Tumor and Stem Cell Biology shRNA Kinome Screen Identifies TBK1 as a Therapeutic Target for HER2þ Breast Cancer

HER2þ breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapsewith aggressivemetastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2þ breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that "immortalize" the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neuþ tumorsphere versusmonolayer cells with a lentivirus short hairpin RNA kinome library.We identified kinases such as the mitogen-activated protein kinase and the TGFbR protein family, previously implicated in HER2þ breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IkB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2þ breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKe, suppressed growth of both mouse and human HER2þ breast cancer cells. TBK1/IKKe inhibition promoted cellular senescence by suppressing p65–NF-kB and inducing p16. In addition, TBK1/IKKe inhibition cooperated with lapatinib, a HER2/ EGFR1–targeted drug, to accelerate apoptosis and kill HER2þ breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2þ breast cancer may benefit from anti-TBK1/IKKe plus antiHER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2þ breast cancer. Cancer Res; 74(7); 2119–30. 2014 AACR. Introduction HER2þ beast cancer is caused by overexpression/amplification of the HER2/ERBB2/NEU receptor tyrosine kinase and represent approximately 20% of breast tumors (1). About 72% of HER2þ breast cancers contain mutations or deletions in the tumor suppressor p53 (2). Patients with HER2þ breast cancer are treated with chemotherapy plus anti-HER2 inhibitors such as trastuzumab, a monoclonal antibody (mAb) directed against HER2 (3–6). Despite improvement in diseasefree survival, adverse effects and emergence of drug-resistant metastases represent serious limitations. There is, therefore, an urgent need to identify novel drugs that can cooperate with anti-HER therapy to effectively kill HER2þ breast cancer. Many cancer types exhibit hierarchical organization, whereby only a subset of tumor cells, termed tumor-initiating cells (TIC), sustains cancer growth (7, 8). These cells are functionally defined by their ability to induce secondary tumors following transplantation into recipient mice, and in certain cases by their ability to grow as spheres under nonadherent conditions (9, 10). In contrast, the tumor bulk comprises non-TICs, which descended from TICs but show reduced tumorigenic potential. TICs exhibit unique sensitivity to radiation and therapeutic drugs relative to non-TICs (11–13). Indeed, following conventional therapy, residual breast cancers are enriched for TICs (14). It was, therefore, suggested that therapeutic drugs should target TICs. However, there is also evidence that non-TICs can revert, albeit at low frequency, back to TICs (8) and, therefore, curative therapeutic regimens should target both compartments. The identification of effective therapeutics is hindered by difficulty in obtaining primary tumor samples. One approach to circumvent this problem involves mousemodels for specific Authors' Affiliations: Division of AdvancedDiagnostics, Toronto General Research Institute–University Health Network; Medicinal Chemistry Platform, Ontario Institute for Cancer Research; The Donnelly Centre, University of Toronto; Program in Developmental and Stem Cell Biology, Department ofMolecular Genetics, TheHospital for Sick Children; Ontario Cancer Institute, University of Toronto, and Drug Discovery Program, Department of Pharmacology and Toxicology; Toronto, Ontario, Canada Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J.C. Liu, P.E.D. Chung, and D. Uehling contributed equally to this work. Corresponding Author: Eldad Zacksenhaus, Division of Cell and Molecular Biology, Toronto General Research Institute—University Health Network, 67 College Street, Room 407, Toronto, Ontario, Canada M5G 2M1. Phone: 416-340-4800, ext. 5106; Fax: 416-340-3453; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-2138 2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 2119 on September 16, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 31, 2014; DOI: 10.1158/0008-5472.CAN-13-2138