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DUSP1 Expression Induced by HDAC1 Inhibition Mediates Gefitinib Sensitivity in Non–Small Cell Lung Cancers

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DUSP1 Expression Induced by HDAC1 Inhibition Mediates Gefitinib Sensitivity in Non–Small Cell Lung Cancers Cancer Therapy: Preclinical Clinical Cancer Research DUSP1 Expression Induced by HDAC1 Inhibition Mediates Gefitinib Sensitivity in Non–Small Cell Lung Cancers Yun-Chieh Lin1, Yu-Chin Lin1,2,3, Jin-Yuan Shih4, Wei-Jan Huang5, Shi-Wei Chao5, Yih-Leong Chang6, and Ching-Chow Chen1 Abstract Purpose: Non–small cell lung cancer (NSCLC) is a leading Results: Gefitinib-resistant NSCLC cells showed HDAC1 over- cause of cancer-related death worldwide. Patients with NSCLC expression, and its knockdown sensitized resistant cells to gefiti- with EGFR-activating mutation benefit greatly by gefitinib, an nib in vitro and in preclinical models through DUSP1 expression. EGFR tyrosine kinase inhibitor. However, acquired resistance Overexpression of DUSP1 in resistant cells restored gefitinib limits its clinical use. Histone deacetylases (HDAC) are oncopro- sensitivity by inhibiting EGFR signaling and inducing apoptosis, teins associated with cancer progression and drug resistance. Here, whereas its knockdown in sensitive cells conferred gefitinib resis- we disclosed that inhibition of HDAC1 induced protein phos- tance. A novel HDAC inhibitor, WJ-26210-2, in combination with phatase DUSP1 upregulation to overcome gefitinib-acquired gefitinib upregulated DUSP1 expression to exert in vitro and in vivo resistance. synergistic effect on inactivation of EGFR signaling, growth inhi- Experimental Design: The effect of HDAC1 inhibition restored bition, and apoptosis. Clinically, high DUSP1 level was correlated gefitinib sensitivity was assessed by in vitro MTT and apoptotic with delayed emergence of gefitinib-acquired resistance. assays, and in vivo xenograft and orthotopic lung cancer mouse Conclusions: Decreased DUSP1 might be a mechanism models. Protein phosphatase array was used to detect DUSP1 responsible for gefitinib resistance, and DUSP1 might be a bio- expression. Immunohistochemical staining and quantitative PCR marker for gefitinib efficacy. HDAC1 inhibition–induced DUSP1 were used to analyze DUSP1 expression in clinical NSCLC upregulation could be a promising strategy to overcome gefitinib- specimens. acquired resistance. Clin Cancer Res; 21(2); 428–38. Ó2015 AACR. Introduction harboring EGFR-activating mutations (5, 6). However, acquired resistance develops after a period of treatment, which is largely Non–small cell lung cancer (NSCLC) is a main cause of cancer- caused by secondary EGFR T790M point mutation or by activa- related death worldwide (1). It is usually diagnosed at advanced tion of other signaling pathway (7, 8). Although second-gener- stage and has poor prognosis (2). Recently, the discovery of EGFR- ation EGFR TKI, afatinib, could overcome resistance caused by activating mutation not only brings a new insight into molecular T790M mutation to regain therapeutic effect, acquired resistance classification, but also leads to a great success in clinical treatment to afatinib emerges soon. Therefore, acquired resistance to EGFR (3, 4). EGFR tyrosine kinase inhibitor (TKI) gefitinib exerts TKI might involve signaling pathways beyond EGFR (9, 10). excellent efficacy to improve survival of patients with NSCLC Histone deacetylases (HDAC), which remove acetyl groups from histone allowing chromatin compaction and resulting in gene silence, are aberrantly overexpressed in various tumors 1Department of Pharmacology, College of Medicine, National Taiwan leading to carcinogenesis, cancer progression, and clinical poor 2 University, Taipei, Taiwan. Division of Oncology and Hematology, outcome (11). Thus, HDACs can be a therapeutic intervention for Department of Internal Medicine, Far-Eastern Memorial Hospital, New Taipei City, Taiwan. 3Department of Oncology, National Taiwan Uni- cancer treatment to reverse aberrant epigenetic states associated versity Hospital and National Taiwan University, College of Medicine, with cancer (11). HDAC inhibitor (HDACi) Vorinostat (SAHA) Taipei, Taiwan. 4Department of Internal Medicine, National Taiwan was approved by the FDA for the treatment of advanced and University Hospital and National Taiwan University, College of Medi- cine, Taipei, Taiwan. 5Graduate Institute of Pharmacognosy, Taipei refractory cutaneous T-cell lymphoma (12). Although HDACi Medical University,Taipei,Taiwan. 6Department of Pathology, National shows activity as a single agent to inhibit cell growth, prolifera- Taiwan University Hospital and National Taiwan University, College of tion, angiogenesis, and metastasis (13, 14), combination with Medicine, Taipei, Taiwan. other anticancer drugs is proved to be the most useful application Note: Supplementary data for this article are available at Clinical Cancer (11). HDACi synergizes with cisplatin causing cell-cycle pertur- Research Online (http://clincancerres.aacrjournals.org/). bation and apoptosis in lung cancer cells (15). It also shows Corresponding Author: Ching-Chow Chen, Department of Pharmacology, Col- synergistic effect with kinase inhibitors such as PI3K inhibitor lege of Medicine, National Taiwan University, 11F, No.1, Jen-Ai Road, Section 1, LY294002 and imatinib (16, 17). Taipei 10018, Taiwan. Phone: 886-2-23123456, ext. 88321; Fax: 886-2-23947833; Dual specificity phosphatase (DUSP) family proteins, which E-mail: [email protected] can dephosphorylate both serine/threonine and tyrosine residues doi: 10.1158/1078-0432.CCR-14-1150 of their substrates, are made up of 11 members and three classes Ó2015 American Association for Cancer Research. (18, 19). Class I DUSPs (DUSP1, 2, 4, and 5) are found in the 428 Clin Cancer Res; 21(2) January 15, 2015 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. HDAC1 Mediates DUSP1 Expression in Gefitinib Efficacy sulfate, and 2 mmol/L glutamine at 37C humidified incubator Translational Relevance under an atmosphere of 5% CO2 in air. Cells were daily checked Gefitinib is highly effective in non–small cell lung cancer by morphology and tested to be Mycoplasma free by DAPI staining (NSCLC) harboring EGFR-activating mutation. However, within the last 6 months. acquired resistance developing after 1 year of treatment is inevitable. Current understanding of acquired resistance Determination of synergism includes EGFR T790M mutation or MET amplification. Our The medium-effect method was used to analyze dose–response study disclosed that DUSP1 downregulation might be a novel of single drug or multiple drugs, and synergistic effect of multiple mechanism of gefitinib-acquired resistance, and HDAC1 inhi- drugs determined by the definition of Chou and Talalay combi- bition could induce DUSP1 upregulation to sensitize resistant nation index (CI) reflecting the interaction of two drugs at different cells to gefitinib in vitro and in preclinical models. In addition, levels of growth inhibition was calculated with the software DUSP1 expression correlated with gefitinib sensitivity in a package Calcusyn (Biosoft; ref. 20). CI values of <1, 1, and >1 clinical study and could serve as a predictive biomarker. indicate synergistic, additive, and antagonistic effects, respectively. Therefore, our study provides not only mechanistic insights into gefitinib-acquired resistance but also a promising strategy Human protein phosphatases array to overcome it. The RT2 Profiler human protein phosphatases PCR array (SABiosciences) containing 84 genes related to the protein phos- phatases, five housekeeping genes, and three controls. Total RNA was isolated and transcribed into cDNA with the RT2 First Strand nucleus, class II (DUSP6, 7, and 9) in cytoplasm, and class III Kit (SABiosciences) in accordance with the manual. The template 2 (DUSP8, 10, and 16) in both nucleus and cytoplasm (18). The was combined with the RT SYBR Green qPCR Master mix m MAPKs, including ERK, JNK, and p38, are critical regulators for (SABiosciences). Equal amounts of this mixture (25 L) were 2 fi cellular growth and proliferation. They are dephosphorylated by added to each well of the RT Pro ler PCR plate containing the fi DUSPs at phospho-tyrosine and phospho-serine/threonine resi- predispensed gene-speci c primer sets, and the reaction was fi dues (19). ampli ed and detected by ABI Prism 7900 Sequence Detection DDC In this study, we disclosed that gefitinib-resistant NSCLC cells, System. Data analysis was based on the t method normalized b including H1975, PC9/Iressa resistance (PC9/IR), and HCC827/ to -actin. Iressa resistance (HCC827/IR), showed HDAC1 overexpression, and its knockdown could sensitize resistant cells to gefitinib and Animal studies induce DUSP1 expression in vitro and in preclinical models. Nude mice (BALB/cAnN.Cg-Foxnlnu/CrlNarl) and NOD/SCID Prkdcscid Overexpression of DUSP1 in resistant cells restored gefitinib mice (NOD.CB17- /JNarl) with 6 to 8 weeks of age weigh- sensitivity, whereas its knockdown in sensitive cells conferred ing 23 to 25 g were purchased from the National Laboratory resistance. We also developed a novel HDACi, WJ-26210-2, which Animal Center (NLAC, Taiwan). The research protocol was fi was more potent than SAHA to inhibit HDAC activity and cellular approved, and mice were housed and maintained under speci c growth. WJ-26210-2 in combination with gefitinib overcame pathogen-free conditions in the Animal Care Facility of the Nation- gefitinib resistance through upregulation of DUSP1, leading to al Taiwan University (Taiwan) in accordance with the guidelines of the inhibition of cell viability and induction of apoptosis in vitro the Institutional Animal Care and Use Committee. For xenograft  6 and in xenograft and orthotopic
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