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Experimental Therapeutics, Preclinical Pharmacology |
and Akt Inactivation
1 Departments of Medicine, 2 Obstetrics and Gynecology, 3 Microbiology and Immunology, 4 Walther Oncology Center, 5 Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana; and 6 Departments of Medicine, 7 Microbiology, Immunology and Molecular Genetics; and 8 Jonsson Comprehensive Cancer Center, University of California, David Geffen School of Medicine, Los Angeles, California
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PDGFR) as an ovarian cancer-specific gene by microarray hybridization using primary cultures. The purpose of this study is to evaluate whether disruption of the platelet-derived growth factor-regulated growth pathway by Imatinib mesylate (Gleevec), a partially selective PDGFR inhibitor, inhibits growth of ovarian cancer cells expressing PDGFR. Experimental Design: To investigate the effects of Imatinib mesylate in ovarian cancer, we established an in vitro model by immortalizing primary ovarian cells, which express endogenous PDGFR, and we evaluated the effects of Imatinib on cell proliferation. In addition, we investigated the involvement of Akt in mediating Imatinib-inhibited cell growth inhibition.
Results: We found that 39% of ovarian tumors express PDGFR by immunohistochemistry. We showed that Imatinib inhibits the growth of ovarian cancer cells in a PDGFR-specific manner, at clinically relevant concentrations (IC50 < 1 µM). Imatinib inhibits the growth of three primary ovarian cultures and two immortalized cultures (PDGFR positive), but has no effects on SkOv3 and CaOv3 cell lines (PDGFR negative). Imatinib exerts antiproliferative effects by arresting cells at G0-G1 and preventing progression through S phase. Imatinib inhibits both PDGFR and Akt phosphorylation at a concentration of 1 µM. Stable expression of constitutively active Akt induces partial resistance to PDGFR inhibition in ovarian cancer cells, as demonstrated by cell proliferation assay and cell cycle analysis.
Conclusions: Our data indicate that Imatinib mesylate inhibits the growth of ovarian cancer cells through PDGFR inactivation. In addition, our results suggest that constitutive Akt activation modulates sensitivity to Imatinib in ovarian cancer cells.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of microarray gene expression analysis using primary ovarian cells, we identified differential expression of the platelet-derived growth factor receptor (PDGFR) between primary cells derived from epithelial ovarian tumors and primary cells derived from normal ovarian epithelium. The level of PDGFR expression is 6-fold higher in tumor-derived cells than in normal ovarian epithelial cells (P = 0.00137; Ref. 2
). PDGFR is normally expressed in mesenchymal and in glial tissues, and is absent in epithelia. The ovarian epithelium that originates in the coelomic embryonic layer has dual epithelial and mesenchymal characteristics (3)
, and undergoes an epithelio-mesenchymal transition during neoplastic transformation (4)
. These specific characteristics of the ovarian epithelium may explain the aberrant PDGFR expression in EOC-derived cells. In a previous report, immunohistochemical staining demonstrated PDGFR expression in 16 of 45 ovarian tumors (5)
. The level of PDGFR expression correlated with higher histological grade and advanced surgical stages of ovarian tumors. Furthermore, stage for stage, patients with PDGFR-positive tumors had shorter survival times than those with negative tumors. These observations suggested that the platelet-derived growth factor (PDGF) -PDGFR system can play a functional role in the progression of EOC and that autocrine or paracrine activation of PDGFR within the tumor tissue contributes to tumor expansion.
PDGFR activation by the PDGF induces receptor dimerization and autophosphorylation of tyrosine residues in the intracellular kinase domain (6)
. Receptor phosphorylation makes possible the subsequent engagement of several proteins with Src homology domains, including phosphatidylinositol 3'-kinase (PI3k), phospholipase 
C, GTP-ase activating protein, Grb, and Nck, that convey mitogenic signals to the nucleus. Interruption of this growth pathway could be instrumental for inhibiting ovarian tumor growth. Imatinib mesylate (STI 571 or Gleevec; Novartis, East Hanover, NJ) is a 2-phenylaminopyrimidine derivative that selectively inhibits the abl, c-kit, and PDGFR tyrosine kinases, but does not affect signal transduction through related receptors such as c-FMS, Tek, or the Flt family of receptors (7
, 8)
.
There is ample evidence in the literature supporting the critical role played by the PI3k/Akt pathway in ovarian oncogenesis (9
, 10) . This pathway can be activated in ovarian tumors through Akt (protein kinase B; Ref. 11
) or PI3k gene amplification (12
, 13)
, through activating mutations of the p85 regulatory subunit of PI3k (13)
or by down-regulation of the phosphatase, PTEN (14)
. In addition, PI3k and downstream Akt can be activated by various growth factors (15)
, including by PDGF (16)
. Paracrine or autocrine engagement of growth pathways within the tumor bed activates Akt. Phosphorylated Akt (pAkt) modifies the activity of survival-related proteins and proliferation factors. Akt promotes cell survival by inhibiting proapoptotic proteins, such as BAD (17)
, caspase 9 (18)
, apoptosis-signal regulating kinase, forkhead transcription factors (19)
, and the growth-inhibitory protein glycogen synthase kinase-3 (20)
. On the other hand, Akt promotes cell proliferation by up-regulating the translation of proteins required for cell cycle progression. For instance, Akt phosphorylates through an m-TOR/FRAP-dependent mechanism (21
, 22)
, the p70 S6 kinase, and inactivates the translational repressor, 4E-BP1 (23)
. The phosphatase PTEN, which has tumor suppressor properties, inactivates Akt (24, 25, 26)
.
In this study, we confirmed by immunohistochemical staining that PDGFR is expressed in 39% of ovarian tumors. We showed that Imatinib mesylate inhibits PDGFR phosphorylation and the growth of ovarian cancer cells harboring PDGFR. This conclusion was reached by using primary ovarian cells, immortalized primary tumor-derived ovarian cells, and two ovarian cancer cell lines, with different levels of baseline endogenous PDGFR expression. The effects of Imatinib mesylate are restricted to PDGFR-expressing cells, as ovarian cancer cell lines lacking PDGFR expression are insensitive to the drug. Inhibition of the PDGFR-PDGF pathway by Imatinib mesylate inactivates Akt, and loss of Akt phosphorylation parallels growth inhibition. Furthermore, stable expression of constitutively active Akt in ovarian cancer cells harboring PDGFR renders these cells resistant to the growth-inhibitory properties of Imatinib mesylate.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antibody (R&D Systems, Minneapolis, MN) at a concentration of 20 µg/ml, following the manufacturer’s instructions. In brief, the slides were deparaffinized in xylene, treated with proteinase K (Dako, Carpinteria, CA) for 5 min, and incubated with primary antibody overnight at 4°. Secondary labeling was based on the avidin/biotin system (Dako; LSAB2 kit). The slides were stained with 3–3' diaminobenzidine and counterstained with hematoxylin. Negative controls were run in parallel, with omission of the primary antibody. All of the slides were read by a board-certified pathologist.
Cell Lines.
SKOV3 and CaOv3 cells (American Type Culture Collection, Manassas, VA) were grown according to the manufacturer’s instructions. CSOC848, CSOC908, and CSOC918 (generous gift from the Cedars Sinai Ovarian Cancer repository, Dr. Rae Lynn Baldwin, UCLA, Los Angeles, CA) are primary ovarian cancer cultures established as described previously (27
, 28)
. In culture, ovarian epithelial cells tend to assume atypical fibroblast-like morphology and dual epithelio-mesenchymal phenotype, characterized by the expression of both keratin (an epithelial marker) and vimentin (a mesenchymal marker; Ref. 29
). We have relied on homogenous cytokeratin and vimentin staining and the absence of factor VIII staining (an endothelial cell marker) to ensure purity of primary cultures (2
, 27)
. All of the primary cell cultures represent early passage cells (<10 passages for tumor-derived cells and <4 passages for primary cultures derived from normal ovarian epithelium). Three immortal ovarian cell lines C272/hTert/E7, C889/hTert, and H281/hTert/E7 were obtained by transducing the catalytic unit of human telomerase and the papilloma virus subunit E7 in primary cancer cell cultures through retroviral infection, as described previously (30)
. C272/hTert/E7 and C889/hTert are derived from two tumor-derived primary cultures (CSOC272 and CSC889), and H281/hTert/E7 was derived from a normal primary ovarian culture (HOSE281). The primary and immortal ovarian cells were grown at 37°C, 4% CO2 in 1:1 growth medium containing MCDB 105 (Sigma, St. Louis, MO) and M199 (Cellgro, Herndon, VA) supplemented with 10% FCS and 1% penicillin/streptomycin. All of the other cells were grown in the presence of 10% fetal bovine serum, unless otherwise specified, and were harvested during the log growth phase, when reaching 70–80% confluence.
Cell Growth Assay.
Cells were plated in equal numbers in serum-containing culture medium. Imatinib mesylate (Gleevec, STI571), synthesized by Novartis, solubilized in water, was added to the culture medium at different concentrations and was renewed every 72 h. All of the experiments were performed in triplicates and were repeated twice. Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma) colorimetric dye reduction method (31)
. Results are presented as the average of at least three experiments performed in identical conditions.
Apoptosis and Cell Cycle Analysis.
Apoptosis was determined in cells incubated with Imatinib mesylate for 72 h, by staining with FITC annexin V antibody and propidium iodide (Molecular Probes, Eugene, OR) followed by flow cytometry analysis, measuring emission at 530 nm (FL1) and 575 nm (FL3). Cell cycle analysis was performed by DNA staining with propidium iodide in cells, after stimulation with 20% fetal bovine serum and treatment with Imatinib mesylate. Cell cycle profile was determined by flow cytometry with FACScan/CellQuest system (Becton-Dickinson, San Jose, CA). The size of sub-G1, G0/G1, S, and G2-M were determined by analyzing the histograms using ModFitLT software (Verity Software, Topsham, ME). All of the experiments were performed in duplicate.
PDGFR Immunoblotting.
Cells actively growing were lysed using lysis buffer containing leupeptin 1 µg/ml, aprotinin 1 µg/ml, phenylmethylsulfonyl fluoride 400 µM, and 1 mM Na3VO4. Cell lysates were sonicated and incubated on ice for 30 min. Cellular debris was removed by centrifugation for 15 min. Cell lysates were separated by SDS electrophoresis, transferred to nitrocellulose membrane, and immunoblotted with anti-PDGFR antibody (Upstate Technology, Waltham, MA) at 1 µg/ml concentration overnight at 4°.
Phospho-AKT Immunoblotting.
Cells in log phase of growth were serum starved for 48 h, treated with various concentrations of Imatinib mesylate, and stimulated with 20% serum or PDGF BB (Sigma) at a concentration of 5 ng/ml or 25 ng/ml. They were lysed in 1x SDS buffer, containing 62.5 mM Tris (pH 6.8), 2% SDS, 50 mM DTT, and 10% glycerol, and boiled for 10 min. They were then separated by SDS gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with 1 µg/ml polyclonal anti-phospho Akt rabbit antibody (Cell Signaling) able to recognize Akt phosphorylation at position S473. Total anti-Akt antibody (Cell Signaling) was used as a control.
Mitogen-Activated Protein Kinase (MAPK) Immunoblotting.
Equal amount of total protein separated by SDS gel electrophoresis were immunoblotted with anti-phospho-MAPK antibody (Zymed, San Francisco, CA) at 1:2000 dilution and with anti-total MAPK antibody (Cell Signaling) as control.
PDGFR Immunoprecipitation.
Cells were lysed on ice in a buffer containing 20 mM HEPES (pH 7.4), 50 mM ß glycerophosphate, 2 mM EGTA, 1 mM DTT, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, leupeptin 1 µg/ml, aprotinin 1 µg/ml, and phenylmethylsulfonyl fluoride 400 µM, as described previously (32)
. After incubation on ice for 15 min, they were centrifuged at 13,000 rpm for 30 min to remove cellular debris. Five hundred µg of protein was incubated for 90 min at 4°C with 5 µg of anti-PDGFR polyclonal rabbit antibody (Upstate Technology, Waltham, MA), then incubated with 50 µg of Protein G Plus-Agarose beads slurry (Oncogene, Boston, MA) for an additional 90 min at 4°C. The protein-antibody bead complexes were washed twice with a wash buffer containing 0.2% Triton, then boiled for 5 min in 1x SDS protein loading dye. PY20 Antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used to recognize phosphorylated tyrosine residues. As control, input protein for immunoprecipitation reaction was immunoblotted with anti-PDGFR antibody.
Stable Transfection with Myristoylated Akt.
Actively growing C272-hTERT/E7 ovarian cells were transfected with myristoylated Akt1 cloned downstream of cytomegalovirus promoter in pLNCX eukaryotic expression vector (BD Biosciences, Palo Alto, CA), which contains a neomycin resistance marker (generous gift of Prof. William R. Sellers, Dana-Farber Cancer Institute, Boston, MA; Ref. 33
). Empty vector transfection and selection were performed in parallel. Stable clones were selected with neomycin, and Akt expression was verified by immunoblotting with total Akt antibody (Cell Signaling). Akt phosphorylation was confirmed by immunoblotting with phosphorylated Akt antibody (Cell Signaling). Two stable clones expressing Akt (Akt#9 and Akt#16) and a control stable clone (empty vector) were used for cell growth and Akt phosphorylation experiments in the presence of various Imatinib mesylate concentrations, as described. All of the growth experiments were performed in triplicate in serum and were repeated twice. A two-tailed t test was performed for both pairs (Akt stable clone #9 and #16 versus control) at each drug concentration level, and P-value was calculated. Cell cycle profiles after treatment with Imatinib (5 µM) were determined in cells expressing stable myrAkt and compared with cells transfected with empty vector.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression in Ovarian Tumors. expression in ovarian tumors, we used immunohistochemical staining with a specific anti-PDGFR antibody. We identified intense membrane staining in 16 of 41 tumors (39% of tumors, Fig. 1
). When evaluated by histological subtype, membrane staining was noted in 4 of 5 clear cell carcinomas, 3 of 8 endometrioid carcinomas, and 8 of 26 papillary serous tumors (Table 1)
. Patchy cytoplasmatic staining was identified in 11 additional tumors (1 clear cell, 7 serous, and 3 endometrioid tumors). In contrast, normal ovarian epithelium did not stain (2 normal ovaries and 1 borderline tumor). The staining is significant in clear cell carcinomas and in invasive, poorly differentiated foci of other ovarian specimens. These data are consistent with a previous report, which records positive PDGFR staining in 16 of 45 ovarian tumors, but no staining in normal ovaries or in benign tumors (5)
. Pericytes and endothelial cells within vessel walls in the tumor bed react with anti-PDGFR antibody (internal positive control). Control staining (without primary antibody) was consistently negative.
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at Clinically Relevant Concentrations. (Fig. 2, A and B)
. In contrast, normal primary ovarian cells (HOSE302, HOSE305, and HOSE326, generous gift of Dr. Robert Bigsby, Indiana University, Indianapolis, IN) and immortalized cell line H281-hTert/E7 (derived from a normal ovarian primary culture HOSE281) do not express PDGFR. Three ovarian cancer cell lines (Ov90, CaOv3, and SkOv3) from the American Type Culture Collection have no detectable PDGFR expression. In parallel, we determined levels of Akt activation in these cell lines or cell cultures. CaOv3 and SkOv3 (American Type Culture Collection) express high endogenous levels of phospho-Akt, whereas in C272-hTert/E7 and H281-hTert/E7, Akt is minimally activated under basal conditions (10% FCS growth conditions). Constitutive activation of Akt in SkOv3 cells and CaOv3 cells was reported previously (34)
. Activation of Akt is less evident in primary cell cultures (Fig. 2, A and B)
, in basal growth conditions.
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expression and three primary ovarian cancer cultures expressing PDGFR. The growth of C272-hTert/E7 cells and C889/hTert cells, which express high levels of PDGFR and of primary CSOC848, CSOC908, and CSOC918 cells, was inhibited at drug concentrations 
1 µM (Fig. 3, A and B)
. This concentration can be achieved in the clinical setting, being tolerated with minimal toxicity (35
, 36)
. By contrast, SkOv3 and CaOv3 cells that do not express PDGFR are insensitive to the effects of Imatinib up to 10 µM concentration (Fig. 3C)
. Drug concentrations >10 µM are toxic to all ovarian cells, regardless of their PDGFR status. These data suggest that Imatinib mesylate inhibits the growth of ovarian cancer cells in a PDGFR-specific manner.
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. After treatment with Imatinib mesylate, PDGFR-expressing cells (C272hTert/E7) are arrested at G0-G1, and serum-induced S phase progression is prevented. In contrast, when apoptosis was analyzed by propidium iodide/Annexin V staining, we noted that the fraction of apoptotic cells is only minimally increased by treatment with Imatinib (5.5%) versus control (3%) at 72 h, suggesting that apoptosis is not the main mechanism of growth inhibition in this model (data not shown).
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and Akt Phosphorylation in Ovarian Cancer Cells. endogenously. In conditions of serum starvation, the receptor is minimally phosphorylated, but after PDGF stimulation, the receptor is rapidly activated. Serum stimulation modestly increases the phosphorylation of PDGFR over the basal level (Fig. 5A)
. Serum-starved C272-hTert/E7 cells have low or undetectable levels of phospho-Akt, but Akt activation in response to PDGF becomes apparent in 30 min and remains stable for at least 90 min after stimulation of the cells (Fig. 5B)
. Serum and PDGF BB induce rapidly Akt phosphorylation, in a dose-dependent manner (Fig. 5C)
. In this cell line we verified the effects of Imatinib mesylate on inactivation of PDGFR and downstream substrates Akt and MAPK. Imatinib mesylate inhibits tyrosine phosphorylation of PDGFR (7
, 37)
and inactivates the receptor at a concentration of 1 µM, which corresponds to the inhibitory concentration (IC50) in proliferative assays (Fig. 6A)
. In parallel, 1 µM of Imatinib mesylate inactivates Akt, reducing its level of phosphorylation to basal state in serum-stimulated cells. In PDGF-stimulated ovarian cells, partial Akt inactivation occurs at 1 µM of Imatinib mesylate, and complete Akt inactivation is noted at 5 µM (Fig. 6B)
. In contrast, Imatinib mesylate does not affect phospho-Akt levels in ovarian cancer cells that do not express PDGFR (SkOv3; Fig. 6C
). In contrast to complete Akt inactivation, the phosphorylation levels of MAPK in serum-stimulated C272/hTERT/E7 cells treated with Imatinib mesylate at concentrations up to 5 µM are only partially inhibited (data not shown), suggesting that the MAPK pathway remains partially "turned on," despite PDGFR blockade.
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. Similar to parental C272-hTert/E7 cells, clones isolated after transfection with empty vector express low levels of phospho-Akt. In Akt-overexpressing stable clones, phospho-Akt inactivation occurs only at high Imatinib concentrations (>10 µM; Fig. 7B
) and is only partial. In contrast, phospho-Akt in cells transfected with empty vector is rapidly inactivated at concentrations of Imatinib as low as 1 µM (Fig. 7C)
.
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; P <0.05). Furthermore, profiling of cell cycle indicates that whereas Imatinib blocks vector-transfected cells at G0-G1 (82% ± 2.8%), stable Akt expressors are able to progress through S phase after serum stimulation (Fig. 9)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We show that PDGFR expression occurs in 39% of ovarian tumors, suggesting that a subgroup of ovarian carcinomas is amenable to therapeutic PDGFR targeting. Furthermore, our data demonstrate that Imatinib mesylate inactivates Akt in ovarian cancer cells and that expression of constitutively active Akt induces partial resistance to growth inhibition by Imatinib. These results are consistent with two reports indicating the critical role of Akt as a resistance marker to cytokine-induced growth arrest in breast cancer cells (38
, 39)
.
Imatinib mesylate was studied extensively in chronic myelogenous leukemia, which is characterized by the (9
, 22)
chromosomal translocation giving rise to a fusion protein with constitutive tyrosine kinase activity. This protein drives the uncontrolled growth of the chronic myelogenous leukemia cells (40)
, which ultimately overwhelm normal hematopoiesis. Imatinib mesylate blocks the kinase activity of abl and inhibits the growth of bcr-abl driven cell lines in vitro (IC50 of 1 µM; Refs. 41
, 42
). This concentration is readily achieved in vivo, with minimal host toxicity, and accounts for the remarkable activity of the drug in patients with chronic and accelerated phase chronic myelogenous leukemia (35
, 36
, 43)
. Outside this setting, Imatinib mesylate inhibits the growth of c-kit-positive small cell lung cancer cell lines (44
, 45)
, PDGFR-expressing glioblastoma cells (46)
, and c-kit positive gastrointestinal stromal tumor cell lines (47)
. This inhibitory in vitro effect translates into therapeutic efficacy for patients with gastrointestinal stromal tumor tumors (48
, 49)
or with myeloproliferative disorders characterized by activation of PDGFRß (50)
and makes Imatinib mesylate therapeutically interesting for other tumors governed by activated PDGFR or c-Kit pathways (51)
. In solid malignancies, these pathways can be activated through autocrine or paracrine interactions, or through activating mutations. Such PDGFR mutations have been described recently in c-kit negative gastrointestinal stromal tumor tumors (52)
, but not in other malignancies.
We (2)
and others (5)
have shown that PDGFR is up-regulated in ovarian tumors and in primary ovarian cancer cells. In addition, it is known that ovarian tumor cells and cell lines express PDGF (53)
. These reports suggest a functional role for PDGF-PDGFR paracrine or autocrine interactions in the growth of ovarian tumors. Interestingly, ovarian cancer cell lines fail to express PDGFR (53)
, a finding confirmed in our study for three American Type Culture Collection cell lines tested (SkOv3, CaOv3, and Ov90), whereas primary ovarian cancer cells express high levels of PDGFR. It is likely that cancer cell lines acquire additional chromosomal aberrations and/or mutations during continuous subculturing, which confer growth factor independence, whereas primary cells more closely resemble the tissues from which they originate. The immortalized ovarian cancer cell lines (C272-hTert/E7 and C889-hTert) used here preserve endogenous PDGFR expression.
In this study we show that Imatinib mesylate inhibits the growth of PDGFR-expressing ovarian cancer cells in the presence of serum and that the inhibitory concentration is 
1 µM, which is achievable clinically. In contrast, ovarian cancer cells that do not express PDGFR (SkOV3 and CaOv3) are insensitive to this drug at clinically relevant concentrations. In ovarian cancer cells, Imatinib has predominantly antiproliferative effects, arresting cells at G0/G1 (Fig. 4)
, mechanism previously recorded for bcr-abl-positive hematopoietic cells and for PDGFR-expressing glioblastoma cells (46
, 54
, 55)
. Other reports have noted that Imatinib induces a significant increase in apoptosis in different tumor models. For instance, proapoptotic effects after treatment with Imatinib have been recorded in leukemic, gastrointestinal stromal tumor, and in dermatofibroma cell lines (44
, 47
, 56
, 57) . We have not observed an increased apoptotic fraction by Imatinib in ovarian cancer cells. Similarly, no increase in apoptosis has been recorded in glioblastoma and small cell lung cancer cells (45
, 46)
, suggesting that Imatinib inhibits cell growth through different mechanisms in different cell lines.
Our results indicate that Imatinib inactivates Akt in a dose-responsive manner and to a greater degree than MAPK in PDGFR-expressing cells. Overexpression of myristoylated Akt1 in ovarian cancer cells induces partial resistance to growth inhibition by Imatinib, implicating Akt as an important effector of growth promotion downstream of PDGFR. Very interestingly, constitutively active Akt1 prevents the growth arrest induced by Imatinib, by allowing cells to progress through S phase. The role of Akt in regulating cell cycle progression by interference with p27 intracellular trafficking (38)
and by regulation of protein translation via p70-S6 kinase and 4E-BP1 phosphorylation could account for these antiproliferative effects. The partial growth inhibition by Imatinib noted in Akt transfectants probably reflects the role of other signaling pathways downstream of PDGFR (ras-MAPK, phospholipase C , and so forth) that regulate cell growth and proliferation.
Activation of the PI3k-Akt pathway through mechanisms dependent or independent of growth factor stimulation plays a central role in ovarian cancer (9
, 11
, 15)
. It is not known whether the level of baseline Akt activation in ovarian tumors impacts prognosis and/or response to therapy. Our data show that Akt activation induces partial resistance to Imatinib mesylate in vitro. We recognize that myrAkt (used in our experiments) may be more oncogenic than the wild-type Akt, because the myristoylation signal amplifies the oncogenic potential of Akt. Thus, overexpression of myrAkt may not entirely reflect the effects exerted by overexpressed endogenous Akt (58
, 59)
, because overexpression of the membrane-bound form of Akt can nonspecifically activate other colocalized proteins (60)
. We note that ovarian cancer cell lines CaOv3 and SkOv3 that have high levels of endogenous activated Akt (Fig. 2)
are also resistant to Imatinib mesylate, although these cells do not express PDGFR.
The Akt isoform used for transfection in these experiments is Akt1, rather than the ovarian cancer-related isoform, Akt2. Although different Akt isoforms have different tissue distribution (e.g., Akt2 is the isoform overexpressed in ovarian carcinomas; Ref. 61 ), they share common downstream substrates that mediate their oncogenic signals (62) . Therefore, we believe that the effects noted as a consequence of constitutively active Akt1 expression are likely to be very similar to those obtained by activation of Akt2 or Akt3.
In conclusion, our data strongly suggest that PDGFR can be targeted therapeutically in EOC by Imatinib mesylate. In vitro, the inhibitory effects of Imatinib are restricted to PDGFR-expressing cells. We show that cancer cells with high basal levels of Akt (either endogenous or induced) are less sensitive to manipulation of the PDGF-regulated growth pathway. Apart from providing a rationale to further study PDGFR inhibition as a novel therapy for patients with EOC, our findings implicate Akt as a potential indicator of sensitivity to treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Daniela Matei, Indiana University School of Medicine, Division of Hematology-Oncology, RT-457, 535 Barnhill Drive, Indianapolis, IN 46202. Phone: (317) 278-8844; Fax: (317) 278-0074; E-mail: dmatei{at}iupui.edu
Received 5/ 7/03; revised 9/22/03; accepted 9/30/03.
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REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am. J. Pathol., 159: 431-437, 2001. gene is an oncogene in human ovarian and colon tumors. Cancer Res., 61: 7426-7429, 2001.(V)(3) and (V)ß(5) integrins and promotes cell motility. Cancer Res., 62: 5358-5364, 2002. in vascular smooth muscle cells by mechanical stress. FASEB J., 12: 1135-1142, 1998.This article has been cited by other articles:
|
|
 |
|
  T. J. Abouantoun and T. J. MacDonald Imatinib blocks migration and invasion of medulloblastoma cells by concurrently inhibiting activation of platelet-derived growth factor receptor and transactivation of epidermal growth factor receptor Mol. Cancer Ther., May 1, 2009; 8(5): 1137 - 1147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Schilder, M. W. Sill, R. B. Lee, T. J. Shaw, M. K. Senterman, A. J. Klein-Szanto, Z. Miner, and B. C. Vanderhyden Phase II Evaluation of Imatinib Mesylate in the Treatment of Recurrent or Persistent Epithelial Ovarian or Primary Peritoneal Carcinoma: A Gynecologic Oncology Group Study J. Clin. Oncol., July 10, 2008; 26(20): 3418 - 3425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Gioni, T. Karampinas, G. Voutsinas, A. E. Roussidis, S. Papadopoulos, N. K. Karamanos, and D. Kletsas Imatinib Mesylate Inhibits Proliferation and Exerts an Antifibrotic Effect in Human Breast Stroma Fibroblasts Mol. Cancer Res., May 1, 2008; 6(5): 706 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. McCabe, D. D. Spyropoulos, D. Martin, and C. S. Moreno Genome-Wide Analysis of the Homeobox C6 Transcriptional Network in Prostate Cancer Cancer Res., March 15, 2008; 68(6): 1988 - 1996. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Matei, M. Satpathy, L. Cao, Y.-C. Lai, H. Nakshatri, and D. B. Donner The Platelet-derived Growth Factor Receptor {alpha} Is Destabilized by Geldanamycins in Cancer Cells J. Biol. Chem., January 5, 2007; 282(1): 445 - 453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Krueger and S. Srivastava Posttranslational Protein Modifications: Current Implications for Cancer Detection, Prevention, and Therapeutics Mol. Cell. Proteomics, October 1, 2006; 5(10): 1799 - 1810. [Full Text] [PDF]
|
|
|
|
 |
|
 N. Holtkamp, A. F. Okuducu, J. Mucha, A. Afanasieva, C. Hartmann, I. Atallah, L. Estevez-Schwarz, C. Mawrin, R. E. Friedrich, V.-F. Mautner, et al. Mutation and expression of PDGFRA and KIT in malignant peripheral nerve sheath tumors, and its implications for imatinib sensitivity Carcinogenesis, March 1, 2006; 27(3): 664 - 671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Davidson, V. Espina, S. M. Steinberg, V. A. Florenes, L. A. Liotta, G. B. Kristensen, C. G. Trope, A. Berner, and E. C. Kohn Proteomic Analysis of Malignant Ovarian Cancer Effusions as a Tool for Biologic and Prognostic Profiling Clin. Cancer Res., February 1, 2006; 12(3): 791 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. N. Kumar, J. H. Ha, R. Radhakrishnan, and D. N. Dhanasekaran Transactivation of Platelet-Derived Growth Factor Receptor {alpha} by the GTPase-Deficient Activated Mutant of G{alpha}12 Mol. Cell. Biol., January 1, 2006; 26(1): 50 - 62. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Jones, H. Otu, D. Spentzos, S. Kolia, M. Inan, W. D. Beecken, C. Fellbaum, X. Gu, M. Joseph, A. J. Pantuck, et al. Gene Signatures of Progression and Metastasis in Renal Cell Cancer Clin. Cancer Res., August 15, 2005; 11(16): 5730 - 5739. [Abstract] [Full Text] [PDF]
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, untreated (control), 
1µM Imatinib, 
5 µM Imatinib,
10 µM Imatinib mesylate; bars, ±SD.
, serum starved;
, serum stimulation;