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

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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 IGFBP2/FAK Pathway Is Causally Associated with Dasatinib Resistance in Non–Small Cell Lung Cancer Cells 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. Ó2013 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 overexpres- sion 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. Ó2013 American Association for Cancer Research. Indeed, the IGFBP2/integrin/integrin-linked kinase 2864 Mol Cancer Ther; 12(12) December 2013 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst October 15, 2013; DOI: 10.1158/1535-7163.MCT-13-0233 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
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