BMS-754807, a Small-Molecule Inhibitor of Insulin-like Growth Factor-1 Receptor/Insulin Receptor, Enhances Gemcitabine Response in Pancreatic Cancer

This article is featured in Highlights of This Issue, p. 2547

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human cancers with extremely poor prognosis and 5-year survival is less than 5% (1). Each year, more than 40,000 individuals are diagnosed with PDAC and this incidence nearly equals mortality mostly due to late clinical presentation, early and aggressive local and metastatic progression, and high resistance to conventional therapies. Gemcitabine (Gem), a nucleoside analog that exerts its cytotoxic effects mainly by being incorporated into the DNA strand and inhibit DNA synthesis, is still the standard chemotherapeutic treatment of locally advanced and metastatic PDAC (2). However, the efficacy of gemcitabine as a single agent remains modest, with a median progression-free survival of approximately 6 months in randomized clinical trials and a 12-month survival of less than 20% (2–4). Currently, several clinical trials are underway to explore combination treatment benefits of gemcitabine with either cytotoxic agents or targeted agents toward development of more effective and less toxic therapeutic strategies for PDAC.

Although the causes of highly aggressive nature of PDAC are not well-understood, previous studies have shown the association of overexpression of several growth factors and their receptors during the progression of PDAC including EGF, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), VEGF, and insulin-like growth factor (IGF) (5, 6). The IGF system consists of ligands IGF-1, IGF-2, insulin, and IGF type-1 receptor (IGF-1R), insulin receptor (IR), and hybrid receptors. Activation of the IGF system stimulates proliferation, differentiation, angiogenesis, metastasis, survival, and resistance to anticancer therapies in many cancers (7), supporting the IGF system as an attractive therapeutic target. In the IGF system, IGF-1R is the primary mitogenic receptor that is overexpressed and has increased tyrosine kinase activity in several cancer types including PDAC (6, 8, 9). The binding of 2 IGF-1R ligands, IGF-1 and IGF-2, leads to the activation of several downstream signal transduction pathways including the phosphatidylinositol 3′-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) signaling pathways (10, 11). IGF-1R shares significant structural homology with IR. Although the binding of insulin to IR is primarily directed to regulate glucose homeostasis, this binding has also been shown to stimulate mitogenic signaling in cancer cells (12). IR has 2 isoforms, the classic IR-B that binds insulin leading to predominantly metabolic effects (13), and IR-A that binds insulin and IGF-2 causing proliferative effects (14). IR also has the ability to form hybrid receptors with IGF-1R (15, 16), which increases the complexity in targeting the IGF signaling pathway and suggests that the inhibition of both IR and IGF-1R may be beneficial for cancer therapy.

The strategy to target IGF-1R with specific monoclonal antibodies is attractive, as IGF-1R is the primary mitogenic receptor responsible for transducing IGF-1 and IGF-2, and as glucose homeostasis remains intact because of lack of IR-B inhibition (17, 18). However, monoclonal antibodies binding specifically to IGF-1R and not to IR could potentially provide escape mechanism for insulin and IGF-2 signaling, leading to limited clinical response. Interestingly, hyperglycemia and evidence of insulin resistance was observed clinically even with the IGF-1R antibody therapy (19). Tyrosine kinase inhibitors can more indiscriminately regulate the kinase domain activity of all IGF system receptors as their primary sequence share 84% homology in the kinase domain with near complete conservation in the ATP binding pocket (20). BMS-754807 is a potent oral, reversible small molecule tyrosine kinase inhibitor of the IGF-1R/IR family kinases. BMS-754807 has shown antitumor activity in a broad range of tumor types in vitro and in vivo (21–23). This drug is currently under phase I clinical investigation for the treatment of a variety of human cancers. In the present study, we evaluated the in vitro and in vivo efficacy of BMS-754807 alone and in combination with gemcitabine in an attempt to identify a more effective PDAC therapeutic strategy.

The human PDAC cell lines AsPC-1, BxPC-3, MIA PaCa-2, and Panc-1 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium (Sigma Chemical Co.) supplemented with 10% FBS at 37°C in a humidified 5% CO₂ atmosphere. Each cell line was tested and authenticated by ATCC. Cells were initially grown and multiple aliquots were cryopreserved. All the cell lines were used within 6 months after resuscitation. BMS-754807 was purchased from Active Biochemicals Co. Limited. Gemcitabine was purchased from Eli Lilly Corporation. The cell proliferation reagent WST-1 was purchased from Roche Diagnostic Corporation.

Cell viability of PDAC lines was evaluated by the colorimetric WST-1 assay. The measurement is based on the ability of viable cells to cleave the sulfonated tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases. PDAC cells (4,000 cells/well) were plated in a 96-well plate in regular growth medium, and after 16 hours the medium was replaced with 2% FBS containing medium. After 5 hours of incubation, the cells were treated with BMS-754807 and gemcitabine. The range of concentrations used (100 nmol/L to 10 μmol/L) for BMS-754807 and gemcitabine was comparable with their clinically achievable concentrations. After 72 hours, 10 μL WST-1 reagent was added in each well followed by additional incubation for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

Subconfluent monolayers of PDAC cells were treated with BMS-754807 (10 μmol/L) and gemcitabine (10 μmol/L). The cells were lysed after 24 hours as previously described (24). To obtain tumor lysates, tumor tissues obtained from the in vivo tumor growth experiment were immediately snap-frozen in liquid nitrogen and stored at −80°C. These samples were crushed in liquid nitrogen using a sterilized mortar, resuspended in lysis buffer, and extracts were sonicated on ice for 10 minutes. Supernatants were recovered by centrifugation at 13,000 rpm, protein concentrations were measured and equal amounts of total protein were separated by SDS-PAGE. Proteins were transferred to PVDF membranes (Bio-Rad) followed by blocking with 5% milk in TBS-T for 1 hour. The membranes were incubated overnight at 4°C with the following antibodies: total IGF-1R, phospho-IGF-1R (Tyr1135/1136)/IR (Tyr1150/1151; #3024), total AKT, phospho-AKT (Ser473), cleaved poly (ADP-ribose) polymerase-1 (PARP-1), cleaved caspase-3 (all from Cell Signaling Technology), α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; both from Sigma). The membranes were then incubated with the corresponding horseradish peroxidase (HRP)–conjugated secondary antibodies (Pierce Biotechnologies) for 1 to 2 hours. Specific bands were detected using the enhanced chemiluminescence reagent (ECL, PerkinElmer Life Sciences) on autoradiographic film and quantitated by densitometry.

Animal studies were conducted in accordance with the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. Athymic female nude mice (4–6 weeks old) were used to establish the subcutaneous xenograft model for tumor growth as previously described (25). AsPC-1 cells (0.75 × 10⁶) were subcutaneously injected in each mouse. The mice were weighed twice a week. Fourteen days after tumor cell injection, all mice had measurable tumor. Mice were then randomly grouped (n = 6–8 per group) and treated intraperitoneally with PBS (control), BMS-754807 (25 mg/kg in 100 μL PBS, 5 times a week), and gemcitabine (100 mg/kg in 100 μL PBS, twice weekly) for 14 days. The tumor size was measured twice weekly, and tumor volume (V) was calculated using the formula V = ½ (L × (W)², whereby L = length and W = width. Net growth in tumor size for each mouse was calculated by subtracting tumor volume on the first treatment day from that on the last day. After completion of treatment, all mice were euthanized, tumors were dissected, weighed, and processed for histological, immunohistochemical and Western blot analysis.

Tumor tissue specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Intratumoral proliferation was measured by Ki67 nuclear antigen staining as per manufacturer's protocol (Abcam). Briefly, paraffin-embedded tissue sections were cut (5 μm), deparaffinized, and rehydrated. Antigen retrieval was carried out by heating the tissue sections in citrate buffer. The tissue sections were incubated with CAS blocking buffer followed by 1-hour incubation with Ki67 antibody (1:200 dilution) and 40 minutes of incubation with Cy3 (1:200 dilution) secondary antibody. Slides were mounted using mounting solution containing 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Intratumoral proliferative index was evaluated by calculating the Ki67-positive cells from 5 different high-power fields in a blinded manner. Intratumoral apoptotic activity was evaluated by staining tissue sections with the Apoptag Apoptosis Detection Kit according to the manufacturer's instructions. Fluorescence microscopy was used to detect fluorescent signals using the IX81 Olympus microscope equipped with a Hamamatsu Orca digital camera (Hamamatsu Corporation) and a DSU spinning confocal unit using Slidebook software (Intelligent Imaging Innovations).

Animal survival studies were conducted using 6- to 8-week-old female severe combined immunodeficient (SCID) mice (26). The mice were intraperitoneally injected with AsPC-1 (0.75 × 10⁶) cells and body weight was measured twice a week. Two weeks after tumor cell injection, mice were randomly grouped (n = 6–8 per group) and treated intraperitoneally with PBS (control), BMS-754807 (25 mg/kg in 100 μL PBS, 5 times a week), and gemcitabine (100 mg/kg, twice weekly) for 2 weeks. Animals were euthanized when turning moribund according to predefined criteria (27, 28). Animal survival was evaluated from the first day of treatment until death.

Statistical significance was analyzed by the 2-tailed Student t test using GraphPad Prism 4 Software (GraphPad Software). In vitro cell proliferation data are expressed as mean ± SD. Statistical analysis for normally distributed in vivo study data was conducted by ANOVA for multiple group comparison and Student t test for the individual group comparison. Survival study statistics were evaluated with StatView for Macintosh (SAS) by nonparametric survival statistics and log-rank testing. Values of P < 0.05 were considered to represent statistically significant group differences.

Analysis of human PDAC cells AsPC-1, BxPC-3, MIA PaCa-2, and Panc-1, revealed that all 4 lines expressed IGF-1R and phospho-IGF-1R. Expression of phospho-IGF-1R was in the following order: AsPC-1>Panc-1>MIA PaCa-2>BxPC-3 (Fig. 1). BMS-754807 dose-dependently inhibited PDAC cell proliferation, with an inhibition in cell proliferation at 10 μmol/L of 54%, 37%, 49%, and 39% in AsPC-1, BxPC-3, MIA PaCa-2, and Panc-1 cells, respectively. Interestingly, inhibition in cell proliferation of PDAC cells by BMS-754807 seems to correlate to their expression of phospho-IGF-1R (Fig. 1).

Gemcitabine, a standard anti-PDAC chemotherapy agent, inhibited cell proliferation of PDAC lines in a dose-dependent manner. At 500 nmol/L, the inhibition in cell proliferation was 27%, 65%, 55%, and 29% in AsPC-1, BxPC-3, MIA PaCa-2, and Panc-1 cells, respectively (Fig. 2). The combination of BMS-754807 with gemcitabine had enhancing inhibitory effects. Including BMS-754807 at its IC₂₅ decreased the gemcitabine IC₅₀ from 9.7 μmol/L to 75 nmol/L for AsPC-1, from 28 to 16 nmol/L for BxPC-3 cells, from 72 to 16 nmol/L for MIA PaCa-2, and from 3 μmol/L to 70 nmol/L for Panc-1 cells, respectively (Fig. 2).

The effect of BMS-754807 on the IGF/receptor axis was investigated using AsPC-1 and Panc-1 PDAC cell lines that were most responsive to this drug in terms of causing a shift in the gemcitabine IC₅₀. Immunoblot analysis revealed that BMS-754807 blocked the expression of phosphorylated IGF-1R and downstream signaling protein phospho-AKT in both cells lines. The effect of BMS-754807 on chemotherapy-induced apoptosis was also evaluated by analyzing cleavage of caspase-3 and PARP-1 proteins as markers of apoptosis. Gemcitabine and BMS-754807 as single agent induced expression of cleaved caspase-3 and PARP-1, whereas the combination of BMS-754807 with gemcitabine caused additive effects on induction in cleavage of these apoptosis-related proteins (Fig. 3).

BMS-754807 significantly inhibited the growth of AsPC-1 xenografts. Treatment of AsPC-1 tumor-bearing mice with BMS-754807 (25 mg/kg, 5 times a week for 2 weeks) resulted in statistically significant net tumor growth inhibition of 59% (P = 0.02) as compared with the PBS-treated control group (Fig. 4A and B). The evaluation of gemcitabine alone treatment in this model resulted in net tumor growth inhibition of 35% (P = 0.05). The combination treatment of AsPC-1 tumor-bearing mice with BMS-754807 and gemcitabine resulted in a 94% inhibition in net tumor growth (P = 0.0007) as compared with controls (Fig. 4A and B). The difference in net tumor growth inhibition in the combination group was significantly different as compared with gemcitabine monotherapy (P = 0.003) or BMS-754807 monotherapy (P = 0.04).

Mechanisms of antitumor activity of BMS-754807, either alone or in combination with gemcitabine, were examined by Western blot analysis of protein lysates from AsPC-1 xenografts. A significant decrease in the expression of phospho-IGF-1R was observed in the BMS-754807–treated groups. BMS-754807 treatment also decreased the expression of the downstream signaling protein phospho-AKT (Fig. 4C). Evaluation of intratumoral apoptosis by analyzing expression of cleaved caspase-3 and cleaved PARP-1 proteins revealed that BMS-754807 and gemcitabine both induced cleavage of caspase-3 and PARP-1, and that the combination of these 2 agents had additive effects on cleavage of these apoptosis-related proteins (Fig. 4C).

No significant change in mice body weight was observed in the BMS-754807, gemcitabine, or combination therapy groups (Supplementary Fig. S1).

Further investigation of mechanisms of the antitumor activity of BMS-754807 by immunohistochemical analyses of tumor tissues revealed that the tumors of BMS-754807–treated mice presented a decreased tumor cell proliferation rate. Intratumoral proliferative index decreased by 29% (P = 0.038) in the BMS-754807–treated group as compared with controls. Gemcitabine monotherapy caused a 37% decrease in intratumoral proliferation as compared with controls (P = 0.0005). The combination of BMS-754807 and gemcitabine resulted in 39% decrease in proliferative activity as compared with control group (P = 0.001). However, the decrease in the intratumoral proliferative index in the combination treatment group was not significantly different than either therapy alone (Fig. 5).

Examination of intratumoral apoptosis in tumor tissues revealed a 3.9-fold increase in apoptotic index in the BMS-754807 monotherapy group (P = 0.004) and a 4.4-fold increase in the gemcitabine-treated group (P = 0.003) as compared with the control group. The combination of BMS-754807 and gemcitabine led to a 6.4-fold induction in intratumoral apoptosis (P = 0.0005). Induction in intratumoral apoptosis in the combination treatment group was significantly higher than in the BMS-754807 monotherapy (P = 0.037) or gemcitabine monotherapy (P = 0.003) groups (Fig. 5).

In AsPC-1 PDAC murine xenograft study, the median survival of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was 21 days in the control group. Median survival of mice was increased by BMS-754807 treatment to 27 days (P = 0.03 vs. controls). Gemcitabine monotherapy resulted in a median survival of 28 days (P = 0.05 vs. controls). The combination of BMS-754807 and gemcitabine had additive effects on mouse survival, with the median survival extended to 41 days. The median survival in the BMS-754807+gemcitabine group was significantly higher as compared with controls (P = 0.007) or the 2 monotherapy groups (P = 0.01; Fig. 6).

Most PDAC cases are unresectable at the time of presentation, due to unspecific and indistinct early symptoms. Also, local recurrence rate is very high in completely resected PDAC patients and they eventually require palliative treatment. Intrinsic and acquired chemoresistance to conventional chemotherapy continues to impact devastating prognosis in PDAC. Gemcitabine, the first-line adjuvant treatment of advanced and metastatic PDAC, provided some clinical benefits but a modest survival advantage (2). The majority of the gemcitabine based-combination chemotherapy regimens for PDAC have failed to show any survival benefit (29, 30), except the EGF receptor inhibitor erlotinib that was approved in combination with gemcitabine based on a small but statistically significant improvement in overall survival (31). Recent advances in understanding of the molecular mechanisms of PDAC progression, however, have highlighted potential therapeutic targets for which combination therapy strategies seem promising. IGF-1R activation seems to be such a mechanism important for progression of several malignancies, and disruption of IGF-1R activation has been shown to inhibit growth and motility of a wide range of cancer cells, both in vitro and in animal models (32, 33). IGF-1R overexpression has also been correlated with tumor aggressiveness (34, 35), whereas IGF-1-deficient mice have reduced capacity to support tumor growth and metastasis (36). Several approaches have been used to block IGF-1R-related tumorigenicity including antisense strategies, RNA interference, blocking antibodies, and tyrosine kinase inhibitors (37, 38). The blockage of IGF-1R signaling has been shown to enhance chemotherapy response in several cancer types in laboratory studies (39–41). Because IGF-1R and IR are highly homologous, antagonist development was initially focused toward monoclonal antibodies that selectively target IGF-1R and not affect IR signaling, which could lead to dysregulation of glucose homeostasis (42). Initial clinical studies with IGF-1R antibodies showed some responses in monotherapy (43) and in combination with cytotoxic chemotherapy (44). However, patients receiving antibody therapy have hyperglycemia indicating the potential of undesirable metabolic side effects of such therapy (45). Several reports showed the involvement of stimulation of IR by insulin or IGF-2 in cancer cell progression (14, 46) suggesting that IGF-1R and IR both are therapeutic targets and inhibition of both receptors may be required for maximum tumor growth inhibition. Our present study thus explored the effects of BMS-754807, a small-molecule inhibitor of both the receptors IGF-1R/IR, in combination with gemcitabine in PDAC.

Previous studies have shown the expression of IGF-1R in pancreatic cancer cells (6). We observed a differential expression of phospho-IGF-1R and IGF-1R in AsPC-1, BxPC-3, MIA PaCa-2, and Panc-1 cells, indicating a possible involvement of aberrant IGF signaling in PDAC progression. Interestingly, BMS-754807 inhibited the proliferation of all PDAC cells tested; importantly, in all lines there were additive inhibitory effects in combination with gemcitabine. The sensitivity of PDAC cells against BMS-754807 seemed to be directly correlated with the expression of phospho-IGF-1R. We found interesting that the 2 cell lines with greater resistance to gemcitabine had greater IGF-1R expression and greater inhibition after exposure to BMS-754807; a rationale to combine these 2 agents may therefore be specifically sensible to enhance clinical PDAC treatment effects. Additional validation of involvement of IGF signaling in PDAC comes from our finding that a PI3K/mTOR inhibitor (NVP-BEZ235) enhances gemcitabine response as PI3K/mTOR may serve as downstream component of IGFR signaling pathway (27). Therefore, further evaluation of combination of NVP-BEZ235 and BMS-754807 seems promising for PDAC clinical application. As previously reported in a human rhabdomyosarcoma cell line (21), we observed that in PDAC cells BMS-754807 treatment caused a decrease in phosphorylated IGF-1R and its downstream signaling protein phospho-AKT, and increased cleavage of apoptosis-related caspase-3 and PARP-1 proteins. These molecular signaling changes are likely operational in the BMS-754807-induced inhibition in cell proliferation and induction in apoptosis, and seem to represent good markers of in vivo activity testing and for clinical validation.

A recent study reported that BMS-754807 has some antitumor activity in multiple tumor models including epithelial, mesenchymal, and hematopoietic cancer cells (21). We observed that in a PDAC murine xenograft model, BMS-754807 inhibited local tumor growth as single agent and more importantly, caused additive effects in combination with gemcitabine. Analysis of intratumoral proliferation and apoptosis seem to be correlated with tumor growth inhibition data. Evaluation of target proteins in tumor tissue lysates showed that BMS-754807 treatment decreased levels of the activated form of IGF-1R and AKT, and induced levels of apoptosis-related proteins, indicating that the BMS-754807 therapy is indeed managing to affect these targets within the local tumor model.

Active PDAC progression involves multiple distinct mechanisms including induction in cancer cell proliferation, migration, differentiation, angiogenesis, and inhibition of cancer or stromal cell apoptosis. Previous studies in our laboratory have shown the benefits of targeting multiple pathways in different cellular compartments including epithelial, vascular, and stromal elements for PDAC treatment (27, 47–49). Gemcitabine itself has antiproliferative and proapoptotic effects on tumor cells, endothelial cells, and fibroblasts (27, 28). The molecular mechanisms for the enhancement in antitumor activity of gemcitabine by BMS-754807 addition is not completely clear, but it is likely caused by augmentation of its antiproliferative and proapoptotic activities and possibly not just restricted to the tumor cells of epithelial origin. Because of the multifactorial nature of the disease and limited effectiveness of gemcitabine, combination therapies targeting multiple pathways provide promising strategies for clinical evaluation. There is some evidence that combination therapy is superior to monotherapy for the management of pancreatic cancer (31). Recently, combination of gemcitabine with nab-paclitaxel has been shown to have substantial antitumor activity in patients with advanced pancreatic cancer in a phase I/II trial (50). On the basis of our findings, combination of gemcitabine and BMS-754807 is a reasonable clinical trial option for phase II evaluation after establishing toxicity of this combination.

Dysregulation of glucose homeostasis is an important concern of using BMS-754807 and therefore the advantage of inhibition of both IGF-1R and IR signaling might have to be balanced with the potential for metabolic side effects. We have not obtained any evidence for in vivo toxicity during a 2-week treatment course, but have no data on whether long-term inhibition of the IGF-1R and IR functions remains tolerable. A previous study reported only a short-term increase in serum glucose or insulin levels at doses up to 12.5 mg/kg, with an average weight change of −1.5 g at 25 mg/kg, but no mortality (21). In our study, no significant change in mouse body weight was observed by BMS-754807 therapy, but blood glucose measurements were not obtained. In conclusion, our study indicates that BMS-754807 can mediate mechanism-specific antitumor activity in experimental PDAC, and it significantly improves gemcitabine response. These findings corroborate the rationale of blocking multiple pathways of IGF and insulin signaling, and support the potential of BMS-754807 as targeting agent for clinical PDAC therapy.

No potential conflicts of interest were disclosed.

Conception and design: N. Awasthi, R.E. Schwarz

Development of methodology: N. Awasthi, R.E. Schwarz

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Awasthi, C. Zhang, W. Ruan, M.A. Schwarz, R.E. Schwarz

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Awasthi, C. Zhang, W. Ruan, R.E. Schwarz

Writing, review, and/or revision of the manuscript: N. Awasthi, R.E. Schwarz

Study supervision: R.E. Schwarz

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.