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

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. 2014 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 chemotherapeutic and CRPC progression remain to be resolved; however, 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 2014 American Association for Cancer Research. insulin and IGF1 receptors (IGF1R) to elicit a cellular

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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 80C. 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, 1 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 University, Nashville, TN) were maintained in DMEM imaging system with GeneSnap Ver. 7.04 software (Syn- plus 10% FBS as above. Gene). Immunoblot images are representative of at least three independent experiments. Small molecule tyrosine kinase inhibitor The small molecule tyrosine kinase inhibitor targeting Flow cytometric analysis the IGF1R–IRS signaling pathway, NT157, was kindly Flow cytometric analysis of propidium iodide (PI)– provided by TyrNovo, Ltd. and used for in vitro and in stained nuclei was done, as described previously (22). vivo studies as previously described (20), wherein the In brief, cells were plated in 10-cm2 dishes and, on the chemical characterization, including molecular structure, day after, were treated as described above. The cells is reported. Briefly, for in vitro studies, NT157 was dis- were trypsinized, washed twice, and incubated in PBS solved in dimethyl sulfoxide (DMSO) as a 10 mmol/L containing 0.12% Triton X-100, 0.12 mmol/L EDTA, stock solution and stored at 4C. For the in vivo studies, and 100 mg/mL ribonuclease. PI (50 mg/mL) was then NT157 was dissolved in 20% 2-hydroxypropyl-b-cyclo- added to each sample for 20 minutes at 4C. Cell-cycle

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distribution was analyzed by ﬂow cytometry (Beckman test as indicated in respective legends. Tumor growth Coulter Epics Elite; Beckman, Inc.) based on G1–G0 and rates were compared using the Kruskal–Wallis test. P < G2–M DNA content. Each assay was performed in Levels of statistical signiﬁcance were set at 0.05. triplicate.

Assessment of in vivo tumor growth Results For xenograft studies, 2 106 LNCaP (suspended in IRS1 and IRS2 expression are increased in prostate 0.1 mL Matrigel; BD Biosciences) or PC3 cells (sus- cancer, downregulated after hormone therapy, and pended in 0.1 mL serum-free DMEM) were inoculated increased with disease progression s.c. in the flank of 6- to 8-week-old male athymic nude Previous reports indicated a slight, but nonsignificant, mice (Harlan Sprague–Dawley, Inc.) via a 27-gauge increase in percentage of IRS1-positive cells (23), and needle under isoflurane anesthesia. For LNCaP xeno- significantly increased IRS2 expression (24) in prostate grafts, when tumor volume exceeded 200 mm3,mice cancer tumors compared with benign epithelium; how- were castrated and randomly selected for treatment ever, what influence ADT might have on IRS expression in with 50 mg/kg NT157 or vehicle (10% 2-HP-beta-CD prostate cancer has not been reported. We therefore, þ 0.67% NaCl/H2O) injected i.p. three times per week. examined expression of IRS1 and IRS2 in the prostate Each experimental group consisted of 12 mice. Tumor cancer microenvironment of untreated, short-term (<6 volume was measured twice weekly (length width months) neoadjuvant hormone therapy (NHT), long-term depth 0.5432). Data points were expressed as average (6–12 months) NHT, and CRPC disease by IHC (Fig. 1). In tumor volume SEM. When PC3 tumors reached 100 this prostate cancer specimen series, IRS1 expression was mm3, mice were randomly selected for treatment with elevated approximately 20% in therapy-na€ve prostate 50 mg/kg NT157 or vehicle (10% 2-HP-beta-CD cancer and CRPC when compared with benign epitheli- þ 0.67% NaCl/H2O) injected i.p. three times per week um, but was indistinguishable from benign epithelium in or treated with 10 mg/kg docetaxel injected i.p. three NHT-treated specimens. IRS2 expression was upregu- times a week for 1 week or combination of 50 mg/kg lated >2-fold in therapy-na€ve prostate cancer and CRPC NT157 and 10 mg/kg docetaxel as indicated. Experi- specimens, and while indistinguishable from na€ve speci- mental groups consisted of 7 mice (vehicle), 9 mice mens in short-term NHT specimens, was increased 2- (NT157), 10 mice (docetaxel), and 16 mice (combina- fold compared with benign epithelium in the long-term tion). Tumor volume and body weight (BW) were NHT cohort. These results indicate that IRS1 and IRS2 measured once weekly. Data points were expressed as expression may be linked to increased AR activity in average tumor volume SEM.Allanimalprocedures prostate cancer, downregulated as a consequence of were performed according to the guidelines of the androgen ablation, and reemerge during CRPC progres- Canadian Council on Animal Care and with appro- sion and suggest that IRS1 and IRS2 expression may be priate institutional certification. important for maintenance of malignant cell viability in androgen-responsive and CRPC states. Immunohistochemistry Immunohistochemistry (IHC) analysis of IRS1 and Dose-dependent and time-dependent suppression of IRS2 in prostate cancer was performed on formalin- IGF1R/PI3K signaling correlates with enhanced fixed, paraffin-embedded 4-mm sections of tissue ERK activation, and IRS1/2 serine phosphorylation microarrays composed of duplicate 5-mm cores from and degradation in NT157-treated LNCaP and PC3 tumor samples from 92 patients distributed, as cells described in Supplementary Table S1. Immunohisto- IGF signaling has previously been implicated in growth chemical staining was conducted using IRS1- and 2- and survival signaling in prostate cancer models, includ- specific primary antibodies in the Ventana autostainer ing LNCaP and PC3 cells (7–10). The small molecule Discover XT (Ventana Medical System) with enzyme- tyrphostin, NT157, is a novel potential therapeutic agent labeled biotin streptavidin system and solvent resistant designed to disrupt IGF signaling that promotes degra- 3,30-diaminobenyidine map kit. Antibody validation dation of IRS1 and IRS2 (20). To evaluate whether NT157 was performed by immunogenic peptide competition might have utility as an anticancer agent for prostate (Supplementary Fig. S1). All comparisons of staining cancer, we first tested whether NT157 was capable of intensities were made at 200 magnification. Staining antagonizing IGF1-mediated signaling in vitro as a pre- intensity was scored by a blinded pathologist (L. Fazli) cursor to assessing the physiologic impact of IGF1R using 0–3 scale. expression on these prostate cancer models (Fig. 2). We observed that 24 hours of pretreatment with NT157 dose- Statistical analysis dependently suppressed IGF1-stimulated activation Immunohistochemical staining comparisons were site tyrosine phosphorylation of the IGF1R without sig- made by the ANOVA and Dunn multiple comparisons. nificantly affecting IGF1Rb subunit expression in In vitro data comparisons were assessed using the both LNCaP and PC3 cells (Fig. 2A). In LNCaP cells, Student t test or ANOVA and Mann–Whitney posthoc IGF1R Y980 phosphorylation was suppressed >60%

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Figure 1. Expression of IRS1 and IRS2 is elevated in prostate cancer specimens. A, representative IHC photomicrographs are presented showing the extent and intensity IRS1 and IRS2 immunoreactivity in benign, therapy-naïve, post short-term ( 6 months) and long-term (> 6 months) NHT, and CRPC tissues. Sections are sequential. B, the mean intensity score for IRS1- and IRS2-positive cells is expressed as the average SEM for each tissue cohort as detailed in the Materials and Methods. Expression of both proteins is not normally distributed in any cohort and the Dunn multiple comparisons test indicates that signiﬁcant median variation for IRS1 expression is between (i) benign versus therapy-naïve (, P < 0.05) and (ii) benign versus CRPC (, P < 0.01) and IRS2 expression is (i) benign versus therapy-naïve and (ii) benign versus CRPC (, P < 0.001), (iii) benign versus NHT > 6 months (, P < 0.05), and (iv) NHT 6 months versus CRPC (, P < 0.05).

at 2.5 mmol/L NT157 and >80% at 5 mmol/L NT157. In PC3 NT157 exposure and maximal decreased IRS2 levels by 8 cells, IGF1R Y980 phosphorylation was suppressed hours. Similar kinetics was observed in PC3 cells but with approximately 40% at 2.5 mmol/L NT157, and >90% at maximal decreased IRS1 and IRS2 expression by 2 hours. 5 mmol/L. This effect generally correlated with decreased These results indicate that NT157 can mediate suppres- IGF1-stimulated Akt and GSK3b phosphorylation and, as sion of IGF1R-mediated survival signaling through the previously noted (20), increased ERK phosphorylation established mechanism for negative feedback of IGF1R and suppressed IRS2 expression in LNCaP cells and IRS1 signaling: targeting IRS1/2 for serine phosphorylation and -2 expression in PC3 cells. and subsequent degradation (25, 26). We next evaluated how NT157 pretreatment might affect IRS1/2 expression at 5 mmol/L from 0.5 to 8 NT157 treatment induces cell-cycle arrest and hour-pretreatment (Fig. 2B and C). In both LNCaP and apoptosis in LNCaP and PC3 Cells PC3 cells, prolonged NT157 treatment resulted in To assess the effect of NT157 on cell viability, LNCaP increased IRS serine phosphorylation and decreased cells cultured in 5% of FBS media or 5% of charcoal- steady-state IRS protein levels. In LNCaP, these effects stripped serum (CSS) media were treated with varying were observed by 30 minutes, with increased IRS2 serine doses of NT157 for 3 days and population density was phosphorylation dramatically increasing by 2 hours of compared with that of DMSO-treated cells using crystal

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Figure 2. Dose- and time- dependent suppression of IRS and intracellular signaling in NT157- treated LNCaP and PC3 cell lines in vitro. A, LNCaP and PC3 cells were cultured in serum-free media for 24 hours with or without indicated NT157, stimulated for 5 months with 50 ng/mL IGF1, and lysed in RIPA. One day after the treatment, whole-cell detergent lysates were prepared and 30 mg of total protein was subjected to SDS-PAGE and immunoblotted for p-IGF1Rb,total IGF1Rb, p-Akt, total-Akt, p-Gskb, total-Gskb, p-Erk, total- Erk, IRS1, IRS2, and vinculin expression. B, LNCaP cells were cultured in serum-free media for 24 hours, then cultured 5 mmol/L NT157 for indicated times followed by stimulation with 50 ng/mL IGF1 for 20 minutes. Whole-cell detergent lysates were prepared and 30 mgof total protein was subjected to SDS- PAGE and immunoblotted for pS- IRS2, total IRS2, and vinculin expression. C, PC3 cells were cultured in serum-free media and treated as in B and lysates immunoblotted for pS-IRS1, total IRS1, pS-IRS2, total IRS2, and vinculin expression.

> m m violet assay (Fig. 3A). The density of LNCaP cells grown in and 70% at 5 mol/L; IC50, 2.5 mol/L). These observa- FBS was decreased approximately 20% at 1 mmol/L, tions are in contrast with the dose-dependent effect approximately 70% at 2 mmol/L, and >90% at 5 mmol/L of NT157 on viability of an immortalized, but benign, m (IC50, 1.4 mol/L). Growth of LNCaP cells is suppressed prostate epithelial cell line (RWPE1) and immortalized >60% when cultured in CSS but still exhibited significant prostate fibroblasts (Supplementary Fig. S2). Although density at 2 mmol/L and maximal decreased density at 5 RWPE1 cells exhibit some cytotoxicity above 5 mmol/L, mmol/L. The density of FBS-cultured PC3 cells was sim- viability was not inhibited by >50% at doses up to ilarly decreased by NT157 treatment (40% at 2 mmol/L 10 mmol/L, while the prostatic fibroblasts exhibit no more

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than a 20% decrease in viability at 10 mmol/L. These cultured LNCaP cells. Cyclin B1 expression was not results indicate that the tumor models exhibit an increased directly correlated to cdc2/CDK1 expression in NT157- sensitivity to NT157, consistent with previous reports treated LNCaP cells (Fig. 3 and Supplementary Fig. S3). indicating a key role for IGF1R signaling for their viability NT157 treatment suppressed both cyclin B1 and cdc2/ (4–6). CDK1 levels in PC3 cells while cdc2/CDK1 expression Decreased density of FBS-cultured LNCaP and PC3 persisted in CSS-cultured LNCaP cells even though cyclin cells treated with NT157 was observed to be proportional B1 levels were suppressed by NT157 treatment. We con- to apoptotic induction as assessed by flow cytometry of sistently observed decreased cyclin B1 expression in FBS- fixed, PI–stained cells (Fig. 3B) and by increased levels of cultured LNCaP cells treated with 2.5 mmol/L NT157, but cleaved PARP (Fig. 3D). The fraction of cells undergoing not at higher doses. We speculate that this may be due to apoptosis (sub G0–G1 fraction or cleaved PARP) was the rapid activation/stabilization of p53 and subsequent significantly increased after treatment of LNCaP and PC3 apoptotic induction indicated by PARP cleavage (Fig. 3D) m cells with 2.5 mol/L NT157 for 2 days, except that and decreased G2–M population (Fig. 3B). The accumu- no increase in cleaved PARP was detected in NT157- lation of a G2–M population, and proportionally lower treated, dextran-coated, activated charcoal-stripped FBS apoptotic induction in PC3 cells would therefore be con- (CSS)-cultured LNCaP cells below 10 mmol/L. Although sistent with their p53-null status. this correlated with decreased numbers of LNCaP cells in G0–G1 and G2–M fractions under both FBS and CSS con- NT157 enhances docetaxel-induced apoptosis in PC3 ditions, in PC3 cells there was a dose-dependent increase cells þ in the G2–M fraction. When analyzing the G0–G1 to S G2 A recent report indicates that IRS1 enhances sensitivity –M fractions of nonapoptotic cells, no obvious differences to the chemotherapy (27). Given the above data indicating were observed in the NT157-treated, FBS or CSS LNCaP that NT157 treatment results in suppressed IRS expres- cultures; however, PC3 cells treated with 2.5 mmol/L sion, we evaluated the efficacy of NT157 in combination in vitro NT157 exhibited a significantly increased G2–M fraction therapy with docetaxel on PC3 cells and determine indicative of G2–M arrest (Fig. 3C). whether this effect was additive or synergistic using the The above results suggest that suppression of IGF1R- dose-dependent effects with constant ratio design accord- mediated activation of IRS proteins promotes cell-cycle ing to the Chou and Talalay median effect principal (28). arrest and induction of apoptosis in these prostate cancer Docetaxel decreased PC3 viability at 1 nmol/L, whereas models. The impact of NT157 treatment on cell-cycle NT157 decreased PC3 viability at 1 mmol/L (Fig. 4A). At progression was analyzed by immunoblotting against a all concentrations tested, when combined, NT157 treat- panel of mitotic and apoptotic regulatory factors (Fig. 3D). ment significantly enhanced docetaxel cytotoxicity such Induction of G1–S arrest in NT157-treated, FBS-cultured that cell density was decreased 60% for cells treated with 1 LNCaP and PC3 cells is indicated by the dose-dependent nmol/L docetaxel and 1 mmol/L NT157; more than 2-fold increased p21 and p27 (LNCaP only) expression, greater than either agent at these doses alone (30% and decreased cyclin D1 and E2 expression, and decreased 20%, respectively). Comparing the combination index (CI) RB phosphorylation (P-RB). Decreased cyclin D3 and value for each constant ratio combination and assessing CDK4 expression was also observed (Supplementary Fig. the ED50 and ED75 CIs revealed that NT157 synergistically S3); however, expression of the E cyclin–linked cyclin- enhances the cytotoxic effect of docetaxel on PC3 cell dependent kinase, CDK2 was low and no apparent growth (Fig. 4B). Moreover, this effect was found to be changes in response to NT157 treatment were noted. In due to enhanced apoptotic induction as measured by both cell types, the dose-dependent decrease in G0–G1 PARP cleavage (Fig. 4C) and accumulation in the sub populations (Fig. 3B) indicated that cells were undergoing G0–G1 fraction (Fig. 4 D). This enhanced apoptotic induc- apoptosis from the G1–S checkpoint. tion be a consequence of apoptotic induction from cells Induction of G2–M arrest was indicated by elevated p53 arresting at both the G1–S and G2–M. expression in LNCaP cells and decreased cdc2/CDK1, cdc25c, and Wee1 expression in both cell lines when NT157 suppresses growth of LNCaP xenografts cultured in FBS. The relatively low expression of cyclin following castration D1, cdc25c, Wee1, and lack of detection of cyclin E2 and P- We next evaluated the efficacy of NT157 as a combina- RB as well as increased p27 and cleaved PARP only at the tion therapy with castration on growth of LNCaP xeno- 10 mmol/L NT157 dose is indicative of senescence of CSS- grafts (Fig. 5). When tumor volume exceeded 200 mm3,

Figure 3. Effect of NT157 on LNCaP and PC3 cell apoptosis and cell cycle. A, LNCaP cells were treated with the indicated concentrations of NT157 or DMSO cultured in 10% of FBS media or 10% CSS media. PC3 cells were treated with indicated NT157 or DMSO cultured in 10% FBS media. Two days after treatment, cells were ﬁxed and permeabilized and then stained with PI and cell-cycle population was analyzed by ﬂow cytometry. B, the ratio of G0–G1 to G2–M was analyzed. Histograms represent the means of three independent triplicate analysis SE. , P < 0.05; , P < 0.01 difference from DMSO by the Student t test. C, LNCaP and PC3 cells were treated for 2 days with indicated NT157 and DMSO. LNCaP cells were cultured in 10% FBS media or 10% CSS media and PC3 cells were cultured in 10% of FBS media. D, whole-cell detergent lysates (30 mg) were subjected to SDS-PAGE and immunoblotted to assess cyclin D1, cyclin E2, p-RB, total-RB, p21, p27, p53, cdc2, cdc25, wee1, and vinculin levels.

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Figure 4. IRS knockdown enhances effects of docetaxel treatment in PC3 cells. A, PC3 cells were treated for 72 hours with indicated docetaxel and NT157. Cell viability was determined by crystal violet assay. Viable cell density was normalized to that of cells treated with DMSO. All experiments were repeated at least three times. B, dose-dependent effects and CI values calculated by CalcuSyn software were assessed in PC3 cells treated for 72 hours with NT157 alone, docetaxel alone, or combined treatment at indicated concentration with constant ratio design between both drugs. The CI for ED50 and ED75 was 0.57 and 0.77, respectively, indicative of a synergistic effect of this combined treatment. C, PC3 cells were treated with NT157 alone, docetaxel alone, or combined treatment at indicated concentration for 48 hours. Whole-cell detergent lysates (30 mg) were subjected to SDS-PAGE and immunoblotted to assess IRS1, IRS2, cleaved-PARP, PARP, and vinculin expression. D, PC3 cells were treated with NT157 alone, docetaxel alone, or combined treatment at indicated concentration for 48 hours. The proportion of cells in sub-G1,G0–G1, S, and G2–M was determined by PI staining. All experiments were repeated at least three times.

animals were castrated and randomly assigned to groups 129.5 mm3/day versus NT157-treated growth rate ¼ treated with either NT157 or vehicle. The mean tumor 3.5 mm3/day). The tumor volume change waterfall plot volume of the two groups was indistinguishable. Animals analysis demonstrated that only one vehicle-treated ani- were treated for 6 weeks as described in the Materials and mal exhibited a tumor growth change that was less than Methods. In castrated mice bearing LNCaP xenografts, the greatest increase in tumor volume of the NT157- NT157 signiﬁcantly delayed tumor growth (Fig. 5A). treated cohort and that half of the NT157-treated cohort Although tumors in vehicle-treated mice continued to tumor volumes decreased over the course of the study grow following castration, average tumor volume (Fig. 5B). No signiﬁcant effect on animal body weight was decreased at 1 week following castration and NT157 observed in the vehicle- or NT157-treated cohorts during treatment, remained static through 4 weeks of treatment, this treatment period (Supplementary Fig. S5). Immuno- and did not exceed precastrate tumor volume at end histochemical analysis reveals decreased P-IGF1R, IRS2, of study. Average tumor growth rate in the NT157 and Ki67 detection and higher apoptotic rates, as shown treated cohort was decreased >35-fold (Supplementary by increased terminal deoxynucleotidyl transferase– Table S2 and Supplementary Fig. S4; vehicle growth rate ¼ mediated dUTP nick end labeling (TUNEL) staining, in

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AB 4

1,200 %) Vehicle 2 )

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Figure 5. NT157 treatment inhibits LNCaP xenograft growth and delays castration-resistant progression. A, LNCaP cells were inoculated s.c. and, when tumor volume exceeded 200 mm3, mice were castrated and randomly selected for treatment with 50 mg/kg of NT157 or vehicle (10% 2-HP-beta-CD þ 0.67% NaCl in DDW) injected i.p. three times per week. Each data point represents the mean tumor volume in each group containing 12 mice SEM. B, the percentage change in volume for each tumor (12 tumors per treatment group) after 6 weeks is shown as a waterfall plot. C, tumors were collected 42 days following treatment initiation and P-IGF1R, IRS2, Ki67, and TUNEL were evaluated by immunohistochemical analysis (original magnification, 200). D, total proteins were extracted from tumors and IRS2 was analyzed by immunoblotting. Total RNAs were extracted from the xenografts treated with castration, NT157 as indicated. IRS2 mRNA expression levels were determined by quantitative RT-PCR. Experimental groups are as indicated under each data column, representing relative levels of expression normalized to the mRNA level of GAPDH. , P < 0.05, significant difference from vehicle-treated group (Student t test). the NT157-treated group as compared with the vehicle- NT157 potentiates docetaxel activity in PC3 treated group (Fig. 5C). Decreased IRS2 expression in the xenografts NT157-treated group was corroborated by immunoblot The cooperative activity of NT157 and docetaxel on PC3 and qPCR of selected tumors (Fig. 5D). These studies growth in vitro prompted an evaluation of the combined indicate that NT157 treatment affects IGF1R and IRS treatment on PC3 xenografts (Fig. 6). Once PC3 tumor targets in xenografts and significantly delays castration- volume exceeded 100 mm3, animals were randomly resistant progression of LNCaP androgen-responsive assigned for treatment with vehicle, NT157, docetaxel, or xenografts when combined with castration. NT157, and docetaxel in combination as described in the

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A B 20 %) 2 3,000 Vehicle 15 2,500 NT157 ) Docetaxel 3 Combination 2,000 10

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Relative IRS1 mRNA expression 0 Vehicle NT157 Docetaxel Combination

180 160 Ki67 140 120 100 80 60 40 TUNEL 20 Relative IRS2 mRNA expression 0 Vehicle NT157 Docetaxel Combination

Figure 6. NT157 potentiates docetaxel activity in PC3 xenograft model. A, PC3 cells were inoculated s.c. and when tumors reached 100 mm3, mice were randomly selected for treatment with the same protocol as LNCaP. Each point represents the mean tumor volume in each group containing 7 mice in vehicle,9 mice in NT157, 10 mice in docetaxel, and 16 mice in combination-treated as described in Materials and Methods (SEM). B, the percentage change in volume for each tumor after 6 weeks is shown as a waterfall plot. C, tumors were collected after 42 days and P-IGF1R, IRS2, Ki67, and TUNEL were evaluated by immunohistochemical analysis (original magnification, 200). D, total proteins were extracted from tumors, and IRS1 and IRS2 were analyzed by immunoblotting. Total RNAs were extracted from the xenografts treated with vehicle, NT157, docetaxel, and the combination of NT157 and docetaxel as indicated. IRS1 and IRS2 mRNA expression levels were determined by quantitative RT-PCR. Experimental groups are as indicated under each data column, representing relative levels of expression normalized to the mRNA level of GAPDH. , P < 0.05, significant difference from vehicle-treated group; , P < 0.05, significant difference from Docetaxel group (Dunn multiple comparisons test).

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NT157 Suppresses IRS1/2 and Augments Prostate Cancer Therapeutic Response

Materials and Methods. Both NT157 and docetaxel sig- progression. Although we observed that IRS1 and -2 nificantly suppressed PC3 xenograft growth rate. Growth expressions are elevated in cancer versus benign prostatic rate of tumors in the NT157 (106.4 mm3/day) and doc- epithelium and remain expressed at elevated levels in etaxel (87.7 mm3/day) cohorts were suppressed approx- CRPC, there is no further elevation in expression in imately 4-fold relative to that of the vehicle cohort (387.2 advanced disease. Intriguingly, consistent with a previ- mm3/day) while the growth rate of the tumors in the ous report indicating that the IRS2/IRS1 transcript ratio is combination cohort (12.5 mm3/day) were suppressed increased in benign versus prostate cancer (32), we >30-fold relative to the vehicle cohort and approximate- observed that increased IRS2 expression is greater than ly 7-fold relative to the NT157 and docetaxel alone that of IRS1 due primarily to the relatively low basal level cohorts (Fig. 6A; Supplementary Table S3; Supplemen- of IRS2 in benign prostatic epithelium. tary Fig. S6). The tumor volume change waterfall plot Although these findings are suggestive of a role for IRS1 analysis demonstrated that while all NT157 and doc- and IRS2 in CRPC progression, mechanisms for how IRS etaxel tumors grew less than half as large as the average proteins impact prostate cancer remains to be resolved. vehicle tumors, all increased in volume over the course Although steroid pathways have been implicated in their of the study; however, in the combination cohort, half expression, IRSes are ubiquitously expressed. Of the IRS the tumors exhibited no growth or decreased in size family, only IRS4 was identified as an AR target gene by over the course of the study (Fig. 6B). No significant ChIP analysis (33); however, in the Androgen Responsive effect on animal body weight was observed between Gene Database (http://argdb.fudan.edu.cn/), IRS1, but NT157, docetaxel or combination, and vehicle-treated not IRS2, is identified as possessing a nominal androgen- mice bearing PC3 xenografts during this treatment response element consensus in its upstream noncoding period (Supplementary Fig. S7). By immunohistochem- region. Furthermore, the AR-null PC3 cell line robustly ical analysis, detection of P-IGF1R, IRS1, IRS2, and Ki67 expresses IRS1 and -2 as well, so their expression in were lower in the NT157 treatment cohort. In the doc- prostate cancer would not appear to be dependent on AR etaxel-treated group, while Ki67 index was decreased, activity. Although there is a trend in IRS2 (and indeed in P-IGF1R, IRS1, and IRS2, staining intensities were indis- IRS1) expression with regard to putative AR activity tinguishable from that of the vehicle cohort. The com- during progression to CRPC, we consider the most prob- bination therapy cohort staining for P-IGF1R, IRS1, and able reason being attributable to the overall effect of IRS2 paralleled that of the NT157 treatment cohort, but androgens on prostatic epithelia and na€ve prostate cancer Ki67 index was lower than in the NT157 and docetaxel quiescence response to hormone withdrawal (34, 35). alone cohorts and only in this cohort was there a Several small molecule tyrosine kinase inhibitors and substantial increase in TUNEL-positive cells in the antibodies targeting IGF1R are in clinical development in xenograft samples (Fig. 5C). Decreased IRS1 and IRS2 prostate and many other cancers; however, until the expression in the NT157-treated groups was corrobo- recent identification of a novel investigational IRS1/2 rated by immunoblot and qPCR of selected tumors (Fig. small molecule inhibitor, NT157 (20), there have been 5D). Intriguingly, docetaxel alone appeared to increase no anticancer drugs targeting IRS proteins. Here, we expression of IRS2 that was reversed when combined demonstrate that NT157 decreases expression of IRS pro- with NT157. These studies again indicate that NT157 teins and downregulates IGF1R-mediated AKT activation treatment affects IGF1R and IRS targets of xenografts in the prostate cancer models, LNCaP and PC3 cell. and indicate that NT157 significantly delays growth of Although androgen-independent PC3 cells express both androgen-independent PC3 xenografts alone or in com- IRS1 and -2 (29) and androgen-dependent LNCaP cells bination with the standard of care chemotherapeutic for express IRS2, but not IRS1 (36), NT157 treatment similarly CRPC, docetaxel. disrupted IRS-mediated signaling, inducing cell-cycle arrest and apoptosis of both cell lines. Although expression of cell-cycle regulatory factors is confounded by the Discussion induction of apoptosis during the course of these studies, There have been few reports that attempt to link IRS1 we conclude that these results correlate NT157-mediated and IRS2 expression to prostate cancer etiology. Early suppressed IRS expression with decreased IGF1-mediat- studies indicated that there is a trend toward increased ed PI3K signaling, cell-cycle arrest, and apoptotic activa- IRS1 expression in malignant biopsies (29) and that IRS1 tion in androgen-responsive and -independent prostate expression is slightly but nonsignificantly increased in cancer cells indicative of apoptosis due to suppressed prostate cancer when compared with benign prostate prosurvival signaling and induction of cell-cycle arrest. tissue (23). More recently, strong IRS2 expression has Furthermore, NT157 treatment delayed growth of LNCaP been reported in prostate cancer (24), and increased xenografts following castration and suppressed growth of expression of IRS2 and insulin receptor is correlated with PC3 xenografts cotreated with docetaxel. As we have CRPC progression (30, 31). In this work, we confirm that previously described (37), p53 wild-type LNCaP cells IRS1 and IRS2 are elevated in prostate cancer, then go on to readily respond to cell-cycle stress by inducing apoptosis demonstrate that expression tends to be decreased in from both G1 and G2–M stages, while p53-mutant PC3 response to hormone therapy, and increased with disease cells accumulate in G2–M before apoptotic induction.

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 2837

Ibuki et al.

Consistent with the model proposed by Reuveni and observations indicate that expression of IRS proteins is colleagues (20), NT157 treatment also results in elevated tightly regulated and can be increased in response to a ERK1/2 activation that may mediate phosphorylation variety of stressors that may lead to resistance or reduced and degradation of IRS proteins. Although activation of effect of the therapies. both the PI3K–AKT and Ras–ERK pathways have been There is precedence for targeted agents to restore ther- implicated in controlling IGF1R-mediated growth and apeutic sensitivity. Suppressing clusterin either through survival signaling (38), these results are consistent with direct antisense targeting (44) or intriguingly through our previous reports indicating that activation of PI3K/ STAT1 interfering RNA treatment (45), has been demon- AKT, not ERK1/2, is primarily responsible for growth and strated to restore taxane sensitivity in prostate cancer survival responses to IGF1 (39–41). Therefore, the ability models. These responses appear to be due to the ability of NT157 to disrupt IGF1R-mediated signaling via IRS of clusterin to protect against cellular stress responses. IGF adaptor proteins, delay CRPC progression, and suppress signaling, particularly through the PI3K axis, can also growth of CRPC tumors is at least, in part, due to inhi- provide cytotoxic protection from a variety of stressors. bition of prosurvival signaling of the PI3K–AKT pathway. The ability of NT157 to downregulate IRS1/2 expression Furthermore, treatment of LNCaP cells with NT157 does in malignancies provides a novel therapeutic approach to not impact the ability of epidermal growth factor to enhance the sensitivity of cancers to other cytotoxic agents activate PI3K-mediated signaling to drive pS473 on AKT as established. or to drive pTEpY of ERK1 and -2 (Supplementary Fig. S8). We conclude that this supports our contention that NT- Disclosure of Potential Conﬂicts of Interest 157 selectivity targets IGF axis signaling regarding the No potential conﬂicts of interest were disclosed. selectivity of NT157 for the IGFIR versus other tyrosine kinases (20). Authors' Contributions This ability of NT157 to promote apoptotic induction Conception and design: N. Ibuki, M. Ghaffari, H. Reuveni, H. Azuma, A. Levitzki, M.E. Cox from G1–G0 arrest compliments the apoptotic induction of Development of methodology: N. Ibuki, M. Ghaffari, H. Reuveni, taxane-treated cells from G2–M arrest and provides a A. Levitzki, M.E. Cox rationale for combining the two therapeutic modalities Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Ibuki, M. Ghaffari, H. Azuma, M.E. Gleave to improve response in patients with CRPC. A range of Analysis and interpretation of data (e.g., statistical analysis, biostatis- therapeutic approaches including reducing ligand avail- tics, computational analysis): N. Ibuki, M. Ghaffari, M. Pandey, M.E. Gleave, A. Levitzki, M.E. Cox ability by IGF1 antibodies and recombinant IGFBPs, Writing, review, and/or revision of the manuscript: N. Ibuki, M. Ghaffari, reducing IGF1R expression by antisense and RNA inter- H. Reuveni, M. Pandey, M.E. Gleave, M.E. Cox ference, or inhibiting of IGF1R signaling by IGF1R anti- Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): N. Ibuki, M. Ghaffari bodies and small molecule tyrosine kinase inhibitors are Study supervision: M. Ghaffari, M.E. Cox under investigation in CRPC (42). Although several of Other (pathology and IHC): L. Fazli these, particularly the humanized IGF1R antibodies, are showing great promise, continued research to understand Acknowledgments how the IGF axis functions to promote prostate cancer The authors thank Virginia Yago, Darrell Trendall, Estelle Li, Mary Bowden, Alice Tai, and Yubin Guo for their excellent technical assistance, growth and progression and to identify new ways to Manuel Altamirano-Dimas for editorial assistance. target the IGF axis in prostate cancer should be pursued. Targeting IRS expression is one such opportunity. Reu- Grant Support veni and colleagues (20) reported that increased IRS This study was supported by operating grant funding from the Cana- expression promotes vemurafenib resistance in several dian Cancer Society to M.E. Cox and a Canadian Institute for Health Research Doctoral Fellowship to M. Ghaffari. cases, while Buck and colleagues (43) reported a require- The costs of publication of this article were defrayed in part by the ment for IRS proteins in resistance to tyrosine kinase payment of page charges. This article must therefore be hereby marked receptor, and mTOR inhibitors. Similarly, we observe advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that IRS2 expression is elevated in prostate cancer, rebounds during CRPC progression, and in PC3 xeno- Received October 4, 2013; revised August 11, 2014; accepted August 27, grafts, increases in response to docetaxel treatment. These 2014; published OnlineFirst September 29, 2014.

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www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 2839

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

Naokazu Ibuki, Mazyar Ghaffari, Hadas Reuveni, et al.

Mol Cancer Ther 2014;13:2827-2839. Published OnlineFirst September 29, 2014.

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