Published OnlineFirst June 29, 2018; DOI: 10.1158/1078-0432.CCR-17-3697 Cancer Therapy: Preclinical Clinical Cancer Research Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells Fiona Simpkins1,2,3, Kibeom Jang1,4, Hyunho Yoon1, Karina E. Hew1,2, Minsoon Kim1,4, Diana J. Azzam1,4, Jun Sun1, Dekuang Zhao1, Tan A. Ince1,5,6, Wenbin Liu7, Wei Guo7, Zhi Wei8, Gao Zhang9, Gordon B. Mills7, and Joyce M. Slingerland1,4,10 Abstract Purpose: Rational targeted therapies are needed for treat- selumetinib, respectively, showed target kinase inhibition and ment of ovarian cancers. Signaling kinases Src and MAPK are synergistic induction of apoptosis and cell-cycle arrest in vitro, activated in high-grade serous ovarian cancer (HGSOC). and tumor inhibition in xenografts. Gene expression and Here, we tested the frequency of activation of both kinases proteomic analysis confirmed cell-cycle inhibition and in HGSOC and the therapeutic potential of dual kinase autophagy. Dual therapy also potently inhibited tumor- inhibition. initiating cells. Src and MAPK were both activated in tumor- Experimental Design: MEK and Src activation was assayed initiating populations. Combination treatment followed by þ in primary HGSOC from The Cancer Genome Atlas (TGGA). drug washout decreased sphere formation and ALDH1 cells. Effects of dual kinase inhibition were assayed on cell-cycle, In vivo, tumors dissociated after dual therapy showed a marked apoptosis, gene, and proteomic analysis; cancer stem cells; and decrease in ALDH1 staining, sphere formation, and loss of xenografts. tumor-initiating cells upon serial xenografting. Results: Both Src and MAPK are coactivated in 31% of Conclusions: Selumetinib added to saracatinib over- HGSOC, and this associates with worse overall survival on comes EGFR/HER2/ERBB2–mediated bypass activation of multivariate analysis. Frequent dual kinase activation in MEK/MAPK observed with saracatinib alone and targets HGSOC led us to assay the efficacy of combined Src and MEK tumor-initiating ovarian cancer populations, supporting inhibition. Treatment of established lines and primary ovarian further evaluation of combined Src–MEK inhibition in cancer cultures with Src and MEK inhibitors saracatinib and clinical trials. Clin Cancer Res; 1–13. Ó2018 AACR. Introduction 1Braman Family Breast Cancer Institute at Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida. 2Department of Obstetrics & Ovarian cancer is the most lethal gynecologic cancer (1). Gynecology, University of Miami, Miami, Florida. 3Ovarian Cancer Research Despite introduction of targeted therapies, survival has not sig- Center, Division of Gynecology Oncology, Department of Obstetrics & nificantly improved in the last decade (2). Patients with most Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania. 4Depart- advanced ovarian cancers relapse within two years (1), and ment of Biochemistry and Molecular Biology, University of Miami, Miami, tumors become therapy resistant, underscoring the need for new 5 Florida. Department of Pathology and Laboratory Medicine, University of treatment options. High-grade serous ovarian cancer (HGSOC), Miami, Miami, Florida. 6Interdisciplinary Stem Cell Institute, University of Miami, Miami, Florida. 7Department of Systems Biology, The University of the most common and aggressive subtype, is characterized by Texas MD Anderson Cancer Center, Houston, Texas. 8Department of Com- genomic instability and few targetable genetic mutations (3). We puter Science, New Jersey Institute of Technology, Newark, New Jersey. previously showed that Src (4) and the MAPK (5) are frequently 9Wistar Institute, Philadelphia, Pennsylvania. 10Department of Medicine, activated in HGSOC from The Cancer Genome Atlas (TCGA) by University of Miami, Miami, Florida. reverse-phase proteomic analysis (RPPA), identifying these as Note: Supplementary data for this article are available at Clinical Cancer potential therapeutic targets. Research Online (http://clincancerres.aacrjournals.org/). Saracatinib (AZD0530), a potent Src family kinase inhibitor K. Jang, H. Yoon, and K.E. Hew contributed equally to this article. (6), has preclinical antitumor activity in HGSOC (4). Our prior Corresponding Authors: Joyce M. Slingerland, University of Miami Miller School work showed elevated expression of MAPKpT202pY204 is of Medicine, 1501 Northwest 10th Avenue, BRB 708 (C227), Miami, FL 33136. frequent and is an independent prognostic factor for decreased Phone: 305-243-7265; Fax: 305-243-6170; E-mail: [email protected] survival in HGSOC (5). Selumetinib (AZD6244), a noncom- ; and Fiona Simpkins, Division of Gynecology Oncology, Department of OB-GYN, petitive MEK1/2 inhibitor, has clinical activity in low-grade University of Pennsylvania, 3400 Civic Center Boulevard, South Tower, Suite 10- ovarian cancer (7), suppresses serous and clear cell ovarian 176, Philadelphia, PA 19104. Phone: 215-662-3318; Fax: 215-349-5849; E-mail: cancer xenografts (5, 8), and may prove to have clinical utility [email protected]. in HGSOC (5). doi: 10.1158/1078-0432.CCR-17-3697 Stem-like or tumor-initiating cancer cells are emerging as Ó2018 American Association for Cancer Research. critical mediators of drug resistance (9). The cancer stem cell www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst June 29, 2018; DOI: 10.1158/1078-0432.CCR-17-3697 Simpkins et al. Translational Relevance Materials and Methods Cell culture Ovarian cancers present late and rapidly develop chemo- PEO1R, an estrogen receptor (ER)-positive antiestrogen-resis- therapy resistance. Targeted therapies have yielded modest tant variant of PEO1 was cultured as described in ref. 4. Short gains, with short responses often due to bypass pathway tandem repeat (STR) profiling verified the unique identity of this activation. Thus, strategies to target multiple pivotal signaling line. PEO1R-SIR is a stable Src inhibitor resistant variant, derived nodes, such as Src and MEK, which are both activated in by 12 weeks of continuous exposure to 1 mmol/L saracatinib. OCI- HGSOC, are attractive. We found 31% of HGSOC have acti- E1P was cultured from an ER-positive, primary endometrioid vation of both of these kinases. To subvert therapy resistance, ovarian cancer, OCI-C5x from a primary clear cell, OCI-P5x from a we tested antitumor efficacy of combined Src and MEK inhi- primary serous, and OCI-U1a from a HGSOC in OCMI medium bition. Not only did dual therapy in vivo synergistically inhibit and have been extensively characterized as described previously tumor growth, but residual tumors were markedly depleted for þ (27). All were used below passage twelve. OCMI medium was ALDH1 , sphere-forming, and tumor-initiating stem cells in obtained from Interdisciplinary Stem Cell Institute Live Tissue vivo. Selumetinib added to saracatinib inhibits the EGFR-1 and Culture Service Center (LTCC). Cell lines were authenticated EGFR-2–mediated bypass MEK/MAPK activation observed using ATCC guidelines. Asynchronous cultures were treated with with saracatinib alone and appears to effectively target self- vehicle, 1 mmol/L saracatinib, 200 nmol/L selumetinib, or both renewing ovarian cancer subpopulations. These findings will for 48 hours or longer as indicated for drug assays. All in vitro cell stimulate further in vitro experimentation and support initia- assays described below included at least three biologic replicates tion of clinical trials testing dual Src and MEK inhibitor therapy and triplicate technical repeats. for patients with ovarian cancer. Drugs Saracatinib (AZD0530) is a potent inhibitor of Src family kinases and Abl (6). Saracatinib and selumetinib (AZD6244) from AstraZeneca were dissolved in DMSO. Saracatinib did not (CSC) hypothesis proposes tumors are heterogeneous and exceed 1 mmol/L in vitro to avoid off-target effects (4). Optimal contain a subpopulation of self-renewing cells that give rise selumetinib concentrations were titrated in vitro and in vivo in fl to progeny with reduced proliferation (9, 10). Ascites uid xenografts (5). Dual therapy concentrations were titrated in a from patients with ovarian cancer contains sphere-forming preliminary in vivo experiment (not shown). For xenografts, drugs cells (11) and tumor-initiating cells that are demonstrable in were suspended in sterile 0.5% hydroxypropyl methyl cellulose xenograft models (12). Various surface markers identify ovar- with 0.1% polysorbate (Tween 80). ian cancer stem cell–enriched populations (13–16). Aldehyde þ dehydrogenase activity (ALDH1 )identifies a population Cell-cycle analysis enriched for tumor-initiating cells in both ovarian cancer lines þ Cells were bromodeoxyuridine (BrdU)-labeled, stained with (16–18) and primary tumors (19). ALDH1 cells are increased anti-BrdU antibodies, and propidium iodide (PI) and cell cycle in populations surviving platinum chemotherapy, suggesting assayed as described previously (28). CSCs survive to repopulate after treatment (20). Although 50%–70% of patients with advanced HGSOC achieve a com- Effects of siRNA-mediated Src and MEK knockdown on cell plete clinical response to initial cytoreductive surgery and cycle chemotherapy, 70% will suffer recurrence and ultimately die Three different antisense oligos to either SRC (sc-29228 for of the disease (1). Thus, treatments that target resistant CSCs siSRC), or MEK1 (sc-29396 for