Diarylureas Are Small-Molecule Inhibitors of Insulin-Like Growth Factor I Receptor Signaling and Breast Cancer Cell Growth

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Diarylureas are small-molecule inhibitors of insulin-like growth factor I receptor signaling and breast cancer cell growth

Karissa L. Gable,1,2 Betty A. Maddux,1 Akt antiapoptotic pathway. Twenty-four hours of treat- Cristina Penaranda,1 Marianna Zavodovskaya,1 ment with 15 Mmol/L PQ401 induced caspase-mediated Michael J. Campbell,2 Margaret Lobo,2 apoptosis. In vivo, treatment with PQ401 (i.p. injection Louise Robinson,4 Steven Schow,4 thrice a week) reduced the growth rate of MCNeuA cells John A. Kerner,3 Ira D. Goldfine,1 implanted into mice. These studies indicate that diaryl urea compounds are potential new agents to test in the and Jack F. Youngren1 treatment of breast and other IGF-I-sensitive cancers. 1Diabetes and Endocrine Research and 2Department of [Mol Cancer Ther 2006;5(4):1079–86] Surgery, University of California, San Francisco/Mt. Zion Medical Center; 3Departments of Medicine, and Obstetrics, Gynecology, and Reproductive Sciences, University of California, Introduction San Francisco, San Francisco, California; and 4Telik, Inc., Palo Receptor tyrosine kinases (RTK) play a critical role in breast Alto, California cancer growth and survival (1–6), and thus have become targets for antitumor therapy (7–10). Like other RTKs, the Abstract insulin-like growth factor receptor (IGF-IR) is a transmem- brane protein containing an extracellular ligand binding In breast and certain other cancers, receptor tyrosine domain and an intracellular tyrosine kinase domain. kinases, including the insulin-like growth factor I receptor Signaling via the tyrosine kinase domain of the IGF-IR is (IGF-IR), play an important role in promoting the oncogenic important for normal cell growth and differentiation (11). process. The IGF-IR is therefore an important target for In addition, the IGF-IR stimulates mitogenesis and sup- developing new anti–breast cancer therapies. An initial presses apoptosis of cancer cells (11). screening of a chemical library against the IGF-IR in breast A critical initial step in IGF-IR signal transduction cancer cells identified a diaryl urea compound as a potent resulting from ligand binding is the conformational change inhibitor of IGF-IR signaling. This class of compounds has of the receptor that results in trans-autophosphorylation of not been studied as inhibitors of the IGF-IR. We studied the the h-subunits on select tyrosine residues. This autophos- effectiveness of one diaryl urea compound, PQ401, at phorylation step is required for the subsequent activation antagonizing IGF-IR signaling and inhibiting breast cancer of IGF-IR tyrosine kinase activity (11). Phosphorylation of in vivo cell growth in culture and . PQ401 inhibited several target substrates activates divergent signaling autophosphorylation of the IGF-IR in cultured human cascades, although many of the biological effects of IGF-I M MCF-7 cells with an IC50 of 12 mol/L and autophosphor- are mediated by tyrosine phosphorylation of the insulin ylation of the isolated kinase domain of the IGF-IR with an receptor substrate (IRS) family of proteins. Phosphorylation M IC50 <1 mol/L. In addition, PQ401 inhibited the growth of IRS-1 and IRS-2 allows binding of the regulatory subunit M of cultured breast cancer cells in serum at 10 mol/L. of phosphatidylinositol 3-kinase via SH2 domains. Activat- PQ401 was even more effective at inhibiting IGF-I- ed phosphatidylinositol 3-kinase serine phosphorylates and M stimulated growth of MCF-7 cells (IC50,6 mol/L). activates the serine kinase Akt (12). The antiapoptotic Treatment of MCF-7 cells with PQ401 was associated effects of the IGF-IR are primarily mediated via the Akt/ with a decrease in IGF-I-mediated signaling through the protein kinase B pathway (13), as Akt phosphorylates the protein BAD, which prevents BAD from forming a proapoptotic complex with Bcl-2 proteins (14). Received 9/30/05; revised 1/20/06; accepted 2/15/06. Interruption of the IGF-IR signaling system, either by Grant support: California Breast Cancer Research Program grant reducing effective IGF-I levels or targeting the receptor, can 8WB-0099 (J.F. Youngren), the University of California, San Francisco Academic Senate Committee on Research (J.F. Youngren), National block growth and proliferation of cancer cells (15–19). Cancer Institute grant 5 U-56 CA92616-04 (J.F. Youngren), the Whereas overexpression of the IGF-IR can drive transforma- John Kerner Fund, and the Jay Gershow Fund. tion and mitogenesis, a more important feature of the IGF-IR The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked is the requirement for its constitutive presence in cancer cells advertisement in accordance with 18U.S.C. Section 1734 solely to (20). Thus, studies employing a variety of strategies to block indicate this fact. IGF-IR signaling in a range of cancer cell types show that the Requests for reprints: Jack Youngren, Division of Diabetes and Endocrine IGF-IR is an effective target in cells even when tumorigenesis Research University of California, San Francisco/Mt. Zion Medical Center, Box 1616, San Francisco, CA 94143-1616. Phone: 415-885-7725; is driven by a variety of molecular mechanisms. Fax: 415-885-7727. E-mail: [email protected] Due to this requirement for IGF-IR signaling in the Copyright C 2006 American Association for Cancer Research. establishment and maintenance of the transformed pheno- doi:10.1158/1535-7163.MCT-05-0397 type and its major role in regulating cancer cell survival,

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inhibitors of the IGF-IR and related RTKs are important IGF-IR Peptide Autophosphorylation targets for drug development (9, 21, 22). We have recently One microgram of constitutively active IGF-IR kinase identified a new class of small-molecule compounds, domain peptide was incubated +/À varying concentrations diaryl ureas, which inhibit the IGF-IR. Herein we describe of PQ401 in 2% DMSO in 40 mmol/L Tris (pH 7.4), 80 the biological effects of the diaryl urea compound PQ401 Amol/L EGTA, 0.25% 2-mercaptoethanol, 80 Amol/L on the growth and survival of breast cancer cells in culture Na3VO4, 10 mmol/L MgCl2, and 2 mmol/L MnCl2 for and in vivo. 20 minutes. ATP was then added at a final concentration of 20 Amol/L. Autophosphorylation of the kinase domain Materials and Methods peptide was allowed to occur for 20 minutes at 22jC. The Materials reaction was stopped by the addition of SDS-reducing Compounds that compose the chemical library employed buffer and the samples were run on SDS-PAGE. Following in an initial screen against the IGF-IR, as well as additional transfer to nitrocellulose membrane, peptide autophos- diaryl urea compounds, were obtained from Telik Corp. phorylation was determined by Western blotting employ- (Palo Alto, CA). IGF-IR kinase domain peptide was obtained ing an antibody against phosphotyrosine (PY20). from Upstate USA (Charlottesville, VA). Antibodies against Effects of Diaryl Ureas on Proliferation of Breast the IGF-IR (C-20), phosphospecific antibodies recognizing Cancer Cells phosphotyrosine (PY20), and horseradish peroxidase– Proliferation assays were conducted with MCF-7or conjugated antiphosphotyrosine antibody (PY20HRP) were MCNeuA breast cancer cells. The MCNeuA cell line is a obtained from Santa Cruz Biotechnology (Santa Cruz, CA). mammary carcinoma cell line we have recently established aIR3, a monoclonal antibody against the IGF-IR, was from a spontaneously arising tumor in a neu transgenic obtained from Calbiochem (San Diego, CA) and the total female mouse (24) and is thus driven by HER2/Neu protein and phosphospecific antibodies (Ser473) against Akt overexpression but which also expresses IGF-IR. The were obtained from Cell Signaling (Beverly, MA). All other inhibitory effects of diaryl urea on breast cancer cell growth reagents were from Sigma (St. Louis, MO) except where were determined using a CyQuant cell proliferation assay kit indicated. (Molecular Probes, Eugene, OR). MCF-7or MCNeuA cells Determination of Ligand-Stimulated IGF-IR Auto- were plated in 96-well plates (5 Â 103 per well) in phenol red– phosphorylation free DMEM supplemented with 10% FCS. One plate was To study the effects of diaryl ureas on IGF-IR signaling in prepared for each harvest day. Cells were allowed to adhere breast cancer cells, MCF-7cells were grown in either six- overnight and were then treated with various concentrations well or 96-well plates. Upon reaching 80% confluence, cells of diaryl urea or DMSO as a vehicle control. Microplate were serum starved for 18 hours. diaryl urea compounds cultures were harvested on days 0, 1, 2, and 3 by inverting the were dissolved in DMSO and diluted with culture medium microplate onto paper towels with gentle blotting to remove before being added to cells for 2.5 hours at 37jC. The final growth medium without disrupting adherent cells. Each concentration of DMSO during the incubation was 0.3%. plate was kept at À80jC until the end of the experiment Cells were then stimulated with 3 nmol/L IGF-I for 10 (day 3) when all of the plates were thawed and assayed minutes at 37jC. Reactions were terminated by rapidly together. After thawing, 200 AL of CyQuant GR solution were aspirating medium and washing cells thrice with ice-cold added to each well and the plates were incubated in the dark PBS. Cells were harvested and solubilized in 50 mmol/L for 2 to 5 minutes. Fluorescence was measured with a HEPES, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L SpectraMax Gemini XS fluorescence microplate reader phenylmethylsulfonyl fluoride, and 2 mmol/L vanadate (Molecular Devices, Sunnyvale, CA) with 480-nm excitation for 1 hour at 4jC. Protein was determined by bicinchoninic and 520-nm emission. Proliferation index was calculated as acid assay (Pierce, Rockford, IL). the percent of nucleotide content versus control cells at day 0. IGF-IR autophosphorylation was then determined by Effects of Diaryl Urea on IGF-I Stimulated Prolifera- ELISA as previously described for the IR (23). Briefly, 20 Ag tion of Breast Cancer Cells of lysate protein were added to duplicate wells in a 96-well MCF-7cells were harvested by washing thrice with PBS plate coated with monoclonal antibody to the IGF-IR (aIR3; and dissociating with 1 mL 0.05% trypsin. Cells were 2 Ag/mL) and incubated for 18 hours at 4jC. Plates were resuspended in 5 mL defined medium (1:1 Ham’s F12/ washed five times, and then horseradish peroxidase– DMEM 4.5 g/L glucose; 1 mg/mL bovine serum albumin; conjugated antiphosphotyrosine antibody (0.3 Ag/mL), 10 Ag/mL transferrin; 15 mmol/L HEPES pH 7.2; 2 mmol/L diluted in Solution B [50 mmol/L HEPES (pH 7.6), 150 L-glutamine; 100 units/mL penicillin G; 100 Ag/mL strep- A A mmol/L NaCl, 0.05% Tween 20, 1 mmol/L phenylmethyl- tomycin SO4;2.5 g/mL fungizone) containing 200 g sulfonyl fluoride, 2 mmol/L vanadate and 1 mg/mL soybean trypsin inhibitor. Cells were plated in 96-well bacitracin], was added for 2 hours at 22jC. Plates were collagen-coated plates (Sigma) at a density of 5,000 per well washed five times before color development with 3,3V,5,5V- in 100-AL medium. Twenty hours later, defined medium tetramethly benzidine peroxidase substrate, which was with or without IGF-I (10 nmol/L) was added. Four hours terminated with 1.0 mol/L H3PO4. Values for receptor later, PQ401 diluted in defined medium was added. Plates autophosphorylation were determined by measuring absor- were harvested on day 3, as described above, for determi- bance at 450 nm. nation of cell number by CyQuant assay.

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Activation of the Akt Apoptotic Pathway 80 and ethanol). Treated mice received 50 or 100 mg/kg Inhibition of IGF-I-stimulated activation of the serine PQ401 prepared in 8% polysorbate 80/ethanol/PBS, ad- kinase Akt was determined from the lysates prepared from ministered i.p. thrice a week. Control mice received i.p. MCF-7cells grown in six-well plates and employed in the injections of the polysorbate 80/ethanol vehicle only. IGF-IR phosphotyrosine ELISA described above. Twelve Tumor growth was measured at the time of drug delivery micrograms of sample were subjected to SDS-PAGE, on each treatment day with calipers and tumor volume was transferred to a nitrocellulose membrane, and phosphory- calculated using the equation (length Â width2) Â (k/6). lated Akt was quantified by blotting with a phosphospecific Statistics antibody to Akt(Ser473). Total Akt content was determined in Statistics were calculated using MedCalc statistical these samples by blotting with an antibody against the Akt software (MedCalc Software, Mariakerke, Belgium). protein. Growth of breast cancer cells in culture and in vivo was Caspase Activation analyzed by two-way ANOVA for treatment, time, and MCF-7cells were plated in 96-well plates (5 Â 103 per interaction effects with post hoc analysis by Student’s t test. well) in a volume of 200 AL. After 24 hours, cells were Dose effects of diaryl urea on receptor phosphorylation washed thrice with PBS and medium containing diaryl were analyzed by one-way ANOVA with post hoc analysis urea (final concentrations, 7.5, 15, or 30 Amol/L) or by Student’s t test. Significance was set at P < 0.05. doxorubicin (final concentration, 10 Amol/L) diluted in DMSO, or DMSO alone (final concentration, 0.5%), was Results added. After 24 hours of incubation with the compounds, Screening Assay 10 AL of 10% NP40 were added per well and the plate was To identify novel inhibitors of the IGF-IR, a chemical placed on a rotary shaker as lysis occurred for 5 minutes. library was generated via the target-related affinity profiling The cell extract and supernatant were transferred to lead identification technology (26, 27). These compounds Eppendorf tubes and debris was removed by spinning in were employed in initial screening assays for inhibitory a microcentrifuge for 2 minutes at 5,000 rpm at 4jC. activity against the IGF-IR in MCF-7breast cancer cells. In an General caspase activity was ascertained by employing the initial screening of 100 compounds at 5 to 25 Ag/mL each, a M30 Apoptosense ELISA kit (Alexis Biochemicals, Lausen, compound with a general diaryl urea structure produced a Switzerland) which measures the level of a neo-epitope in dose-dependent inhibition of IGF-I-stimulated IGF-IR auto- the COOH-terminal domain of cytokeratin 18 (amino acids phosphorylation (data not shown). To identify an effective 387–396) that is revealed after apoptotic cleavage. Briefly, diaryl urea compound for early biological studies, we 25 AL of culture extract plus supernatant were added to initially constructed a small diaryl urea library consisting duplicate wells in a 96-well plate coated with mouse of four basic families, each with a distinct structure or monoclonal antibody to CK18. Then, horseradish peroxi- linkage of the second aromatic ring. Based on results from a dase–conjugated M30 antibody, diluted in phosphate subsequent screen of these related diaryl ureas, we chose one buffer with protein stabilizers (included in kit), was added compound from the phenyl-4-quinoyl urea class, PQ401 immediately afterwards, and plate was incubated with (Fig. 1A), to study the biological effects of this class of small agitation for 4 hours at 22jC. Plates were washed five times molecules based on its significant potency against the IGF-IR and 3,3V,5,5V-tetramethly benzidine peroxidase substrate in cell-based assays. In a series of studies in MCF-7cells, was added, and plates were then incubated in darkness PQ401 inhibited IGF-I-stimulated IGF-IR autophosphoryla- j F A for 20 minutes at 22 C. The reaction was stopped with 1.0 tion with an IC50 of 12.0 0.9 mol/L (Fig. 1B). IGF-IR mol/L H2SO4. Values for caspase activity were determined inhibition was minimal or absent in eight compounds with a by measuring absorbance at 450 nm. 3-quinoyl group replacing the methyl-substituted 4-quinoyl In vivo Studies group seen in PQ401, suggesting the more linear conforma- Our syngeneic model studies used the FVB/N- tion of the 3-linkage of these compounds reduced effective- TgN(MMTVneu)202 mouse strain developed by Guy et al. ness against the target (data not shown). From this family of (25). This strain, denoted hereafter as neuTg, expresses the inactive compounds, PQ20 (Fig. 2), which showed no IGF-IR wild-type rat neu proto-oncogene (a homologue of human inhibiting capacity (Fig. 2), was subsequently employed as HER2) under the control of the mouse mammary tumor an IGF-IR kinase-inactive control due to its first aromatic virus (MMTV) long terminal repeat on an FVB mouse back- ring being identical to PQ401. PQ20 was thus employed to ground. The MCNeuA mammary carcinoma cell line test the specificity of the IGF-IR inhibitory effects of PQ401 employed in these studies was derived from a spontane- on cell growth. ously arising tumor in a neuTg female mouse (23). These Direct Effects of PQ401 on IGF-IR Kinase Domain cells are tumorigenic when transplanted back into neuTg To determine if PQ401 was acting directly against the mice. Female mice were injected s.c. with 105 MCNeuA kinase domain of the IGF-IR, we incubated a constitutively tumor cells on day 0. Treatment with PQ401 began on day 3 active synthetic peptide consisting of amino acids 959-end of postinoculation. For injection, PQ401 was dissolved into the IGF-IR with increasing concentrations of PQ401 before 50% polysorbate 80/50% ethanol at a concentration of the addition of 20 Amol/L ATP. PQ401 inhibited autophos- 25 mg/mL. This mixture was then diluted with PBS to a phorylation of the IGF-IR kinase domain at concentrations A final concentration of 4 mg/mL PQ401 (8% polysorbate <100 nmol/L, with an IC50 <1 mol/L (Fig. 3).

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inhibit growth of these cells mediated by IGF-I alone. Supplementing serum-free medium with 10 nmol/L IGF-I produced a 3-fold increase in cell number over the 3 days of growth, showing the responsiveness of these cells to IGF-I (Fig. 4C). In MCF-7cells grown in serum-free medium supplemented only with 10 nmol/L IGF-I, PQ401 inhibited f A proliferation with an IC50 of 6 mol/L (Fig. 4C). The addition of PQ401 to cells incubated in nonsupplemented, serum-free medium produced a less dramatic inhibition of growth. At concentrations of PQ401 z15 Amol/L, the growth stimulatory effects of IGF-I over the serum-free condition were fully abolished. Effects of PQ401on Apoptosis To explore the mechanisms linking the IGF-IR inhibitory actions of diaryl ureas and their cytotoxic effects in cells, we studied the effects of PQ401 on the cell cycle and apoptotic pathways that are regulated by the IGF-IR. Exposure of MCF-7cells to varying concentrations of PQ401 for 2 hours produced a significant reduction in the IGF-I-stimulated phosphorylation of the serine kinase Akt/protein kinase B (Fig. 5). Next, we employed an ELISA for the apoptosis- associated M30 neo-epitope in total cellular extracts plus supernatant medium to assess activation of the caspase apoptotic pathway. Twenty-four hours of incubation with PQ401 induced a dose-dependent increase in caspase activation (Fig. 6) in MCF-7cells. Figure 1. Inhibition of IGF-IR signaling by PQ401 in MCF-7 cells. A, Effects of PQ401on BreastTumors in Mice chemical structure of PQ401. MCF-7 breast cancer cells were incubated with varying concentrations of PQ401 for 1 h. Cells were then incubated F We employed MCNeuA breast cancer cells in a syngeneic 3 nmol/L IGF-I. B, tyrosine phosphorylation of IGF-IR was determined from mouse model of breast cancer. This cell line is a mammary soluble extracts by specific ELISA. Columns, mean of four experiments, carcinoma cell line that we have recently established from a normalized to cells treated with vehicle alone; bars, SE. *, values significantly reduced versus vehicle-treated controls. spontaneously arising tumor in a neuTg female mouse (23). These cells display the characteristics of an estrogen- insensitive, p53-null, HER2-positive breast tumor. In Effects of PQ401 on Growth of MCF-7 Cells To test our hypothesis that inhibition of the IGF-IR by PQ401 would inhibit growth of breast cancer cells, we studied the effects of exposure to both PQ401 and the kinase inactive PQ20 on proliferation of MCF-7breast cancer cells. Employing a CyQuant assay, which estimates cell number by quantifying nucleic acid content, we examined MCF-7 proliferation across 3 days of incubation with various concentrations of either diaryl urea. The compounds were added to normal medium on day 0 and the medium was not changed over the course of the study. With this protocol, PQ401, at concentrations in the range of 1 Amol/L, A significantly reduced proliferation (IC50,8 mol/L), and at concentrations >10 Amol/L, produced a dramatic reduction in cell number from pretreatment levels (Fig. 4A). In contrast, PQ20 had no effect on proliferation of MCF-7cells at any concentration tested (Fig. 4B). PQ401 was also able to inhibit growth of MCNeuA cells with somewhat less A potency (IC50,15 mol/L; data not shown), suggesting that inhibition of IGF-IR by diaryl ureas can reduce growth in breast tumors driven by overexpression of the HER2/neu Figure 2. Characteristics of negative control PQ20. A, chemical receptor. structure of PQ20. B, despite similar structure as PQ401, PQ20 has no To determine further whether the inhibitory effects of ability to inhibit the IGF-IR in MCF-7 cells. Tyrosine phosphorylation of IGF- IR was determined from soluble extracts by specific ELISA. Columns, PQ401 on MCF-7cells were specific for the effects on the mean of four experiments, normalized to cells treated with vehicle alone; IGF-IR, we examined the ability of PQ401 to specifically bars, SE.

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Figure 3. Direct inhibition of IGF-IR kinase domain by PQ401. Peptides corresponding to the kinase domain of IGF-IR were incubated with varying concentrations of PQ401 before the addition of ATP. Reduction of tyrosine phosphorylation of peptides by PQ401 was determined by Western blot that employed an antiphosphotyrosine antibody. Representative blot of three experiments. culture, these cells displayed similar sensitivity as MCF-7 cells to growth inhibition by PQ401 as well as inhibition of IGF-IR autophosphorylation (data not shown). Thus, we employed these cells for in vivo studies to examine PQ401 effects against an aggressive tumor phenotype observed in f30% of human breast cancers that is recalcitrant to antiestrogen therapy. In addition, when these cells are implanted s.c. into the strain from which they were derived, all injected animals develop tumors. Thus, we could initiate treatment 3 days following implantation, before the development of palpable tumors. Injection of either 50 or 100 mg/kg PQ401 thrice per week in a polysorbate 80/ethanol vehicle (28) resulted in a significant dose-dependent reduction in tumor growth over the course of the study (Fig. 7). Tumor growth in the animals treated with 100 mg/kg PQ401 thrice a week was 20% of that in the vehicle-treated controls. This dosing protocol was well tolerated by the animals.

Discussion In the present study, we observed that diaryl ureas are direct inhibitors of the IGF-IR in isolated receptor prepa- rations and cultured breast cancer cells. Diaryl urea treatment reduces breast cancer cell growth in cell culture in a manner specific for IGF-IR inhibition and reduces tumor growth in vivo. These studies suggest that diaryl ureas are potential anticancer agents acting via inhibition of the IGF-IR. The IGF-IR is an essential component of tumorigenesis and an active target in the development of anticancer therapies (5). Expression of the IGF-IR is a requirement for Figure 4. PQ401 inhibits proliferation of cultured MCF-7 cells grown in transformation in cultured cells (29). IGF-IR overexpression serum or IGF-I. MCF-7 cells were incubated with varying concentrations is a feature of many types of transformed cells and tumors of PQ401 (A), PQ20 (B), or DMSO alone beginning on day 0. Cell (20, 30). The IGF-IR is overexpressed and hyperphosphory- number was estimated by determination of nucleotide content (CyQuant in vivo assay) on days 0, 1, 2, and 3. Proliferation index was calculated as the lated in breast tumors (31), and hyperactivation of percent difference in cell number versus day 0. Columns, mean of three the IGF-IR is associated with the early stages of breast experiments; bars, SE. *, proliferation significantly reduced versus cancer (3, 32, 33). vehicle-treated controls. c, cell number significantly reduced from day A number of approaches have been employed to target 0. C, for studies of IGF-I-mediated growth, MCF-7 cells were initially plated in basal medium. Growth continued in basal medium or medium the IGF-IR in cultured breast cancer cells. Antisense RNA supplemented with 10 nmol/L IGF-I. Twenty-four hours after plating (day for the IGF-IR (16), monoclonal antibodies (34, 35), 0), cells were incubated with varying concentrations of PQ401 or DMSO transfection with a dominant negative IGF-IR (17), and alone. Plates were harvested on day 3 for CyQuant assay. Values shown small-molecule catechol mimics (21, 36) have all shown are absorbance values reflecting total nucleic acid content per well. Columns, mean of four experiments; bars, SE. c, proliferation significantly effectiveness at inhibiting proliferation of breast cancer reduced versus vehicle-treated controls. *, proliferation significantly cells in vitro. Expressing inactive IGF-IR in breast cancer greater than serum-free condition.

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IGF-IR that is greater against isolated receptors than in cell- based assays (44, 45). Both compounds showed effectiveness at inhibiting proliferation of multiple cancer cell lines, and each compound inhibited the growth of xenograft models of multiple myeloma or fibroid sarcoma. One compound, NVP-ADW-742, showed cell growth–inhibitory activity similar to that observed with a monoclonal antibody against the IGF-IR, suggesting IGF-IR-specific effects (44). The mechanism whereby diaryl urea inhibits RTK Figure 5. PQ401 inhibits the IGF-I-mediated antiapoptotic pathway in activity has not yet been elucidated. Whereas most small- MCF-7 cells. MCF-7 breast cancer cells were incubated with varying molecule RTK inhibitors are competitive inhibitors of ATP concentrations of PQ401 for 1 h. Cells were then incubated F 3 nmol/L binding, diaryl urea does not share general structural IGF-I. A, serine phosphorylation of Akt/protein kinase B determined by Western blot that employed a phosphospecific (Ser473) antibody against homology with ATP analogues. Others have reported that the activated Akt/protein kinase B protein. B, total Akt/protein kinase B heterocyclic ureas inhibit the p38 mitogen-activated protein content determined as a control. The Western blot employed an antibody serine threonine kinase by interacting with a conserved against Akt/protein kinase B protein. Representative blots of three experiments. hydrophobic pocket that is spatially distinct from the ATP binding site (46, 47). In their model, binding of heterocyclic ureas within the pocket causes a side chain of Phe to block cells inhibits tumor growth in vivo (17). The effectiveness of access to the ATP binding site. Thus, the compounds act as these anti-IGF-I therapies has also been shown in a variety indirect inhibitors of ATP binding. of other tumor cell types including human prostate cancers Our data are consistent with an IGF-I-specific effect of (37), human lung cancers (38), human papillomavirus– diaryl urea molecules on tumor growth. We employed a positive and –negative endometrial cancers (39), ovarian negative control diaryl urea compound, PQ20, which has cancer (40), glioblastomas (41), melanomas (42), and an identical structure of both the aryl ring and the urea rhabdomyosarcomas (43). Nakamura et al. (39) showed linker but features an alternate substitution position on that initial overexpression of IGF-IR was not required for the quinoyl ring. This compound displayed no activity growth inhibition by antisense RNA-mediated down- against IGF-I-stimulated receptor autophosphorylation in regulation of the IGF-IR. In their studies, C33a cervical MCF-7cells and, correspondingly, had no negative effects cancer cells, which have a p53 mutation, very low IGF-IR on growth of MCF-7cells in culture. Thus, it is unlikely expression, and less IGF-I-stimulated growth than other that any reduction in cell proliferation by PQ401 was due cervical cancer cell lines, showed significant reductions in to general toxicity of the compound. Further, inhibition of monolayer growth and soft agar colony formation after IGF-IR activation by PQ401 was accompanied by a IGF-IR down-regulation (39). These results highlight the coordinate decrease in the phosphorylation state of Akt requirement of IGF-IR signaling for survival of cells of and an activation of the caspase apoptotic pathway. which the growth may be driven primarily by a variety of Together, these data show a specific blockade of the other kinases or oncogenes (3). In the present study, PQ401 was able to inhibit growth of both MCF-7and MCNeuA breast cancer cells. Whereas transformation and growth of MCNeuA cells is driven by overexpression of the HER2/Neu receptor, the effectiveness of IGF-IR inhibition is not surprising considering the generalized requirement of IGF-I signaling for cancer cell growth and survival. It is not clear, however, the extent to which inhibition of the IGF-IR by PQ401 bypasses the stimulatory effects of HER2 in these cells, whether cross talk between the RTKs might modulate the HER2 signal, or whether effects beyond those observed against the IGF-IR might block growth of the MCNeuA cells in vivo. Various other RTKs have been employed as targets in recent and ongoing attempts to create anticancer pharma- ceutical agents (reviewed in ref. 9). Many of these compounds have been generated against the platelet- Figure 6. PQ401 increases caspase-mediated apoptotic activity in MCF- derived growth factor receptor, vascular endothelial growth 7 cells. MCF-7 cells were incubated with either various concentrations of factor receptor, and epidermal growth factor receptor PQ401, 10 Amol/L doxorubicin, or DMSO vehicle alone for 24 h. Cells and family of kinases, either as antiproliferative or antiangio- medium were collected and cellular caspase activity determined by an genic agents (9). Recently, the anticancer effects of a small- ELISA that quantifies the apoptosis-associated M30 neo-epitope in both total cellular extracts plus medium. Columns, mean of three experiments; molecule inhibitor of the IGF-IR have been reported (44, 45). bars, SE. *, caspase activity significantly increased versus vehicle-treated These compounds are reported to show selectivity for the controls.

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The pharmacology of the diaryl urea compounds employed in these studies has not yet been studied. We cannot rule out the possibility that a metabolite or degradation product, rather than the PQ401 compound, is responsible for the effects on cells and in vivo. However, the ability of PQ401 to directly inhibit autophosphorylation of the isolated kinase domain suggests a direct effect of this compound on the IGF-IR. Overall, the metabolism of diaryl ureas has not been studied extensively. Pyridines (51) and quinolines (52) are known to be metabolically labile and tend to produce 1- and 2-oxides, as well as other metabolites. The possibility that the 4-aminoquinaldine heterocycle present in PQ401 is processed or activated in a similar fashion has not been shown but cannot be ruled out in the

Figure 7. Growth inhibition of MCNeuA cells in vivo by PQ401. current study. MCNeuA cells were injected into neuTg mice on day 0. Treatment with In summary, we have identified a novel class of IGF-IR- PQ401 began on day 3, with the compound administered thrice per week inhibiting compounds that are potential anticancer agents. in a polysorbate 80/ethanol vehicle injected i.p. Animals received 50 or The lead diaryl urea compound, PQ401, inhibits growth of 100 mg/kg PQ401 or vehicle alone. Columns, mean tumor volume for five animals per group; bars, SE. *, tumor volume significantly reduced for MCF-7breast cancer cells both in culture and in a syngeneic PQ401 treatment versus vehicle-treated controls. mouse model of breast cancer. PQ401 acts, at least in part, by inducing caspase-mediated apoptosis via inhibition of the Akt/protein kinase B pathway. 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