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Cancer Therapy: Preclinical |
Authors' Affiliation: Department of Cancer Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts
Requests for reprints: Dario C. Altieri, Department of Cancer Biology, LRB428, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605. Phone: 508-856-5775; Fax: 508-856-5792; E-mail: dario.altieri{at}umassmed.edu.
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Abstract |
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Experimental Design: We used molecular, genetic, and pharmacologic antagonists of GSK3ß in p53+/+ or p53–/– colorectal cancer cells. We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, and cell death by multiparametric flow cytometry. A xenograft colorectal cancer model was used to study antitumor activity in vivo.
Results: Treatment of p53+/+ colorectal cancer cells with pharmacologic inhibitors of GSK3ß resulted in sustained elevation of p53, with up-regulation of p21Waf1/Cip1 and loss of survivin levels. Molecular targeting of GSK3ß by overexpression of a GSK3ß dominant-negative mutant, or acutesilencing of GSK3ß by RNA interference, reproduced the induction of transcriptionally active p53 in colorectal cancer cells. This pathway was recapitulated by deregulated Wnt/T-cell factor signaling, with elevation of the tumor suppressor p14ARF, and reduced expression of the p53 antagonist, MDM2. Rather than cell cycle arrest, GSK3ß blockade resulted in p53-dependent apoptosis, which was contributed by acute loss of survivin and inhibition of colorectal cancer growth in mice.
Conclusions: Acute ablation of GSK3ß in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. This pathway may be exploited for rational treatment of colorectal cancer patients retaining wild-type p53.
Key Words: Apoptosis • GSK3ß • p53 • survivin • colorectal cancer
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Materials and Methods |
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Modulation of p53 expression and function. p53+/+ or p53–/– HCT116 or SW48 cells were incubated with inhibitors of GSK3ß, LiCl (30 mmol/L, Sigma, St. Louis, MO), thiadiazolidinones (0-20 µmol/L, Calbiochem, San Diego, CA), or SB216763 (20 µmol/L, Sigma); alternatively, cells were incubated with increasing concentrations (0-20 µmol/L) of dual-specificity antagonists of Cdk (most notably p34cdc2) and GSK3ß, purvalanol A, Olomoucine, or Alsterpaullone (all from Calbiochem, San Diego, CA) for 6 to 48 hours at 37°C. After washes, the cell pellet was solubilized in lysis buffer [20 mmol/L Tris (pH 7.2), 0.5% deoxycholate, 1% Triton X-100, 0.1% SDS, 150 mmol/L NaCl, 1 mmol/L EDTA] containing protease inhibitors (Complete, Roche Applied Science, Indianapolis, CA) for 30 minutes at 4°C. The lysate was cleared by centrifugation and protein-normalized aliquots (30 µg) were separated by SDS-PAGE, transferred to nylon membranes (Millipore, Billerica, MA), and incubated with primary antibodies to p53 (Oncogene Research Products, San Diego, CA; FL-393, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p21Waf1/Cip1 (Oncogene), XIAP (BD Transduction Laboratories, San Jose, CA), survivin (NOVUS Biologicals, Littleton, CO), MDM2 (Santa Cruz Biotechnology), p14ARF (NOVUS), caspase-3 (Cell Signaling Technology, Beverly, MA), ß-catenin (BD Transduction Laboratories), cyclin B1 (GNS1, Santa Cruz Biotechnology), GSK3ß (Santa Cruz Biotechnology), or ß-actin (Sigma) with detection of reactive bands by chemiluminescence (Amersham Corp., Piscataway, NJ). For all experiments, quantification of ß-actin–normalized immunoblotting data was carried out by densitometry.
Kinase assays and transfection experiments. p53+/+ HCT116 cells were treated with vehicle or purvalanol A for 6 to 24 hours, solubilized in lysis buffer containing 50 mmol/L Tris (pH 7.5), 0.1% deoxycholate, 1% NP40, 0.1% SDS, 50 mmol/L NaCl, 1 mmol/L protease inhibitors (Roche Applied Science), and 1 mmol/L Na3Vo4 for 30 minutes at 4°C. Lysates were centrifuged at 15,000 x g for 15 minutes at 4°C, and 200 µg of protein were precleared with protein A-Sepharose beads (Amersham Bioscience). The p34cdc2-cyclin B1 complex was immunoprecipitated with an antibody to cyclin B1 (6 µg/mL) for 16 hours at 4°C under constant agitation. The immune complexes were recovered by addition of protein-A Sepharose beads, washed in lysis buffer, and incubated in a kinase buffer containing 10 µCi of [
-32P]ATP (Amersham) and histone H1 (1 µg) for 30 minutes at 30°C. Samples were separated by SDS gel electrophoresis and radioactive bands were visualized by autoradiography. Equal protein loading was confirmed by Coomassie blue staining of the gel. In other experiments, p53+/+ HCT116 cells were incubated with inhibitors LiCl (30 mmol/L), thiadiazolidinones (20 µmol/L), or purvalanol A (10 µmol/L), harvested after 6 to 12 hours and immunoprecipitated with an antibody to GSK3ß as described above. The immune complexes were mixed with a synthetic peptide containing a GSK3ß phosphorylation site derived from the sequence of glycogen synthase (Upstate Biotechnology, Charlottesville, VA) in the presence of 10 µCi of [-32P]ATP (Amersham). After 30-minute incubation at 30°C, the reaction mixtures were centrifuged briefly and 15 µL of the supernatant was spotted onto P81 phosphocellulose paper (Whatman, Florham Park, New Jersey), washed thrice in 175 mmol/L phosphoric acid, rinsed once in acetone, dried, and changes in peptide phosphorylation were determined by scintillation counting. In other experiments, p53+/+ HCT116 cells were transfected with control pcDNA3 or a p34cdc2Asp146
Asn dominant-negative mutant (16 µg) characterized in previous studies (14) by LipofectAMINE. After 36 hours, cells were harvested and analyzed for DNA content by propidium iodide staining and flow cytometry, or by changes in expression of p53, p34cdc2-reactive material, or ß-actin by Western blotting. Alternatively, p53+/+ HCT116 cells were transfected with wild-type ß-catenin or a truncated TCF-4 dominant-negative mutant lacking the first 31 amino acids and characterized previously (15), treated with purvalanol A (10 µmol/L), and analyzed for changes in expression of p53, p21Waf1/Cip1, or ß-actin at 6- to 12-hour time intervals by Western blotting. For silencing of GSK3ß by RNA interference (RNAi), p53+/+ colorectal cells were seeded in McCoy's medium containing 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin in six-well plates at a density of 1 x 105 cells/well. Cells were transfected after 24 hours with SiControl (nontargeting SiRNA pool) or SiRNA SMRT pool human GSK3ß (both from Dharmacon, Lafayette, CO) using OligofectAMINE (3 µL/well) reagent in 1 mL of Opti-MEM medium (both from Invitrogen, San Jose, CA). Four hours after transfection, 500 µL of McCoy's medium containing 30% FCS, 300 units/mL penicillin, and 300 µg/mL streptomycin were added to each well. Cells were harvested after 48 hours and analyzed by Western blotting.
Analysis of apoptosis. p53+/+ or p53–/– HCT116 cells were treated with purvalanol A (0-20 µmol/L), harvested after 48 hours, and analyzed for hypodiploid DNA content by propidium iodide staining and flow cytometry as previously described (16). Alternatively, cells were incubated with LiCl (30 mmol/L) or thiadiazolidinones (20 µmol/L), harvested after 48 hours, and analyzed for plasma membrane integrity (propidium iodide, red channel) and active caspase-3/7 activity (CaspaTag, Intergen, Burlington, MA; green channel) by multiparametric flow cytometry as described (15). In addition, p53+/+ HCT116 cells transfected with the SiControl (nontargeting SiRNA pool) or SiRNA SMRT pool human GSK3ß were harvested after 48 hours and analyzed by simultaneous multiparametric flow cytometry for caspase activity (CaspaTag, Intergen) and plasma membrane integrity (propidium iodide).
In other experiments, purvalanol A (10 µmol/L)–treated p53+/+ or p53–/– HCT116 cells were harvested after a 6- to 48-hour culture at 37°C, and extracts were analyzed with an antibody to caspase-3 by Western blotting. To determine the role of survivin in modulation of p53-dependent apoptosis, p53+/+ HCT116 cells were transduced with replication-deficient adenoviruses encoding GFP (pAd-GFP) or survivin (pAd-Survivin) at multiplicity of infection of 50 for 24 hours as described (17). Transduced cultures were treated with vehicle or purvalanol A (10 µmol/L) and analyzed for hypodiploid DNA content by propidium iodide staining and flow cytometry after an additional 24-hour incubation at 37°C. Alternatively, survivin levels in p53–/– HCT116 cells were acutely ablated by RNAi as previously described (13). Cells were transfected with control (VIII) or survivin-derived (S4) double-stranded RNA (dsRNA) oligonucleotides (50 nmol/L) using OligofectAMINE reagent (3 µL/well) in 1 mL of Opti-MEM medium (both from Invitrogen). Four hours after transfection, cells were replenished with growth medium containing 125 µL of McCoy's medium, 30% FCS, 300 units/mL penicillin, and 300 µg/mL streptomycin. For double transfection, p53+/+ HCT116 cells were loaded twice with dsRNA oligonucleotides at a 24-hour interval between transfection, and incubated with vehicle or a suboptimal concentration of purvalanol A (5 µmol/L) for an additional 24-hour culture at 37°C before analysis of hypodiploid DNA content by flow cytometry and survivin expression by Western blotting.
Xenograft colorectal cancer model. All animal experiments were approved by the Institutional Animal Care and Use Committee. p53+/+ or p53–/– HCT116 cells (2.5 x 106) were injected in the flank of CB17 SCID/beige mice (Taconics, Germantown, NY) and allowed to form palpable tumors for 4 to 6 days (two tumors per animal, four animals per group). When tumors reached 
50 mm3 in volume, animals were randomized and given vehicle or purvalanol A (25 mg/kg) daily as i.p. injections (200 µL per injection). Tumor measurements were taken with a caliper and tumor volume was calculated according to the formula 1/2 [length (mm)] x [width (mm)]2.
Statistical analysis. Data were analyzed using the unpaired t test on a Graphpad software package for Windows (Prism). A P value of 0.05 was considered as statistically significant.
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Results |
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2-fold, induction of p53 in HCT116 cells, detectable at 24 hours after treatment (not shown). Exposure of p53+/+ HCT116 cells to purvalanol A (18), a dual-specificity inhibitor of GSK3ß and Cdk, most notably p34cdc2, reproduced the rapid induction of p53, which was associated with up-regulation of p21Waf1/Cip1 and time-dependent loss of survivin levels (Fig. 1B), consistent with the ability of wild-type p53 to repress survivin gene transcription (19, 20). Conversely, purvalanol A did not affect the expression of another IAP family protein, XIAP (Fig. 1B; ref. 21). To further establish the p53 dependence of the observed response, we analyzed p53–/– HCT116 cells. Exposure of these cells to thiadiazolidinones, SB216763, or purvalanol A did not result in significant modulation of survivin or p21Waf1/Cip1 levels and p53-reactive material was undetectable (Fig. 1C). Similar to purvalanol A, other dual-specificity GSK3ß/Cdk inhibitors Alsterpaullone and Olomoucine (7) also strongly induced p53 expression in HCT116 cells (Fig. 1D).
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5-fold less potently than purvalanol A at the concentrations tested (Fig. 3A). To determine whether induction of p53 by dual-specificity Cdk/GSK3ß kinase inhibitors was contributed by deregulated cell cycle progression, we immunoprecipitated p34cdc2 from HCT116 cells and analyzed its kinase activity for histone H1 phosphorylation. Consistent with its specificity as a high-affinity p34cdc2 inhibitor (18), purvalanol A suppressed histone H1 phosphorylation at 24 and 48 hours after treatment (Fig. 3B). However, there was no decrease in p34cdc2 activity at 6 or 12 hours after purvalanol A treatment (Fig. 3B), which coincide with peak up-regulation of p53 (Fig. 1B). Next, we expressed a p34cdc2Asp146Asn dominant-negative mutant that interferes with mitotic progression (14) and looked for changes in p53 levels. Transfection of HCT116 cells with p34cdc2 dominant-negative mutant caused cell cycle arrest at G2-M, by DNA content analysis (Fig. 3C), and accumulation of p34cdc2-reactive material, by Western blotting (Fig. 3D). However, no changes in p53 expression were observed in HCT116 cells transfected with control vector or p34cdc2 dominant-negative mutant (Fig. 3D). Taken together, we conclude from these experiments that induction of p53 by dual-specificity Cdk/GSK3ß kinase inhibitors (7), including purvalanol A, does not involve inhibition of mitotic Cdks or deregulation of cell cycle progression, and is solely mediated by inhibition of GSK3ß.
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25% of the cell population), expression of GSK3ß dominant-negative mutant may result in a 7-fold increase in p21Waf1/Cip1 levels (Fig. 4A), comparable with that obtained with pharmacologic antagonists of GSK3ß (Fig. 1A). One of the molecular regulators of p53 is the p14ARF tumor suppressor gene, which is thought to up-regulate p53 levels by controlling its nuclear export and potential association with the negative regulator, MDM2 (23). Transfection of HCT116 cells with GSK3ß dominant-negative mutant also resulted in increased expression of p14ARF, whereas wild-type GSK3ß did not affect endogenous p14ARF levels (Fig. 4B).
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was only moderately affected (Fig. 4C). In contrast, untreated cells or HCT116 cells transfected with a control dsRNA oligonucleotide exhibited no changes in expression of GSK3 or GSK3ß by Western blotting (Fig. 4C). Under these experimental conditions, acute ablation of GSK3ß by RNAi was associated with strong induction of p53 in HCT116 cells compared with control cultures (Fig. 4C). Consistent with the results obtained with GSK3ß dominant-negative mutant (Fig. 4B), acute ablation of GSK3ß by RNAi also resulted in up-regulation of p14ARF in HCT116 cells, whereas control dsRNA oligonucleotide had no effect (Fig. 4D). Lastly, we analyzed nonimmortalized primary GSK3ß–/– MEFs for potential changes in p53 expression. Loss of GSK3ß resulted in constitutively elevated p53 levels in these cells, compared with wild-type MEF (Fig. 4E). In addition, purvalanol A did not further modulate p53 expression in wild-type or GSK3ß–/– MEF by Western blotting (Fig. 4E), thus suggesting cell type specificity of the observed response.
Molecular requirements of p53 induction by glycogen synthase kinase-3ß targeting. Because inhibition of GSK3ß causes deregulated TCF/ß-catenin signaling (24), and because p14ARF is a TCF/ß-catenin target gene (25), we asked whether forced expression of ß-catenin reproduced the induction of p53 observed after molecular or pharmacologic inhibition of GSK3ß. Overexpression of wild-type ß-catenin in HCT116 cells resulted in sustained induction of p53 and time-dependent up-regulation of p21Waf1/Cip1 compared with vehicle-treated cultures (Fig. 5A). Conversely, expression of a TCF-4 dominant-negative mutant that lacks the DNA binding domain and interferes with TCF/ß-catenin signaling (15) blunted purvalanol A induction of p53 at 12 hours after drug treatment, compared with vector control transfectants (Fig. 5B). This was associated with reduced expression of p21Waf1/Cip1 (Fig. 5B), which at this early time point precedes p53-dependent transcription and may, thus, reflect a role of TCF signaling in steady-state maintenance of p21Waf1/Cip1 in these cells. Consistent with a potential role of p14ARF in modulation of p53 levels (Fig. 4B and D), exposure of HCT116 cells to purvalanol A or thiadiazolidinones resulted in a 2.4-fold increased expression of p14ARF by Western blotting (Fig. 5C and not shown). Increased p14ARF expression in purvalanol A- or thiadiazolidinones-treated cells was also associated with reduced expression of the negative p53 regulator, MDM2, compared with vehicle-treated HCT116 cells (Fig. 5D and data not shown).
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4.5-fold in p53+/+ cultures and only by 1.7-fold in p53–/– cells, compared with vehicle treatment; similar results were obtained with LiCl (Fig. 6C). To independently validate the specificity of pharmacologic inhibitors in p53-dependent apoptosis, we acutely suppressed GSK3ß levels in colorectal cancer cells by RNAi (Fig. 4C and D). Transfection of HCT116 cells with control dsRNA oligonucleotide did not result in increased caspase activity or loss of plasma membrane integrity compared with untreated cultures (Fig. 6D). Conversely, RNAi suppression of GSK3ß resulted in enhanced caspase activity in HCT116 cells (Fig. 6D), with a magnitude comparable with that induced by pharmacologic inhibitors (Fig. 6C).
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Antitumor activity of glycogen synthase kinase-3ß targeting in vivo. To test whether acute inhibition of GSK3ß could exert p53-dependent anticancer activity in vivo, we used a colorectal cancer xenograft model with p53+/+ or p53–/– HCT116 cells. These studies were carried out with purvalanol A, which showed considerable increased efficacy of GSK3ß inhibition in vivo compared with other kinase antagonists (Fig. 3A). Injection of p53+/+ or p53–/– HCT116 cells in CB-17 SCID/beige mice gave rise to comparable exponentially growing tumors within a 1-week period (Fig. 7). Daily administration of purvalanol A (25 mg/kg/i.p.) after establishment of palpable masses (50 mm3) significantly inhibited the growth of p53+/+ HCT116 tumors compared with vehicle-treated animals (Fig. 7A). In contrast, purvalanol A treatment (25 mg/kg/daily/i.p.) of animals carrying p53–/– xenograft HCT116 tumors was completely ineffective at inhibiting tumor growth compared with the vehicle-treated group (Fig. 7B). Throughout the experiment, purvalanol A–treated animals exhibited no weight loss or other signs of local or systemic toxicity.
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Discussion |
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With 146,000 new cases and an estimated 57,000 deaths every year, colorectal cancer remains a major contributor of cancer-related mortality in the United States. Although considerable progress has been made in the understanding of the basic biology of adenomatous polyposis coli and aberrant ß-catenin signaling in disease pathogenesis (24), there have been far fewer concrete prospects for rational i.e., targeted, therapeutic intervention. Despite its importance as the kinase that phosphorylates, and thus destabilizes, ß-catenin (24), a direct role of GSK3ß in the molecular pathogenesis of colorectal cancer and its potential relevance in tumor cell survival have not been widely investigated. Previous data have suggested that GSK3ß may exert disparate effects on cell viability, potentially in a highly context- or cell-dependent fashion. In neuronal cells (5), GSK3ß inhibition is associated with PI3 kinase/Akt-dependent cell survival (22), and small molecule inhibitors of GSK3ß may be beneficial for neuroprotection (8). Conversely, in addition to the apoptosis-prone phenotype of GSK3ß knockout mice, potentially reflecting deregulated nuclear factor-
B signaling (6), inhibitors of GSK3ß have been associated with induction of p53 and cellular senescence in endothelial cells (26) and strong inhibition of cell proliferation in prostate cancer cells (27). These findings fit well with the data presented here, demonstrating that, at variance with the paradigm of neuronal cells (8), acute ablation of GSK3ß in colorectal cancer cells activates a p53-dependent cell death pathway with all the biochemical hallmarks of apoptosis. Consistent with current models of GSK3ß signaling (24), the molecular requirements of this response involved deregulated Wnt/TCF/ß-catenin activity, which has been previously linked to modulation of p53 expression. Accordingly, accumulation of p53 by transfection or DNA damage resulted in down-regulation of ß-catenin (28), whereas forced expression of ß-catenin was associated with stabilization of p53 through induction of the p14ARF tumor suppressor, which has been proposed as a TCF/ß-catenin target gene (25). A similar circuitry has emerged here, with pharmacologic, genetic, and molecular inhibition of GSK3ß inducing increased expression of p14ARF and attenuation of the p53-negative regulator, MDM2. Although HCT116 cells were reported to contain methylated/mutated alleles of p14ARF (29), modulation of endogenous p14ARF expression in this cell type has been independently shown (30), in agreement with the data presented here. Taken together, these data suggest that deregulated ß-catenin activity in response to acute inhibition of GSK3ß may act as a bona fide oncogenic signal, thus stabilizing p53 in a pathway ideally poised to counter aberrant cell proliferation and cell survival mediated by TCF target genes (24). This may also explain why colorectal cancer cells remain under selective pressure to eliminate p53 after the onset of adenomatous polyposis coli/ß-catenin alterations (31), thus removing an antiproliferative and proapoptotic response activated by aberrant ß-catenin activity.
Although associated with strong up-regulation of the cell cycle inhibitor, p21Waf1/Cip1, p53 activation by GSK3ß targeting led exclusively to apoptosis in HCT116 cells (32), and one of the effectors of this pathway was identified as the acute loss of survivin levels (33). Although it is still debated whether this involves a direct repressive action of p53 on the survivin promoter (19), or an indirect mechanism of transcriptional squelching (20), the abrupt decrease in survivin expression has been shown to contribute to p53-dependent apoptosis, with spontaneous loss of cell viability and enhanced sensitivity to cell death stimuli (33).
To probe the suitability of the GSK3ß-p53 axis described here for novel cancer therapeutic strategies, we used bona fide pharmacologic antagonists of GSK3ß (LiCl, thiadiazolidinones, SB216763), as well as dual-specificity Cdk/GSK3ß kinase inhibitors (7). Although both classes of antagonists reproduced the up-regulation of p53 and the activation of p53-dependent apoptosis, purvalanol A, a 2,6,9-trisubstituted ATP inhibitor of p34cdc2 (18), as well as Cdk2 (7), provided the most robust inhibition of GSK3ß activity in vivo, thus at variance with previous in vitro conditions (7). Also, contrary to previous findings (34), p53 induction by dual-specificity Cdk/GSK3ß kinase antagonists was independent of Cdk inhibition and did not involve deregulation of mitotic progression. As a single agent, purvalanol A significantly inhibited HCT116 xenograft tumor growth in vivo and this activity was strictly p53-dependent (i.e., purvalanol A was inactive against p53–/– HCT116 tumors). Previously, purvalanol A was largely devoid of anticancer activity in mice and was unable to induce significant apoptosis in tumor cell lines, including MCF-7 breast carcinoma cells carrying wild-type p53 (16, 35), as well as p53-negative HT29 colorectal carcinoma cells (35). This suggests that purvalanol A may exert optimal antitumor activity selectively in colorectal cancer cells retaining the functional GSK3ß-p53 axis described here. Whereas these data should help guiding ongoing clinical trials using dual-specificity Cdk/GSK3ß inhibitors (i.e., roscovitine), the restoration of p53-dependent apoptosis by acute ablation of GSK3ß may provide a novel rational approach for the treatment of colorectal cancer patients retaining wild-type p53 (31).
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12/21/04; revised 3/ 2/05; accepted 3/29/05.
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B activation. Nature 2000;406:86–90.[CrossRef][Medline]
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