(2014) 33, 5415–5423 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE The role of p21waf1/cip1 and p27Kip1 in HDACi-mediated tumor cell and arrest in the Em- model of B-cell

A Newbold1, JM Salmon1, BP Martin1, K Stanley1 and RW Johnstone1,2

Following the establishment of histone deacetylases (HDACs) as promising therapeutic targets for the reversal of aberrant epigenetic states associated with , the development of HDAC inhibitors (HDACi) and their underlying mechanisms of action has been a significant area of scientific interest. HDACi induce diverse biological responses including the inhibition of cell proliferation by blocking progression through the G1 or G2/M phases of the cell cycle. As a putative tumor-suppressor , p21waf1/cip1 influences cell proliferation by inhibiting the activity of –cyclin-dependent (CDK) complexes at the G1/S and G2/M cell cycle checkpoints. HDACi transcriptionally activate CDKN1A, and it has been proposed that induction of p21waf1/cip1 can determine if a cell undergoes or cell cycle arrest following HDACi treatment. In the Em-myc transgenic mouse model of B-cell lymphoma, knockout of cdkn1a had no effect on latency, indicating that p21waf1/cip1 did not function as a tumor suppressor in this system. Although HDACi robustly induced expression of p21waf1/cip1 in wild-type Em-myc , deletion of cdkn1a did not sensitize the lymphoma cells to HDACi-induced apoptosis and HDACi-induced cell cycle arrest still occurred. However, knockdown of in cdkn1a knockout lymphomas resulted in defective vorinostat-mediated arrest at G1/S indicating an essential role of p27Kip1 in mediating this biological response to vorinostat. These data demonstrate that induction of cdkn1a does not regulate HDACi-mediated tumor cell apoptosis and refute the notion that p21waf1/cip1 is an obligate mediator of HDACi-induced cell cycle arrest.

Oncogene (2014) 33, 5415–5423; doi:10.1038/onc.2013.482; published online 2 December 2013 Keywords: inhibitors; CDKN1A; tumor-suppressor protein; cell cycle arrest

INTRODUCTION cyclin-dependent kinase inhibitors (CDKi) p21waf1/cip1, p27Kip1 and Kip2 8 Kip2 Kip1 Histone deacetylase (HDAC) inhibitors (HDACi) have been p57 , and A and D. Both p57 and p27 have been 9,10 successfully used to treat patients with hemopoietic malignancies, reported to have tumor-suppressor functions, however, a waf1/cip1 11 although the molecular and biological events that underlie their similar role for remains controversial. Deletion of therapeutic effects have not been clearly delineated.1 HDACi cdkn1a in mice can result in spontaneous tumors with late onset,12 induce a range of tumor cell intrinsic biological outcomes such whereas Ras-induced transformation of from cdkn1a as induction of cell cycle arrest, senescence, apoptosis and knockout mice resulted in an increase in proliferation and a differentiation, any one or more of which may have a primary role downregulation of the differentiation markers compared with in reducing tumor burden.1 Interestingly, HDACi can concurrently effects in wild-type keratinocytes.13 Conversely, other studies induce both cell cycle arrest and apoptosis,2 and inhibition of using a model of tumorigenesis mediated by of newborn apoptosis through forced expression of pro-survival Bcl-2 mice with Moloney murine showed that knockout results in HDACi-mediated cell cycle arrest, becoming the of cdkn1b encoding p27Kip1 but not cdkn1a increased tumor dominant biological outcome.3,4 Although we have demon- onset.14 strated that induction of tumor cell apoptosis is requisite for As a consequence of altering cyclin-CDK activity, treatment of diverse HDACi such as vorinostat, and to tumor cells with HDACi can result in cell cycle arrest at the G1/S mediate significant therapeutic benefit in pre-clinical models,4–6 it checkpoint concomitant with the dephosphorylation of retino- remains possible that the interplay between inhibition of the cell blastoma protein (pRb).1 Interestingly, HDACi can induce cycle and initiation of apoptosis ultimately determines the relative expression of p21waf1/cip1 in a -independent manner,15 and it sensitivity of tumor cells to HDACi-induced death. Indeed, has been proposed that p21waf1/cip1 can influence the decision of a overexpression of p21waf1/cip1 can confer resistance to HDACi- tumor cell to undergo apoptosis and/or cell cycle arrest following mediated apoptosis7 providing some evidence that cell cycle HDACi treatment. For example, overexpression of p21waf1/cip1 can regulatory proteins may influence the sensitivity of tumor cells to confer resistance to HDACi-mediated apoptosis,7 however, the HDACi-mediated apoptosis. effects of HDACi on tumor cell apoptosis in the absence of HDACi can affect the cell cycle by up- and/or downregulating p21waf1/cip1 are inconsistent. For example, while one study the expression of a number of cell cycle proteins including the reported the loss of p21waf1/cip1-abolished butyrate-induced G1

1Cancer Therapeutics Program, Gene Regulation Laboratory, The Peter MacCallum Cancer Institute, St Andrews Place, East Melbourne, Victoria, Australia and 2The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. Correspondence: Professor RW Johnstone, Cancer Therapeutics Program, Gene Regulation Laboratory, The Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne 3002, Victoria, Australia. E-mail: [email protected] Received 16 April 2013; revised 2 October 2013; accepted 7 October 2013; published online 2 December 2013 Regulation of HDACi antitumor activity A Newbold et al 5416 arrest,16 another study reported that (TSA) was still Em-myc background did not affect the onset or rate of able to induce a G1 arrest in human colon HCT116 cells deficient tumorgenesis with median survival of Em-myc þ ve/cdkn1a þ / þ of of p21waf1/cip1.17 112.5 days compared with 155 days for Em-myc þ ve/cdkn1a À / À Thus, in this study, Em-myc transgenic mice were crossed with mice (Figure 1a). Taken together, our results indicated that the cdkn1a knockout mice to determine the role of p21waf1/cip1 in absence of p21waf1/cip1 did not increase the onset of myc-induced regulating myc-induced lymphomagenesis. The Em-myc lympho- tumorgenesis and, if anything, the opposite trend was apparent. mas derived from that experiment were assessed for their Therefore, p21waf1/cip1 did not function as a tumor-suppressor sensitivity to vorinostat-mediated apoptosis and cell cycle arrest. protein in the Em-myc B-cell lymphoma model. Our results indicate that p21waf1/cip1 does not have a major role in Tumor cells from the lymph nodes of Em-myc þ ve/cdkn1a þ / þ regulating the oncogenic activities of c-myc nor is it important for and Em-myc þ ve/cdkn1a À / À mice were harvested and verified for vorinostat-induced apoptosis or cell cycle arrest. their expression of p21waf1/cip1 by . Treatment with HDACi induces p21waf1/cip1 expression,19 and as shown in Figure 1b, p21waf1/cip1 was only detected in Em-myc þ ve/cdkn1a þ / þ RESULTS lymphoma cells following vorinostat treatment. As expected, Deletion of cdkn1a does not increase the rate of tumorigenesis in tumors from Em-myc þ ve/cdkn1a À / À mice were negative for Em-myc transgenic mice p21waf1/cip1 expression in the presence and absence of vorinostat To determine the role of p21waf1/cip1 in regulating myc-induced treatment (Figure 1b). tumorigenesis, cdkn1a À / À mice were crossed with Em-myc transgenic mice,18 and subsequent intercrossing was performed to establish Em-myc À ve/cdkn1a À / À , Em-myc þ ve/cdkn1a þ / þ and Knockout of p21waf1/cip1 does not affect the sensitivity of the Em-myc þ ve/cdkn1a À / À colonies. As shown in Figure 1a, mice that lymphoma cells to vorinostat did not express the myc transgene but had deletion of cdkn1a did Tumor cells overexpressing p21waf1/cip1 can have reduced sensitivity not develop lymphoma nor did they display any signs of illness to HDACi-induced apoptosis.7 We therefore determined whether throughout the 400-day study period. Conversely, mice harboring Em-myc lymphomas deficient in p21waf1/cip1 were hypersensitive the c-myc transgene that were wild type for cdkn1a developed to HDACi-induced apoptosis. Lymphomas from Em-myc þ ve/ lymphoma with kinetics consistent with previously published cdkn1a þ / þ and Em-myc þ ve/cdkn1a À / À mice were tested for Em-myc mouse studies.18 Homozygous deletion of cdkn1a on an their sensitivity to vorinostat-induced apoptosis, and as shown in

100 E-myc-ve/cdkn1a-/-  +ve +/+ 80 E -myc /cdkn1a E-myc+ve/cdkn1a-/-

60

robability (%) 40

Survival p 20

0 100 200 300 400 Time (Days)

(1) (2) (3) (2) (1) (3) (4) -/- -/- +/+ +/+ -/- -/- +/+

1a 1a 1a 1a 1a

/cdkn1a /cdkn1a /cdkn /cdkn /cdkn /cdkn /cdkn e

+ve +ve +ve +ve +ve +ve +v

c y

-myc -myc -myc -myc -myc -m -myc        E E E E E E E

p21waf1/cip1

-

Vorinostat: - + - + - + - + - + -++ - Figure 1. Homozygous deletion of cdkn1a on an Em-myc background does not increase tumorgenesis. (a) Em-myc transgenic mice were crossed with cdkn1a knockout mice, and the resulting genotypes (Em-myc À ve/cdkn1a À / À , Em-myc þ ve/cdkn1a þ / þ and Em-myc þ ve/cdkn1a À / À ) were compared in a survival study from 2 weeks of age. A minimum of 15 mice per genotype were entered into the study and mice were genotyped at the beginning and at the end of the study to verify their genotypic status. The study was terminated at 400 days, Kaplan–Meier analysis was performed and comparisons were made using the log-rank (Mantel–Cox) test (MedCalc software Version 8.0.2.0). (b) Lymph nodes were harvested from Em-myc/cdkn1a þ / þ and Em-myc/cdkn1a À / À mice and cultured in vitro. Cells were tested by western blot for the waf1/cip1 þ / þ presence of p21 expression following exposure to 2 mM vorinostat for 6 h. Cells obtained from Em-myc/cdkn1a mice are highlighted in red.

Oncogene (2014) 5415 – 5423 & 2014 Macmillan Publishers Limited Regulation of HDACi antitumor activity A Newbold et al 5417  +ve +/+  +ve -/- 100 E -myc /cdkn1a (1) E -myc /cdkn1a (1) 100 N.S Vehicle 100 Vorinostat 80 80 80

60 60 60 E-myc +ve /cdkn1a +/+ (1)  +ve +/+ 40 40 E -myc /cdkn1a (3) 40 E-myc +ve /cdkn1a -/- (2) % PI Uptake 20 20 E-myc +ve /cdkn1a -/- (1) Vehicle 20 Vorinostat Survival probability (%) Survival probability (%) E-myc +ve /cdkn1a -/- (4) 0 0 0 0 102030405060 0 10 20 30 40 50 60 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Days Days Vorinostat (M) E-myc +ve /cdkn1a +/+ (3) E-myc +ve /cdkn1a -/- (2) 100 Vehicle 100 Vehicle  +ve +/+ 100 E -myc /cdkn1a (1) Vorinostat Vorinostat E-myc +ve /cdkn1a +/+ (3) 80 80 80 E-myc +ve /cdkn1a -/- (2) 60 60 E-myc +ve /cdkn1a -/- (1) 60 E-myc +ve /cdkn1a -/- (4) 40 40

40 20 20 % MOMP Survival probability (%) Survival probability (%) 0 0 20 0 102030405060 0 102030405060 Days Days 0 0 0.05 0.1 0.5 1 1.5 E-myc +ve /cdkn1a -/- (4) Vorinostat (M) 100 Vehicle À / À Vorinostat Figure 2. Em-myc/cdkn1a lymphoma cells are not sensitized to 80 apoptosis following vorinostat treatment. Em-myc/cdkn1a þ / þ and Em-myc/cdkn1a À / À lymphomas were incubated in vitro for 24 h with 60 the indicated doses of vorinostat. Cell viability was assessed by: 40 (a) PI , and (b) mitochondrial outer membrane permeabiliza- tion (MOMP) was determined by TMRE staining. Data shown is the 20

± Survival probability (%) mean of three independent experiments s.e.m. Levels of 0 significance were determined by a Student’s two-tailed t-test; 0 102030405060 NS, not significant. Days Figure 3. Absence of p21waf1/cip1 does not affect the therapeutic Figure 2, all tumors were equivalently sensitive to vorinostat efficacy of vorinostat in vivo. C57BL/6 mice (10 mice per group) regardless of cdkn1a status. We next wanted to compare the injected with the following Em-myc lymphomas: (a) Em-myc/cdkn1a antitumor effects of vorinostat in vivo in mice bearing Em-myc/ þ / þ (tumor no. 1), (b) Em-myc/cdkn1a þ / þ (tumor no. 3), (c) Em-myc/ þ / þ À / À cdkn1a À / À ( tumor no. 1), (d) Em-myc/cdkn1a À / À (tumor no. 2) or cdkn1a and Em-myc/cdkn1a tumors. Consistent with À / À previous studies that showed treatment of mice bearing Em-myc (e) Em-myc/cdkn1a (tumor no. 4). Tumor-bearing mice were lymphomas with vorinostat significantly increased their life span,4 treated with vorinostat or vehicle (DMSO) after WBC counts reached B13 Â 103/ml. The therapy consisted of 200 mg/kg vorinostat intra- the survival of vorinostat-treated mice used in this study was also peritoneal injections (i.p) daily for 7 days, then 150 mg/kg for 3 significantly extended compared with vehicle-treated mice weeks or until mice succumbed to disease. Kaplan–Meier analysis irrespective of the cdkn1a status of the tumors (Figures 3a–e, was made using the log-rank (Mantel–Cox) test (MedCalc software Table 1). Taken together, our results indicated that although cdkn1a version 8.0.2.0). was an HDACi-responsive gene, loss of expression of p21waf1/cip1 did not affect the apoptotic or therapeutic effects of vorinostat. vorinostat were analyzed (Figure 4c). As expected, knockout of cdkn1a resulted in the cells proliferating slightly faster than HDACi-induced cell cycle arrest still occurs in the absence of Em-myc tumors wild type for cdkn1a (unpublished observations), waf1/cip1 p21 and this was reflected by the relatively greater proportion We have previously shown that cells resistant to vorinostat- of Em-myc/cdkn1a À / À /Bcl-2 cells in S-phase compared with induced apoptosis through overexpression of the pro-survival Em-myc/cdkn1a þ / þ /Bcl-2 cells. Treatment with vorinostat, how- protein, Bcl-2, undergo arrest at the of the cell cycle.4 To ever, induced a reduction in the proportion of cells in S- and determine the importance of p21waf1/cip1 in mediating this cell G2/M phases of the cell cycle and an increase in cells in G1 that cycle arrest, we overexpressed Bcl-2 in Em-myc/cdkn1a þ / þ and was not dependent on the presence of wild-type cdkn1a Em-myc/cdkn1a À / À cells (Figure 4a). Em-myc/cdkn1a þ / þ /Bcl-2 and (Figure 4c). The vorinostat-induced cell cycle arrest at the G1/S Em-myc/cdkn1a À / À /Bcl-2 cells had equivalent levels of over- checkpoint was confirmed biochemically by analysis of the expressed Bcl-2, and p21waf1/cip1 was induced by vorinostat in phosphorylation state of Rb. Consistent with the cell cycle Em-myc/cdkn1a þ / þ /Bcl-2 cells (Figure 4a). The anti-apoptotic data shown in Figure 4c, greater levels of hyperphosphorylated activity of Bcl-2 was confirmed by exposing the cells to vorinostat Rb (ppRb) was observed in untreated Em-myc/cdkn1a À / À /Bcl-2 and measuring uptake of propidium iodide (PI) as a readout for cells compared with Em-myc/cdkn1a þ / þ /Bcl-2 cells (Figure 4d). cell viability. As shown in Figure 4b, Em-myc/cdkn1a þ / þ /Bcl-2 and Treatment with vorinostat resulted in the appearance of Em-myc/cdkn1a À / À /Bcl-2 cells were resistant to vorinostat- hypophosphorylated Rb (pRb) in both Em-myc/cdkn1a À / À / induced apoptosis, whereas Em-myc/cdkn1a þ / þ and Em-myc/ Bcl-2 and Em-myc/cdkn1a þ / þ /Bcl-2 cells (Figure 4d). Taken cdkn1a-/ cells were equivalently sensitive to vorinostat. together, these data demonstrate that vorinostat-induced The cell cycle profiles of Em-myc/cdkn1a þ / þ /Bcl-2 and Em-myc/ cell cycle arrest at the G1/S checkpoint still occurs in the cdkn1a À / À /Bcl-2 cells grown in the presence and absence of absence of p21waf1/cip1.

& 2014 Macmillan Publishers Limited Oncogene (2014) 5415 – 5423 Regulation of HDACi antitumor activity A Newbold et al 5418 Table 1. Therapeutic response of mice bearing Em-myc tumors with a E-myc/ E-myc/ cdkn1a +/+ (1) cdkn1a -/- (1) the designated genotypes to vorinostat waf1/cip1 Tumor genotype Median Median Statistical p21 survival survival significance Bcl-2 vehicle vorinostat (P) -Actin treated treated Bcl-2 --++- - + + (days) (days) 2M Vorinostat --+++- - +

Em-myc/cdkn1a þ / þ (1) 22 35 o0.0001 b 100 Em-myc/cdkn1a þ / þ (3) 15 33 o0.0001 80 Em-myc/cdkn1a À / À (2) 22 37 o0.0001 À / À Em-myc/cdkn1a (1) 23 47.5 o0.0001 60 À / À Em-myc/cdkn1a E-myc/cdkn1a +/+/MSCV (1) (4) 20.5 37 o0.0001 ** Em-myc/cdkn1a À / À 23 36.5 o0.0001 40 E-myc/cdkn1a +/+/Bcl2 (1) E-myc/cdkn1a -/-/MSCV (1) shRNAmiR-Scr % PI Uptake E-myc/cdkn1a -/-/Bcl2 (1) Em-myc/cdkn1a À / À 23 36.5 o0.0001 20 shRNAmiR-cdkn1b#1 0 0 0.5 1 1.5 2 Vorinostat (M) Kip1 P27 has an important role in vorinostat-mediated cell cycle c * * arrest at G1/S but not vorinostat-induced apoptosis 100% G2M HDACi have been reported to upregulate the expression of both S p57kip2 and p27Kip1, and induce a G1 cell cycle arrest preceding 80% G1 apoptosis.20 Thus, we tested for expression of p57Kip2 and p27Kip1 60% in Em-myc/cdkn1a þ / þ /Bcl-2 and Em-myc/cdkn1a À / À /Bcl-2 cells following vorinostat treatment. There was no apparent increase 40% in expression of p57Kip2 following treatment with vorinostat in either lymphoma (Figure 5a); however, the expression of p27Kip1 20% was substantially increased in vorinostat-treated cells (Figure 5b). 0% Kip1 E E E E To determine whether increased expression of p27 following -myc/cdkn1a -myc/cdkn1a -myc/cdkn1a -myc/cdkn1 vorinostat treatment occurred at the transcriptional or post- + vorinostat + vorinost transcriptional level, we assessed the expression of both cdkn1a a þ / þ +/+ +/+ -/ - -/- and cdkn1b mRNA in Em-myc/cdkn1a /Bcl-2 and Em-myc/ /Bcl-2 (1) /Bcl-2 (1) /Bcl-2 (1) /Bcl-2 (1) cdkn1a À / À /Bcl-2 cells following vorinostat treatment. Consistent at with previous reports,19 cdkn1a mRNA was markedly induced in a time-dependent manner following treatment of Em-myc/ þ / þ cdkn1a /Bcl-2 cells with vorinostat (Figure 5c). In contrast, the d E-myc/cdkn1a +/+ E-myc/cdkn1a -/- levels of cdkn1b mRNA remained relatively unchanged in Em-myc/ /Bcl-2 (1) /Bcl-2 (1) þ / þ À / À Hyperphosphorylated-Rb (ppRb) cdkn1a /Bcl-2 and Em-myc/cdkn1a /Bcl-2 cells treated with Hypophosphorylated-Rb (pRb) vorinostat (Figure 5d). These data indicate that the increase in  p27Kip1 expression following vorinostat treatment occurs only at a -Actin  translational level and is not a transcriptional event. 2 M Vorinostat - ++- p21waf1/cip1 ++-- To determine the potential functional role of p27Kip1 in response to vorinostat, we targeted cdkn1b in Em-myc/ Figure 4. Deletion of p21waf1/cip1 does not inhibit vorinostat-mediated À / À cell cycle arrest. (a) Em-myc/cdkn1a þ / þ (tumor no. 1) and Em-myc/ cdkn1a cells overexpressing Bcl-2 using RNA interference to À / À knockdown p27Kip1. As shown in Figure 6a, cells transduced with cdkn1a (tumor no. 1) lymphoma cells were retrovirally transduced miR30-based vectors expressing two different short hairpin RNA with MSCV-Bcl-2 or MSCV alone. Tumor cells were incubated for 6 h À / À with or without 2 mM vorinostat. Cells were harvested and analyzed by (shRNA) sequences targeting cdkn1b (Em-myc/cdkn1a /shRNA- western blot. Expression of p21waf1/cip1 and Bcl-2 was measured with miR-cdkn1b#1/Bcl-2 and Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#2/ Kip1 anti-mouse p21 and anti-mouse Bcl-2 antibody, respectively. Bcl-2) had a dramatic decrease in expression of p27 . In contrast, Anti-b-actin was used as a loading control. (b) Lymphomas developed cells transduced with a control miR30 vector with a non-targeting in a were incubated with increasing doses of vorinostat for 24 h. Cell scrambled shRNA sequence (Em-myc/cdkn1a À / À /shRNAmiR-Scr/ death was analyzed by PI staining and data shown is the mean of Bcl-2) expressed wild-type levels of p27Kip1. These cells were then three independent experiments ±s.e.m. Levels of significance were determined by a Student’s two-tailed t-test; **Po0.01. (c) Em-myc/ exposed to vorinostat and cell cycle progression was assessed. þ / þ À / À Knockdown of p27Kip1 in cdkn1a knockout cells resulted in a loss cdkn1a /Bcl-2 and Em-myc/cdkn1a /Bcl-2 lymphoma cells were of vorinostat-mediated arrest at the G1/S checkpoint that was cultured with or without 0.5 mM vorinostat for 24 h. Cell cycle status waf1/cip1 was determined by PI staining. The percentage of cells in G1, S and observed in p21 -competent cells (Figure 6b). Remarkably, G2/M phases of the cell cycle are shown and SubG1 was not the Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b/Bcl-2 cells were arrested Kip1 measured. Data shown is the mean of three independent experi- in G2/M following exposure to vorinostat, indicating that p27 ments ±s.e.m. Levels of significance were determined by a Student’s has a key role in mediating vorinostat-induced cell cycle arrest two-tailed t-test; *Po0.05 for the percentage decrease of cells in at G1/S. S-phase. (d) Mouse Rb expression in Em-myc/cdkn1a þ / þ /Bcl-2 and We next determined if knockdown of cdkn1b in cdkn1a-deleted Em-myc/cdkn1a À / À /Bcl-2 lymphoma cells was detected by western Em-myc cells affected vorinostat-induced apoptosis. As shown in blot following a 24-h incubation in vitro with 2 mM vorinostat. Anti-b- Figures 7a–c, Em-myc lymphomas were equivalently sensitive to actin antibody was used as a loading control. vorinostat regardless of p27kip1 expression. We also tested the importance of p27Kip1 in regulating the in vivo therapeutic effects tumors. Interestingly, the antitumor efficacy of vorinostat of vorinostat using C57B/6 mice mice bearing Em-myc/cdkn1a À / À remained fully intact in mice bearing Em-myc tumors with shRNAmiR-Scr and Em-myc/cdkn1a À / À shRNAmiR-cdkn1b#1 concomitant deletion of cdkn1a and shRNAmiR-mediated

Oncogene (2014) 5415 – 5423 & 2014 Macmillan Publishers Limited Regulation of HDACi antitumor activity A Newbold et al 5419  +/+  -/- treatment resulted in Rb hypophosphorylation regardless of the a E -myc/cdkn1a E -myc/cdkn1a waf1/cip1 kip1 /Bcl-2(1) /Bcl-2(1) expression status of p21 or p27 . p57Kip2 DISCUSSION -Actin 2M Vorinostat -+ - + In this study, we determined whether the loss of cdkn1a affected p21waf1/cip1 ++ - - the rate of lymphomageneis in the Em-myc mouse, and by using Em-myc/cdkn1a À / À tumor cells, we determined whether loss of b E-myc/cdkn1a+/+ E-myc/cdkn1a-/- cdkn1a affected vorinostat-mediated apoptosis or cell cycle arrest. /Bcl-2(1) /Bcl-2(1) Homozygous deletion of cdkn1a in C57BL/6 mice did not result in

Kip1 spontaneous tumor development. Our results are in contrast to the p27 study by Martı´n–Caballero et al.,12 who reported that cdkn1a–/– -Actin mice developed spontaneous tumors with a late onset of around 16 months. One explanation could be difference in genetic 2M Vorinostat -+ -+ backgrounds of the cdkn1a À / À mice with the mice used in our p21waf1/cip1 ++ -- study being on a pure C57BL/6 background, whereas the Martı´n– Caballero study used mice on a mixed 129Sv/C57BL/6 background. c  +/+ 10 E -myc/cdkn1a /Bcl-2(1) We also demonstrated that deletion of cdkn1a did not accelerate E-myc/cdkn1a-/-/Bcl-2(1) myc-induced tumorigenesis and these studies align with others 8 * demonstrating that loss of cdkn1a alone did not affect tumor 6 development driven by Moloney murine leukemia virus infection.14 4 In a previous study, deletion of cdkn1a enhanced the rate of 14 2 tumorigenesis only in the context of loss of one allele of cdkn1b, Fold Change 0 demonstrating a level of functional redundancy between different CDKi that we hypothesize may also be apparent in vorinostat- Time (hours) 246246 mediated cell cycle arrest as discussed below. d It has been well documented that transcriptional induction of 10 E-myc/cdkn1a+/+/Bcl-2(1) cdkn1a resulting in increased expression of p21waf1/cip1 is a 8 E-myc/cdkn1a-/-/Bcl-2(1) common molecular response to treatment of cells with HDACi;1 waf1/cip1 6 however, the importance of p21 in regulating HDACi- N.S induced apoptosis or cell cycle arrest remains controversial. 4 N.S N.S Although some studies have proposed that HDACi-mediated 2 upregulation of p21waf1/cip1 has an essential role in subsequent Fold Change cell cycle arrest at the G1/S transition1 and may either protect 0 7,21 Time (hours) 2 4 6246 cells from HDACi-mediated apoptosis or promote the apoptotic effects of HDACi,22 we saw no such effects using Kip1 Kip2 Figure 5. Vorinostat induces p27 but not p57 .(a) Mouse Em-myc/cdkn1a À / À tumors. Our results are supported by other p57Kip2 expression in Em-myc/cdkn1a þ / þ /Bcl-2 versus Em-myc/ À / À studies demonstrating that knockdown of cdkn1a in colon cdkn1a /Bcl-2 lymphoma cells was detected by western blot carcinoma lines did not sensitize cells to death following following a 6-h incubation in vitro with 2 mM vorinostat. Anti-b-actin 23 antibody was used as a loading control. (b) Mouse p27Kip1 treatment with a vorinostat/ combination. þ / þ À / À Moreover, the HDACi trichostatin A induced equivalent cell cycle expression in Em-myc/cdkn1a /Bcl-2 versus Em-myc/cdkn1a / þ / þ À / À Bcl-2 lymphoma cells was detected by western blot following a 6-h arrest in human isogenic CDKN1A and CDKN1A cell lines 17 incubation in vitro with 2 mM vorinostat. Anti-b-actin antibody was produced by homologous recombination. used as a loading control. All westerns are a representative of at All previous studies performed to date that aimed to determine least four independent experiments. (c, d) The levels of cdkn1a and the functional role of CDKN1A/cdkn1a in mediating biological cdkn1b mRNA following vorinostat treatment were measured by responses to HDACi were performed using tumor cell lines grown quantitative real-time PCR in Em-myc/cdkn1a þ / þ /Bcl-2 and Em-myc/ À / À in culture. A key component of our study were in vivo experiments cdkn1a /Bcl-2 lymphoma cells after incubation with 2 mM vorino- using primary lymphomas to determine whether the loss of stat. Time points were taken at 2, 4 and 6 h, and RNA and cDNA were made according to standard methods (refer to Materials and cdkn1a altered the therapeutic effects of vorinostat in a methods for details). Fold change in (c) cdkn1a mRNA or (d) cdkn1b physiologically relevant setting. As shown in Figure 3, deletion mRNA levels normalized to HPRT mRNA levels are shown. Data of cdkn1a had little or no effect on the ability of vorinostat to shown represents mean of three independent experiments ±s.e.m. mediate therapeutic responses as measured by increased survival Levels of significance were determined by a Student’s two-tailed of mice bearing Em-myc lymphomas. Using the Em-myc system, we t-test; *Po0.05. have clearly demonstrated that induction of tumor cell apoptosis is key for different HDACi to provide therapeutic benefit.4,5,6 Moreover, using the same system, we have recently shown that depletion of cdkn1b (Figure 7d, Table 1). Our results therefore HDACi with broad HDAC specificity (for example, vorinostat) and indicated that while both cdkn1a and cdkn1b were HDACi- agents that predominantly inhibited selected class I HDACs (for responsive genes, the loss of expression of p21waf1/cip1 or example, romidepsin) mediate effects on tumor cell proliferation p27kip1, individually or combined, did not affect the apoptotic or and survival in vitro and in vivo.24 Our studies using Em-myc/ therapeutic effects of vorinostat. cdkn1a À / À tumors clearly showed that even though p21waf1/cip1 As mentioned previously in Figure 6b, the p21/p27 double- was robustly induced by vorinostat in Em-myc cells, it did not have deficient cells were arrested in G2/M following exposure to an important role in regulating vorinostat-induced apoptosis vorinostat, indicating that p27Kip1 had a key role in mediating in vitro or therapeutic efficacy in vivo. Interestingly, when we vorinostat-induced cell cycle arrest at G1/S. We therefore assessed blocked vorinostat-mediated apoptosis in Em-myc cells through the phosphorylation status of Rb in Em-myc tumors with deletion overexpression of Bcl-2, the cells went into cell cycle arrest at the of cdkn1a and shRNAmiR-mediated depletion of cdkn1b following G1/S transition that was not dependent on p21waf1/cip1. We note treatment with vorinostat. As shown in Figure 7e, vorinostat that in other tumor settings driven by oncogenic proteins and

& 2014 Macmillan Publishers Limited Oncogene (2014) 5415 – 5423 Regulation of HDACi antitumor activity A Newbold et al 5420 pathways other than Myc, upregulation of cdkn1a and other CDKi ligase complex.26 To address the role of p27kip1 in may regulate the cellular responses to HDAC inhibition. Moreover, mediating vorinostat-induced cell cycle arrest, we knocked down the Em-myc model requires secondary genetic ‘hits’ that cdkn1b in cdkn1a knockout cells. Consistent with the notion that co-operate with c-Myc to drive B-cell transformation.25 Although p21waf1/cip1 and p27kip1 share functionally redundant roles in we functionally assessed the role of p21waf1/cip1 in HDACi-induced regulating transition through G1/S, we found that vorinostat- apoptosis and cell cycle arrest in multiple primary Em-myc tumors, treated cells passed through the G1/S checkpoint but halted in we cannot rule out the possibility that in certain genetic settings, G2/M. These data demonstrate that p27kip1 is essential for cdkn1a may have a role in mediating antitumor responses vorinostat-mediated cell cycle arrest at G1/S, but other cell cycle mediated by HDACi. regulatory proteins other than p21waf1/cip1 and p27kip1 are We observed a strong upregulation of p27kip1 but not p57kip2 important for arrest at later stages of the cell cycle. in vorinostat-treated Em-myc/cdkn1a þ / þ /Bcl-2 and Em-myc/ In conclusion, we have shown that loss of cdkn1a did not cdkn1a À / À /Bcl-2 cells, indicating that p27kip1 may provide a level accelerate tumorigenesis in the Em-myc transgenic mouse model. of functional redundancy for loss of cdkn1a. The increased Furthermore, we found that although p21waf1/cip1 was robustly expression of p27kip1 following vorinostat treatment occurred in induced in response to vorinostat, it had little if any role in the the absence of an increase in cdkn1b mRNA pointing to a post- apoptotic- or cell cycle regulatory activities of the compound, and translational effect. Consistent with our data, it was recently loss of cdkn1a did not affect the therapeutic response to vorinostat. shown that vorinostat increased the expression of p27kip1 through Finally, we demonstrate that p27kip1, a CDKi that is most likely post- downregulation of S-phase kinase-associated protein 2 (Skp2) and transcriptionally upregulated by vorinostat, has a fundamental role CDK subunit 1 (Cks1), the components of the SCFSkp2-Cks1 in mediating vorinostat-induced cell cycle arrest at G1/S.

Vehicle Vorinostat

l-2 l-2 500 1.0k  -/- E -myc/cdkn1a /Bcl-2 400 800 l-2 300 600 Count Count 400 / Bcl-2 / Scr / Bc/ cdkn1b#1/ cdkn1b#2 / Bc / Bc 200 -/- -/- -/- -/- 100 200 0 0 100 101 102 103 100 101 102 103 -myc/cdkn1a-myc/cdkn1a-myc/cdkn1a PI PI -myc/cdkn1a   E E E E p27 500 mBcl-2 E-myc/cdkn1a-/-/ shRNAmiR- 600 400 -Actin Scr/Bcl-2 300 400 Count 200 Count 200 100 0 0 100 101 102 103 100 101 102 103 PI PI

1.0k

400 800 E-myc/cdkn1a-/-/ shRNAmiR- cdkn1b#1/Bcl-2 300 600

200 Count 400 Count 100 200

0 0 100 101 102 103 100 101 102 103 PI PI

800 E-myc/cdkn1a-/-/ shRNAmiR- 400 cdkn1b#2/Bcl-2 600 300 400

200 Count Count 100 200

0 0 100 101 102 103 100 101 102 103 PI PI Figure 6. p27Kip1 has an important role in vorinostat-mediated cell cycle arrest at G1/S. (a) Western blot analysis of whole-cell lysates from Em-myc/cdkn1a À / À /Bcl-2, Em-myc/cdkn1a À / À /shRNAmiR-Scr/Bcl-2, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1/Bcl-2 and Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#2/Bcl-2 cells probed with against p27Kip1, Bcl-2 and b-actin. (b) Em-myc/cdkn1a þ / þ /Bcl-2, Em-myc/cdkn1a À / À / shRNAmiR-Scr/Bcl-2, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1/Bcl-2 and Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#2/Bcl-2 cells were treated with vehicle of vorinostat (2 mM) for 24 h. was performed using PI staining and the percentage of cells in SubG1, G1, S and G2/M phases of the cell cycle are shown. The profiles shown are a representative of three independent assays.’

Figure 7. Depletion of cdkn1b does not affect the apoptotic or therapeutic responses to vorinostat. (a) Western blot analysis of whole-cell lysates from Em-myc/cdkn1a À / À ,Em-myc/cdkn1a À / À /shRNAmiR-Scr, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1 and Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#2 cells probed with antibodies against p27Kip1 and b-actin. (b, c) Em-myc/cdkn1a À / À ,Em-myc/cdkn1a À / À /shRNAmiR-Scr and Em-myc/cdkn1a À / À / shRNAmiR-cdkn1b#1 lymphomas were incubated in vitro for 24 h with the indicated doses of vorinostat. Cell viability was assessed by: (b)PIstaining, and (c) mitochondrial outer membrane permeabilization (MOMP) was determined by TMRE staining. Data shown is the mean of three independent experiments ±s.e.m. Levels of significance were determined by a Student’s two-tailed t-test; NS, not significant. (d) C57BL/6 mice (12 mice per group) were injected intra-venously (i.v.) with Em-myc/cdkn1a À / À ,Em-myc/cdkn1a À / À /shRNAmiR-Scr or Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1 lymphomas. Tumor-bearing mice were treated with vorinostat or vehicle (DMSO) after WBC counts reached B13 Â 103/ml. Therapy consisted of 200 mg/kg vorinostat i.p daily for 7 days, then 150 mg/kg for 3 weeks or until mice succumbed to disease. Kaplan–Meier analysis was made using the log-rank (Mantel–Cox) test (MedCalc software version 8.0.2.0). (e) Mouse Rb expression in Em-myc/cdkn1a À / À ,Em-myc/cdkn1a À / À /shRNAmiR- Scr, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1 and Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#2 lymphoma cells was detected by western blot following a 24-h incubation in vitro with 2 mM vorinostat. Anti-b-actin antibody was used as a loading control.

Oncogene (2014) 5415 – 5423 & 2014 Macmillan Publishers Limited Regulation of HDACi antitumor activity A Newbold et al 5421 MATERIALS AND METHODS genotype were entered into a survival study from 2 weeks of age. Mice were Mice genotyped at the beginning and end of the study to verify their genotypic Em-myc transgenic male mice were crossed with cdkn1aÀ / À female mice to status. Mice were palpated every 2–4 days for enlarged nodes, white blood produce Em-myc þ ve/cdkn1a þ / À and Em-myc-ve/cdkn1a þ / À offspring. The cell counts were monitored fortnightly (Advia 120 Hematology Analyzer, offspring were then brother/sister mated to produce the following Siemans, Melbourne, VIC, Australia) and the overall health of the mice was genotypes: Em-myc þ ve/cdkn1a þ / þ , Em-myc þ ve/cdkn1a þ / À , Em-myc þ ve/ monitored daily. When an ethical endpoint was reached, final spleen weights cdkn1a À / À and Em-myc-ve/cdkn1a À / À mice. A minimum of 15 mice per and white blood cell counts were taken along with a necropsy report.

/ / / a -/- -/- -/-

-myc/cdkn1a -myc/cdkn1a -myc/cdkn1a E E E shRNAmiR-Scr shRNAmiR-cdkn1b#1shRNAmiR-cdkn1b#2

p27

β-actin

b 100 N.S

E-myc/cdkn1a-/- E-myc/cdkn1a-/- 50 shRNAmiR-Scr

% PI uptake E-myc/cdkn1a-/- hRNAmiR-cdkn1b#1

0 0 0.05 0.1 0.5 1.5 2 Vorinostat (M) c E-myc/cdkn1a-/- 100 E-myc/cdkn1a-/- shRNAmiR-Scr E-myc/cdkn1a-/- shRNAmiR-cdkn1b#1 50 % MOMP

0 0 0.05 0.1 0.5 1.5 2 Vorinostat (M) d

E-myc/cdkn1a-/- shRNAmiR-Scr

E-myc/cdkn1a-/- shRNAmiR-cdkn1b#1

- - - e iR iR iR m m m A A A N N N R cl-2 shR B / sh 1a-/- / shR n1a-/- / l-2 k l-2 n1a-/- / c c k dkn1a-/-

yc/cdknl-2 yc/c yc/cd yc/cd c -m -m -m -m  E cr/B E E E S cdkn1b#1/B cdkn1b#2/B Hyperphosphorylated-Rb (ppRb) Hypophosphorylated-Rb (pRb) -Actin 2M Vorinostat -+-+-+-+

& 2014 Macmillan Publishers Limited Oncogene (2014) 5415 – 5423 Regulation of HDACi antitumor activity A Newbold et al 5422 Western blotting and antibodies Lymph nodes were harvested and cells were frozen from five mice per Western blot protein lysates from Em-myc lymphomas were lysed in ice- genotype and a number of suspension cell lines were established in vitro. cold lysis buffer (0.15 M NaCl, 10 mM Tris-Cl, pH 7.4, 5 mM EDTA and 1% Em-myc þ ve/cdkn1a þ / þ , Em-myc þ ve/cdkn1a À / À , Em-myc þ ve/cdkn1a þ / þ / Triton X-100) and supplemented with inhibitors (Leupeptin, Bcl-2 and Em-myc þ ve/cdkn1a À / À /Bcl-2 lymphomas were cultured in six- Pepstatin and phenylmethylsulfonyl fluoride (PMSF), Sigma-Aldrich) as well plates (Greiner Bio-One, Frickenhausen, Germany) in the high-glucose previously described.27 Proteins (30–100 mg) were separated on 15% SDS- version of DMEM supplemented with 10% fetal calf serum, 2mM penicillin/ polyacrylamide gels and electroblotted onto Immobilon-P nylon streptomycin, 0.1 mM L- and 50 mM 2-mercaptoethanol. All cell membranes (Millipore, Bedford, MA, USA). Membranes were incubated lines were cultured in a humidified 10% CO2 atmosphere at 37 1C. Cultured with the following antibodies: a-mBcl-2, mouse monoclonal (1:1000; BD cell lines were further verified by western for the presence and absence of Pharmingen, North Ryde, NSW, Australia; no. 554218); a-p21 (F-5), mouse p21waf1/cip1 expression. The following cell lines were developed from monoclonal (1:300; Santa Cruz Technology, Santa Cruz, CA, USA; no. sc- individually derived lymphomas Em-myc þ ve/cdkn1a À / À (tumors no. 1–4) 6246); a-p27 (c-19), rabbit polyclonal (1:200; Santa Cruz Technology; no. sc- and 2 x Em-myc þ ve/cdkn1a þ / þ (tumors no. 1–3). 528), a-p57kip2, rabbit polyclonal (1:200; Abcam, Cambridge, CA, USA) and a-human Rb, mouse monoclonal (1:200; BD Pharmingen; no. 554136). Anti- b-actin, mouse monoclonal (1:10 000; Sigma; no. A2228, clone AC-74) was Reagents used as a loading control. All antibodies were incubated overnight at 4 1C Vorinostat (Merck, Boston, MA, USA) was dissolved in DMSO for the followed by subsequent incubation with horseradish peroxidase- preparation of 10 mM stock solutions. was supplied as a 34 mM conjugated secondary antibodies (DAKO, Glostrup, Denmark). Immuno- stock (Mayne Pharma Pty Ltd, Mulgrave, VIC, Australia), TMRE (tetra- reactive bands were visualized by enhanced chemiluminescence (Amersham, methylrhodamine ethyl ester) was purchased from Invitrogen Australia Pty Castle Hill, NSW, Australia). All western blots were repeated 3–5 times using Ltd (Mulgrave, VIC, Australia), and PI solution was purchased from Sigma cell lysates obtained from biologically repeated experiments. (St Louis, MO, USA) Retroviral transductions Em-myc/Bcl-2/cdkn1a þ / þ /Bcl-2, Em-myc/Bcl-2/cdkn1a À / À /Bcl-2, Em-myc/ Therapy studies cdkn1a À / À /shRNAmiR-Scr/Bcl-2, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1/ Six to eight week-old C57BL/6 mice were injected with Em-myc lymphomas Bcl-2, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#2/Bcl-2, Em-myc/cdkn1a À / À / (5 Â 105 cells per animal). Peripheral white blood cell (WBC) counts were shRNAmiR-Scr, Em-myc/cdkn1a À / À /shRNAmiR-cdkn1b#1 and Em-myc/ monitored until they exceeded 13 Â 103/ml (Advia 120 Hematology cdkn1a À / À /shRNAmiR-cdkn1b#2 tumors were engineered by retroviral Analyzer, Siemens) and then therapy commenced. Therapy consisted of transduction. Retrovirus-containing supernatant was produced by transfect- vorinostat (200 mg/kg for 7 days, then 150 mg/kg daily for 21 days as ing Phoenix E packaging cells with murine stem cell virus (MSCV)-IRES-GFP/ determined previously4). Mice in the control cohort received the Bcl-2, MSCV-IRES-GFP or MSCV-IRES-GFP/miR30 constructs by standard corresponding amount of DMSO. Cohorts consisted of 10 mice each. phosphate transfection methods. Viral supernatant was used to Peripheral WBC counts and body weights were recorded weekly. Upon transduce Em-myc/cdkn1a þ / þ and Em-myc/cdkn1a À / À cells. Seventy two signs of major distress or when lymphomas were relapsing as indicated by hours after transduction, GFP-positive cells were isolated by flow cytometry- enlarged brachio-axial lymph nodes, mice were euthanized and a necropsy mediated cell sorting and were cultured. Targeting shRNA sequences for was performed. For analysis of therapeutic efficacy, tumor-induced cdkn1b are available upon request. mortality ‘events’ were recorded. Kaplan–Meier analysis was performed and comparisons were made using the log-rank (Mantel–Cox) test MedCalc RNA isolation, cDNA and quantitative real-time PCR analysis software Version 8.0.2.0 (Ostend, Belgium). Total RNA from Em-myc/Bcl-2/cdkn1a þ / þ /Bcl-2 and Em-myc/Bcl-2/ À / À cdkn1a /Bcl-2 cells treated with 2 mM vorinostat or vehicle (DMSO) In vitro assays were isolated using an RNeasy mini kit (Qiagen, Valencia, CA, USA). Time points were taken at 2, 4 and 6 h. Following this, cDNA synthesis was Cells (5–8 Â 105 cells/ml) were incubated for 24 h with increasing doses of performed according to manufacturer’s instructions (Promega, Sydney, vorinostat or etoposide in 1ml cell culture media in 24-well plates (Greiner NSW, Australia). Quantitative PCR analysis of samples was performed on a Bio-One). After incubation, a sample of cell suspension from each sample 7900HT Fast Real-Time PCR System (Applied Biosystems, Mulgrave, VIC, well was harvested into Eppendorf tubes and centrifuged at 13 000 r.p.m. Australia) with SYBR-green ROX mix (Agilent, Mulgrave, VIC, Australia). for 30 s. The pellets were then prepared for a number of apoptosis assays Primers were specific for cdkn1a and cdkn1b, using the housekeeping gene including PI uptake, cell cycle analysis or TMRE staining (see below). All hprt. Primer sequences can be requested from the author. samples were analyzed by flow cytometry. Statistics PI uptake All experiments claiming significant results were analyzed for confidence A 200-ml volume of cell suspension from each sample well was harvested intervals with P-values calculated using a two-tailed Student’s t-test with into Eppendorf tubes and centrifuged at 13 000 r.p.m. for 30 s. The pellets Po0.05 considered statistically significant. were resuspended in 100 ml PBS and 10 ml of PI solution (Sigma) was added from a diluted PI stock (1 mg/ml PI in PBS). CONFLICT OF INTEREST The authors declare no conflict of interest. Cell cycle assay A cell suspension of 200–500 ml was transferred to Eppendorf tubes and centrifuged at 13 000 r.p.m. for 30 s. The supernatant was discarded ACKNOWLEDGEMENTS and pellets were fixed in 200 ml PBS þ 200 ml 100% ice-cold ethanol. Fixed RWJ is a Principal Research Fellow of the National Health and Medical Research cells were stored at 4 1C for 1–7 days. Following storage of fixed cells, Council of Australia (NHMRC) and supported by NHMRC Program and Project Grants, samples were centrifuged at 13 000 r.p.m. for 30 s and washed once the Cancer Council Victoria, The Leukemia Foundation of Australia, the Victorian in PBS. The pellets were resuspended with 200 ml of PI/cell cycle solution Cancer Agency and Victorian Research Consortium. and transferred to fluorescent activated cell sorter (FACS) tubes for analysis. REFERENCES 1 Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase TMRE assay inhibitors. Nat Rev Drug Discov 2006; 5: 769–784. A cell suspension of 100 ml was harvested into FACS tubes and 100 mlof 2 Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of TMRE (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) was cancer. Nat Rev Drug Discov 2002; 1: 287–299. added from a diluted stock (1/10 000 TMRE in PBS) 10–15 min before 3 Ruefli AA, Ausserlechner MJ, Bernhard D, Sutton VR, Tainton KM, Kofler R et al. The analysis. histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide

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& 2014 Macmillan Publishers Limited Oncogene (2014) 5415 – 5423