A novel small molecule inhibitor of FAK decreases growth of human pancreatic cancer

Abstract

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed in many types of tumors, including pancreatic cancer, and plays an important role in cell adhesion and survival signaling. Pancreatic cancer is a lethal disease and is very resistant to chemotherapy, and FAK has been shown recently to assist in tumor cell survival. Therefore, FAK is an excellent potential target for anti-cancer therapy. We identified a novel small molecule inhibitor (1,2,4,5-Benzenetetraamine tetrahydrochloride, that we called Y15) targeting the main autophosphorylation site of FAK and hypothesized that it would be an effective treatment strategy against human pancreatic cancer. Y15 specifically blocked phosphorylation of Y397-FAK and total phosphorylation of FAK. It directly inhibited FAK autophosphorylation in a dose- and time-dependent manner. Furthermore, Y15 increased pancreatic cancer cell detachment and inhibited cell adhesion in a dose-dependent manner. Y15 effectively caused human pancreatic tumor regression in vivo, when administered alone and its effects were synergistic with gemcitabine chemotherapy. This was accompanied by a decrease in Y397-phosphorylation of FAK in the tumors treated with Y15. Thus, targeting the Y397 site of FAK in pancreatic cancer with the small molecule inhibitor, 1,2,4,5-Benzenetetraamine tetrahydrochloride, is a potentially effective treatment strategy in this deadly disease.

Introduction

Pancreatic cancer is a lethal disease resulting in an overwhelming majority of patients dying within a year of diagnosis. To date, few treatment options exist besides surgical resection and this is frequently not possible due to the extent of disease at diagnosis. Novel therapeutic approaches are urgently needed in relation to this disease.¹

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 extracellular matrix (ECM). Tyrosine phosphorylation of FAK occurs in response to clustering of integrins,² during formation of focal adhesions and cell spreading,^3–5 and upon adhesion to fibronectin.⁶ When FAK is subsequently activated by integrin binding to the ECM, it becomes tyrosine phosphorylated at its major site, Tyr397.⁷ This autophosphorylation site of FAK is a critical component in downstream signaling,⁸ providing a high-affinity binding site for the SH2 domain of Src family kinases.^9,10 The interaction between Y397-activated FAK and Src leads to a cascade of tyrosine phosphorylation of multiple sites in FAK, as well as other signaling molecules such as p130^CAS and paxillin, resulting in cytoskeletal changes and activation of other downstream signaling pathways.¹¹

FAK is involved in multiple cellular functions such as cell proliferation, survival, motility and invasion.¹² FAK inhibition through multiple approaches has been shown to decrease cellular viability and cause growth inhibition or apoptosis in tumor cells, including pancreatic cancer. FAK is important as a therapeutic target because FAK inhibition appears to have minimal effect on non-transformed cells. Antisense inhibition of FAK does not induce loss of adhesion or apoptosis in normal fibroblasts.¹³ In addition, overexpression of the carboxy-terminal domain (FAK-CD) which inhibits FAK function by competing for proteins normally associating with the FAK protein does not induce apoptosis in normal cells.¹⁴ Furthermore, 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.¹⁵

The majority of tumors from pancreatic cancer patients overexpress FAK and the subsequent silencing of the FAK gene has been shown to suppress metastasis in pancreatic cancer cells and xenograft models.¹⁶ Previously, we have demonstrated that pancreatic cancer cells have survival signals operating through FAK activity since FAK inhibition was effective in inducing cell detachment, decreasing cell proliferation and increasing apoptosis in pancreatic cancer cell lines.¹⁷ While emerging data strongly suggests that FAK is an excellent target for developmental therapeutics of cancer,¹⁸ specific inhibitors of FAK have been difficult to obtain. Recently, a FAK kinase inhibitor was reported from Novartis (NVP-TAE 226).^19,20 However, the main problem with this and other kinase inhibitors is their lack of specificity. In fact, we and others have identified that in addition to its effects on FAK function, it inhibited IGF-1R kinase activity.^17,21 Small organic molecules are particularly attractive as inhibitors of molecular targets because of the ability to modify their structures to achieve optimal target binding, and because of their ease of delivery in in-vivo systems.²²

Our goal in this study was to develop a novel and specific small molecule inhibitor of FAK that would have anti-neoplastic activity. Our hypothesis is that an inhibitor targeting the Y397 site of FAK would inhibit pancreatic tumor growth. Since the Y397 site is important for FAK survival function, we performed computer and functional modeling approaches which have been previously described.^22,23 Y15 specifically has been shown to decrease phosphorylation of Y397 and did not affect other kinases and decreased breast tumorigenesis.²³ This allows us to specifically target the Y397-site of FAK and to find small-molecule compounds that inhibit FAK function and decrease cell viability and tumor growth.

Since pancreatic cancer is very resistant to chemotherapy, and FAK has been shown to play major role in its survival, the aim of the study was to decrease tumorigenesis by targeting the Y397 site. We tested 140,000 small molecule compounds against the Y397 site of FAK. We found that 1,2,4,5-Benzenetetraamine tetrahydrochloride, called Y15, targets the Y397 site, directly and specifically decreases Y397-phosphorylation of FAK in vitro, inhibits pancreatic cancer cell viability, causes detachment, decreases cell adhesion and blocks tumor growth in vivo. Thus, targeting the Y397 site can be an effective therapy approach for developing future novel FAK inhibitors.

Results

Targeting Y397 site of FAK by structure-based molecular docking approach and NCI database screening reveals Y397 compound that significantly decreased cell viability

The crystal structure of the N-terminal (FERM) domain of FAK has been recently identified²⁴ and was available in the Protein Data Bank. We used a rapid structure-based approach combining molecular docking and functional testing to identify molecules that bound to FAK. More than 140,000 compounds with known three-dimensional structure were docked into the structural pocket of FAK containing Y397 site (Fig. 1A). This approach combined the NCI/DTP (atomic coordinates and small molecules) database with molecular docking and scoring algorithms of DOCK 5.1 program.²² Each of the 140,000 small-molecule compounds was docked in 100 different orientations using DOCK 5.1.0. Y15 had a high score of binding energy of interaction with the Y397 site of FAK. The orientation of the Y15 compound docking to Y397 site is shown in Figure 1B. The chemical name of this compound is 1,2,4,5-Benzenetetraamine tetrahydrochloride and its structure is shown in Figure 1C.²³

Figure 1.

Targeting of the Y397 site of FAK by structure-based molecular docking approach. (A) The crystal structure of FAK (FERM) domain,²⁴ with the Y397 pocket demonstrated by orange color. (B) A diagram of the Y15 compound positioned in the FAK Y397 pocket. The secondary structure of alpha helices and beta sheets of the FAK-N-terminal domain are shown. Y15 compound is shown in purple. The red dashed lines show hydrogen bonds and blue dashed line shows hydrophobic interaction. Y397 residue is shown in orange. (C) Structure of benzene-1,2,4,5-tetraamine tetrahydrochloride is shown.

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) inhibits cell viability in a dose-dependent manner

Using MTT, the effect of Y15 on pancreatic tumor cell viability at 72 hours was studied. Y15 inhibited pancreatic cancer cell viability starting at a dose of 1 μM and increased with higher doses. (Fig. 2A) Thus, Y15 inhibits viability of pancreatic cells in a dose-dependent manner. Similar results were seen for Miapaca-2 cells (data not shown).

Figure 2.

(A) The effect of Y15 on viability of pancreatic cancer cell lines. Y15 was added to Panc-1 cells for 72 hours at increasing doses and MTT assay was performed, as described in Materials and Methods. Known FAK inhibitor, TAE226 (Novartis), was used as a control. Bars show means ± standard deviations, *p < 0.05 vs. untreated. Similar results found for Miapaca-2 cells. (B) Y15 directly blocks in vitro kinase activity of FAK. In vitro kinase assay was performed with γ-ATP³², 0.1 μg of purified recombinant FAK protein and 1 μM of Y15 inhibitor for 10 minutes at room temperature, as described in Materials and Methods. Y15 directly blocks FAK kinase activity.

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) is a direct FAK autophosphorylation inhibitor

To test, whether Y15 is a direct inhibitor of FAK, we performed an in vitro kinase assay with purified FAK protein as previously described.²⁵ We performed an in vitro kinase assay with a 1 μM dose. We used a FAK kinase inhibitor (TAE226, Novartis) as a positive control (data not shown). Y15 directly blocked autophosphorylation activity of FAK (Fig. 2B). In addition, we have previously shown that Y15 did not significantly decrease kinase activity of the other kinases.²³ Thus, Y15 is a direct and specific inhibitor of FAK auto-phosphorylation.

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) blocks Y397-FAK phosphorylation in a dose dependent fashion

To test the effect of Y15 on Y397 phosphorylation, we treated Panc-1 pancreatic cancer cells with Y15 at increasing doses or TAE226 for 24 hours and performed western blotting with Y397 FAK antibody (Fig. 3A). Y15 inhibited Y397 phosphorylation of FAK starting at 0.1 μM in Panc-1 cells. At a dose of 100 μM, Y15 had the same or better inhibition as TAE226. Of note, total FAK is downregulated at higher doses of Y15. Y15 also blocked phosphorylation of the FAK downstream substrate, paxillin. Total paxillin was decreased at higher doses similar to FAK. Thus, Y15, 1,2,4,5-Benzenetetraamine tetrahydrochloride, inhibits FAK phosphorylation in a dose-dependent manner. Similar results were seen for Miapaca-2 cells (data not shown).

Figure 3.

Effect of Y15 on FAK and ERK phosphorylation. (A) Y15 decreases Y397 FAK phosphorylation in a dose-dependent manner. Y15 decreases Y397 phosphorylation in a dose-dependent manner. Cells were treated with different doses of Y15 or TAE226 inhibitor for 24 hours and western blotting was performed with Y-397 and then with FAK antibody and antibody to p-paxillin and paxillin. Western blotting with β-Actin antibody was performed to control for equal protein loading. Similar results were seen with Miapaca-2 cells (data not shown). (B) Y15 inhibits FAK autophosphorylation in a time-dependent manner. Panc-1 cells were treated with 100 μM of Y15 for 1, 4, 6 and 24 hours. Western blotting with Y397 was performed to detect Y397-FAK level. Then the blot was stripped and western blotting with FAK and β-Actin was performed. Y15 inhibits Y397-FAK phosphorylation by 24 hours. (C) Y15 blocks ERK 1/2 phosphorylation. Panc-1 cells were treated with increasing doses of Y15 or TAE 226. Western blotting after 24 hours was performed to detect p-ERK 1/2 and total ERK protein. Y15 inhibits ERK 1/2 phosphorylation in a dose dependent fashion. Similar results found in Miapaca-2 cells (data not shown).

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) blocks FAK autophosphorylation by 24 hours

Next we analyzed whether Y15 inhibits FAK Y397 phosphorylation in time-dependent manner. We treated pancreatic tumor cells with 100 μM of Y15 for 0, 1, 4, 6 and 24 hours, and then western blotting was performed with Y397 antibody (Fig. 3B). The results demonstrate that treatment with Y15 for 6 hours or less did not significantly decrease Y397 phosphorylation, but 24 hours was enough to completely block Y397-phosphorylation and to downregulate FAK.

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) blocks ERK1/2 phosphorylation

ERK1/2 is known to be a downstream player from FAK in survival signaling.²⁶ To demonstrate the effect of Y15 on FAK signaling, we tested its effect on ERK1/2 phosphorylation. The ability of Y15 to downregulate p-ERK was evaluated. Consistent with the effects of Y15 on p-FAK, p-ERK is downregulated in both cell lines in a dose dependent fashion (Fig. 3C). Similar results were seen in Miapaca-2 cells (data not shown).

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) causes dose-dependent cell detachment

To test the cytotoxic effect of Y15 inhibitor on pancreatic cancer cells, we treated Panc-1 cells with the Y15 inhibitor at increasing doses for 24 and 48 hours. We performed analysis of detachment and apoptosis at time points shown in Figure 4A. Y15 caused a dose-dependent increase in detachment. After 48 hours with a 10 μM dose, Y15 caused 13% detachment in Panc-1 cells, while at a 50 μM dose, detachment was equal to 32%. Y15 caused more detachment than TAE226 (Novartis) inhibitor. Thus, Y15 effectively caused dose-dependent cellular detachment. Similar results were seen in Miapaca-2 cells (data not shown).

Figure 4.

Y15 causes dose-dependent cell detachment with no significant apoptosis in pancreatic cancer cells. (A) Panc-1 cells were treated with different doses of Y15 for 24 or 48 hours. The detachment was determined on a hemacytometer, as described in Materials and Methods. Bars show means ± standard deviations in three independent experiments. Y15 significantly decreased cell detachment (left, *p < 0.05 vs. untreated). Hoechst staining was performed on Panc-1 cells with different doses of Y15 and TAE226 inhibitors, as described in Materials and Methods. No significant apoptosis was detected with Y15. Bars represent means ± standard deviations in three independent experiments (right). Similar results for detachment and apoptosis were seen in Miapaca-2 cells (data not shown). (B) Hoechst staining of Y15-treated Panc-1 cells. Apoptotic nuclei stained with Hoechst are shown. No apoptotic nuclei were observed with Y15 inhibitor at a 50 μM dose compared to TAE226 inhibitor at the same dose. (C) Y15 causes a dose dependent decrease in cell viability, possibly via necrosis. Panc-1 cells were treated with increasing doses of Y15 for 24 hours. Trypan blue staining showed decreased viability with no significant increase in apoptosis. Similar results were seen in Miapaca-2 cells (data not shown).

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) treatment does not result in significant apoptosis

To test the effect of Y15 on apoptosis, we performed Hoechst staining on untreated and Y15-treated cells. At high dose (50–100 μM), Y15 caused a small and not significant (less than 10%) increase in apoptosis in Panc-1 cells. The same effect was seen in Miapaca-2 cells (data not shown). TAE226 (Novartis) caused a slightly higher level of apoptosis after 48 hours of treatment compared to similar doses of Y15 (Fig. 4A). Hoechst stained nuclei of Y15 and TAE226-treated cells are shown in Figure 4B. No apoptotic nuclei were detected in Y15-treated cells at a 50 μM dose in contrast to TAE226-treated cells at 50 μM doses (Fig. 4B). Since Y15 does not cause significant apoptosis at high doses, the mechanism of intracellular FAK kinase inhibition is independent of apoptotic cell death. Trypan blue staining in Panc-1 cells shows decreased cell viability indicative of necrosis with increasing doses of Y15 (Fig. 4C). Similar results were seen in Miapaca-2 cells.

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) inhibits cell adhesion in a dose-dependent manner

To test the effect of Y15 on cell adhesion, we treated pancreatic tumor cells with different doses of Y15 and with 50 μM TAE226 on collagen-coated plates and measured adhesion. Y15 inhibited cell adhesion in a dose-dependent manner (Fig. 5). Starting with a dose of 10 μM, cell adhesion was significantly decreased consistent with Y397-decreased FAK phosphorylation at these doses. A 50 μM dose of Y15 decreased adhesion in a similar fashion to 50 μM of TAE226. Thus, Y15 effectively blocks cell adhesion in a dose-dependent manner.

Figure 5.

Y15 blocks cell adhesion in a dose-dependent manner. Panc-1 cells were treated with Y15 at different concentration and cell adhesion was measured as described in Materials and Methods. TAE226 inhibitor at 50 μM was used as a control. Y15 significantly blocked cell adhesion in a dose dependent manner. Bars show means ± standard errors in four independent experiments *p < 0.05 vs. untreated cells. Similar results seen in Miapaca-2 cells (data not shown).

1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) inhibits human pancreatic tumor growth in vivo and decreases Y397-FAK phosphorylation

Gemcitabine is considered to be the most active agent in the treatment of patients with pancreatic cancer.²⁷ Thus, we evaluated pancreatic cancer cell viability in-vitro with Y15 or with gemcitabine alone or in the presence of the combination of Y15 plus gemcitabine chemotherapy. As shown in Figure 6A, the combination of gemcitabine (10 μM) chemotherapy + Y15 (10 μM) treatment significantly decreased cell viability compared to gemcitabine (10 μM) or Y15 treatment (10 μM) alone. Following this,to evaluate the in-vivo effect of Y15, we introduced pancreatic tumor cells subcutaneously into nude mice. Initially we determined that a dose of 30 mg/kg was the optimal non-toxic dose. We treated mice with intraperitoneal Y15 (30 mg/kg) for 5 days/week and compared tumor growth to mice receiving a placebo saline control. No animal weight loss or death was observed in any tumor growth inhibition experiment for 36 days (data not shown). Next, nude mice had Panc-1 tumor cells injected into the subcutaneous position. After one week of tumor growth, animals (n = 5/group) were randomized to receive daily intraperitoneal injections of PBS, Y15 alone (30 mg/kg), gemcitabine alone (30 mg/kg) or Y15 (30 mg/kg) + gemcitabine (30 mg/kg). As shown in Figure 6B, when given alone, Y15 or gemcitabine inhibited tumor growth. Y15 inhibited tumor growth even better than gemcitabine alone. Importantly, the combination of Y15 + gemcitabine treatment significantly inhibited tumor growth compared to either one alone. In addition, combined treatment with Y15 + gemcitabine caused a significant decrease in tumor weight compared to the other groups (Fig. 6C). On day 36, following the last treatment, mice were sacrificed and tumors were analyzed for FAK-Y397 levels by western blotting. Tumors from Y15 treated mice had lower levels of Y397 phosphorylation than tumors treated with PBS (control) (Fig. 6D). Thus, Y15 significantly suppressed pancreatic cancer tumorigenesis and had a synergistic effect with gemcitabine chemotherapy. These findings are consistent with the in-vitro viability data.

Figure 6.

In vitro and in vivo effects of Y15 when combined with gemcitabine. (A) Y15 potentiates gemcitabine activity in vitro. Panc-1 cells were treated with gemcitabine alone (10 μM), Y15 alone (10 μM) or the combination of both gemcitabine (10 μM) and Y15 (10 μM) for 72 hours. Cell viability was determined by MTT assay. *p < 0.05 vs. Y15 or gemcitabine alone. (B) Y15 significantly blocks tumor growth in vivo and its effects are synergistic with gemcitabine treatment. Mice (n = 5/group) were subcutaneously injected with Panc-1 cells. The day after injection, mice were treated with daily intraperitoneal PBS, intraperitoneal Y15 (30 mg/kg), intraperitoneal gemcitabine alone (30 mg/kg) or Y15 (30 mg/kg) + gemcitabine (30 mg/kg). The combination of Y15 + gemcitabine significantly decreased tumor volume compared to Y15 or gemcitabine (Gen) alone. *p < 0.05 vs. Y15 or gemcitabine alone. (C) Y15 + gemcitabine significantly decreases tumor weight. At day 36 after pancreatic cancer cell injection, tumors were extracted, and weight was determined in grams. Y15 + gemcitabine significantly decreased tumor weight. Bars represent means ± standard deviations. *p < 0.05 vs. gemcitabine (Gem) or Y15 alone. (D) Y15 decreases Y397-FAK phosphorylation in tumors. Tumors were excised from PBS treated (control) mice and from mice treated with Y15. Cell lysates were prepared, and western blotting was performed with Y397 antibody. β-actin antibodies were used to evaluate loading. (E) Y15 + gemcitabine decreases tumorigenesis in vivo. Immunohistochemistry for Ki67 staining was performed from excised tumors and quantitated. Tumors from animals treated with Y15 + gemcitabine had significantly less Ki67 positive cells than all other groups.

Immunohistochemistry to evaluate caspase-3 and Ki67 were performed from tumors in all four groups. Caspase-3 staining revealed no significant increase in apoptotic cells in tumors treated with Y15 alone (6%), gemcitabine alone (2%) or Y15 + gemcitabine (2%) (data not shown). However, Ki67 staining decreased the most in tumors treated with Y15 + gemcitabine (Fig. 6E).

Discussion

FAK appears to have many functions in cells, linking integrin signaling to downstream targets,^28,29 acting as part of a survival signal pathway^30,31 and having a connection with cell motility.^32,33 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-ECM signaling events. Tyrosine phosphorylation of FAK occurs in response to clustering of integrins, during formation of focal adhesions and cell spreading, and upon adhesion to fibronectin.⁶

Using computer modeling and functional approaches, we developed a novel approach to target the main autophosphorylation site on FAK. We screened 140,000 compounds from the National Cancer Institute repository of small molecules against the Y397 pocket of FAK. Among these compounds 1,2,4,5-Benzenetetraamine tetra-hydrochloride (called Y15 compound) was identified and found to be the most effective in decreasing cell viability in our pancreatic cancer cell lines. Y15 decreased pancreatic cancer cell Y397 phosphorylation and total FAK phosphorylation. Importantly, it directly decreased FAK autophosphorylation in vitro and decreased FAK phosphorylation in a dose- and time dependent manner. This compound decreased viability through necrosis and not apoptosis, since apoptosis was minimal but trypan blue positive cells increased with increasing doses. In addition, Y15 increased cell detachment and decreased cell adhesion in a dose-dependent manner. Finally, the Y15 compound significantly inhibited tumor growth in nude mice and demonstrated synergistic activity with gemcitabine chemotherapy. Tumors from mice treated with Y15 demonstrated decreased Y397-phosphorylation of FAK. Thus, utilization of the Y15 inhibitor, targeting the Y397 site of FAK, may be an effective novel strategy in pancreatic cancer.

We showed that by using computational modeling and in silico screening in conjunction with functional testing, we can successfully identify novel small molecule inhibitors of FAK. This method has been successfully used before to identify novel inhibitors of Jak2 kinase;²² however, there have been no previous reports of its use for targeting the main phosphorylation site of FAK. It is possible to use this approach for identifying small compounds that interact with other important functional sites on FAK such as the amino terminus where interaction with many growth factors occurs. It is also possible to synthesize derivatives of Y15 which may allow increased efficacy at lower doses.

Of importance is that Y15 blocked tumorigenesis in mice in vivo, showing its potential as a novel agent for therapy. The effect of Y15 can be compared to two other novel FAK kinase inhibitors that have been recently reported, TAE226 (Novartis) and PF-573,228 (Pfizer).^{17,19–21,34} Although TAE226 inhibits pancreatic cancer cell growth in-vitro, it has been shown to have limited specificity for FAK as low doses have been shown to inhibit the kinase activity of IGF-1R.¹⁷ Y15 has been shown to specifically block the Y397 phosphorylation of FAK and not inhibit other kinases.²³ Despite this, in our study at comparable doses, Y15 causes a similar inhibition of cell adhesion and increase in cell detachment to TAE226. In addition, Y15 blocked tumorigenesis at lower doses than those used for TAE226 in gliomas (50–75 mg/kg).¹⁹

Y15 significantly inhibited Y397 phosphorylation in pancreatic cancer cells at doses of <50 μM, and a 1 μM dose was enough to significantly block FAK autophosphorylation in an in vitro kinase assay. Importantly, both assays demonstrate FAK as a specific target of Y15. The FAK kinase inhibitor, PF562,271, was effective in human xenograft models at doses of 25–50 mg/kg but has no effect on cell viability and no report on tumorigenesis is known.³⁵ Thus, developing novel, more specific inhibitors of FAK that will block tumorigenesis is of critical importance.

Pancreatic cancer is a lethal malignancy and standard chemotherapy and radiation treatments have only improved survival by weeks.²⁷ There is an urgent need for novel therapies in this disease. The results demonstrated here represent the first published study targeting FAK in pancreatic cancer utilizing a novel small molecule inhibitor. Our in vitro and in vivo studies clearly show that small molecules targeting FAK synergize with standard chemotherapy in pancreatic cancer. This provides rationale for further development and study of such inhibitors in pre-clinical models and possibly in patients with pancreatic cancer.

In summary, we have identified a novel small molecule inhibitor of the Y397 autophosphorylation site on FAK. This compound, Y15 or 1,2,4,5-Benzenetetraamine tetrahydrochloride inhibits pancreatic cancer cell viability and adhesion in-vitro and blocks tumorigenesis in vivo and its effects synergize with gemcitabine chemotherapy. Thus, this compound and its derivatives may be important for future targeted therapy in pancreatic cancer.

Materials and Methods

Cell lines and culture

Panc-1 and Miapaca-2 carcinoma cells were obtained from ATCC and maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 5 μg/ml insulin and 1 μg/ml penicillin/streptomycin.

Structure-based in silico molecular docking of FAK small-molecule inhibitors

We used a structure-based approach combining molecular docking with functional testing. 140,000 small-molecule inhibitors following the Lipinski rules were docked into the N-terminal domain of FAK domain of the human FAK crystal structure in 100 different orientations using DOCK5.1 program, as described previously.²² The crystal structure of FAK, N-terminal FERM domain²⁴ (PDB ID:2AL6) was used for docking of FAK inhibitors. All water molecules were removed from the crystal structure, and SYBYL (Tripos, St. Louis, MO) was used to protonate and add charges to the residues. The spheres describing the target pocket of FAK were created using DOCK 5.1 suite program SPHGEN. 24 spheres were ultimately selected to constrain small molecule orientations to within 5 angstroms of Y397 site. Docking calculations were performed on the University of Florida High Performance Computing supercomputing cluster using16 processors (http://hpc.ufl.edu).

Computational docking

All docking calculations were performed with the University of California, San Francisco DOCK 5.1. program, using a clique-matching algorithm to orient small molecule structures with sets of spheres that describe the target Y397 site. Orientations were optimized using a simplex minimization algorithm, 100 orientations were created for each small molecule in the target site that were independently scores using DOCK5.1 grid-based scoring function. Briefly the three dimensional coordinates of the 140,000 compounds of the National Cancer Institute, Developmental Therapeutics Program (NCI/DTP) database were obtained from NCI. The files for hydrogen atoms and partial charges were created using SYBDB program.

Small-molecule compounds

The Y15 small molecule was identified as one of the top compounds that were detected by the DOCK 5.1 program to best fit into the Y397 site of FAK. The Y15 compound was ordered from Sigma for biochemical analyses in vitro and injection into mice for in-vivo studies. Y15 was solubilized in water at concentration of 25 mM and stored at −20°C and −80°C.

FAK inhibitors

The FAK kinase inhibitor, TAE226 was obtained from Novartis Inc.²⁰ TAE226 was dissolved in DMSO at 25 mM. The structure and the therapeutic effect of compound have been previously described.^{17,19–21,34} The TAE-226 inhibitor was used as a control for FAK inhibition in the in vitro experiments.

Antibodies

Monoclonal anti-FAK (4.47) antibody to N-terminal FAK, monoclonal and ERK antibodies were obtained from Upstate Biotechnology, Inc., (Lake Placid, NY). Polyclonal anti-phospho-Tyr397-FAK antibody was from Biosource Inc., (Carlsbad, CA). Monoclonal anti-β-actin antibody was obtained from Sigma. Antibodies to caspase-3 and Ki67 were obtained from DAKO (Carpinteria, CA) and Cell Signaling (Danvers, MA), respectively.

Cell viability assay

The cells were treated with Y15 or TAE226 at different concentrations for 24 hours. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium compound from Promega Viability kit (Madison, IL) was added, and the cells were incubated at 37°C for 1–2 hours. The optical density on 96-plate was analyzed with a microplate reader at 490 nm to determine cell viability. In addition, cells were stained with trypan blue after 24 hours of treatment with Y15 and the percent of cells that stained positive were determined with a hemacytometer.

Cell adhesion assay

The poly-L-Lysine or Collagen (5 μg/ml) coated 96-well plates were blocked with the blocking buffer (medium with 0.5% BSA) for 1 hour at 37°C. The cells were pre-treated with Y15 or TAE226 for 3 hours, collected and plated for adhesion assay at 4 × 10⁵ cells on a 96-well plate. Cells were incubated at 37°C for 1 hour, fixed in 3.7% formaldehyde, washed in 0.1% BSA in PBS and stained with crystal violet (5 mg/ml in 2% ethanol) for 10 minutes. Then 2% SDS was added to the dried plated and OD at 590 nm was measured for detecting cell adhesion.

Western blotting

Cells or homogenized tumor samples were washed twice with cold 1xPBS and lysed on ice for 30 minutes in a buffer containing: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-X, 0.5% NaDOC, 0.1% SDS, 5 mM EDTA, 50 mM NaF, 1 mM NaVO₃, 10% glycerol and protease inhibitors: 10 mg/ml leupeptin, 10 mg/ml PMSF and 1 mg/ml aprotinin. The lysates were cleared by centrifugation at 10,000 rpm for 30 minutes at 4°C. Protein concentrations were determined using a Bio-Rad Kit. The boiled samples were loaded on Ready SDS-10% PAGE gels (Bio Rad, Inc.,) and used for western blot analysis with the protein-specific antibody. Immunoblots were developed with chemiluminescence Renaissance reagent (NEN Life Science Products, Inc.,).

Immunoprecipitation

Immunoprecipitation was performed according to the standard protocol. In brief, the pre-cleared lysates with equal amount of protein were incubated with 1 μg of primary antibody and 30 μl A/G agarose beads overnight at 4°C. The precipitates were washed with lysis buffer three times and re-suspended in 2xLaemmli buffer. The boiled samples were used for western blotting, as described above.

Detachment assay

Cells were plated with and without inhibitors for 24 hours, and detached and attached cells were counted in a hemocytometer. We calculated the percent of detachment by dividing the number of detached cells by the total number of cells. The percent of detached cells was calculated in three independent experiments.

Apoptosis assay

Detached cells were collected and fixed in 3.7% formaldehyde in 1xPBS solution for the apoptosis assay. Detection of apoptosis was done with Hoechst 33342 staining. The percent of apoptotic cells was calculated as a ratio of apoptotic detached cells divided by the total number of cells in three independent experiments in several fields with the fluorescent microscope. For each experiment 300 cells per treatment were counted.

In vitro kinase assay

10 μCi of [γ-³²P]-ATP in a kinase buffer 20 mM HEPES, pH 7.4, 5 mM MgCl₂, 5 mM MnCl₂, 0.1 mM Na₃VO₄ with 0.1 μg of purified FAK protein were incubated in a kinase buffer with 10 μCi of [γ-³²P]-ATP. The kinase reaction was performed for 5 minutes at room temperature and stopped by addition of 2x Laemmli buffer. Proteins were separated on a Ready SDS-10% PAGE gel, and the phosphorylated enolase was visualized by autoradiography.

Tumor growth in nude mice in vivo

Female nude mice, 6 weeks old, were purchased from Harlan Laboratory. The mice were maintained in the animal facility and all experiments were performed in compliance with NIH animal-use guidelines and IACUC protocol approved by the University of Florida Animal Care Committee. Panc-1 cells were injected, 2 × 10⁶ cells/injection, subcutaneously. In preliminary experiment different doses of the compound were introduced into the mice, and 30 mg/kg was chosen as optimal, non-toxic dose. The ability of this dose to inhibit tumor growth was determined. The day after tumor inoculation, the compound was introduced by IP injection at 30 mg/kg dose daily 5 days/week for 3 weeks. Subsequently, the effect of this compound in the presence of gemcitabine chemotherapy was evaluated. Tumor diameters were measured with calipers and tumor volume in mm³ was calculated using this formula = (width)² × Length/2. At the end of experiment, tumor weight and volume was determined.

Immunohistochemistry staining

Immunohistochemistry staining for caspase-3 (1:400 dilution) and Ki67 (1:500 dilution) was performed on slides with paraffin-embedded tumor samples, as described previously.³⁶

Statistical analyses

Student’s t test or ANOVA were performed, when appropriate, to determine significance. The difference between data with p < 0.05 was considered to be significant.