| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cancer Therapy: Preclinical |
Authors' Affiliations: Departments of 1 Systems Biology, 2 Gynecologic Medical Oncology, 3 Experimental Diagnostic Imaging, and 4 Diagnostic Radiology, University of Texas M. D. Anderson Cancer Center, Houston, Texas and 5 Keryx Pharmaceuticals, New York, New York
Requests for reprints: Bryan T. Hennessy, CPB6.3444, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-7960; Fax: 713-745-1541; E-mail: bhennessy{at}mdanderson.org.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Experimental Design: A novel functional proteomics technology, reverse phase protein array, was used to establish and quantify pharmacodynamic markers of perifosine efficacy.
Results: Perifosine selectively prevents AKT recruitment to the membrane and blocks activation of downstream effectors. Perifosine inhibited breast, ovarian, and prostate cancer models. Growth inhibition was associated with apoptosis. Activation of AKT as a consequence of genomic aberrations predicted perifosine efficacy. In cell lines and xenografts, there was a highly statistically significant correlation between the degree of antitumor efficacy of different perifosine doses and quantified down-regulation of phosphorylation of AKT and of its downstream targets, particularly S6.
Conclusions: Because of a strong correlation between proportional modulation of PI3K pathway biomarkers and quantified perifosine efficacy, it is likely that early measurement of such pharmacodynamic biomarkers with reverse phase protein array will optimize selection of responding patients and guide perifosine dosing. Furthermore, PI3K pathway activation status may allow baseline selection of patients most likely to respond to perifosine alone or in combination with other therapies.
The PI3K/AKT pathway regulates many "hallmarks of cancer," including survival, proliferation, and metastasis (3). PDK1, TSC2, glycogen synthase kinase 3 (GSK3), mTOR, p70S6 kinase, and S6 mediate many effects of PI3K/AKT and participate in feedback loops that regulate pathway homeostasis. These feedback loops may play a critical role in patient management potentially limiting, for example, rapamycin efficacy (8).
Herein, we apply novel quantitative reverse phase protein lysate microarrays (RPPA) to objective quantification of the pharmacodynamic effects of the targeted therapeutic perifosine in vitro and in vivo (9–16). We show that perifosine selectively inhibits PI3K/AKT signaling and that quantification of this inhibition with RPPA actually predicts the degree of perifosine efficacy in cell lines and xenografts. We, thus, establish potentially useful pharmacodynamic markers of perifosine efficacy using RPPA as a proof of principle for making clinical drug development more efficient by facilitating early determination of efficacy and optimal biological dosing. For selection of patients likely to respond, we show that cell lines with activating PI3K/AKT genomic abnormalities are more sensitive to perifosine than lines without such aberrations. Enforced PI3K/AKT pathway activation also renders cells more sensitive to perifosine. Thus, perifosine may be particularly useful in treating tumors with PI3K/AKT-activating genomic anomalies.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
/GSK3β (Ser21/9), total and phosphorylated p70S6K (Thr389), phosphorylated S6 (Ser235/236), phosphorylated S6 (Ser240/244), phosphorylated MEK (Thr217/221), phosphorylated c-Jun-NH2-kinase (JNK; Thr183/Tyr185), phosphorylated epidermal growth factor receptor (Tyr992), Jun, p38, and cleaved caspase-7. Rabbit anti-JNK1 and anti-ERK2 antibodies and mouse anti-GSK3 antibody were purchased from Santa Cruz Biotechnology. Rabbit anti–cyclin B1 was obtained from Epitomics. Horseradish peroxidase–conjugated goat anti-rabbit and anti-mouse IgGs were purchased from Bio-Rad. Rabbit antiactive mitogen-activated protein kinase (MAPK) antibody and a nonradioactive cell proliferation assay (Celltiter 96 for MTS assay) were purchased from Promega. APO-BRDU reagents for cell cycle and apoptosis assay were purchased from Phoenix Flow Systems, Inc.
Expression vectors. Human AKT was cloned from OVCAR3 cells by reverse transcription–PCR (Titan One Tube RT-PCR System from Roche Applied Science). The myristylation site (ATG-GGG-AGC-AGC-AAG-AGC-AAG-CCC-AAG) and HA-tagged sequence (ATG-TAC-CCA-TAC-GAT-GTT-CCA-GAT-TAC-GCT) were attached to the NH2 terminus of AKT by PCR. Constitutively activated AKT (T308D/S473D) and dominant-negative AKT (K179A/T308A/S473A) were generated by site-directed mutagenesis (Stretagene) on the backbone of human myristylated wild-type AKT. The constructs were entirely sequenced and were without error. Adenovirus-containing kinase inactive (dominant-negative) AKT (K179M) was provided by Dr. Ho-Young Lee (M. D. Anderson Cancer Center). Adenovirus-containing β-gal Lac-Z was prepared in the Core Facility at M. D. Anderson Cancer Center. DNA plasmids containing green fluorescent protein (GFP)-AKT-PH and GFP-PLC
-PH domain were gifts from Dr. Tobias Meyer (Stanford University).
Cell lines and transfection. The breast and ovarian cancer cell lines MDA-MB-468 (PTEN-mutant), MDA-MB-231 (RAS/RAF-mutant), SKOV3 (HER2 amplification), OVCAR3 (p85-mutant), and DOV13 (no known PI3K pathway aberrations) were cultured in complete medium [RPMI 1640 supplemented with 10% fetal bovine serum (FBS)]. BT474, MCF7 (both PIK3CA mutants), PC3 (PTEN-mutant), DU145 (no known genomic PI3K anomaly), A431, and HeLa cell lines were cultured by Keryx Pharmaceuticals. Isogenic pairs of DOV13 cell lines were established by stable transfection with different cDNA plasmids containing human active PI3K p110 catalytic domain or active or kinase inactive AKT2 sequences using Fugene 6 transfection reagent (Roche Molecular Biochemicals) and Nucleofector reagent (Amaxa Biosystems), respectively, as suggested by the manufacturer and selected by G418 (400 µg/mL). In the experiments indicated, cells were infected with adenovirus containing dominant-negative AKT at different MOIs. Cells were incubated with medium containing adenovirus (25 µL/well of 96-well plate) at 37°C for 1 h followed by 48-h incubation with complete medium to allow transgene expression. Cells were then subjected to proliferation assays or immunoblotting.
Cell lysis and immunoblotting. Cells were treated with inhibitors in serum-free medium and left unstimulated or stimulated with epidermal growth factor (EGF). Cells were washed twice with PBS and lysed in ice-cold lysis buffer [1% Triton X-100, 50 mm HEPES (pH 7.4), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 100 mmol/L NaF, 10 mmol/L Na pyrophosphate, 1 mmol/L Na3VO4, 10% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL aprotinin]. Protein concentration was determined by bicinchoninic acid (Pierce). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin and incubated at 4°C overnight with anti–phosphorylated p70S6K, anti–phosphorylated GSK3/β, anti–phosphorylated AKT, anti-AKT, anti–phosphorylated S6 (all 1:1,000 dilution), antiactive MAPK (1:5,000), anti-GSK3, or anti-ERK2 (both 0.2 µg/mL) antibodies. The membranes were washed in TBS-T [10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Tween 20] and incubated with horseradish peroxidase–conjugated goat anti-rabbit or goat anti-mouse IgG (1:2,500) at room temperature for 1 h. The membranes were washed, and the proteins were visualized by enhanced chemiluminescence detection (Amersham Pharmacia Biotech).
RPPA. Lysis buffer was used to lyse cell lines or frozen mice xenografts by homogenization. Cell/tumor lysates were normalized to 1 µg/µL concentration using bicinchoninic acid assay and boiled with 1% SDS, and the supernatants were manually diluted in six or eight 2-fold serial dilutions with lysis buffer. A GeneTAC arrayer (Genomic Solutions, Inc.) created 1,152 spot arrays on nitrocellulose-coated FAST slides (Schleicher & Schuell BioScience, Inc.) from the serial dilutions. Each slide was probed with a validated primary antibody (listed above), and the signal was amplified using a DakoCytomation–catalyzed system. A secondary antibody (antimouse or antirabbit) was used as a starting point for amplification. The slides were scanned, analyzed, and quantitated using Microvigene software (VigeneTech Inc.) to generate serial dilution–signal intensity curves for each sample with the logistic fit model: ln(y) = a + (b – a) / (1 + exp {c*[d – ln(x)]}). A representative natural logarithmic value of each sample curve on the slide (curve average) was then used as a relative quantification of the amount of each protein in each sample. Protein loading was corrected across samples by correction of the linear expression values using the average expression levels of at least four proteins (e.g., ERK2, GSK3, JNK, mTor) to calculate a loading correction factor for each sample. These proteins were found to be stably expressed and not altered on Western blotting and RPPA during perifosine treatment. In the PC3 mouse model, as an example, we observed a 22-fold (2,200%) decrease in AKT phosphorylation at Ser473 with perifosine, and the range of loading correction factors was 0.4 to 2.4. As an alternative, all measured proteins were used to calculate loading correction factors, and results did not differ. To generate heat maps, Treeview (University of Glasgow) and X-cluster softwares were used.
AKT localization assay. GFP fusion proteins containing the AKT-PH or PLC-PH domain were transfected into the cell line OVCAR3 by Nucleofector (Amaxa Biosystems). Eighteen hours later, cells were serum starved for 2 h and incubated with perifosine (2.5, 5, and 10 µmol/L) or LY294002 (20 µmol/L) for 6 h in serum-free medium. Cells were left unstimulated or stimulated with EGF (20 ng/mL) for 30 min. Cells were washed and fixed with 4% paraformaldehyde at room temperature for 30 min, and the cell nuclei were counterstained by 4',6-diamidino-2-phenylindole. GFP fusion protein localization was observed using a fluorescence microscope.
Cell proliferation assay. Cells were seeded in 96-well plates (5,000/well) and serum starved overnight before incubation with or without perifosine in 0.5% FBS for 72 h, followed by 3-h incubation with MTS reagents (Promega). Alternatively, cells were washed at the end of treatment with ice-cold PBS and stained with 0.5% crystal violet containing 20% methanol for 30 min at room temperature followed by dissolution of the dye in Sorenson's buffer for 1 h. For some experiments, cells were seeded in 96-well plates and infected with adenovirus containing dominant-negative AKT. Forty-eight hours after infection, cells were washed and incubated in medium supplemented with 0.5% FBS for 72 h, followed by 3-h incubation with MTS reagents. In all proliferation assays, triplicates were done. The quantity of formazan product converted by metabolically active cells was measured at 490 nmol/L absorbance. For the crystal staining assay, absorbance was read at 570 nmol/L.
Colony formation assay. OVCAR3 cells were incubated with different perifosine concentrations (5 and 10 µmol/L) for 24 h in 0.5% FBS, harvested, and washed. The same number of cells was seeded in culture dishes for an additional 15-day incubation in 10% FBS in the absence of perifosine. The number of colonies was subsequently counted and used as a reference variable for cell growth.
Cell cycle and apoptosis analysis. Cells were cultured in 60-mm dishes, serum starved overnight, and incubated with perifosine in the presence of 0.5% FBS for the time period indicated. Both floating and attached cells were collected and fixed using 4% paraformaldehyde. Cells were labeled by APO-BRDU reagents (Phoenix Flow Systems, Inc.), a two-color TUNEL assay for labeling DNA breaks and total DNA. Cell cycle change and apoptosis were measured by flow cytometry and analyzed by CellQuest (Becton Dickinson).
Tumor growth in vivo. BALB-c nude mice (female, 3 weeks old) were purchased from Harlan and adapted in the M.D. Anderson Cancer Center animal facility for a week. SKOV3 cells were injected orthotopically into the peritoneal cavity at 8 to 10 x 106 cells/0.2 mL medium. Each mouse received one injection. Each treatment group included five mice. Seven days later, perifosine (in vehicle PBS) was administrated orally as 200 mg/kg weekly or 70 mg/kg thrice weekly. Vehicle only was given to control mice. Mouse abdominal circumference was measured every 2 days. Mice were euthanized on the 24th day; tumors were excised and measured by weight, herein represented using the mean weight from each group. In a separate study, for pharmacodynamic measurements, mice were inoculated with PC3 or DU145 prostatic cells. Docetaxel was given i.v. at 12.5 mg/kg every other day for three treatments, or perifosine was administrated orally by gavage beginning when the tumor grew to 5 mm3 in volume (day 1). In the control group, vehicle (sterile water) was given daily. The tumor was monitored until day 24 (PC3) or day 106 (DU145). Tumor weight was calculated by weight (mg) = [width2 (mm2) x length (mm)] / 2. Mice were then euthanized, and the tumors were excised and homogenized in lysis buffer for RPPA. In a third study, mice were inoculated with PC3, A431, or BT474 cells and treated with vehicle or perifosine. The tumor was monitored until day 28 (PC3 and A431) or day 20 (BT474), and the animals were sacrificed. Additional mice were sacrificed at days 0, 1, 2, 7, 14 (BT474 only), and 21 (PC3/A431 only), and tumor was extracted for RPPA.
Mouse magnetic resonance imaging. Initial magnetic resonance imaging of six mice bearing i.p. tumors was done 7 days after injection of SKOV3 cells. On the same day, three animals were treated with perifosine (200 mg/kg orally weekly) and three with vehicle. Imaging was repeated on day 14. For magnetic resonance, animals were anesthetized with 2% isoflurane. Magnetic resonance was done using a 4.7 T Biospec small animal imager (Bruker Biospin USA). Images from a coronal T2-weighed fast-spin echo sequence (echo time, 70 ms; repetition time, 4,000 ms; nex, 4; slice thickness, 1.3 mm; field of view, 3.7 x 5 cm; matrix, 256 x 256; spatial resolution, 195 µm) were used for tumor measurements with Image J program (Version 1.33u, NIH, 2004; ref. 17). To assess therapeutic effect, tumor weight from the early time point was subtracted from that at the later time point.
Measurement of vascular endothelial growth factor production. Supernatants of cultured cells were quantified for vascular endothelial growth factor by ELISA using the human vascular endothelial growth factor–specific QuantiGlo vascular endothelial growth factor kit (R&D Systems). Vascular endothelial growth factor concentration was calculated by comparing sample absorbance to standard curves.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
, which preferentially binds PI(4,5)P2 (18, 19). In OVCAR3 cells expressing GFP-PH domain fusion constructs, perifosine induced a concentration-dependent inhibition of EGF-induced AKT-PH domain membrane translocation without altering PLC-PH domain localization (Fig. 1
). Thus, perifosine has selectivity for the AKT-PH domain, potentially by preventing interaction with PI(3,4,5)P3.
|
|
RPPA also has the potential to provide quantitative analysis. Perifosine at 5 µmol/L reduced AKTp473 by 65% (±27%) in resting MDAMB468 cells and by 77% (±6%) in EGF-stimulated cells. We previously found, by comparing lysates derived from resting and EGF-stimulated MDAMB468 cells to phosphopeptide controls using RPPA, that these cells have 0.2 and 0.87 amol AKTp473 per cell, respectively (21). Thus, 5 µmol/L perifosine reduces AKTp473 in each resting MDAMB468 cell by 0.13 amol (±0.05 amol) and in each EGF-stimulated cell by 0.67 amol (±0.05 amol).
Perifosine-induced growth inhibition correlates with PI3K/AKT pathway genomic status. To determine if the PI3K/AKT pathway mutational status alters perifosine sensitivity, we used RPPA to assess four ovarian/breast cancer cell lines that have previously been comprehensively characterized for PI3K/AKT genomic aberrations, three with activating anomalies (OVCAR3-p85 mutation, PIK3CA amplification; SKOV3-HER2/neu amplification; MDAMB468-PTEN mutation, epidermal growth factor receptor amplification), and one without known aberrations in the core PI3K/AKT pathway (MDAMB231-RAS/RAF mutations).6 Baseline AKTp473 levels correlated with growth inhibition induced by a 72-h treatment with 2.5 µmol/L (Fig. 3A
) and 5 µmol/L perifosine in 0.5% FBS. Higher AKTp473 levels were associated with increased sensitivity to perifosine-induced growth inhibition in cells possessing PI3K/AKT-activating genomic abnormalities versus MDAMB231, the latter having low basal AKTp473 levels despite possessing RAS/RAF mutations. Perifosine concentrations of 
10 µmol/L induced marked growth inhibition of all four cell lines in 0.5% FBS, possibly reflecting "off-target" effects or an AKT inhibition threshold, even in cells without PI3K/AKT pathway aberrations. Thus, genomic PI3K/AKT pathway activation seems to predict the likelihood of response to perifosine. In support of the effects of perifosine being dependent on PI3K/AKT inhibition and of genomic pathway activation being an indicator of perifosine sensitivity, a dominant-negative AKT (K179M) transgene significantly reduced growth of MDAMB468 cells with little effect on MDAMB231, although both expressed equivalent transgene levels (not shown). Thus, genomic PI3K/AKT pathway activation sensitizes tumor cells to pathway inhibition and could potentially be used to select patients for clinical trials with perifosine.
|
(DOV13mp110) sensitizes PI3K/AKT wild-type DOV13 cells to PI3K inhibition by LY294002 while rendering them taxane resistant (21). The effects of LY294002 are mimicked by introduction of a dominant-negative AKT, confirming that DOV13mp110 cells are "addicted" to PI3K signaling (not presented). DOV13mp110 cells are also more sensitive to perifosine than parental cells (Fig. 3B). Furthermore, perifosine effectively reversed the effects of activated p110 on paclitaxel sensitivity, particularly at low concentrations. Low concentrations of perifosine (
5 µmol/L) also sensitized three cell lines with PI3K/AKT-activating genomic aberrations (MDAMB468, OVCAR3, SKOV3) to 0.1 µmol/L paclitaxel (not presented). Because perifosine selectively inhibits AKT membrane translocation (Fig. 1), membrane-targeted AKT should bypass growth inhibitory effects if the latter are mainly due to PI3K/AKT inhibition. Indeed, a membrane-targeted (myristylated) activated AKT (DOV13AKT2dd) almost completely bypassed the effects of perifosine on DOV13 proliferation (Fig. 3B). Furthermore, perifosine failed to sensitize DOV13AKT2dd cells to paclitaxel.
In these assays, perifosine was present throughout incubation. To determine the effects of brief exposure, which may occur during patient treatment, OVCAR3 cells were incubated with perifosine for 24 h, washed, and replated in the absence of perifosine. Perifosine induced a dose-dependent irreversible inhibition of proliferative competence (Supplementary Fig. S1). In contrast, LY294002 did not significantly alter colony formation. This suggests that perifosine induces irreversible growth inhibition, potentially through induction of apoptosis. The differing effects of perifosine and LY294002 are possibly due to selective inhibition of AKT by perifosine while leaving other parts of the PI3K pathway (e.g., PDK1) intact or, alternatively, to AKT-independent effects of perifosine. In contrast, LY294002, by inhibiting PI3K, blocks the complete pathway, resulting in reversible growth arrest.
Perifosine induces apoptosis. Based on the ability of a brief perifosine incubation to block colony formation, we characterized its effects on cell cycle arrest and apoptosis in OVCAR3. After incubation with perifosine for 72 h in medium containing 0.5% FBS, cells were harvested and apoptosis was determined by propidium iodide and BrdUrd staining. Both perifosine and LY294002 induced G1 arrest, more marked with equimolar LY294002 concentrations (Supplementary Table S1). Unlike LY294002, perifosine, particularly at 10 µmol/L, dramatically increased apoptosis. Similar effects occurred in SKOV3 (not shown). Thus, the selective ability of a brief perifosine incubation to block colony formation is likely due to apoptosis rather than replicative arrest.
Perifosine inhibits xenograft growth. To explore the effects of perifosine on orthotopic tumor growth, SKOV3 cells were inoculated i.p. in athymic nude mice. Seven days later, perifosine was administrated orally (70 mg/kg thrice or 200 mg/kg once weekly). Perifosine at 200 mg/kg weekly significantly inhibited tumor growth (Supplementary Fig. S2A). This was also shown using T2-weighed magnetic resonance images at baseline and 14 days into study treatment (Supplementary Fig. S2B).
Biologically relevant dosing. It is critical to develop methods that assess the pharmacodynamic effects of targeted therapeutics to improve drug development and ensure that patients receive a biologically active dose. Because the antitumor efficacy of perifosine seems dependent on PI3K/AKT inhibition, we used RPPA to quantify effects of perifosine on activation of components of this pathway to identify biomarkers of efficacy that may facilitate early identification of responding patients. The studies described with the two breast and two ovarian cancer cell lines above suggested that PI3K/AKT pathway aberrations, activation status, and inhibition may be accurate predictive and response markers. We sought to test these contentions in a series of additional cell lines across multiple tumor lineages. Thus, IC25, IC50, and IC75 values were determined for six human cancer cell lines representing a range of epithelial tumors, some possessing genomic PI3K/AKT pathway aberrations [BT474 (HER2-amplified PIK3CA-mutant), MCF7 (PIK3CA-mutant), PC3 (PTEN-mutant), DU145 (no detectable aberrations), A431, and HeLa (not fully characterized)]. This approach allows correction for individual variations in perifosine uptake, transport, and metabolism, allowing correlation of changes in proliferation with degree of pathway activation. Each cell line was treated with vehicle or perifosine (IC25, IC50, and IC75; Supplementary Table S2) for 0.5, 1, 4, and 8 h in serum-replete conditions, and phosphoprotein levels were determined by RPPA (4 h is presented in Fig. 4 ; similar results were observed at each time point). Despite the wide range of concentrations required to achieve IC25, IC50, and IC75, AKTp473 inhibition induced by IC25, IC50, and IC75 doses averaged across all six lines approximated growth inhibition (Fig. 4A). The signaling effects of perifosine IC25, IC50, and IC75 were remarkably consistent with higher doses blocking PI3K/AKT pathway activity as measured by phosphorylation of AKT, GSK3, and S6, particularly at later time points (Fig. 4B). However, there were subtle differences at midrange perifosine concentrations, with the IC50 decreasing AKTp473 but not AKTp308 in MCF7/A431 and inhibiting both sites in PC3/BT474. Perifosine did not consistently alter MAPK signaling. The correlation between growth and PI3K/AKT inhibition suggests that phosphorylation of pathway components may be useful early biomarkers of perifosine efficacy.
|
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
In assessing novel therapies in human cancer trials, pharmacodynamic markers of drug activity must be defined in preclinical models to make clinical development more efficient and ensure that patients are receiving a biologically relevant dose. Maximal inhibition of the target may be a more important end point than maximum tolerated dose per se. This approach may optimize efficacy, decrease toxicity, in particular off-target toxicity, and facilitate early identification of nonresponders. In this regard, perifosine-induced PI3K/AKT inhibition correlated remarkably well with tumor growth inhibition using multiple dosing schedules. Furthermore, assessment of multiple PI3K/AKT pathway members may prove superior to single markers. RPPA facilitates analysis of multiple PI3K/AKT pathway components and identification of mechanisms associated with inhibition.
RPPA (9–16, 20) is particularly applicable to assessment of on-target and off-target effects of novel therapies. It is inexpensive, high-throughput, applicable to small tissue amounts (e.g., from fine needle aspiration) and, unlike immunohistochemistry/Western blotting, can concurrently quantitate multiple markers and pathways. We expect further RPPA validation to support applicability to in vitro drug development, optimal biological dose determination, and early identification of nonresponders. RPPA also has the potential to identify and validate pathways wherein a molecular imaging approach is applicable. By quantifying functional proteomics, RPPA may prove essential in efforts to develop molecular markers for optimal biological dosing and early response detection.
When we determined perifosine IC25, IC50, and IC75 across multiple cell lines (Supplementary Table S2), there was wide variability in perifosine concentrations, likely reflecting differences in uptake, transport, and/or metabolism. Despite this variability, decreases in cell growth and PI3K/AKT signaling were concordant. Thus, it may be possible to achieve optimal personalized patient doses that account for individual differences in perifosine pharmacology and pharmacodynamics. This may improve the real but limited response rates observed in perifosine trials (1, 26). More benefit may result from individual patient dose "titration" to maximal PI3K/AKT inhibition than to maximum tolerated dose per se, potentially facilitating dose limitation, for example, in patients with sensitive cancers (e.g., having lost PTEN). Studies of this approach necessitate biopsies. However, as glucose uptake is dependent on PI3K/AKT activity, fluorodeoxyglucose-positron emission tomography may be a useful surrogate (3). We believe that such an approach is critical in clinical trials of targeted therapies.
The signaling effects of perifosine and LY294002 are attenuated by growth factors (Fig. 2A) and high FBS concentrations. Although it is difficult to determine the concentration of growth factors at the tumor interface, they are likely to be much lower than those in 10% FBS. Most of our studies were thus done in 0.5% FBS. Impaired signaling inhibition in 10% FBS markedly decreased perifosine-induced growth inhibition and apoptosis, particularly at low concentrations. Whether serum growth factor levels or perifosine binding in serum limits its effectiveness remains to be determined.
The use of cell lines to predict drug efficacy and molecular markers has many concerns. The comparative utility of two-dimensional/three-dimensional culture, representative cell lines, coculture of tumor with stroma, and appropriate growth factor concentrations remains unknown. However, the use of multiple cell lines possessing baseline and induced PI3K/AKT aberrations may alleviate some problems and render our results more applicable to human trials.
Perifosine-induced AKT inhibition was relatively specific at low drug concentrations. However, perifosine also mediates its effects through other mechanisms that become apparent at concentrations of 10 µmol/L in vitro. We also showed that perifosine effects on motility, invasion, and neovascularizing factors are likely PI3K/AKT independent. Although effects of perifosine on MAPK and JNK phosphorylation have been shown in other studies, we found that these changes lacked the consistency or predictive value of PI3K/AKT inhibition. However, these effects may also contribute to perifosine activity.
In contrast to LY294002, a brief perifosine incubation prevented subsequent colony formation. Furthermore, perifosine, but not LY294002, induced marked apoptosis under anchorage-dependent conditions. The different effects of perifosine and LY294002 on cell survival remain unexplained; however, they may relate to the differential effects of targeting PI3K versus AKT or to off-target effects. Furthermore, this suggests that perifosine may show increased or broader spectrum activity in human tumors than PI3K inhibitors.
In conclusion, perifosine has significant antitumor activity that is attributable, at least in part, to AKT inhibition. Perifosine, particularly at lower concentrations, may have greater inhibitory effects on tumors with PI3K/AKT-activating genomic abnormalities. As a proof of principle, we have established the potential practical utility of novel RPPA for quantifying the pharmacodynamic effects of targeted therapies in vivo. AKTp473 and S6p240-244 are potentially the most useful pharmacodynamic markers of early efficacy and of biologically optimal perifosine dosing in human trials. Because of a very strong correlation between proportional modulation of these PI3K pathway biomarkers and quantified perifosine efficacy, measurement of such pharmacodynamic biomarkers with RPPA may maximize our ability to select patients who are responding to perifosine and guide optimal perifosine dosing. This approach requires investigation in clinical trials. Furthermore, PI3K pathway activation status may allow baseline selection of patients most likely to respond to perifosine alone or in combination with other therapies.
|
Footnotes |
|---|
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Y. Lu and B.T. Hennessy contributed equally as first authors of this manuscript.
Received 4/ 2/07; revised 6/22/07; accepted 7/18/07.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. L. Dillon, R. Marcotte, B. T. Hennessy, J. R. Woodgett, G. B. Mills, and W. J. Muller Akt1 and Akt2 Play Distinct Roles in the Initiation and Metastatic Phases of Mammary Tumor Progression Cancer Res., June 15, 2009; 69(12): 5057 - 5064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Neeley, S. M. Kornblau, K. R. Coombes, and K. A. Baggerly Variable slope normalization of reverse phase protein arrays Bioinformatics, June 1, 2009; 25(11): 1384 - 1389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Agarwal, A.-M. Gonzalez-Angulo, S. Myhre, M. Carey, J.-S. Lee, J. Overgaard, J. Alsner, K. Stemke-Hale, A. Lluch, R. M. Neve, et al. Integrative Analysis of Cyclin Protein Levels Identifies Cyclin B1 as a Classifier and Predictor of Outcomes in Breast Cancer Clin. Cancer Res., June 1, 2009; 15(11): 3654 - 3662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Niedermeier, B. T. Hennessy, Z. A. Knight, M. Henneberg, J. Hu, A. V. Kurtova, W. G. Wierda, M. J. Keating, K. M. Shokat, and J. A. Burger Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach Blood, May 28, 2009; 113(22): 5549 - 5557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. T. Hennessy, A.-M. Gonzalez-Angulo, K. Stemke-Hale, M. Z. Gilcrease, S. Krishnamurthy, J.-S. Lee, J. Fridlyand, A. Sahin, R. Agarwal, C. Joy, et al. Characterization of a Naturally Occurring Breast Cancer Subset Enriched in Epithelial-to-Mesenchymal Transition and Stem Cell Characteristics Cancer Res., May 15, 2009; 69(10): 4116 - 4124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, N. Guggisberg, M. Jorda, A. Gonzalez-Angulo, B. Hennessy, G. B. Mills, C.-K. Tan, and J. M. Slingerland Combined Src and Aromatase Inhibition Impairs Human Breast Cancer Growth In vivo and Bypass Pathways Are Activated in AZD0530-Resistant Tumors Clin. Cancer Res., May 15, 2009; 15(10): 3396 - 3405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Aziz, M. Davies, E. Pick, C. Zito, L. Jilaveanu, R. L. Camp, D. L. Rimm, Y. Kluger, and H. M. Kluger Phosphatidylinositol-3-Kinase as a Therapeutic Target in Melanoma Clin. Cancer Res., May 1, 2009; 15(9): 3029 - 3036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Gonzalez-Angulo, K. Stemke-Hale, S. L. Palla, M. Carey, R. Agarwal, F. Meric-Berstam, T. A. Traina, C. Hudis, G. N. Hortobagyi, W. L. Gerald, et al. Androgen Receptor Levels and Association with PIK3CA Mutations and Prognosis in Breast Cancer Clin. Cancer Res., April 1, 2009; 15(7): 2472 - 2478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. S. Boyd, Q. J. Wu, C. O'Brien, J. Spoerke, H. Savage, P. J. Fielder, L. Amler, Y. Yan, and M. R. Lackner Proteomic analysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays Mol. Cancer Ther., December 1, 2008; 7(12): 3695 - 3706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Stemke-Hale, A. M. Gonzalez-Angulo, A. Lluch, R. M. Neve, W.-L. Kuo, M. Davies, M. Carey, Z. Hu, Y. Guan, A. Sahin, et al. An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer Cancer Res., August 1, 2008; 68(15): 6084 - 6091. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
