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Cancer Research 67, 10976, November 15, 2007. doi: 10.1158/0008-5472.CAN-07-2667
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Therapeutic Efficacy of a Novel Focal Adhesion Kinase Inhibitor TAE226 in Ovarian Carcinoma

Jyotsnabaran Halder1, Yvonne G. Lin1, William M. Merritt1, Whitney A. Spannuth1, Alpa M. Nick1, Toshiyuki Honda6, Aparna A. Kamat1, Liz Y. Han1, Tae Jin Kim1,7, Chunhua Lu1, Ana M. Tari3, William Bornmann2, Ariel Fernandez3,5, Gabriel Lopez-Berestein3 and Anil K. Sood1,4

Departments of 1 Gynecologic Oncology, 2 Experimental Diagnostic Imaging, Chemistry Section, 3 Experimental Therapeutics, and 4 Cancer Biology, The University of Texas M. D. Anderson Cancer Center, and 5 Department of Bioengineering and Division of Applied Physics, Rice University, Houston, Texas; 6 Discovery Biology, Novartis Institutes for BioMedical Research, Tsukuba, Ibaraki, Japan; and 7 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Cheil General Hospital, Kwandong University College of Medicine, Seoul, South Korea

Requests for reprints: Anil K. Sood, Departments of Gynecologic Oncology and Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1155 Herman Pressler, Unit 1362, Houston, TX 77030. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: asood{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Focal adhesion kinase (FAK) overexpression is frequently found in ovarian and other cancers and is predictive of poor clinical outcome. In the current study, we characterized the biological and therapeutic effects of a novel FAK inhibitor, TAE226. Taxane-sensitive (SKOV3ip1 and HeyA8) and taxane-resistant (HeyA8-MDR) cell lines were used for in vitro and in vivo therapy experiments using TAE226 alone and in combination with docetaxel. Assessment of cytotoxicity, cell proliferation [proliferating cell nuclear antigen (PCNA)], angiogenesis (CD31), and apoptosis (terminal nucleotidyl transferase–mediated nick end labeling) were done by immunohistochemistry and immunofluorescence. In vitro, TAE226 inhibited the phosphorylation of FAK at both Y397 and Y861 sites, inhibited cell growth in a time- and dose-dependent manner, and enhanced docetaxel-mediated growth inhibition by 10- and 20-fold in the taxane-sensitive and taxane-resistant cell lines, respectively. In vivo, FAK inhibition by TAE226 significantly reduced tumor burden in the HeyA8, SKOV3ip1, and HeyA8-MDR models (46–64%) compared with vehicle-treated controls. However, the greatest efficacy was observed with concomitant administration of TAE226 and docetaxel in all three models (85–97% reduction, all P values <0.01). In addition, TAE226 alone and in combination with chemotherapy significantly prolonged survival in tumor-bearing mice. Even in larger tumors, combination therapy with TAE226 and docetaxel resulted in tumor regression. The therapeutic efficacy was related to reduced pericyte coverage, induction of apoptosis of tumor-associated endothelial cells, and reduced microvessel density and tumor cell proliferation. The novel FAK inhibitor, TAE226, offers an attractive therapeutic approach in ovarian carcinoma. [Cancer Res 2007;67(22):10976–83]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ovarian cancer is the fifth most common cause of cancer-related deaths in women and the most frequent cause of death from a gynecologic malignancy (1). Despite advances in surgical and chemotherapeutic approaches, the resistance of cancer cells to traditional cytotoxic agents is a major obstacle in clinical cancer therapy, and most patients eventually succumb to their disease (2). Therefore, novel therapeutic approaches for ovarian cancer are needed.

Focal adhesion kinase (FAK) is a 125-kDa non-receptor protein tyrosine kinase that was first identified in Src-transformed fibroblasts (3). FAK is overexpressed in many tumors, including those derived from the head and neck, colon, breast, prostate, liver, and thyroid (4–13). We have previously shown that FAK is overexpressed in a substantial proportion of ovarian cancers and is predictive of poor clinical outcome. The overexpression of FAK may be related to FAK gene amplification (14). FAK has been shown to play a significant role in cell survival, migration, and invasion (15–19). For example, decreased FAK phosphorylation by the dominant negative FAK-related non-kinase resulted in reduced migration and invasion of ovarian cancer cells in vitro (18, 20, 21). FAK overexpression has also been suggested to protect cells from stressors such as chemotherapy by activation of the phosphoinositide-3-kinase–AKT survival pathway, activation of nuclear factor-{kappa}B, and induction of inhibitor-of-apoptosis proteins (22). We have previously shown that FAK is cleaved after treatment with docetaxel chemotherapy in a caspase-3–dependent manner, and that FAK down-regulation promoted docetaxel cytotoxicity in ovarian cancer cells (23). Moreover, in vivo FAK silencing using small interfering RNA (siRNA) incorporated in a neutral nanoparticle resulted in antitumor and antiangiogenic effects in orthotopic models of ovarian carcinoma (24). In both studies, FAK silencing enhanced the effects of chemotherapy (23, 24). Although siRNA-based therapeutic approaches are gradually being developed for human use, small-molecule inhibitors have been developed against many targets and have been integrated into clinical trials. Based on the known role of FAK in ovarian cancer pathogenesis and the encouraging results with FAK gene silencing, we tested the efficacy of a novel FAK inhibitor, TAE226, in multiple ovarian cancer models.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. The derivation, source, and propagation of the human ovarian cancer cell lines, SKOV3ip1 and HeyA8, have been described previously (25). The taxane-resistant cell line, HeyA8-MDR, was a kind gift from Dr. Isaiah Fidler (The University of Texas M. D. Anderson Cancer Center) and was maintained in RPMI 1640 supplemented with 300 µg/mL of paclitaxel (Bristol-Myers Squibb Company). Because there are no true human pericyte cell lines, we used a pericyte-like cell line, 10T1/2. These cells represent undifferentiated mesenchymal cells and have previously been used as presumptive mural cell precursors (26). All in vitro experiments were conducted with 70% to 80% confluent cultures.

Reagents. Leupeptin, aprotinin, and sodium orthovanadate were obtained from Sigma-Aldrich Company; EDTA was obtained from Life Technologies Invitrogen; and docetaxel was obtained from Sanofi-Aventis. The FAK inhibitor, TAE226, was obtained from Novartis Pharma AG (Fig. 1A ; ref. 27). TAE226 inhibits the FAK kinase domain with an IC50 of 0.0055 µmol/L in in vitro kinase assays (27). The primary antibodies used were mouse anti-FAK (Biosource International), anti–phospho-FAK [Y397] (Biosource), anti–vascular endothelial growth factor (anti-VEGF; Santa Cruz Biotechnology), anti–matrix metalloproteinase-9 (anti–MMP-9; Chemicon-Millipore), mouse anti-proliferating cell nuclear antigen (anti-PCNA) clone PC10 (DAKO), mouse anti-CD31 (PharMingen) and mouse anti-desmin (DAKO). The following secondary antibodies were used for colorimetric immunohistochemical (IHC) analysis: horseradish peroxidase (HRP)–conjugated goat anti-rabbit immunoglobulin G (IgG) F(ab')2 and HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc.), biotinylated mouse anti-goat antibody (Biocare Medical), HRP-conjugated streptavidin (DAKO), HRP-conjugated rat anti-mouse IgG2a (Serotec Harlan Bioproducts for Science, Inc.), and fluorescent Alexa 488–conjugated goat anti-rabbit IgG (Molecular Probes Invitrogen).


Figure 1
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  		Figure 1. In vitro cytotoxicity and dose and time kinetics of TAE226. A, chemical structure of TAE 226. B, ovarian cancer cell lines plated on collagen I–coated and non-coated plates were exposed to increasing doses of TAE226. Cell lysates were collected and examined by Western blot analysis for pFAKY397 and total FAK. Time-kinetic experiments were done to determine the onset and duration of action of TAE226 in down-regulating FAK phosphorylation (c, vehicle control). After treating cells with 1 µmol/L of TAE226, lysates were collected at 3, 24, 48, and 72 h and then analyzed for down-regulation of pFAK at residue Y397. C, lysates from collagen I–coated and noncoated plates were also examined by Western blot analysis for pFAKY861. B and C, show band intensity of pFAK relative to FAK. D, cytotoxicity was ascertained for docetaxel both alone (hatched line) and in conjunction with TAE226 (solid line) in HeyA8 and HeyA8-MDR cell lines. Points, means of three independent experiments. Bars, SE.

 
Western blot analysis. Whole cell lysate for SDS-PAGE and Western blot analysis for FAK expression was prepared as previously reported (24). To prepare lysate from dissected in vivo tumors, samples were snap frozen in liquid nitrogen immediately after sacrificing the animals and stored at –80°C. The lysate was incubated on ice in radioimmunoprecipitation assay buffer for 2 h before being homogenized using a mortar and pestle. The homogenized sample was centrifuged, and the supernatant was collected and stored at –80°C. Protein quantification and Western blot for FAK were done as previously reported (24). Equal loading was confirmed with ß-actin (0.1 µg/mL, Sigma Chemical). Densitometric analysis was done using the Scion Imaging software (Scion Corporation), using total FAK or actin as a control for each sample.

Cytotoxicity assay. The IC50 was determined as described previously (23). Briefly, 2,000 cells per well were seeded onto 96-well plates and allowed to adhere overnight, after which the FAK inhibitor, TAE226, was added. Twenty-four hours after incubation, the media was exchanged for one containing increasing concentrations of docetaxel dissolved in ethanol. After 96 h of docetaxel exposure, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done. The absorbance at 570 nm was recorded, and the IC50 was determined.

Animal care and orthotopic implantation of tumor cells. Female athymic mice (NCr-nu) were purchased from the National Cancer Institute–Frederick Cancer Research and Development Center. The mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with the current regulations and standards of the U.S. Department of Agriculture, the U.S. Department of Health and Human Services, and the NIH. All studies were approved and supervised by the University of Texas M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. The mice used in these experiments were 8 to 12 weeks old.

To produce tumors, SKOV3ip1, HeyA8-MDR cells (both 1 x 106 cells per 0.2 mL HBSS; Life Technologies Invitrogen), or HeyA8 cells (2.5 x 105 cells per 0.2 mL HBSS) were injected i.p. into the mice. For in vivo experiments, cells were collected by trypsinization and centrifugation at 1,000 rpm for 7 min at 4°C. Cells were then washed twice before being reconstituted in HBSS. Only single-cell suspensions with >95% viability, as determined by trypan blue exclusion, were used for the in vivo injections. Mice (n = 10 per group) were monitored daily for adverse effects of therapy and were sacrificed on day 35 (SKOV3ip1), day 28 (HeyA8 or HeyA8-MDR), or when any of the mice seemed moribund. Total body weight, tumor incidence and mass, and the number of tumor nodules were recorded. In animals bearing SKOV3ip1 tumors, the volume of malignant ascites was recorded. Tumors were either fixed in formalin and embedded in paraffin or snap frozen in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Inc.) in liquid nitrogen.

Therapy for established i.p. tumors in nude mice. Before evaluating the therapeutic effect of TAE226 combined with docetaxel in our mouse model, we first did preliminary dose-response experiments for TAE226. Seventeen days after HeyA8 cell inoculation (when i.p. tumors were palpable), a single i.p. dose (PBS or TAE226 15, 30, and 100 mg/kg) was given. Three mice from each treatment group were randomly sacrificed 8, 24, or 48 h after treatment, and tumors were collected for IHC.

To determine the antitumor effects of TAE226, SKOV3ip1, HeyA8, and HeyA8-MDR, tumor cells were injected i.p., and therapy with one of four regimens was initiated 7 days later. Treatment regimens were as follows: vehicle, TAE226 30 mg/kg p.o. daily, docetaxel 2 mg/kg (SKOV3ip1) or 2.5 mg/kg (HeyA8 and HeyA8-MDR) i.p. weekly, or TAE226 daily combined with docetaxel weekly.

Immunohistochemistry. Phosphorylated FAK (pFAK[Y397]) was assessed by IHC. Formalin-fixed, paraffin-embedded tissues were heated and then deparaffinized using xylene and declining grades of ethanol before being rehydrated in 0.1% Triton X-100 for 10 min. Endogenous peroxidases were blocked with 6% hydrogen peroxide for 30 min. Nonspecific epitopes were blocked with 5% normal horse serum and 1% normal goat serum for 30 min. All sections were then incubated with anti-pFAK[Y397] antibody (1:25) overnight at 4°C. After washing with PBS followed by Optimax buffer, the appropriate secondary antibody was applied, and visualization was done using the Vectastain ABC detection kit (Vector Labs) according to the manufacturer's instructions. The chromogenic reaction was done with 3,3'-diaminobenzidine (DAB, Phoenix Biotechnologies).

VEGF, MMP-9, and PCNA IHC analyses were done as previously described (28). Briefly, after deparaffinization and rehydration, antigen retrieval was done using either heated citrate buffer (0.1 mol/L; pH, 6.0) in a microwave (MMP-9, PCNA) or pepsin in a 37°C humidified incubator (VEGF). Endogenous peroxidases and nonspecific epitopes were blocked with 3% H2O2/methanol and 5% normal horse serum with 1% normal goat serum, respectively. Slides were then incubated with the appropriate primary antibody at the following dilutions: VEGF, 1:100; MMP-9, 1:400; anti-PCNA, 1:50, in blocking solution overnight at 4°C. After incubating with the appropriate secondary antibody conjugated to HRP, detection was achieved with DAB substrate and counterstained with Gill's No. 3 hematoxylin (Sigma). The proliferative index was calculated as the percentage of PCNA-positive cells in 10 randomly selected high-power field (HPF) at 100x per slide.

CD31 IHC was done using freshly cut frozen tissue as previously described (23). Briefly, slides were fixed in cold acetone and then incubated with anti-CD31 [platelet/endothelial cell adhesion molecule 1 (PECAM-1)]. Microvessel density (MVD) was quantified by counting the number of microvessels per 100x HPF over 10 randomly selected 0.159-mm2 fields. A microvessel was defined as a discrete CD31+ cluster or single cell adjacent to a lumen.

Terminal nucleotidyl transferase–mediated nick end labeling assay. Immunofluorescence staining for terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) was done on frozen sections as previously described (29). To quantify TUNEL-positive cells, the number of green fluorescence–positive cells was counted in 10 random 0.011-mm2 fields at 400x magnification.

Bioluminescence imaging. The luciferase-transfected HeyA8 cell line (HeyA8-Luc) was established using a Lentivirus system, as previously described (29). Treatment with TAE226 and docetaxel was initiated 17 days after i.p. injection with HeyA8-Luc cells (2.5 x 105 cells per mouse). Bioluminescence imaging and data acquisition were done twice weekly as previously reported (29) using the IVIS 100 imaging system coupled to the Living Image software (Xenogen).

Immunofluorescence staining for CD31 and desmin. Freshly cut, snap-frozen sections were double stained for CD31 and desmin as described previously (28). Briefly, after fixation in cold acetone and blocking of nonspecific epitopes, sections were incubated with CD31 antibody followed by the fluorescence-conjugated antibody. After washing with PBS, samples were incubated with desmin antibody (1:400), followed by incubation with fluorescence-conjugated antibody. Samples were counterstained with Hoeschst for 5 min and mounted. Pericyte coverage was determined by the percent of vessels with 50% or more coverage by the green fluorescence of associated desmin-positive cells in five random fields at 200x magnification for each tumor.

Microscopic analysis. IHC-stained slides were examined with a 10x objective on a Microphot-FX microscope (Nikon) equipped with a three-chip charge-coupled device color video camera (model DXC990, Sony Corp.). Immunofluorescence microscopy was done using a 20x objective on a Microphot-FXA microscope (Nikon) equipped with an HBO 100 mercury lamp and narrow band-pass filters to individually select for green, red, and blue fluorescence (Chroma Technology). Images were captured using a cooled charge-coupled device camera (model 5810, Hamamatsu) and Optimas Image Analysis software (Media Cybernetics).

Quantitative real-time reverse transcription-PCR. RNA was extracted, and reverse transcription was done using an oligo(dT) primer and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies). After PCR amplification, quantitative values were obtained, as previously described (30). The primers were obtained from Applied Biosystems.

Statistical analyses. Differences in continuous variables were analyzed using Student's t test or ANOVA as appropriate. A P value ≤0.05 was considered statistically significant. For values that were not normally distributed, the Mann-Whitney rank sum test was used. Survival analyses were done using the Kaplan-Meier method. The Statistical Package for the Social Sciences (SPSS, SPSS Inc.) was used for all statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of TAE226 on FAK phosphorylation and ovarian cancer growth. Before in vivo experiments, we first tested the in vitro effects of TAE226 on FAK phosphorylation and growth of ovarian cancer cell lines. Given that FAK is known to bind to the ß1 integrins, which consequently leads to FAK activation and autophosphorylation, we tested the effects of TAE226 on ovarian cancer cells plated on collagen I–coated plates (16). The expression of pFAKY397 was significantly inhibited by TAE226 in a dose-dependent manner on collagen I–coated plates and completely suppressed on noncoated plates following treatment with TAE226 for 1 h (Fig. 1B). Although decreases in FAK phosphorylation were noted at the 0.5-µmol/L dose, the maximal suppression occurred at 1 µmol/L. TAE226 inhibited pFAKY397 expression up to 24 h, with gradual return of expression by 72 h (Fig. 1B). Similar results were obtained with the HeyA8 cell line (data not shown). Because FAK phosphorylation at Y861 is also known to potentially influence tumor vasculature and, hence, tumorigenicity (31), we tested the efficacy of TAE226 in inhibiting pFAKY861. TAE226 decreased pFAKY861 in a dose-dependent manner; however, doses up to 5 µmol/L were required to decrease phosphorylation at this site (Fig. 1C).

Based on our prior data with sensitization of ovarian cancer cells to taxanes following FAK silencing, we tested the effect of TAE226 on ovarian cancer cell growth. The IC50 with TAE226 alone was 1 µmol/L. Docetaxel cytotoxicity was 10-fold greater in combination with TAE226 compared with docetaxel alone in HeyA8 cells (Fig. 1D) and 20-fold greater in the taxane-resistant HeyA8-MDR cells (Fig. 1D). Similar results were observed with the SKOV3ip1 and SKOV3-TR cells (data not shown).

Effect of TAE226 on ovarian tumor growth in vivo. In light of our in vitro data on the effect of TAE226 in sensitizing ovarian cancer cells to docetaxel, we examined the in vivo therapeutic potential of TAE226. Before therapy experiments, dose-finding studies were done. Mice bearing HeyA8 tumors were given a single p.o. dose of TAE226 at 15, 30, or 100 mg/kg and then sacrificed 8, 24, or 48 h later. The tumors were harvested, and pFAKY397 expression was assessed by Western blot and IHC. TAE226 doses of 30 mg/kg or greater significantly inhibited pFAKY397 by 24 h (Fig. 2A ), with the return of expression noted by 48 h. Total FAK levels were not affected by TAE226. Therefore, daily administration of TAE226 at 30 mg/kg was selected as the dosing schedule for subsequent therapy experiments.


Figure 2
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  		Figure 2. In vivo effects of TAE226 alone and in combination with docetaxel. A, down-regulation of pFAKY397 is assessed by Western blot analysis in an in vivo dose- and time-kinetic experiment with TAE226 (or vehicle) in HeyA8 tumors collected from animals 8, 24, and 48 h after a single TAE226 administration (15, 30, or 100 mg/kg). IHC staining for pFAKY397 shows down-regulation of pFAKY397 by 24 h with return of expression by 48 h from TAE226 administration (30 mg/kg). Pictures are taken at 100x. Graph shows band intensity of pFAK relative to FAK. B, mice injected with HeyA8, SKOV3ip1, and HeyA8-MDR underwent treatment with vehicle alone (Control), TAE (30 mg/kg p.o. daily), docetaxel (2 mg/kg for SKOV3ip1 or 2.5 mg/kg for HeyA8 and HeyA8-MDR) weekly, or combination TAE226 plus docetaxel. Columns, mean tumor weights; bars, SDs. *, P < 0.01, versus control; {dagger}, P < 0.05, compared with the docetaxel group. Compared with all other groups, combination TAE226 and docetaxel shows superiority in overall survival rates. C and D, based on photon measurements from bioluminescence.

 
To determine the in vivo therapeutic potential of TAE226, a series of in vivo experiments were carried out. Female nude mice bearing HeyA8 tumors in peritoneal cavity were randomly allocated to one of four treatment groups (n = 10 per group): (a) daily PBS p.o. and weekly PBS i.p. (control), (b) daily p.o. TAE226, (c) weekly i.p. docetaxel, and (d) both TAE226 and docetaxel. Therapy experiments were terminated when the control animals developed signs of significant tumor burden and seemed moribund (3 to 4 weeks of therapy depending on the cell line). There was a 54% to 79% reduction in tumor burden either by TAE226 or docetaxel monotherapy (Fig. 2B). Combination therapy resulted in the greatest tumor reduction, with 89% reduction in the HeyA8 model (P < 0.002 when compared with controls). The same therapy schema was carried out in the SKOV3ip1 model, and a 97% tumor reduction was observed in the combination group compared with controls (P < 0.001). These were no differences in body weight, feeding habits, or mobility between the groups.

Because chemotherapy resistance is a common problem in ovarian carcinoma, we also did experiments with the HeyA8-MDR model, which is resistant to several chemotherapy agents including taxanes. Interestingly, TAE226 alone showed 46% reduction in tumor weight even in this model (P < 0.04; Fig. 2B), but docetaxel alone was not effective. The combination of TAE226 and docetaxel yielded an 85% reduction in tumor growth in this model (P = 0.01) compared with controls.

To further evaluate the effects of TAE226 therapy on metastatic tumor growth, tumor incidence and number of nodules were examined from the therapy experiments described above (Table 1 ). Monotherapy with either TAE226 or docetaxel resulted in a modest reduction in the number of tumor nodules in the HeyA8 and SKOV3ip1 models, but had no effect in the HeyA8 MDR model. However, combination therapy consistently produced fewer tumor nodules in all groups (HeyA8: 78% reduction, P = 0.006; SKOV3ip1: 92%, P = 0.001; HeyA8-MDR: 14% reduction, P = NS). In addition, all treatments were effective in blocking the development of ascites in the SKOV3ip1 model: mean volume, 1.93 ± 0.58 mL in controls compared with the TAE226 (mean volume, 0.26 ± 0.11 mL), docetaxel (mean volume, 0.31 ± 0.13 mL), or combination groups (mean volume 0; P < 0.001). There were no significant differences in body weight, feeding habits, or mobility between the groups.


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  		Table 1. Tumor characteristic after TAE226/docetaxel therapy

 
Based on the encouraging results with TAE226-based therapy, we next examined its effects on survival using a moribund state as the end point. TAE226 and docetaxel each prolonged survival compared with controls, but the combination resulted in the greatest prolongation of survival (median 96 days; P < 0.001; Fig. 2C).

Effects of TAE226 on established ovarian tumors. Although the experiments described above used a model of small volume disease, patients with recurrent cancer will frequently have larger tumor burden. To test the effects of TAE226-based therapy on larger tumors, we used the HeyA8-Luc cell line. Therapy was started 2 weeks after tumor cell injection when the palpable tumor size ranged between 0.5 and 1 cm. In the TAE226 or docetaxel monotherapy groups, the rate of tumor growth was slower, but no apparent tumor regression was noted. Remarkably, in the combination group, there was 60% tumor regression noted by 15 days after initiating therapy (Fig. 2D).

Effect of pFAKY397 targeting on apoptosis, angiogenesis, and cell proliferation. To examine potential mechanisms responsible for the therapeutic effects of TAE226 and docetaxel, a series of experiments were done. First, we examined tumor samples at the conclusion of therapy for sustained FAK suppression. Indeed, TAE226 treatment resulted in decreased pFAKY397 compared with controls at the conclusion of therapy (Fig. 3A ). Because of recent studies (4, 32) related to FAK and tumor angiogenesis, we next examined the effects on vessel density using CD31 immunostaining (Fig. 3B). TAE226 and docetaxel each reduced MVD compared with controls (P = 0.007 and 0.008, respectively). The combination of these agents had an even greater effect on vessel density compared with either treatment alone (P < 0.002). Based on the known FAK-mediated regulation of VEGF and MMP-9 (32–34), we examined the expression of these angiogenic factors in tumors harvested from the various therapy groups (Fig. 3B). The expression of both VEGF and MMP-9 was significantly lower in the TAE226-based therapy groups, suggesting that the antitumor effect may be mediated, in part, by suppression of angiogenesis. To further characterize the anti-vascular mechanism, we also evaluated the effects of therapy on tumor cell and tumor-associated endothelial cell apoptosis (Fig. 3C and D). Tumors from vehicle-treated animals had largely absent apoptosis in the endothelium. Apoptotic endothelial cells were apparent in the TAE226-treated group and the docetaxel-treated group. In the combination therapy group, both endothelial cells and tumor cells had significantly greater apoptosis compared with all other groups (Fig. 3C).


Figure 3
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  		Figure 3. In vivo effects of TAE226 on pFAKY397, angiogenesis, invasion, and apoptosis. IHC of SKOV3ip1 tumors from mice receiving long-term TAE226 therapy: (A) pFAKY397 expression; (B) CD31/MVD; VEGF; MMP-9 expression; (C) CD31 (red)/TUNEL (green) immunofluorescence demonstrating increased endothelial cell apoptosis (yellow) with TAE226 treatment; (D) PCNA representing therapeutic effect of reducing the proliferative index with TAE266 treatment; and tumor cell apoptosis (green) by TUNEL staining. *, P < 0.05, versus control. The columns in all graphs correspond to the labeled columns in the picture.

 
To assess the effects of TAE226 therapy on tumor proliferation, we did IHC analysis for PCNA (Fig. 3D). Compared with tumors from mice receiving vehicle alone, the percentage of PCNA-positive cells was reduced to 22 ± 1.8% in tumors from mice that received both TAE226 and docetaxel (P < 0.005). The number of PCNA-positive cells was minimally reduced by treatment with TAE226 alone or docetaxel alone compared with vehicle. Finally, we examined tumor cell apoptosis by using the TUNEL method (Fig. 3D). Minimal apoptosis occurred in the single-agent treatment groups. Combination therapy resulted in substantially more apoptotic cells than vehicle, TAE226 alone, or docetaxel alone (Fig. 3D).

Effect of anti-FAK therapy on vessel maturation. Based on the efficacy of TAE226 in causing tumor regression, we next asked whether the mature tumor vasculature was also affected. FAK is known to play a role in the migration of vascular smooth muscle cells (35). However, the effects of FAK inhibition on perivascular cells are not known. Therefore, we examined the extent of pericyte coverage in the tumors harvested from the therapy experiments described above. Docetaxel alone had no effect on pericyte coverage, but the extent of pericyte coverage was significantly lower in the TAE226 groups (Fig. 4A ). It is known that pericytes may reduce the effectiveness of antiangiogenic therapy by providing local survival signals for endothelial cells. One of the mechanisms by which pericytes may protect tumor-associated endothelial cells is by producing VEGF locally in response to platelet-derived growth factor-BB (PDGF-BB)–mediated stimulation. To address whether FAK may play a role in this pathway, we used the pericyte-like 10T1/2 cells. As expected, both PDGF-BB and SKOV3ip1 conditioned medium (CM) stimulated VEGF mRNA production (Fig. 4B). Remarkably, TAE226 blocked the production of VEGF by the 10T1/2 cells despite stimulation with PDGF-BB or SKOV3ip1-CM. Similarly, PDGF-BB treatment resulted in a significant increase in VEGF protein production (1,055 pg/mL versus 312 pg/mL, P < 0.01) in 10T1/2-derived conditioned media. This increase was blocked by TAE226.


Figure 4
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  		Figure 4. Effect of FAK inhibition on vascular maturation in vitro and in vivo. A, TAE226 therapy alone and in combination with docetaxel decreased pericyte coverage. B, TAE226 abrogated the stimulatory effect of PDGF-BB and SKOV3ip1 conditioned media on VEGF expression as determined by real-time PCR. *, P < 0.05, versus control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main findings from our study are that FAK inhibition by TAE226 has significant therapeutic efficacy in both chemotherapy-sensitive and chemotherapy-resistant ovarian cancer models. The therapeutic efficacy was the greatest when FAK inhibition was combined with docetaxel and occurred by both direct effects on tumor cells and indirect effects on the tumor vasculature.

Ovarian cancer remains the most deadly gynecologic malignancy despite advances in chemotherapy and surgery. Although alterations in dosing and routes of administration for chemotherapy have provided incremental improvements in the survival of patients (36), the addition of more chemotherapy agents does not seem to provide any further benefit. Therefore, attention has turned toward targeting key biological pathways in both tumor cells and the microenvironment. Targeting the VEGF pathway has provided promising proof that antiangiogenic therapies can indeed improve therapeutic outcomes among cancer patients (37). Initial clinical trials with bevacizumab in ovarian cancer have shown encouraging results (38, 39). However, it is likely that additional targets may be required to achieve further gains in patient survival. FAK is an attractive therapeutic target because it is a key convergence point for many growth factor pathways required for survival and metastatic functions of cancer cells. FAK is overexpressed in a substantial proportion of ovarian cancers and is predictive of poor clinical outcome (11, 14). FAK is also overexpressed in tumor-associated endothelial cells (40) and represents a unique opportunity for concomitant targeting of tumor cells and the microenvironment.

FAK is an important regulator of signaling processes between the extracellular matrix and tumor cells (41, 42). FAK phosphorylation at Y397 follows integrin stimulation or ligand binding by growth factor receptors (43, 44). Subsequently, phosphorylation at Y397 promotes the Src homology domain 2 (SH2)–dependent binding of Src-family tyrosine kinases and the formation of an activated FAK-Src complex. FAK activation at focal adhesion sites enhances cytoskeletal reorganization, cellular adhesion, and cell survival (15, 45). FAK also functions in tumor cell migration and invasion (11). However, there are a limited number of approaches currently available for targeting FAK (31). TAE226 is a novel potent inhibitor of FAK activity that has been shown to have therapeutic efficacy in a glioma model (27). Remarkably, our study shows that TAE226-based therapy was indeed effective even in chemotherapy-resistant ovarian cancer models.

FAK is a particularly appealing target because it is overexpressed in both tumor cells and tumor-associated endothelial cells (14, 40). The effects of tumor cell FAK on the tumor microenvironment can be mediated by the regulation of angiogenic cytokines. Sheta et al. (46) showed that transcriptional induction of VEGF occurs via FAK. Subsequently, Mitra et al. (33) showed that 4T1 breast cancer cells have reduced levels of VEGF due to the inhibition of FAK activity. We have shown that either FAK silencing or suppression of FAK activity reduced VEGF and MMP-9 expression in vivo. This reduction contributed to apoptosis of tumor-associated endothelial cells. However, FAK is likely to have direct functional significance for the tumor vasculature as well. FAK is expressed in angiogenic blood vessels of malignant astrocytomas where it facilitates angiogenesis by enabling haptotactic migration toward ECM proteins (47). Recently, we isolated tumor-associated endothelial cells from human ovarian cancers and showed that FAK was overexpressed in these cells (40). Moreover, FAK inhibition in endothelial cells inhibited cell motility and tube formation assays in vitro. Therefore, the therapeutic efficacy of FAK inhibition likely results from both direct antitumor and anti-vascular effects. We extend our previous findings by demonstrating an additional mechanism whereby pericytes were also affected by the anti-FAK therapy. Pericytes provide survival signals for endothelial cells (26, 48). FAK is known to play an important role in PDGF-stimulated pericyte migration (35). Our results indicate that FAK inhibition results in reduced pericyte coverage, thereby providing an additional explanation for the observed anti-vascular effects.

A potential confounding factor with the use of small-molecule inhibitors is that several targets may be affected. For example, TAE226 can also inhibit the insulin-like growth factor-I receptor (IGF-IR) and the proline-rich tyrosine kinase 2 (Pyk2), which may play a role in the growth of ovarian and other cancers (27, 49). However, we have previously shown that FAK silencing in vivo using siRNA incorporated in neutral liposomes results in substantial antitumor activity, suggesting that FAK is indeed an important therapeutic target (24).

In summary, we have shown that TAE226 is an effective inhibitor of FAK activation. Moreover, inhibition of FAK phosphorylation in combination with docetaxel effectively inhibited ovarian cancer growth by both direct and indirect anti-vascular mechanisms. Based on these findings, FAK represents an attractive therapeutic target in ovarian cancer and supports further consideration of anti-FAK therapies for clinical development.


   Acknowledgments
 
Grant support: Y.G. Lin, W.M. Merritt, A.M. Nick, and W.A. Spannuth are supported by the National Cancer Institute (NCI)/Department of Health and Human Services/NIH Training of Academic Gynecologic Oncologist Grant (T32-CA101642). The research of A. Fernandez was supported by NIH grant GM072614. This research was supported in part by NCI grants CA110793 and CA109298, the University of Texas M. D. Anderson Cancer Center Specialized Programs of Research Excellence in Ovarian Cancer (1P50CA083639), the Marcus Foundation, and a Program Project Development Grant from the Ovarian Cancer Research Fund, Inc. to A.K. Sood.

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.

We thank Donna Reynolds and Dr. Robert Langley for assistance with immunohistochemistry and helpful discussions.


   Footnotes
 
Note: J. Halder and Y.G. Lin contributed equally to this work.

Received 7/16/07. Revised 9/ 4/07. Accepted 9/21/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30.[Abstract/Free Full Text]
  2. McGuire WP, Hoskins WJ, Brady MF, et al. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer [comment]. N Engl J Med 1996;334:1–6.[Abstract/Free Full Text]
  3. Kanner SB, Reynolds AB, Vines RR, Parsons JT. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A 1990;87:3328–32.[Abstract/Free Full Text]
  4. Kornberg LJ. Focal adhesion kinase expression in oral cancers. Head Neck 1998;20:634–9.[CrossRef][Medline]
  5. Owens LV, Xu L, Craven, RJ, et al. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 1995;55:2752–5.[Abstract/Free Full Text]
  6. Owens LV, Xu L, Dent GA, et al. Focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer. Ann Surg Oncol 1996;3:100–5.[CrossRef][Medline]
  7. Tremblay L, Hauck W, Aprikian AG, Begin LR, Chapdelaine A, Chevalier S. Focal adhesion kinase (pp125FAK) expression, activation and association with paxillin and p50CSK in human metastatic prostate carcinoma. Int J Cancer 1996;68:164–71.[Medline]
  8. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of focal adhesion kinase gene and invasive cancer. Lancet 1993;342:1024–5.[CrossRef][Medline]
  9. Glukhova M, Koteliansky V, Sastre X, Thiery JP. Adhesion systems in normal breast and in invasive breast carcinoma. Am J Pathol 1995;146:706–16.[Abstract]
  10. Han NM, Fleming RY, Curley SA, Gallick GE. Overexpression of focal adhesion kinase (p125FAK) in human colorectal carcinoma liver metastases: independence from c-src or c-yes activation. Ann Surg Oncol 1997;4:264–8.[CrossRef][Medline]
  11. Sood AK, Coffin JE, Schneider GB, et al. Biological significance of focal adhesion kinase in ovarian cancer: role in migration and invasion. Am J Pathol 2004;165:1087–95.[Abstract/Free Full Text]
  12. Judson PL, He X, Cance WG, Van Le L. Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma. Cancer 1999;86:1551–6.[CrossRef][Medline]
  13. Cance WG, Harris JE, Iacocca MV, et al. Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clin Cancer Res 2000;6:2417–23.[Abstract/Free Full Text]
  14. Bonome T, Lee JY, Park DC, et al. Expression profiling of serous low malignant potential, low-grade, and high-grade tumors of the ovary. Cancer Res 2005;65:10602–12.[Abstract/Free Full Text]
  15. Sieg DJ, Hauck CR, Ilic D, et al. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2000;2:249–56.[CrossRef][Medline]
  16. Schaller MD. Focal adhesion kinase: an integrin-linked protein tyrosine kinase. Trends Cell Biol 1993;3:258–62.[CrossRef][Medline]
  17. Schaller MD, Hildebrand JD, Parsons JT. Complex formation with focal adhesion kinase: a mechanism to regulate activity and subcellular localization of Src kinases. Mol Biol Cell 1999;10:3489–505.[Abstract/Free Full Text]
  18. Kohno M, Hasegawa H, Miyake M, Yamamoto T, Fujita S. CD151 enhances cell motility and metastasis of cancer cells in the presence of focal adhesion kinase. Int J Cancer 2002;97:336–43.[CrossRef][Medline]
  19. Hsia DA, Mitra SK, Hauck CR, et al. Differential regulation of cell motility and invasion by FAK. J Cell Biol 2003;160:753–67.[Abstract/Free Full Text]
  20. Schaller MD, Borgman CA, Parsons JT. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK. Mol Cell Biol 1993;13:785–91.[Abstract/Free Full Text]
  21. Richardson A, Parsons T. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 1996;380:538–40.[CrossRef][Medline]
  22. Sonoda A, Sonoda Y, Muramatu R, Streilein JW, Usui M. ACAID induced by allogeneic corneal tissue promotes subsequent survival of orthotopic corneal grafts. Invest Ophthalmol Vis Sci 2000;41:790–8.[Abstract/Free Full Text]
  23. Halder J, Landen CN, Jr., Lutgendorf SK, et al. Focal adhesion kinase silencing augments docetaxel-mediated apoptosis in ovarian cancer cells. Clin Cancer Res 2005;11:8829–36.[Abstract/Free Full Text]
  24. Halder J, Kamat AA, Landen CN, et al. Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy. Clin Cancer Res 2006;12:4916–24.[Abstract/Free Full Text]
  25. Landen CN, Jr., Chavez-Reyes A, Bucana C, et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 2005;65:6910–8.[Abstract/Free Full Text]
  26. Reinmuth N, Liu W, Jung YD, et al. Induction of VEGF in perivascular cells defines a potential paracrine mechanism for endothelial cell survival. FASEB J 2001;15:1239–41.[Free Full Text]
  27. Liu TJ, LaFortune T, Honda T, et al. Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo. Mol Cancer Ther 2007;6:1357–67.[Abstract/Free Full Text]
  28. Lin YG, Kunnumakkara AB, Nair A, et al. Curcumin inhibits tumor growth and angiogenesis in ovarian carcinoma by targeting the nuclear factor-{kappa}B pathway. Clin Cancer Res 2007;13:3423–30.[Abstract/Free Full Text]
  29. Lu C, Kamat AA, Lin YG, et al. Dual targeting of endothelial cells and pericytes in antivascular therapy for ovarian carcinoma. Clin Cancer Res 2007;13:4209–17.[Abstract/Free Full Text]
  30. Thaker PH, Han LY, Kamat AA, et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian cancer. Nat Med 2006;12:939–44.[CrossRef][Medline]
  31. McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role of focal-adhesion kinase in cancer—a new therapeutic opportunity. Nat Rev Cancer 2005;5:505–15.[CrossRef][Medline]
  32. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005;6:56–68.[CrossRef][Medline]
  33. Mitra S, Molina J, Hanson D, et al. Focal adhesion kinase (FAK) activity promotes tumor growth and neo-vascularization via up-regulation of angiogenic factors [abstract 2029]. Proc Am Assoc Cancer Res 2005;46:475.
  34. Shibata K, Kikkawa F, Nawa A, et al. Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretion by fibronectin in ovarian cancer cells. Cancer Res 1998;58:900–3.[Abstract/Free Full Text]
  35. Hauck CR, Hsia DA, Schlaepfer DD. Focal adhesion kinase facilitates platelet-derived growth factor-BB–stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells. J Biol Chem 2000;275:41092–9.[Abstract/Free Full Text]
  36. Markman M, Walker JL. Intraperitoneal chemotherapy of ovarian cancer: a review, with a focus on practical aspects of treatment. J Clin Oncol 2006;24:988–94.[Free Full Text]
  37. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005;23:1011–27.[Abstract/Free Full Text]
  38. Burger R, Sill M, Monk B, Greer B, Sorosky J. Phase II trial of bevacizumab in persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer: a Gynecologic Oncology Group study. In: Orlando (FL): American Society of Clinical Oncology Annual Meeting Proceedings; 2005;23:165, Part I of II:5009.
  39. Frumovitz M, Sood AK. Vascular endothelial growth factor (VEGF) pathway as a therapeutic target in gynecologic malignancies. Gynecol Oncol 2007;104:768–78.[CrossRef][Medline]
  40. Lu C, Bonome T, Li Y, et al. Gene alterations identified by expression profiling in tumor-associated endothelial cells from invasive ovarian carcinoma. Cancer Res 2007;67:1757–68.[Abstract/Free Full Text]
  41. Schlaepfer DD, Hauck CR, Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol 1999;71:435–78.[CrossRef][Medline]
  42. Schaller MD. Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim Biophys Acta 2001;1540:1–21.[Medline]
  43. Schlaepfer DD, Hunter T. Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol 1998;8:151–7.[CrossRef][Medline]
  44. Casamassima A, Rozengurt E. Insulin-like growth factor I stimulates tyrosine phosphorylation of p130(Cas), focal adhesion kinase, and paxillin. Role of phosphatidylinositol 3'-kinase and formation of a p130(Cas).Crk complex. J Biol Chem 1998;273:26149–56.[Abstract/Free Full Text]
  45. Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 1996;134:793–9.[Abstract/Free Full Text]
  46. Sheta EA, Harding MA, Conaway MR, Theodorescu D. Focal adhesion kinase, Rap1, and transcriptional induction of vascular endothelial growth factor. J Natl Cancer Inst 2000;92:1065–73.[Abstract/Free Full Text]
  47. Haskell H, Natarajan M, Hecker TP, et al. Focal adhesion kinase is expressed in the angiogenic blood vessels of malignant astrocytic tumors in vivo and promotes capillary tube formation of brain microvascular endothelial cells. Clin Cancer Res 2003;9:2157–65.[Abstract/Free Full Text]
  48. Lu C, Sood AK. Role of pericytes in angiogenesis. 2nd ed. In: Angiogenesis agents in cancer therapy. The Humana Press, Inc. In press.
  49. Resnicoff M, Ambrose D, Coppola D, Rubin R. Insulin-like growth factor-1 and its receptor mediate the autocrine proliferation of human ovarian carcinoma cell lines. Lab Invest 1993;69:756–60.[Medline]



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