Proteasome-Mediated Destruction of the Cyclin A/Cyclin-Dependent Kinase 2 Complex Suppresses Tumor Cell Growth in Vitro and in Vivo

[CANCER RESEARCH 64, 3949–3957, June 1, 2004] Proteasome-Mediated Destruction of the Cyclin A/Cyclin-Dependent Kinase 2 Complex Suppresses Tumor Cell Growth in Vitro and in Vivo

Wei Chen, Jeongwu Lee, Steve Y. Cho, and Howard A. Fine Neuro-Oncology Branch, National Cancer Institute, National Institutes of Neurological Disorder and Stroke, NIH, Bethesda, Maryland

ABSTRACT phorylation of substrate by cyclin A/cdk2 and cyclin E/cdk2 and preferentially induces apoptosis in transformed cells (24, 50). As a small Cyclin-dependent kinases (cdks) represent potentially promising mo- peptide, however, the clinical use of LFG may be limited by pharmaco- lecular targets for cancer therapeutic strategies. To evaluate the antitu- logical constraints, leading to inefficient delivery in vivo. Furthermore, a mor activity of selective cyclin/cdk inhibition, we constructed a chimeric protein composed of a F-box protein (TrCP) fused to a peptide comprising recent study questions whether selective cdk2 inhibition is a useful cancer the cyclin/cdk2 binding motif in p21-like cdk inhibitors (TrCP-LFG). We strategy (51, 52). Additionally, the Barbacid laboratory has shown no cell now demonstrate that endogenous cyclin A and its binding substrate, cycle abnormalities in either a cdk2 null mouse or following acute cdk2, can be tethered to ␤-TrCP, ubiquitinated, and effectively degraded. ablation of cdk2 in primary cells by Cre-loxP-mediated recombination, Degradation of cdk2 and cyclin A together, but not cdk2 alone, results in thereby raising the question of the true importance of cdk2 in cellular massive tumor cell apoptosis in vitro and in vivo in a proteasome-depend- proliferation (53). Thus, there is an urgent need for the evaluation of other ent manner with no toxicity to normal tissue. These data demonstrate that potentially more efficient strategies aimed at selectively targeting cdk2 cyclin A and/or the cyclin A/cdk2 complex is a promising anticancer target and its binding partners in tumor cells. with a high therapeutic index. One of the potential reasons for the inefficiency of the LFG peptide approach may be that the peptide merely competes with substrate for INTRODUCTION cdk2 binding sites, thus requiring high, continuous intracellular concentration of the peptide. We reasoned that destruction of the cdk2 protein, Most cyclin-dependent kinases (cdks) are involved in the orderly with or without its binding partners, might represent a more efficient progression of the cell cycle. Dysregulation of cell cycle progression can approach. A major pathway used by eukaryotic cells to degrade specific result in disordered and uncontrolled cell growth, which is characteristic proteins is ubiquitin-dependent proteolysis, which involves a cascade of of neoplasia (1–6). A number of cdks (i.e., cdk4/6, cdk2) target the enzymatic reactions catalyzed by the E1 ubiquitin-activating enzyme, the retinoblastoma protein (pRb), leading to its inactivation through phos- E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin-protein ligases. phorylation (7–12). One critical set of downstream targets of pRb are the The substrate specificity of the ubiquitin pathway is conferred by the E3. family of cell-cycle regulatory transcription factors, E2F (13–15). In One of the best characterized ubiquitin ligase complexes is the Skp1- mitotically quiescent normal cells, hypophosphorylated pRb complexed Cullin-F-box (SCF) complex (reviewed in Ref. 54). Within the SCF to E2F forms a transcriptional repressor, whereas phosphorylation of pRb complex, the F-box-containing proteins serve as the receptors for specific leads to reduced levels of functional pRb/E2F repressor complexes and substrates (55). The F-box protein contains two essential modular do- activation of E2F-responsive promoters important for moving the cell mains: the F-box that is required for binding to Skp1 and a protein-protein through the G1-S phase of the cell cycle (16–22). interaction domain for binding distinct substrates (56). It has been pre- In addition to pRb, E2F is also negatively regulated by cdk activity, viously reported that specific proteins can be degraded when a F-box particularly cyclinA/cdk2 (19, 23–27). E2F-1, E2F-2, and E2F-3 each protein is engineered to contain a specific protein interaction domain contain a short, collinear cyclin A/cdk2 binding motif that is required for without affecting degradation of normal endogenous substrates (57, 58). the phosphorylation-mediated neutralization of E2F as a cell prepares to We now report the construction of a chimeric protein composed of a exit S phase (26, 28). Mutation of this motif abrogates phosphorylation of F-box protein (TrCP) fused to the LFG peptide and demonstrate that E2F, leading to unopposed E2F activity, which in the proper cellular endogenous cdk2 and its binding substrate, cyclin A, can be tethered to context can lead to apoptosis (25). Previous studies suggested that in the ␤-TrCP, ubiquitinated, and degraded. Degradation of cdk2 and cyclin A absence of functional pRb, as is found in most human cancer (29–31), together, but not cdk2 alone, results in massive tumor cell apoptosis in inhibition of cyclin A/cdk2 should additionally increase unphosphoryl- vitro and in vivo in a proteasome-dependent manner. ated E2F-1 and thus E2F-1 activity (32, 33). Furthermore, because some E2F family members, including E2F-1, are themselves transcribed from MATERIALS AND METHODS E2F-responsible promoters, unopposed activation of E2F activity should establish a positive feedback loop, leading to very high intracellular E2F Reagents. N-Acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) were pur- activity and ultimately apoptotic cell death (34–45). chased from Sigma (St. Louis, MO). All other reagents were purchased either Thus, cdk2 inhibition, particularly in the setting of functional pRb from Invitrogen (Gaithersburg, MD) or from the sources stated in the text. inactivation, has the potential as a selective cancer therapeutic strategy. Monoclonal antibody specific to Flag was purchased from Sigma. All of the other antibodies used in immunoblotting and immunoprecipitation were pur- Small molecule cdk inhibitors such as flavopiridol have been developed, chased from Pharmingen (San Jose, CA). although their specificity for any particular cdk (i.e., cdk2) is limited (33, Cell Culture. U87, 293, U2OS, SAOS, HeLa, HCT116, and SW480 cells 46, 47). In the search for a more specific cdk2 inhibitor, a short peptide were purchased from American Type Culture Collection (Manassas, VA). (PVKRRLFG) (“LFG”) was found on the cyclin/cdk2 binding motif in Cells were cultured in the media as described in the manufacturer’s data sheet. p21-like cdk inhibitors (28, 48, 49). LFG specifically blocks the phos- Plasmids and Adenovirus. The Flag-tagged ␤-TrCP-LFG (TrCP-LFG), Flag-tagged ⌬TrCP-LFG (⌬TrCP-LFG), and Flag-tagged TrCP (TrCP) were ⌬ Received 12/14/03; revised 2/24/04; accepted 3/24/04. generated by PCR using TrCP/PcDNA3 or F-TrCP/PcDNA3 (a gift from Dr. The costs of publication of this article were defrayed in part by the payment of page Klaus Strebel) as a template. The final PCR fragments were cloned into the charges. This article must therefore be hereby marked advertisement in accordance with BglII/NotI sites of pAdTrack plasmid. All PCR products were verified by auto- 18 U.S.C. Section 1734 solely to indicate this fact. mated sequencing at the National Institute of Neurological Disorders and Stroke Note: W. Chen and J. Lee contributed equally to the work. Requests for reprints: Howard A. Fine, Neuro-Oncology Branch, NIH, 9000 Rock- core facility at the NIH. These plasmids were recombined with pAdEasy1 and ville Pike, Rockville, MD 20892. transfected into 293 cells to make recombinant adenoviruses (59). Ad/p73dd was 3949

generated by PCR using PcDNA3-p73dd as a template (a gift from Dr. William G. phosphate was determined by 10% NuPAGE. Analysis of the dried gel was Kaelin). Recombinant adenovirus for p73dd was generated using the same strategy performed using a PhosphoImager (Molecular Dynamics, Piscataway, NJ). as recombinant adenovirus for TrCP. Animal Studies. For the s.c. tumor model, U87 cells (1 ϫ 107) were s.c. Western Blots and Immunoprecipitation. For Western blots, protein ex- injected into the right flank of 4-week-old female nude mice (National Cancer tracts of cells were prepared in lysis buffer [20 mM HEPES (pH 7.4), 50 mM Institute-Frederick Cancer Research & Development Center, Frederick, MD). 3 ␤-glycerol phosphate, 2 mM EGTA, 1 mM DTT, 10 mM NaF, 1 mM sodium Once tumor size had reached 200 mm , the mice were treated with adenovirus orthovanadate, 1% Triton X-100, and 10% glycerol with proteinase and phospho- expressing either green fluorescent protein (GFP) or TrCP-LFG or PBS. The mice rylation inhibitors]. The protein concentration of the cell lysates was measured by were injected with 100 ␮l of Ad/CMV-GFP (109 pfu) or Ad/TrCP-LFG (109 pfu) the Bradford assay (Bio-Rad, Hercules, CA), and 20–30 ␮g of protein/sample (70 every 3–4 days for a total of six treatments. Tumor size was measured every 3–4 2 ␮g for p73) were loaded onto SDS/PAGE gels or NuPAGE gels (Invitrogen). days, and tumor volume was calculated from the formula (length) ϫ (width) , Proteins were transferred to polyvinylidene difluoride membranes and immuno- where length and width are perpendicular. The student t test was used to assess blotted with specific antibodies. For immunoprecipitation, cells were lysed using statistical significance. PsofϽ0.05 were considered significant. immunoprecipitation buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton For the intracranial U87 glioma model, 4-week old nude mice were anesthe- X-100, 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium orthovanadate, 0.2 tized with ketamine and xylazine and immobilized in a rodent stereotactic frame mM phenylmethylsulfonyl fluoride, and 0.5% NP40]. Anti-Flag or cdk2 antibodies (Stoetling Co., Wood Dale, IL). Through a small scalp incision, an intracranial were added to 500-␮g lysates and incubated at 4°C for 1 h. Protein A- or opening was made using an 18-gauge needle tip in a position 2 mm right and 1 mm G-agarose beads were added to the antigen-antibody mixture and additionally anterior from the bregma. A single guide cannula (with a 4-mm pedestal and a incubated for 30 min at 4°C. Beads were washed (twice) with immunoprecipitation 1-mm subpedestal; Plastic-One, Roanoke, VA) was installed and secured onto the buffer and subjected to Western blot analysis using anti-cyclin A, cdk2, and Flag scalp at the stereotactic opening using medical grade cyanoacrylate gel. The ϫ 4 ␮ antibodies as described above. following day, U87 cells (5 10 ) suspended in 5 l of PBS were injected Transient Transfection in HeLa Cells. Various plasmids were introduced stereotactically into the right parietal region to a depth of 2.5 mm using a Hamilton into HeLa cells using Lipofectamine Plus reagent following manufacturer’s syringe(Hamilton Co, Reno, NV) guided through the secured cannula. Three days instructions (Invitrogen). Forty-eight h after transfection, cells were harvested, after tumor implantation, animals were randomly chosen to receive recombinant adenoviruses expressing either GFP or TrCP-LFG through the installed cannula. and protein lysates were resolved via gel-electrophoresis and blotted onto Adenovirus (5 ϫ 109 pfu) in 5 ␮l of PBS was injected into the substance of the polyvinylidene difluoride membrane. The expression of Flag-tagged TrCP- intracranial tumor using the guide cannula. The mice received three total intracra- conjugated protein was confirmed by immunoblotting using the anti-Flag nial viral injections at 3–4-day intervals. These mice were examined daily and monoclonal antibody. after death, and the brains were examined to confirm that the cause of death was Small Interfering (si)RNA Transfection. The siRNA sequences used for tumor progression, not infection or treatment toxicity in each case. Survival for the targeted silencing of cdk2 (5Ј-aagctgctggatgtcattcac-3Ј), cyclin A (5Ј-aaccattggtc- treated and control animals were compared using a log-rank analyses from the cctcttgatt-3Ј), and p27 (5Ј-aagtacgagtggcaagaggtg-3Ј) were chosen as described by Kaplan-Meier survival curves. Elbashir et al. (60) and recommended by the siRNA supplier (Xeragon, German- town, MD). Human genome database (BLAST) searches were carried out to ensure that the sequences would not target other gene transcripts. siRNAs were RESULTS introduced to the cells using Oligofectamine according to the protocol of Elbashir The Engineered ␤-TrCP-LFG Targets Cdk2 for Degradation. et al. (60) and manufacturer’s instructions (Invitrogen). Briefly, two siRNA To explore the effects of inducing proteasome-mediated degradation of transfections were sequentially performed at 24 and 48 h after cells were plated. Four h after the second transfection, cells were infected with the recombinant cdk2, we engineered chimeric proteins with the full-length flag-tagged ␤ adenovirus. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays -TrCP (TrCP) protein fused at the protein-protein interaction domain to (Roche, Germany) were carried out 96 h after adenovirus treatment. Two days the cdk2 binding 8 amino acid peptide LFG (TrCP-LFG; Fig. 1A). As after treatment, cells were harvested for Western blotting analysis. shown in Fig. 1B, expression of TrCP-LFG in HeLa cells resulted in a Apoptosis and Cell Cycle Analysis. Cells (3 ϫ 105/well) were seeded in decrease in the endogenous level of cyclin A and cdk2. The expression of 6-well plates and infected 16–18 h later with adenoviruses at a multiplicity of TrCP or F-box-truncated ␤-TrCP fused to LFG (⌬TrCP-LFG) did not infection of 200. Forty-eight h after infection, both attached cells and floating reduce cyclin A protein levels nor cdk2 protein levels. The faster migrat- cells were harvested, washed in PBS, and fixed with 70% ethanol. Apoptotic ing phosphorylated (Thr160) active form of cdk2 (lower band; Refs. 48, cells were detected by using ApopTag peroxidase in situ apoptosis detection 61–63) showed a preferential and marked reduction. Cyclin E protein kit (Intergen, Norcross, GA). Cell cycle analysis was prepared by first fixing levels remained constant (Fig. 1B). To confirm that the TrCP-LFG- ␮ cells and treating with RNase A, followed by staining with 5 g/ml propidium mediated decrease in the endogenous cyclin A and cdk2 protein levels iodide in PBS. Cell cycle analysis was performed on a FACS brand flow was through a ubiquitin-dependent proteolysis pathway, we performed a cytometer (Becton Dickinson, San Jose, CA); the data were analyzed by MULTI-FIT software. series of experiments where TrCP-LFG treated cells were exposed to Determination of Cell Growth Rate. Cell growth was assessed using the N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, a peptide inhibitor of prote- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Roche). olysis by the 26S proteasome (Fig. 1C). N-Acetyl-L-leucinyl-L-leucinyl- U87 cells were plated on 96-well plates at a density of 10,000 cells/well; all L-norleucinal inhibited the degradation of TrCP-LFG in a dose-dependent other cells were plated at a density of 5000 cells/well. One day after plating, manner and prevented the TrCP-LFG-mediated down-regulation of cy- cells were treated with adenovirus at a multiplicity of infection of 200–500 or clin A and cdk2 protein levels. The specificity of this effect for cdk2 and transfected with siRNA. Cell viability was determined 96 h after adenovirus its binding partners was demonstrated by the fact that cdk4 protein levels infection or siRNA transfection. Each experiment was repeated at least three remained unchanged under identical experimental conditions (Fig. 1C). times with each experiment done in a set of 6 wells. The student t test was used Specific binding of chimeric TrCP-LFG protein to cyclin A and cdk2 was Ͻ to assess statistical significance. Psof 0.05 were considered significant. shown by coimmunoprecipitation in that anti-Flag antibodies precipitated ␮ Cdk Kinase Activity Assay. Two hundred g of protein were immuno- TrCP-LFG and coprecipitated cyclin A and cdk2 (Fig. 1D). The faster precipitated for each sample by anti-cdk2 or anti-cdk4 antibodies. The immu- migrating active form of cdk2 was preferentially precipitated when anti- noprecipitated protein was resuspended in 50 ␮l of kinase buffer [20 mM Flag antibodies were used for immunoprecipitation. Coimmunoprecipi- HEPES (pH 7.2), 25 mM ␤-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 7.5 mM MgCl , and 50 mM ATP] containing 10 tation with anti-cdk2 antibody also showed cyclin A and TrCP-LFG (Fig. 2 ␤ ␮Ci of 32P-dATP (3000 Ci/mmol; Amersham, Piscataway, NJ) and 5 ␮gof 1D). These results indicate that -TrCP fused with the cdk2-specific Rb-C fusion protein (Cell Signaling Technology, Beverly, MA), and incubated binding motif, LFG, can selectively interact with cyclin A/cdk2 complex for2hat30°C. Reaction was stopped by adding 15 ␮lof4ϫ NuPAGE SDS and result in the degradation of cyclin A and cdk2 through a proteasome- sample buffer. Samples were boiled for 5 min, and incorporation of radioactive dependent mechanism. 3950

Fig. 1. TrCP-mediated degradation of cyclin A and cyclin-dependent kinase 2 (cdk2). A, strategy for the selective degradation of cdk2 and cyclin A through TrCP- LFG-mediated ubiquitination (Ub). TrCP-LFG is desig- nated as E3. Cyclin A/cdk2 complex is associated with the LFG binding motif in TrCP-LFG, whereas TrCP domain binds to its specific E2 partner. The cognate substrate cyclin A/cdk2 is thus targeted for degradation through the ubiquitination/proteasome system. B, TrCP- LFG induces degradation of cyclin A and cdk2. HeLa cells were transfected with indicated constructs. Forty- eight h after transfection, cellular lysates were generated, resolved by SDS-PAGE, and visualized using specific antibodies. Expression of flag-tagged recombinant proteins is shown in the top panel. Changes in the expression level of cyclin A, cyclin E, cdk2, and ␤-actin are shown. The two bands on cdk-2 Western represent two forms of cdk2. C, proteasome-dependent degradation of cdk2 and cyclin A. HeLa cells were transfected with TrCP-LFG in the presence of proteasome inhibitor N-acetyl-L-leucinyl- L-leucinyl-L-norleucinal (ALLN) at indicated concentra- tions. The change in level of cdk2, TrCP-LFG, cyclin A, cdk4, and ␤-actin protein is demonstrated by Western. D, endogenous cyclin A/cdk2 interacts with TrCP-LFG. Ly- sates were generated 48 h after HeLa cell transfection with TrCP-LFG. Cells were incubated with or without 100 ␮M ALLN for 6 h before harvesting. The ability of TrCP-LFG to bind cyclin A/cdk2 was determined by coimmunoprecipitation using either anti-Flag or cdk2 antibody and subsequent Western for TrCP-LFG, cdk-2, and cyclin A. WL indicates whole lysates before immunoprecipitation.

Inhibition of Tumor Cell Growth and Induction of Cell Death ptosis in normal human fibroblasts or astrocytes (Fig. 3B and data not by Adenovirus-Mediated Transduction of TrCP-LFG. Recombi- shown). nant adenoviral vectors are a commonly used delivery system for Induction of p73 Is Required for Ad/TrCP-LFG-Induced Cell cancer gene therapy. We constructed a replication defective recom- Death in Rb-Negative Cell Lines. Cdk2 kinase activity has been binant adenovirus that expresses TrCP-LFG (Ad/TrCP-LFG). West- shown to be important for phosphorylating and inactivating E2F ern blot analysis was performed to examine the effect of Ad/TrCP- activity. High E2F activity can induce apoptosis by a p73-dependent LFG on cyclin A and cdk2 protein level in different cell lines. In all mechanism. Thus, one might predict that inactivation of the Cdk2 tested cell lines, both cyclin A and cdk2 protein levels were signifi- complex would lead to unopposed E2F activity resulting in apoptosis. cantly reduced following Ad/TrCP-LFG transduction (Fig. 2A). Con- To test this hypothesis, we evaluated the levels of p73 in each of our sistent with the effects on protein levels, transduction by Ad/TrCP- cell lines (Fig. 4A). Basal expression of p73 was much higher in LFG resulted in significantly reduced intracellular cdk2 activity (Fig. U2OS cells compared with the other cell lines. Ad/TrCP-LFG treat- 2B). Ad/TrCP-LFG transduction of various cell lines in vitro mediated ment induced expression of p73 in SAOS cells and, to a much lesser a significant cytotoxic effect in most tumor cell lines examined, extent, in U87 cells but failed to induce expression in other cell lines, whereas there were minimal effects on the survival and growth of including U20S (Fig. 4A, top panel). The increased expression level of normal human fibroblasts and astrocytes (Fig. 2, B and C). Control p73 in the Rb-negative cell line SAOS and Rb-dysregulated U87 in adenoviral vectors expressing TrCP or ⌬TrCP-LFG caused no signif- response to the treatment of Ad/TrCP-LFG suggested that the induc- icant change in cdk2 protein level nor cytotoxicity in any of the cell tion of p73 might contribute to Ad/TrCP-LFG-mediated cell death. To lines (Fig. 2D). These data suggest a differential cytotoxic response test this hypothesis, we constructed a recombinant adenoviral vectors between cancer and normal cells to cyclin A/cdk2 down-regulation. expressing a dominant-negative p73 construct (Ad/p73dd). Preinfect- Induction of Apoptosis in Tumor Cells Transduced with Ade- ing SAOS cells with Ad/p73dd partially protected cells from apoptotic novirus-Expressing TrCP-LFG. We performed experiments to eluci- death induced by Ad/TrCP-LFG (from 42.6% cytotoxicity to 11% date the mechanisms responsible for Ad/TrCP-LFG-mediated death in cytotoxicity; P Ͻ 0.01; Fig. 4B). Ad/p73dd also partially protected tumor cells. Tumor cells treated with Ad/TrCP-LFG exhibited membrane U87 cells (Fig. 4B, from 82.3% cytotoxicity to 37.5% cytotoxicity; blebbing and nuclear condensation (data not shown), suggestive of apo- P Ͻ 0.01). Interestingly, Ad/p73dd also protected against Ad/TrCP- ptotic cell death. To confirm that apoptosis had occurred in Ad/TrCP- LFG-induced cell death in another Rb-negative cell line, H596 (Fig. LFG-transduced cells, we performed terminal deoxynucleotidyl transfer- 4B, from 62.35 cytotoxicity to 39.3% cytotoxicity; P Ͻ 0.01), despite ase dUTP nick end-labeling assay. Two days after Ad/TrCP-LFG the fact that no p73 induction was noted in these cells after infection transduction, U2OS, SAOS, and U87 tumor cells all demonstrated sig- by Ad/TrCP-LFG. By contrast, Ad/p73dd offered no protection nificant terminal deoxynucleotidyl transferase dUTP nick end-labeling against Ad/TrCP-LFG-induced cell death in the two Rb-positive cell staining, whereas there were little or no terminal deoxynucleotidyl trans- lines we tested (U2OS and A549; Fig. 4B). These data suggest that ferase dUTP nick end-labeling-positive normal cells (Fig. 3A). We quan- p73 may play a preferential role in Ad/TrCP-LFG-induced apoptotic titated the degree of apoptosis induced by adenoviral-mediated transduc- cell death in Rb-negative cell lines. To explore other potential mech- tion of TrCP-LFG by flow cytometric analysis (fluorescence-activated anisms involved in TrCP-LFG-mediated cell death, we examined the cell sorting). The percentage of apoptotic cells (sub-G1 fraction) in- changes of protein expression of the cdk inhibitors p27 and p21 in creased steadily over the first 96 h after Ad/TrCP-LFG transduction in all response to Ad/TrCP-LFG treatment. Induction of p27 was evident in tumor cell lines, reaching a high of 40–90% in all transduced cells (Fig. all cell lines 48 h after transduction by Ad/TrCP-LFG (Fig. 4A). By 3B). By contrast, Ad/TrCP-LFG transduction caused essentially no apo- contrast, p21 levels were either not or only minimally increased after 3951

Fig. 2. Effects of TrCP-LFG on cell survival. A, reduction of cyclin A and cyclin-dependent kinase 2 (cdk2) expression levels in cells treated with Ad/TrCP-LFG. Cells were transduced with Ad/TrCP-LFG or Ad/GFP (control) vectors [multiplicity of infection (m.o.i.) of 50]. Cell lysates were generated 48 h after adenoviral transduction. Cyclin A and cdk2 expression was detected by Western blot using anti-cyclin A or cdk2 antibody. ␤-Actin was shown to ensure equal loading. B, comparison of the effects of Ad/TrCP-LFG on U87 versus two colon cancer cell lines (HCT116 and SW480). Top panel: U87, HCT116, and SW480 cells were infected with Ad/TrCP-LFG or Ad/GFP. Similar viral transduction efficiency was observed in all cell lines (m.o.i. of 200). Cell survival was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 96 h after viral treatment. The relative survival for the Ad/TrCP-LFG-treated cells is normalized by the control (Ad/GFP) vector-treated cells. Values shown are the mean Ϯ SD from one representative experiment, n ϭ 6. Bottom panel: analysis of cdk2 activity in cells treated with Ad/TrCP-LFG. Cells (indicated at the bottom of the figure) were treated with either control virus (Ad/GFP) or Ad/TrCP-LFG (m.o.i. of 50). Cell extracts were generated 48 h after viral infection and followed by immunoprecipitation with anti-cdk2 antibody. Kinase assay was performed using Rb-C fusion protein as substrate (see “Materials and Methods”). C, the effect of Ad/TrCP-LFG on cell survival. Transformed or nontransformed cells were treated with Ad/TrCP-LFG or Ad/GFP adenoviral vectors (m.o.i. of 200), and cell killing was measured by the MTT assay at indicated times after vector transduction. Survival index was calculated for the Ad/TrCP-LFG treatment group by normalizing to survival in the control (Ad/GFP) adenoviral vector group. The data shown here is the representative of three independent experiments. Values shown are the mean Ϯ SD, n ϭ 6. P was determined using the student t test by comparing experimental group (Ad/TrCP-LFG) to control (Ad/GFP) group at the 96-h time point. Top panel: time course of growth inhibition of U2OS osteosarcoma; SAOS osteosarcoma and U87 glioma induced by Ad/TrCP-LFG (m.o.i. of 200). Bottom panel: time course of survival rate of Hs27 normal human fibroblasts and normal human astrocytes (NHA) treated with either the control or Ad/TrCP-LFG. The transduction efficiency observed in nontransformed (i.e., normal) cells was similar to that in the transformed cell lines. D, the effect of control viral constructs. Top panel: the effect of control viral constructs on cell growth. U2OS, U87, and SAOS cells were infected with various control viral constructs at m.o.i. of 200 (Ad/GFP; Ad/TrCP; and Ad/⌬TrCP-LFG). Cell growth was measured by the MTT assay 96 h after viral infection. Similar results were observed in several independent experiments, n ϭ 6. Bottom panel: the effect of control viral constructs on the expression of cdk2. U2OS was infected by indicated adenoviruses. Forty-eight h after viral infection, cell lysates were generated, and cdk2 expression level was examined by Western blot analysis using anti-cdk2 antibody. The data shown is the representative result from at least three independent experiments. 3952

Fig. 3. Induction of apoptosis in tumor cells transduced with Ad/TrCP-LFG. A, terminal deoxynucleotidyl transferase-mediated nick end labeling staining of various cell lines after treatment of Ad/TrCP-LFG. Forty-eight h after treatment with Ad/GFP (control) or Ad/TrCP-LFG, U2OS, SAOS, U87, and Hs27, cells were evaluated for apoptotic death by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. B, time course of TrCP-LFG induced apoptosis by fluorescence-activated cell sorting analysis. The percentage of apoptotic cells (sub-G1 fraction) induced by Ad/GFP or Ad/TrCP-LFG treatment at indicated time points was determined by fluorescence-activated cell sorting. The data shown here are the representative data from at least three independent experiments.

exposure to TrCP-LFG, except for a large increase seen in SAOS Suppression of Tumor Growth by Intratumoral Injection of cells. Knockdown of p27 induction by p27 siRNA treatment did not Ad/TrCP-LFG. Given the pronounced and selective cytotoxic effects protect the cell death induced by Ad/TrCP-LFG in the Rb-negative of Ad/TrCP-LFG on tumor cells but not normal cells in vitro,we cell lines, whereas it afforded some protection from Ad/TrCP-LFG- evaluated the efficacy of Ad/TrCP-LFG administration on glioma tumor induced cell death in Rb-positive cells (data not shown). Taken growth in vivo. Subcutaneous U87 gliomas were treated with intratu- together, these data suggest that the molecular mechanisms responsi- moral injections of Ad/TrCP-LFG or a control adenoviral vector starting ble for TrCP-LFG-mediated cell death may be different in Rb-positive day 3 after tumor implantation and then every 3–4 days for a total of six cells compared with Rb-negative cells. treatments. All tumors in the mice treated with Ad/TrCP-LFG showed Both Cyclin A and Cdk2 Degradation Are Necessary for Max- significant suppression of growth compared with tumors in PBS-treated imal Tumor Cell Apoptosis. A recent study (51) suggests that cdk2 and Ad/GFP-treated mice (Fig. 6A). In a separate experiment, we eutha- is not required for tumor cell survival and growth and cdk2-knockout nized a subgroup of animals 24 h after viral injection to examine the mice and cells do not show abnormality in cell cycle progression. effects of Ad/TrCP-LFG on tumor cells in vivo. The transduction effi- Because TrCP-LFG mediates destruction of both cdk2 and cyclin A, ciency of the different adenoviral vectors was roughly equivalent as we performed a series of experiments designed to individually eval- evidenced by the fact that the intensity and distribution of GFP expression uate the effects of isolated cdk2 and cyclin A down-regulation. was similar in both the control GFP-expressing and GFP/TrCP-LFG- siRNA-mediated down-regulation of cdk2 alone did not result in expressing adenoviral vector-treated tumors. By contrast, there were cytotoxicity in any of the tumor cell lines evaluated (Fig. 5). siRNA significantly more terminal deoxynucleotidyl transferase dUTP nick end- down-regulation of cyclin A did, however, have a small but statisti- labeling-labeled apoptotic nuclei in the Ad/TrCP-LFG-treated group cally significant cytotoxic effect in U87 glioma cells and HCT116 compared with the control group (Fig. 6B). colon carcinoma cells lines (Fig. 5B). By contrast, simultaneous We further assessed the potential of Ad/TrCP-LFG as a therapeutic down-regulation of cyclin A and cdk2 by siRNAs caused a greater strategy for human brain cancers using an orthotopic intracranial U87 effect on cell killing than seen with down-regulation of either protein glioma model. To evaluate the effects of the TrCP-LFG construct on alone (Fig. 5B). Of note, SW480 were totally resistant to TrCP-LFG- normal cerebral tissue, Ad/TrCP-LFG or the control Ad/GFP virus was and siRNA-mediated cell killing despite the effective down-regulation stereotactically injected into the right caudate of nontumor-bearing mice. of cyclin A and cdk2 protein levels (Fig. 5A) and diminished cdk2 All animals remained clinically well with no signs of neurological tox- kinase activity to levels seen in all of the other sensitive cell lines we icity. The brains of the injected animals were examined at 1, 7, and 28 examined (Fig. 2B). These data demonstrate that the cytotoxic effects days after viral transduction. There was no histological evidence of of simultaneous cdk2/cyclin A down-regulation are more profound neurotoxicity in any of the examined brains with only a minimal mono- than down-regulation of either protein alone in all cell lines tested nuclear cell infiltrate within the injected area, characteristic of the effect with one notable exception. seen after adenoviral injection into the brain (data not shown). There was 3953

Fig. 4. The role of p73 in TrCP-LFG-mediated tumor cell death. A, expression levels of p73, p27, and p21 in cells transduced with Ad/TrCP-LFG. Cells were harvested 48 h after transduction with Ad/GFP or Ad/TrCP-LFG, and the total number protein extracts was used for immunoblotting. The specific antibodies used for immunoblotting are indicated at the right of each panel. The data represented is a typical experiment conducted three times with comparable results. B, the effect of Ad/p73dd on Ad/TrCP-LFG mediated cell death. Different cell lines (indicated on the top of each panel) were infected with either the control adenoviral vector (Ad/GFP) with or without adenovirus-expressing dominant-negative p73 (Ad/p73dd) or Ad/TrCP-LFG with or without Ad/p73dd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed 96 h after viral infection. Shown are mean Ϯ SD from one representative experiment; n ϭ 6. no difference between the histological appearance of the brains injected animals (P Ͻ 0.01). More impressively, 50% of the animals receiving with the either the Ad/GFP or Ad/TrCP-LFG virus. We next stereotac- Ad/TrCP-LFG were ultimately cured of their tumors (Ͼ120-day sur- tically inoculated U87 glioma cells into the right parietal region of the vival) with no neurological or physical evidence of tumor nor toxicity. brain of nude mice after 3 days later by stereotactic administration of The long-term survivors were ultimately euthanized, and microscopic recombinant adenovirus expressing either GFP or TrCP-LFG. Vector examination of their brains revealed no evidence of either residual tumor administration was repeated three times at 3–4-day intervals, and animals or normal tissue toxicity (Fig. 7B). were followed for survival. Kaplan-Meier survival curves for the control animals and those treated with Ad/TrCP-LFG are shown in Fig. 7A. The DISCUSSION median survival of control vector treated animals was 34 days, whereas the median survival of the Ad/TrCP-LFG-treated animals was 50 days We demonstrate that a cyclin A/cdk2 inhibitor binding peptide fused to (P Ͻ 0.01). Log-rank analysis of Kaplan-Meier survival curves demon- a recombinant TrCP protein can selectively direct cyclin A and its strated a significant survival advantage for the Ad/TrCP-LFG treated binding partner, cdk2, toward proteasome-mediated degradation resulting

Fig. 5. The effect of cyclin A and cyclin-dependent kinase 2 (cdk2) down-regulation on tumor cell death. A, Western blot analysis of cdk2 and cyclin A. U87 cells and SW480 cells (indicated in the bottom of the figure) were transfected with either mismatched control small interfering (si)RNA (control siRNA) or siRNA specific to cdk2 or cyclin A (indicated on top of each panel) twice within a 24-h interval. Cell extracts were generated 48 h after the second transfection. Cdk2 or cyclin A expression level was examined by Western blots using specific antibodies (indicated at the right of each panel). Specificity of siRNA treatment was examined by checking the expression level of cyclin E (shown in the right panel). B, the effect of cdk2 and/or cyclin A depletion on cell survival. Endogenous cdk2 and cyclin A expression was depleted by their respective siRNAs. The effect of siRNA transfection on cell survival was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which was performed 96 h after second transfection. The data represents a typical experiment conducted three times with comparable ,ء .results. Data shown is mean Ϯ SD, n ϭ 6 P Ͻ 0.01. P was determined using the student t test by comparing experimental siRNA group to the control siRNA group.

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Fig. 6. In vivo antitumor effects of Ad/TrCP- LFG on s.c. tumors. A, nude mice with established s.c. U87 tumors were intratumorally injected with Ad/TrCP-LFG, Ad/GFP (control), or PBS at a dose of 109 pfu in 100 ␮l/injection (n ϭ 6) over a 25-day period, beginning on day 3 after U87 implantation (arrows). Tumor volumes were calculated as described in “Materials and Methods.” Each point represents the mean Ϯ SD of tumor volume mice in each group (n ϭ 9 mice/group). At day 42, there was a significant difference in the size of the Ad/ TrCP-LFG-treated tumors compared with the control Ad/GFP (P ϭ 0.0087) or saline-treated tumors (P ϭ 0.0093) (P ϭ 0.27 for PBS treatment group versus Ad/GFP group). B, distribution of GFP expression and apoptosis. Twenty-four h after intratumoral viral vector injection, GFP expression in both Ad/GFP- and Ad/TrCP-LFG-treated tumors was examined by immunostaining using anti-GFP antibody. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated nick end labeling staining.

in tumor cell apoptosis in vitro and in vivo. This strategy may represent Furthermore, we did not see effects of the transected TrCP-LFG on a more efficient way of inhibiting the cyclin A/cdk2 pathway than the endogenous substrates for the ␤-TrCP E3 ligase such as nuclear LFG peptide approach because, instead of only competitively inhibiting factor-␬␤ (data not shown). At first glance, the highly specific nature the kinase activity, the TrCP-LFG fusion protein targets the kinase of the TrCP fusion peptide approach might appear surprising. Given complex for destruction. the central role of proteasome-mediated proteolysis for processes as Not only was the TrCP-LFG strategy highly efficient for cell important and sensitive as cell cycle regulation and cell survival, killing, but the strategy also appears to be specific for targeting the however, E3 ligases are by necessity highly specific for their targeted cyclin A/cdk2 complex given that we did not see effects of TrCP- binding proteins. Thus, it is not all that unexpected that a TrCP- LFG on cyclin B or D protein levels or associated kinase activity peptide fusion approach might so specific for its targeted protein (Figs. 1 and 2 and data not shown). Consistent with these obser- binding partner. vations is the fact that previous work has demonstrated that LFG Much attention has recently focused on interfering RNAs as a revo- and similar peptides can inhibit cyclin A-associated kinase activity lutionary strategy for uniquely down-regulating-specific target mRNAs. but not cyclin B or D-associated kinase activity (28, 42, 48–50). Recent experience, however, has demonstrated siRNAs to be problematic 3955

Fig. 7. In vivo antitumor effects of Ad/TrCP- LFG on intracranial tumors. A, effect of Ad/TrCP- LFG treatment on survival of mice bearing intracranial U87 glioma. Viral vector treatment (5 ϫ 109 pfu) was started 3 days after intracranial tumor implantation and then repeated for a total of 3 times at 3–4-day intervals. The Kaplan-Meier survival curve is shown for both the Ad/GFP-treated (n ϭ 4) and Ad/TrCP-LFG-treated animals (n ϭ 6). B, pho- tomicrographs show either Ad/GFP (left panel)- treated or Ad/TrCP-LFG-treated (right panel) animal brain. Animal brain specimens were evaluated histologically by H&E staining.

relative to intracellular transduction inefficiencies, their unpredictable mediated down-regulation of cyclin A/cdk2 remain to be fully effectiveness depending on the specific sequence that is used, and the elucidated, although our data suggest that those mechanisms may difficulties in efficiently delivering these molecules in vivo. Additionally, differ depending on the pRb status of the cell. TrCP-LFG-treated recent data has shown that siRNAs may not be as specific as previously tumor cells, which have deregulated Rb function, appear to un- thought resulting in interference in a number of off-target mRNAs (64). dergo apoptosis in a p73-dependent manner. This is consistent with By contrast, an approach such as our TrCP-LFG strategy that targets the previous studies (41, 65, 66) demonstrating that unopposed E2F specific protein of interest, rather than an intermediate RNA species, is activity results in p73-dependent apoptosis. Because cyclin A/cdk2 theoretically attractive. Thus, we believe our data supports the premise phosphorylates E2F-1, thereby inhibiting its activity, TrCP-LFG- that the TrCP-fusion peptide approach represents a potentially powerful mediated down-regulation of cyclin A/cdk2 would be expected to strategy for targeting specific proteins both in vitro and in vivo (57). result in even higher E2F activity in cells with baseline deregulated Given the abundance of data suggesting that cdk2 is a potentially pRb activity compared with those that have intact pRb pathways. promising cancer therapeutic target, we initially devised the TrCP- Our data does in fact demonstrate that Rb-deregulated cells gen- LFG strategy with the expectation that a cytotoxic affect would be a erate high E2F transcriptional activity after TrCP-LFG-mediated consequence of cdk2 down-regulation. McCormick et al. (51), how- cyclin A/cdk2 down-regulation and that the resultant apoptosis can ever, have recently published data using principally colon cancer cell be partially inhibited by a dominant-negative p73 mutant. By lines, questioning the need for cdk2 in tumor cell proliferation and contrast, tumor cells with intact Rb are not rescued by the domi- survival. Barbacid et al. (53) also showed that cdk2 may not be nant-negative p73 after TrCP-LFG transduction. Although our necessary for normal cell cycle progression by using cdk2-knockout siRNA experiments suggest that inhibiting the p27 induction that mice and Cre-loxP-mediated somatic cell knockout. Because cdk2 occurs after TrCP-LFG treatment in pRb intact tumor cells can binds with several different protein partners, however, loss of cdk2 reduce the amount of apoptotic cell death, these effects are modest function may not be synonymous with targeted destruction of the and suggest another prominent mechanism for TrCP-LFG-medi- cdk2/cyclin complex. Because TrCP-LFG down-regulates both cyclin ated apoptosis in pRb intact cells. Whether this mechanism A and cdk2, it was possible that the observed cytotoxic effects were involves an E2F-mediated, p73-independent apoptotic pathway a consequence of the destruction of either cdk2, cyclin A, or both. We, remains to be elucidated. therefore, performed experiments using siRNAs to specifically target The efficiency with which TrCP-LFG-induced tumor cell death cdk2 and cyclin A mRNA and demonstrate that, indeed, down- appeared to be cell type dependent, although all cell lines tested regulation of cdk2 alone had no effects on cell viability, whereas (n ϭ 10) were sensitive except for SW480. One of the most impres- cyclin A down-regulation did mediate a cytotoxic effect. Interestingly, sive features of TrCP-LFG-mediated cell death was the apparent however, it appeared that the combination of both cyclin A and cdk2 tumor cell selectivity. There was no significant toxicity to normal cell inhibition led to the most potent cytotoxic effect in sensitive cell lines. lines in vitro or normal brain tissue in vivo after transduction by Although more in-depth studies will be required to fully understand TrCP-LFG-expressing vectors. The fact that cyclin A/cdk2 inhibition these observations, it appears that the cyclin A or the cyclin A/cdk2 did not cause toxicity to mitotically quiescent normal cells in the brain complex may be a preferable to cdk2 as a therapeutic tumor target. is perhaps not surprising given the dominant effect of the cdk inhib-

The downstream mediators of cytotoxicity after TrCP-LFG- itors on G1 cyclins, thereby maintaining pRb in a predominantly 3956

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. Proteasome-Mediated Destruction of the Cyclin A/Cyclin-Dependent Kinase 2 Complex Suppresses Tumor Cell Growth in Vitro and in Vivo

Wei Chen, Jeongwu Lee, Steve Y. Cho, et al.

Cancer Res 2004;64:3949-3957.

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