells were harvested and were stained with propidium iodide and cell-cycle distribution was analyzed by ﬂow cytometry. Top, Charts, the numbers indicate the proportion of cells in the different phases of the cell cycle. Bottom, Ki-67 and SA-b-gal stainings were performed on the indicated cells. Scale bars, 50 mm.

(Fig. 3A). This indicates that these two miRNAs are potential HFSN1C cells, which express both miRNAs, and a nonspecific negative regulators of MDM2 and mediates the p16-dependent sequence was used as control. Figure 3C shows that the inhibition negative regulation of the gene. To confirm this, HFSN1p16sh cells of miR-141 or miR-146b-5p increased the level of the MDM2 were transfected with plasmids bearing pre-miR-141, pre-miR- mRNA in HFSN1C cells. These results indicate that, like p16, miR- 146b-5p, or an empty vector used as control and the level of the 141 and miR-146b-5p negatively regulate the MDM2 expression. MDM2 mRNA was assessed by qRT-PCR. Figure 3B shows that ectopic expression of pre-miR-141 or pre-miR-146b-5p in miR-141 and miR-146b-5p control the expression of the MDM2 HFSN1p16sh cells significantly reduced the level of the MDM2 mRNA via its 30UTR mRNA 2.4- and 4-fold as compared with controls (p16sh-C; The analysis of miRNA databases showed that the MDM2 Fig. 3B). This indicates that miR-141 and miR-146b-5p are poten- 30UTR contains three potential binding sites for miR-141 located tial negative regulators of MDM2 and mediate the p16-dependent at bases 2066, 3706, and 4289 (mirSVR score ¼0.0002, negative regulation of the gene. To confirm this, miR-141 and miR- 0.0019, and 0.1846, respectively) and one potential binding 146b-5p were inhibited by specific anti-miRNAs (miRZips) in site for miR-146b-5p with high complementarity located at bases

OF4 Mol Cancer Res; 2018 Molecular Cancer Research

p16 Stabilizes p53 via MDM2 Destabilization

A B HFSN1C HFSN1p16sh 100 Time in minutes 0 20 40 60 90 120 0 20 40 60 90 120

p53 50 HFSN1C GAPDH

HFSN1p16sh

CD % p53 Protein remaining 10 0 20 40 60 90 120 p16ORF p16ORF Control Control Time in minutes

HFSN1C HFSN1p16sh MDM2 E MDM2 GAPDH 100 HFSN1p16sh GAPDH 0.030 0.10 0.025 0.08 50 0.0015 0.020 0.06 HFSN1C 0.015 0.0010 0.04 0.010

abundance 0.02 0.0005 0.005 10 mRNA Remaining % MDM2 mRNA abundance 0.000 p16ORF0.00 p16ORF 0 1 2 4 6 Relative MDM2 mRNA Control Control 0.0000 Time in hours

Relative MDM2 mRNA HFSN1CHFSN1p16sh MCF10A U2OS

Figure 2. p16 stabilizes the p53 protein and destabilizes the MDM2 mRNA. A, Exponentially growing cells were treated with cycloheximide (20 mg/mL) for the indicated periods of time, and then cell lysates were prepared and utilized for immunoblotting. B, The graph is showing the proportions of protein remaining. Dotted lines indicate the corresponding protein half-lives. Error bars represent means SD, values of three independent experiments (, P ¼ 7.06 105). C and D, whole-cell lysates were prepared from the indicated cells, and were utilized for immunoblotting using speciﬁc antibodies for the indicated proteins (top). Bottom, total RNA was prepared from the indicated cells and utilized to assess the level of the MDM2 mRNA by qRT-PCR. Error bars represent means SD, values of three independent experiments. , P 0.002395. E, Cells were treated with actinomycin D (5 mg/mL) for the indicated periods of time. Total RNA was prepared and the remaining amount of the MDM2 mRNA was assessed by qRT-PCR. The values at time 0 were considered as 100%. The dashed lines reveal the half-life (50% mRNA remaining) using regression analysis. Error bars represent means SD, values of three independent experiments (, P ¼ 0.000257).

4557–4575 (mirSVR score ¼0.0005; Fig. 3D, top). These was assessed by immunoblotting. Figure 3G shows that similar regions are highly conserved among different species (Fig. 3D, to p16 downregulation, inhibition of miR-141 or miR-146b-5p bottom). strongly increased the level of the MDM2 protein. However, the Next, we evaluated the potential contribution of the miR-141 expression of pre-miR-141 or pre-miR146b-5p in HFSN1p16sh and miR-146b-5p binding sites in the MDM2 mRNA 30UTR on the cells decreased the level of the MDM2 protein. These results control of the MDM2 expression. To this end, intact MDM2 30UTR confirm the role of miR-141 and miR-146b-5p as negative or a mutated sequence for these binding sites were inserted into a regulators of MDM2. To further confirm this, we investigated luciferase/Renilla reporter vector (Fig. 3E) and were introduced the effect of these miRNAs on p53. Figure 3G shows that the into HFSN1C cells. Figure 3F shows that the reporter activity fused increase in the level of MDM2 with inhibition of miR-141 or to the mutated sequence of miR-141 or miR-146b-5p binding miR-146b-5p led to a strong decrease in the expression of the sites on the MDM2 30UTR was significantly increased in HFSN1C p53 protein. However, the level of p53 was upregulated in cells as compared with the wild-type sequence. Furthermore, the response to the expression of pre-miR-141 or pre-miR-146b-5p reporter activity was increased when miR-141 or miR-146b-5p in HFSN1p16sh cells, as compared with its level in the control were inhibited in HFSN1C cells as compared with the control cells cells (Fig. 3G). These results indicate that miR-141 and miR- (Fig. 3F). This shows that the effect of miR-141 and miR-146b-5p 146b-5p target MDM2 at its 30UTR. on MDM2 is mediated through interaction with their seeding sequence in the MDM2 30UTR. p16 stabilizes the p53 protein in an MDM2-dependent manner To further elucidate the implication of miR-141 and Next, we sought to investigate the implication of MDM2 in the miR-146b-5p in the negative regulation of MDM2 by p16, p16-dependent stabilization of p53. To this end, p16-deficient whole-cell extracts were prepared from HFSN1 expressing cells (HFSN1p16sh) were transfected with a plasmid expressing either miRZip-141 or miRZip-146b-5p, an empty vector was either specific MDM2 shRNA (4 different sequences) or a scram- used as control. Subsequently, the level of the MDM2 protein bled sequence used as negative control. Total RNA was prepared

www.aacrjournals.org Mol Cancer Res; 2018 OF5

Al-Khalaf and Aboussekhra

A B C

0.012 U2OS 0.0014 0.0010 MCF10A 1.0 0.0012 0.010 0.0008 0.8 0.008 0.0010 0.6 0.0006 0.006 0.0008

0.004 0.4 0.0006 0.0004

0.002 0.2 0.0004 0.0002

Relative miR-141 expression 0.000 0.0 Controlp16ORFControlp16ORF Relative miR-146b-5p expression Controlp16ORFControlp16ORF 0.0002 0.0000 mRNA level Relative MDM2 mRNA 0.0000 Controlp16sh-Cp16sh-Pre-miR-141p16sh-Pre-miR-146b-5p mRNA abundance Relative MDM2 mRNA Control D miRZip-141 miRZip-146b-5p F WT MDM2 3'UTR G Mut MDM2 3'UTR/miR-141 Mut MDM2 3'UTR/miR-146b-5p

100

80 $ $ 60 miRZip141 p16sh-Pre-miR-141 Control p16sh 40 miRZip146b-5p p16sh-Pre-miR-146b-5p

activity (%) 20 MDM2 E 0 p53 Relative luciferase/Renilla Control GAPDH miRZip-141 miRZip-146b-5p

Figure 3. p16-dependent repression of MDM2 is mediated through miR-141 and miR-146b-5p. A–C, Total RNA was prepared from the indicated cells and utilized to assess the level of mature miR-141, mature miR-146b-5p, and the MDM2 mRNA by qRT-PCR. Error bars represent means SD, values of three independent experiments (, P 8.78 103). D, Sequence alignment of human miR-141 and miR-146b-5p binding sites in the MDM2 30UTR in different species. E, Schematic representation of the luciferase/Renilla reporter vector bearing the MDM2 30UTR. F, HFSN1 cells expressing miRZip-141, miRZip-146b-5p, or a control plasmid were stably transfected with the luciferase/Renilla reporter vector bearing either the wild type or a mutated sequence of the MDM2 30UTR. The reporter activity was assessed at 48 hours posttransfection. Data (mean SEM, n ¼ 4) were presented as % change in reporter activity as compared to the negative control cells () or to the wild-type 30UTR ($) (, P 0.0074; $, P 0.0004). G, Whole-cell lysates were prepared from the indicated cells, and were utilized for immunoblotting using speciﬁc antibodies for the indicated proteins.

from these cells and used to assess the level of the MDM2 mRNA utilized as control. The immunoblotting with anti-p53 antibody by qRT-PCR. Figure 4A shows that although the four sequences of showed high molecular weight p53 bands corresponding to the MDM2-shRNA decreased the level of the MDM2 mRNA, MDM2- ubiquitinated form of the protein that was increased in the shRNA-C showed the strongest effect. Subsequently, these cells HFSN1p16sh cells as compared with HFSN1C cells (Fig. 4D). were either sham-treated or challenged with cyclohexamide Interestingly, upon downregulation of MDM2 in the (20 mg/mL) for different periods of time, and the level of the HFSN1p16sh cells, the high molecular weight p53 band became p53 protein was assessed by immunoblotting. Figure 4B shows almost undetectable (Fig. 4D). Consequently, while the level of that the specific MDM2 downregulation stabilized p53 in p53 decreased in the input from HFSN1p16sh cells as compared HFSN1p16sh cells as compared with control cells. Indeed, while with HFSN1C cells, it increased upon downregulation of MDM2 p53 half-life was 35 minutes in control cells, it reached 110 in HFSN1p16sh cells (Fig. 4D). This shows that p16 controls p53 minutes in HFSN1p16sh-MDM2sh-C cells (Fig. 4C). This indi- stability in an MDM2-dependent manner. cates that p16 stabilizes the p53 protein through MDM2 repression. To further confirm this, and as MDM2 targets the p53 protein for ubiquitination, whole-cell extracts were prepared from Discussion HFSN1C as well as HFSN1p16sh cells expressing either Several lines of evidence indicate the presence of a positive MDM2-shRNA or a control plasmid, and specific anti-ubiquitin correlation between the tumor suppressor proteins p16 and p53. antibody was used for immunoprecipitation, while IgG was Indeed, both are upregulated during aging, senescence as well as

OF6 Mol Cancer Res; 2018 Molecular Cancer Research

p16 Stabilizes p53 via MDM2 Destabilization

ABHFSN1p16sh-Ctrl HFSN1p16sh-MDM2sh-C Time in minutes 0 20 40 60 90 120 0 20 40 60 90 120 0.0025 p53 0.0020 GAPDH

0.0015

0.0010 C

100 HFSN1p16sh-MDM2sh-C 0.0005 $

0.0000 ControlshRNA-AshRNA-BshRNA-CshRNA-D 50 mRNA expression level Relative MDM2 mRNA

HFSN1p16sh-Ctrl % p53 Protein remaining D IP: IgG Ub 10 Input 0 20 40 60 90 120 Time in minutes Control p16shRNA p16sh- MDM2sh-C Control p16shRNA p16sh- MDM2sh-C

70 Ub-p53 p53 50 IgG

Figure 4. p16 stabilizes the p53 protein in an MDM2-dependent manner. A, HFSN1p16sh cells were transfected with plasmids bearing either MDM2-shRNA (HFSN1p16sh-MDM2sh) or control plasmid (HFSN1p16sh-Ctrl). Total RNA was prepared from the indicated cells and utilized to assess the level of the MDM2 mRNA by qRT-PCR. Error bars represent means SD, values of three independent experiments ($, P ¼ 0.003). B and C, Figure legends are as in Fig. 2A and B, respectively. D, Whole-cell extracts were prepared from the indicated cells and utilized for immunoprecipitation using anti-ubiquitin antibody (mouse IgG was used as control). Immunoblotting was performed using specific p53 antibody. The input is the starting protein lysates was used as a positive control. in response to different cytotoxic agents (5–8, 22). Furthermore, effective positive regulator of p53 expression at the protein we have recently shown that the levels of p16 and p53 as well as level in both human and mice cells. This effect is mediated via their common target p21 are reduced in breast cancer-associated p16-dependent stabilization of the p53 protein through suppres- fibroblasts as compared with their adjacent counterparts from sing the expression of MDM2, which promotes the ubiquitin- histologically normal tissues (9, 10). Further investigations dependent degradation of p53 (35). This indicates that, in showed that p16 and p53 have non-cell–autonomous tumor absence of cellular stress, the presence of p16 is necessary for suppressor functions in stromal fibroblasts (9, 11, 12). In another the stability of the p53 protein, which becomes vulnerable to recent study, it has been reported that p16 and p53 are both MDM2-related degradation upon p16 downregulation. To this downregulated in breast stromal fibroblasts when exposed to the end, p16 destabilizes the MDM2 mRNA, which keeps the MDM2 cytokine IL6 (13). In addition, it has also been reported that the protein level under control and the consequent accumulation of levels of the p53 and p16 proteins are positively correlated in p53. These effects were shown during replicative aging of both triple-negative breast cancers (14, 15). However, the mechanisms human and mouse cells (Fig. 1A and C). underlying this positive correlation between these two important Importantly, specific downregulation of MDM2 in the tumor suppressor proteins are still not well defined. In this report, p16-deficient cells alleviated the stability and ubiquitination of we addressed this important question using different molecular the p53 protein and restored its normal level, which indicates the and cellular approaches, and have shown that p16 is indeed an capital role of MDM2 in mediating the p16-dependent regulation

www.aacrjournals.org Mol Cancer Res; 2018 OF7

Al-Khalaf and Aboussekhra

of p53 stability. It has been previously shown that p14ARF directly interacts with MDM2 to inhibit MDM2-dependent degradation of p53 (41, 42). However, we have shown here that the p16-dependent regulation of p53 during aging is ARF-independent because while p16 knockdown inhibited p53 accumulation, the levels of both p14 in human and p19 in mice cells were not affected by the status of p16 (Fig. 1). In addition, p14 level was not affected when p16 was specifically knocked down. In fact, aging-dependent p16 upregulation and p14 downregulation was previously present in MEF cells (8). We also present here the first indication that p16 negatively regulates MDM2 through miR-141 and miR-146b-5p, two important tumor suppressor miRNAs that are positively regulated by p16 (23). In fact, there is high complementarity between mature miR-141/miR-146b-5p and the MDM2 30UTR, which was respon- sive to both miRNAs. In line with these findings, we have shown the presence of an inverse correlation between the expression of Figure 5. Schematic representation of the p16-dependent regulation of the p53 p16/miR-141/miR-146b-5p and the level of their target MDM2. protein through miR-141- and miR-146b-5p–dependent inhibition of MDM2. Recent studies have demonstrated that miRNAs play an important See text for details. role in regulating the p53 function in tumor suppression through regulating the balance between p53 and MDM2 (46). Indeed, several miRNAs have been shown to activate p53 by repressing aging, p16 and p53 do not have antagonistic nor compensatory MDM2. Most of these miRNAs have been demonstrated to be relationship. Indeed, the levels of both proteins increase during able to inhibit cancer cell proliferation through promoting aging, senescence and upon genotoxic stress, while both decrease p53-mediated apoptosis, cell-cycle arrest, and/or senescence during cellular transdifferentiation into pluripotent stem cells (47–50). Some of them were also reported to repress the migra- (55–57). These results and others favor the presence of a positive tion and invasion of cancer cells and reverse epithelial-to- relationship between p16 and p53. In conclusion, we have mesenchymal transition to inhibit cancer metastasis (51, 52). shown here that, like p14ARF, p16 also positively regulates p53 However, no study to date has identified miR-141 or through inhibition of MDM2. While p14ARF exerts its effect at the miR-146b-5p as negative regulators of MDM2. Thus, these two protein level through formation of a tertiary complex with MDM2 miRNAs are new components of the p53 signaling pathway and p53 (42), p16 destabilizes the MDM2 mRNA in a miR-141/ through regulating the p53/MDM2 feedback loop. Importantly, 146b-5p–dependent manner, leading to p53 stabilization and we have recently shown that the levels of both miRNAs miR-141 accumulation (Fig. 5). and miR-146b-5p increase during aging of human HFSN1 cells in a p16-dependent manner, which also explains MDM2 down- Disclosure of Potential Conflicts of Interest regulation during the aging of these cells (Fig. 1A). However, our No potential conflicts of interest were disclosed. data do not exclude other mechanism(s) by which p16 may negatively regulate MDM2. Nonetheless, the relationship Authors' Contributions between p16 and MDM2 is of great importance because, besides Conception and design: H.H. Al-Khalaf, A. Aboussekhra p53, MDM2 influences the expression of many other genes Development of methodology: H.H. Al-Khalaf Acquisition of data (provided animals, acquired and managed patients, including p73, NFkB, TGFb, and E2F1 (53). These genes are fl provided facilities, etc.): important for apoptosis, tumorigenesis, viral replication, in am- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, mation, and various immune responses, which suggests a key role computational analysis): H.H. Al-Khalaf, A. Aboussekhra of p16 in all these processes, and explains p16-dependent regu- Writing, review, and/or revision of the manuscript: H.H. Al-Khalaf, lation of a large number of genes (22, 24). A. Aboussekhra Collectively, the current findings show another layer of cross- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H.H. Al-Khalaf talk between p16 and p53 through MDM2. However, they do not Study supervision: H. AlKhalaf, A. Aboussekhra exclude that p16 may regulate the level of the p53 protein in an MDM2-independent manner via targeting and/or association Acknowledgments with some of the p53 regulators. The authors are very thankful to the Office of Research Affairs (ORA) at the On the other hand, it has been previously shown that there is an KFSH&RC for the continuous support. This work was carried out under the RAC antagonistic relationship between p16 and p53 in epithelial but proposal #2090027. This work was entirely supported by King Faisal Specialist not in fibroblast cells, and that reduced levels of the p16 protein Hospital and Research Centre. stabilizes the p53 protein through inhibition of its proteolytic degradation (54). The reasons of this discrepancy are unclear, but The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked could be owing to the use of different cells and/or different advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate knockdown systems. Nonetheless, based on our current data this fact. indicating the presence of positive link between p16 and p53, as well as data from other studies showing that during several Received January 8, 2018; revised March 22, 2018; accepted April 24, 2018; physiologic processes, such as cellular response to stresses and published first May 8, 2018.

OF8 Mol Cancer Res; 2018 Molecular Cancer Research

p16 Stabilizes p53 via MDM2 Destabilization

References 1. Ruas M, Peters G. The p16INK4a/CDKN2A tumor suppressor and its 24. Vernell R, Helin K, Muller H. Identification of target genes of the relatives. Biochim Biophys Acta 1998;1378:F115–77. p16INK4A-pRB-E2F pathway. J Biol Chem 2003;278:46124–37. 2. Rocco JW, Sidransky D. p16(MTS-1/CDKN2/INK4a) in cancer progression. 25. Shapiro GI, Edwards CD, Ewen ME, Rollins BJ. p16INK4A participates in a Exp Cell Res 2001;264:42–55. G1 arrest checkpoint in response to DNA damage. Mol Cell Biol 1998; 3. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in 18:378–87. human cancers. Science 1991;253:49–53. 26. Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC. Involve- 4. Al-Khalaf HH, Hendrayani SF, Aboussekhra A. The atr protein kinase ment of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative controls UV-dependent upregulation of p16INK4A through inhibition of senescence of normal human fibroblasts. Proc Natl Acad Sci U S A 1996; Skp2-related polyubiquitination/degradation. Mol Cancer Res 2011; 93:13742–7. 9:311–9. 27. Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, et al. 5. Lu X, Lane DP. Differential induction of transcriptionally active p53 Oncogene-induced senescence is a DNA damage response triggered by following UV or ionizing radiation: defects in chromosome instability DNA hyper-replication. Nature 2006;444:638–42. syndromes? Cell 1993;75:765–78. 28. Chavey C, Bibeau F, Gourgou-Bourgade S, Burlinchon S, Boissiere F, Laune 6. Hendrayani SF, Al-Khalaf HH, Aboussekhra A. Curcumin triggers p16- D, et al. Oestrogen receptor negative breast cancers exhibit high cytokine dependent senescence in active breast cancer-associated fibroblasts and content. Breast Cancer Res 2007;9:R15. suppresses their paracrine procarcinogenic effects. Neoplasia 2013;15: 29. Al-Mohanna MA, Manogaran PS, Al-Mukhalafi Z, A Al-Hussein K, 631–40. Aboussekhra A. The tumor suppressor p16(INK4a) gene is a regulator 7. Rodier F, Campisi J, Bhaumik D. Two faces of p53: aging and tumor of apoptosis induced by ultraviolet light and cisplatin. Oncogene suppression. Nucleic Acids Res 2007;35:7475–84. 2004;23:201–12. 8. Zindy F, Quelle DE, Roussel MF, Sherr CJ. Expression of the p16INK4a 30. Papazoglu C, Mills AA. p53: at the crossroad between cancer and ageing. tumor suppressor versus other INK4 family members during mouse J Pathol 2007;211:124–33. development and aging. Oncogene 1997;15:203–11. 31. Rufini A, Tucci P, Celardo I, Melino G. Senescence and aging: the critical 9. Al-Ansari MM, Hendrayani SF, Shehata AI, Aboussekhra A. p16(INK4A) roles of p53. Oncogene 2013;32:5129–43. represses the paracrine tumor-promoting effects of breast stromal fibro- 32. Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-medi- blasts. Oncogene 2013;32:2356–64. ated tumour suppression. Nat Rev Cancer 2014;14:359–70. 10. Hawsawi NM, Ghebeh H, Hendrayani SF, Tulbah A, Al-Eid M, Al-Tweigeri 33. Zhang, Wang H. MDM2 oncogene as a novel target for human cancer T, et al. Breast carcinoma-associated fibroblasts and their counterparts therapy. Curr Pharm Des 2000;6:393–416. display neoplastic-specific changes. Cancer Res 2008;68:2717–25. 34. Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW, Vogelstein B. 11. Kiaris H, Chatzistamou I, Trimis G, Frangou-Plemmenou M, Pafiti-Kondi Oncoprotein MDM2 conceals the activation domain of tumour suppressor A, Kalofoutis A. Evidence for nonautonomous effect of p53 tumor sup- p53. Nature 1993;362:857–60. pressor in carcinogenesis. Cancer Res 2005;65:1627–30. 35. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation 12. Lujambio A, Akkari L, Simon J, Grace D, Tschaharganeh DF, Bolden JE, of p53. Nature 1997;387:296–9. et al. Non-cell-autonomous tumor suppression by p53. Cell 2013;153: 36. Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM. Mdm2 is a RING 449–60. finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 13. Hendrayani SF, Al-Khalaf HH, Aboussekhra A. The cytokine IL-6 reactivates 2000;275:8945–51. breast stromal fibroblasts through transcription factor STAT3-dependent 37. Ghebeh H, Sleiman GM, Manogaran PS, Al-Mazrou A, Barhoush E, up-regulation of the RNA-binding protein AUF1. J Biol Chem 2014;289: Al-Mohanna FH, et al. Profiling of normal and malignant breast tissue 30962–76. show CD44high/CD24low phenotype as a predominant stem/progenitor 14. Shan M, Zhang X, Liu X, Qin Y, Liu T, Liu Y, et al. P16 and p53 play distinct marker when used in combination with Ep-CAM/CD49f markers. BMC roles in different subtypes of breast cancer. PLoS One 2013;8:e76408. Cancer 2013;13:289. 15. Silva J, Silva JM, Dominguez G, Garcia JM, Cantos B, Rodriguez R, et al. 38. Bond J, Jones C, Haughton M, DeMicco C, Kipling D, Wynford-Thomas D. Concomitant expression of p16INK4a and p14ARF in primary breast Direct evidence from siRNA-directed "knock down" that p16(INK4a) is cancer and analysis of inactivation mechanisms. J Pathol 2003;199: required for human fibroblast senescence and for limiting ras-induced 289–97. epithelial cell proliferation. Exp Cell Res 2004;292:151–6. 16. Sarkisian CJ, Keister BA, Stairs DB, Boxer RB, Moody SE, Chodosh LA. Dose- 39. Al-Mohanna MA, Al-Khalaf HH, Al-Yousef N, Aboussekhra A. The dependent oncogene-induced senescence in vivo and its evasion during p16INK4a tumor suppressor controls p21WAF1 induction in response to mammary tumorigenesis. Nat Cell Biol 2007;9:493–505. ultraviolet light. Nucleic Acids Res 2007;35:223–33. 17. Koh J, Enders GH, Dynlacht BD, Harlow E. Tumour-derived p16 alleles 40. Al-Khalaf HH, Nallar SC, Kalvakolanu DV, Aboussekhra A. p16INK4A encoding proteins defective in cell-cycle inhibition. Nature 1995;375: enhances the transcriptional and the apoptotic functions of p53 through 506–10. DNA-dependent interaction. Mol Carcinog 2017;56:1687–1702. 18. Lukas J, Parry D, Aagaard L, Mann DJ, Bartkova J, Strauss M, et al. 41. Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour et al. The Ink4a tumor suppressor gene product, p19Arf, interacts suppressor p16. Nature 1995;375:503–6. with MDM2 and neutralizes MDM20s inhibition of p53. Cell 1998;92: 19. Sherr CJ. The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell 713–23. Biol 2001;2:731–7. 42. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and 20. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 control causing specific inhibition of cyclin D/CDK4. Nature 1993;366: tumor suppression pathways. Cell 1998;92:725–34. 704–7. 43. Itahana K, Dimri G, Campisi J. Regulation of cellular senescence by p53. 21. Giacinti C, Giordano A. RB and cell cycle progression. Oncogene 2006;25: Eur J Biochem 2001;268:2784–91. 5220–7. 44. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 22. Al-Khalaf HH, Colak D, Al-Saif M, Al-Bakheet A, Hendrayani SF, Al-Yousef for tumor suppressor p53. FEBS Lett 1997;420:25–7. N, et al. p16(INK4a) positively regulates cyclin D1 and E2F1 through 45. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by negative control of AUF1. PLoS One 2011;6:e21111. Mdm2. Nature 1997;387:299–303. 23. Al-Khalaf HH, Mohideen P, Nallar SC, Kalvakolanu DV, Aboussekhra A. 46. Zhang C, Liu J, Wang X, Feng Z. The regulation of the p53/MDM2 feedback The cyclin-dependent kinase inhibitor p16INK4a physically interacts loop by microRNAs. RNA Dis 2015;2:e502. with transcription factor Sp1 and cyclin-dependent kinase 4 to trans- 47. Hermeking H. MicroRNAs in the p53 network: micromanagement of activate microRNA-141 and microRNA-146b-5p spontaneously and in tumour suppression. Nat Rev Cancer 2012;12:613–26. response to ultraviolet light-induced DNA damage. J Biol Chem 48. Rokavec M, Li H, Jiang L, Hermeking H. The p53/microRNA connection in 2013;288:35511–25. gastrointestinal cancer. Clin Exp Gastroenterol 2014;7:395–413.

www.aacrjournals.org Mol Cancer Res; 2018 OF9

Al-Khalaf and Aboussekhra

49. Hoffman Y, Pilpel Y, Oren M. microRNAs and Alu elements in the 53. Biderman L, Manley JL, Prives C. Mdm2 and MdmX as Regulators of Gene p53-Mdm2-Mdm4 regulatory network. J Mol Cell Biol 2014;6: Expression. Genes Cancer 2012;3:264–73. 192–7. 54. Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and 50. Zhang C, Liu J, Wang X, Wu R, Lin M, Laddha SV, et al. MicroRNA-339–5p tumor suppressors. Dev Biol 2007;302:1–12. inhibits colorectal tumorigenesis through regulation of the MDM2/p53 55. Li H, Collado M, Villasante A, Strati K, Ortega S, Canamero M, et al. The signaling. Oncotarget 2014;5:9106–17. Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 2009;460: 51. Dar AA, Majid S, Rittsteuer C, de Semir D, Bezrookove V, Tong S, et al. The 1136–9. role of miR-18b in MDM2-p53 pathway signaling and melanoma pro- 56. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, et al. gression. J Natl Cancer Inst 2013;105:433–42. Linking the p53 tumour suppressor pathway to somatic cell reprogram- 52. Fortunato O, Boeri M, Moro M, Verri C, Mensah M, Conte D, et al. Mir-660 ming. Nature 2009;460:1140–4. is downregulated in lung cancer patients and its replacement inhibits 57. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, et al. lung tumorigenesis by targeting MDM2-p53 interaction. Cell Death Dis Suppression of induced pluripotent stem cell generation by the p53-p21 2014;5:e1564. pathway. Nature 2009;460:1132–5.

OF10 Mol Cancer Res; 2018 Molecular Cancer Research

p16 Controls p53 Protein Expression Through miR-dependent Destabilization of MDM2

Huda H. Al-Khalaf and Abdelilah Aboussekhra

Mol Cancer Res Published OnlineFirst May 8, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-0017

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/early/2018/06/13/1541-7786.MCR-18-0017. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.