Clinical Cancer Research The Science of Cancer Health Disparities Infection and Cancer: Biology, Therapeutics, and Prevention
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
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, H.-Y.
Right arrow Articles by Hong, W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, H.-Y.
Right arrow Articles by Hong, W. K.
Clinical Cancer Research Vol. 11, 6065-6074, August 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Response of Non–Small Cell Lung Cancer Cells to the Inhibitors of Phosphatidylinositol 3-Kinase/Akt- and MAPK Kinase 4/c-Jun NH2-Terminal Kinase Pathways: An Effective Therapeutic Strategy for Lung Cancer

Ho-Young Lee1, Seung-Hyun Oh1, Young-Ah Suh2, Jin Hyen Baek4, Vali Papadimitrakopoulou1, Suyun Huang3 and Waun Ki Hong1

Authors' Affiliations: Departments of 1 Thoracic/Head and Neck Medical Oncology, 2 Molecular Genetics, and 3 Neurosurgery-Research, University of Texas M.D. Anderson Cancer Center, Houston, Texas and 4 Institute for Cell Engineering, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

Requests for reprints: Ho-Young Lee, Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Unit 432, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-796-8655; E-mail: hlee{at}mdanderson.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We previously showed that phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways cooperate to promote non–small cell lung cancer (NSCLC) cell proliferation in vitro. This study was designed to explore whether inhibition of these pathways effectively inhibits NSCLC tumor growth in vivo.

Experimental Design: The effects of PI3K/Akt inhibitors {LY294002, adenoviruses expressing dominant-negative mutant of the p85{alpha} adaptor subunit of PI3K (Ad-dnp85{alpha}), dominant-negative Akt [Ad-HA-Akt(KM)], or PTEN (Ad-PTEN)}, MKK4/c-jun NH2-terminal kinase (JNK) inhibitor [SP600215, adenovirus expressing dominant-negative MKK4, Ad-MKK4(KR)], and their combinations on proliferation and apoptosis in NSCLC cells were tested in vitro and in vivo using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a flow cytometry-based terminal deoxynucleotidyl transferase–mediated nick-end labeling assay, Western blot and immunohistochemical analyses, and an NSCLC xenograft tumor model.

Results: Ad-dnp85{alpha} significantly inhibited proliferation of a subset of NSCLC cell lines used in our study. Intratumoral injection of Ad-dnp85{alpha} induced a significant decrease in the growth of H1299 NSCLC xenograft tumors. Concurrent inhibition of the PI3K/Akt and MKK4/JNK pathways showed enhanced antiproliferative effects on H1299 cells in vitro and in vivo by increasing apoptosis.

Conclusions: PI3K/Akt and MKK4/JNK pathways cooperate to stimulate NSCLC cell proliferation by maintaining cell survival, suggesting that simultaneously targeting these two pathways might be an effective therapeutic strategy against NSCLC.

Lung cancer is the leading cause of cancer death in the United States and worldwide, a fact that underscores the need for more effective treatment strategies. Because tumorigenic transformation results from the deregulation of cell cycle control and provides tumor cells with constitutive mitogenic and survival signals, selectively inhibiting growth factor signaling pathways could be an efficient strategy to inhibit tumor progression (1). The phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathway is an attractive target because it is important in the transformation, proliferation, survival, and metastatic potential of a variety of cancer cells, including lung cancer cells (2–6).

Class I PI3K consists of a family of heterodimeric complexes, each composed of a p110 catalytic subunit and an adaptor subunit that exists predominately as a p85 form (7, 8). PI3K phosphorylates the D3 position of phosphatidylinositol on PI(4)P and PI(4,5)P to produce PI(3,4)P2 and PI(3,4,5)P3 (9), which recruit the pleckstrin homology domains of specific intracellular proteins, such as PDK-1 and Akt/PKB (7), to the cytoplasmic membrane. This mechanism is regulated by the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene (10). Akt phosphorylates and inactivates several proapoptotic proteins, including the Bcl-2 family member BAD and caspase-9 (11–13). Akt also phosphorylates proteins that indirectly inhibit apoptosis, such as forkhead transcription factors, I{kappa}B kinase, and mdm-2 (14–16). In addition, Akt affects cell proliferation by regulating the cell cycle machinery; Akt induces the accumulation of cyclin D1 via inactivation of glycogen synthase kinase-3 and expression of the cyclin-dependent kinase inhibitors p27 and p21WAF1 (17–20).

We and others have suggested that the PI3K/Akt signaling pathway is involved in the early stage of lung cancer progression; increases in gene copy number of the PI3K catalytic subunit {alpha} and phosphorylated Akt expression have been observed in premalignant and malignant human bronchial epithelial cells and non–small cell lung cancer (NSCLC) cells (21–26). In addition, inhibition of PI3K/Akt through pharmacologic and genetic approaches induces antiproliferative effects on certain NSCLC cell lines (21–25). We found that inhibition of the PI3K/Akt pathway by overexpression of the dominant-negative mutant form of the p85{alpha} adaptor subunit induced NSCLC cells to undergo apoptosis in vitro, whereas cell cycle arrest in NSCLC cells was induced by overexpression of PTEN. Because the dominant-negative mutant form of p85{alpha} inhibits both Akt and c-jun NH2-terminal kinase (JNK) activation in NSCLC cells and treatment with the PI3K inhibitor LY294002 induced apoptosis in mitogen-activated protein kinase (MAPK) kinase 4 (MKK4)-null cells (23), we hypothesized that the PI3K/Akt- and MKK4/JNK-dependent pathways cooperate to maintain the survival of NSCLC cells. In this study, we undertook a systematic approach to determine whether a dominant-negative mutant form of p85{alpha} inhibits tumor growth and whether inhibition of both PI3K/Akt and MKK4/JNK pathways exhibits enhanced antitumor activities in vivo in mouse xenograft models.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals, cell lines, and reagents. Female nude mice, 4 weeks old, were purchased from Harlan-Sprague-Dawley (Indianapolis, IN). H1299 NSCLC cells and 293 cells were maintained in RPMI 1640 and DMEM supplemented with 10% FCS (Life Technologies, Inc., Gaithersburg, MD), respectively, in a humidified environment with 5% CO2. We purchased insulin-like growth factor (IGF)-I (R&D Systems, Minneapolis, MN), the class I PI3K inhibitor LY294002, JNK inhibitor SP600215 and its companion [(–)SP600215)], MAPK kinase (MEK) inhibitor PD98059, and p38 MAPK inhibitor SB202190 (Calbiochem, La Jolla, CA), recombinant GST-c-Jun and H2B (Santa Cruz Biotechnology, Santa Cruz, CA), and basic protein A-G agarose beads (Santa Cruz Biotechnology). We also purchased rabbit polyclonal antibodies against human phosphorylated Akt (Ser473) and Akt (Cell Signaling Technology, Beverly, MA), rabbit polyclonal anti-Bax and anti-caspase-3 antibodies (PharMingen, San Diego, CA), and rabbit polyclonal anti-Bcl-xL and rabbit polyclonal anti-poly(ADP-ribose) polymerase antibodies (VIC 5, Roche Molecular Biochemicals, Indianapolis, IN). Murine monoclonal anti-PTEN and anti-ß-actin antibodies (Santa Cruz Biotechnology) were used for Western blot analyses. Adenoviral vectors that express the dominant-negative 85{alpha} (Ad-dnp85{alpha}) or PTEN (Ad-PTEN) were previously described (23, 24, 27). Adenoviral vectors expressing full-length human Akt-KM, a kinase-dead, dominant-negative Akt, with a hemagglutinin (HA) tag, in which the lysine (Lys179) in the ATP binding site has been mutated to methionine [Ad-HA-Akt(KM)], or expressing dominant-negative MKK4 in which the lysine (Lys129) in the ATP binding site has been mutated to arginine [Ad-MKK4(KR)] were generated and amplified as previously described (24, 28). The presence of human Akt-KM or MKK4(KR) was confirmed by dideoxy-DNA sequencing and Western blot analysis. Viral titers were determined by plaque assays and spectrophotometric analysis.

Cell proliferation analysis. For cell proliferation assays, NSCLC cell lines were seeded at 1 to 2 x 103 cells per well in 96-well plates. After 24 hours, cells were uninfected or infected in serum-free media with different doses of Ad-dnp85{alpha}, Ad-MKK4(KR), Ad-HA-Akt-KM, Ad-PTEN, or their combinations. Empty virus was used to adjust the total doses of adenoviruses in each infection. After 2 hours, cells were changed to RPMI 1640 supplemented with 10% FCS containing different concentrations of LY294002, SP600215 or its companion [(–)SP600215)], SB202190, PD98059, or their combinations. After 3 days of incubation, cell number was measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described (24). Six replicate wells were used for each analysis, and data from replicate wells are presented as mean values with 95% confidence intervals. At least three independent experiments were done to obtain each result, and the mean was calculated.

Inhibition of tumor growth in vivo. The effects of Ad-dnp85{alpha}, Ad-MKK4(KR), and Ad-HA-Akt(KM) on growth of established s.c. tumor nodules were determined in athymic nude mice in a defined pathogen-free environment as described elsewhere (28). Briefly, 1 day after irradiation with 350 Gy (137Cs source), mice were s.c. injected with H1299 cells (5 x 106) in 100 µL of PBS at a single dorsal site. After tumor volume reached about 100 mm3, 12 groups of five mice were randomized. The six groups were given intratumoral injections of 100 µL of 1x PBS containing 1 x 1010 viral particles of empty virus, Ad-dnp85{alpha}, Ad-MKK4(KR), Ad-HA-Akt(KM), or a combination of Ad-MKK4(KR) and Ad-HA-Akt(KM). The other six groups of five mice were injected with the indicated viruses twice, days 0 and 5 (for Ad-dnp85{alpha} or empty virus) or days 0 and 7 [for empty virus, Ad-MKK4(KR), Ad-HA-Akt(KM), or combined injection of Ad-MKK4(KR) and Ad-HA-Akt(KM)]. Tumor size was measured everyday for 11 days (mice injected with empty virus or Ad-dnp85{alpha}) or 14 days [mice injected with single or combination of empty virus, Ad-MKK4(KR), or Ad-HA-Akt(KM)], when all mice were sacrificed. Tumor volumes were calculated by 0.5 x a x b2, where a and b are the long and short diameters, respectively. Mice with necrotic tumors or tumors of >1.5 cm in diameter were euthanized. Results were expressed as the mean (±SD) tumor volumes (calculated in each group of five mice) relative to the volume of tumors injected with control adenovirus. Immunohistochemical staining for phosphorylated Akt and Akt and Western blot analysis on caspase-3 and ß-actin were done on tumors collected from mice at the end of measurement of tumors injected with indicated viruses twice.

Immunoblotting. For immunoblotting, whole cell lysates were prepared in lysis buffer, as described previously (21). Equivalent amounts of protein were resolved using SDS-PAGE (7.5-12%) and transferred to nitrocellulose membranes. After being blocked in TBS containing 0.05% Tween 20 (TBST) and 5% w/v nonfat powdered milk, the membranes were incubated with primary antibody at the appropriate dilution in TBS with 5% nonfat milk at 4°C for 16 hours. The membranes were then washed several times with TBST and incubated with the appropriate horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. The protein-antibody complexes were detected using enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, IL) according to the manufacturer's recommended protocol.

Immune complex kinase assays. H1299 cells untreated, treated with various concentrations of LY294002 and SP600215, or infected with several doses of empty virus, Ad-PTEN, Ad-Akt(KM), and Ad-MKK4(KR) were incubated in RPMI supplemented with 10% FCS for 1 day. The cells were serum starved for 24 hours, treated with insulin-like growth factor-I (50 ng/mL) for 15 minutes, lysed, and then subjected to the Western blot analysis and immune complex kinase reactions. Total cell extracts (100 µg) were subjected to immunoprecipitation with a JNK1 antibody or Akt antibodies by rotation at 4°C overnight. Protein A-G agarose beads (20 µL) were added, and the solution was incubated at 4°C for 1 hour. The beads were washed thrice with lysis buffer and once with kinase buffer [20 mmol/L HEPES (pH 7.5), 20 mmol/L ß-glycerol phosphate, 10 mmol/L MgCl2, 1 mmol/L DTT, and 50 mmol/L sodium orthovanadate]. Kinase assays were done by incubating the beads with 30 µL kinase buffer to which 20 µmol/L cold ATP, 5 µCi [{gamma}32P]ATP (2,000 cpm/pmol), and 2 µg of GST-c-Jun or H2B as a substrate were added, as previously described (21). The kinase reaction was done at 30°C for 20 minutes. The samples were suspended in 1x Laemmli buffer and boiled for 5 minutes, and the samples were analyzed by 12% SDS-PAGE. The gel was dried and autoradiographed.

Apoptosis assays. H1299 cells were plated at a concentration of 5 x 105 cells on 60-mm plates. H1299 cells were untreated, treated with LY294002 (10 µmol/L), Ad-PTEN [10 plaque-forming units (pfu) per cell], Ad-Akt(KM) (20 pfu per cell), Ad-MKK4(KR) (20 pfu per cell), or their combinations, incubated in RPMI supplemented with 10% FCS for 3 days, and then used for Western and fluorescence-activated cell sorting (FACS) assays to analyze apoptosis. For FACS analysis, all cells (i.e., nonadherent and adherent) were harvested, fixed with 1% paraformaldehyde and 70% ethanol, processed using the APO-bromodeoxyuridine staining kit (Phoenix Flow Systems, San Diego, CA), and subjected to a flow cytometry-based, modified terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assay as described previously (21). The number of apoptotic cells is represented by the number of FITC-positive cells of the total 10,000 gated cells. The percentage of dead cells was determined using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cells treated with empty virus were used as a control for nonapoptotic populations and as a reference for cells treated with Ad-MKK4(KR) or Ad-HA-Akt(KM). As an internal control, HL-60 cells provided in the apoptosis detection kit were treated with camptothecin to induce apoptosis to ensure that the TUNEL reaction was occurring during the staining procedure. Representative results from two independent experiments were presented.

Statistical analysis. All data are expressed as means ± SD. Cell proliferation among groups was compared using Student's t tests. Synergistic indexes of combination treatment was calculated by growth inhibition rate, as previously described (29). Ps < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of dominant-negative p85{alpha} on non–small cell lung cancer cell growth in vitro and in vivo. We previously showed that inhibition of the PI3K/Akt pathway by Ad-dnp85{alpha} efficiently induced expression of dominant-negative p85{alpha} in the H1299 NSCLC cell line and markedly inhibited cell proliferation (23). In the present study, we further tested the antiproliferative effects of Ad-dnp85{alpha} on a number of NSCLC cell lines. Relative to the control, Ad-dnp85{alpha} infection inhibited the growth of NSCLC cells (Fig. 1A). To determine the effects of Ad-dnp85{alpha} on the growth of NSCLC cells in vivo, H1299 xenograft tumor nodules established in athymic nude mice were injected with Ad-dnp85{alpha}. After a single injection of Ad-dnp85{alpha}, tumor growth was decreased, although statistically not significant (Fig. 1B); the mean volume of tumors injected with Ad-dnp85{alpha} (mean volume, 656.3 ± 208.8 mm3) reduced by 39.8% compared with tumors injected with empty virus (mean volume, 1,089.3 ± 439.8 mm3) at day 11(P = 0.084). Compared with a single injection, however, double injections of Ad-dnp85{alpha} significantly reduced tumor volume by 58.3% (mean volume, 190.1 ± 87.0 mm3) compared with tumors injected with empty virus twice (mean volume, 560.7 ± 233.5 mm3; P = 0.025; Fig. 1C). According to the immunohistochemical analysis, the level of phosphorylated Akt was reduced dramatically in the tumors injected with Ad-dnp85{alpha} and not in the tumors injected with empty virus or 1x PBS alone (Con; Fig. 1D), showing the specific in vivo activity of Ad-dnp85{alpha} on Akt activity.



View larger version (57K):
[in this window]
[in a new window]
  		Fig. 1. Ad-dnp85{alpha} inhibits the growth of NSCLC cells. A, effects of Ad-dnp85{alpha} on the growth of NSCLC cell lines were measured. NSCLC cell lines were uninfected or infected with 20 pfu per cell of Ad-dnp85{alpha} or empty virus (EV) and incubated in a growth medium for 3 days. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and the results are expressed relative to the number of uninfected control cells incubated in the same medium. Column, mean from six identical wells; bars, ±SD. **, P < 0.01; ***, P < 0.001 compared with control cells. B-C, effects of Ad-dnp85{alpha} on the growth of H1299 NSCLC xenograft tumors were tested. H1299 cells were injected into the dorsal flank of athymic nude mice. Once tumor volume reached about ~100 mm3, 1 x 1010 viral particles of Ad-dnp85{alpha} were intratumorally injected. These viruses were injected (B) once (day 0)or (C) twice (days 0 and 5). The growth of H1299 NSCLC xenograft tumors were measured every day. Point, mean tumor volume (calculated from five mice) relative to the volume of tumors injected with empty virus; bars, ±SD. *, P < 0.05 compared with control tumors. D, immunohistochemical analysis for phosphorylated Akt (pAkt) and Akt expression was done on the xenograft tumor tissues that were injected with empty virus or Ad-dnp85{alpha} twice (days 0 and 5) and then collected at 11 days, when mice were sacrificed.

 
Response of non–small cell lung cancer cells to the inhibition of both phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase kinase 4/c-jun NH2-terminal kinase pathways. Our previous work showed that PI3K/Akt- and MKK4/JNK-dependent pathways interact to maintain cell survival (23); the PI3K inhibitor LY294002 induced apoptosis in MKK4-null (–/–) cells but not in wild-type (+/+) MEF cells. We also observed that overexpression of dominant-negative p85{alpha} inhibited activation of Akt and JNK, whereas PTEN suppressed activation of Akt but not JNK (23). Therefore, we hypothesized that PI3K/Akt and MKK4/JNK pathways cooperate to maintain NSCLC cell survival and the antitumor activities of Ad-dnp85{alpha} on NSCLC cells are due to its inhibitory effects on these two pathways.

To test our hypothesis, we tested the effects of PI3K inhibitors (i.e., LY294002, Ad-PTEN, and Ad-Akt(KM)], JNK inhibitors [i.e., SP600215 and Ad-MKK4(KR)], and their combinations on the proliferation of NSCLC cells in vitro and in vivo. We previously found that these inhibitors regulate JNK or PI3K/Akt pathways in NSCLC cells incubated in complete medium (data not shown). We then tested the activities of these inhibitors on growth factor–stimulated JNK and PI3K/Akt activities in H1299 cells 2 days after the drug treatment. According to the Western blot analysis (Fig. 2A-B) and in vitro immune complex kinase assay (Fig. 2C), IGF-I increased phosphorylation and activation of AKt, which were suppressed by the treatment with LY294002 (Fig. 2A), Ad-PTEN (Fig. 2B), or Ad-Akt(KM) (Fig. 2C). IGF-I also induced JNK activities, which were suppressed by SP600215 (Fig. 2D) or Ad-MKK4(KR) (Fig. 2E). Western blot analysis of PTEN, HA, and MKK4 revealed that Ad-PTEN, Ad-HA-Akt(KM), and Ad-MKK4(KR) increased the expression of the adenoviral gene products. Equal amounts of protein loading for Akt and JNK1 were confirmed by Western blot analysis. These results indicate that the pharmacologic and genetic approaches used in our study effectively blocked the PI3K/Akt or MKK4/JNK signaling pathways.



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 2. Effect of inhibitors of PI3K/Akt and MKK4/JNK pathways on Akt and JNK activity. A-B, H1299 cells were untreated, treated with the indicated concentrations of LY294002 (A), or infected with the indicated doses of empty virus (EV) or Ad-PTEN (B). One day after incubation in complete medium, cells were serum-starved for on day and unstimulated or stimulated with IGF-I (100 ng/mL) for 15 minutes. Expression of phosphorylated Akt (pAkt), Akt, and PTEN was analyzed by Western blot analysis (W). C-E, H1299 cells were untreated, infected with the indicated dose of empty virus, Ad-Akt(KM) (C), or Ad-MKK4(KR) (D), or treated with 10 or 20 µmol/L of SP600215 (E). One day after incubation in complete medium, cells were serum-starved and then stimulated with IGF-I (100 ng/mL) for 15 minutes as described above. Akt or JNK activity was determined by immune complex kinase assays using H2B or GST-c-Jun, respectively, as a substrate. HA, MKK4, and JNK1 expression was examined by Western blot analysis.

 
We then tested the effects of single or combined treatment of these inhibitors on the proliferation and apoptosis of NSCLC cells. To avoid nonspecific effects of these inhibitors on other signaling pathways, we used 10 or 20 µmol/L of LY294002, 10 pfu per cell of Ad-PTEN, 20 pfu per cell of Ad-Akt(KM), 20 pfu per cell of Ad-MKK4(KR), 10 µmol/L of SP600215 or its companion [(–)SP600215] that lacks the ability to inhibit JNK (30, 31); the minimum doses that were enough to inhibit the respective kinases after 3 days of treatment (data not shown). H1299 cells infected with Ad-PTEN, Ad-MKK4(KR), or Ad-Akt(KM), or treated with LY294002 or SP600215 showed mild but significant antiproliferative effects compared with control (Fig. 3; Table 1). Ad-PTEN/Ad-MKK4(KR) (Fig. 3A), LY294002/Ad-MKK4(KR) (Fig. 3B), SP600215/Ad-PTEN (Fig. 3C), and Ad-MKK4(KR)/Ad-Akt(KM) (Fig. 3D) showed synergistically enhanced antiproliferative effects on H1299 cells compared with single-agent treatment (Table 1). Similar results of significantly decreased H1299 cell proliferation were observed after the combined treatment of chemical inhibitors, LY294002 (20 µmol/L) and SP600215 (10 µmol/L), compared with the effects of single-agent treatment (Fig. 3E). In contrast, (–)SP600215 did not enhance the effects of LY294002 on proliferation of NSCLC cells (Fig. 3F). MEK inhibitor (PD98059, 10 µmol/L) alone decreased H1299 cell proliferation (Fig. 3G) to the similar degree as the combination of JNK and Akt inhibitors (compared with the Fig. 3B, D, and E). Moreover, PD98059 and p38 MAPK inhibitor (SB202190) mildly enhanced the antiproliferative effects of Ad-PTEN (Fig. 3G and H). However, the cooperativity between Ad-PTEN with PD98059 or SB202190 in inhibiting NSCLC cell proliferation was less than additive (Table 1). Similarly, combinations of chemical inhibitors of PI3K/Akt and MEK (LY294002/PD98059) or PI3K/Akt and p38 MAPK (LY294002/SB202190) did not show enhanced antiproliferative effects on NSCLC cells (Table 1). Because PD98059 inhibits more than MEK depending on cellular context, we also tested the effects of combined treatment with U0126, another MEK inhibitor, and PI3K/Akt inhibitors. Similar to PD98059, U0126 showed antiproliferative effects on NSCLC cells at <10 µmol/L, however, it did not enhance the antiproliferative effects of PI3K/Akt inhibitors on NSCLC cells (data not shown). These findings suggest that the specific MAPK pathway MKK4/JNK and PI3K/Akt cooperate to stimulate NSCLC cell proliferation.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 3. Effects of the PI3K/Akt inhibitor, MKK4/JNK inhibitors, or their combination on the proliferation of NSCLC cells; 1-2 x 103 H1299 cells per well in 96-well plate were uninfected (E, F) or infected (A-D, G, H) with indicated doses of Ad-MKK4(KR), Ad-PTEN, Ad-HA-Akt(KM), or their combination in serum-free condition for 2 hours. Empty virus (EV) was used to adjust the total doses of adenoviruses same in each infection. Then, cells were changed to RPMI 1640 supplemented with 10% FCS containing LY294002 (10 or 20 µmol/L), SP600215 (10 µmol/L), (–)SP600215 (10 µmol/L), PD98059(10 µmol/L), SB202190(10 µmol/L), or their combinations. Cells treated with same doses of empty virus (A-D, G, H) or 0.1% DMSO (E, F) were used as a control. After 3 days, cell number was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. % Cell proliferation relative to the proliferation of control cells (–). Column, mean value of six identical wells from a representative single experiment (n = 3); bars, upper 95% confidence intervals. At least three independent experiments were done to obtain each result, and the mean was calculated.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Synergistic indexes of combination treatment calculated by growth inhibition rate

 
We then studied whether inhibition of PI3K/Akt- and MKK4/JNK-dependent pathways enhanced apoptosis in H1299 cells that were simultaneously treated with Ad-Akt(KM)/Ad-MKK4(KR), Ad-MKK4(KR)/Ad-PTEN, or Ad-MKK4(KR)/LY294002. The cells simultaneously treated with these inhibitors showed typical morphologic changes of apoptosis, including membrane blebbing, increased refractoriness, and chromatin condensation (data not shown). To measure apoptotic cell population, we did terminal deoxynucleotidyl transferase–mediated nick-end labeling staining of H1299 cells. One representative data out of two separate experiments with similar results are shown in Fig. 4. Compared with 0.5% of H1299 cells infected with empty virus, 15.3% of H1299 cells infected with Ad-Akt(KM), and 5.3% of H1299 cells infected with Ad-MKK4(KR), 62.4% of H1299 cells treated with Ad-Akt(KM)/Ad-MKK4(KR) underwent apoptosis (Fig. 4A, top). Similarly, cells treated with Ad-MKK4(KR)/Ad-PTEN or Ad-MKK4(KR)/LY294002 showed a marked increase in apoptotic populations compared with those treated with a single agent (Fig. 4A, bottom). Western blot analysis revealed that the combined treatment of Ad-MKK4(KR) and Ad-Akt(KM) induced decreased levels of procaspase-3 (Mr = 32 kDa) and antiapoptotic Bcl-xL protein expression and increased levels of poly(ADP-ribose) polymerase cleavage (113-kDa to the 89-kDa fragment) and proapoptotic Bax protein expression (Fig. 4B), suggesting that a change in the ratio of antiapoptotic protein (i.e., Bcl-xL) to proapoptotic protein (i.e., Bax) might have contributed to the apoptotic activities of Ad-Akt(KM)/Ad-MKK4(KR) in NSCLC cells. All of these results support our hypothesis that the PI3K/Akt and MKK4/JNK pathways cooperate to maintain NSCLC cell survival.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 4. Effects of the PI3K/Akt inhibitor, MKK4/JNK inhibitors, or their combination on the apoptosis of NSCLC cells. A, H1299 cells were treated with empty virus (EV), Ad-MKK4(KR) (20 pfu per cell), Ad-HA-Akt(KM) (20 pfu per cell), LY294002 (10 µmol/L), Ad-PTEN(10 pfu per cell), or their combinations. Flow cytometry was done using APO-BRDU staining. Living gating of the forward and orthogonal scatter channels was used to exclude debris and to selectively acquire cell events. All values reflect the percentage of cells as determined by light scatter. The percentage of dead cells was determined by fluorescence-activated cell sorting analysis of propidium iodide–stained nuclei. Top, representative cytogram from the cells infected with empty virus, Ad-MKK4(KR), Ad-Akt(KM), or Ad-MKK4(KR)/Ad-Akt(KM). The number of apoptotic cells is represented by the number of FITC-positive cells of the total 10,000 gated cells. Two separate experiments were done with similar results. B, H1299 cells infected with empty virus, Ad-Akt(KM), Ad-MKK4(KR), or Ad-Akt(KM)/Ad-MKK4(KR) were subjected to Western blot analysis for the expression of caspase-3, poly(ADP-ribose) polymerase (PARP), Bax, and BcL-xL. ß-Actin was included as an indicator of equal protein loading.

 
Effect of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase kinase 4/c-jun NH2-terminal kinase pathways on tumor growth. The cooperative growth and regulatory effects of PI3K/Akt- and MKK4/JNK-dependent pathways were further explored in vivo using s.c. H1299 tumor nodules established in athymic nude mice. Ad-MKK4(KR) and Ad-Akt(KM), either individually or in a combination, were injected into the tumors once (day 0) or twice (days 0 and 7). Mean volumes of tumors injected with empty virus were used as a control. A single treatment with Ad-MKK4(KR) or Ad-Akt(KM) reduced tumor volume by 29.70% (mean volume, 1,131.22 ± 561.14 mm3; P = 0.3506) or 16.50% (mean volume, 1,344.09 ± 559.56 mm3; P = 0.5922), respectively, compared with the empty virus-injected tumor (mean volume, 1,609.65 ± 638.25 mm3). Suppression of tumor growth was significantly enhanced by the concurrent injection of Ad-MKK4(KR) and Ad-Akt(KM) (58.86%; mean volume, 662.21 ± 322.69 mm3; P = 0.0381) compared with the volume of tumors injected with Ad-MKK4(KR) or Ad-Akt(KM) alone (Fig. 5A). Compared with a single injection, double injections significantly enhanced the inhibitory effects on tumor growth. Double injections of Ad-MKK4(KR) or Ad-Akt(KM) significantly reduced the tumor volume by 53.46% (mean volume, 389.26 ± 90.03 mm3; P = 0.0297) or 80.80% (mean volume, 160.56 ± 91.29 mm3; P = 0.0074), respectively, compared with the volume of tumors injected with empty virus (mean volume, 829.60 ± 225.27 mm3) twice (Fig. 5B). Concurrent double injections of Ad-MKK4(KR) and Ad-Akt(KM) resulted in significantly enhanced inhibitory effects on NSCLC tumor growth (95.25%; mean volume, 39.69 ± 7.22 mm3; P = 0.0031) compared with the tumors injected with Ad-MKK4(KR) or Ad-Akt(KM) alone. Representative tumors injected with empty virus, Ad-MKK4(KR), Ad-Akt(KM), or combination of Ad-MKK4(KR) and Ad-Akt(KM) twice are shown in Fig. 5C. Enhanced apoptosis by concurrent injection of Ad-MKK4(KR) and Ad-Akt(KM) was evident by Western blot analysis on caspase-3 using the H1299 xenograft tumor tissues injected with indicated viruses twice and then collected at 14 days, when mice were cleaved sacrificed. Densitometric analysis of the levels of cleaved active caspase-3 (Ac-caspase3/uncleaved pro-caspase-3 [pro-caspase3]) revealed that injection of both Ad-MKK4(KR) and Ad-Akt(KM) induced activation of caspase-3 compared with the levels induced by empty virus, Ad-MKK4(KR), or Ad-Akt(KM) alone (Fig. 5D). These findings indicate that inhibition of both the PI3K/Akt and MKK4/JNK signaling pathways markedly enhance antitumor activities in vivo.



View larger version (42K):
[in this window]
[in a new window]
  		Fig. 5. Effect of simultaneous treatment with Ad-Akt(KM) and Ad-MKK4(KR) on the growth of NSCLC xenografts. H1299 cells were injected into the dorsal flank of athymic nude mice. Once tumor volume reached ~100 mm3, 1 x 1010 viral particles of single or combination of the indicated adenovirus were intratumorally injected. These viruses were injected (A) once (day 0) or (B and C) twice (days 0 and 7). Tumors were measured everyday. Column, mean tumor volume (calculated from five mice) relative to the volume of tumors injected with empty virus; bars, ±SD (A, B). *, P < 0.05; **, P < 0.01 compared with control tumors. D, Western blot analysis was done on the tissues from H1299 xenograft tumor nodules injected with indicated viruses twice and then collected at 14 days, when mice were sacrificed. ß-Actin was included as a loading control.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The studies reported herein show that intratumoral injection of the dominant-negative mutant form of p85{alpha} inhibited NSCLC xenograft tumor growth. Simultaneous inhibition of the PI3K/Akt and MKK4/JNK pathways also efficiently inhibited NSCLC cell proliferation in vitro and in vivo by inducing apoptosis. These findings indicate cooperative activities of the PI3K/Akt and MKK4/JNK pathways in promoting the growth of NSCLC.

Tumor cells have devised several mechanisms to inhibit apoptosis and prolong their survival. Clear evidence exists for the involvement of the PI3K/Akt signaling pathway in lung carcinogenesis. Akt is constitutively active in premalignant and malignant human bronchial epithelial cells (21, 32) as well as in NSCLCs (23, 25, 26), probably owing to the activating mutations of ras (33, 34), overexpression of the epidermal growth factor receptor family and one of its ligands, transforming growth factor {alpha} (35, 36), increased copy number of PI3K catalytic subunit {alpha} (26), elevated levels of the subunits of PI3K (37), and reduced level of PTEN expression (38). Activation of Akt has been suggested an early biochemical effect of tobacco constituents on normal human bronchial epithelial cells (39). These findings indicated that Akt activation is an early event in lung tumorigenesis and, as a corollary, that the PI3K/Akt signaling pathway is a potential target for chemoprevention and treatment of lung cancer. In fact, our recent attempts to interfere with the PI3K/Akt pathway have shown some promise in lung cancer prevention (21). Moreover, inhibition of the PI3K/Akt signaling pathway by treatment with PI3K inhibitor LY294002 or by overexpression of dominant-negative p85{alpha} or PTEN lipid phosphatase effectively inhibited H1299 NSCLC cell lines (23). Based on our previous findings that (a) overexpression of a dnp85{alpha} regulatory subunit of PI3K induced apoptosis, whereas LY294002 or overexpression of the PTEN induced proliferative arrest in H1299 NSCLC cells; (b) dnp85{alpha} inhibited JNK activity and Akt activities, whereas LY294002 or overexpression of the PTEN inhibited Akt activity with no effect on JNK activity in H1299 cells; (c) constitutively active Rac-1 (Val12) blocked dnp85{alpha}-induced apoptosis in these cells; and (d) LY294002 treatment induced apoptosis in MKK4-null MEF cells but not in wild-type MEF cells, we hypothesized that the PI3K/Akt signaling pathway cooperates with other mitogen-activated signaling pathways to maintain cell survival and that dnp85{alpha} induces apoptosis through the combined inhibition of MKK4/JNK- and PI3K/Akt-dependent pathways. This hypothesis is further supported by the previous finding that MAPK family members play distinct biological roles in tumor cells (40, 41). Therefore, we set out the current study to evaluate the antitumor activities of dnp85{alpha} as well as that of combined inhibition of PI3K/Akt and MKK4/JNK pathways in NSCLC in vitro as well as in vivo.

Overexpression of dominant-negative p85{alpha} inhibited proliferation of most of the NSCLC cell lines used in this study and was shown to reduce tumor growth in the H1299 NSCLC xenograft tumor model. We previously found that the introduction of wild-type p85{alpha} did not recapitulate the effects of dnp85{alpha} on JNK and Akt, suggesting that dnp85{alpha} functions through mechanisms other than increasing intracellular levels of p85{alpha}, which inactivates PI3K lipid kinase activity through changes in the stoichiometry of p85/p110 (42). In this study, we also found that wile-type p85 PI3K adenovirus had no effects on the induction of cell death and adenovirus expressing wild-type p85 did not enhance apoptotic activities in NSCLC cells, when simultaneously treated with MKK4/JNK inhibitors (data not shown). Moreover, supporting our hypothesis, enhanced regression of tumor volume was also observed in the H1299 xenograft tumors injected with Ad-Akt(KM) and Ad-MKK(KR) compared with tumors injected with each virus alone, providing an in vivo evidence of the cooperative activities between these two pathways in maintaining survival of NSCLC cells. These findings also suggest that the MKK4 gene is a potential tumor promoter in NSCLC. Evidence relating to the role of MKK4 as a tumor promoter or suppressor is contradictory. Deletion or mutation of the MKK4 gene has been observed in several types of carcinomas, and reintroduction of MKK4 inhibits the metastatic ability of certain tumor cells, indicating the activity of MKK4 as a tumor suppressor (43–45). In contrast, MKK4 has been shown to play a role in cellular transformation by mediating the effects of Rac-1 that contribute to Ras-induced cellular transformation (46). Similarly, the role of JNK in apoptosis has been in debate. JNK is involved in the apoptotic response of cells exposed to stress, but in some studies in tumor cells, the JNK signal transduction pathway has been implicated in cell survival. The possibility that JNK might mediate a survival signal in tumor cells is further supported by the finding that it is activated in response to some oncoprotein, such as BCR-ABL (47). In addition, JNK has been implicated in the phosphorylation of Bcl-2, which may be required for Bcl-2 to achieve its full potent antiapoptotic activity (48). Therefore, it is plausible that the MKK4/JNK pathway can act as both a promoter and a suppressor of human carcinogenesis, depending on the presence of cell type-specific factors that induce or inhibit specific functions of the pathway.

We found that induction of apoptosis by Ad-MKK4(KR) and Ad-Akt(KM) has been associated with an abrogation of caspase-3 activity and changes in the expression of Bcl-2 family members, including Bax and Bcl-xL. The balance between antiapoptotic and proapoptotic proteins has been shown to determine whether a cell survives or undergoes apoptosis (49). These findings suggest that the enhanced antiapoptotic activities of Ad-MKK4(KR) and Ad-Akt(KM) resulted, in part, from changes in the regulation of the antiapoptotic to proapoptotic protein ratio. Because Bcl-xL also functions independently in regulating cell death (50), the reduction in Bcl-xL protein expression could, in part, account for the increased apoptosis in NSCLC cells treated with these adenoviral vectors.

In conclusion, the findings presented here indicate for the first time that the PI3K/Akt and MKK4/JNK pathways cooperate to promote the survival of NSCLC cells in vitro and in vivo. Our findings illustrate the importance of understanding the role of interactions between different signaling pathways, especially PI3K/Akt and MKK4/JNK, in the growth of NSCLC cells, providing a rationale for designing effective therapeutic strategies for lung cancer. Whereas we showed the cooperativity between the Akt and JNK pathways in NSCLC cell proliferation and tumor growth, this effect could be specific to the H1299 cell line. Therefore, intensive studies using several NSCLC cell types with the multiple individual pathway inhibitors are required to investigate how variations in PI3K and Akt signaling can predetermine the efficacy of this treatment strategy. In addition, further studies are warranted to determine the optimal sequence of the administration of PI3K/Akt and MKK4/JNK pathway inhibitors, which would affect the degree of synergism and thus the therapeutic efficacy of these treatments (51).


   Footnotes
 
Grant support: NIH grants R01 CA109520 (H-Y. Lee) and CA100816 (H-Y. Lee), American Cancer Society grant RSG-04-082-01-TBE (H-Y. Lee), U.S. Department of Defense grant DAMD17-01-1-0689 (W. K. Hong), and American Cancer Society Clinical Research professorship (W.K. Hong).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/ 3/05; revised 5/ 9/05; accepted 5/17/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gibbs JB. Anticancer drug targets: growth factors and growth factor signaling. J Clin Invest 2000;105:9–13.[Medline]
  2. Kennedy SG, Kandel ES, Cross TK, Hay N. Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol Cell Biol 1999;19:5800–10.[Abstract/Free Full Text]
  3. Hausler P, Papoff G, Eramo A, Reif K, Cantrell DA, Ruberti G. Protection of CD95-mediated apoptosis by activation of phosphatidylinositide 3-kinase and protein kinase B. Eur J Immunol 1998;28:57–69.[CrossRef][Medline]
  4. Brodt P, Samani A, Navab R. Inhibition of the type I insulin-like growth factor receptor expression and signaling: novel strategies for antimetastatic therapy. Biochem Pharmacol 2000;60:1101–7.[CrossRef][Medline]
  5. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501.[CrossRef][Medline]
  6. Brazil DP, Hemmings BA. Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci 2001;26:657–64.[CrossRef][Medline]
  7. Toker A, Newton AC. Cellular signaling: pivoting around PDK-1. Cell 2000;103:185–8.[CrossRef][Medline]
  8. Wymann MP, Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1998;1436:127–50.[Medline]
  9. Toker A, Cantley LC. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 1997;387:673–6.[CrossRef][Medline]
  10. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997;275:1943–7.[Abstract/Free Full Text]
  11. Di Cristofano A, Pandolfi PP. The multiple roles of PTEN in tumor suppression. Cell 2000;100:387–90.[CrossRef][Medline]
  12. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:231–41.[CrossRef][Medline]
  13. Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318–21.[Abstract/Free Full Text]
  14. Kops GJ, Burgering BM. Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J Mol Med 1999;77:656–65.[CrossRef][Medline]
  15. Romashkova JA, Makarov SS. NF-{kappa}B is a target of AKT in anti-apoptotic PDGF signalling. Nature 1999;401:86–90.[CrossRef][Medline]
  16. Mayo LD, Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A 2001;98:11598–603. Epub 2001 Aug 14.[Abstract/Free Full Text]
  17. Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC. Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol 2001;3:245–52.[CrossRef][Medline]
  18. Collado M, Medema RH, Garcia-Cao I, et al. Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1. J Biol Chem 2000;275:21960–8.[Abstract/Free Full Text]
  19. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995;378:785–9.[CrossRef][Medline]
  20. Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3ß regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 1998;12:3499–511.[Abstract/Free Full Text]
  21. Chun KH, Kosmeder JW II, Sun S, et al. Effects of deguelin on the phosphatidylinositol 3-kinase/Akt pathway and apoptosis in premalignant human bronchial epithelial cells. J Natl Cancer Inst 2003;95:291–302.[Abstract/Free Full Text]
  22. Lee HY. Molecular mechanisms of deguelin-induced apoptosis in transformed human bronchial epithelial cells. Biochem Pharmacol 2004;68:1119–24.[CrossRef][Medline]
  23. Lee HY, Srinivas H, Xia D, et al. Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent pathways cooperate to maintain lung cancer cell survival. J Biol Chem 2003;278:23630–8. Epub 2003 Apr 24.[Abstract/Free Full Text]
  24. Lee HY, Moon H, Chun KH, et al. Effects of insulin-like growth factor binding protein-3 and farnesyltransferase inhibitor SCH66336 on Akt expression and apoptosis in non-small-cell lung cancer cells. J Natl Cancer Inst 2004;96:1536–48.[Abstract/Free Full Text]
  25. Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 2001;61:3986–97.[Abstract/Free Full Text]
  26. Massion PP, Taflan PM, Shyr Y, et al. Early involvement of the phosphatidylinositol 3-kinase/Akt pathway in lung cancer progression. Am J Respir Crit Care Med 2004;170:1088–94.[Abstract/Free Full Text]
  27. Hara K, Yonezawa K, Sakaue H, et al. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A 1994;91:7415–9.[Abstract/Free Full Text]
  28. Lee HY, Chun KH, Liu B, et al. Insulin-like growth factor binding protein-3 inhibits the growth of non-small cell lung cancer. Cancer Res 2002;62:3530–7.[Abstract/Free Full Text]
  29. Dings RP, Yokoyama Y, Ramakrishnan S, Griffioen AW, Mayo KH. The designed angiostatic peptide anginex synergistically improves chemotherapy and antiangiogenesis therapy with angiostatin. Cancer Res 2003;63:382–5.[Abstract/Free Full Text]
  30. Bennett BL, Sasaki DT, Murray BW, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci U S A 2001;98:13681–6.[Abstract/Free Full Text]
  31. Shin M, Yan C, Boyd D. An inhibitor of c-jun aminoterminal kinase (SP600125) represses c-Jun activation, DNA-binding and PMA-inducible 92-kDa type IV collagenase expression. Biochimica et Biophysica Acta 2002;1589:311–6.[Medline]
  32. Tsao AS, McDonnell T, Lam S, et al. Increased phospho-AKT (Ser(473)) expression in bronchial dysplasia: implications for lung cancer prevention studies. Cancer Epidemiol Biomarkers Prev 2003;12:660–4.[Abstract/Free Full Text]
  33. Mitsudomi T, Steinberg SM, Oie HK, et al. ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res 1991;51:4999–5002.[Abstract/Free Full Text]
  34. Mills NE, Fishman CL, Rom WN, Dubin N, Jacobson DR. Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res 1995;55:1444–7.[Abstract/Free Full Text]
  35. Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld J, Dmitrovsky E. Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor {alpha} is frequent in resectable non-small cell lung cancer but does not predict tumor progression. Clin Cancer Res 1997;3:515–22.[Abstract]
  36. Fontanini G, De Laurentiis M, Vignati S, et al. Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival. Clin Cancer Res 1998;4:241–9.[Abstract]
  37. Lin X, Bohle AS, Dohrmann P, et al. Overexpression of phosphatidylinositol 3-kinase in human lung cancer. Langenbecks Arch Surg 2001;386:293–301.[CrossRef][Medline]
  38. Soria JC, Lee HY, Lee JI, et al. Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation. Clin Cancer Res 2002;8:1178–84.[Abstract/Free Full Text]
  39. West KA, Brognard J, Clark AS, et al. Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest 2003;111:81–90.[CrossRef][Medline]
  40. Gupta S, Plattner R, Der CJ, Stanbridge EJ. Dissection of Ras-dependent signaling pathways controlling aggressive tumor growth of human fibrosarcoma cells: evidence for a potential novel pathway. Mol Cell Biol 2000;20:9294–306.[Abstract/Free Full Text]
  41. Shields JM, Mehta H, Pruitt K, Der CJ. Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin. Mol Cell Biol 2002;22:2304–17.[Abstract/Free Full Text]
  42. Cuevas BD, Lu Y, Mao M, et al. Tyrosine phosphorylation of p85 relieves its inhibitory activity on phosphatidylinositol 3-kinase. J Biol Chem 2001;276:27455–61.[Abstract/Free Full Text]
  43. Su GH, Hilgers W, Shekher MC, et al. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene. Cancer Res 1998;58:2339–42.[Abstract/Free Full Text]
  44. Yoshida BA, Dubauskas Z, Chekmareva MA, Christiano TR, Stadler WM, Rinker-Schaeffer CW. Mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1), a prostate cancer metastasis suppressor gene encoded by human chromosome 17. Cancer Res 1999;59:5483–7.[Abstract/Free Full Text]
  45. Teng DH, Perry WL III, Hogan JK, et al. Human mitogen-activated protein kinase kinase 4 as a candidate tumor suppressor. Cancer Res 1997;57:4177–82.[Abstract/Free Full Text]
  46. Qiu RG, Chen J, Kirn D, McCormick F, Symons M. An essential role for Rac in Ras transformation. Nature 1995;374:457–9.[CrossRef][Medline]
  47. Hess P, Pihan G, Sawyers CL, Flavell RA, Davis RJ. Survival signaling mediated by c-Jun NH(2)-terminal kinase in transformed B lymphoblasts. Nat Genet 2002;32:201–5.[CrossRef][Medline]
  48. Deng X, Xiao L, Lang W, Gao F, Ruvolo P, May WS Jr. Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem 2001;276:23681–8. Epub 2001 Apr 25.[Abstract/Free Full Text]
  49. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–19.[CrossRef][Medline]
  50. Coll ML, Rosen K, Ladeda V, Filmus J. Increased Bcl-xL expression mediates v-Src-induced resistance to anoikis in intestinal epithelial cells. Oncogene 2002;21:2908–13.[CrossRef][Medline]
  51. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
M. Toulany, R. Kehlbach, U. Florczak, A. Sak, S. Wang, J. Chen, M. Lobrich, and H. P. Rodemann
Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair
Mol. Cancer Ther., July 1, 2008; 7(7): 1772 - 1781.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Q. Jin, L. Feng, C. Behrens, B. N. Bekele, I. I. Wistuba, W.-K. Hong, and H.-Y. Lee
Implication of AMP-Activated Protein Kinase and Akt-Regulated Survivin in Lung Cancer Chemopreventive Activities of Deguelin
Cancer Res., December 15, 2007; 67(24): 11630 - 11639.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. Angelo, M. Anita, A. Amalia, and T. Stefania
Phosphatidylinositol 3-Kinase in Breast Cancer: Where from Here?
Clin. Cancer Res., October 15, 2007; 13(20): 5988 - 5990.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
L. Ying, A. B. Hofseth, D. D. Browning, M. Nagarkatti, P. S. Nagarkatti, and L. J. Hofseth
Nitric Oxide Inactivates the Retinoblastoma Pathway in Chronic Inflammation
Cancer Res., October 1, 2007; 67(19): 9286 - 9293.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, H.-Y.
Right arrow Articles by Hong, W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, H.-Y.
Right arrow Articles by Hong, W. K.

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