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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (4) : 420-429     DOI: 10.1007/s11684-016-0478-3
Repression of CDKN2C caused by PML/RARα binding promotes the proliferation and differentiation block in acute promyelocytic leukemia
Xiaoling Wang1,Yun Tan1,Yizhen Li1,Jingming Li1,Wen Jin1,2,3(),Kankan Wang1,2,3()
1. State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China
3. Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Inappropriate cell proliferation during oncogenesis is often accompanied by inactivation of components involved in the cell cycle machinery. Here, we report that cyclin-dependent kinase inhibitor 2C (CDKN2C) as a member of the cyclin-dependent kinase inhibitors is a target of the PML/RARα oncofusion protein in leukemogenesis of acute promyelocytic leukemia (APL). We found that CDKN2C was markedly downregulated in APL blasts compared with normal promyelocytes. Chromatin immunoprecipitation combined with quantitative polymerase chain reaction demonstrated that PML/RARα directly bound to the CDKN2C promoter in the APL patient-derived cell line NB4. Luciferase assays indicated that PML/RARα inhibited the CDKN2C promoter activity in a dose-dependent manner. Furthermore, all-trans retinoic acid treatment induced CDKN2C expression by releasing the PML/RARα binding on chromatin in NB4 cells. Functional studies showed that ectopic expression of CDKN2C induced a cell cycle arrest at the G0/G1 phase and a partial differentiation in NB4 cells. Finally, the transcriptional regulation of CDKN2C was validated in primary APL patient samples. Collectively, this study highlights the importance of CDKN2C inactivation in the abnormal cell cycle progression and differentiation block of APL cells and may provide new insights into the study of pathogenesis and targeted therapy of APL.

Keywords CDKN2C      acute promyelocytic leukemia      cell cycle arrest      differentiation     
Corresponding Authors: Wen Jin,Kankan Wang   
Just Accepted Date: 25 October 2016   Online First Date: 23 November 2016    Issue Date: 01 December 2016
URL:     OR
Fig.1  Repression of CDKN2C by PML/RARα in APL cells. (A) CDKN2C was significantly lower expressed in APL patient cells compared with the normal promyelocytes. We analyzed the mRNA expression levels of CDKN2C in APL patient samples (n = 14) and normal promyelocytes (n = 5) and compared their absolute intensities of mRNA expression values after log transformation. ** P<0.01. (B) The expression level of CDKN2C was repressed by PML/RARα. The mRNA expression levels of PML/RARα and CDKN2C were examined five days after transfection of the expression plasmid of PML/RARα or the empty vector into U937 cells.
Fig.2  Direct binding of PML/RARα on the CDKN2C promoter in APL cells. (A) Overview of PML/RARα binding sites on the CDKN2C promoter in NB4 cells. ChIP assays were carried out in NB4 cells using anti-PML and anti-RARα antibodies. The arrow denotes the transcription direction of CDKN2C. The peaks represent the enriched regions ChIPed by anti-PML and anti-RARα antibodies. (B) PML/RARα was significantly enriched on the CDKN2C promoter in NB4 cells. The DNA binding of PML/RARα on the CDKN2C promoter was analyzed by ChIP-qPCR assays. NC was amplified by a pair of negative primers for an irrelevant region. ChIP with the normal rabbit immunoglobulin G (IgG) was used as a negative control. The fold change of PML/RARa enrichment was calculated relative to normal IgG. Data represent the average of three replicates±SD.
Fig.3  PML/RARα repressing the promoter activity of CDKN2C. (A) Schematic diagram showing the promoter region of the CDKN2C constructed into the luciferase report vector (pGL3-CDKN2C). The gray rectangles represent the peak regions bound by PML/RARα. (B) PML/RARα represses the activity of CDKN2C promoter in a dose-dependent manner. Increasing quantities of the PML/RARα expression plasmid (pSG5-PML/RARα) were cotransfected into 293T cells with the CDKN2C luciferase reporter plasmid (pGL3-CDKN2C). The relative luciferase activity was standard with the activity of pGL3-CDKN2C cotransfected with the pSG5 empty expression vector. Data represent the average of three replicates±SD.
Fig.4  Induction of CDKN2C expression by ATRA in APL cells. (A, B) The relative mRNA and protein levels of CDKN2C were increased after ATRA treatment of NB4 cells for the indicated times. The mRNA levels of CDKN2C were detected by qRT-PCR assays and normalized to GAPDH expression. The relative mRNA expressions were all compared with the values in ATRA-untreated NB4 cells (Ctrl). (C) ATRA severely decreased the binding capacity of PML/RARα on the CDKN2C promoter after ATRA treatment of NB4 cells for 24 h. ChIP-qPCR assays were carried out using anti-PML and anti-RARα antibodies before and after ATRA treatment of NB4 cells for 24 h. ChIP using normal IgG was used as a negative control. The fold change of PML/RARα enrichment by anti-PML and anti-RARα antibodies is calculated relative to normal IgG. (D) The repression of CDKN2C was only conferred by PML/RARα. NB4 cells were pretreated with or without CHX at a final concentration of 10 mg/ml for 30 min and then treated with 1 mmol/L ATRA for 8 h. Data represent the mean of three replicates±SD. * P<0.05, ** P<0.01, *** P<0.001. ns, non-significant.
Fig.5  Role of CDKN2C in cell cycle arrest and differentiation of APL cells. (A) The mRNA and protein levels of CDKN2C after overexpression. (B) Restored expression of CDKN2C induced the G0/G1 phase arrest. GFP-positive NB4 cells were sorted by FACS three days after infection. Then, the distribution of cell cycle phases was analyzed by flow cytometry analysis. Data represent the mean of three replicates±SD. (C) Ectopic expression of CDKN2D induced a partial differentiation. Flow cytometry analysis of cell surface marker CD11b in GFP-positive NB4 cells is shown on the left. Wright’s staining of GFP-positive NB4 cells is shown on the right. Data represent the mean of three replicates±SD. *** P<0.001.
Fig.6  Validation of the PML/RARα binding to the CDKN2C locus and the increase of CDKN2C expression by ATRA in primary APL patient cells. (A) Overview of the PML/RARα binding sites on CDKN2C in APL patient blast cells. Blue represents the PML ChIP-seq signals retrieved from the ChIP-seq profiling performed by Martens et al. (GSE18886) [6]. (B) ATRA increased the mRNA level of CDKN2C expression in primary APL patient cells. The gene expression data for CDKN2C were retrieved from microarray gene expression profiling performed by Stegmaier et al. (GSE976) using U133A Affymetrix microarrays [26]. (C) A schematic illustration of CDKN2C expression in an APL patient sample before and after ATRA treatment. The peak represents the RNA-seq signals for CDKN2C. The RefSeq annotation for CDKN2C is shown at the bottom. The expression values are the absolute intensities.
1 Lo-Coco F, Di Donato L; GIMEMA, Schlenk RF; German–Austrian Acute Myeloid Leukemia Study Group and Study Alliance Leukemia. Targeted therapy alone for acute promyelocytic leukemia. N Engl J Med 2016; 374(12): 1197–1198
doi: 10.1056/NEJMc1513710 pmid: 27007970
2 Burnett AK, Russell NH, Hills RK, Bowen D, Kell J, Knapper S, Morgan YG, Lok J, Grech A, Jones G, Khwaja A, Friis L, McMullin MF, Hunter A, Clark RE, Grimwade D; UK National Cancer Research Institute Acute Myeloid Leukaemia Working Group. Arsenic trioxide and all-trans retinoic acid treatment for acute promyelocytic leukaemia in all risk groups (AML17): results of a randomised, controlled, phase 3 trial. Lancet Oncol 2015; 16(13): 1295–1305
doi: 10.1016/S1470-2045(15)00193-X pmid: 26384238
3 Mi JQ, Chen SJ, Zhou GB, Yan XJ, Chen Z. Synergistic targeted therapy for acute promyelocytic leukaemia: a model of translational research in human cancer. J Intern Med 2015; 278(6): 627–642
doi: 10.1111/joim.12376 pmid: 26058416
4 de Thé H, Chen Z. Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat Rev Cancer 2010; 10(11): 775–783
doi: 10.1038/nrc2943 pmid: 20966922
5 Wang K, Wang P, Shi J, Zhu X, He M, Jia X, Yang X, Qiu F, Jin W, Qian M, Fang H, Mi J, Yang X, Xiao H, Minden M, Du Y, Chen Z, Zhang J. PML/RARalpha targets promoter regions containing PU.1 consensus and RARE half sites in acute promyelocytic leukemia. Cancer Cell 2010; 17(2): 186–197
doi: 10.1016/j.ccr.2009.12.045 pmid: 20159610
6 Martens JH, Brinkman AB, Simmer F, Francoijs KJ, Nebbioso A, Ferrara F, Altucci L, Stunnenberg HG. PML-RARα/RXR alters the epigenetic landscape in acute promyelocytic leukemia. Cancer Cell 2010; 17(2): 173–185
doi: 10.1016/j.ccr.2009.12.042 pmid: 20159609
7 Hoemme C, Peerzada A, Behre G, Wang Y, McClelland M, Nieselt K, Zschunke M, Disselhoff C, Agrawal S, Isken F, Tidow N, Berdel WE, Serve H, Müller-Tidow C. Chromatin modifications induced by PML-RARα repress critical targets in leukemogenesis as analyzed by ChIP-Chip. Blood 2008; 111(5): 2887–2895
doi: 10.1182/blood-2007-03-079921 pmid: 18024792
8 Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development 2013; 140(15): 3079–3093
doi: 10.1242/dev.091744 pmid: 23861057
9 Franklin DS, Godfrey VL, O’Brien DA, Deng C, Xiong Y. Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity. Mol Cell Biol 2000; 20(16): 6147–6158
doi: 10.1128/MCB.20.16.6147-6158.2000 pmid: 10913196
10 Ramsey MR, Krishnamurthy J, Pei XH, Torrice C, Lin W, Carrasco DR, Ligon KL, Xiong Y, Sharpless NE. Expression of p16Ink4a compensates for p18Ink4c loss in cyclin-dependent kinase 4/6-dependent tumors and tissues. Cancer Res 2007; 67(10): 4732–4741
doi: 10.1158/0008-5472.CAN-06-3437 pmid: 17510401
11 Franklin DS, Godfrey VL, Lee H, Kovalev GI, Schoonhoven R, Chen-Kiang S, Su L, Xiong Y. CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev 1998; 12(18): 2899–2911
doi: 10.1101/gad.12.18.2899 pmid: 9744866
12 Drexler HG. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells. Leukemia 1998; 12(6): 845–859
doi: 10.1038/sj.leu.2401043 pmid: 9639410
13 Guo SX, Taki T, Ohnishi H, Piao HY, Tabuchi K, Bessho F, Hanada R, Yanagisawa M, Hayashi Y. Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia. Leuk Res 2000; 24(1): 39–46
doi: 10.1016/S0145-2126(99)00158-7 pmid: 10634644
14 Ragione FD, Iolascon A. Inactivation of cyclin-dependent kinase inhibitor genes and development of human acute leukemias. Leuk Lymphoma 1997; 25(1-2): 23–35
doi: 10.3109/10428199709042493 pmid: 9130611
15 Casini T, Pelicci PG. A function of p21 during promyelocytic leukemia cell differentiation independent of CDK inhibition and cell cycle arrest. Oncogene 1999; 18(21): 3235–3243
doi: 10.1038/sj.onc.1202630 pmid: 10359529
16 Wang Y, Jin W, Jia X, Luo R, Tan Y, Zhu X, Yang X, Wang X, Wang K. Transcriptional repression of CDKN2D by PML/RARa contributes to the altered proliferation and differentiation block of acute promyelocytic leukemia cells. Cell Death Dis 2014; 5(10): e1431
doi: 10.1038/cddis.2014.388 pmid: 25275592
17 Thullberg M, Bartkova J, Khan S, Hansen K, Rönnstrand L, Lukas J, Strauss M, Bartek J. Distinct versus redundant properties among members of the INK4 family of cyclin-dependent kinase inhibitors. FEBS Lett 2000; 470(2): 161–166
doi: 10.1016/S0014-5793(00)01307-7 pmid: 10734227
18 Pei XH, Bai F, Tsutsui T, Kiyokawa H, Xiong Y. Genetic evidence for functional dependency of p18Ink4c on Cdk4. Mol Cell Biol 2004; 24(15): 6653–6664
doi: 10.1128/MCB.24.15.6653-6664.2004 pmid: 15254233
19 Bai F, Pei XH, Godfrey VL, Xiong Y. Haploinsufficiency of p18(INK4c) sensitizes mice to carcinogen-induced tumorigenesis. Mol Cell Biol 2003; 23(4): 1269–1277
doi: 10.1128/MCB.23.4.1269-1277.2003 pmid: 12556487
20 Latres E, Malumbres M, Sotillo R, Martín J, Ortega S, Martín-Caballero J, Flores JM, Cordón-Cardo C, Barbacid M. Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis. EMBO J 2000; 19(13): 3496–3506
doi: 10.1093/emboj/19.13.3496 pmid: 10880462
21 Leone PE, Walker BA, Jenner MW, Chiecchio L, Dagrada G, Protheroe RK, Johnson DC, Dickens NJ, Brito JL, Else M, Gonzalez D, Ross FM, Chen-Kiang S, Davies FE, Morgan GJ. Deletions of CDKN2C in multiple myeloma: biological and clinical implications. Clin Cancer Res 2008; 14(19): 6033–6041
doi: 10.1158/1078-0432.CCR-08-0347
22 Jalili A, Wagner C, Pashenkov M, Pathria G, Mertz KD, Widlund HR, Lupien M, Brunet JP, Golub TR, Stingl G, Fisher DE, Ramaswamy S, Wagner SN. Dual suppression of the cyclin-dependent kinase inhibitors CDKN2C and CDKN1A in human melanoma. J Natl Cancer Inst 2012; 104(21): 1673–1679
doi: 10.1093/jnci/djs373 pmid: 22997239
23 Cui H, Zhao C, Gong P, Wang L, Wu H, Zhang K, Zhou R, Wang L, Zhang T, Zhong S, Fan H. DNA methyltransferase 3A promotes cell proliferation by silencing CDK inhibitor p18INK4C in gastric carcinogenesis. Sci Rep 2015; 5: 13781
doi: 10.1038/srep13781 pmid: 26350239
24 Payton JE, Grieselhuber NR, Chang LW, Murakami M, Geiss GK, Link DC, Nagarajan R, Watson MA, Ley TJ. High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples. J Clin Invest 2009; 119(6): 1714–1726
doi: 10.1172/JCI38248 pmid: 19451695
25 Qian M, Jin W, Zhu X, Jia X, Yang X, Du Y, Wang K, Zhang J. Structurally differentiated cis-elements that interact with PU.1 are functionally distinguishable in acute promyelocytic leukemia. J Hematol Oncol 2013; 6(1): 25
doi: 10.1186/1756-8722-6-25 pmid: 23547873
26 Stegmaier K, Ross KN, Colavito SA, O’Malley S, Stockwell BR, Golub TR. Gene expression-based high-throughput screening (GE-HTS) and application to leukemia differentiation. Nat Genet 2004; 36(3): 257–263
doi: 10.1038/ng1305 pmid: 14770183
27 Forget A, Ayrault O, den Besten W, Kuo ML, Sherr CJ, Roussel MF. Differential post-transcriptional regulation of two Ink4 proteins, p18 Ink4c and p19 Ink4d. Cell Cycle 2008; 7(23): 3737–3746
doi: 10.4161/cc.7.23.7187 pmid: 19029828
28 Zindy F, den Besten W, Chen B, Rehg JE, Latres E, Barbacid M, Pollard JW, Sherr CJ, Cohen PE, Roussel MF. Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d). Mol Cell Biol 2001; 21(9): 3244–3255
doi: 10.1128/MCB.21.9.3244-3255.2001 pmid: 11287627
29 Kim WY, Sharpless NE. The regulation of INK4/ARF in cancer and aging. Cell 2006; 127(2): 265–275
doi: 10.1016/j.cell.2006.10.003 pmid: 17055429
30 Ruas M, Peters G. The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1998; 1378(2): F115–F177
pmid: 9823374
31 Phelps DE, Hsiao KM, Li Y, Hu N, Franklin DS, Westphal E, Lee EY, Xiong Y. Coupled transcriptional and translational control of cyclin-dependent kinase inhibitor p18INK4c expression during myogenesis. Mol Cell Biol 1998; 18(4): 2334–2343
doi: 10.1128/MCB.18.4.2334 pmid: 9528803
32 Morse L, Chen D, Franklin D, Xiong Y, Chen-Kiang S. Induction of cell cycle arrest and B cell terminal differentiation by CDK inhibitor p18(INK4c) and IL-6. Immunity 1997; 6(1): 47–56
doi: 10.1016/S1074-7613(00)80241-1 pmid: 9052836
33 Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nat Cell Biol 2004; 6(5): 436–442
doi: 10.1038/ncb1126 pmid: 15122268
34 Yu H, Yuan Y, Shen H, Cheng T. Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners. Blood 2006; 107(3): 1200–1206
doi: 10.1182/blood-2005-02-0685 pmid: 16234365
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