p28-mediated activation of p53 in g m phase of the cell...

Therapeutics, Targets, and Chemical Biology

p28-Mediated Activation of p53 in G2–M Phase ofthe Cell Cycle Enhances the Efficacy of DNADamaging and Antimitotic ChemotherapyTohru Yamada, Tapas K. Das Gupta, and Craig W. Beattie

Abstract

p28 is an anionic cell-penetrating peptide of 28 amino acidsthat activates wild-type andmutated p53, leading subsequently toselective inhibition of CDK2 and cyclin A expression and G2–Mcell-cycle arrest. In this study, we investigated the cytotoxic effectsof p28 treatment alone and in combination with DNA-damagingand antimitotic agents on human cancer cells. p28 enhanced thecytotoxic activity of lower concentrations (IC20-50) of DNA-dam-aging drugs (doxorubicin, dacarbazine, temozolamide) or anti-mitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells

expressing wild-type or mutated p53. Mechanistic investigationsrevealed that p28 induced a post-translational increase in theexpression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at theG2–Mphase. The enhanced activity of theseanticancer agents in combination with p28 was facilitatedthrough the p53/p21/CDK2 pathway. Taken together, theseresults highlight a new approach to maximize the efficacy ofchemotherapeutic agents while reducing dose-related toxicity.Cancer Res; 76(8); 2354–65. �2016 AACR.

IntroductionCurrent cytotoxic, chemotherapeutics include DNA-damaging

and antimitotic agents acting singly or in combination. DNA-damaging agents, such as doxorubicin, intercalate with DNA,inducing double strand breaks that induce ataxia-telangiectasiamutated (ATM)-dependent nuclear accumulation of p53 (1).Additional targets for DNA-damaging agents include the apopto-tic pathway via Bcl-2/Bax and the caspase cascade, the necroticpathway through Toll-like receptors, etc. Unfortunately, similardamage to normal cells results in adverse effects and possibly life-threatening toxicity (e.g., cardiotoxicity) to major organs. Incontrast to intercalating agents, the DNA alkylating agent dacar-bazine (DTIC) and its derivative temozolamide (TMZ) inhibitcancer growth by methylating guanine nucleotides, preventingDNA replication, and inducing G2–M arrest via p53, respectively(2). This suggests that the integrity of the G2–M cell-cycle check-point may be important in the cytotoxicity of these agents and asite for intervention (3). However, the multiple roles of p53 inregulating DNA-repair enzymes may be dependent on tumortype, and inherent and acquired resistance to alkylating agentspresent major obstacles to successful treatment (4).

Taxanes, mitotic inhibitors such as paclitaxel and docetaxel,used to treat a variety of malignant tumors, bind to the beta-

tubulin subunits of microtubules. This induces a prolongedactivation of the mitotic spindle checkpoint and mitotic arrestfollowed by mitotic slippage and induction of apoptosis (5).Although considered cytotoxic, they are in fact cytostatic, with thearrest triggering cell death (6). Taxanes also induce post-transcrip-tional acetylation and phosphorylation of p53. This increasesintracellular p53, upregulating its target proteins p21 and Bax,inhibiting the cell cycle, and increasing apoptosis (5). Unfortu-nately, cancer cells have weak spindle checkpoints and activatevarious prosurvival signals that lead to clinical resistance towardtaxanes. In addition to resistance, taxanes, like DNA-damagingagents, can also induce significant toxicity that may force treat-ment to become dose limiting.

Increasing the concentration of DNA-damaging and antimitot-ic agents or combining them to increase cytotoxicity generallyenhances antitumor activity by activating multiple pathways (7).For example, the cyclinD–CDK4/6-retinoblastoma protein (RB)–INK4 axis is universally disrupted, facilitating cancer cell prolif-eration and prompting the targeting of CDK4/6 as an anticancerstrategy (8). The dependence of subsets of melanomas andneuroblastomas on CDK2 (9) suggests that the CDK2 pathwaymay also significantly affect proliferation. For example, inhibitorsof CDK2 predispose cells to DNA damage and activate p53 (10),suggesting the G2 checkpoint andDNA-damage pathwaysmay belinked (11). As direct CDK inhibitors abrogate DNA damage–induced checkpoint and repair pathways, they provide an oppor-tunity to potentiate the response to DNA-damaging agents (12)and potentially antimitotics. Specific, non-toxic cell-cycle inhibi-tors that maintain or increase efficacy while reducing the toxicityof either DNA-damaging or antimitotic agents, alone or in com-bination, would clearly be welcome.

This suggests adding aCDK inhibitor to either a taxane orDNA-damaging agent would potentially decrease chemoresistance andincrease efficacy (13). However, becausemanyCDK inhibitors arenot selective, their use in concert with DNA-damaging agents canbe complicated by a direct cell-cycle arrest superimposed on the

Department of Surgery, Division of Surgical Oncology, University ofIllinois at Chicago College of Medicine, Chicago, Illinois.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Tohru Yamada, University of Illinois at Chicago Collegeof Medicine, Department of Surgery, Division of Surgical Oncology, 840 SouthWood Street, Chicago, IL 60612. Phone: 312-413-1156; Fax: 312-996-9365; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-2355

�2016 American Association for Cancer Research.

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modulation of upstream components of the checkpoint andrepair pathways (12). Selective (14) and pan-CDK inhibitors,such as flavopiridol (15), inhibit CDKs 1, 2, 4, 6, and 9, andinduce cell-cycle arrest at G1 and G2 through competitive inhibi-tion of the ATP-binding site, they also have a synergistic effectwhen combinedwith aDNA-damaging agent (11) or taxane (13).This effect is complicated as direct inhibitors of CDK1 and CDK2arrest the cell cycle in G1 andG2, andmay also inhibit the effect ofproliferation-dependent agents (11). The absence of pharmaco-dynamic endpoints for pan-CDK inhibitors complicates confir-mation of target inhibition, cytotoxicity is not limited to cyclingcells, and appears independent fromwild-type or mutated p53 orRb (16).

Although exposure to a direct cell-cycle inhibitor in combina-tion with chemotherapeutic agents may increase chemosensitiv-ity, evidence remains limited for enhanced activity of either aDNA-damaging or antimitotic agent via a p53-regulated, CDK-mediated pathway, independent of cell type or agent (17).

p28, amino acids 50 to 77 of azurin, a cupredoxin secreted byPseudomonas aeruginosa, is a non-toxic, amphipathic, anionic cell-penetrating peptide that preferentially enters a wide variety ofcancer cells (18, 19). Upon entry, p28 binds to amutational "coldspot" (20) within the DNA-binding domain (DBD) of p53wt,mut,where it blocks the binding of the E3 ligase Cop1, inducing asignificant post-translational increase in the level and activity ofwild-type and mutant p53 (21–26). This increase in p53wt,mut

levels and activity upregulates p21 and p27, inducing a significantdecrease in the intracellular level of the CDK2–cyclin A complex,essential proteins in the mitotic process (27), as well as FoxM1, atranscription factor for G2–M progression (24, 26). As a targetedcytostatic therapy, p28 exhibits no significant adverse effects ortoxicity (28), exerts a prolonged block at G2–M that leads to asignificant apoptotic cell death in breast cancer cells (26) andprevents the carcinogen-induced transformation of mouse mam-mary epithelial ducts (29). Chemopreventive agents such as theantimitotic Vinca alkaloids that inhibit cellular proliferation alsoindirectly lead to apoptosis (6, 30). As such, p28 appears as a truecell-cycle–specific cytostatic agent that induces cytotoxicity indi-rectly via a p53wt,mut-mediated block at G2–M (18, 26).

In this study, we assessed the effects of p28 alone and incombination with IC20 and IC50 concentrations of standardDNA-damaging and antimitotic agents on the degree of cytotoxiceffect in p53wt,mut,null-matched human prostate, breast cancer,glioblastoma, melanoma, and neuroblastoma cell lines as afunction of cell proliferation and p53 status. The results suggestthat a sustained increase in p53wt,mut in the presence of lowerdoses of either DNA-damaging or antimitotic agents is a prom-ising avenue to reduce the overall toxicity of chemotherapeuticagents while maximizing their efficacy.

Materials and MethodsPeptide synthesis

p28 (Leu50-Asp77 LSTAADMQGVVTDGMASGLDKDYLKPDD,2914Da)was synthesized byCSBio, Inc. at>95%purity andmassbalance.

Cell cultureTemozolomide (>98%) and docetaxel (>97%) were obtained

from Sigma-Aldrich, dissolved in DMSO and diluted in MEM-E(Invitrogen; final conc of DMSO <3% and <0.1%, respectively).Clinical grade of doxorubicin hydrochloride and paclitaxel was

obtained fromBenVenue Laboratories andTeva Pharmaceuticals,respectively. DTIC was obtained from APP Pharmaceuticals, dis-solved in sterile water and diluted in MEM-E.

Human fibroblast, mammary MCF-10A cell line, and humancancer cell lines were obtained from American Type CultureCollection (authenticated using morphology, karyotyping, PCR,and STR profile): prostate cancer, LNCaP (p53wt, ARþ), DU145(p53mut, AR�), and PC-3 (p53null, AR�); neuroblastoma, IMR-32(p53wt), and SK-N-BE2 (p53mut); breast cancer, ZR-75 (p53wt)andMDA-MB-231 (p53mut); and glioblastoma, U87 (p53wt), andLN229 (p53mut).Humanmelanoma,Mel-29 (p53wt), andMel-23(p53mut) were established and characterized in our laboratory(authenticated usingmorphology and karyotyping; ref. 31). Cellswere cultured inMEME supplemented with 10% heat-inactivatedFBS (Atlanta Biological, Inc.) at 37�C in 5% CO2 (18, 24, 25).

Cell proliferation assaysHuman cancer cells were cultured in MEM-E containing

2 mmol/L L-glutamine, 0.1 mmol/L essential amino acids sup-plemented with 10% heat-inactivated fetal bovine serum (18).The doubling time of each cancer cell line (LNCaP, DU145, IMR-32, SK-N-BE2, Mel-29, Mel-23, U87, LN229, ZR-75, and MDA-MB-231)was determined as follows. Cellswere seeded (triplicate)at 20,000 cells/well and cultured in MEM-E for 72 hours in theabsence of cytotoxic agent or p28 changed daily as describedpreviously (18, 26, 32, 33). Proliferative rate was expressed asdoubling time in hours. The doubling time of each cancer cell linewas determined from semi-logarithmic plots of initial cell num-ber versus cell counts at 24, 48, and 72 hours. The relationshipbetween doubling time and cytotoxic activity or difference incytotoxic activity between agents alone and in combination wasanalyzed by nonlinear regression. Curves were fitted usingGraph-Pad PRISM (version 5.0) as a function of a nonlinear one-phasedecay (maximum iterations: 1,000, Cl: 95%).

We initially determined the concentration of p28 (�100mmol/L) that inhibited cell proliferation 20% to 50% (IC20-50)across the p53wt,mut cell lines (18, 24–26) over 72 hours bydirect count, then established the IC20-50 for cytotoxicity using astandard MTT assay (ref.18; Table 1). Each cell line was thenexposed to a cytotoxic agent or p28 alone or p28 in combina-tion with IC20-50 dose of doxorubicin, DTIC, TMZ, paclitaxel, ordocetaxel to establish whether cytotoxicity was enhanced. Cul-ture medium was replaced with fresh media containing p28(quadruplicate), chemotherapeutic agent, or an equal volumeof medium (control; 8 replicates) daily for 72 hours. Resultswere compared by ANOVA (Newman–Keuls multiple compar-isons; GraphPad InStat ver. 3.0). Methods for Western blottingand pull-down assay are described in Supplementary Methods.

Xenograft tumor modelsHuman breast cancer MDA-MB-231andmelanomaUISO-Mel-

23 were injected s.c. on the right flank of 5- to 6-week-old femaleandmale athymicmice, respectively.When tumors reached 3mmin diameter, animals were randomized into control and treatmentgroups that received: PBS i.p. (control); paclitaxel 12.8 mg/kg (15mmol/kg) i.p. ondays 10, 14, 21, and25 after tumor development;DTIC (dacarbazine), 4 mg/kg (22 mmol/kg), 3�/week; or p28 10mg/kg (3.4 mmol/kg) i.p. daily for 30 days. Tumor volume wasdetermined 3�weekly andnormalized to bodyweight.Micewerenecropsied on either days 16 or 31 (24 hours). At necropsy,

Effect of p53 Activation on Chemotherapeutic Agents

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tumors were dissected, weighed, and frozen prior to preparationof lysates (26). Statistical comparisons were performed byANOVA (control vs. each treatment group). Similarly, humanmelanoma UISO-Mel-2 cells were injected s.c. in the rightflank ofmale athymicmice. Animals were injected i.p. with DTIC,4mg/kg, 3�/week; p28 4mg/kg (1.4 mmol/L) i.p. daily; or p28 incombination with DTIC for 16 days. Statistical comparisons wereperformed by ANOVA.

ResultsEffect of p28 treatment on the growth of human cancer cells

p28 inhibits the proliferation of a wide variety of p53þ humantumor cells in vitro (18, 26) and in vivo (34). Figure 1 illustrates theinhibitory effect of 50 and 100 mmol/L p28 on the proliferation ofhistogenetically diverse human cancer cell lines matched for p53(wt vs. mut) expression (25, 35). The cytostatic effect on prostatecancer cell lines LNCaP (p53wt, ARþ) and DU145 (heterozygousp53mut P223L,V274F, AR�)was time- (data not shown) anddose-dependent, decreasing the proliferation of LNCaP andDU145 celllines at 72 hours by 18% and 22% (P < 0.001) at 100 mmol/L,respectively (Fig. 1). p28was not active against the p53null cell linePC3 (AR�), confirming our earlier report (24). Exposure to 100mmol/L p28 for 72 hours decreased the proliferation of neuro-blastoma IMR-32 (p53wt) and SK-N-BE2 (p53mut C135F) cells�35% from control as well as inhibiting the proliferation ofhuman glioblastoma U87 (p53wt), LN229 (p53mut K164E),breast cancer ZR-75 (p53wt), MDA-MB-231 (p53mut R280K), andmelanoma Mel-29 (p53wt) and Mel-23 (p53mut internal deletionat 178–183) cell lines, consistent with p28 inhibition of Mel-2(p53wt; ref.18; Fig. 1). In contrast, exposure to 100 mmol/L p28 for72 hours did not decrease the proliferation (<5 %) of normalhuman cells;fibroblasts (control vs. p28, P¼ 0.77), andMCF-10Abreast cells (P ¼ 0.48) as previously reported (24). Collectively,the data demonstrate that p28 is an effective cytostatic agentagainst p53wt and p53mut cancer cell lines that do not harborDNA contact mutations or a completely unfolded DNA bindingdomain (DBD; refs. 24, 25). These results are also consistent withsuggestions that a potential gain-of-function associated withmutations in p53 is not a universal phenomenon for p53mutants(24, 25, 34).

Similar results were observed in MDA-MB-231 and Mel-23xenograft tumors (Fig. 2A and D), where 10 mg/kg p28 inhibitedtumor growth equal to or better than the IC50 dose of paclitaxelin MDA-MB-231 cells and induced a dose-related inhibition of

Mel-23 proliferation comparable with an IC20 dose of DTIC. Thep28-induced inhibition of tumor proliferation (24, 25) wasclearly associated with an increase in tumor p53 and p21 levels(Fig. 2B, C, E and F). In an additional study, p28 4 mg/kg alone(20%–60% less than the optimumdose; Fig. 2A andD; ref. 34) i.pdaily inhibited the growth of p53wt human melanoma (UISO-Mel-2) xenografts (Fig. 2G). Tumor growth in animals treatedwith p28 in combination with DTIC (4 mg/kg each) i.p. wassignificantly decreased from DTIC alone and p28 alone after 14days of exposure (Fig. 2G) without any loss in body weight(control group: 25.7 � 0.5; DTIC: 25.4 � 0.6; combination:25.9 � 0.8; p28: 26.9 � 0.9 g) or alteration in behavior (datanot shown).

p28 enhances the activity of DNA-damaging and antimitoticagents

We determined the degree of cytotoxicity on the same set ofcancer cell lines exposed to either p28 alone (Fig. 1) or incombination with either DNA-damaging or antimitotic agents.p28 was not cytotoxic against LNCaP (p53wt, ARþ) and DU145(p53mut, AR�) or PC3 (p53null, AR�) prostate cancer cells (Fig. 3A,G and H) after 72-hour exposure, with marginal effect on IMR-32and SK-N-BE2 neuroblastoma and ZR-75, MDA-MB-231 breastcancer cells (Fig. 3B and F). However, p28 did induce significantcytotoxicity (25%–30%) in U87 and LN229 glioblastoma cellsand p53wt Mel-29 melanoma after a 72-hour exposure withoutsignificantly affecting p53mutMel-23melanoma cells (Fig. 3C–E).Additional MTT assays demonstrated that exposure to p28 at 100mmol/L for 72 hours did not induce cytotoxicity in fibroblasts(control vs. p28, P ¼ 0.12) and MCF-10A (control vs. p28, P ¼0.67). It is noteworthy that, in general, the induction of cytotox-icity in response to p28 alone was inversely correlated with thedoubling time of each cell line (pair), suggesting that the degree ofcytotoxicity following exposure to p28 alone was related to thedoubling time (Fig. 4A; ref. 36) of the cell line exposed andduration of that exposure (Fig. 3). This was not true of eitherDNA-damaging or antimitotic agents at their IC20 and IC50

concentrations (Table 1 and Fig. 4B), illustrating the differencebetween a true cytostatic and the cytostatic/cytotoxic agentscurrently in use as therapeutics (6). It has also been suggestedthat it is difficult to discern whether the cytotoxic activity ofstandard chemotherapeutic agents is mediated via a differenttarget (off-target; ref. 6). Figure 4C illustrates the correlationbetween the cytotoxic activity of a p28/agent combination versusthe agent alone and doubling time of each cell line treated. A

Table 1. IC20–50 values for DNA-damaging and antimitotic agents determined by the MTT assay

Prostate cancer Glioblastoma Melanoma Neuroblastoma Breast cancerCell line LNCaP DU145 U87 LN229 Mel-29 Mel-23 IMR32 SK-N-BE ZR-75 MDA-MB-231

p53 statusa p53wt P223L,V274F p53wt K164E p53wt D178-183 p53wt C135F p53wt R280KDoxorubicin 8, 83

nmol/L7, 60nmol/L

N.D. N.D. N.D. N.D. 1.2, 8nmol/L

46, 158nmol/L

N.D. N.D.

DTIC N.D. N.D. 0.3, 1.6mmol/L

1.8, >2mmol/L

0.4, 1.4mmol/L

0.5, 1.5mmol/L

N.D. N.D. N.D. N.D.

TMZ N.D. N.D. 0.1, 1mmol/L

0.1, 0.8mmol/L

N.D. N.D. N.D. N.D. N.D. N.D.

Paclitaxel 0.8, 5nmol/L

2, 5nmol/L

N.D. N.D. N.D. N.D. N.D. N.D. 5.1, 34nmol/L

3.8, 14nmol/L

Docetaxel 0.3, 1.1nmol/L

1.8, 7.5nmol/L

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Abbreviations: N.D., not determined; DTIC, dacarbazine; TMZ, temozolomide.aYamada et al. (25)

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shorter doubling time was again correlated with the increase incytotoxic activity observed with the combination versus the agentalone. There was no apparent relationship (P¼ 0.26) between theincrease in cytotoxicity and p53 mutation status as they did notinclude DNA contact mutations or mutations that produce acomplete unfolding of the p53 molecule (Fig. 4D).

Although all DNA-damaging and antimitotic agents induced acytotoxic effect in all five p53wt,mut,null cell lines treated (Table 1and Fig. 3), there were significant differences in the concentrationof doxorubicin and p28 to effect a similar inhibitory response

between p53wt, p53mut, and p53null cell lines of the same type(Table 1 and Fig. 3A). In contrast, the IC20-50 concentrations ofDTIC and TMZ were similar across cell lines and p53 status. Ingeneral, concentrations of the antimitotic paclitaxel and docetaxelthat achieved an IC20–50 were also cell line dependent, but not asdependent on p53 status as doxorubicin (Table 1; Fig. 3A, F–H).

Exposure to p28 significantly increased cytotoxicity of doxo-rubicin on p53wt, ARþ LNCaP, and p53mut, AR� DU145 prostatecancer cells, but did not increase the cytotoxicity from either agentin p53null, AR� PC3 cells (Fig. 3A, G and H). A similar effect

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Figure 1.Growth-inhibitory effect of p28. The effect of p28 on cell growth was determined by direct cell counting. Prostate (A, LNCaP, DU145, and PC-3), neuroblastoma(B, IMR-32 and SK-N-BE2), melanoma (C, Mel29 and Mel23), glioblastoma (D, U87 and LN229), and breast (E, ZR-75 and MDA-MB-231) cancer cells wereincubated with increasing concentrations of p28 (gray bar, 50 mmol/L; white bar, 100 mmol/L; black bar, control) at 37�C for 72 hours. Cells were then counted in aBeckman Coulter (Z1 coulter particle counter). Cell numbers in control wells were considered as 100%. Mean � SE. Compared with control; � , P < 0.05;�� , P < 0.01; �� , P < 0.001. Compared between p28 doses; #, P <0.05; ##, P < 0.01.






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Figure 2.Effect of p28 as a single agent on xenograft growth. A, p53mut MDA-MB-231 human breast cancer cells. Tumor volumewas determined 3�weekly and normalized tobody weight for 30 days. Control (n ¼ 9), paclitaxel (n ¼ 6), p28 (n ¼ 6). B, p53 and p21 expression. Tumor lysates were subjected to Western analysis(three randomly selected tumors/group). C, treatment levels are expressed as a percentage of control. Mean � SE; � , P < 0.05. D, p53mut Mel-23 humanmelanoma cells. Tumor volume was determined 3�weekly for 30 days and normalized to body weight. Control (n¼ 19), DTIC (n¼ 10), p28 (n¼ 10). E. expressionof p53 and p21 in DTIC- and p28-treated mice. Lysates of excised tumors were subjected to Western analysis (three randomly selected tumors/group).F, each experimental level is expressed as a percentageof control. � ,P<0.05. G, treatment groups injected i.p. withDTIC, 4mg/kg, 3�/week (n¼ 7); p28, 4mg/kg i.p.daily (n ¼ 7); or p28 in combination with DTIC under an identical dosage (n ¼ 7) for 16 days. Control group received PBS i.p. (n ¼ 13). Statistical comparisonswere performed by ANOVA (control vs. each treatment group; � , P < 0.05, combination vs. each treatment group; #, P < 0.05).

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was observed for p28 in combination with doxorubicin inp53wt IMR-32 and p53mut SK-N-BE2 neuroblastoma cells (Fig.3B). The increase in cytotoxicity inducedbyp28wasnot limited tothe DNA-intercalating agent doxorubicin. When p53wt,mut glio-blastoma and human melanoma cells were exposed to combina-tions of p28 and the methylating agents DTIC and TMZ, cyto-toxicity was also significantly enhanced overDTIC and TMZ alone(Fig. 3C–E), suggesting that the effect is not limited to either asingle type of DNA-damaging agent, p53 status, or the embryonicorigin of a cell line.

While exposure to p28 did not increase the cyctotoxic effect ofpaclitaxel on normal MCF-10A breast cells (paclitaxel vs. combi-nation; P¼ 0.18) and fibroblasts (paclitaxel vs. combination: P¼0.98), exposure of p53wt ZR-75 and p53mut MDA-MB-231 breastcancer cells, and p53wt LNCaP and p53mut DU145 prostate cancercells to p28 in combination with paclitaxel significantly increasedthe cytotoxicity (Fig. 3F and G). A similar response was observedfor p28 in combination with docetaxel (Fig. 3H). This againsuggests that a p28-induced increase in the expression of p53mediates the increase in cytotoxic effect irrespective of the type ofagent it is combined with.

p53 and downstream targetsExposure of p53wt LNCaP and p53mut DU145 cells (but not

p53null PC3 cells) to doxorubicin increased p53, p21, and FoxM1,but not CDK2, levels relative to control. p28 alone also elevatedp53 and p21 levels, but, in contrast to doxorubicin, decreasedFoxM1 and CDK2 levels (Fig. 5A) and had no effect on p53null

PC3 cells. The increase in p53 in LNCaP and DU145 cells treatedwith p28 combined with doxorubicin was similar (LNCaP 242%vs. DU145 223%), as was the relative increase above doxorubicinalone. A similar increase in p21 levels was observed over doxo-rubicin, although the relative value compared with control washigher in DU145 cells (Fig. 5A), suggesting that either DNA-damaging agents or p28 may be more effective in increasingp21 in AR� prostate cancer cells. Doxorubicin also increasedFoxM1 levels significantly over control in LNCaP and DU145cells (Fig. 5A). FoxM1, an essential transcription factor for G2–Mprogression in LNCaP and DU145 cells, is reportedly stabilizedthrough the checkpoint pathway by DNA damage–sensingkinases (e.g., ATM) anddownstream signaling effectors, includingcheckpoint kinase 2, in response to DNA damage (37). Anincrease in FoxM1 suggests that the G2–M transition is enhanced.As the expression level of CDK2 reportedly correlates with FoxM1expression (38), the increase in CDK2 inDU145 cells treated withdoxorubicin was associated with the increase in FoxM1 (Fig. 5A).In contrast to doxorubicin, the p28-induced increase in p53reduced the levels of FoxM1 in LNCaP and DU145 cells (Fig.5A) as previously reported (25). This is not surprising as p53negatively regulates FoxM1 expression independently of thechecking pathway (39). Exposure of LNCaP and DU145 cells top28 alone or in combination with doxorubicin reduced CDK2levels below that of control or doxorubicin alone, respectively, inparallel with FoxM1.

In contrast to doxorubicin, DTIC did not significantly increasethe level of p53, p21, or FoxM1 in p53wt U87 glioblastoma cellsrelative to control, but did reduce CDK2 levels. However, unlikep53mut Mel-23 melanoma cells (Fig. 2E and F; 3C) and p53wt

U87 glioblastoma (Fig. 3D), DTIC did increase p53mut and p21significantly in p53mut LN229 cells (Fig. 5B) without altering

either FoxM1 or CDK2. p28 alone also increased the levels ofp53 and p21 in p53wt U87 and p53mut LN229 cells whiledecreasing the levels of FoxM1 and CDK2 in both lines. Expo-sure to p28 in combination with DTIC increased the levels ofp53 and p21 above that of either control or DTIC alone withoutaltering the levels of FoxM1 and decreasing CDK2 in both celllines, mirroring the increase in efficacy (Fig. 3D). A similarpattern was observed in the responses of p53, p21, FoxM1, andCDK2 for TMZ in U87 and LN229 cells. The addition of p28produced a pattern of expression similar to that of DTIC thatparalleled the activity of the combination in both lines. In sum,the increased activity of DNA-damaging agents (Fig. 5A–C) incombination with p28 appeared to result from the activation ofthe p53/p21 axis, significantly reducing CDK2 levels relative toeach agent alone and enhancing the cytotoxicity of lower con-centrations of these agents.

Exposure of p53wt LNCaP cells to the antimitotic paclitaxelincreased the levels of p53 and p21, but did not alter FoxM1 orCDK2 (Fig. 5D). The increase in p53 and p21 levels in p53wt

LNCaP cells treated with p28 in combination with paclitaxel wasqualitatively similar to cells treated with paclitaxel alone (Fig.5D). Although the level of FoxM1 was not altered by p28 andpaclitaxel in LNCaP cells, CDK2was significantly reduced to levelsbelow those observed with p28 alone. The paclitaxel-inducedcytotoxic effect in p53mut DU145 cells (Fig. 3D)was accompaniedby a decrease in p53mut relative to control, while slightly increas-ing p21 and significantly elevating FoxM1 and CDK2 abovecontrol levels (Fig. 5D). The increase in FoxM1 in p53mut DU145cells treated with paclitaxel alone (Fig. 5D) reportedly occurs inresponse to the paclitaxel-induced decrease in p53, a paclitaxel-induced early activation of p53/p21 pathway (40), or activationvia a p53-independent pathway (41). As p28 in combinationwithpaclitaxel increased p53 and p21 while significantly decreasingFoxM1 and CDK2 levels below those observed with paclitaxelalone and control, it suggests that themutations in p53 in this cellline do not prevent p28 from enhancing the generalized cytotox-icity observedwithpaclitaxel alone (Figs. 2B, 3F andG; refs. 5, 42).The increased activity of paclitaxel in combination with p28,coupled with the increase in p53 and p21 and decrease in FoxM1and CDK2 compared with that of paclitaxel alone, was likely dueto the post-translational increase in p53 and p21 induced by p28(Fig. 5D) that enhanced the cytotoxic activity of paclitaxel in thiscell line.

Unlike paclitaxel, docetaxel, the taxane prescribed for advancedAR� prostate cancer, increased p53 and FoxM1 without signifi-cantly altering either p21 or CDK2 levels in LNCaP cells (Fig. 5E).Here again, p28 in combination with docetaxel further increasedthe level of p53 andp21while inhibiting FoxM1 andCDK2 levels.Docetaxel, unlike paclitaxel, did not significantly alter either p53or p21 levels inDU145 cells, but did increase FoxM1while leavingCDK2unchanged, suggesting subtle differences in themechanismof action of these two taxanes. However, p28 in combinationwithdocetaxel increasedp53andp21 levelswhile significantly decreas-ing the expression of FoxM1 and CDK2 below that in docetaxel-treated and control DU145 cells (Fig. 5E), a pattern similar to thatobserved with paclitaxel (Fig. 5D).

Chemotherapeutic agents do not alter the interaction of p28with p53

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Yamada et al.





activity. For example, doxorubicin in combination with a recom-binant human IL1a induces a significant synergistic antiprolifera-tive effect in humanmelanoma cells by increasing IL1abinding tothe receptor on melanoma (43). Genotoxic drugs can also triggermultiple molecular events, including activation of p53-indepen-dent checkpoints, partially protecting cancer cells during chemo-therapy (44). The effect can be avoided with agents targetedspecifically at enhancing p53 activity. Treatment of tumor cellswith doxorubicin causes phosphorylation and acetylation of p53,transcriptional upregulation of p21 and other target genes, andgrowth arrest (45). These observations, coupled with the ATP-dependent, nuclear pore-mediated transport of the doxorubicin–proteasome complex, suggest the possibility that doxorubicincould directly or indirectly alter the affinity of p53 for p28. We

explored this possibility with competitive pull-down assays ofp28 binding to p53 in lysates from cells previously treated witheither doxorubicin or paclitaxel. GST coupled to p28 (GST-p28)pulled down endogenous p53 from p53wt LNCaP and p53mut

DU145 cells; GST alone did not (Fig. 6). The L1 (aa 112–124) andS7–S8 (aa 214–236) loops and T140, P142, Q144, W146, R282,andL289of thep53DBDhavebeen identified as potential sites forp28 binding (24). Although DU145 is heterozygous for muta-tions in p53 (P223L, V274F), p28 pulled down p53mut fromDU145 more effectively than did p53wt in LNCaP (Fig. 6, lanes 2and 6, top). This confirms our earlier results showing that theheterozygous mutant p53 (P223L, V274F) had a higher affinityfor p28 (�59 kJ/mol) than wt p53 (25). Neither paclitaxel nordoxorubicin altered the interaction of p28with p53 in LNCaP and

Figure 3.The effect of combination of p28 and chemotherapeutic agents on cancer cells was determined by the MTT assay. Chemotherapeutic agent p28 or p28 incombination was added daily for 72 hours. Prostate (A, G, H), neuroblastoma (B), melanoma (C), glioblastoma (D and E), and breast (F) cancer cells were incubatedwith chemotherapeutic agent, p28 or p28 in combination for 72 hours. Concentrations are shown above each bar. MTT reagent was added to each well,and the percentage of change in absorbance at 570 nm in treated cells relative to untreated controls was determined. Values, mean� SE. Combination versus eachsingle agent; a, P < 0.05; a, P < 0.01; A, P < 0.001. Compared with control; b, P < 0.01; B, P < 0.001.

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Figure 4.Relationship between proliferative rate, drug sensitivity, and p53 status. All cell lines were cultured for 72 hours in complete media in the absence of cytotoxicagent or p28 and their doubling time determined. A, percent increase in the cytotoxic effect in p28-treated cells relative to untreated controls plotted as afunction of the respective doubling time for each cell line (control considered as 100%). Mean values were calculated from independent replicates in the samecell lines exposed to p28 at 50 mmol/L (e.g., Fig. 3A, G, H and D, E). B, percent increase in cytotoxic activity as a function of the respective doubling time foreach cell line in agent-treated cells relative to untreated controls. C, percent increase in cytotoxic activity determined by the MTT assay (percent decreaseof p28:agent combination subtracted from percent decrease of the agent alone) plotted against doubling time (hours). D, scatter dot plots of percent increase incytotoxic activity and p53 status (wt vs. mut) in cancer cell lines tested.






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Figure 5.Effect of the combination of p28 and chemotherapeutic agents on the p53 pathway. Prostate cancer cells (LNCaP, DU145, and PC-3) and glioblastoma (U87 andLN229) were treated with chemotherapeutic agent p28 or combination added daily for 72 hours. Concentrations used as follows: A, p28 (LNCaP: 50 mmol/L,DU145 and PC-3: 100 mmol/L), doxorubicin (LNCaP: 1 nmol/L, DU145: 5 nmol/L, and PC-3: 100 nmol/L), or combination. B, p28 (U87 and LN229: 50 mmol/L), DTIC (U87and LN229: 1 mmol/L), or combination. C, p28 (U87 and LN229: 50 mmol/L), TMZ (U87 and LN229: 100 mmol/L), or combination. D, p28 (LNCaP andDU145: 50 mmol/L,PC-3: 100 mmol/L), paclitaxel (LNCaP and DU145: 1 nmol/L, PC-3: 2 nmol/L), or combination. E, p28 (LNCaP and DU145: 50 mmol/L), docetaxel (LNCaP: 0.1 nmol/Land DU145: 1 nmol/L), or combination. Whole-cell lysates were subjected to Western analysis. Each band was normalized by calculating the ratio of p53, p21,FoxM1, or CDK2 over actin. The histograms are representative of at least two independent experiments (relative intensity to control expressed as 100%).


Yamada et al.




DU145 cells (Fig. 6). Although p28 in combination with pacli-taxel and doxorubicin enhanced cytotoxic effect in LNCaP andDU145 (Fig. 3), the enhancement was not the result of an alteredaffinity between p28 and p53 (Fig. 6).

DiscussionThe majority of expressed p53 mutations reportedly exhibit

gain-of-function properties actively promoting prosurvival sig-nals and tumorigenesis, independent of the loss of wild-type p53function (46). Although gain-of-function effects are complex(46), an apoptosis-resistance feature appears to be directly linkedto high expression ofmutant p53 (47). Tumors that depend on aninactivated, e.g., mutated, p53 for continued proliferation areinhibited if p53 is reactivated. Because suppression of at leastcertain types ofmutant p53 renders cancer cells highly susceptibleto apoptosis, it affords the opportunity to combine restoration ofp53 activity with lower doses of DNA-damaging and antimitotictherapy to further increase efficacy and reduce toxicity. Thisapproach would appear to bemost effective in high-grade tumorswith rapid doubling times, as an increase in the overall effective-ness of multiple combinations of p28 with DNA-damaging orantimitotic agents appears dependent on a rapid proliferative rate(Fig. 4).DNA-damaging agents induce apoptosis in cancer cells byactivating p53 through the ATM pathway (48). Taxanes alsoinduce p53 via post-transcriptional modifications that increaseboth its level and activity (5). To date, strategies directed atactivating p53 in tumors have largely focused on targetingwild-type p53, rather than themutated p53present in a significantpercentage of most solid tumors. Fortunately, a significant per-centage of these mutations are single-point, missense mutationsthat allow the expression of a p53mut that essentially remainsfunctional (49). This is in sharp contrast to other tumor suppres-sor proteins (e.g., the cyclin D–CDK4/6–retinoblastoma protein(RB)–INK4 and p16) that are generally inactivated by homozy-gous deletion, additional smaller deletions or promoter methyl-ation that produce a truncated unstable protein or complete lackof expression. These observations also suggest that drugging p53wt

and a significant number of p53mut tumors to induce p53-depen-dent suppression of cell growth is a viable option for treatment.

The post-translational increase in p53 levels and activityinduced by a non-genotoxic p28 significantly increases the down-streamCDK inhibitors p21 and p27, inhibiting CDK2 and the cellcycle at G2–M (26) in virtually all p53wt and p53mut tumorsdevoid of DNA contact mutations or mutations that produce acomplete unfolding of the p53 molecule (25, 26). Neither theCDK4/6 pathway, p16INK4A nor senescence is invoked (26). For

example, p28 increased the level of p53 and p21 in p53wt LNCaPand p53mut DU145 prostate cancer and p53wt U87 and p53mut

LN229 glioblastoma cells, but decreased the p53 negativelyregulated FoxM1 and CDK2 proteins (Fig. 5A–C). In contrast top28, doxorubicin significantly increased FoxM1 levels in LNCaPand DU145 prostate cancer cells (Fig. 5A). Because several DNAmismatch repair proteins (MLH1, PMS1, and PMS2) are inacti-vated in DU145 cells (50), the level of FoxM1 in response toDNAdamage was higher than that in LNCaP cells (Fig. 5A). FoxM1 isalso transcriptionally regulated by E2F1, which induces the accu-mulation of FoxM1 in response to DNA damage, suggesting thatthe DNA-damage–response pathway also regulates FoxM1 inde-pendent of p53 (51). CDK2 expression was not altered by doxo-rubicin alone in either cell line, but significantly reduced whencombined with p28, reflecting the increase in levels of p53 andp21 above either agent alone (Fig. 5A). The levels of CDK2 incancer cells treatedwithDNA-damaging agents paralleled those ofFoxM1, as CDK2 expression transcriptionally correlates withFoxM1 expression, supporting a role for CDK2 during FoxM1-induced cell-cycle progression (38). These data clearly suggest thatthe enhanced cytotoxicity with p28 and doxorubicin in combi-nation in prostate cancer and neuroblastoma (Fig. 3A and B) is adirect result of a p28-induced increase in p53.

Expression of DNA repair proteins also plays a decisive role inprotecting cells against the effect of alkylating agents. The DNArepair enzymeO6-alkylguanineDNA alkyltransferase (AT) repairsO6-methylguanine in DNA by transferring the methyl group to acysteine acceptor site on the protein itself (52). Expression of AT isreportedly mediated through p53 (52), suggesting that inductionof thisDNA repair pathway inp53wtU87 cells in response toDTICor TMZ treatment fails to alter p53 levels (Fig. 5B and C).However, p21 expression was increased following exposure ofU87 cells to TMZ, but not DTIC, suggesting a difference in themechanism of action of these two similar agents. This was not thecase with either agent in p53mut LN229 cells (Fig. 5B and C; Table1), where DTIC and TMZ increased p53 and p21. p53 affects boththe duration of G2–M arrest and the fate of TMZ-treated humanglioblastoma cells (3). If resistance to TMZ is promoted byenhanced DNA repair activity in the G2–M transition, a CDKinhibitor could suppress this activity, leading to potentiating ofTMZ action on glioma cells (11). This is essentially what weobserved (Fig. 3D and E; Fig. 5B and C). p28 alone increasedp53 levels in U87 and LN229 cells (Fig. 5B and C), in turndecreasing the level of FoxM1 in U87 and LN229 cells. As acell-cycle–specific inhibitor at G2–M, p28 exhibits antiprolifera-tive activity independent of the DNA repair pathway. Because p28in combination with DTIC or TMZ increases the levels of p53 andp21 in U87 and LN229 cells, it suggests that the increase incytotoxicity induced by p28 in combination versus the agentalone extends to a variety of DNA-damaging agents and may alsobe independent of cancer cell phenotype (Fig. 3A–E; 5A–C).

The repeated, significant decrease in CDK2 in response to anincrease in p53 and p21, irrespective of cancer cell type, isimportant as CDKs are also involved in activation of DNA-damage checkpoint signaling and the initiation of DNA repair.Primary resistance to DNA-damaging agents is partly due to theactivation of checkpoint and repair pathways (53). The inhibitionof CDKs may prevent the activation of DNA-damage–inducedcheckpoint and repair pathways. It has been suggested that CDK1(cdc2) and CDK2 may be the most attractive family members ofCDK to target in combination with DNA-damaging agents

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because abrogation of individual CDK1 and CDK2 activity resultsin defective DNA-damage–response pathways in several types ofhuman cancer cells (12). However, because many inhibitors ofCDK inhibit multiple CDK family members that carry a similarATP-binding site, their use in combination with DNA-damagingagents is complicated by a cell-cycle arrest that may be super-imposed on the checkpoint and repair pathways.

Unlike competitive small-molecule inhibitors of the ATP-bind-ing pocket on CDKs, p28 specifically inhibits the cancer cell cycleat G2–M by inducing the endogenous CDK inhibitors, p21 andp27. This leads to lower levels of CDK2 and cyclin A, and a higherlevel of phosphorylated CDK1 (Thr14/Thr15; inactive form) in ap53-dependentmanner (24–26). Theminimal response of CDK1following exposure to p28 is also accompanied by a significantincrease in the level of cyclin B1, indicating p28 inhibits the cancercell cycle at G2–M (26), as degradation of these two proteins isrequired to exit frommitosis (54). Specific inhibition of CDK2 byp28 avoids the activation of checkpoint and repair pathwaysobserved following DNA-damaging agents.

It is also likely that a p53/p21–mediated inhibition of CDK2plays a significant role in the enhanced effect when p28 is used incombination with antimitotic agents such as paclitaxel (55) anddocetaxel in p53-positive cell lines irrespective of p53 status (Fig.3F–H). The increase in activity in p53wt LNCaP cells correlatedwith apaclitaxel or docetaxel induced increase in p53 andp21 anddecrease in FoxM1 and CDK2. This was not the case for DU145cells, particularly with paclitaxel, which significantly decreasedp53 levels (Fig. 5D and E). This was reversed with the addition ofp28, which increased p53 and p21 and significantly reducedFoxM1 and CDK2 levels when combined with either paclitaxelor docetaxel. Although the potential for taxane resistance existswhen cancer cells are exposed to taxanes in combination withCDK2 inhibitors, the addition of p28 to a taxane regimen wouldavoid such resistance because CDK2 activity appears unrelated topaclitaxel sensitivity (56). Moreover, as p28 increases the level ofphosphorylated cdc2 (CDK1) leaving the level of cdc2 virtuallyunchanged (26), the cytotoxic activity of a taxane is enhanced, anobvious benefit.

What is also clear is that the increase in cytotoxic activityfollowing exposure of cancer cells to p28 in combination witheither a DNA-damaging or antimitotic agent does not result fromeither class of agents increasing (or decreasing) the affinity of p53for p28 (Fig. 6). TheDNA-damaging agent doxorubicin reportedlyinduces significant acetylation of p53 C-terminal domain (CTD;

refs. 45, 57). Although such post-translational modificationswithin the p53CTD induce conformational changes of thep53DBD (57), doxorubicin did not alter the p28:p53 interactionirrespective of p53 mutation status (Fig. 6). This suggests themultiple molecular events, including activation of p53-indepen-dent checkpoints triggered by exposure to genotoxic drugs thatpartially protect cancer cells during chemotherapy can be miti-gated with p28, which directly enhances p53 activity (44).

In sum, p28, in combination with DNA-damaging and anti-mitotic agents, increased their cytotoxicity by activating p53wt,mut,which subsequently induced the endogenous CDK inhibitor p21.The increase in p21 significantly enhanced their cytotoxic activity,independent of cancer cell type. Our results demonstrate thepotential therapeutic value in targeting the p53/p21/CDK2 path-way in combinationwith lower doses of chemotherapeutic agentsto improve anticancer efficacy while reducing toxicity.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Yamada, T K. Das Gupta, C W. BeattieDevelopment of methodology: C W. BeattieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. YamadaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Yamada, C W. BeattieWriting, review, and/or revisionof themanuscript: T. Yamada, T K.DasGupta,C. W. BeattieStudy supervision: T. Yamada, C W. Beattie

AcknowledgmentsThe authors thank Albert Green, Anne Shilkaitis, and Laura Bratescu for

technical assistance and Scott Kennedy for editorial assistance and a criticalreviewof themanuscript. They thankCDGTherapeutics, Inc., for thep28used inthese studies.

Grant SupportThis work was partially supported by a research grant from the Musella

Foundation to T. Yamada.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 28, 2015; revised January 11, 2016; accepted January 15,2016; published OnlineFirst February 26, 2016.

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2016;76:2354-2365. Published OnlineFirst February 26, 2016.Cancer Res Tohru Yamada, Tapas K. Das Gupta and Craig W. Beattie ChemotherapyEnhances the Efficacy of DNA Damaging and Antimitotic

M Phase of the Cell Cycle−2p28-Mediated Activation of p53 in G

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