PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of carcinLink to Publisher's site
 
Carcinogenesis. 2008 June; 29(6): 1096–1107.
Published online 2008 February 7. doi:  10.1093/carcin/bgn026
PMCID: PMC2902396

FAK and IGF-IR interact to provide survival signals in human pancreatic adenocarcinoma cells

Abstract

Pancreatic cancer is a lethal disease accounting for the fourth leading cause of cancer death in USA. Focal adhesion kinase (FAK) and the insulin-like growth factor-I receptor (IGF-1R) are tyrosine kinases that activate common pathways, leading to increased proliferation and cell survival. Sparse information is available regarding their contribution to the malignant behavior of pancreatic cancer. We analyzed the relationship between FAK and IGF-1R in human pancreatic cancer cells, determined which downstream signaling pathways are altered following kinase inhibition or downregulation and studied whether dual kinase inhibition represents a potential novel treatment strategy in this deadly disease. Using immunoprecipitation and confocal microscopy, we show for the first time that FAK and IGF-1R physically interact in pancreatic cancer cells and that inhibition of tyrosine phosphorylation of either kinase disrupts their interaction. Decreasing phosphorylation of either FAK or IGF-1R alone resulted in little inhibition of cell viability or increased apoptosis. However, dual inhibition of FAK, using either a dominant-negative construct (FAK-CD) or small interfering RNA, and IGF-1R, using a specific small molecule tyrosine kinase inhibitor (AEW-541) or stable expression of a truncated, mutated IGF-1R, led to a synergistic decrease in cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) and increase in cell detachment and apoptosis compared with inhibition of either pathway alone. Dual kinase inhibition with FAK-CD and AEW-541 resulted in a marked increase in apoptosis when FAK was displaced from the focal adhesions. Inhibition of both tyrosine kinase activities via a novel single small molecular inhibitor (TAE 226), at low doses specific for FAK and IGF-1R, resulted in significant inhibition of cell viability, decrease in phosphorylation of ERK and Akt and increase in apoptosis accompanied by cleavage of Poly (ADP-ribose) polymerase (PARP) and activation of caspase-3 in pancreatic cancer cells. Thus, simultaneous inhibition of both tyrosine kinases represents a potential novel therapeutic approach in human pancreatic adenocarcinoma.

Introduction

Focal adhesion kinase (FAK) is a protein tyrosine kinase that, as its name suggests, is localized to focal adhesions, which are contact points between a cell and its extra cellular matrix. FAK appears to have many functions in cells, linking integrin signaling to downstream targets (1,2), acting as part of a survival signal pathway (3,4) and having a connection with cell motility (5,6). Importantly, FAK appears to play a role in tumorigenesis as it is overexpressed in cancers and inhibition of FAK sensitizes tumor cells to apoptotic cell death (79).

Insulin-like growth factor-I receptor (IGF-IR) is a transmembrane receptor, that once activated by ligand binding will activate insulin receptor substrate-1, which then initiates a cascade of events that have mitogenic, metabolic and antiapoptotic effects (10). Several studies support the significance of the IGF-1 receptor-mediated mitogenic signal in pancreatic cancer. Both IGF-1 receptor antisense oligonucleotides and anti-IGF-IR antibodies have been shown to inhibit the proliferation of human pancreatic cancer cells (1114).

It has been shown that FAK activates proliferation and inhibits apoptosis in cancer cells (15,16). The signaling pathways of FAK and IGF-IR converge since activation of either FAK or IGF-IR induces cell survival. Cross talk has been proposed between the two pathways as induction of the IGF-IR results in mitogen-activated protein kinase pathway activation and, recently, it has been shown that a member of the mitogen-activated protein kinase pathway (mitogen-activated protein kinase kinase 1) binds to FAK, linking FAK to possible activation of this pathway (17,18). Despite a greater understanding of the multiple and complex pathways that FAK or IGF-IR signal to, the investigations have been done in artificial systems and are lacking in cancer cells.

Human pancreatic cancer is an aggressive and complex malignancy with redundant survival pathways. Multiple inhibitors are utilized in this present study including dominant negatives to FAK and IGF-1R, small interfering RNA (siRNA) to FAK, a specific kinase inhibitor of IGF-1R (AEW541) (1921), and a FAK and IGF-1R dual kinase inhibitor (TAE226) (22) to emphasize the relevance of FAK and IGF-1R signaling in human pancreatic cancer cells. Previous work in our laboratory has validated the concept of FAK interacting with a receptor tyrosine kinase, epidermal growth factor receptor (EGFR), to enhance survival signaling (7). Here, we have shown for the first time that FAK and IGF-IR interact in pancreatic cancer cells, that tyrosine phosphorylation is necessary for their interaction and that inhibition of FAK and IGF-IR expression and kinase activity through multiple methods leads to decreased extracellular signal-regulated kinase (ERK) and Akt activation and a synergistic decrease in cell adhesion and viability and increased apoptosis compared with inhibition of FAK or IGF-IR alone. These data may provide the basis for future therapy targeting FAK and IGF-1R in pancreatic cancer.

Materials and methods

Cell lines and cell culture

Panc-1 and MiaPaca-2 cells were obtained from American Type Culture Collection (Rockville, MD). Panc-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1 μg/ml penicillin–streptomycin. MiaPaca-2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2.5% horse serum and 1 μg/ml penicillin–streptomycin. The L3.6pl cell lines, kindly provided by Lee Ellis (The University of Texas MD Anderson Cancer Center, Houston, TX), were maintained in modified Eagle medium supplemented with 10% FBS, 1 μg/ml penicillin–streptomycin, vitamins, 1 mmol/l sodium pyruvate, 2 mmol/l L-glutamine and non-essential amino acids. All cell lines were incubated at 37°C in a 5% CO2 humidified incubator.

Reagents and antibodies

FAK siRNA was purchased from Dharmacon RNA Technologies (Lafayette, CO). NVP-AEW 541 and TAE226 were obtained from Novartis (East Hanover, NJ). Anti-FAK monoclonal (4.47) and anti-phospho-tyrosine monoclonal (4G10) antibodies were obtained from Upstate (Lake Placid, NY). Anti-IGF-IR antibody was from Calbiochem (San Diego, CA). Anti-phospho-FAK (Tyr397) and anti-phospho-Src antibody were from Biosource (Camarillo, CA). Anti-phospho-EGFR, anti-EGFR, anti-phospho-Akt, anti-Akt, anti-phospho-ERK1/2, anti-ERK1/2, anti-cyclin B1 and anti-Aurora B were from Cell Signaling Technology (Beverly, MA). Anti-caspase 3 and anti-PARP antibodies were from BD Biosciences (San Jose, CA, catalogue #611038). Anti-β-actin antibodies were from Sigma (St Louis, MO). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were from Advanced ImmunoChemical (Long Beach, CA).

Adenoviral infections

Recombinant adenovirus carrying the LacZ or the dominant-negative FAK construct coding for amino acids 693–1052 of FAK (Ad-FAK-CD) were propagated by the Gene Therapy Center Virus Vector Core Facility of the University of North Carolina. Cells were plated at a density of 6 × 103 or 2 × 105 into culture plates and allowed to attach for 24 h. The cells were then infected with adenovirus at a viral concentration of 50–500 multiplicity of infection or focus-forming units (FFU) per cell as we have described previously (7,8,23,24). This optimal viral titer was determined by infecting cells with various doses of Ad-GFP and visualizing the percent infection by fluorescent microscopy. Treatment with 100 FFU of Ad-GFP per cell resulted in >95% infection rate. Cells were used 48 or 72 h after infection for further experiments.

siRNA transfection

Cells were plated at a density of 6 × 103 cells for 60 mm diameter or 2 × 105 cells for 100 mm diameter culture plates and allowed to attach for 24 h. The cells were then transfected with 1–10 nM of FAK siRNA or non-specific siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, California) according to the manufacturer's protocol. Several FAK siRNA sequences were utilized to screen for knock down of FAK (see supplementary Figure 1 is available at Carcinogenesis Online). The sequences of FAK siRNA found to be most effective (#5 and #8) and resulting in reproducible inhibition of FAK and, therefore, utilized in our cell lines were 5′-GAAGUUGGGUUGUCUAGAAUU-3′ and 5′-GGUUCAAGCUGGAUUAUUUUU-3′. Cells were incubated 48–72 h after transfection and then used for experiments. FAK inhibition by siRNA was verified with western blotting. The data shown are representative of experiments done in triplicate.

Immunoprecipitation and western blotting

Cells were washed twice with ice cold 1× phosphate-buffered saline (PBS) and lysed on ice for 30 min in buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 5 mM ethylenediaminetetraacetic acid, protease inhibitors (Complete™ Protease Inhibitor Cocktail from Roche, Nutley, New Jersey) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set I and Set II from Calbiochem). The lysates were centrifuged at 10 000 r.p.m. for 30 min at 4°C and the supernatants were analyzed. Protein concentration was determined by using Bio-Rad Protein Assay. For immunoprecipitation, 1 mg of total cell extract was used for each sample. The extracts were incubated with 1 μg of the appropriate antibody overnight at 4°C. Twenty-five microliters of protein A/G-agarose beads (Oncogene Research Products, La Jolla, California) were added and the samples were incubated with rocking for an additional 2 h at 4°C. The precipitates were washed three times with lysis buffer, resuspended in 40 μl Laemmli buffer and 35 μl was removed for western blotting. For western blotting, boiled samples containing 30 μg of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transferring to polyvinylidene difluoride membrane (Bio-Rad, Hercules, California). Western blotting was carried out according to the protocol supplied with each antibody. The immunoblots were developed with the Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Waltham, Massachusetts). The intensity of the bands in the western blots was measured with an image analysis software program (image J).

Immunofluorescent staining and confocal microscopy

Cells were fixed in 3.7% paraformaldehyde in 1× PBS for 10 min and permeabilized with 0.5% Triton X-100 for 5 min on ice. Cells were then washed with 1× PBS, blocked with 25% normal goat serum in 1× PBS for 20 min and incubated with primary antibody (1:200 dilution in 25% goat serum) for 30 min at room temperature. After washing three times with 1× PBS, cells were incubated with a Texas Red-conjugated secondary antibody (1:400 dilution in 25% goat serum) for 30 min at room temperature and washed another three times with 1× PBS before observed under the microscope. For coimmunostaining experiments, cells were incubated with another primary antibody diluted 1:100 in 25% goat serum for 1 h. After washing three times with 1× PBS, a fluorescein isothiocyanate-conjugated secondary antibody (1:100 dilution) was applied to the coverslip. Cells immunostained with FAK and IGF-IR antibodies were evaluated for colocalization with a Leica confocal microscope and the MRC-1024 confocal laser scanning system. Cells treated with FAK-CD or FAK siRNA with or without NVP-AEW541 were stained with FAK antibody and evaluated for displacement of FAK from the focal adhesions with a Zeiss microscope.

Inhibition of FAK and IGF-1R function

Cells were plated at a density of 6 × 103 cells for 60 mm3 or 2 × 105 cells for 100 mm3 culture plates and allowed to attach for 24 h. The cells were then treated following different protocols: in FAK-CD + NVP-AEW541 groups, cells were infected with Ad-FAK-CD and exposed to NVP-AEW541 at the same time; in FAK siRNA + NVP-AEW541 groups, cells were first transfected with FAK siRNA for 24 h, and then NVP-AEW541 was added to the cells. Cells were analyzed for detachment, viability and apoptosis 72 h after the siRNA transfection.

Cell viability and detachment assays

After cells were treated, cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (CellTiter 96® AQueous, Promega, Madison, WI). Briefly, 20 μl of the tetrazolium compound was added to each well. The cells were then incubated at 37°C for 1 h. The plate was read at 490 nm with a plate reader to determine the viability. In detachment assays, detached and attached cells were harvested separately and counted in a hemocytometer. The percentage of detachment was calculated by dividing the number of detached cells by the total number of cells.

Apoptosis assays

After treatment, attached and detached cells were collected, counted and prepared for terminal uridine deoxynucleotidyl transferase (TUNEL) assay by utilizing an APO-BRDU kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. Stained cells were analyzed with a fluorescence-activated cell sorting-Calibur flow cytometer (BD Biosciences). Calculation of the percentage of apoptotic cells in the sample was completed with CellQuest software (BD Biosciences). Apoptotic cells were also analyzed by Hoechst staining. Hoechst 33342 (1 μg/ml) was added to the fixed cells and the specimens were mounted on glass coverslips. The slides were viewed under the Zeiss microscope for apoptotic nuclei. The percent of apoptotic cells was calculated as the ratio of apoptotic cells to total number of cells as we have described previously (7,25). Over 300 cells per sample were analyzed.

Statistical analysis

Student's t-test was used to determine significance. Data was significant if P < 0.05. Data are shown from representative experiments performed in triplicate.

Results

IGF-1 stimulation of cell proliferation is dependent on FAK. FAK is known to be an integrator of signals from integrin activation and receptor tyrosine kinase/growth factors (26,27). To directly test a relationship between IGF-IR and FAK, we used normal and FAK-null mouse embryonic fibroblasts to determine whether IGF-1 stimulation of cell proliferation through IGF-1R activation was dependent on the presence of FAK. Western blot analysis demonstrates absence of FAK in the FAK −/− cells (Figure 1A). While basal tyrosine phosphorylation of IGF-1R was noted in both FAK −/− and FAK +/+ cells due to growth in 10% FBS, stimulation with exogenous IGF-1 induced more IGF-1R phosphorylation in FAK +/+ (lanes 2 and 4) than in FAK −/− cells (lanes 1 and 3) (Figure 1B). Furthermore, cell proliferation was significantly increased (1.5-fold) with IGF-1 stimulation in FAK wild-type fibroblasts (FAK +/+) compared with FAK-null cells (FAK −/−) (Figure 1C). This demonstrates the importance of the presence of FAK for IGF-1 to exert its proliferative effects and confirms interaction between the two kinases.

Fig. 1.
IGF-1 stimulation of mouse embryo fibroblast proliferation is dependent on FAK. (A) Whole-cell extracts from untreated mouse embryo fibroblasts were analyzed by western blot for FAK expression. (B) Untreated and treated (100 ng/ml IGF-1) mouse embryo ...

FAK and IGF-IR interact in pancreatic cancer cells. One of the possible mechanisms for the cross talk between FAK and IGF-1R involve a physical interaction between these two kinases. For the next series of experiments, two pancreatic cancer cell lines were studied that both constitutively expressed FAK and IGF-1R but the relative amount of FAK and IGF-1R are increased in Panc-1 compared with MiaPaca-2 cells (Figure 2A). To determine if FAK and IGF-1R physically interact, FAK or IGF-1R was immunoprecipitated from Panc-1 and MiaPaca-2 whole-cell extracts. The immunoprecipitated complexes were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed for the presence of FAK and IGF-1R (Figure 2B). FAK was present in the samples immunoprecipitated with the IGF-1R antibody. Similarly, IGF-1R was present in the samples immunoprecipitated with the FAK antibody. Importantly, the complex of FAK and IGF-1R is more immunoprecipitated in Panc-1 cells with increased FAK and IGF-1R expression than in MiaPaca-2 cells (Figure 2B). These results demonstrate for the first time that FAK and IGF-1R physically interact in two pancreatic adenocarcinoma cells.

Fig. 2.
FAK and IGF-1R physically interact and colocalize in the focal adhesions. (A) Panc 1 and Mia Paca-2 pancreatic cancer cell lines were analyzed by western blot for expression of FAK and IGF-1R. (B) FAK or IGF-1R was immunoprecipitated from whole-cell extracts. ...

FAK and IGF-1R colocalize to evaluate whether FAK and IGF-1R are located in the same cellular compartments, immunofluorescence and confocal microscopy were utilized. FAK and IGF-1R were visualized in the focal adhesions and cytoplasm of Panc-1 (Figure 2C) and MiaPaca-2 (data not shown) cells. FAK was stained with Texas red, IGF-1R was stained with fluorescein isothiocyanate and the nucleus was stained with DAPI. The areas of colocalization appear yellow in the merged (green arrow) and cross-sectional (black arrow) images. Both FAK and IGF-1R were present in the focal adhesions indicating, for the first time in pancreatic cancer cells, that they colocalize in this region (Figure 2C).

Inhibition of either IGF-1R or FAK activity with dominant negatives, siRNA or a small molecule inhibitor, does not significantly alter cell viability. To determine the optimal conditions for the combination inhibition of both FAK and IGF-1R pathways, different approaches targeting the two pathways were screened to test their effects on cell viability and the activities of FAK and IGF-1R. A small molecule kinase inhibitor, NVP-AEW541, was used to block the IGF-1R-signaling pathway (1921). NVP-AEW541 inhibited the viability of pancreatic cancer cells in a dose-dependent manner. One millimolar of NVP-AEW541, a sublethal concentration that inhibited cell viability by ~25% for MiaPaca-2 cells and 10% for Panc-1 cells, effectively blocked the cells response to IGF-1 (Figure 3A and B), therefore, this concentration was used for the dual inhibition experiments.

Fig. 3.
Inhibition of IGF-1R or FAK. (A) Panc-1 (top two panels) and MiaPaca-2 cells (bottom two panels) were treated with various doses of NVP-AEW541, Ad-FAK-CD or Ad-LacZ for 72 h. Cell proliferation was determined by CellTiter 96® AQueous. Ad-FAK-CD ...

To inhibit FAK function, cells were treated with FAK-CD, the C-terminal domain of FAK, which has previously been shown to act as a dominant negative for FAK. FAK-CD causes dephosphorylation of FAK and its relocalization from the focal adhesions, and as a result, degradation of FAK, loss of cell adhesion and cell detachment (7,8,23,24). Initially, our studies determined that 100 FFU per cell of Ad-GFP infected the pancreatic cancer cells with >95% efficiency (data not shown). Subsequently, FAK-CD caused a dose-dependent inhibition of cell viability of both cell lines (Figure 3A), whereas control Ad-LacZ did not. One hundred FFU per cell of Ad-FAK-CD, a concentration that inhibited cell viability by ~10% in both cell lines caused significant decreases in both total and phosphorylated FAK (Figure 3A and C), therefore, this dose was used later for the dual inhibition experiments. Several FAK siRNA sequences were tested for their ability to knock down FAK with variable results. The representative data shown are from sequences of FAK siRNA that caused decreases of total FAK protein levels in a dose-dependent manner and FAK dephosphorylation (Figure 3D). FAK siRNA (10 nM) was used for dual inhibition experiments since maximal FAK knock down was achieved with this dose. In all experiments, viability was inhibited to a greater extent in MiaPaca-2 than Panc-1 cells, probably due to greater constitutive presence of FAK and IGF-1R in Panc-1 cells.

Interaction of FAK and IGF-1R depends on the phosphorylation status of both kinases. To determine if inhibitors of FAK or IGF-1R tyrosine phosphorylation altered their binding, Panc-1 or MiaPaca-2 cells were treated with 100 FFU per cell of Ad-FAK-CD and/or 1 mM NVP-AEW541 for 48 h. Immunoprecipitation was performed from cell lysates and probed for FAK or IGF-1R. As shown in Figure 4A, binding of complexes containing FAK and IGF-1R was significantly decreased with inhibition of FAK or IGF-1R tyrosine phosphorylation. In Panc-1 cells, which express more FAK and IGF-1R, complexes were decreased with Ad-FAK-CD or NVP-AEW541 alone but the combination of the two inhibitors led to near complete absence of any binding. Degradation of FAK is seen frequently following treatment with Ad-FAK-CD as shown in the FAK immunoblot from the Panc-1 cells. In MiaPaca-2 cells, due to the presence of less constitutive FAK and IGF-1R, complexes were reduced at baseline and inhibition of either FAK or IGF-1R phosphorylation effectively eliminated any binding. The presence of more FAK protein in MiaPaca-2 cells treated with Ad-FAK-CD can be explained by the variability in protein loading due to different levels of complex formation in cells and overall efficacy of the immunoprecipitation.

Fig. 4.
Dual inhibition of FAK and IGF-1R disrupts binding and has synergistic effects on cell viability, detachment and apoptosis. (A) To determine if inhibitors of FAK or IGF-1R phosphorylation altered binding, Panc-1 or MiaPaca-2 cells were treated with 100 ...

Dual inhibition of FAK and IGF-1R decreases cell viability and increases detachment and apoptosis. To compare the effect of dual inhibition of FAK and IGF-1R-signaling pathways on cell viability, detachment and apoptosis, with inhibition of each pathway alone, the two pancreatic cancer cell lines were treated with NVP-AEW541, Ad-FAK-CD or FAK siRNA alone or the combination of NVP-AEW541 with Ad-FAK-CD or FAK siRNA as described in the Materials and Methods. With the combination of NVP-AEW541 and Ad-FAK-CD, synergistic effects on the inhibition of cell viability were observed in both cell lines. In Panc-1 cells, a concentration of 100 FFU per cell of Ad-FAK-CD or 1 mM of NVP-AEW541 caused 15 and 3.4% growth inhibition, respectively, whereas the combination treatment caused 38% growth inhibition. In MiaPaca-2 cells, similar findings were observed (Figure 4B). Synergistic effects of combining NVP-AEW541 with Ad-FAK-CD on cell detachment were also observed in both cell lines (Figure 4C).

In Panc-1 cells, 1 mM of NVP-AEW541 or 100 FFU per cell of Ad-FAK-CD induced 1.1 and 11.9% cell apoptosis, respectively, whereas the combination treatment caused 27.2% cell apoptosis. In MiaPaca-2 cells, 1 mM of NVP-AEW541 induced 4.3% cell apoptosis and 100 FFU per cell of Ad-FAK-CD induced 28.6% cell apoptosis, whereas the combination caused 60.1% cell apoptosis (Figure 4D). The synergistic effects of the combination of NVP-AEW541 with Ad-FAK-CD on cell apoptosis were confirmed by western blot, demonstrating that there were more PARP cleavages in the dual inhibition groups than in single treatment groups (Figure 4E).

To further prove that dual inhibition of FAK- and IGF-1R-signaling pathways has synergistic effects, the two cell lines were treated with NVP-AEW541 in combination with FAK siRNA. Utilizing MTT assay or Hoechst staining, significant effects on cell viability or apoptosis with combination treatments were found in both cell lines. MiaPaca-2 cells were most sensitive to dual inhibition with a 37% decrease in cell viability compared with control siRNA + NVP-AEW541 (Figure 5A). NVP-AEW541 combined with FAK siRNA increased apoptosis by 1.7-fold in Panc-1 cells and by 2-fold in MiaPaca-2 cells compared with single treatments (Figure 5B). These effects were confirmed by western blot showing that there were more PARP and caspase-3 cleavage in the combination treatment groups than in single treatment groups (Figure 5C). In addition, there was a greater decrease in p-ERK in the combination treatment groups than in single treatment groups with no changes in the level of total ERK (Figure 5C). The differential effects on apoptosis seen following FAK inhibition with Ad-FAK-CD compared with siRNA maybe explained by the ability of FAK-CD to displace FAK from the focal adhesions. Treatment with siRNA reduces but does not eliminate FAK expression and FAK is still observed in the adhesions. This is demonstrated by immunofluorescence studies in the Panc-1 cells (Figure 5D).

Fig. 5.
Dual inhibition of FAK and IGF-1R through different methods alters focal adhesions, decreases cell viability and increases apoptosis through downregulation of ERK. (A and B) Cells were transfected with 10 nM of control siRNA or FAK siRNA for 24 h and ...

Pancreatic cancer cells expressing a dominant-negative IGF-1R are sensitive to FAK inhibition. In the next experiment, we utilized a dominant-negative model for IGF-1R inhibition to confirm the synergistic effect of simultaneous FAK and IGF-1R inhibition on cell apoptosis. L3.6pl pancreatic adenocarcinoma cells were transfected with a vector expressing a dominant-negative IGF-IR construct, L3-DN, or an empty vector (pcDNA), L3-Mock. Expression of the dominant-negative IGF-IR, truncated at position 952, results in inhibition of IGF-IR phosphorylation and signal transduction (14). As can be seen in Figure 5E, untreated L3-Mock and L3-DN cells express similar amounts of total FAK, p-FAK and total IGF-1R. However, following a 30 min stimulation with IGF-1 (100 ng/ml), L3-Mock have a significant increase in phosphorylation of the IGF-1R compared with L3-DN.

Subsequently, L3-Mock and L3-DN cells were treated with FAK-CD. The treatment caused a significant increase in detachment (5 versus 30%) and apoptosis (5 versus 50%) in the L3-DN cells, whereas only minor effects were observed in L3-Mock cells (Figure 5F and G). These results demonstrated the importance of the interaction of FAK with IGF-1R in pancreatic cancer cells and indicated the significance of simultaneous inhibition of their activities in these cells.

A small molecule tyrosine kinase inhibitor, TAE226, causes a dose-dependent inhibition of FAK and IGF-1R phosphorylation in pancreatic cancer cells. To test another inhibitor of FAK, in addition to the dominant-negative FAK-CD or siRNA, and another inhibitor of IGF-1R, in addition to the dominant negative or AEW541 kinase inhibitor, we utilized a novel small molecule tyrosine kinase inhibitor, TAE226, shown to inhibit both FAK and IGF-1R activities (22). This inhibitor has been shown to inhibit human glioma tumor cell growth but has not been studied in pancreatic cancer cells. We first investigated the specificity of TAE226 in pancreatic cancer cells. In both cell lines, 1 h treatment of TAE226 significantly decreased the activity of FAK in a dose-dependent manner, demonstrated by decreases in Tyr397 phosphorylation (Figure 6A). One hour of TAE226 treatment effectively blocked both cell lines' responses to IGF-1 without changing total IGF-1R levels (Figure 6B). Of note, similar to the results seen previously regarding the increased resistance of Panc-1 cells to FAK or IGF-1R inhibition (Figure 3), TAE226 treatment had greater effects in decreasing FAK and IGF-1R phosphorylation in MiaPaca-2 cells than Panc-1 cells. In contrast to the changes observed in phosphorylated FAK and IGF-1R, TAE226 treatment had no effect on basal levels of p-Src (Figure 6A, lower two panels). In addition, both cell lines have high levels of EGFR, and their responses to epidermal growth factor were not affected by TAE226 (Figure 6C). These data show for the first time the specificity of TAE226 to target FAK and IGF-1R activities in pancreatic cancer cells.

Fig. 6.Fig. 6.
Effect of TAE226 on phosphorylation of tyrosine kinases, cell viability, apoptosis and signaling to ERK and Akt. (A) Cells were treated with different doses of TAE226 for 1 h and harvested. Cell extracts were analyzed by western blot for the changes in ...

TAE226 causes decreases in cell viability and increases in cell detachment and apoptosis in pancreatic cancer cells. Since FAK and IGF-IR are important survival signals in cancer cells, and FAK also is involved in cancer cell adhesion, the effects of TAE226 on cell viability, detachment and apoptosis were further explored. The Inhibitory concentration 50% for TAE226 is cell type specific and the doses utilized in our pancreatic cancer cells were the lowest doses that had the effects demonstrated.

Dose- and time-dependent effects on cell viability were found in both cell lines. MiaPaca-2 cells were more sensitive to TAE226 for inhibition of cell viability than Panc-1 cells. After 72 h of treatment, to induce 50% growth inhibition, >5 μM of TAE226 was needed in Panc-1 cells versus only 1 μM in MiaPaca-2 cells (Figure 6D). Therefore, for subsequent studies involving detachment, apoptosis and biochemistry, 5 and 3 mM were the maximal doses of TAE226 utilized in Panc-1 and MiaPaca-2 cells, respectively. Similar patterns of sensitivities were seen in both cell lines in regards to the cell detachment induced by TAE226. TAE226 caused a dose-dependent increase in cell detachment. Treatment with 1 μM of TAE226 for 72 h caused twice as much cell detachment in MiaPaca-2 cells as in Panc-1 cells (Figure 6E). These results are consistent with the inhibitory effect of TAE226 on FAK and IGF-IR activities in pancreatic cancer cells and indicate that TAE226 inhibits cell proliferation by blocking FAK- and IGF-IR-signaling pathways.

To further characterize the effect of TAE226, pancreatic cancer cells were analyzed for induction of apoptosis following TAE226 treatment. TAE226 induced apoptosis in both cell lines in a dose-dependent manner. Two approaches were used for evaluating cellular apoptosis. Utilizing Hoechst staining, after 72 h of treatment, a dose of 3 μM of TAE226 caused twice as much apoptosis in MiaPaca-2 cells compared with a dose of 5 μM TAE226 in Panc-1 cells, demonstrated by changes in the characteristics of the nuclei (Figure 6F). In addition, after 72 h of treatment, TAE226 induced the activation of caspase-3 and cleavage of PARP in both cell lines in a dose-dependent manner. Western blot also demonstrated that TAE226 caused more apoptosis in MiaPaca-2 cells than Panc-1 cells (Figure 6G). Thus, for the first time we have demonstrated that dual inhibition of both FAK and IGF-1R phosphorylation with a small molecule kinase inhibitor leads to decreased cell viability and increased detachment and apoptosis in human pancreatic cancer cells.

TAE226 decreases ERK and Akt phosphorylation in pancreatic cancer cells. We studied the effects of TAE226 on signaling markers of cell proliferation, ERK, and cell survival, Akt, to understand the mechanism of cell apoptosis induced by TAE226. Dose-dependent decreases in the phosphorylation of Akt and ERK were found after TAE226 treatment in both cell lines with a more pronounced effect in MiaPaca-2 cells (Figure 6). This result is consistent with the antiproliferative effect of TAE226 in pancreatic cancer cells and confirms that TAE226 inhibits cell proliferation by inducing cell apoptosis through blocking FAK and IGF-IR activities and downstream signals including phosphorylation of Akt and ERK. Of note, treatment with TAE226, at similar doses to NVP-AEW541, resulted in a greater effect on proliferation and apoptosis possibly due to its effects on both FAK and IGF-1R kinase activities.

Discussion

Pancreatic cancer cells show increased resistance to chemotherapeutic agents compared with other cancer cells and the molecular mechanisms of this resistance are not fully known. However, it is known that this tumor possesses among the highest number of genetic alterations of any cancer cell and this may explain the difficulties encountered in the treatment of this aggressive malignancy (28,29). In the present study, we have demonstrated for the first time that FAK and IGF-1R physically interact in human pancreatic cancer cells and that these cells have survival signals operative through FAK and IGF-IR activities. We have shown through the use of models in fibroblast and cancer cells, and with the use of multiple inhibitors including transient expression of a FAK dominant negative (Ad FAK-CD), FAK knock down with siRNA, stable expression of an IGF-1R dominant negative, a selective small molecule inhibitor of IGF-1R (AEW-541) and a novel small molecule inhibitor of both FAK and IGF-1R (TAE226) that dual inhibition of both kinases synergistically induces cell detachment, decreases cell viability and increases apoptosis through pathways involving ERK and Akt. In addition, their interaction is dependent on tyrosine phosphorylation as inhibitors of phosphorylation prevent binding.

IGF-IR has been shown to provide important survival signals in many types of cancers. Several studies support the significance of the IGF-1 receptor-mediated mitogenic signal in pancreatic cancer. In a nude mouse model of human pancreatic cancer, it was shown that IGF-IR inhibition leads to decreases in pancreatic tumor volume and weight, vessel density and tumor cell proliferation (14).

In contrast to the more extensive evaluation of IGF-IR, FAK expression and its importance in pancreatic cancer as a survival signal has not been fully elucidated. FAK localizes to human chromosome 8q24 and may be upregulated in pancreatic cancer due to the increased copy number of this chromosome that has been demonstrated in this malignancy (30). Of note, a recent study did show that inhibition of FAK with siRNA sensitized pancreatic cancer cells and tumor xenografts to gemcitabine chemotherapy (31).

FAK and IGF-IR have redundant signaling pathways since activation of either kinase will signal to the PI3K–Akt pathway. We demonstrate that these kinases interact in fibroblasts since cell proliferation is significantly increased following phosphorylation of the IGF-1R in FAK wild-type compared with FAK-null cells. This demonstrates the importance of the presence of FAK for downstream proliferative effects following activation of the IGF-1R.

There have been no previous studies which have examined the effect of strategies that target FAK and IGF-1R interactions in human pancreatic cancer cells. Utilizing multiple inhibitors of IGF-1R (dominant negative and kinase inhibitors) and FAK (dominant negative, siRNA and kinase inhibitor), we have shown that inhibition of the activity of both tyrosine kinases resulted in a synergistic decrease in cell proliferation, increase in cell detachment and increase in apoptosis. The mechanism for this synergistic effect appears to be through pathways that involve ERK and Akt. Both p-ERK and p-Akt were decreased following dual inhibition of FAK and IGF-1R. We found that siRNA to FAK does not appear to be as effective as Ad-FAK-CD in inducing apoptosis when combined with IGF-1R inhibition. It appears that displacement of FAK from the focal adhesions, which occurs to a greater extent in the presence of Ad-FAK-CD compared with FAK siRNA, is important for a synergistic effect on apoptosis. Alternatively, the differential effects observed between the two approaches may be related to the efficiency of infection with it being >90% for adenovirus and less for siRNA. Furthermore, TAE226 which inhibited both FAK and IGF-1R tyrosine kinase activities led to the inhibition of cell growth and increased apoptosis at lower doses than individual FAK or IGF-1R kinase inhibitors. The marked effects of the small molecule kinase inhibitor, TAE226, on apoptosis suggest that it is the kinase activity of FAK and IGF-IR that is important for cell survival.

Finally, treatment with TAE226, at similar doses to NVP-AEW541, resulted in a greater effect on proliferation and apoptosis possibly due to its effects on both FAK and IGF-1R kinase activities. The reduction in phosphorylation by TAE226 appears to be specific for FAK and IGF-1R signaling as concentrations of TAE226 which inhibited FAK and IGF-1R phosphorylation did not inhibit EGFR or Src phosphorylation. However, we cannot entirely rule out the possibility that TAE226 may affect the phosphorylation of other tyrosine kinases in addition to FAK and IGF-1R.

While siRNA and dominant negative constructs are good experimental approaches to study the mechanisms and signaling pathways in an in vitro model system, their availability in the clinic is very limited. Many small molecule tyrosine kinase inhibitors have been found to have good clinical profiles. Targeting more than one signaling pathway in cancer cells with a single drug is a new direction for the development of anticancer drugs. TAE226 may fit into this category.

It appears that FAK cooperates with IGF-IR signaling to suppress apoptosis and to enhance survival in pancreatic cancer cells. Studies of FAK in some malignancies have shown that up-regulation of FAK expression is an early event in tumorigenesis, occurring before the tumor has developed the capacity for invasion and metastasis (32). FAK functions not only as a kinase but also as a scaffolding protein for the assembly of a number of cellular signaling molecules, suggesting that FAK is a critical mediator of cell-extra cellular matrix signaling events. This scaffolding function appears to be important in preventing apoptosis since knocking FAK out of the focal adhesions with Ad-FAK-CD leads to a marked increase in apoptosis. This finding has been seen in previous studies from our laboratory (33,34).

Understanding and targeting molecular alterations in human pancreatic cancer will be essential to improve patient survival. However, a recent trial that targeted a single molecular alteration in pancreatic cancer showed minimal improvement in patient survival (35). Clearly, rationale combinations of molecular targeted inhibitors will be important in the design of future trials. This study in human pancreatic cancer cells, for the first time, has shown through several approaches that inhibition of FAK and IGF-1R activities leads to a synergistic decrease in cell viability and increase in cell detachment and apoptosis. Therefore, this demonstrates clear evidence that FAK and IGF-IR represent suitable targets worthy of evaluation in preclinical models of pancreatic cancer.

Supplementary material

Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/

Funding

Career Development Award from the University of Florida to SNH; National Institutes of Health (CA113766-02) to SNH.

Supplementary Material

[Supplementary Data]

Acknowledgments

We would like to thank Dr Lee Ellis (University of Texas, MD Anderson Cancer Center) for providing L3.6pl parental, mock and IGF-IR dominant-negative cells. We also thank members of the Flow Cytometry Core Laboratory at the University of Florida for help with confocal microscopy and fluorescence-activated cell sorting techniques.

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

EGFR
epidermal growth factor receptor
ERK
extracellular signal-regulated kinase
FAK
focal adhesion kinase
FBS
fetal bovine serum
FFU
focus-forming unit
IGF-IR
insulin-like growth factor-I receptor
PBS
phosphate-buffered saline
siRNA
small interfering RNA

References

1. Schlaepfer DD, et al. Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol. Cell. Biol. 1998;18:2571–2585. [PMC free article] [PubMed]
2. Schlaepfer DD, et al. Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 1998;8:151–157. [PubMed]
3. Frisch SM, et al. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol. 1996;134:793–799. [PMC free article] [PubMed]
4. Ilic D, et al. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J. Cell Biol. 1998;143:547–560. [PMC free article] [PubMed]
5. Cary LA, et al. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 1996;109:1787–1794. [PubMed]
6. Ilic D, et al. Impairment of mobility in endodermal cells by FAK deficiency. Exp. Cell Res. 1996;222:298–303. [PubMed]
7. Golubovskaya V, et al. Dual inhibition of focal adhesion kinase and epidermal growth factor receptor pathways cooperatively induces death receptor-mediated apoptosis in human breast cancer cells. J. Biol. Chem. 2002;277:38978–38987. [PubMed]
8. Golubovskaya VM, et al. Simultaneous inhibition of focal adhesion kinase and SRC enhances detachment and apoptosis in colon cancer cell lines. Mol. Cancer Res. 2003;1:755–764. [PubMed]
9. Xu LH, et al. Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth Differ. 1996;7:413–418. [PubMed]
10. Vincent AM, et al. Control of cell survival by IGF signaling pathways. Growth Horm. IGF Res. 2002;12:193–197. [PubMed]
11. Ishiwata T, et al. Altered expression of insulin-like growth factor II receptor in human pancreatic cancer. Pancreas. 1997;15:367–373. [PubMed]
12. Bergmann U, et al. Increased expression of insulin receptor substrate-1 in human pancreatic cancer. Biochem. Biophys. Res. Commun. 1996;220:886–890. [PubMed]
13. Bergmann U, et al. Insulin-like growth factor I overexpression in human pancreatic cancer: evidence for autocrine and paracrine roles. Cancer Res. 1995;55:2007–2011. [PubMed]
14. Stoeltzing O, et al. Regulation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and angiogenesis by an insulin-like growth factor-I receptor autocrine loop in human pancreatic cancer. Am. J. Pathol. 2003;163:1001–1011. [PubMed]
15. Yamamoto D, et al. FAK overexpression upregulates cyclin D3 and enhances cell proliferation via the PKC and PI3-kinase-Akt pathways. Cell. Signal. 2003;15:575–583. [PubMed]
16. McLean GW, et al. The role of focal-adhesion kinase in cancer—a new therapeutic opportunity. Nat. Rev. Cancer. 2005;5:505–515. [PubMed]
17. Arbet-Engels C, et al. C-terminal Src kinase associates with ligand-stimulated insulin-like growth factor-I receptor. J. Biol. Chem. 1999;274:5422–5428. [PubMed]
18. Yujiri T, et al. MEK kinase 1 interacts with focal adhesion kinase and regulates insulin receptor substrate-1 expression. J. Biol. Chem. 2003;278:3846–3851. [PubMed]
19. Garcia-Echeverria C, et al. In vivo antitumor activity of NVP-AEW541—a novel, potent, and selective inhibitor of the IGF-1R kinase. Cancer Cell. 2004;5:231–239. [PubMed]
20. Scotlandi K, et al. Antitumor activity of the insulin-like growth factor-1 receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors. Cancer Res. 2005;65:3868–3876. [PubMed]
21. Hopfner M, et al. Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma cells. Biochem. Pharmacol. 2006;71:1435–1448. [PubMed]
22. Liu TJ, et al. Inhibition of both focal adhesion kinase and insulin-like growth factor-1 receptor kinase suppresses glioma proliferation in vitro and in vivo. Mol. Cancer Ther. 2007;6:1357–1367. [PubMed]
23. Park HB, et al. Activated Src increases adhesion, survival and alpha2-integrin expression in human breast cancer cells. Biochem J. 2004;378((Pt 2)):559–567. [PubMed]
24. Garces CA, et al. Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells. Cancer Res. 2006;66:1446–1454. [PubMed]
25. Kurenova E, et al. Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol. Cell. Biol. 2004;24:4361–4371. [PMC free article] [PubMed]
26. Sieg DJ, et al. FAK integrates growth-factor and integrin signals to promote cell migration. Nat. Cell Biol. 2000;2:249–256. [PubMed]
27. Baron V, et al. p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors. J. Biol. Chem. 1998;273:7162–7168. [PubMed]
28. Jimeno A, et al. Molecular biomarkers: their increasing role in the diagnosis, characterization, and therapy guidance in pancreatic cancer. Mol. Cancer Ther. 2006;5:787–796. [PubMed]
29. Maitra A, et al. Molecular pathogenesis of pancreatic cancer. Best Pract. Res. Clin. Gastroenterol. 2006;20:211–226. [PubMed]
30. Mahlamaki EH, et al. Frequent amplification of 8q24, 11q, 17q, and 20q-specific genes in pancreatic cancer. Genes Chromosomes Cancer. 2002;35:353–358. [PubMed]
31. Duxbury MS, et al. RNA interference targeting focal adhesion kinase enhances pancreatic adenocarcinoma gemcitabine chemosensitivity. Biochem. Biophys. Res. Commun. 2003;311:786–792. [PubMed]
32. Cance WG, 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;6:2417–2423. [PubMed]
33. Xu LH, et al. The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth Differ. 1998;9:999–1005. [PubMed]
34. Xu L-H, et al. The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. J. Biol. Chem. 2000;275:30597–30604. [PubMed]
35. Moore MJ. Brief communication: a new combination in the treatment of advanced pancreatic cancer. Semin. Oncol. 2005;32:5–6. [PubMed]

Articles from Carcinogenesis are provided here courtesy of Oxford University Press