The phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway is often constitutively activated in various human cancers, providing validated targets for cancer therapy. Among a series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs designed and synthesized based on PI3K target, 4-(2-(dimethylamino)vinyl)-2-(3-hydroxyphenyl)-6-morpholinopyrimidine-5-carbonitrile (WJD008) was selected for further pharmacological characterization because of its potent activity against PI3K signaling. WJD008 inhibited kinase activity of PI3Kα and mTOR with less activity against PIKK family members. In cellular settings, WJD008 abrogated insulin-like growth factor-I-activated PI3K-Akt-mTOR signaling cascade and blocked the membrane translocation of a pleckstrin homology domain containing enhanced green fluorescent protein-general receptor for phosphoinositides, isoform 1-pleckstrin homology fusion protein, suggesting down-regulation of phosphatidylinositol (3,4,5)-trisphosphate output induced by WJD008 resulted in inactivation of PI3K pathway. Consequently, WJD008 arrested cells in G1 phase without induction of apoptosis. Furthermore, WJD008 reversed the hyperactivation of the PI3K pathway caused by the oncogenic mutation of p110α H1047R and suppressed the proliferation and clonogenesis of transformed RK3E cells harboring this mutant. WJD008 was superior to the pan-PI3K inhibitor wortmannin against proliferation of a panel of cancer cells independently of their status of PI3K pathway or tissue originations. In summary, WJD008 is a potent dual PI3K/mTOR modulator with antiproliferative and anticlonogenic activity in tumor cells and transformed cells with PIK3CA mutant, which provides new clues for the design and development of this chemical scaffold as an anticancer drug.
Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases that are responsible for the generation of 3-phosphorylated inositides and play central roles in diverse cellular responses, such as proliferation, survival, mobility, and metabolism (Vivanco and Sawyers, 2002). The PI3K pathway is frequently deregulated in a wide range of tumor types as a result of genetic and epigenetic aberrations. Except for hyperactivation of the upstream growth factor signaling (Douglas et al., 2006; Hollestelle et al., 2007) and mutation in or loss of PTEN (Harima et al., 2001; Byun et al., 2003), oncogenic mutations in PIK3CA itself provide further evidence of the role of PI3K in tumorigenesis and validation of PI3K as an attractive target for cancer therapy (Samuels et al., 2004). Mutations in PIK3CA, which encodes the p110α catalytic subunit, have been reported to occur at high frequency in several human cancers (27% glioblastomas, 25% gastric cancers, 8% breast cancers, and 4% lung cancers). It is interesting to note that more than 80% of the mutations in PIK3CA cluster in two small conserved regions, i.e., E545K in helical domain and H1047R (HR) in kinase domain (Samuels et al., 2004). Despite the suggested different functions (Zhao and Vogt, 2008; Chaussade et al., 2009), mutations at these two PIK3CA hot spots lead to full activation of the catalytic subunit (Samuels et al., 2004) and transformation (Zhao et al., 2005; Bader et al., 2006). Genetic ablation of the PI3K mutant allele in the colorectal cancer cell lines HCT116 and DLD-1 resulted in the reduction of their oncogenic properties (Samuels et al., 2005). Therefore, discovery and development of the anticancer drugs targeting PI3K, especially mutated PI3K, has been an attractive strategy in tumor chemotherapy.
With multiple efforts under way in academia and industry to develop clinically relevant inhibitors of this signaling pathway, several pan-specific or isoform-specific PI3K inhibitors have been identified and developed as potential molecularly targeted anticancer therapies. The first set of PI3K inhibitors has entered clinical trials. Of them, 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile (NVP-BEZ235) (Maira et al., 2008), N-(3-(N-(3-(3,5-dimethoxyphenylamino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (XL765), and 4-(17-carboxy-14-(carboxymethyl)-8-(3-guanidinopropyl)-18-hydroxy-3,6,9,12,15-pentaoxo-2-oxa-7,10,13,16-tetraazaoctadecyl)-4-(4-oxo-8-phenyl-4H-chromen-2-yl)morpholin-4-ium (SF1126) (Garlich et al., 2008; for review, see Brachmann et al., 2009) all are reported active against phosphatidylinositol-3 kinase related kinases (PIKKs), including mTOR, which is also the downstream effector of PI3K and participates in the negative feedback loop targeting an upstream component of the signaling chain (Wullschleger et al., 2006).
Despite the effort made in the past decade, the identified PI3K inhibitors are far from enough to meet clinical needs as well as for probing the complicated PI3K/mTOR pathway. To this end, we designed and synthesized a series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs in an aim of discovering new modulators of the PI3K-Akt-mTOR pathway using a structure-based design approach. Of these analogs, 4-(2-(dimethylamino)vinyl)-2-(3-hydroxyphenyl)-6-morpholinopyrimidine-5-carbonitrile (WJD008) distinguished itself as a potent dual PI3K/mTOR inhibitor with potential against PIK3CA mutant-transformed cells. Highlighted by the methodology of target-based synthesis, the discovery of WJD008 provides a promising novel chemical template for discovery and development of PI3K inhibitors, especially those targeting mutated PI3K.
Materials and Methods
A series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs were synthesized as potential PI3K inhibitors based on the structures reported previously (Hayakawa et al., 2006). Wortmannin and camptothecin (Furuta et al., 2003) was purchased from Sigma-Aldrich (St. Louis, MO). All compounds were prepared at 10 or 20 mM in 100% DMSO, and aliquots were stored at −20°C. All compounds were diluted to the desired concentrations immediately before each experiment. Final DMSO concentration was kept below 0.2% in control and compound-treated cells.
Cell Lines and Cell Culture.
The transformed rat kidney epithelial cell lines RK3E/NT and RK3E/p110α(H1047R) were kindly provided by Dr. Peter K. Vogt (The Scripps Research Institute, La Jolla, CA). The Chinese hamster ovary cell line CHO-K1, human breast cancer cell lines MDA-MB-468 and BT-474, breast cell line MCF-10A, squamous carcinoma cell line HeLa, colon cancer cell line SW1116, prostate cancer cell lines PC-3 and DU-145, and glioblastoma cell lines M059J and M059K were obtained from the American Type Culture Collection (Manassas, VA). The human breast cancer cell line MCF-7, ovarian cancer cell line SKOV-3, colon cancer cell line HCT-116, and stomach cancer cell lines MKN28 and MKN45 were obtained from the Japanese Foundation of Cancer Research (Tokyo, Japan). Rhabdomyosarcoma cell line Rh30 was a gift from the St. Jude Children's Research Hospital (Memphis, TN). Human lung adenocarcinoma cell line A549 was from the National Cancer Institute (Bethesda, MD). Human hepatocellular carcinoma cell lines SMMC-7721 and Zip-177 were gifts from the Second Military Medical School (Shanghai, China).
MKN28, MKN45, HCT-116, SKOV-3, and MCF-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 10% FBS (Invitrogen), penicillin (100 IU/ml), and streptomycin (100 μg/ml), and additional 3 μg/ml puromycin was included in the medium for RK3E. A549 and PC-3 cells were maintained in Ham's F-12 medium (Invitrogen) with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml). CHO-K1 cells transfected with pEGFP-C1-Grp1-PH were selected in the presence of 700 ng/ml G418 (Calbiochem, San Diego, CA) and maintained in the same medium containing 350 ng/ml G418. SMMC-7721, Zip-177, M059J, M059K, DU-145, Rh30, SW1116, HeLa, and MDA-MB-468 cells were maintained in RPMI medium 1640 (Invitrogen) supplemented with 10% FBS, l-glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 μg/ml), and HEPES (10 mM; pH 7.4). BT-474 cells were incubated in the aforementioned medium supplemented with 1 mM sodium pyruvate. All cells were cultured in humidified atmosphere of 95% air and 5% CO2 at 37°C.
In Vitro PI3K Kinase Assay.
The catalytic activity of PI3K was measured by Kinase-Glo Assay as described previously (Serra et al., 2008). In brief, the kinase reactions were preformed in 96-well white plates (Greiner Bio-One, Tokyo, Japan). Each well was loaded with 1 μl of test compounds (in 10% DMSO) and 25 μl of reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 3 mM MgCl2, 1 mM 1,4-dithiothreitol, and 0.05% CHAPS) containing 10 μg/ml substrate d-myo-phosphatidylinositol 4,5-bisphosphate (Echelon Biosciences Inc., Salt Lake City, UT) and 100 μM PI3K (p110α/p85α; Millipore, Billerica, MA). The reaction was initiated by the addition of 25 μl of 1 μM ATP prepared in the reaction buffer and terminated by the addition of 50 μl of Kinase-Glo buffer (Promega, Madison, WI) after incubation for 60 min. The plates were read in a Novostar microplate reader (BMG Labtech GmbH, Offenburg, Germany) for luminescence detection.
ELISA-Based mTOR Kinase Assay.
A glutathione transferase-tagged truncated human TOR (amino acids 1360–2549) was expressed by using the Bac-to-Bac baculovirus expression system (Invitrogen) and purified in glutathione-Sepharose 4B (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The kinase activity of the purified truncated mTOR kinase was determined by an ELISA-based assay. The kinase reaction was performed in 96-well plates precoated with 10 μg/ml, p70S6 kinase 1 (p70S6K1; amino acids 332–415) as a substrate. Ninety microliters of kinase assay buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM 1,4-dithiothreitol, 0.25 μM microcystin-LR, and 100 μg/ml bovine serum albumin) containing 5 μM ATP was added to each well. The reaction was initiated by the addition of 1.8 μg/ml glutathione transferase-tagged truncated human TOR. After incubation for 2 h at 37°C, the plate was washed three times with phosphate-buffered saline containing 0.1% Tween 20 (PBST). Next, 100 μl of antibody against phosphorylated p70S6K1 at Thr389 (1A5; Cell Signaling Technology, Danvers, MA) in a 1:4000 dilution was added into each well. After 1-h incubation at room temperature, the plate was washed three times. Then, mouse anti-mouse IgG horseradish peroxidase (100 μl; 1:2000 dilution) diluted in PBST containing 5 mg/ml bovine serum albumin was added. The plate was incubated at room temperature for 1 h and then with a 100-μl solution of 0.1% H2O2 and 2 mg/ml o-phenylenediamine in 0.1 M citrate buffer, pH 5.5, after secondary antibody was washed out. The reaction was terminated by adding 100 μl of 2 M H2SO4, and an OD value at 490 nm was measured using a multiwell spectrophotometer (VersaMax; Molecular Devices, Sunnyvale, CA).
Western Blot Analysis.
Rh30 and PC-3 cells grown to 50% confluence in 12-well plates continued to be incubated in serum-free medium for 24 h. After exposure to tested compounds for 1 h, cells were stimulated with 50 ng/ml IGF-I for 10 min. Cells were then collected and subjected to Western blot analysis as described previously (Zhang et al., 2005) with the antibodies against phosphorylated phosphatidylinositol 3-phosphate-dependent kinase 1 (PDK1; Ser241), phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-mTOR (Ser2448), phospho-p70S6K (Thr389) (Cell Signaling Technology), and phospho-eukaryotic initiation factor 4E-binding protein 1 (4E-BP1; Thr70) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Membrane-detected phosphorylated proteins were stripped with Re-Blot Plus Mild solution (Millipore Bioscience Research Reagents) and reblotted with antibodies against corresponding total PDK1, Akt, and mTOR (Cell Signaling Technology) and S6K and 4E-BP1 (Abcam plc, Cambridge, UK). To detect γ-H2AX (a member of the histone H2A family), Rh30 cells grown in 12-well plates were pretreated with WJD008 or wortmannin for 0.5 h and then cotreated with 1 μM camptothecin for 2 h. Cells were collected and subjected to Western blot analysis using antibodies against phospho-H2AX (Ser139) (Cell Signaling Technology). GAPDH (KangChen Bio-tech Inc., Shanghai, China) were used as loading control.
Phosphorylated Akt (Ser473) ELISA Assay.
Rh30 cells were grown to 50% confluence in 12-well plates and continued to be incubated in serum-free medium for 24 h. After exposed to test compounds for 1 h, cells were stimulated with 50 ng/ml IGF-I for 10 min at 37°C. In parallel, ELISA test plates (Corning Life Sciences, Lowell, MA) were prepared with the anti-Akt coating antibody (Santa Cruz Biotechnology, Inc.). At the end of the treatment, cells were lysed and the cell lysate (50 μl) was transferred into ELISA plates and incubated for 3 h at 4°C. After three washes, 50 μl of diluted antibody against phosphorylated Akt at Ser473 (Cell Signaling Technology) was added, and plates were incubated overnight at 4°C. After the plate was washed with PBST for three times, horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) was added and incubated for 2 h, and the immune complexes were assayed with 100 μl of substrate (tetramethylbenzidine). The OD value at 450 nm was read on a multiwell spectrophotometer (VersaMax), and the IC50 value was determined with the sigmoid dose-response curves by Prism 4 software (GraphPad Software Inc., San Diego, CA).
Grp1-PH Translocation Assay.
The CHO-K1 cells were transfected with pEGFP-C1-Grp1-PH (a gift from Dr. Tamas Balla, National Institutes of Health, Bethesda, MD) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. CHO-K1 cells stably expressing pEGFP-C1-Grp1-PH were selected in the presence of 700 ng/ml G418 and maintained in the same medium containing 350 ng/ml G418. Cells (5 × 103) were seeded in a 96-well black plate (Greiner Bio-One). The next day, cells were incubated in serum-free media for 24 h followed by treatment of WJD008 or wortmannin for 1 h. After addition of IGF-I (250 ng/ml) for 10 min to trigger PI3K activation, the cells were washed with ice-cold phosphate-buffered saline and fixed with 4% paraformaldehyde for 10 min at room temperature. The fluorescent images were captured with a fluorescence microscope (BX51; Olympus, Tokyo, Japan).
Cell Cycle Analysis.
Exponentially growing Rh30 were seeded in a six-well plate at a density of 2 × 105 (Corning Life Sciences). The next day, cells were treated with WJD008 at the indicated concentrations for 72 h. Cells were collected for cell number counting (Z1 Coulter particle counter; Beckman Coulter, Fullerton, CA), and the rest of the cells were fixed in ice-cold 70% ethanol. DNA content was measured with an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), and cell cycle distribution was analyzed by using ModFit LT software (Verity Software House, Topsham, ME). In total, 10,000 cells were analyzed from each sample.
Cell proliferation was evaluated by sulforhodamine B (Sigma-Aldrich) staining assay (Skehan et al., 1990). In brief, cells seeded in 96-well plates were treated with test compounds for 72 h. The medium was removed, and the cells adhered to the plate were then fixed with 10% trichloroacetic acid and stained with sulforhodamine B. After three washings using 1% acetum, sulforhodamine B was dissolved in 100 μl of buffer containing 10 mM Tris-base, and the OD value was measured at 520 nm with a multiwell spectrophotometer (VersaMax). The inhibitory rate on cell proliferation was calculated by using the formula (OD520 nmcontrol cells − OD520 nmtreated cells)/OD520 nmcontrol cells × 100%. The average IC50 values were from at least three independent tests.
Colony Formation Assay.
RK3E/NT and RK3E/p110α(H1047R) (single-cell suspension) cells were seeded in six-well plates at a density of 200 cells per well. The next day, cells were treated with test compounds at indicated concentrations for 1 week. Adherent cells were stained with 0.1% violet after fixation of methanol, and visible colonies (≥50 cells) were counted.
Data are presented as mean ± S.D. from at least three independent experiments, and differences were considered significant at P < 0.05 as determined by Student's t test.
Structures of WJD Series Compounds and Their Effects on PI3K-Mediated Signaling.
Among the PI3K inhibitors recently reported in the literature (Marone et al., 2008; Yap et al., 2008), tricyclic pyridofuropyrimidine PI-103 strongly inhibits all four class I isoforms and mTOR at nanomolar range (Hayakawa et al., 2007; Raynaud et al., 2007; Fig. 1A). Its thienopyrimidine analog 2 is a selective p110α inhibitor that inhibits tumor cell proliferation (Hayakawa et al., 2006). In this study, we chose compound 2 as our lead compound for further optimization because of its good selectivity for p110α. It has been reported that the cyano group and thio atom have similar lipophilicity properties, and they were used as bioisosteres in estrogen receptor ligand design successfully (Meyers et al., 2001; Schopfer et al., 2002). Here, we simplified the thienopyrimidine scaffold into a 5-cyanopyrimidine ring and kept the morpholine and 3-substituted-phenyl ring that bound to the ATP binding pocket (Knight et al., 2006) of PI3K. We then synthesized a series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs 3, named WJD, as potential PI3K inhibitors (Fig. 1A).
To determine whether WJD series compounds have any effect on PI3K-mediated signaling in Rh30 cells, the effect of these compounds on IGF-I-induced Akt phosphorylation was evaluated. As shown in Fig. 1B, WJD008 distinguished itself among the WJD series compounds with potent activity in inhibiting phosphorylation of Akt at Ser473 upon the stimulation of IGF-I. To further confirm this effect, ELISA assay was used to measure phosphorylation Akt triggered by IGF-I in the presence of WJD008, and we found that WJD008 significantly suppressed this process, with an IC50 value of 0.32 ± 0.02 μM (Fig. 1C).
WJD008 Potently Inhibits the Catalytic Activity of PI3Kα and mTOR.
Because WJD008 displayed most potent activity in blocking IGF-I-triggered Akt activation among this series of compounds, we examined whether this effect is caused by its inhibition on the catalytic activity of PI3K by using an ATP depletion (Kinase-Glo) assay (Koresawa and Okabe, 2004). As shown in Fig. 2A, WJD008 dose-dependently inhibited the kinase activity of PI3Kα, with an IC50 value of 1.72 ± 0.66 nM. Considering the important role of mTOR in the PI3K-Akt-mTOR pathway and its catalytic domain being highly homologous to PI3K, we detected whether WJD008 inhibits the activity of mTOR by an ELISA-based assay. WJD008 inhibited the kinase activity of mTOR, with an IC50 value of 3.42 μM (Fig. 2B). Further study indicated that that increasing concentrations of ATP reduced the inhibitory activity of WJ008 against mTOR (data not shown), suggesting that WJD008 is an ATP-competitive inhibitor. It should be noted that the concentration of ATP used in mTOR assay is much higher than that used in the PI3K assay, which might result in less potency of WJD008 against mTOR. Therefore, WJD008 is probably a dual PI3K/mTOR inhibitor.
In addition to mTOR, DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) are three serine/threonine protein kinases sharing highly homologues catalytic domain with PI3K (Jackson, 1997), and they are therefore assigned as members of the PIKK family. Several PI3K inhibitors, such as LY294002 and wortmannin, have been reported to inhibit these PIKK kinases (Izzard et al., 1999). We next determined the effect of WJD008 on the activity of DNA-PK, ATM, and ATR by measuring the level of phosphorylated H2AX (γ-H2AX) in DNA-damaged cells. DNA-PK, ATM, and ATR are involved in the DNA repair machinery, and their activation is required for the phosphorylation of H2AX at Ser139 upon DNA strand breaks induced by camptothecin (CPT) (Furuta et al., 2003). Therefore, the cellular readouts were used to evaluate the potential activity of WJD008 against these kinases. As shown in Fig. 2C, Rh30 cells displayed a robust increase of γ-H2AX after treatment with CPT (10 μM). Similar to the effect induced by wortmannin, cotreatment with WJD008 abrogated the up-regulation of γ-H2AX at the concentration up to 40 μM, whereas the effect was mild at the concentrations below 8 μM. The result indicates that high concentrations of WJD008 also inhibit the kinase activity of ATM, ATR, and DNA-PK in Rh30 cells.
WJD008 Blocks the PI3K-Akt-mTOR Signaling in Tumor Cells.
Because WJD008 had been demonstrated to potently inhibit the kinase activity of PI3K and mTOR and abrogate activation of Akt, we next determined in detail how WJD008 could abrogate the PI3K/mTOR-mediated signaling pathway stimulated by growth factors in tumor cells. Serum-deprived Rh30 cells with an intrinsically hyperactivated PI3K-Akt-mTOR pathway were treated with various concentrations of WJD008 followed by the stimulation of IGF-I. As illustrated in Fig. 3A, left, the level of phosphorylated PDK1 was not affected by the stimulation of IGF-I or preincubation of WJD008, which is similar with the previous report that phosphorylation of PDK1 at Ser241 was insensitive to serum deprivation or treatment with wortmannin (Pullen et al., 1998; Casamayor et al., 1999). In contrast, WJD008 inhibited phosphorylation of Akt at Thr308 and Ser473 in a dose-dependent manner. Moreover, the phosphorylation levels of mTOR at Ser2448 and its two best-characterized downstream effective molecules, p70S6K and 4E-BP1, all reduced in the presence of increasing concentrations of WJD008. Meanwhile, the total protein level of the aforementioned downstream components in the PI3K pathway remained unchanged, suggesting that down-regulation of phosphorylated proteins by WJD008 is not due to loss of total proteins. The inhibitory activity of WJD008 on the PI3K-Akt-mTOR signaling was also confirmed using prostate cancer PC-3 cells (Fig. 3A, right).
To further verify that down-regulated PI3K signaling is caused by inhibition of PI3K activity in tumor cells, the effect of WJD008 on the redistribution of Grp1-PH domain after stimulation of IGF-I was determined in CHO-K1 cells expressing Grp1-PH fused to GFP. Grp1-PH domain is shown a specific binding affinity to PtdIns(3,4,5)P3 rather than phosphatidylinositol 4,5-bisphosphate in cells and aggregates around the cell membrane responding to the recruitment of PtdIns(3,4,5)P3 (Gray et al., 1999). As shown in Fig. 3B, activation of PI3K and production of PtdIns(3,4,5)P3 resulted in the translocation of GFP-Grp1-PH to cellular membrane, which was indicated as the fluorescent foci near the membrane. However, preincubation of 10 μM WJD008 for 1 h before IGF-I stimulation prevented the translocation of GFP-Grp1-PH fusion protein to the plasma membrane, which was consistent with its inhibition of PI3K signaling in Rh30 cells. A similar effect was observed when cells were treated with wortmannin but not with rapamycin (data not shown). Thus, it appears that the inhibition of the activity of PI3K by WJD008 results in down-regulation of PtdIns(3,4,5)P3 and further blocks PI3K-Akt-mTOR signaling.
WJD008 Induces G1-Phase Arrest without Apoptosis in Tumor Cells.
It has been reported that some PI3K inhibitors induces apoptosis, although most of them, such as PI-103 and ZSTK474, induce G1-phase arrest instead of apoptosis (Yaguchi et al., 2006; Raynaud et al., 2007; Maira et al., 2008). Hence, we examined the effect of WJD008 on the cell cycle distribution of Rh30 cells. As presented in Fig. 4A, WJD008 arrested cells in G1 phase in a dose-dependent manner. Cell population in G1 phase increased from 43.60% (DMSO control) to 55.09 and 63.74% upon treatment of the cells with 5 or 10 μM WJD008, respectively. Similarly, cell population in G1 phase increased in the presence of wortmannin but to a less extent. To exclude WJD008-induced cytotoxicity, we counted the cell number in parallel while performing fluorescence-activated cell sorting. As shown in Fig. 4B, fold change in cell number relative to that seeded was plotted. This is no loss in cell number after treatment with WJD008 up to 10 μM, and more than 95% of cells were viable observed by trypan blue staining (data not show). However, an obvious sub-G1 peak failed to appear after WJD008 treatment even at 10 μM for 72 h, indicating that WJD008 is unable to induce apoptosis in Rh30 cells and its ability to induce G1-phase arrest may at least partially result in inhibition of proliferation of tumor cells.
WJD008 Inhibits p110α Exhibiting Oncogenic H1047R Mutation in Cells.
H1047R is one of the mutation hot spots in the PIK3CA, which results in hyperactivation of PI3K and transformation. To evaluate the potential ability of WJD008 against cells harboring mutated p110α H1047R, we examined the antiproliferative activity of WJD008 in transformed RK3E cells (Fu et al., 2005) stably expressing p110α H1047R by retroviral infection. The oncogenic mutation H1047R induced constitutive phosphorylation of Akt even when cells were deprived of serum (Fig. 5A), which was also observed in the breast cancer cells exogenously expressing the mutant (Serra et al., 2008). When treated with WJD008 for 1 h followed by IGF-I stimulation, the phosphorylation level of Akt was more vulnerable to WJD008 in H1047R mutant cells than that in nontransfected (NT) cells (Fig. 5A), suggesting that WJD008 was potent against p110α H1047R.
To determine whether blockage of PI3K signaling result in the inhibition of proliferation of RK3E cells with mutant PI3K, an SRB assay was used to measure the effect of WJD008 on the proliferation of these cells. Although WJD008 and wortmannin displayed similar potency against RK3E/NT cells, with IC50 values of 16.29 ± 7.72 or 25.79 ± 4.35 μM, respectively, there is a sharp divergence in their activities against H1047R mutant cells (Fig. 5B). WJD008 was active to inhibit the proliferation of H1047R mutant cells, with an IC50 value of 23.06 ± 9.52 μM, whereas wortmannin had little effect on the proliferation of this cell line and the inhibitory rate was approximately 25% even as the concentration reached 40 μM.
The antiproliferative activity of WJD008 was also detected in H1047R mutant cells with colony formation assays. As expected, the mutant transfected cells are more clonogenic than NT cells (Fig. 5C). The colonies of mutant H1047R cells formed decreased in a concentration-dependent manner in the presence of WJD008, with an IC50 of 12.91 μM, and almost no colony could be detected upon treatment with 40 μM WJD008 (with inhibition rate of 98.77%). Wortmannin was much less potent against colony formation of mutant H1047R cells compared with WJD008 and induced an inhibition by 66.83% at 40 μM (P < 0.01; Fig. 4, C and D). Collectively, these findings substantiate that WJD008 is capable of repressing the proliferation and clonogenesis potential of both RK3E/NT and RK3E/p110α(H1047R) cells.
WJD008 Possesses Potent Antiproliferative Activity in Cancer Cells.
The antiproliferative activity of WJD008 was measured with an SRB assay against a panel of human tumor cells, including liver cancer, lung cancer, stomach cancer, glioblastoma, prostate cancer, rhabdomyosarcoma, colon cancer, ovarian cancer, squamous carcinoma, and breast cancer (Fig. 6). The average IC50 values of WJD008 and wortmannin against the 17 human tumor cell lines tested were 16.45 and 23.73 μM (P < 0.05), respectively. Although WJD008 and wortmannin shared similar profile of antiproliferative activity in some cell lines, such as Zip-177, A549, and Rh30, they did display distinctive activities in certain cell types. To gain insight into the antiproliferative properties of WJD008 in the tumor cells with aberrant PI3K signaling, the cell lines were classified into five groups according to their PI3K status: PTEN-negative (PC-3 and MDA-MB-468), upstream deregulation of growth factor (Rh30), H1047R mutant (HCT-116 and SKOV-3), E545K mutant (MCF-7), and other cell lines without any reported deregulation of PI3K pathway so far (Ikediobi et al., 2006; Marty et al., 2008). WJD008 is active in cells exhibiting deregulation of the PI3K pathway by various means (Fig. 6). Of particular note, WJD008 is more potent than wortmannin against MCF-7 cells with the oncogenic E545K mutation in PI3K (Fig. 6). Together with the fact that WJD008 is active in H1047R mutant cells (Figs. 5 and 6), oncogenic mutations in PI3K known at present are unlikely to confer resistance to this inhibitor.
WJD008, among a series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs, stood out for its potent activity against PI3K/mTOR axis. In this study, we identified the cellular target of WJD008 and characterized its mechanism of action. Although WJD008 was designed based on a p110α-specific inhibitor, WJD008 potently inhibited the kinase activity of both p110α and mTOR, with less activity against PIKK family members. In cellular context, WJD008 inhibited several key components in PI3K-Akt-mTOR pathway in cascade, such as PtdIns(3,4,5)P3, Akt, and mTOR, as well as its downstream effectors p70S6K and 4E-BP1, which resulted in the G1-phase arrest. WJD008 was further noted for its antiproliferative activity against a panel of tumor cells and transformed cells expressing PIK3CA H1047R mutant.
It has been reported that mTOR allosteric inhibitor rapamycin analogs are cytostatic in tumor cells (Easton and Houghton, 2004; Takeuchi et al., 2005). However, it was somewhat disappointing when these inhibitors were tested clinically, in part because of retrograde hyperphosphorylation of Akt (Margolin et al., 2005). The recent discovery of the dual PI3K/mTOR inhibitors, such as NVP-BEZ235 and PI-103 (Raynaud et al., 2007; Maira et al., 2008; Serra et al., 2008), has shed the light on vertical blockade of multiple molecules in the PI3K pathway to overcome the feedback loops and achieve the effective cytostasis in cancers exhibiting deregulation of this pathway. In this study, as a novel dual PI3K/mTOR inhibitor, WJD008 well translated its potent activity into cells against the PI3K-Akt-mTOR axis. WJD008 prevented PtdIns(3,4,5)P3 production and Akt and mTOR activation as well as its downstream effectors p70S6K and 4E-BP1. It is noteworthy that WJD008 abrogated the phosphorylation of Akt at Ser473 in spite of its activity in inhibiting the catalytic activity of TOR complex 1, which might be caused by simultaneous inhibition of PI3K and possibly TOR complex 2. Thus, WJD008 functions on several levels to block both the growth factor- and nutrient-sensing pathways and the inhibitory efficacy of PI3K and mTOR was augmented mutually. The characterization of WJD008 so far supports the emerging consensus of inhibition of PI3K and mTOR in combination as a mechanistic rationale for the cancer therapeutic options.
WJD008 impeded the PI3K signaling in cells with a hyperactivated pathway either because of enhanced growth factor signaling (demonstrated as Rh30 cells) (Minniti et al., 1995) or loss of PTEN (demonstrated as PC-3 cells). In addition, WJD008 not only displayed potent antiproliferative activity in cells that harbor PIK3CA mutants (HCT-116, SKOV-3, and MCF-7) or PTEN-negative cells (PC-3 and MDA-MB-468) but also was active in cells with no reported deregulated PI3K pathway so far (Ikediobi et al., 2006; Marty et al., 2008). Thus, WJD008 blocked proliferation in all the cancer cell lines tested irrespective of their PI3K status. This notion was further supported by the experiments using isogenic PIK3CA wild-type or H1047R mutant RK3E cells. WJD008 blocked the PI3K signaling pathway and proliferation in both wild-type or H1047R mutant RK3E cells. Differentiated from WJD008, wortmannin displayed less potency in RK3E/p110α(H1047R) cells compared with WJD008, even though it possessed similar activity with WJD008 in RK3E/NT cells. Because increased PI3K signaling in tumor cells may result from vertical (such as hyperactivated receptor signaling), horizontal (such as PTEN loss), or presence of activating PI3K mutation, WJD008 was inferred to lead to antiproliferative outcome regardless of activation modes of PI3K and target both p110α wild-type and H1047R mutant. This is of great importance because it indicates WJD008 may have the potential for wider applicability in cancer therapy. In contrast, because WJD008 possessed a wide spectrum of cytostatic activity, its potential side effects should be carefully monitored.
In summary, WJD008 functions as a dual PI3K/mTOR inhibitor and inhibits activated signaling in both wild type and mutated p110α in cellular models. With research continuing to provide insight into the structure-based machinery of these kinds of compounds, we look forward to discovering PI3K inhibitors with more potent efficacy in cancer therapy.
We thank Peter K. Vogt for the RK3E/NT and RK3E/p110α(H1047R) cells and Tamas Balla for the pEGFP-C1-Grp1-PH plasmid.
This work was supported by the National Science and Technology Major Project Key New Drug Creation and Manufacturing Program [Grant 2009ZX09301-001]; the Knowledge Innovation Program of Chinese Academy of Sciences [Grant KSCX2-YW-R-25]; the National Natural Science Foundation of China [Grants 30721005, 90713034]; and the Science and Technology Commission of Shanghai Municipality Pujiang Talent Program (08PJ14114) [Grants 07dz05906, 09JC1416600].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- phosphatidylinositol 3-kinase
- phosphatase and tensin homolog deleted from chromosome 10
- phosphatidylinositol 3-kinase-related protein kinase
- mammalian target of rapamycin
- protein kinase B
- dimethyl sulfoxide
- Chinese hamster ovary
- fetal bovine serum
- enhanced green fluorescent protein
- general receptor for phosphoinositides, isoform 1
- pleckstrin homology
- enzyme-linked immunosorbent assay
- target of rapamycin
- phosphate-buffered saline containing 0.1% Tween 20
- optical density
- insulin-like growth factor
- glyceraldehyde-3-phosphate dehydrogenase
- sulforhodamin B
- DNA-dependent protein kinase
- phosphatidylinositol 3-phosphate-dependent kinase 1
- ataxia telangiectasia mutated
- ataxia telangiectasia mutated- and Rad3-related
- eukaryotic translation initiation factor 4E binding protein 1
- the 70-kDa ribosomal S6 kinase
- green fluorescent protein
- phosphatidylinositol (3,4,5)-trisphosphate
- Received March 22, 2010.
- Accepted June 2, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics