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The Tyrphostin NT157 Suppresses Insulin Receptor Substrates and Augments Therapeutic Response of Prostate Cancer

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The Tyrphostin NT157 Suppresses Insulin Receptor Substrates and Augments Therapeutic Response of Prostate Cancer Published OnlineFirst September 29, 2014; DOI: 10.1158/1535-7163.MCT-13-0842 Molecular Cancer Small Molecule Therapeutics Therapeutics The Tyrphostin NT157 Suppresses Insulin Receptor Substrates and Augments Therapeutic Response of Prostate Cancer Naokazu Ibuki1,2, Mazyar Ghaffari1,3, Hadas Reuveni4,5, Mitali Pandey1, Ladan Fazli1, Haruhito Azuma2, Martin E. Gleave1,6, Alexander Levitzki5, and Michael E. Cox1,6 Abstract Insulin-like growth factor (IGF) signaling is associated with castrate-resistant prostate cancer (CRPC) progression. Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from IGF1 receptor (IGF1R), insulin receptor, and other oncoproteins. This study demonstrates that IRS1/2 expression is increased in prostate cancer, and persists in CRPC. Furthermore, this study assesses the anticancer activity of NT157, a small molecule tyrphostin targeting IRS proteins, using androgen-responsive (LNCaP) and -independent (PC3) prostate cancer cells in vitro and in vivo. NT157 treatment resulted in dose- dependent inhibition of IGF1R activation, suppression of IRS protein expression, inhibition of IGF1-induced AKT activation, but increased ERK activation in NT157-treated cells in vitro. These effects were correlated with decreased proliferation and increasing apoptosis of LNCaP cells and increasing G2–M arrest in PC3 cells. NT157 also suppressed androgen-responsive growth, delayed CRPC progression of LNCaP xenografts, and suppressed PC3 tumor growth alone and in combination with docetaxel. This study reports the first preclinical proof-of-principle data that this novel small molecule tyrosine kinase inhibitor suppresses IRS1/2 expression, delays CRPC progression, and suppresses growth of CRPC tumors in vitro and in vivo. Demonstration that IRS expression can be increased in response to a variety of stressors that may lead to resistance or reduced effect of the therapies indicate that NT157-mediated IRS1/2 downregulation is a novel therapeutic approach for management of advanced prostate cancer. Mol Cancer Ther; 13(12); 2827–39. Ó2014 AACR. Introduction androgen synthesis (2). Even though current second- Prostate cancer is the most frequently diagnosed line ADTs, such as abiratarone and enzalutamide, pro- cancer in men and second leading cause of cancer- vide palliative responses in men who have failed stan- related death in Western countries (1). For patients with dard ADT and chemotherapies, lethal disease progres- advanced, extracapsular disease, androgen-deprivation sion still occurs. Although androgen ablation prolongs therapy (ADT) is the primary disease management life in men with advanced prostate cancer, remissions option. Although 80% of the patients initially respond are temporary because surviving tumor cells progress to to ADT, remission generally lasts only 18 to 36 months establish castration-resistant prostate cancer (CRPC; and disease progresses despite inhibition of gonadal refs. 2, 3). The mechanisms underlying chemotherapeu- tic and CRPC progression remain to be resolved; how- ever, aberrant phosphatidylinositol-3-kinase (PI3K)/ 1The Vancouver Prostate Centre, Vancouver General Hospital, Vancouver, Akt and ERK/mitogen-activated protein kinase signal- British Columbia, Canada. 2Department of Urology, Osaka Medical Col- 3 ing mediated by insulin-like growth factor (IGF), epi- lege, Osaka, Japan. Department of Medicine, University of British Colum- bia, Vancouver, British Columbia, Canada. 4TyrNovo Ltd., Herzliya, Israel. dermal growth factor, vascular endothelial growth fac- 5Unit of Cellular Signaling, Department of Biological Chemistry, The tor, and MET/hepatocyte growth factor tyrosine kinase Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel. 6Depart- ment of Urologic Sciences, University of British Columbia, Vancouver, receptors are strongly implicated candidate pathways British Columbia, Canada. that augment or bypass androgen receptor (AR) signal- Note: Supplementary data for this article are available at Molecular Cancer ing (4–6). Therapeutics Online (http://mct.aacrjournals.org/). Of these, particular attention has been focused on per- N. Ibuki and M. Ghaffari contributed equally to this article. turbations in the IGF axis. This axis is a key regulator of cell growth, survival, and metastatic potential in a variety Corresponding Author: Michael E. Cox, The Vancouver Prostate Centre, Vancouver General Hospital, 2660 Oak Street, Vancouver, BC, Canada of malignancies that have been strongly implicated in V6H3Z6. Phone: 604-875-4818; Fax: 604-875-5654; E-mail: prostate cancer etiology and progression (7–10). The insu- [email protected] lin receptor substrate (IRS) proteins are a family of cyto- doi: 10.1158/1535-7163.MCT-13-0842 plasmic adaptor proteins that transmit signals from the Ó2014 American Association for Cancer Research. insulin and IGF1 receptors (IGF1R) to elicit a cellular www.aacrjournals.org 2827 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst September 29, 2014; DOI: 10.1158/1535-7163.MCT-13-0842 Ibuki et al. response (11, 12). Once tyrosine is phosphorylated, the IRS dextrin (2-HP-b-CD; Sigma) at 5 mg/mL and stored at proteins function as adaptors to organize signaling À80C. complexes predominantly through activation of PI3K (13). IRS protein expression can be regulated by steroi- Cell growth assays dal, cytokine, and integrin signaling (14–16). There are Cell growth was assessed using crystal violet assay, as five human IRS genes (reviewed in ref. 11). IRS1 and described previously (21). Cells were plated in 24-well IRS2 are ubiquitously expressed and are the primary plates and treated with NT157 as described. Crystal mediators of IGF-dependent mitogenesis and insulin- violet staining was carried out for time course, and mediated regulation of glucose metabolism in most cell 72 hours after treatment. The absorbance was deter- types (17). IRS1 plays a central role in cancer cell mined with a microtiter plate reader (Becton Dickinson proliferation, its expression is increased in many human Labware) at 562 nm. Cell survival after NT157 treatment malignancies and its upregulation in human hepatocel- was calculated as the percentage of the absorbance in lular carcinoma mediates resistance to anticancer drugs vehicle-treated cells. (18). IRS2 is associated with cancer cell motility and metastasis (19). These findings make the IRS proteins Immunoblot analysis potential targets of anticancer therapies. Indeed, we After the indicated treatments, detached cells were have recently reported that tyrphostins targeting IRS pelleted from media and pooled with attached cells and proteins possess antitumor effects (20). lysed in RIPA buffer (50 mmol/L Tris, pH 7.2, 1% NP-40, Here, we report elevated IRS1/2 expression in prostate 0.1% deoxycholate, 0.1% SDS, 100 mmol/L NaCl, 1Â cancer and CRPC and characterize the preclinical anti- Roche complete protease inhibitor cocktail). Samples con- cancer activity of NT157, a small molecule tyrosine kinase taining equal amounts of whole-cell lysate protein (30 mg) inhibitor that downregulates IRS expression. We assess were subjected to SDS–PAGE, transferred to nitrocellu- the potency of this tyrphostin drug candidate by charac- lose filters, blocked in PBS containing 5% nonfat milk terizing its effects on IRS knockdown, suppressed prolif- powder for 1 hour, and incubated at 4C overnight with erative signaling, induction of apoptosis, and cell-cycle primary antibody: anti-phosho-IGF1Rb subunit (Y980) arrest using LNCaP and PC3 prostate cancer models. We rabbit monoclonal antibody (mAb), anti-IGF1Rb subunit demonstrate the ability of NT157 to delay CRPC progres- rabbit polyclonal antibody (PAb), anti-phosho-AKT sion of LNCaP and to augment taxane sensitivity of PC3 (S473) rabbit PAb, anti-AKT rabbit PAb, anti-phospho- cells in vitro and in vivo. ERK1/2 (T202/Y204) rabbit PAb, anti-ERK1/2 rabbit PAb, anti-phosho-IRS1 (S636/639) rabbit PAb, anti- phosho-IRS1 (Thr1222) rabbit PAb, anti-IRS1 rabbit PAb, Materials and Methods anti-phosho-IRS2 (Ser731) rabbit PAb (Abcam), anti-IRS2 Prostate cancer cell lines rabbit PAb (Abcam), anti-cleaved PARP (N214) rabbit LNCaP cells were maintained in RPMI-1640 (Invitro- PAb, anti-cyclin D1 (M-20) rabbit PAb (Santa Cruz Bio- gen) supplemented with 5% fetal bovine serum (FBS) and technology), anti-phosho-RB (Ser807/811) rabbit PAb, 2 mmol/L L-glutamine. PC3 cells were purchased from anti-RB mouse mAb, anti-p21 mouse mAb (Santa Cruz the American Type Culture Collection and maintained in Biotechnology), anti-p27 mouse mAb (Santa Cruz Bio- Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen technology), anti-p53 rabbit PAb, anti-cdc2 rabbit PAb, Life Technologies, Inc.) containing 5% FBS. Cell lines were anti-cdc25C rabbit mAb, and anti-wee1 rabbit. All anti- cultured in a humidified 5% CO2 atmosphere at 37 C. Cell bodies were from Cell Signaling Technology unless oth- line identities were verified by short tandem repeat (STR) erwise indicated. Immunoblots were incubated horserad- fingerprinting by grcf DNA Services (Johns Hopkins, ish peroxidase–conjugated anti-mouse or rabbit IgG anti- Baltimore, MD) in January 2013, and passaged for less body (Santa Cruz Biotechnology) and visualized with than 3 months after resurrection. Immortalized normal enhanced chemiluminescence immunoblotting analysis prostatic fibroblasts (courtesy of Dr. S. Hayward, Vander- system (Amersham Life Science) using a DYVERSITY bilt
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