IGFBP2/FAK Pathway Is Causally Associated with Dasatinib Resistance in Non–Small Cell Lung Cancer Cells

Published OnlineFirst October 15, 2013; DOI: 10.1158/1535-7163.MCT-13-0233

Molecular Cancer Cancer Therapeutics Insights Therapeutics

Haibo Lu1,2, Li Wang1, Wen Gao1, Jieru Meng1, Bingbing Dai1, Shuhong Wu1, John Minna3, Jack A. Roth1, Wayne L. Hofstetter1, Stephen G. Swisher1, and Bingliang Fang1

Abstract Insulin-like growth factor (IGF)-binding protein-2 (IGFBP2) expression is increased in various types of cancers, including in a subset of patients with lung cancer. Because IGFBP2 is involved in signal transduction of some critical cancer-related pathways, we analyzed the association between IGFBP2 and response to pathway- targeted agents in seven human non–small cell lung cancer (NSCLC) cell lines. Western blot analysis and ELISA showed that four of the seven NSCLC cell lines analyzed expressed high levels of IGFBP2, whereas the remaining three had barely detectable IGFBP2. Susceptibilities of those seven cell lines to nine anticancer agents targeting to IGF1R, Src, FAK, MEK, and AKT were determined by a dose-dependent cell viability assay. The results showed that high IGFBP2 levels were associated with resistance to dasatinib and, to a lesser degree, to sacaratinib, but not to other agents. Ectopic IGFBP2 overexpression or knockdown revealed that changing IGFBP2 expression levels reversed dasatinib susceptibility phenotype, suggesting a causal relationship between IGFBP2 expression and dasatinib resistance. Molecular characterization revealed that focal adhesion kinase (FAK) activation was associated with increased IGFBP2 expression and partially contributed to IGFBP2- mediated dasatinib resistance. Treatment with a combination of dasatinib and FAK inhibitor led to enhanced antitumor activity in IGFBP2-overexpressing and dasatinib-resistant NSCLC cells in vitro and in vivo. Our results showed that the IGFBP2/FAK pathway is causally associated with dasatinib resistance and may be used as biomarkers for identiﬁcation of dasatinib responders among patients with lung cancer. Simultaneous targeting on Src and FAK will likely improve the therapeutic efﬁcacy of dasatinib for treatment of lung cancer. Mol Cancer Ther; 12(12); 2864–73. 2013 AACR.

Introduction in various cancers (5, 6), IGFBP2 has been shown to pro- Insulin-like growth factor (IGF)–binding protein-2 mote tumorigenesis (7), metastasis (4, 8), cancer stem cell (IGFBP2) is a member of the IGFBP family of proteins, expansion (9), and tumor angiogenesis (8, 10). Overexpres- which function as carriers of IGF-I and IGF-II in blood and sion of IGFBP2 has been reported in glioma (11), breast extracellular fluid, and is the second most abundant IGFBP cancer (12), ovarian cancer (13), prostate cancer (14), colo- in the circulation (after IGFBP3; ref. 1). In addition to IGF- rectal cancer (15), gastric cancer (16), lung cancer (17), binding domains that are common to all IGFBPs, IBFBP2 leukemia (18), and astrocytoma (19). Moreover, increased contains Gly-Arg-Asp (RGD; ref. 2) and heparin-binding expression of IGFBP2 is implicated in shorter overall sur- motifs (3, 4) that directly bind to integrins and extracellular vival time (20–22) and in resistance to chemotherapy matrix and trigger biologic actions independent of IGFs. (18, 23). Overexpression of IGFBP2 has been associated Unlike IGFBP3, which induces tumor-suppressive activity with resistance to docetaxel or paclitaxel (23, 24) and anti- hormone therapy (25), suggesting that IGFBP2-induced functional changes in cancer cells could play a critical role fi 1 Authors' Af liations: Department of Thoracic and Cardiovascular Sur- in the efficacy of anticancer therapy. gery, The University of Texas MD Anderson Cancer Center, Houston, Texas; 2The 8th Department of Internal Medicine, The Third Affiliated In our recent study, we found that IGFBP2 expression Hospital of Harbin Medical University, Harbin, China; and 3Hamon Center was drastically increased in a portion of primary lung for Therapeutic Oncology, The Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas cancer tumor tissues (17), suggesting that overexpression of IGFBP2 could be a marker for a subset of lung Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). cancers. As a secreted protein that can activate both IGF1R by increasing local IGF concentration through its Corresponding Author: Bingliang Fang, Department of Thoracic and Cardiovascular Surgery, Unit 445, The University of Texas MD Anderson IGF-binding domains and integrins by direct interaction Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: with them through its RGD and/or heparin-binding 713-563-9147; Fax 714-794-4901; E-mail: [email protected] motifs, IGFBP2 is likely to promote tumor progression doi: 10.1158/1535-7163.MCT-13-0233 through either the IGF1R or the integrin pathway. 2013 American Association for Cancer Research. Indeed, the IGFBP2/integrin/integrin-linked kinase

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IGFBP2/FAK Pathway Associated with Dasatinib Resistance

(ILK)/NF-kB network is reported to be a key player in Cell viability assays progression and poor outcome of glioma (26). Increased The inhibitory effects of pathway-targeted anticancer expression of IGFBP2 promotes breast cancer cell metas- agents on cell growth were determined by using the tasis by recruitment of endothelial cells to the cancer site sulforhodamine B assay, as described previously (29). through upregulation of the IGFI/IGF1R signaling Each experiment was carried out in quadruplicate and pathway (8). repeated at least 3 times. The relative cell viability (%) was Because both the IGF1R and integrin pathways fre- calculated using the equation ODT/ODC 100% (where quently cooperate with other growth factor pathways in ODT represents the absorbance of the treatment group and signal transduction of cancer cells (27, 28), we hypoth- ODC represents the absorbance of the control group). The esized that overexpression of IGFBP2 induces autocrine median inhibitory concentration (IC50) values were cal- and/or paracrine effects through IGF1R, integrins, or culated by using CurveExpert 1.3 software. both, thereby triggering alteration of cellular signal transduction and resistance to pathway-targeted thera- Western blot analysis py in lung cancer. To test whether overexpression of Whole-cell lysates were prepared by washing the cells IGFBP2 induces resistance to targeted therapy in lung with PBS and subjecting them to lysis with radioimmu- cancer cells, we evaluated responses to pathway-tar- noprecipitation assay (RIPA) buffer supplemented with geted anticancer agents in a panel of lung cancer cell the protease inhibitor cocktail. After the lysates were lines with high or low IGFBP2 expression. Our results sonicated for 15 seconds, the protein concentrations were show that a high level of IGFBP2 expression in lung quantified using the Bio-Rad Protein Assay Kit. Equiva- cancer cell lines is causally associated with increased lent amounts of each protein were loaded, separated by phosphorylation of focal adhesion kinase (FAK) and 10% or 12% SDS-PAGE and then transferred to polyvi- resistance to dasatinib. nylidene fluoride membranes at 80 V for 2 hours. The membranes were blocked for 1 hour with 5% nonfat dried milk in PBS buffer containing 0.1% Tween-20 (PBST) and Materials and Methods probed with diluted primary antibody at 4C overnight. Chemicals and antibodies The membranes were then washed 3 times in the PBST Small-molecule IGFR1 inhibitors picropodophyllin, buffer and probed with infrared dye–labeled secondary GSK 1904529A, BMS-754807, and OSI-906 (linsitinib) were antibodies. The immunoreactive bands were visualized obtained from Chemie Tek. Src inhibitors saracatinib and with the Odyssey Imager (Li-COR Biosciences). dasatinib were obtained from Selleck Chemical. PF- 562271, a FAK inhibitor, was obtained from MedKoo ELISA for IGFBP2 Biosciences Inc. AZD6244, a mitogen-activated protein IGFBP2 concentrations in media from cell cultures were kinase kinase (MEK) inhibitor, and MK2206, an Akt determined with the IGFBP2 DuoSet ELISA kit according inhibitor, were obtained from the Translational and Ana- to the manufacturer’s protocol. For this purpose, cells (5 lytical Chemistry Core facility of The University of Texas 104 per well) were cultured in 24-well plates with 0.5 mL of MD Anderson Cancer Center (Houston, TX). Antibodies medium for 48 hours. The ELISA capture antibody was m for total and phosphorylated FAK (p-FAK; pY397) were diluted to 6 g/mL in 0.1 mol/L NaHCO3, pH 9.5, and purchased from Epitomics. Antibodies for IGFBP2, total coated on 96-well ELISA plates. After incubation over- Src, phosphorylated Src (p-Src; Y527 and Y416), total Akt, night at room temperature, the plates were washed in TBS and phosphorylated Akt (p-Akt; S473) were obtained buffer (50 mmol/L Tris, pH7.5, 100 mmol/L NaCl, 0.05% from Cell Signaling Technology. ILK antibody and Tween-20) and blocked with TBS containing 1% bovine IGFBP2 DuoSet ELISA kit were obtained from R&D Sys- serum albumin. Culture medium supernatant (100 mL) or tems. Protease inhibitor cocktail, b-actin antibody, and IGFBP2 standard was added to each plate and the plates sulforhodamine were obtained from Sigma Chemical were incubated for 2 hours at room temperature, followed Corporation. Protein assay reagents were purchased from by immunoreaction with horseradish peroxidase–labeled Bio-Rad Laboratories. IGFBP2 detection antibody and colorimetric detection with the substrate 3,30,5,50-tetramethyl benzidine dihy- Cell lines and cell culture drochloride (Sigma). IGFBP2 concentration was calculat- Human non–small cell lung cancer (NSCLC) cell lines ed from the standard curve. All experiments were carried were maintained in our laboratories as previously out in duplicate and repeated twice. described (29). The authentication for each cell line was conducted by short tandem repeat–based DNA finger- Lentiviral vector–mediated gene knockdown or print analysis within 12 month. The cells were cultured in overexpression RPMI-1640 or high-glucose Dulbecco’s Modified Eagle’s Plasmids for lentiviral vectors expressing cDNA or Medium supplemented with 10% FBS, 100 mg/mL ampi- short hairpin RNA (shRNA) used in this study were cillin, and 0.1 mg/mL streptomycin; they were main- obtained from Open Biosystems through the shRNA and tained at 37 C in a humidified atmosphere containing 5% ORFeome Core facility in The University of Texas MD CO2 and 95% air. Anderson Cancer Center. Lentiviral vectors were

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packaged in 293 cells after cotransfection with lentiviral H1944 cells (Fig. 1B). Together, those results show that packaging plasmids by using Fugene 6 as instructed by IGFBP2 is differentially expressed in NSCLC cell lines. the manufacturer (Promega). The medium from trans- Because IGFBP2 can modulate functions of the IGF1R fected 293 cells was filtered through a sterile 0.22-mm filter and integrin pathways, both of which have been explored and used to infect target cells in the presence of 8 mg/mL as targets for anticancer therapy and/or implicated in the polybrene. After selection with puromycin (1–2 mg/mL) mechanisms of other pathway-targeted therapies (27, 30), or blasticidin (2–10 mg/mL), the cells were pooled togeth- we analyzed responses to various clinically relevant path- er for subsequent studies. The proportion of stably trans- way-targeted anticancer agents in the same seven NSCLC fected cells (expressing GFP or RFP in the lentiviral lung cancer cell lines, which express either high or low backbone) was usually more than 85% after selection. levels of IGFBP2. The anticancer agents used are listed in Materials and Methods. The antitumor activity of each Animal experiments agent at various doses ranging from 0.03 to 30 mmol/L was Animal experiments were carried out in accordance determined by a cell viability assay. The IC50 of each agent with Guidelines for the Care and Use of Laboratory Animals in each cell type was calculated from the dose–response (NIH publication number 85-23) and the institutional curve. The results show that the level of IGFBP2 expres- guidelines of MD Anderson Cancer Center. Subcutaneous sion in NSCLC cell lines was not associated with tumors were established in 6- to 8-week-old female nude responses to most of the agents tested; the exceptions mice (Charles River Laboratories Inc.) by inoculation of were dasatinib and saracatinib (Table 1). The NSCLC cell 2 106 H460 cells into the dorsal flank of each mouse. lines expressing high levels of IGFBP2 were highly resis- After the tumors grew to 3 to 5 mm in diameter, the mice tant to dasatinib, whereas the cell lines expressing low were grouped randomly into four groups and treated with levels of IGFBP2 were quite sensitive to dasatinib (Fig. oral administration of (i) dasatinib (25 mg/kg/d), (ii) PF- 1C). This relationship was also observed for saracatinib, 562271 (25 mg/kg/d), (iii) both dasatinib and PF-562271 although this agent was less active in the sensitive cells. In as in the groups 1 and 2, and (iv) solvent (10% dimethyl contrast, there was no noticeable correlation between sulfoxide and 10% polyethylene glycol 400). Tumor IGFBP2 expression level and responses to other anticancer volumes were calculated by using the formula a b2 agents (Table 1). The dose responses for BMS-754807 and 0.5, where a and b represented the larger and smaller PF562271 are shown in Fig. 1C and D, respectively, as diameters, respectively. Mice were killed when the examples. The inverse association of IGFBP2 expression tumors grew to 15 mm in diameter. Blood samples were with sensitivity to dasatinib observed in this study is collected from the tail vein one day after the last treatment, consistent with a published report showing that global and serum alanine transaminase, aspartate transaminase, gene expression profiling analysis identified IGFBP2 as and creatinine levels were determined at the Research one of six genes that can effectively predict in vitro dasa- Animal Support Facility of our institution. tinib response in breast cancer and lung cancer cell lines (31). Statistical analysis Each experiment or assay was conducted at least 2 IGFBP2 overexpression is causally related to times, and representative examples are shown. Data are resistance to dasatinib reported as mean SD or SE. Statistical significance of the A recent study showed that kinase-impaired BRaf differences between treated samples was determined by mutation is associated with dasatinib sensitivity in some using the two-tailed Student t test and one-way ANOVA NSCLC cells (32). Nevertheless, the gene mutation status analysis. Differences were considered statistically signif- of the cell lines used in the present study, obtained from icant at P < 0.05. the Sanger Institute Catalogue Of Somatic Mutations In Cancer web site, http://cancer.sanger.ac.uk/cancergen- ome/projects/cosmic (33), and our lung cancer database, Results showed that all cell lines tested except H3122, for which IGFBP2 expression in lung cancer cells is associated BRaf mutation status is not known, have the wild-type with sensitivity to dasatinib and saracatinib BRaf gene (Table 1). There is no obvious association Our recent study showed that expression of IGFBP2 is between dasatinib sensitivity and mutation in the KRas, dramatically increased in some primary lung cancer tis- NRas, EGFR,orp53 genes. sues (17). To test whether IGFBP2 is also increased in We then investigated whether overexpression of cultured lung cancer cell lines, we conducted Western blot IGFBP2 was the culprit in resistance to dasatinib. To this analysis on seven NSCLC cell lines. IGFBP2 was highly end, we stably transfected H1299 cells with a lentiviral expressed in Calu3, H460, H1437, and H3122 cells but was vector expressing IGFBP2 or a control lentiviral vector. barely detectable in H1299, H1792, and H1944 cells (Fig. The expression of IGFBP2 in H1299/IGFBP2 cells, but not 1A). Moreover, ELISA detected high levels of IGFBP2 in parental H1299 or H1299/vector cells, was confirmed (150 ng/mL) in the culture media collected from Calu3, by Western blot analysis of cell lysates and ELISA of the H460, H1437, and H3122 cells, whereas IGFBP2 was not culture media collected from those cells (Fig. 2A and B). detectable in the media collected from H1299, H1792, and Cell viability analysis showed that ectopic expression of

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A B

H1792 H1944 H1299 Calu3 H460 H1437 H3122 1.4 IGFBP2- 1.2 H1792 b-Actin- 1.0 H1944 300 0.8 H1299 250 Calu3 0.6 200 H460

150 Cell viability 0.4 H1437 H3122 100 0.2

IGFBP2 (ng/mL) 50 0 0 0 0.03 0.1 0.3 1 3 10 30 Dasatinib (μmol/L)

C D 1.4 1.4 H1792 1.2 H1792 1.2 H1944 H1944 H1299 1.0 H1299 1.0 Calu3 Calu3 H460 0.8 H460 0.8 H1437 H1437 0.6 H3122 H3122

0.6 Cell viability Cell viability 0.4 0.4

0.2 0.2

0 0 0 0.03 0.1 0.3 1 3 10 30 0 0.03 0.1 0.3 1 3 10 30 BMS754807 (μmol/L) PF567721 (μmol/L)

Figure 1. IGFBP2 expression and susceptibility to pathway-targeted agents in NSCLC. A, IGFBP2 expression in seven NSCLC cell lines. Top, intracellular IGFBP2 protein expression in seven lung cancer cell lines was detected by Western blot analysis. b-Actin was used as loading control. Bottom, IGFBP2 in cell culture media was detected by ELISA. Values represent mean SD. B to D, dose–response curves were plotted for seven NSCLC cell lines treated with dasatinib (B), BMS754807 (C), or PF562271 (D) for 72 hours. Cell viability was determined with the sulforhodamine B assay. The values in control cells were set as 1. Data presented are representative of two independent experiments.

IGFBP2 in H1299 resulted in resistance to dasatinib (Fig. knockdown cells. In contrast, lentiviral vector–mediated 2C). The IC50 was more than 100-fold greater in H1299/ knockdown of Src had no effect on dasatinib dose IGFBP2 cells than in parental or vector-transfected H1299 response in Calu3 cells (data not shown). Together, these cells, suggesting that IGFBP2 alone is sufficient to induce results show that cellular IGFBP2 levels were causally resistance to dasatinib. associated with dasatinib sensitivity in NSCLC cells. To further validate a role for IGFBP2 in the response to dasatinib, we knocked down IGFBP2 in H1437 and Calu3 IGFBP2 overexpression induces FAK cells through lentiviral vector–mediated expression of phosphorylation IGFBP2 shRNA. The knockdown of IGFBP2 was con- To identify pathways or downstream mediators that firmed by Western blot and ELISA analyses (Fig. 2D, E, might have been associated with IGFBP2 expression and G, and H). The cell viability assay showed that knock- dasatinib susceptibility in the NSCLC, we analyzed the down of IGFBP2 in H1437 and Calu3 cells was sufficient to expression and/or phosphorylation of several key sensitize these cells to dasatinib (Fig. 2F and I). Whereas IGFBP2 downstream molecules reported in literature. m the dasatinib IC50 was 4.5 mol/L for parental and Among them were IGF1R (8), Akt (34), ILK (26), FAK, control shRNA-transfected H1437 and Calu3 cells, it was and Src. Lysates from the seven NSCLC cell lines were about 0.07 and 0.006 mmol/L, respectively, in the IGFBP2- analyzed for expression and/or phosphorylation of these

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Table 1. IC50 of nine anticancer agents and gene mutation data in seven NSCLC cell lines

Compounds Targets H1792 H1944 H1299 Calu3 H460 H1437 H3122 Picropodophyllin IGF1R 0.85 0.69 0.87 0.26 0.3 0.22 0.16 GSK1904529A IGF1R >31 23.99 >31 >31 >31 >31 >31 BMS-754807 IGF1R >31 1.45 1.17 3.02 0.35 0.56 1.55 Linsitinib IGF1R 15.49 4.57 7.94 10.96 7.59 2.19 10.96 Dasatinib Src 0.35 0.001 0.015 5.49 9.12 10.23 15.85 Saracatinib Src 4.68 2.63 12.02 14.79 >31 >31 23.99 PF-562271 FAK 2.63 1.99 7.08 4.57 3.16 3.24 2.34 AZD6244 MEK 12.3 22.91 >31 >31 >31 2.09 6.17 MK2206 AKT 7.41 7.41 15.49 8.13 13.8 5.25 3.31 Mutation status KRas G12C G13D WT WT Q61H WT NRas WT WT Q61K WT WT WT BRaf WT WT WT WT WT WT EGFR WT WT WT WT WT TW WT p53 Intron M237I WT R267P

proteins. The results show that, of the molecules tested, concentration had only mild effects (14%–28% reduction) only the pattern of FAK phosphorylation (p-Y397) was on cell viability in those cells, its presence dramatically consistent with IGFBP2 expression in the seven NSCLC shifted the dose–response curves and enhanced the cell lines (Fig. 3A). All four cell lines expressing high levels response to dasatinib in all four cell lines. On the basis of IGFBP2 had high levels of p-FAK, whereas all 3 of the of the dose response to single-agent dasatinib or cell lines expressing low levels of IGFBP2 had low levels of PF562271, as shown in Fig. 1 and Table 1, we conducted p-FAK, strongly suggesting a functional association cell viability analysis for the combined effects of dasatinib between IGFBP2 expression and FAK phosphorylation. and PF562271 at various concentrations. The ratio of the 2 We then tested whether ectopic expression of IGFBP2 in compounds was based on the IC50 value of each. The cell H1299 cells and knockdown of IGFBP2 in Calu3 and viability assay was conducted in parallel in cells treated H1437 cells would elicit changes of FAK phosphorylation with each single agent. The combined effect of the 2 agents in those cells. Western blot analysis revealed that ectopic was determined by calculating the combination index expression of IGFBP2 in H1299 substantially increased the according to the Chou–Talalay method (36), using Calcu- levels of FAK phosphorylation, whereas knockdown of Syn software (Biosoft) as we reported previously (37). The IGFBP2 in Calu3 and H1437 cells reduced FAK phosphor- results show that the combination led to synergistic or ylation (Fig. 3B and C)and also suggest that FAK may be a additive effects at various dasatinib:PF562271 ratios rang- potential downstream mediator of IGFBP2. ing from 1:1 to 3.3:1 (Fig. 5A–C, Supplementary Table S1). This result suggests that for the cell lines having high FAK activity is associated with dasatinib resistance levels of IGFBP2 and FAK phosphorylation that were To characterize the possible roles of cellular FAK activ- resistant to dasatinib, treatment with a combination of ity in dasatinib resistance, we stimulated FAK activity by dasatinib and FAK inhibitor would improve the thera- using fibronectin as reported elsewhere (35). H1299 and peutic effects. H1972 cells were cultured in plates coated with fibronec- We also tested the combination effects of dasatinib and tin and then harvested for Western blot analysis. The PF562271 in vivo in a xenograft tumor model–derived results show that culturing those cells in the fibronec- H460 cells. Dasatinib (25 mg/kg) and PF562271 (25 tin-coated plates led to dramatic increases in phosphor- mg/kg) alone or in combination were administrated daily ylated FAK in those cells (Fig. 4A). The cell viability assay to mice bearing H460 tumors. Animals treated with sol- showed that cells cultured in the fibronectin-coated plates vent were used as controls. The tumor volumes were were less sensitive to dasatinib than the cells cultured in monitored during the treatment. The results showed that uncoated plates (Fig. 4B and C). This result suggests, at there was no obvious difference in tumor growth between least, that increased FAK activity partially contributes to control and PF562271-treated animals. Treatment with resistance to dasatinib in NSCLC cells. dasatinib resulted in a mild but insignificant suppression To further examine the role of FAK in dasatinib sus- of H460 tumors. Nevertheless, the combination led to ceptibility of NSCLC, we determined the combined effects significant growth suppression of H460 tumors in vivo of dasatinib and a FAK inhibitor in the dasatinib-resistant (Fig. 5D). No weight loss or abnormality in serum liver NSCLC. We first tested the dose response to dasatinib in enzyme and creatinine levels was detected in all treatment Calu3, H460, H1437, and H3122 cells in the presence or groups, suggesting that the combination therapy is effec- absence of PF562271 (1 mmol/L). While PF562271 at this tive and well tolerated.

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IGFBP2/FAK Pathway Associated with Dasatinib Resistance

A B 3,000 C 1.2 Parental 2,500 Vector control 1.0 IGFBP2 2,000 0.8

1,500 0.6 H1299

Parental control Vector IGFBP2 1,000 0.4 IGFBP2 (ng/mL) Cell viability -IGFBP2 500 0.2 -β-Actin 0 0 Parental Vector Ctr IGFBP2 0 0.03 0.1 0.3 1 3 10 30

D E 300 F 1.2

250 1.0

200 0.8

150 0.6 Calu3 100 0.4 IGFBP2 (ng/mL) Control shRNA Parental IGFBP2 shRNA

Cell viability Parental -IGFBP2 50 0.2 Control shRNA -β-Actin IGFBP2 shRNA 0 0 Parental Ctr shRNA IGFBP2 shRNA 0 0.03 0.1 0.3 1 3 10 30

G H I 300 1.2

250 1.0

200 0.8 shRNA shRNA 150 0.6 H1437 100 0.4 IGFBP2 Control Parental IGFBP2 (ng/mL) Cell viability Parental 0.2 -IGFBP2 50 Control shRNA -β-Actin IGFBP2 shRNA 0 0 0 0.03 0.1 0.3 1 3 10 30 Parental Ctr shRNA IGFBP2 shRNA Dasatinib (μmol/L)

Figure 2. Association between IGFBP2 expression and susceptibility to dasatinib in NSCLC. Top, IGFBP2 was transfected into H1299 cells by lentivector- mediated gene transfer. Middle, Calu3 cells; bottom, H1437 cells. IGFBP2 was knocked down by lentivector-mediated transfection of IGFBP2 shRNA in Calu3 and H1437 cells. The expression of IGFBP2 in parental, control vector, and IGFPB2-transfected (in H1299) or IGFBP2 shRNA-transfected (Calu3 and H1437) cells was determined by Western blot analysis (A, D, G) and ELISA (B, E, H), and their susceptibility to dasatinib by a cell viability assay (C, F, I). The expression of IGFBP in cells and media and the cell viabilities of parental, control vector, and IGFBP2 shRNA–transfected cells were determined as described for Fig. 1.

Discussion including Src family kinase members (LYN, SRC, FYN, The drastic increase of IGFBP2 expression in a subset of LCK, and YES), nonreceptor tyrosine kinases (FRK, BRK, lung cancer tissues and potential roles of IGFBP2 in signal and ACK), and receptor tyrosine kinases (ephrin recep- transduction in cancer cells led us to investigate whether tors, DDR1, EGFR, and PDGFR; ref. 39). Dasatinib is overexpression of IGFBP2 is associated with response to currently approved for treatment of chronic myeloid pathway-targeted therapy in lung cancer. Our results leukemia (40) and has been investigated in clinical trials show that IGFBP2 overexpression is causally associated for treatment of lung cancer (41–43). The results from with resistance to dasatinib in NSCLC and that FAK, a phase I and II trials suggest that a subpopulation of downstream signaling molecule of IGFBP2, may contrib- patients with lung cancer will benefit from treatment with ute to dasatinib resistance. dasatinib and that a biomarker-directed personalized Dasatinib was initially identified as a dual Src/Abl strategy will be required for the success of future studies kinase inhibitor with potent antitumor activity (38), but (41–43). Thus, identification of biomarkers that are asso- subsequently it was found to inhibit multiple kinases, ciated with dasatinib susceptibility in lung cancer will

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AB H1299 Figure 3. FAK as a downstream molecule in IGFBP2-mediated H460 H1792 H1299 H3122 H1944 Calu3 H1437 signaling. A, basal levels of Src, IGFBP2 p-Src, FAK, p-FAK, AKT, p-AKT, p-Src(527) and ILK were determined in the Parental Control Vector IGFBP2 p-FAK seven NSCLC cell lines shown p-Src(416) in Fig. 2. Level of p-FAK was high in Total FAK all four cell lines that expressed Total-Src β-Actin high levels of IGFBP2. B and C, p-FAK C changes in p-FAK levels were Calu3 H1437 measured in H1299, Calu3, Total-FAK and H1437 cells after ectopic expression or knockdown of p-AKT(473) IGFBP2 as described for Fig. 2. Notably, p-FAK increased Total-AKT dramatically in IGFBP2- Control shRNA Parental IGFBP2 shRNA Parental IGFBP2 shRNA β-Actin Control shRNA overexpressing H1299 cells and p-FAK decreased dramatically in IGFBP2- ILK Total FAK knockdown Calu3 and H1437 cells. β-Actin β-Actin

have an impact on the future success of treating patients is one of six genes whose levels in breast and lung cancer with lung cancer with this agent. cell lines could predict cancer cells’ response to dasatinib Effort has been made to identify molecular markers (31). Interestingly, IGFBP2 was also found to be associated associated with dasatinib susceptibility in lung cancer with susceptibility to saracatinib, another Src inhibitor, in cells. Recent studies showed that mutations in BRaf (32) pancreatic cancer (45). We observed a similar tendency for and DDR2 (44) may render lung cancer cells susceptible to an association between IGFBP2 level and saracatinib sus- dasatinib. As all cell lines used in this study are BRaf wild- ceptibility in our study, although saracatinib was less type, the susceptibility differences observed in those cell active than dasatinib in dasatinib-sensitive NSCLC. lines are unlikely to be associated with BRaf mutations. We also found that FAK is a candidate downstream Because we do not have data on DDR2 gene mutations in mediator in IGFBP2-induced dasatinib resistance. our lung cancer cell lines, it is unclear whether DDR2 Although little has been reported about FAK activation mutations contribute to the dasatinib susceptibility in by IGFBP2, activation of FAK by integrin (46) and activa- those cell lines. Nevertheless, the ectopic gene overexpres- tion of integrin by IGFBP2 (2, 47) are well documented in sion and gene knockdown experiments in the present the literature. The activation of FAK by the integrin study show that IGFBP2 level is causally associated with pathway led to autophosphorylation of Y397 and promot- resistance to dasatinib in NSCLC. This observation is ed interaction of FAK and Src (46, 48). Interestingly, consistent with a previous report by others that IGFBP2 dasatinib is reported to suppress FAK by acting on the

A B H1792 C H1299 1.2 With fibronectin 1.0 No fibronectin

0.8 H1792 H1299-FN H1299 H1792-FN 0.6 p-FAK

Cell viability 0.4 Total-FAK 0.2 β-Actin 0 0 0.03 0.1 0.3 1 3 10 30 0 0.03 0.1 0.3 1 3 10 30 Dasatinib (mmol/L) Dasatinib (mmol/L)

Figure 4. Effect of fibronectin on p-FAK level and susceptibility to dasatinib. H1792 and H1299 cells were cultured in plates with (FN) or without fibronectin coating. Expression of p-FAK (A) and susceptibility to dasatinib (B and C) were determined by the Western blot analysis and cell viability assay, respectively. Culturing cells in fibronectin-coated plates induced increases in p-FAK expression and partial resistance to dasatinib.

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A B 1.2 Calu3 Calu3+ 1.0 H460 H460+ H460 H1437 0.8 H1437 0 2 24 0 2 24 h H1437+ 0.6 p-FAK H3122 H3122+ 0.4 Total-FAK Cell viability

β-Actin 0.2

0 0 0.1 0.3 1 3 10 30 Dasatinib (μmol/L) 1.2 H1437 C D 1,800 H460 1.0 1,600 )

3 1,400 Combination 0.8 1,200 Dasatinib 0.6 1,000 PF562271 800

Cell viability 0.4 Combination 600 Dasatinib * 0.2 (mm Tumor volume 400 PF562271 * 200 * 0 0 PF562271 0 0.39 0.78 1.56 3.13 6.25 12.5 (μmol/L) 5 8 13 16 19 21 Dasatinib 0 0.78 1.56 3.13 6.25 12.5 25.0 (μmol/L) Days after tumor inoculation

Figure 5. Effect of FAK inhibitor on dasatinib-mediated antitumor activity. A, H460 and H1437 cells were treated with FAK inhibitor PF562271 (1 mmol/L) for the times indicated. p-FAK and FAK levels were determined by Western blot analysis. B, dose responses of Calu3, H460, H1437, and H3122 cells to dasatinib in the absence or presence (þ)of1mmol/L PF562271 were plotted. PF562271 alone had only mild effects on cell viability (cell viability about 72%–86% in those cells). C, dose responses of H1437 cells to dasatinib as a single agent and combined with PF562271. Synergistic effects were detected for the combination therapy. D, in vivo growth of H460 tumors. The mice were treated as indicated. The values are means SE of data from 5 mice per group. , P < 0.05 when compared with other groups. upstream kinases Src and Lyn (49, 50). Nevertheless, our Writing, review, and/or revision of the manuscript: H. Lu, J. Minna, J.A. Roth, W.L. Hofstetter, S.G. Swisher, B. Fang results indicate that elevated IGFBP2 expression and Administrative, technical, or material support (i.e., reporting or orga- activated FAK in cancer cells are causally associated with nizing data, constructing databases): H. Lu, L. Wang, J.A. Roth, W.L. Hofstetter dasatinib resistance and that combination of dasatinib Study supervision: H. Lu, W.L. Hofstetter, B. Fang with a FAK inhibitor may overcome this resistance. These Provided grant support for the project: J. Minna results may have an impact on design of future clinical trials to identify effective regimens combining these inno- Acknowledgments vative anticancer agents for the treatment of lung cancer. The authors thank Kathryn L. Hale for editorial review on the manuscript. Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed. Grant Support This work was supported in part by the NIH through grant and R01 Authors' Contributions CA124951 to B. Fang, the Specialized Program of Research Excellence Conception and design: H. Lu, W.L. Hofstetter, B. Fang (SPORE) in Lung Cancer grant CA070907 to J. Minna and J.A. Roth, and Development of methodology: H. Lu, W.L. Hofstetter The University of Texas MD Anderson Cancer Center Support Grant Acquisition of data (provided animals, acquired and managed patients, CA016672; the shRNA and ORFeome Core, the Translational and provided facilities, etc.): H. Lu, L. Wang, W. Gao, B. Dai, W.L. Hofstetter, Analytical Chemistry Core, the Research Animal Support Core, and S.G. Swisher the Flow Cytometry Core facilities of The University of Texas MD Analysis and interpretation of data (e.g., statistical analysis, biostatis- Anderson Cancer Center; and by endowed funds to The University of tics, computational analysis): H. Lu, W. Gao, J. Meng, S. Wu, J. Minna, Texas MD Anderson Cancer Center, including the Homer Flower Gene J.A. Roth, B. Fang Therapy Research Fund to S.G. Swisher, the Charles Rogers Gene

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Lu et al.

Therapy Fund to S.G. Swisher, the Flora & Stuart Mason Lung Cancer The costs of publication of this article were defrayed in part by the pay- Research Fund to W.L. Hofstetter, the Charles B. Swank Memorial Fund ment of page charges. This article must therefore be hereby marked adver- for Esophageal Cancer Research to S.G. Swisher, the George O. Sweeney tisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Fund for Esophageal Cancer Research to S.G. Swisher, the Phalan Thoracic Gene Therapy Fund to S.G. Swisher, the M.W. Elkins Endowed Fund for Thoracic Surgical Oncology to S.G. Swisher, and Received April 2, 2013; revised October 1, 2013; accepted October 2, 2013; the Chapman Foundation to W.L. Hofstetter. published OnlineFirst October 15, 2013.

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IGFBP2/FAK Pathway Is Causally Associated with Dasatinib Resistance in Non−Small Cell Lung Cancer Cells

Haibo Lu, Li Wang, Wen Gao, et al.

Mol Cancer Ther 2013;12:2864-2873. Published OnlineFirst October 15, 2013.

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