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Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (P.-H.T., Y.-C.W., S.-C.W., C.-S.C., R.W.B., C.-S.C.), and Division of Hematology and Oncology, Department of Internal Medicine (C.L.S.), the Ohio State University, Columbus, Ohio; Department of Biological Science and Technology, China Medical University and Hospital, Taichung, Taiwan (J.-R.W.); Department of Family Medicine, College of Medicine, National Taiwan University, and the Gerontology Research Division, the National Health Research Institutes, Taipei, Taiwan (C.-Y.C.); British Columbia Research Institute for Children's and Women's Health, Department of Pediatrics, University of British Columbia. Vancouver, British Columbia, Canada (S.E.D.); and Department of Oncology, Jewish General Hospital, and McGill University, Montreal, Canada (M.P.)
Received February 28, 2006; accepted August 3, 2006
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Abstract |
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), down-regulation of phosphatidylinsitol 3-kinase (PI3K)/Akt signaling (Clark et al., 2002), and increases in the expression of the cyclin-dependent kinase inhibitor p27kip1. Therefore, HER2-positive breast cancer cells are able to evade the antiproliferative action of trastuzumab by adopting mechanisms to counter these antiproliferative mechanisms (Hynes and Lane, 2005), including up-regulation of insulin-like growth factor-I receptor (IGF-IR) expression (Lu et al., 2001), loss of PTEN function (Nagata et al., 2004), and down-regulation of the cyclin-dependent kinase (CDK) inhibitor p27kip1 expression (Nahta et al., 2004). In light of the important role of Akt signaling in the antitumor action of trastuzumab (Yakes et al., 2002), these mechanistic findings provide a rationale for the use of an Akt signaling inhibitor to enhance the sensitivity to trastuzumab and, more important, to overcome trastuzumab resistance (Arteaga, 2003; Nagata et al., 2004).
On the basis of our finding that the cyclooxygenase-2 (COX-2) inhibitor celecoxib mediates apoptosis in cancer cells, in part, by blocking phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling independently of COX-2 inhibition, we have used celecoxib as a scaffold to develop a novel class of PDK-1 inhibitors devoid of COX-2 activity (Zhu et al., 2004). The novel compound OSU-03012 exhibits high potency in deactivating Akt and inducing apoptosis at low micromolar concentrations in cancer cells through PDK-1 inhibition. Because PDK-1 is a proximal mediator of PI3K signals, PDK-1 inhibitors target a large portion of the PI3K/Akt pathway and can potentially offer an effective treatment (Crowder and Ellis, 2005). This celecoxib-derived PDK-1 inhibitor has been demonstrated to be effective in inhibiting the proliferation of a panel of breast cancer cell lines (Kucab et al., 2005) and is currently undergoing preclinical testing under the Rapid Access to Intervention Development program at the National Cancer Institute.
We hypothesized that concurrent inhibition of PDK-1/Akt signaling would sensitize HER2-positive breast cancer cells, even those with the resistant phenotype, to trastuzumab's antiproliferative effect. A similar strategy has been used to overcome imatinib mesylate resistance in Bcr-Abl mutant cell lines (Tseng et al., 2005). Here, we demonstrate that OSU-03012 interacted synergistically with trastuzumab to enhance cell killing in two HER2-positive breast cancer cells (BT474 and SKBR3) and to reverse the resistant phenotype in SKBR3 cells that overexpress IGF-IR (SKBR3/IGF-IR). This synergistic effect, however, was not observed in MDA-MB-231 cells that lack HER2 expression.
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Materials and Methods |
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). Cells were grown in 75-cm2 plastic tissue culture flasks at 37°C in a humidified incubator in Dulbecco's minimal essential medium/Ham's F-12 (DMEM/F12; 1:1) medium containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), and 10 µg/ml gentamicin (Sigma, St. Louis, MO) at 37°C in 5% CO2. Cells were passaged at 1:4 dilution with fresh medium once every 4 days. Trastuzumab, also known as Herceptin (Genentech Inc., San Francisco, CA), was obtained from The Ohio State University Hospital Pharmacy. The PDK-1 inhibitor OSU-03012 was synthesized as described previously (Zhu et al., 2004). Mouse monoclonal anti-poly-(ADP-ribose) polymerase (PARP) was purchased from Cell Signaling Technology Inc. (Beverly, MA). Rabbit antibodies against HER2, phospho-Thr308-Akt, Akt, and p27kip1, and the mouse antibody against p70S6K were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-phospho-Thr229-p70S6K and anti-phospho-Thr157-p27 were from R&D Systems Inc. (Minneapolis, MN). Mouse monoclonal antiactin was purchased from MP Biomedicals Inc. (Irvine, CA). Goat anti-rabbit and goat anti-mouse IgG-horse-radish peroxidase conjugates were from Jackson ImmunoResearch Laboratories (West Grove, PA).
Cell Viability Assay. Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (TCI America; Portland, OR) in six replicates. Cells (3000) were grown in 10% FBS-supplemented DMEM/F12 medium in 96-well, flat-bottomed plates and were exposed to various concentrations of individual agents or combination of drugs dissolved in DMSO (final concentration, 
0.1%) in the same medium. Control groups received DMSO vehicle at a concentration equal to that in drug-treated cells. After 72-h treatment, medium was removed and replaced by 200 µl of MTT (0.5 mg/ml) in DMEM/F12 medium, and cells were incubated in the CO2 incubator at 37°C for 3 h. After discarding the MTT solution, the reduced MTT dye was solubilized with 200 µl/well DMSO and was analyzed by measuring the absorbance at 570 nm in a plate reader. The IC50 value, which represents the drug concentration required for 50% growth inhibition, was calculated with the Calcusyn software (Biosoft, Cambridge, UK) by using the median-effect method (Chou and Talalay, 1984).
Immunoblotting. The general procedure for the Western blot analysis was performed as follows. Cells were scratched from culture flasks and collected by centrifugation at 2000g and resuspended in RIPA lysis buffer consisting of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and a mixture of protease inhibitor cocktail (Calbiochem, La Jolla, CA) and phosphatase inhibitors (10 µM sodium fluoride, 5 µM sodium vanadate, and 10 µM 
-glycerol phosphate). The mixture was sonicated for 5 s, and protein contents were analyzed by using the Bradford assay kit (Bio-Rad, Hercules, CA). Fifty micrograms of total proteins was resolved in SDS-polyacrylamide gels on a Minigel apparatus and transferred to a nitrocellulose membrane using a semidry transfer cell. After blocking with Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% nonfat milk for 60 min, the membrane was incubated with the appropriate primary antibody at 1:1000 dilution in TBST-1% nonfat milk at 4°C overnight and washed three times with TBST. The membrane was probed with horseradish peroxidase-conjugated secondary antibodies at 1:3000 for 1 h at room temperature and washed with TBST three times. The immunoblots were visualized by enhanced chemiluminescence.
Western Blot Analysis of PARP Cleavage. Drug-treated cells for 72 h were collected, washed with ice-cold PBS, and resuspended in the aforementioned RIPA lysis buffer. Soluble cell lysates were collected after centrifugation at 10,000g for 5 min. Equivalent amounts of proteins (50 µg) from each lysate were resolved in 10% SDS-polyacrylamide gels. Bands were transferred to nitrocellulose membranes and analyzed by immunoblotting.
Analysis of DNA Fragmentation by ELISA. Induction of apoptosis was assessed with a Cell Death Detection ELISA kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instruction. This test is based on the quantitative determination of cytoplasmic histone-associated DNA fragments in the form of mononucleosomes and oligonucleosomes after induced apoptotic death. In brief, 1 x 106 cells were exposed to individual treatments in a T-25 flask in 10% FBS-supplemented DMEM/F12 medium for 72 h. Both floating and adherent cells were collected; cell lysates equivalent to 5 x 105 cells were used in the ELISA.
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Immunocytochemical Analysis. SKBR and SKBR3/IGF-IR cells were treated with 5 µM OSU-03012 in 10% FBS-supplemented DMEM/F12 medium for 24 h, washed with Dulbecco's PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and then washed with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 in 1% FBS-containing PBS, stained with HER2 antibodies at 1:100 dilution (Santa Cruz Biotechnology) for 24 h at 4°C, and treated with Alexa Fluor 647 goat anti-rabbit IgG (Molecular Probes) in the aforementioned PBS for conjugation. The nuclear counter staining was performed using a 4,6-diamidino-2-phenylindole-containing mounting medium (Vector Laboratories, Burlingame, CA) before examination. Images of immunocytochemically labeled samples were observed using a Zeiss confocal microscope (LSM510; Carl Zeiss Inc., Thornwood, NY) with an argon laser and a helium-neon laser and appropriate filters (excitation wavelength, 633 nm for HER2 and 543 nm for 4,6-diamidino-2-phenylindole).
Statistical Analysis. Each experiment was performed in triplicate. All experiments were carried out at least two times on different occasions. The medium-effect method was used to analyze dose-response data for single or multiple drugs. The synergistic effect of multiple drugs was determined by the definition of Chou and Talalay (1984). Cells were treated with series dilution of trastuzumab and OSU-03012 at fixed concentration ratios. The concentration of trastuzumab ranged from 1 to 10 µg/ml, whereas that of OSU-03012 was 0.5 to 5 µM. These two drugs were assumed as totally independent modes of action and were therefore mutually nonexclusive. By using the software package Calcusyn (Biosoft, Cambridge, UK), the values of combination index (CI) were calculated. The values of CI at different levels of growth inhibition were calculated based on the formula for mutually nonexclusive mechanism: (D1/Dx1) + (D2/Dx2) + (D1D2/DxlDx2), where Dx1 and Dx2 are the doses of drug 1 and drug 2 alone required to produce x percentage effect, and D1 and D2 are the doses of drug 1 and drug 2 in combination required to produce the same effect. The CI values less than 1, equal to 1, and greater than 1 are indicative of synergism, additive effect, and antagonism, respectively.
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Results |
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Among these four cell lines, MDA-MB-231 cells were insensitive to trastuzumab because of lack of HER2 expression. Exposure of HER2-overexpressing BT474 and SKBR3 cells to trastuzumab caused a dose-dependent decrease in cell proliferation, which leveled off at the maximum cell reduction of approximately 50% (Fig. 1B, left). However, SKBR3/IGF-IR cells acquired a resistant phenotype as a result of IGF-IR overexpression (Lu et al., 2001). Despite the discrepancy in trastuzumab sensitivity, these four cell lines were equally susceptible to the antiproliferative effect of OSU-03012, indicating a lack of cross-resistance to OSU-03012 in trastuzumab-resistant SKBR3/IGF-IR cells (Fig. 1B, right). The respective IC50 values were the following: MDA-MB-231, 3.8 ± 0.4 µM; BT474, 3.9 ± 0.4 µM; SKBR3, 3.0 ± 0.2 µM; and SKBR3/IGF-IR, 3.2 ± 0.1 µM.
To shed light onto the mechanism responsible for this differential response, we examined the effects of trastuzumab and OSU-03012 on the expression and/or phosphorylation status of a number of pertinent signaling markers in individual cell lines. These biomarkers included HER2, Akt, the CDK inhibitor p27kip1, and the PDK1 substrate p70S6K. p27kip1 is known to be phosphorylated by Akt, which impairs the nuclear import and action of this CDK inhibitor (Liang et al., 2002). In BT474 and SKBR3 cells, trastuzumab caused a dose-dependent decrease in HER2 expression, accompanied by increased p27kip1 expression and decreased phosphorylation of Akt, p27kip1, and, to a lesser extent, p70S6K (Fig. 2). None of these effects, however, was noted in low HER2-expressing MDA-MB-231 or trastuzumab-resistant SKBR3/IGF-IR cells within the dose range examined, underlying the lack of antiproliferative effects of trastuzumab in these two cell lines.
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). Immunocytochemical analysis indicates that OSU-03012 decreased the cell surface expression of HER2 and facilitated the internalization of the receptor in both SKBR3 and SKBR/IGF-IR cells, which was also noted with the PI3K inhibitor LY294002 (Fig. 3). This finding suggests a link between receptor internalization and HER2 down-regulation. The underlying mechanism is currently under investigation.
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OSU-03012 Restores the Sensitivity of SKBR/IGF-IR Cells to Trastuzumab's Antiproliferative Effect. In light of the important role of Akt in the antitumor action of trastuzumab, we hypothesized that concurrent inhibition of PDK-1/Akt signaling by OSU-03012 could sensitize trastuzumab-resistant cells to the residual antiproliferative activity of this antibody. To test this hypothesis, MDA-MB-231, BT474, SKBR3, and SKBR3/IGF-IR cells were exposed to varying concentrations of trastuzumab in the presence of 2.5 or 5 µM OSU-03012 versus DMSO control for 72 h. Figure 4 demonstrates the ability of OSU-03012 to improve the sensitivity of HER2-overexpressing cells to trastuzumab-induced cell death. For example, 10 µg/ml trastuzumab and 2.5 µM OSU-03012 alone caused approximately 25 and 30%, respectively, cell reduction in BT474 and SKBR3 cells, and the combination of these two agents could achieve 65% cell death. Especially noteworthy, OSU-03012 was able to overcome the resistant phenotype in SKBR3/IGF-IR cells. Although trastuzumab alone at concentrations lower than 50 µg/ml was ineffective in inhibiting SKBR3/IGF-IR cell proliferation, OSU-03012 restored the sensitivity of these resistant cells to trastuzumab's antiproliferative effects to an extent similar to that of the parent cell line SKBR3 (Fig. 4A). This sensitizing effect, however, was not noted in the low HER2-expressing MDA-MB-231 cells.
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We used SKBR3 and SKBR3/IGF-IR cells to obtain evidence that the chemosensitizing effect of OSU-03012 was attributable to its ability to enhance the susceptibility of these HER2-overexpressing cells to trastuzumab-induced apoptosis. DNA fragmentation ELISA indicates that trastuzumab alone did not cause appreciable increase in nucleosome formation even at 20 µg/ml. However, in the presence of 5 µM OSU-03012, trastuzumab significantly increased the extent of DNA fragmentation in a dose-dependent manner (*, P < 0.005 compared with 5 µM OSU-03012 alone) (Fig. 5A). The effect of 5 µM OSU-03012 on sensitizing both cell lines to trastuzumab-induced apoptosis was also confirmed by the annexin V/PI staining (Fig. 5B). As shown, 10 µg/ml trastuzumab and 5 µM OSU-03012 alone gave rise to 12 and 35%, respectively, apoptosis compared with 13% for the vehicle control in SKBR3 cells (top). In contrast, treatment of SKBR3 cells with a combination of 5 and 10 µg/ml trastuzumab with 5 µM OSU-03012 led to 60 and 89%, respectively, apoptotic death. A similar finding was also noted with the IGF-IR-overexpressing SKBR3 cells (bottom).
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). Figure 6A depicts the dose-response curves of these four cell lines after 72-h treatment of five different dose combinations of trastuzumab (nanomolar)/OSU-03012 (micromolar) at a fixed ratio (7/0.5, 14/1, 28/2, 42/3, 56/4, and 70/5; 
) versus varying concentrations of trastuzumab (
) and OSU-03012 (
) alone. These dose-response data were used to determine CI values in relation to the fraction affected (Fig. 6B). With the exception of MDA-MB-231, the other three cell lines exhibited CI values all lower than 1, which was indicative of synergism between trastuzumab and OSU-03012 in HER2-overexpressing cells. For example, the CI values calculated for the combination treatment that caused 50% cell death (fractional effect, 0.5) were 0.96, 0.50, 0.71, and 0.68 for MDA-MB-231, BT474, SKBR3, and SKBR3/IGF-IR cells, respectively.
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Discussion |
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).
The present study indicates that concurrent inhibition of PDK-1 with OSU-03012 reverses the trastuzumab-resistant phenotype in IGF-IR-overexpressing SKBR3 cells. These trastuzumab-resistant cells exhibited the same susceptibility to OSU-03012 as their parent (SKBR3) cells as well as BT474 and MDA-MB-231 cells irrespective of the resistance status. The lack of cross-resistance underscores the functional relevance of targeting PDK-1/Akt signaling in trastuzumab-resistant cells. Despite the resistant phenotype, trastuzumab still retains its ability to bind HER2 and to activate downstream apoptotic signaling in resistant cells. Therefore, OSU-03012 could sensitize trastuzumab-resistant cells to trastuzumab's antitumor action through PDK-1 inhibition, as evidenced by the dephosphorylation of Akt, p27kip1, and p70S6K. It is noteworthy that Western blot analysis indicates that OSU-03012 caused the repression of HER2 expression in a manner similar to that of trastuzumab. Immunocytochemical analysis shows that OSU-03012 treatment led to HER2 internalization in both SKBR3 and SKBR3/IGF-IR cells, which presumably led to receptor degradation. The effect of OSU-03012 on HER2 down-regulation is reminiscent of that of the Hsp90 inhibitor geldanamycin on HER-family tyrosine kinases((Zheng et al., 2000), although the underlying mechanisms might differ. It was speculated that geldanamycin might repress the expression of HER kinases through the disruption of the interaction between Hsp90 and the catalytic domain of HER kinases (Zheng et al., 2000). We rationale that the effect of OSU-03012 on HER2 down-regulation might be attributable to the inhibition of PDK-1/Akt signaling because LY294002 could also achieve a similar effect. OSU-03012's ability to suppress HER2 expression is noteworthy because HER2 degradation represents a major mechanism underlying the antiproliferative effect of trastuzumab and other anti-HER2 antibodies (Hurwitz et al., 1995; Frankel, 2002). A correlation has been shown between the effectiveness in HER2 down-regulation and the in vivo potency of different anti-HER2 antibodies in tumor growth inhibition (Harwerth et al., 1993). Reduced HER2 at the cell surface allows less HER2 heterodimer formation, resulting in reduced growth factor-induced signaling and proliferation.
The combination of trastuzumab and an epidermal growth factor receptor inhibitor such as gefitinib (Iressa; AstraZeneca Pharmaceuticals LP, Wilmington, DE) or erlotinib (Tarceva; OSI Pharmaceuticals, Melville, NY) is being evaluated as a strategy to prevent or delay resistance in HER2-positive patients (Miller, 2004). The ability of gefitinib to enhance the efficacy of trastuzumab has been demonstrated in several sensitive breast cancer cell systems (Moulder et al., 2001). However, in a phase I trial in patients with pre-treated metastatic breast cancer, the combination of trastuzumab and gefitinib was well tolerated but showed poor antitumor activity.
Because PDK-1/Akt signaling represents important down-stream effectors of many receptor tyrosine kinases, use of the PDK-1 inhibitor OSU-03012 enhances the efficacy in sensitive cells, and, more importantly, restores sensitivity in resistant cells. It is noteworthy that OSU-03012 is currently undergoing preclinical testing under the Rapid Access to Intervention Development program at the National Cancer Institute. Our data indicate that oral administration of this agent in tumor-bearing nude mice at 200 mg/kg for 1 month achieves a peak serum concentration of more than 20 µM with no apparent toxicity at necropsy (S.K. Kulp and C.-S. Chen, unpublished data). Our data indicate that low doses of OSU-03012 were able to synergize with trastuzumab in inducing apoptotic death in HER2-positive cells.
The potential clinical relevance of the trastuzumab-OSU-03012 combination is considerable. Not only do our data suggest that the trastuzumab-OSU-03012 combination represents a novel strategy to overcome trastuzumab resistance, but the in vitro evidence also suggests that OSU-03012 can increase the efficacy of trastuzumab in HER2-positive breast cancer treatment. The regimen of weekly trastuzumab dosing at 2 mg/kg or every 3-week dosing at 6 mg/kg will achieve a steady-state serum level of 60 to 80 µg/ml (Leyland-Jones et al., 2003). In vitro cell viability assays using BT474 and SKBR3 cells indicate that the maximum cell reduction attainable at this range was approximately 50%. However, trastuzumab in this combination range in combination with OSU-03012 at 2.5 µM achieved greater than 90% killing.
Recent data provide evidence that development of resistance to trastuzumab in vivo is often associated with increased IGF-I receptor level (Cohen and Jani, 2005). This suggests that the SKBR3/IGFIR cell line we used may be a relevant model for at least a subset of spontaneously arising trastuzumab-resistant breast cancers. We are currently evaluating OSU-03012 in combination with trastuzumab in pre-clinical in vivo animal models. If the results of in vivo testing are promising, then this combination may represent a viable strategy for clinical testing in both trastuzumab-sensitive and -resistant HER2-positive breast cancer.
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Footnotes |
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ABBREVIATIONS: PI3K, phosphatidylinositol 3-kinase; PDK-1, phosphoinositide-dependent kinase-1; IGF-IR, insulin-like growth factor-I receptor; SKBR3/IGF-IR, insulin-like growth factor-I receptor-overexpressing SKBR3; CDK, cyclin-dependent kinase; COX-2, cyclooxygenase-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; CI, combination index; PARP, poly(ADP-ribose) polymerase; OSU-03012, 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide; DMEM/F12, Dulbecco's minimal essential medium/Ham's F-12; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; TBST, Tris-buffered saline/Tween 20; PBS, phosphate-buffered saline; Hsp90, 90-kDa heat shock protein; ELISA, enzyme-linked immunosorbent assay; PI, propidium iodide; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride.
Address correspondence to: Dr. Ching-Shih Chen, at College of Pharmacy, 336 Parks Hall, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210-1291. E-mail: chen.844{at}osu.edu
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