Purpose: To evaluate 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography imaging (18FDG-PET) as a predictive, noninvasive, pharmacodynamic (PD) biomarker of response following administration of a small-molecule insulin-like growth factor-1 receptor and insulin receptor (IGF-1R/IR) inhibitor, OSI-906.

Experimental Design:In vitro uptake studies of 3H-2-deoxy glucose following OSI-906 exposure were conducted evaluating correlation of dose with inhibition of IGF-1R/IR as well as markers of downstream pathways and glucose metabolism. Similarly, in vivo PD effects were evaluated in human tumor cell line xenografts propagated in athymic nude mice by 18FDG-PET at 2, 4, and 24 hours following a single treatment of OSI-906 for the correlation of inhibition of receptor targets and downstream markers.

Results: Uptake of 3H-2-deoxy glucose and 18FDG was significantly diminished following OSI-906 exposure in sensitive tumor cells and subcutaneous xenografts (NCI-H292) but not in an insensitive model lacking IGF-1R expression (NCI-H441). Diminished PD 18FDG-PET, collected immediately following the initial treatment agreed with inhibition of pIGF-1R/pIR, reduced PI3K (phosphoinositide 3-kinase) and MAPK (mitogen activated protein kinase) pathway activity, and predicted tumor growth arrest as measured by high-resolution ultrasound imaging.

Conclusion:18FDG-PET seems to serve as a rapid, noninvasive PD marker of IGF-1R/IR inhibition following a single dose of OSI-906 and should be explored clinically as a predictive clinical biomarker in patients undergoing IGF-1R/IR–directed cancer therapy. Clin Cancer Res; 17(10); 3332–40. ©2011 AACR.

Translational Relevance

The development of inhibitors targeting the insulin-like growth factor-1 receptor and insulin receptor (IGF-1R/IR) is a clinically important area of cancer research. OSI-906 is a potent and highly selective tyrosine kinase inhibitor now being evaluated in clinical studies that exhibits similar biochemical potency against IGF-1R (8 nmol/L) and IR (14 nmol/L), and is greater than 4 orders of magnitude more selective for IGF-1R/IR compared with a wide number of other receptor and nonreceptor kinases. Objective means to assess pharmacodynamic response to OSI-906 therapy in tumors remains challenging. To this end, we evaluated 18FDG-PET as a clinically relevant molecular imaging metric to quantify and predict pharmacodynamic response to OSI-906 in preclinical mouse models of lung cancer.

The insulin-like growth factor-1 receptor (IGF-1R) is a tetrameric transmembrane receptor tyrosine kinase that is widely expressed in normal human tissues and is upregulated in a number of human cancers including colorectal, non–small cell lung, ovarian, and pediatric cancers. The receptor is composed of 2 α- and 2 β-subunits linked by disulfide bonds in which the extracellular α-subunit is responsible for ligand binding and the β-subunit consists of a transmembrane domain and a cytoplasmic tyrosine kinase domain. Ligand binding activates the tyrosine kinase activity of IGF-1R and results in trans-β-subunit autophosphorylation and stimulation of signaling cascades that include PI3K-mTOR and MAPK (mitogen activated protein kinase) pathways. Activation of IGF-1R has been reported to stimulate proliferation, survival, transformation, metastasis, and angiogenesis, whereas inhibition of IGF-1R has been shown to impede tumorigenesis in several human xenograft models (1).

Increased expression of IGF-1R and its cognate ligands, IGF-I and IGF-II, has been shown in a wide range of solid tumors and hematologic neoplasias relative to normal tissue levels. Epidemiologic studies have shown an increased risk for the development of colon, lung, breast, and bladder cancers with increased circulating levels of IGF-I (2–5). Additionally, IGF-1R expression levels have been correlated to poor prognosis in renal cell carcinoma (6, 7). IGF-1R signaling mechanism has also been linked to resistance to various antitumor therapies including epidermal growth factor receptor inhibitors (1, 6, 8, 9).

Similarly, the insulin receptor (IR) is composed of a heterotetramer consisting of 2 extracellular α-subunits and 2 transmembrane β-subunits. Binding of insulin to the IR extracellular α-subunit causes a conformational change bringing together the 2 β-subunits. Activated IR tyrosine kinase phosphorylates several intracellular substrates including IRS-1-4, Shc, Gab1, and Cbl. These phosphorylated proteins provide a docking site for effector proteins containing Src homology 2 (SH2) domains further linking IR to phosphoinositide 3-kinase (PI3K) via the regulatory p85 subunit. Homology between IR and IGF-IR ranges from 45% to 65% in the ligand binding domains to 60% to 85% in tyrosine kinase domains. Expression of IR is highest in adipose tissue and to a lesser extent in liver, heart, and muscle (10). Overexpression of IR in breast, colon, lung, ovarian, and thyroid cancers suggest a role of IR in tumor progression (10). More recently we have shown that forced overexpression of IR is tumorigenic in mice (11).

OSI-906 is a potent and highly selective tyrosine kinase inhibitor that exhibits similar biochemical potency against IGF-1R (8 nmol/L) and IR (14 nmol/L) and is greater than 4 orders of magnitude more selective for IGF-1R/IR compared with a wide number of other receptor and nonreceptor kinases (12). Within a panel of more than 180 kinases, only IGF-1R and IR were inhibited by greater than 50% at 1.0 μmol/L OSI-906. Inhibition of cell proliferation and induction of apoptosis following exposure to OSI-906 seems to be directly linked to inhibition of AKT in colorectal, lung, and pancreatic cancer cell lines (1, 12). In addition, OSI-906 has shown potent antitumor activity in vivo in several xenograft models (1). Because IGF-1R and IR pathway signaling is linked to glucose metabolism, we asked whether 18FDG-PET could function as a surrogate pharmacodynamic (PD) marker for OSI-906. To this end, we employed in vitro cell culture assays and in vivo animal models measuring uptake of radioactive glucose analogues as a function of treatment by OSI-906. Our data show that glucose uptake is rapidly inhibited in vitro and in vivo and tracks with IGF-1R, IR, and AKT inhibition after OSI-906 treatment in sensitive tumors. Moreover, reduced glucose uptake was readily observed after OSI-906 treatment in tumor tissues by using 18FDG-PET imaging methodologies. Hence, 18FDG-PET may function as a rapid, noninvasive tumor-specific PD marker for OSI-906 in the clinical setting where accurate assessment of PD effects is often limited by the lack of readily accessible tumor samples. Thus, 18FDG-PET may be a useful clinical tool in identifying active doses and patients potentially sensitive to this novel antitumor agent warranting further clinical investigation of this approach.

Cell lines

Human non–small cell lung carcinoma cell lines (NCI-H292, NCI-H441) were obtained from American Type Culture Collection. All cell lines were maintained in RPMI 1640 media (Mediatech) supplemented with 10% FBS (Sigma) and 1% sodium pyruvate (Mediatech) and maintained at 37°C and 5.0% CO2. Cells were propagated to 80% to 90% confluency prior to in vitro and in vivo assays.

3H-2-deoxy glucose uptake assay

Cells were seeded in 12-well tissue culture plates (Becton Dickinson) at a density of 9.0 × 105 cells per well in normal glucose (11.1 mmol/L) media and allowed to attach for 6 to 8 hours at 37°C (n = 3 wells/group). The media was then changed to 5.5 mmol/L glucose media and the cells were allowed to equilibrate overnight. Three hours prior to the assay, the media was again removed and replaced with media containing 0.0 mmol/L glucose (glucose starvation). The cells were then treated with varying concentrations of OSI-906 (0.0–30 μmol/L) and 0.15 mCi of 3H-2-deoxy glucose (Perkin Elmer). After 30 minutes, the media was removed, the cells placed on ice, and washed once with ice-cold PBS (Mediatech). The PBS was then removed and the cells were lysed in radioimmunoprecipitation assay buffer (Sigma) for 15 minutes on ice. The lysates were harvested and counted in a Beckman LS6500 Liquid Scintillation counter (Fullerton). 3H-2-deoxy glucose uptake was calculated as raw counts and normalized to control samples (0.0 μmol/L OSI-906). As a positive control of glucose uptake inhibition, NCI-H292 cells were treated with increasing concentrations (2.5–10 μmol/L) of cytochalasin B (Sigma), a known inhibitor of GLUT1 and GLUT4 glucose transporters.

Mouse models

Studies involving mice were conducted in accordance with federal and institutional guidelines. NCI-H292 and NCI-H441 non–small cell human xenograft tumors were generated as described (13). Briefly, 4 × 106 cells were injected subcutaneously on the right flank of 5- to 6-week-old female athymic nude mice (Charles Rivers). Using this method, palpable tumors were typically observed within 2 weeks following injection of cells and were allowed to progress until approximately 150 to 200 mm3, and then randomized for treatment studies. Measurement of volume was done by using high-resolution ultrasound imaging as described (14). Mice were treated when the tumors reached approximately 200 mm3 in volume. Blood glucose was measured by a Freestyle digital glucose meter and test strips (Abbott) before and at 2 hours, and 4 hours after treatment with 60 mg/kg OSI-906 or 25 mmol/L tartaric acid vehicle.

Procurement of 18FDG

18FDG was synthesized in the Vanderbilt University Medical Center Radiopharmacy and distributed by PETNET. The average radiochemical purity of the product was 98.5% and specific activity was more than 1,000 Ci/mmol.

18FDG-PET imaging

Animal handling methods in preparation for and during 18FDG-PET imaging were similar to the published protocols (15–17). Briefly, before imaging, mice were fasted overnight and allowed to acclimate to the PET imaging facility environment for at least 1 hour in a warmed chamber at 31.5°C. Mice were administered a single dose of OSI-906 at 60 mg/kg in a 25 mmol/L tartaric acid vehicle via oral gavage (n = 8/group). 18FDG was administered via a single retro-orbital injection of approximately 200 μCi (100 μL) and imaged 2, 4, and 24 hours postdosing of OSI-906, or 4 hours after tartaric acid vehicle. Mice were conscious during the uptake period and maintained in a warmed chamber. Following a 50-minute uptake period, 10-minute static PET scans were collected on a Concorde Microsystems micro-PET Focus 220 (Siemens). Mice were maintained under 2% isofluorane anesthesia in 100% O2 at 2 L/min and kept warm via a circulating water heating for the duration of the scan. Immediately following imaging, mice were sacrificed and tissues collected for molecular analysis. PET images were reconstructed by the ordered subsets expectation maximization algorithm. The percent injected dose per gram of tissue (%ID/g) was calculated from analysis of tumor regions of interest by ASIPro software (Concorde Microsystems Inc.).

Statistical analysis of data

Wilcoxon rank-sum (Mann–Whitney U) tests were carried out to compare each treatment time point to vehicle-treated mice. Comparisons were unadjusted for the multiplicity of testing and were deemed significant if P < 0.05.

Pharmacokinetic analysis in vivo

At 2, 4, and 24 hours after administration of OSI-906, blood was collected via cardiac puncture and placed in BD Microtainer EDTA collection tubes (Becton Dickinson). The samples were centrifuged at 1,500 × g for 10 minutes and plasma protein precipitated with methanol. Analysis of drug concentration was done by high-performance liquid chromatography/tandem mass spectroscopy (Applied Biosystems).

Immunoprecipitation/Western blot analysis

Phosphorylation of IGF-1R and IR in cells and tumor samples were analyzed by immunoprecipitation/Western blotting. Cells were lysed by using NP-40 lysis buffer (Sigma). Tumor samples were homogenized by using Precellys 24 (MO BIO Laboratories Inc.) in tumor lysis buffer [1% Triton X-100, 10% glycerol, 50 mmol HEPES (pH 7.4), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA supplemented with fresh protease inhibitor cocktail (Sigma), phosphatase inhibitor cocktail (Sigma), 10 mmol/L NaF, and 1 mmol/L sodium orthovanadate]. After preclearing by centrifugation (14,000 rpm for 15 minutes), 1 mg of total protein was immunoprecipitated with anti-phosphotyrosine antibody (pY20; Exalpha) at 4°C overnight. The immunoprecipitates were separated on SDS-PAGE and immunoblotted with a total IGF-1R antibody (Cell Signaling) followed by detection by enhanced chemiluminescence (GE Healthcare Life Sciences). The blots were reprobed with total IR antibody (Cell Signaling). Phosphorylated IGF1-R and IR bands were quantified by an Image Quant LAS 4000 with Image Quant TL 7.0 software (GE Healthcare Life Sciences).

Markers of altered glycolysis were analyzed by Western blot analysis. Tumor or cell lysate samples were separated on SDS-PAGE, immunoblotted, and detected by using enhanced chemiluminescence (GE Healthcare Life Sciences). The antibodies included pAKT (Ser473), total AKT, pS6 (Ser235/236), pERK 1/2, total ERK 1/2, (Cell Signaling), and β-actin (Sigma). The phosphorylated to total signal intensities were quantified as described earlier.

Receptor tyrosine kinase analysis

Tumor lysates were prepared according to the manufacturer's protocol (Proteome Profiler; R&D Systems) in NP-40 lysis buffer and clarified by centrifugation. The samples were incubated with the Human Phospho-RTK Array at 2,000 μg total protein overnight at 4°C with rocking. The arrays were developed by SuperSignal FEMTO ECL detection (Pierce). The phospho-spots on the receptor tyrosine kinase (RTK) blot were quantified by using Image Quant LAS 4000 with Image Quant TL 7.0 software (GE Healthcare Life Sciences)

Sensitivity of NCI-H292 and NCI-H441 to OSI-906

Non–small cell lung cancer is a potentially attractive indication for OSI-906 due to the implication of IGF1R/IR as a driver in this, as well as drug resistance in this setting. We established sensitivity of the NCI-H292 and NCI-H441 xenograft models to OSI-906 in vivo by measuring tumor volumes longitudinally with high-resolution ultrasound imaging. Daily treatment with 60 mg/kg OSI-906 over 10 days resulted in tumor growth inhibition in the NCI-H292 xenografts compared with controls (Fig. 1A), but no growth changes were observed in the nonresponsive NCI-H441 xenografts (Fig. 1B). We found that NCI-H292 tumors had considerably higher levels of pIGF-1R and pIR than NCI-H441 tumors (Fig. 1C).

Figure 1.

Daily treatment of mice bearing NCI-H292 xenografts with 60 mg/kg OSI-906 results in significant tumor growth inhibition (A) compared with analogously treated vehicle controls. In contrast, NCI-H441 xenografts (B) do not exhibit a difference in tumor growth when comparing OSI-906–treated and vehicle-treated cohorts. RTK arrays (C) illustrate that NCI-H292 cells possess relatively high levels of pIGF-1R and pIR compared with the barely detectable levels of pIGF-1R and pIR in NCI-H441 cells. P-Y control, Phospho-Tyrosine positive control.

Figure 1.

Daily treatment of mice bearing NCI-H292 xenografts with 60 mg/kg OSI-906 results in significant tumor growth inhibition (A) compared with analogously treated vehicle controls. In contrast, NCI-H441 xenografts (B) do not exhibit a difference in tumor growth when comparing OSI-906–treated and vehicle-treated cohorts. RTK arrays (C) illustrate that NCI-H292 cells possess relatively high levels of pIGF-1R and pIR compared with the barely detectable levels of pIGF-1R and pIR in NCI-H441 cells. P-Y control, Phospho-Tyrosine positive control.

Close modal

Inhibition of 3H-2-deoxy glucose uptake in vitro

We assessed the effect of OSI-906 treatment on uptake of 3H-2-deoxy glucose in NCI-H292 and NCI-H441 cells in vitro. Cells were treated for only 30 minutes with OSI-906 to avoid potential antiproliferative effects of the drug to interfere with this endpoint analysis. OSI-906 treatment resulted in a rapid and dose-dependent inhibition of uptake of the radiotracer in the NCI-H292 cell line (Fig. 2A). The percent inhibition ranged from 12% to 60% as the dose increased from 1.0 to 30 μmol/L OSI-906. In comparison, the NCI-H441 cell line showed a reduced sensitivity to OSI-906. For the NCI-H292 cell line a 35% decrease in uptake of 3H-2-deoxy glucose was achieved at 10 μmol/L OSI-906, whereas in the NCI-H441 cell line the same decrease of the radiotracer was observed at only 30 μmol/L OSI-906 (Fig. 2B). Analysis for cell death by fluorescence-activated cell sorting by using the Invitrogen Live/Dead assay determined no significant cell death at all OSI-906 concentrations (1.0–30 μmol/L) tested compared with 0.05% dimethylsulfoxide controls (data not shown). As a positive control, cytochalasin B (2.5–10 μmol/L) was administered to the NCI-H292 cells and evaluated for 3H-2-deoxy glucose uptake in an analogous manner. Figure 3C shows that cytochalasin B significantly inhibits uptake of the radiotracer by 85% to 90% in this cell line, and that the inhibition of 3H-2-deoxy glucose by OSI-906 in NCI-H292 cells represents a rapid PD effect.

Figure 2.

3H-2-deoxy glucose uptake 30 minutes after OSI-906 treatment in NCI-H292 cells showed a dose-dependent decrease (A). Similar decreases in 3H-2-deoxy glucose uptake were seen at higher doses of OSI-906 in the nonresponding NCI-H441 cells compared with the responding NCI-H292 cells (B). Treatment with cytochalasin B as a positive control in NCI-H292 cells showed that 3H-2-deoxy glucose uptake is directly affected by exposure OSI-906, and can be linked directly to cellular pathways associated with glucose metabolism (C). Western blot of NCI-H292 cells following 30 minutes of exposure to OSI-906 shows target inhibition of pIGF-1R and pIR at all doses as well as inhibition of downstream targets pAKT and pS6 (D).

Figure 2.

3H-2-deoxy glucose uptake 30 minutes after OSI-906 treatment in NCI-H292 cells showed a dose-dependent decrease (A). Similar decreases in 3H-2-deoxy glucose uptake were seen at higher doses of OSI-906 in the nonresponding NCI-H441 cells compared with the responding NCI-H292 cells (B). Treatment with cytochalasin B as a positive control in NCI-H292 cells showed that 3H-2-deoxy glucose uptake is directly affected by exposure OSI-906, and can be linked directly to cellular pathways associated with glucose metabolism (C). Western blot of NCI-H292 cells following 30 minutes of exposure to OSI-906 shows target inhibition of pIGF-1R and pIR at all doses as well as inhibition of downstream targets pAKT and pS6 (D).

Close modal
Figure 3.

Western blot of NCI-H292 cells treated with 10 nmol/L, 100 nmol/L, 500 nmol/L, 1 μmol/L, and 5 μmol/L OSI-906 show target inhibition over a 24-hour time course. All concentrations of OSI-906 induce a reduction in pIGF-1R at 2 hours, and inhibition remains through 24 hours in all but the lowest, 10 nmol/L concentration.

Figure 3.

Western blot of NCI-H292 cells treated with 10 nmol/L, 100 nmol/L, 500 nmol/L, 1 μmol/L, and 5 μmol/L OSI-906 show target inhibition over a 24-hour time course. All concentrations of OSI-906 induce a reduction in pIGF-1R at 2 hours, and inhibition remains through 24 hours in all but the lowest, 10 nmol/L concentration.

Close modal

Correlation with target-pathway inhibition in vitro

NCI-H292 cell lysates were treated with an increasing concentration of OSI-906 (0.0–10 μmol/L) for 30 minutes and then analyzed for pIGF-1R, pIR, pERK 1/2, pAKT, pS6, and β-actin as shown in Figure 2D. We observed a significant decrease in phosphorylation of AKT and S6, suggesting a correlation between decreased glucose uptake and inhibition of targets downstream of IGF-1R and IR. NCI-H292 cells treated at lower concentrations (10 nmol/L to 5 μmol/L) over 2, 12, and 24 hours showed target inhibition at all concentrations at 2 hours, and sustained inhibition of pIGF-1R at both 12 and 24 hours for all concentrations except 10 nmol/L (Fig. 3).

Inhibition of 18FDG uptake in vivo

18FDG-PET images of mice bearing the NCI-H292 and NCI-H441 xenografts are shown in Figure 4A. The NCI-H292 xenografts (sensitive to OSI-906 treatment) show a significant decrease (P < 0.05) in 18FDG uptake at 2, 4, and 24 hours postdosing with OSI-906 compared with vehicle-treated controls. NCI-H441 xenografts (insensitive to OSI-906 treatment) did not show a significant change in uptake of 18FDG at any time point evaluated. Graphically, these results are shown in Figure 4B and C. The decreased %ID/g in the NCI-H292 xenografts is suggestive of a rapid PD effect observed by 18FDG imaging mediated by the inhibition of IGF-1R and IR pathways by OSI-906. Conversely, for the NCI-H441 xenograft model no difference in uptake of the radiotracer was observed in the tumor samples between vehicle controls and the OSI-906 treatment group.

Figure 4.

Representative transverse 18FDG-PET images of NCI-H292 and NCI-H441 tumor xenografts (A) show that 18FDG uptake is significantly reduced (P < 0.05) in the NCI-H292 xenografts at all time points following a single treatment of 60 mg/kg OSI-906 (B) whereas NCI-H441 xenografts show no changes in 18FDG uptake (C). Veh, vehicle.

Figure 4.

Representative transverse 18FDG-PET images of NCI-H292 and NCI-H441 tumor xenografts (A) show that 18FDG uptake is significantly reduced (P < 0.05) in the NCI-H292 xenografts at all time points following a single treatment of 60 mg/kg OSI-906 (B) whereas NCI-H441 xenografts show no changes in 18FDG uptake (C). Veh, vehicle.

Close modal

Correlation with target-pathway inhibition

Target inhibition of both pIGF-1R and pIR by a single dose of OSI-906 at 60 mg/kg in vivo in NCI-H292 xenograft tumors is shown in Figure 5A. The data show that at 2 and 4 hours posttreatment target inhibition of pIGF-1R is greater than 80%, with 30% inhibition observed at 24 hours (Fig. 5B). The effect on pIR is equally pronounced, showing significant target inhibition of this receptor. Target inhibition of pIR was greater than 80% at 4 hours posttreatment with 40% inhibition observed at 24 hours. Inhibition of both target receptors correlated with decreased uptake of 18FDG in the same tumor samples analyzed. Figure 5C shows the results of a Western blot from tumor lysates at selected time points from mice bearing the NCI-H292 xenografts that were treated with 60mg/kg OSI-906 (n = 4/group). We found reduced activation levels of targets involved in glycolysis that are downstream of IGF-1R and IR, including pAKT, pS6, and pERK 1/2 as measured 4 hours posttreatment with OSI-906 compared with untreated control lysates. Importantly, Western blot analysis of OSI-906–treated NCI-H441 tumor xenografts, which do express very low levels of the target receptor, showed no reduction in pAKT levels at any time point compared with control (Supplementary Fig. S1)

Figure 5.

RTK array analysis shows strong target inhibition of both pIGF-1R and pIR in NCI-H292 tumor lysates at 2, 4, and 24 hours after a single 60 mg/kg treatment of OSI-906 (A and B). In vivo Western blot of NCI-H292 tumor lysates at 4 and 24 hours shows inhibition of selected markers of altered glycolysis, pERK 1/2, pAKT, and pS6 at 4 hours postdosing that return to baseline levels by 24 hours (C).

Figure 5.

RTK array analysis shows strong target inhibition of both pIGF-1R and pIR in NCI-H292 tumor lysates at 2, 4, and 24 hours after a single 60 mg/kg treatment of OSI-906 (A and B). In vivo Western blot of NCI-H292 tumor lysates at 4 and 24 hours shows inhibition of selected markers of altered glycolysis, pERK 1/2, pAKT, and pS6 at 4 hours postdosing that return to baseline levels by 24 hours (C).

Close modal

Pharmacokinetic analysis

Table S1 shows the drug concentration in the plasma samples from the NCI-H292 xenografts remained at a constant concentration approximately 20 μmol/L for 2 to 8 hours postdosing. By 24 hours postdosing, the level of OSI-906 in the plasma had decreased by approximately 60% to approximately 6.5 μmol/L, resulting in some potential loss of target coverage with time.

Catabolism of glucose through the tricarboxylic acid cycle in normal cells is the preferred method of ATP production leading to cell proliferation and survival. It is now well known that many cancer cells avidly consume glucose and produce lactic acid for ATP production despite the inefficiency of this metabolic pathway. The reason why cancer cells utilize a less efficient means of ATP production remains elusive; however, recent studies suggest that in cancer cells an increase in glycolysis, in addition to respiration, can generate energy more quickly than normal cells that rely on respiration alone. As a result, this high rate of glucose metabolism by cancer cells has resulted in the wide use of 18FDG PET to image and diagnose rapidly dividing cells including tumors (18).

Both IGF-1R and IR signal through the PI3K signaling pathway. PI3K is linked to both growth control and glucose metabolism. PI3K directly regulates glucose uptake and metabolism via AKT mediated regulation of glucose transporter activation and expression (GLUT1 and GLUT4), enhanced glucose capture by increased hexokinase activity, and stimulation of phosphofructokinase activity (19–22). PI3K activation thus renders cells dependent on glucose leading to glucose addiction. In normal cells, activation of PI3K/AKT is highly controlled by dephosphorylation of phosphatidylinositol by PTEN. However, in many cancers, PTEN is lost leading to constitutive activation of the PI3K pathway (23). Moreover, activation of this pathway can be enhanced by other mechanisms, which, when combined, can constitute some of the more prevalent classes of mutations in human malignancy (e.g., PI3CA, AKT2, BCR-ABL, HER2/neu). Therefore, activation of AKT is likely the most important signaling event in relation to cellular metabolism, because AKT is sufficient to drive glycolysis and lactate formation and suppress macromolecular degradation in cancer (23, 24). It has been shown that various therapeutic agents that disrupt the PI3K/AKT pathway, either directly or upstream of PI3K/AKT, lead to decreased glucose uptake in tumors as measured by 18FDG-PET (25). Furthermore, the ability to inhibit FDG uptake in tumors has been shown to correlate well with treatment response in a number of cancers. As a consequence, 18FDG-PET has been used clinically in cancer patients to predict response to various therapies via the ability of agents to disrupt glucose metabolism and glucose uptake in tumors (22, 26–28)

The primary purpose of these studies was to determine whether 18FDG-PET could be used as an early, noninvasive PD biomarker for the dual kinase inhibitor OSI-906. We first determined in vitro using the sensitive cell line, NCI-H292 that a rapid decrease in 3H-2-deoxy glucose uptake was observed in a dose-dependent manner after treatment with pharmacologically relevant concentrations of OSI-906. In the NCI-H441 cell line reduced sensitivity to equimolar concentrations of OSI-906 was observed for the same assay. NCI-H292 cell lysates were then probed for markers of altered glycolysis by Western blot analysis and showed a significant reduction in pIGF-1R, pIR, pAKT, pS6, and pERK 1/2. Target inhibition of these markers strongly link IGF-1R and IR to the PI3 kinase and AKT pathways and resultant changes in metabolic activity of cultured cells when exposed to OSI-906.

In vivo, decreased uptake of 18FDG was observed rapidly at 2, 4, and 24 hours after administration of an efficacious dose of 60 mg/kg of OSI-906 in NCI-H292 tumor–bearing animals. In comparison, the insensitive NCI-H441 xenografts showed no change in uptake of the radiotracer at the same time points and same dosage. Analysis of target inhibition of pAKT, pS6, pERK 1/2, pIGF-1R, and pIR from NCI-H292 tumor lysates was carried out by Western blot and RTK array analysis. The results showed strong target inhibition of these markers at 4 hours postadministration of a single 60 mg/kg dose of OSI-906, further corroborating the link of metabolic activity of tumors with IGF-1R and IR signaling pathways. Specific target inhibition of pIGF-1R and IR by RTK array analysis resulted in significant (P <0.05) reduction of these phospho-targets (>80%) at 2 and 4 hours postadministration of the agent, and correlated to reduced uptake of 18FDG. Blood glucose levels of non–tumor-bearing mice appeared elevated from a baseline, fasted level following 2 and 4 hours of 60mg/kg OSI-906 treatment; however, the increased levels were not statistically significant (P>0.5). As expected, similarly evaluated vehicle-treated mice did not exhibit elevated glucose levels when evaluated at 2 and 4 hours (Supplementary Fig. S2A). Importantly, 18FDG uptake in NCI-H441 tumors, which are insensitive to OSI-906, was similar in both OSI-906–treated and vehicle-treated tumors. The fact that posttreatment 18FDG uptake in these mice was not decreased when compared with baseline imaging suggests that the somewhat elevated circulating glucose levels had no detectable impact on 18FDG uptake in this study. As further evidence, no change in 18FDG uptake was seen in skeletal muscle following OSI-906 (Supplementary Fig. S2B), and only a slight increase in liver 18FDG uptake was seen at 2 and 4 hours before returning to baseline at 24 hours (Supplementary Fig. S2C). Nonetheless, it is possible that human trials incorporating 18FDG PET as a biomarker of response to OSI-906 may benefit from measurement of blood glucose levels, as the effects on 18FDG uptake in patient studies could be larger than we observed in mice.

The present findings support a strong link of rapidly altered metabolic activity in both cultured cells and in vivo tumors induced by target inhibition of the IGF-1R and IR signaling pathways. Though there is still much to be learned how cellular metabolism in proliferating cells is regulated, there is an ever increasing body of information supporting increased communication between signaling pathways and metabolic control of the cell. These studies suggest that 18FDG-PET has potential to serve as a rapid, noninvasive biomarker of PD effects of OSI-906 in patients treated with this dual IGF-1R/IR kinase inhibitor. This method may be most beneficial in early clinical development where accurate assessment of PD effects is often limited by the lack of readily accessible tumor samples. Thus, 18FDG-PET may be a useful clinical tool in identifying active doses and patients potentially sensitive to this novel antitumor agent and perhaps other compounds of this target class. Currently, 18FDG-PET imaging is being employed in several clinical trials as a biomarker for early efficacy of OSI-906.

J.E. Bugaj, C. Mantis, P.C. Gokhale, and R. Wild are employed with OSI Pharmaceuticals. H.C. Manning received a commercial grant from OSI Pharmaceuticals. R. Wild has ownership interest in OSI Pharmaceuticals. The other authors disclosed no potential conflicts of interest.

The authors thank Dr. Andy Cooke and Mr. Mark Bittner for expert analysis of in vivo plasma samples and Dr. M. Noor Tantawy and Ms. Clare A. Osborne for assistance with the PET imaging studies.

This study was supported by the National Cancer Institute (R01-CA140628, U24-CA126588, K25 CA127349, 1RC1 CA145138) and a research grant from OSI Pharmaceuticals. E.T. McKinley was supported by a cellular molecular imaging training grant (R25-CA136440).

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.

1.
Ji
QS
,
Mulvihill
MJ
,
Rosenfeld-Franklin
M
,
Cooke
A
,
Feng
L
,
Mak
G
, et al
A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor dependent tumor growth in vivo
.
Mol Cancer Ther
2007
;
6
:
2158
67
.
2.
Chan
JM
,
Stampfer
MJ
,
Giovannucci
E
,
Gann
PH
,
Ma
J
,
Wilkinson
P
, et al
Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study
.
Science
1998
;
279
:
563
6
.
3.
Hankinson
SE
,
Willett
WC
,
Colditz
GA
,
Hunter
DJ
,
Michaud
DS
,
Deroo
B
, et al
Circulating concentrations of insulin-like growth factor-I and risk of breast cancer
.
Lancet
1998
;
351
:
1393
6
.
4.
Ma
J
,
Pollak
MN
,
Giovannucci
E
,
Chan
JM
,
Tao
Y
,
Hennekens
CH
, et al
Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3
.
J Natl Cancer Inst
1999
;
91
:
620
5
.
5.
Yu
H
,
Spitz
MR
,
Mistry
J
,
Gu
J
,
Hong
WK
,
Wu
X
. 
Plasma levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis
.
J Natl Cancer Inst
1999
;
91
:
151
6
.
6.
LeRoith
D
,
Roberts
CT
 Jr
. 
The insulin-like growth factor system and cancer
.
Cancer Lett
2003
;
195
:
127
37
.
7.
Parker
AS
,
Cheville
JC
,
Janney
CA
,
Cerhan
JR
. 
High expression levels of insulin-like growth factor-I receptor predict poor survival among women with clear-cell renal cell carcinomas
.
Hum Pathol
2002
;
33
:
801
5
.
8.
Baserga
R
,
Peruzzi
F
,
Reiss
K
. 
The IGF-1 receptor in cancer biology
.
Int J Cancer
2003
;
107
:
873
7
.
9.
Riedemann
J
,
Macaulay
VM
. 
IGF1R signalling and its inhibition
.
Endocr Relat Cancer
2006
;
13
Suppl 1
:
S33
43
.
10.
Belfiore
A
,
Frasca
F
,
Pandini
G
,
Sciacca
L
,
Vigneri
R
. 
Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease
.
Endocr Rev
2009
;
30
:
586
623
.
11.
Buck
E
,
Gokhale
P
,
Koujak
S
,
Brown
E
,
Eyzaguirre
A
,
Tao
N
, et al
Compensatory insulin receptor (IR) activation upon inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for co-targeting IGF-1R and IR in cancer
.
Mol Cancer Ther
2010
;
9
:
2652
64
.
12.
Mulvihill
MJ
,
Cooke
A
,
Rosenfeld-Franklin
M
,
Buck
E
,
Foreman
K
,
Landfair
D
, et al
Discovery of OSI-906: a selective and orally efficacious dual inhibitor of the IGF-1 receptor and insulin receptor
.
Future Med Chem
2009
;
1
:
1153
71
.
13.
Manning
HC
,
Merchant
NB
,
Foutch
AC
,
Virostko
JM
,
Wyatt
SK
,
Shah
C
, et al
Molecular imaging of therapeutic response to epidermal growth factor receptor blockade in colorectal cancer
.
Clin Cancer Res
2008
;
14
:
7413
22
.
14.
Ayers
GD
,
McKinley
ET
,
Zhao
P
,
Fritz
JM
,
Metry
RE
,
Deal
BC
, et al
Volume of preclinical xenograft tumors is more accurately assessed by ultrasound imaging than manual caliper measurements
.
J Ultrasound Med
;
29
:
891
901
.
15.
Shah
C
,
Miller
TW
,
Wyatt
SK
,
McKinley
ET
,
Olivares
MG
,
Sanchez
V
, et al
Imaging biomarkers predict response to anti-HER2 (ErbB2) therapy in preclinical models of breast cancer
.
Clin Cancer Res
2009
;
15
:
4712
21
.
16.
Fueger
BJ
,
Czernin
J
,
Hildebrandt
I
,
Tran
C
,
Halpern
BS
,
Stout
D
, et al
Impact of animal handling on the results of 18F-FDG PET studies in mice
.
J Nucl Med
2006
;
47
:
999
1006
.
17.
Dandekar
M
,
Tseng
JR
,
Gambhir
SS
. 
Reproducibility of 18F-FDG microPET studies in mouse tumor xenografts
.
J Nucl Med
2007
;
48
:
602
7
.
18.
Weber
WA
. 
Positron emission tomography as an imaging biomarker
.
J Clin Oncol
2006
;
24
:
3282
92
.
19.
Elstrom
RL
,
Bauer
DE
,
Buzzai
M
,
Karnauskas
R
,
Harris
MH
,
Plas
DR
, et al
Akt stimulates aerobic glycolysis in cancer cells
.
Cancer Res
2004
;
64
:
3892
9
.
20.
Clemmons
DR
. 
Involvement of insulin-like growth factor-I in the control of glucose homeostasis
.
Curr Opin Pharmacol
2006
;
6
:
620
5
.
21.
Deberardinis
RJ
,
Sayed
N
,
Ditsworth
D
,
Thompson
CB
. 
Brick by brick: metabolism and tumor cell growth
.
Curr Opin Genet Dev
2008
;
18
:
54
61
.
22.
Vander Heiden
MG
,
Cantley
LC
,
Thompson
CB
. 
Understanding the Warburg effect: the metabolic requirements of cell proliferation
.
Science
2009
;
324
:
1029
33
.
23.
DeBerardinis
RJ
,
Lum
JJ
,
Hatzivassiliou
G
,
Thompson
CB
. 
The biology of cancer: metabolic reprogramming fuels cell growth and proliferation
.
Cell Metab
2008
;
7
:
11
20
.
24.
Kroemer
G
,
Pouyssegur
J
. 
Tumor cell metabolism: cancer's Achilles' heel
.
Cancer Cell
2008
;
13
:
472
82
.
25.
Engelman
JA
,
Chen
L
,
Tan
X
,
Crosby
K
,
Guimaraes
AR
,
Upadhyay
R
, et al
Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers
.
Nat Med
2008
;
14
:
1351
6
.
26.
DeBerardinis
RJ
. 
Is cancer a disease of abnormal cellular metabolism? New angles on an old idea
.
Genet Med
2008
;
10
:
767
77
.
27.
Kelloff
GJ
,
Hoffman
JM
,
Johnson
B
,
Scher
HI
,
Siegel
BA
,
Cheng
EY
, et al
Progress and promise of FDG-PET imaging for cancer patient management and oncologic drug development
.
Clin Cancer Res
2005
;
11
:
2785
808
.
28.
Mankoff
DA
,
Eary
JF
,
Link
JM
,
Muzi
M
,
Rajendran
JG
,
Spence
AM
, et al
Tumor-specific positron emission tomography imaging in patients: [18F] fluorodeoxyglucose and beyond
.
Clin Cancer Res
2007
;
13
:
3460
9
.