ORIGINAL ARTICLE Cyclin-dependent kinase 1 expression is inhibited by p16 INK4a at the post-transcriptional level through the microRNA pathway WW Chien 1 , C Domenech 1 , R Catallo 1 , T Kaddar 1 , J-P Magaud 1,2 , G Salles 1,3 and M Ffrench 1,2 1 Universite´Claude Bernard Lyon 1, CNRS UMR 5239 ENS—HCL, Faculte´de Me´decine Lyon Sud, Oullins, France; 2 Laboratoire d’He´matologie, CHLS, Hospices Civils de Lyon, 165 Chemin du Grand Revoyet, Pierre-Be´nite, France and 3 Serviced’He´matologie, CHLS, Hospices Civils de Lyon, Pierre-Be´nite, France The p16 INK4a protein regulates cell cycle progression mainly by inhibiting the activity of G1-phase cyclin-dependent kinases (CDKs) 4 and 6, the subsequent retinoblastoma protein (pRb) phosphorylation and E2F transcription factor release. The p16 INK4a protein can also repress the activity of other transcription factors, such as c-myc, nuclear factor- kappaB and c-Jun/AP1. Here, we report that, in two p16 À/À , pRb WT and p53 WT cell lines (MCF7 and U87), p16 INK4a overexpression induces a dramatic decrease in CDK1 protein expression. In response to p16 INK4a , the decreased rate of CDK1 protein synthesis, its unchanged protein half-life, unreduced CDK1 mRNA steady-state levels and mRNA half-life allow us to hypothesize that p16 INK4a could regulate CDK1 expression at the post-transcriptional level. This CDK1 downregulation is mediated by the 3 0 -untranslated region (3 0 UTR) of CDK1 mRNA as shown by translational inhibition in luciferase assays and is associated with a modified expression balance of microRNAs (miRNAs) that potentially regulate CDK1, analyzed by TaqMan Human microRNA Array. The p16 INK4a -induced expression of two miRNAs (miR-410 and miR-650 chosen as an example) in MCF7 cells is confirmed by individual reverse transcription– qPCR. Furthermore, we show the interaction of miR-410 or miR-650 with CDK1-3 0 UTR by luciferase assays. Endogen- ous CDK1 expression decreases upon both miRNA over- expression and increases with their simultaneous inhibition. The induction of miR-410, but not miR-650 could be related to the pRb/E2F pathway. These results demonstrate the post-transcriptional inhibition of CDK1 by p16 INK4a . We suggest that p16 INK4a may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs. Oncogene (2011) 30, 1880–1891; doi:10.1038/onc.2010.570; published online 20 December 2010 Keywords: p16 INK4a ; CDK1; cell cycle; microRNA; E2F; post-transcriptional regulation Introduction The p16 INK4a protein, encoded by the INK4a gene on chromosome 9p21 (Serrano et al., 1993; Kamb et al., 1994), is widely considered a tumor suppressor as its inactivation is frequently observed in a large variety of human malignancies (Cairns et al., 1995), and INK4a knockout also increases cancer incidence in mice (Serrano et al., 1996). p16 INK4a acts as a tumor suppressor through multiple biological functions, in- cluding the inhibition of cell cycle progression (Serrano et al., 1993), the induction of senescence (Hara et al., 1996) and differentiation (Urashima et al., 1997), and its involvement in apoptosis (Kataoka et al., 2000) and DNA repair (Shapiro et al., 1998). The p16 INK4a protein can also impair tumor metastasis by affecting the expression of proteins involved in cell adhesion and angiogenesis (Harada et al., 1999; Nalabothula et al., 2007). Furthermore, an emerging concept about cancer stem cells, which have self-renewal ability to perpetuate the disease, has been reported (Kopper and Hajdu, 2004; Cox et al., 2007): the imbalance in the network, involving p16 INK4a and its transcriptional inhibitor Bmi- 1, can affect stem cells self-renewal and ultimately tumor development (Molofsky et al., 2003, 2006). In the centre of these orchestrated metabolic net- works, p16 INK4a has an important regulatory role in the maintenance of homeostasis. First of all, through the inhibition of the activity of cyclin-dependent kinase 4 (CDK4) and CDK6, G1-phase cell cycle kinases, p16 INK4a is involved in the transcriptional regulation of genes through CDK4/6-Rb-E2F/c-myc pathway (Serrano et al., 1993; Haas et al., 1997). p16 INK4a also prevents transcriptional activity of nuclear factor- kappaB (Wolff and Naumann, 1999) and, through JNK1/3, c-Jun/AP1 (Choi et al., 2005), and is thought to inhibit RNA polymerase II activity by CDK7 (Nishiwaki et al., 2000). A proteomic study has shown potential interactions of p16 INK4a with other proteins involved in the metabolism of RNA (U1 snRNP-A, snRNP-B, heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, hnRNP A2/B1 and RuvB-like2) and of protein (HSP90, HSPA8, HS70RY, USP26 and EF-2), suggesting that p16 INK4a may regulate gene expression at other levels in addition to transcription (Souza-Rodrigues et al., 2007). Received 12 May 2010; revised 13 October 2010; accepted 12 November 2010; published online 20 December 2010 Correspondence: Dr M Ffrench, Laboratoire Pathologies des cellules lymphoı¨des, Universite´ Claude Bernard Lyon 1, CNRS UMR 5239 ENS—HCL, Faculte´ de Me´decine Lyon-Sud, 165 Chemin du Grand Revoyet, Oullins cedex, Rhone Alpes 69495, France. E-mail: martine.ffrench@chu-lyon.fr Oncogene (2011) 30, 1880–1891 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc