Impact of the Anticancer Drug NT157 on Tyrosine Kinase Signaling Networks Shih-Ping Su1,2, Efrat Flashner-Abramson3, Shoshana Klein3, Mor Gal3, Rachel S
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Published OnlineFirst February 12, 2018; DOI: 10.1158/1535-7163.MCT-17-0377 Small Molecule Therapeutics Molecular Cancer Therapeutics Impact of the Anticancer Drug NT157 on Tyrosine Kinase Signaling Networks Shih-Ping Su1,2, Efrat Flashner-Abramson3, Shoshana Klein3, Mor Gal3, Rachel S. Lee1,2, Jianmin Wu4, Alexander Levitzki3, and Roger J. Daly1,2 Abstract The small-molecule drug NT157 has demonstrated promising kinase AXL exhibited a rapid decrease in phosphorylation in efficacy in preclinical models of a number of different cancer response to drug treatment, followed by proteasome-dependent types, reflecting activity against both cancer cells and the tumor degradation, identifying an additional potential target for NT157 microenvironment. Two known mechanisms of action are deg- action. However, NT157 treatment also resulted in increased radation of insulin receptor substrates (IRS)-1/2 and reduced activation of p38 MAPK a and g, as well as the JNKs and specific Stat3 activation, although it is possible that others exist. To Src family kinases. Importantly, cotreatment with the p38 MAPK interrogate the effects of this drug on cell signaling pathways inhibitor SB203580 attenuated the antiproliferative effect of in an unbiased manner, we have undertaken mass spectrometry– NT157, while synergistic inhibition of cell proliferation was based global tyrosine phosphorylation profiling of NT157-trea- observed when NT157 was combined with a Src inhibitor. These ted A375 melanoma cells. Bioinformatic analysis of the resulting findings provide novel insights into NT157 action on cancer cells dataset resolved 5 different clusters of tyrosine-phosphorylated and highlight how globally profiling the impact of a specificdrug peptides that differed in the directionality and timing of on cellular signaling networks can identify effective combination response to drug treatment over time. The receptor tyrosine treatments. Mol Cancer Ther; 17(5); 931–42. Ó2018 AACR. Introduction The small-molecule drug NT157 represents a new class of anticancer agents that affect both the cancer cell and its supportive The past two decades have seen major advances in our under- microenvironment (4–8). NT157 inhibits the proliferation of a standing of the molecular mechanisms that underpin the different wide variety of cancer cell lines in vitro, including those derived hallmarks of cancer, and these have led to the development of new from colon, breast, and prostate cancer and melanoma, and classes of therapies that selectively target molecular mechanisms reduces growth of melanoma and prostate cancer cell line xeno- of specific importance to the survival and proliferation of cancer grafts as well as tumor burden in the CPC-APC mouse colon cancer cells. However, the inherent genomic instability of cancer cells model (6–8). It also inhibits metastasis of A375 melanoma xeno- and their propensity to acquire additional mutations, coupled grafts (7). One mechanism of NT157 function is via degradation of with intratumoral heterogeneity, means that the development of insulin receptor substrate (IRS)-1/2 and hence inhibition of pro- resistance to targeted therapies represents a major clinical prob- proliferative and survival signaling mediated by these docking lem (1). In light of this issue, the development of agents that can proteins. This mechanism involves direct allosteric modulation target several oncogenic signal transduction pathways, and/or of the IGF-1R, uncoupling of the receptor from IRS-1/2, recruit- factors in the tumor microenvironment that contribute to disease ment of Shc1, and activation of the Erk MAP kinases. The latter progression, has recently gained attention (2, 3). kinases then mediate serine phosphorylation of IRS1/2, leading to their degradation by the proteasome (7). However, NT157 also acts by reducing levels of activated, tyrosine-phosphorylated Stat3 via stimulation of an uncharacterized protein tyrosine phosphatase 1Cancer Program, Biomedicine Discovery Institute, Monash University, 2 (4, 8). Stat3 plays a major role in the cross-talk between tumor cells Melbourne, Australia. Department of Biochemistry and Molecular Biology, fl Monash University, Melbourne, Australia. 3Department of Biological Chemistry, and their microenvironment, and re ecting this, NT157 treatment Unit of Cellular Signaling, The Alexander Silberman Institute of Life Sciences, of mouse melanoma and colon cancer models leads to reduced The Hebrew University of Jerusalem, Jerusalem, Israel. 4Key Laboratory of tumor inflammation, reactive stroma, and macrophage infiltration Carcinogenesis and Translational Research (Ministry of Education/Beijing), (4, 8). The ability of NT157 to inhibit two independent signaling Centre for Cancer Bioinformatics, Peking University Cancer Hospital & Institute, pathways critical for cancer cell proliferation, survival, and metas- Beijing, China. tasis, as well as cancer cell interaction with the tumor microenvi- Note: Supplementary data for this article are available at Molecular Cancer ronment, makes it a promising anticancer agent that may prove Therapeutics Online (http://mct.aacrjournals.org/). refractory to development of acquired drug resistance. Corresponding Author: Roger J. Daly, Department of Biochemistry and Molec- To date, interrogation of the mechanism of action of NT157 has ular Biology, School of Biomedical Sciences, Level 1, Building 77, Monash been based on candidate-based approaches. However, applica- University, Melbourne, Victoria 3800, Australia. Phone: 613-9902-9301; Fax: tion of global, unbiased strategies may reveal novel effects of this 613-9902-9500; E-mail: [email protected] drug on cancer cell signaling pathways, thereby identifying addi- doi: 10.1158/1535-7163.MCT-17-0377 tional potential NT157 targets as well as potential ways to Ó2018 American Association for Cancer Research. improve NT157 efficacy. Because mass spectrometry (MS)–based www.aacrjournals.org 931 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 12, 2018; DOI: 10.1158/1535-7163.MCT-17-0377 Su et al. phosphoproteomic profiling represents a powerful strategy to overnight using Sequencing Grade Modified Trypsin (1:100, resolve the impact of particular drugs on oncogenic signaling w/w, Promega). Digested samples were desalted using Sep-Pak networks (9, 10), we have applied this approach to NT157. This columns (Waters) as per the manufacturer's instructions. Two reveals novel potential modes of action of NT157, including "spike-in" heavy peptides with alanine (al) C13N15 modifica- inhibition of AXL and activation of p38 MAPKs, as well as path- tions were added prior to Sep-Pak clean-up (Sep-Pak, Waters). ways that may limit drug efficacy and represent candidates for These peptides were EF1A1 (EHA(al)LLApYTLGVK) (500 fmol) combination treatment. and MAPK14 (HTDDEMTGpYVA(al) (10 pmol; Mimotopes). Phosphopeptides in the samples were enriched through sequen- Materials and Methods tial overnight immunoprecipitations using resin-bound P-Tyr- Cell culture 100 antibody (9411, Cell Signaling Technology), followed by Human A375 melanoma cells were obtained from the ATCC in P-Tyr-20 antibody (610000, BD Biosciences). p-Tyr antibodies 2008. The following human melanoma cell lines were also were prebound to Protein G Sepharose resin (GE Healthcare Life utilized: A375SM from the MDA Cancer Centre, Houston, TX in Sciences) by incubating antibody and resin at a 1:4 ratio (w/v) 2009; and M571 and M2068 provided by Dr. Michal Lotem at overnight at 4 C. Phosphopeptides were eluted from the anti- Hadassah Medical Center, Jerusalem, Israel, in 2012. A375 and body–resin conjugate by adding 0.1 % trifluoroacetic acid, 40% M2068 cells were subject to additional validation in our labora- acetonitrile, after 3 washes with IAP buffer [50 mmol/L Tris-HCl tory by short tandem repeat polymorphism in March 2017. All cell (pH 7.4), 150 mmol/L NaCl, 1% n-octyl-b-D-glucopyranoside, lines were routinely checked for mycoplasma contamination by cOmplete EDTA-free Protease Inhibitor Cocktail Tablet (Roche)], PCR and utilized for experiments within 4 passages. A375 cells and 4 washes with Milli-Q H2O. Phosphopeptide samples were were maintained in RPMI1640 medium supplemented with 10% lyophilized by centrifugal evaporation, and resolubilized in 0.1% FCS and 100 U/mL penicillin and 100 mg/mL streptomycin, and formic acid, 2% acetonitrile for LC/MS-MS analysis. maintained at 37C/5% CO . A375 SM cells were grown in MEM 2 LC/MS-MS identification and quantitation supplemented with 10% FBS, whereas M571 and M2068 cells Enriched peptides were analyzed using a Q Exactive Plus were maintained in RPMI/DMEM/F12 in the ratio 1:3:1, supple- Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher mented with 10% FBS. Scientific) coupled to a Dionex UHPLC. Peptides were separated fl Small-molecule drugs over a gradient of 65 minutes at a ow rate of 250 nL/minute. The The molecular structure of NT157 is reported in Reuveni and scan range for MS1 analysis was 375 to 1,600 m/z, and the top 12 colleagues (7). NT157 was synthesized in-house as described most intense ions were fragmented with higher energy collision- previously (7). The Src inhibitor PP1 and p38 MAPK inhibitor induced dissociation using a target value of 10,000 ions. The scan SB203580 were obtained from Synthos Ltd. and Calbiochem, range for MS/MS analysis was 200 to 2,000 m/z. respectively. Crizotinib and MG132 were purchased from Sell- MS data analysis eckchem