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. 2015 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 2015 American Association for Cancer Research. (18, 19). Class I DUSPs (DUSP1, 2, 4, and 5) are found in the

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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 phosphatases, 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 mouse models. Clinically, animal models, (i) 2 10 H1975 cells (suspended in 0.1 mL PBS fl tumors with gefitinib-acquired resistance had decreased DUSP1 and mixed with 0.1 mL Matrigel) s.c. injected in the right- ank area fi € of mice were randomized into four experimental groups (n ¼ 6) mRNA level compared with those from ge tinib-na ve patients, 3 and patients harboring high DUSP1 expression tumors had when established tumors were palpable (100 mm ). Mice were fi favorable outcome while taking gefitinib as the first-line treat- orally treated with ge tinib every day (150 mg/kg in 1% aqueous ment. Our results not only provide a novel strategy to overcome Tween 80) and/or i.p. administrated with WJ-26210-2 every other 6 gefitinib-acquired resistance, but also imply that DUSP1 might be day (25 mg/kg in DMSO) for 28 days; (ii) 2 10 H1975, H1975- a predictive biomarker for gefitinib treatment. vector, or H1975-shHDAC1 cells inoculated into the nude mice were treated with or without gefitinib for 21 days (n ¼ 7–8) when Materials and Methods tumor established. Tumor size was measured with a caliper twice per week and calculated using the formula V (mm3) ¼ 0.52 [ab2], Cell lines where a is the length and b is the width of the tumor. Body weight Human lung adenocarcinoma cells harboring EGFR-activating was monitored twice per week. For the orthotopic model, 4 106 mutation, PC9 (exon 19 deletion), PC9/Iressa resistance (PC9/IR, luciferase-expressing H1975 cells (suspended in 40 mL PBS) were not T790M mutation), H1975 (L858R/T790M double mutation), orthotopically injected into the right lateral thorax of NOD/SCID and HCC827 (exon 19 deletion) cultured in RPMI-1640 were mice. Tumor formation was monitored by the noninvasive bio- kindly provided by Dr. Chih-Hsin Yang (National Taiwan Uni- luminescence system (IVIS-Spectrum). The tumors established versity, Taiwan). HCC827/Iressa resistance (HCC827/IR, not about 10 days then mice were randomized into four experimental T790M mutation) was generated by exposing to increasing con- groups (n ¼ 6). centrations of gefitinib from 0.1 to 5 mmol/L. A549 human lung carcinoma cells, PC3 human prostate adenocarcinoma, and Real-time quantitative PCR and immunohistochemical HCT116 human colorectal carcinoma from the ATCC were cul- staining of DUSP1 from NSCLC specimens tured in DMEM. All cells were cultured in medium containing Malignant pleura effusions were obtained from 22 patients 10% FBS, 100 U/mL penicillin G, 100 mg/mL streptomycin before taking gefitinib (pre-gefitinib) and from 26 patients

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suffered from disease progression by gefitinib (acquired resis- were found to be overexpressed (Fig. 1B). Knockdown of HDAC1 tance, post-gefitinib). All 48 patients have EGFR-activating muta- by shRNA (clone F2) could decrease the IC50 value of resistant tion. Their pleural fluids were collected in vacuum bottles by H1975, PC9/IR, and HCC827/IR cells to respective 1.22, 0.67, and thoracentesis, and red blood cells in the specimen were hemo- 3.21 mmol/L (Fig. 1C and Supplementary Fig. S2), indicating the lyzed using RBC lysis buffer. The remaining cells were washed involvement of HDAC1 in gefitinib resistance. twice with PBS and cultured in complete RPMI-1640 medium as described previously (21, 22). Medium was replaced every 2 to 3 WJ compounds, novel HDAC inhibitors, inhibited cell growth days and cells were harvested after 10 days. Total RNA was and induced acetylation of histone-H3 and a-tubulin in various extracted from cultured cells using the TRizol reagent (Invitro- cancer cells gen), and the expression of DUSP1 mRNA levels between pre- and We have synthesized a series of HDACis, which showed the post-gefitinib groups was determined by quantitative PCR (Q- ability to suppress in vitro activity of class I (HDAC1 and HDAC8) PCR). In addition, 47 patients with EGFR-activating mutation had and class IIb (HDAC6) HDACs with greater capacity than SAHA undergone surgical resection and took gefitinib after identified (Supplementary Fig. S3A and Supplementary Table S1). To con- tumor recurrence. The tumor specimens were collected to analyze firm their inhibition in cells, H1975, PC9, and PC9/IR cells treated DUSP1 expression by IHC staining. The studies were approved by with these compounds were analyzed by histone and a-tubulin the Institutional Review Board of National Taiwan University acetylation. Among them, WJ-25591, WJ-26208-56, and WJ- Hospital (201304046RIND). Written informed consent was 26210-2 were the most potent to induce acetylation of histone- obtained from all patients. IHC staining was analyzed by H score H3 and a-tubulin (Supplementary Fig. S3B). Parallel to their with the formula: 0 [percentage of no staining (0 þ)] þ 1 HDAC inhibition, WJ compounds effectively inhibited cell [percentage of weakly staining (1 þ)] þ 2 [percentage of growth in PC3 cells (Supplementary Fig. S3C) and WJ-26210-2 moderately staining (2 þ)] þ 3 [percentage of strongly staining was chosen as a lead compound (preparation of WJ-26210-2 was (3 þ)], giving a range from 0 to 300 (23). Moreover, we dichot- provided in Supplementary Materials and Methods). Its effect on H omized score into higher or lower expression by the median the cell viability of NSCLC cells was assessed with IC50 values value. Each IHC specimen was reviewed for 10 high-power fields around 1 mmol/L (Supplementary Fig. S3D). by pathologist Yih-Leong Chang, thoracic oncologist Jin-Yuan Shih, and medical oncologist Yu-Chin Lin to achieve the WJ-26210-2 combined with gefitinib overcame gefitinib consensus. resistance by inhibiting EGFR signaling and inducing apoptosis Statistical analysis Because HDAC1 was overexpressed in resistant H1975, PC9/IR, Data were presented as the mean SD and compared using the and HCC827/IR cells and its knockdown could sensitize resistant Student t test. , P < 0.05; , P < 0.01; and , P < 0.005 were cells to gefitinib, the effect of WJ-26210-2 combined with gefitinib statistically significant. The c2 test was used to compare the on these resistant cells was examined. Cell viability of PC9/IR cells characteristics of patients with high or low DUSP1 mRNA expres- exposed to individual agent or combination at different ratio was sion. Survival curves were plotted by the Kaplan–Meier method analyzed by the median-effect method to determine the IC50 and compared by the log-rank test. values and CI (Supplementary Fig. S4A, left). CI values at different ratio especially 1:1 were smaller than 1, indicating the synergism between WJ-26210-2 and gefitinib (Supplementary Fig. S4A, right Results and S4B). Therefore, 1:1 combination was used. The IC50 and CI HDAC1 was overexpressed in gefitinib-resistant NSCLC cells, values of WJ-26210-2 plus gefitinib were found to be smaller than and its knockdown could overcome gefitinib resistance those of SAHA plus gefitinib, indicating more potent of WJ- Several NSCLC cells, including two gefitinib-sensitive (PC9 and 26210-2 than SAHA to combine with gefitinib (Supplementary HCC827) and four gefitinib-resistant (A549, H1975, PC9/IR, and Table S2 and Supplementary Fig. S4C). Combination of 1 mmol/L HCC827/IR) cells, were used to assess the effect of gefitinib on WJ-26210-2 with 1 mmol/L gefitinib induced 80% reduction of their viability. PC9/IR and HCC827/IR cells did not show T790M cell viability in both H1975 and PC9/IR cells but not normal MEF mutation in EGFR exon 20 as did H1975 cells (Supplementary Fig. cells (Supplementary Fig. S4C). Colony formation in resistant S1A). As expected, gefitinib dramatically inhibited cell viability in cells was also inhibited by combination but not either gefitinib or sensitive PC9 and HCC827 cells with IC50 values smaller than 10 WJ-26210-2 alone (Supplementary Fig. S4D). These data dem- nmol/L. In contrast, H1975, PC9/IR, and HCC827/IR cells were onstrated the synergism between WJ-26210-2 and gefitinib to resistant to gefitinib inhibition with IC50 values of 7.02, 5.52, and suppress cell proliferation in gefitinib-resistant cells. more than 10 mmol/L, respectively (Supplementary Fig. S1B). Corresponding to the inhibition on cell viability, WJ-26210-2 Because EGFR downstream AKT and ERK signalings play impor- plus gefitinib facilitated dephosphorylation of p-EGFR and inhi- tant roles in the proliferation and survival of NSCLC, the effect of bition of AKT and ERK activation in H1975, PC9/IR, and HCC827/ gefitinib was assessed. Gefitinib inhibited activation of EGFR and IR cells (Fig. 1D). Cleavages of PARP and pro–caspase-3, upregula- downstream AKT and ERK as well as induced apoptosis in sen- tion of BIM and downregulation of MCL-1 were also induced by sitive, but not resistant cells (Fig. 1A and Supplementary Fig. S1C). combination but not gefitinib or WJ-26210-2 alone (Supplemen- The apoptosis was evaluated by PARP and pro–caspase-3 clea- tary Fig. S4E). This event occurred in a time-dependent manner vages, BIM upregulation and MCL-1 reduction (Supplementary (Supplementary Fig. S4F). In addition, WJ-26210-2 was also more Fig. S1C). Overexpression of HDACs has been reported to con- potent than SAHA in combination with gefitinib to inhibit EGFR tribute to chemoresistance in many cancer cells (24); we therefore signaling and induce cell apoptosis (Supplementary Fig. S4G). examined their expression in NSCLC cells. HDAC1 in H1975, Therefore, WJ-26210-2 combining with gefitinib is more effective PC9/IR, and HCC827/IR cells, and HDAC6 in HCC827/IR cells than SAHA to overcome gefitinib resistance.

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Figure 1. Overexpression of HDAC1 in gefitinib- resistant NSCLC cells and its effect on gefitinib sensitivity. A, PC9, PC9/IR, HCC827, and HCC827/IR cells were treated with 1 mmol/L gefitinib for the indicated time, and total cell lysates were analyzed by Western blot analysis detected by antibodies against EGFR signaling molecules with b-actin as loading control. B, differential expression of HDAC1, 2, 3, and 6 in various NSCLC cells was examined by Western blot analysis with b-actin as loading control. C, HDAC1 in H1975 cells was knocked down by shRNA (clones F2, G2, and H2), and HDAC1, 2, 3, and 8 were examined by Western blot analysis (left). HDAC1 knocked down cells were treated with gefitinib for 3 days and cell viability was measured by MTT assay. Data, as means SD (n ¼ 5; Student t test; , P < 0.05; , P < 0.01; , P < 0.005; right). D, gefitinib- resistant NSCLC cells were treated with 1 mmol/L WJ-26210-2 for 24 hours and then exposed to 1 mmol/L gefitinib for the indicated time points before cell harvest. Total cell lysates were analyzed by Western blot analysis detected by antibodies against EGFR signaling molecules with b-actin as loading control.

Induction of protein tyrosine phosphatase DUSP1 expression and only DUSP1 was found to be increased in all these cells by WJ- in resistant cells by combination of WJ-26210-2 and gefitinib 26210-2 and enhanced by the cotreatment with gefitinib (Fig. 2B). To explore the mechanism underlining combination-induced RT-PCR also confirmed this effect (Supplementary Fig. S5A). inhibition of EGFR signaling, H1975 cells were treated with a Induction of DUSP1 expression was further demonstrated by protein tyrosine phosphatase (PTP) inhibitor, sodium orthova- immunofluorescence assay. Increased DUSP1 (red) expression nadate (Na3VO4). Inactivations of EGFR, ERK, and AKT were and its colocalization with EGFR (green) were seen (Fig. 2C). reversed by Na3VO4, indicating the involvement of phosphatase SAHA combined with gefitinib and HDAC1 knockdown could in this event (Fig. 2A). To get more insight into which protein also induce DUSP1 expression in gefitinib-resistant H1975 cells phosphatase was involved, the Human Protein Phosphatases RT2 (Fig. 2D). These results suggested that DUSP1 was induced by Profiler PCR array was performed to analyze the mRNA profiles. A HDACi WJ-26210-2 and SAHA through HDAC1 inhibition and high induction of DUSPs (DUSP26, DUSP1, and DUSP4), cyto- enhanced by the cotreatment with gefitinib to overcome gefitinib solic phosphotyrosine-specific PTPs (PTPN7 and PTP4A1), trans- resistance. membrane receptor-like PTPs (PTPRN2, PTPRR, and PTPRE), To further explore how DUSP1 was induced, the protein protein phosphatase (catalytic subunit) PDP1, and PPEF1 was biosynthesis inhibitor, cycloheximide was used. It attenuated the seen after cotreatment (Supplementary Table S3). These genes in upregulated DUSP1 and reversed the inactivated EGFR signaling, H1975, PC9/IR, and HCC827/IR cells were validated by Q-PCR, suggesting the neosynthesis of DUSP1 (Fig. 3A). We have found

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Figure 2. Induction of DUSP1 expression in gefitinib-resistant NSCLC cells by combination of WJ-26210-2 and gefitinib. A, H1975 cells were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 in the presence of Na3 VO4 for 24 hours. Total cell lysates were analyzed by Western blot analysis detected by antibodies against EGFR signaling molecules with b-actin as loading control. B, gefitinib-resistant NSCLC cells were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 for 24 hours, and mRNA levels of protein phosphatases were quantified by Q-PCR; (n ¼ 3; Student t test; , P < 0.05). C, H1975 cells were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 for 24 hours. DUSP1 (red) and EGFR (green) fluorescence staining were shown by confocal photomicrographs, and merged with DAPI staining; scale bar, 20 mm (top). Colocalization coefficient of DUSP1 and EGFR was quantified by ZEN and presented as means SD (n ¼ 3; Student t test; , P < 0.05; , P < 0.005; bottom). D, H1975 cells were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 or 1 mmol/L SAHA for 24 hours. DUSP1 expression was examined by Western blot analysis with b-actin as loading control (top). HDAC1 in H1975 cells was knocked down by shRNA (clones F2, G2, and H2), and DUSP1 expression was examined by Western blot analysis with b-actin as loading control (bottom).

that the binding of SP1 to the EGFR promoter was affected by DUSP1 was downregulated in gefitinib-resistant NSCLC cells SAHA (25), and there are several SP1-binding sites on the DUSP1 and its overexpression sensitized resistant cells to gefitinib promoter. Therefore, SP1 inhibitor mithramycin A was used to Because DUSP1 seems to play an important role in mediating examine whether SP1 was involved in WJ-26210-2 plus gefitinib- gefitinib sensitivity, its expression in five EGFR-mutant–expres- induced DUSP1 expression. Mithramycin A reversed the upregu- sing NSCLC cells was examined. Overexpression of DUSP1 was lation of DUSP1 mRNA and protein (Fig. 3B). Quantitative found in gefitinib-sensitive PC9 and HCC827 cells compared with chromatin immunoprecipitation (ChIP–qPCR) assay was further resistant H1975, PC9/IR, and HCC827/IR cells (Fig. 4A, left). performed to examine the binding of SP1 and HDAC1 to the Three EGFR wild-type A549, CL1-0, and CL1-5 cells also resistant DUSP1 promoter. The results showed increased SP1 and to EGFR TKI were examined with less DUSP1, compatible with the decreased HDAC1 binding to the promoter, indicating the findings in EGFR-mutant cells. However, the expression pattern of involvement of SP1 and HDAC1 in the DUSP1 gene transcription HDAC1 in these three EGFR wild-type cells was not similar to that (Fig. 3C). of in EGFR-activating mutation cells (Fig. 4A, right), suggesting

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Figure 3. Mechanism of DUSP1 induction. A, H1975 cells were treated with 1 mmol/L geﬁtinib and 1 mmol/L WJ-26210-2 in the presence of 10 mmol/L cycloheximide (CHX) for 24 hours. Total cell lysates were analyzed by Western blot analysis detected by antibodies against EGFR signaling molecules with b-actin as loading control. B, H1975 cells were treated with 1 mmol/L geﬁtinib and 1 mmol/L WJ-26210-2 in the presence of various concentrations of mithramycin A (MTM) for 24 hours. The mRNA level and protein expression of DUSP1 were detected by Q-PCR and Western blot analysis, respectively (n ¼ 3; Student t test; , P < 0.05). C, the binding of SP1 and HDAC1 to the DUSP1 promoter was detected by ChIP–qPCR using anti-SP1 and anti-HDAC1 antibodies. Data were analyzed by Q-PCR and plotted as the percentage (%) of input DNA (Student t test; , P < 0.05).

that HDAC1/DUSP1 regulation contributes to acquired EGFR TKI with H1975 and H1975/vector xenograft, tumor growth of resistance specifically in EGFR-mutant–expressing NSCLC cells. HDAC1 knocked down H1975 xenograft was slightly retarded Transfection of DUSP1 into H1975 and PC9/IR cells induced (Fig. 5A, left). Gefitinib treatment further suppressed the growth dephosphorylation of EGFR and its downstream signaling in a of HDAC1 knocked down H1975 xenograft (Fig. 5A, left and dose-dependent manner (Fig. 4B, top). Phosphorylation of Supplementary Fig. S6A), indicating that HDAC1 knockdown ErbB3 but not MET was also decreased (Supplementary Fig. could sensitize H1975 tumor to gefitinib inhibition. DUSP1 S5B). The association between DUSP1 and EGFR was demon- expression was induced in H1975/shHDAC1 tumors and strated by coimmunoprecipitation (Fig. 4B, lower). To further enhanced by the cotreatment with gefitinib (Fig. 5A, right and investigate the role of DUSP1 in gefitinib sensitivity, H1975 Supplementary Fig. S6B), indicating that knockdown of HDAC1 andPC9/IRcellstransfectedwithDUSP1werefoundtobe upregulated DUSP1 expression to overcome gefitinib resistance in sensitized to gefitinib with IC50 values around 0.15 and 0.1 vivo. mmol/L, respectively, which was comparable with the effect of WJ-26210-2 plus gefitinib (Fig. 4C, top). In colony formation Combination of WJ-26210-2 and gefitinib induced in vivo assay, DUSP1 transfection also sensitized H1975 and PC9/IR DUSP1 expression in gefitinib-resistant NSCLC and inhibited cells to gefitinib as the effect of WJ-26210-2 plus gefitinib in lung cancer growth parental cells (Fig. 4C, lower). Similarly, PARP cleavage and To investigate whether combination of WJ-26210-2 and gefi- MCL-1 downregulation were induced by gefitinib in these tinib suppressed gefitinib-resistant tumor growth, the orthotopic resistant cells transfected with DUSP1 or treated with WJ- lung cancer model was used. By injection of luciferase-expressing 26210-2, as the effect of gefitinib in PC9 cells (Fig. 4D, com- H1975 cells into the right lower lobe of lungs, tumor growth in pared lane 8 and lanes 2, 3, 6; compared lane 16 and lanes 10, NOD/SCID mice (n ¼ 6/group) was monitored by biolumines- 11, 14, and 17). On the other hand, knockdown of DUSP1 in cence imaging. IVIS and ex vivo bioluminescence from excised gefitinib-sensitive PC9 and HCC827 cells activated EGFR sig- lungs showed that combination inhibited lung tumor growth and naling and conferred resistance to gefitinib (Supplementary Fig. nodule formation (Fig. 5B and Supplementary Fig. S6C). Hema- S5C). Sensitizing effect of WJ-26210-2 plus gefitinib in H1975 toxylin and eosin (H&E) staining further confirmed this finding, cells was abolished by DUSP1 knockdown (Supplementary Fig. and IHC staining demonstrated DUSP1 upregulation (Fig. 5C). In S5D). All these results indicated that DUSP1 expression addition, the H1975 cells derived xenograft mouse model was induced by HDAC inhibition did mediate gefitinib sensitivity also used to demonstrate that combination synergistically inhib- by inducing apoptosis through inactivation of EGFR signaling. ited tumor growth compared with individual treatment (Fig. 5D, left and Supplementary Fig. S6D). All mice tolerated the treatment HDAC1 knockdown sensitized H1975 cells to gefitinib in the well without significant toxicity and showed stable body weights. mouse xenograft model Increased expression of DUSP1 and reduced activation of EGFR, To validate the in vivo effect of HDAC1 on gefitinib resistance, AKT, and ERK were also seen in tumor lysates from combination H1975 cells infected with a lentivirus-expressing shRNA against treatment (Fig. 5D, right and Supplementary Fig. S6E). These HDAC1 were inoculated into the nude mice. Mice inoculated with results indicated that WJ-26210-2 combined with gefitinib could H1975, H1975/vector, or H1975/shHDAC1 cells were treated overcome gefitinib resistance in vivo via the induction of DUSP1 with vehicle or gefitinib (6 groups, n ¼ 7–8/group). Compared expression to reduce the EGFR signaling.

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Figure 4. Downregulated DUSP1 in gefitinib-resistant NSCLC cells and its effect on gefitinib sensitivity. A, differential expression of DUSP1 in five EGFR-mutant–expressing NSCLC cells was examined by Western blot analysis with b-actin as loading control (left). Expression of DUSP1 and HDAC1 in normal lung BEAS-2B cells; EGFR wild- type A549, CL1-0, and CL1-5 cells; and EGFR-activating mutation H1975, PC9, and PC9/IR cells were examined by Western blot analysis with b-actin as loading control (right). B, H1975 and PC9/IR cells were transiently transfected with 1 to 6 mg of DUSP1 plasmids, and total cell lysates were analyzed by Western blot analysis detected by antibodies against EGFR signaling molecules (top). H1975 cells transiently transfected with 2-mg DUSP1 or 2-mg Flag-DUSP1 plasmids were immunoprecipitated with anti-DUSP1 or anti-Flag antibody, respectively. DUSP1 and EGFR expressions were examined by Western blot analysis (bottom). C, H1975 (top, left) and PC9/IR (top, right) cells transiently transfected with vector or 2-mg DUSP1 plasmids were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 for 3 days, and cell viability was measured by MTT assay. Data, as means SD (n ¼ 5). H1975 (bottom, left) and PC9/IR (bottom, right) cells transiently transfected with vector or 2-mg DUSP1 plasmids were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 for 5 days, and cells were stained with crystal violet as described in the Supplementary Materials and Methods. D, H1975 and PC9/IR cells transiently transfected with vector or 2-mg DUSP1 plasmids were treated with 1 mmol/L gefitinib and 1 mmol/L WJ-26210-2 for 24 hours, and total cell lysates were analyzed by Western blot analysis detected by antibodies against apoptosis markers. PC9 cells were shown as a positive control.

Patients with NSCLC with decreased DUSP1 expression undergone surgical resection taking gefitinib as their first-line associated with gefitinib-acquired resistance and poor treatment after tumor recurrence were collected. DUSP1 expres- progression-free survival sion was analyzed by IHC staining with positive control of To determine the clinical significance of DUSP1 expression in expression in normal kidney tubules and negative control of gefitinib-acquired resistance of patients with NSCLC, 48 speci- staining by normal rabbit IgG (Fig. 6B, a and b). Among 47 mens of malignant pleura effusion, including 22 pre-gefitinib and patients, 26 with higher DUSP1 expression had longer progres- 26 post-gefitinib specimens (acquired resistance), and their sion-free survival (PFS; Fig. 6C, Kaplan–Meier method, P ¼ DUSP1 mRNA levels were analyzed by Q-PCR (Fig. 6A, left; 0.024). Therefore, DUSP1 may serve as a predictive marker for Supplementary Table S4). The DCt value from 0.33 to 6.03 with gefitinib efficacy and its decrease may be associated with gefitinib a median of 3.5 was used to classify patients into the DUSP1 high- resistance. expression or low-expression group. Fourteen of 22 specimens in fi DUSP1 the pre-ge tinib group had higher mRNA, whereas 17 of Discussion 26 in the post-gefitinib group had lower DUSP1 mRNA (Fig. 6A, right). The association of low DUSP1 level and the post-gefitinib EGFR-targeted therapy provide not only a new treatment par- group was significant (c2 test, P ¼ 0.043), indicating the corre- adigm but also a new biologic insight into NSCLC, in which lation between decreased DUSP1 mRNA level and gefitinib- gefitinib exerts a durable PFS and significantly prolongs overall acquired resistance. In addition, 47 specimens from patients survival in patients with EGFR-activating mutation (5, 26).

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Figure 5. HDAC1 inhibition sensitized H1975 cells to gefitinib through in vivo DUSP1 induction in preclinical mouse models. A, H1975, H1975-Vector, or H1975-shHDAC1 cells were s.c. inoculated into the nude mice. Relative growth of each tumor was compared with the original size before gefitinib treatment (150 mg/kg/d, oral gavage). Data, as means SD (n ¼ 7–8; Student t test; , P < 0.05; , P < 0.005; N.S, not significant; left). Tumors dissected from each experimental group were homogenized, and the intratumoral expression of HDAC1 and DUSP1 was examined by Western blot analysis with b-actin as loading control (right). B, NOD/SCID mice orthotopically injected with luciferase-expressing H1975 cells were treated with gefitinib (150 mg/kg/d, oral gavage), WJ-26210-2 (25 mg/kg/every other day, i.p. administration), and combination, and the bioluminescence images of dissected lung are shown (n ¼ 6). C, histologic analyses by H&E staining and immunohistochemical staining of DUSP1 expression in lung tumors are shown; scale bar, 100 mm. D, nude mice s.c. inoculated with H1975 cells were treated with gefitinib (150 mg/kg/d, oral gavage), WJ-26210-2 (25 mg/kg/every other day, i.p. administration), and combination, and the relative growth of each tumor was compared with the original size before drug treatment. Data, as means SD (n ¼ 5–6; Student t test; , P < 0.05; , P < 0.005; left). Tumors dissected from each experimental group were homogenized, and the intratumoral expression of DUSP1 and EGFR signaling molecules was examined by Western blot analysis with b-actin as loading control (right).

However, the emergence of acquired resistance is inevitable and 10, 27). In our study, downregulated DUSP1 was seen in gefitinib- remains the major obstacle. The emergence of acquired resistance acquired–resistant H1975, PC9/IR, and HCC827/IR cells, and to afatinib suggests the involvement of other signaling pathways DUSP1 induction by HDAC1 inhibition could sensitize resistant beyond EGFR per se, such as c-Met amplification, PIK3CA muta- cells to gefitinib in vitro and in vivo. Furthermore, in clinical NSCLC tions, BRAF mutations, or small-cell cancer transformation (9, specimens, DUSP1 mRNA level was decreased in tumor with

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Figure 6. The correlation between DUSP1 expression and gefitinib sensitivity in patients with NSCLC. A, DUSP1 mRNA level in lung specimens from pre- gefitinib (pre-TKI) and post-gefitinib (post-TKI) patients with NSCLC (left). The DUSP1 mRNA level was stratified as high or low by median value (3.5), and the differences between these two groups were compared by the c2 test (right). B, immunohistochemical staining of DUSP1 in patients with NSCLC specimens; a, strong staining of DUSP1 in human kidney tubules as a positive control; b, staining with normal rabbit IgG as a negative control; c, tumor cells without staining (IHC 0þ); d, a partial weak membrane staining (IHC 1þ); e, weak to moderate circumferential membrane staining (IHC 2þ); f, strong circumferential membrane staining (IHC 3þ); scale bar, 100 mm. C, Kaplan–Meier survival plots of PFS for patients with NSCLC grouped by DUSP1 expression. The differences between groups were compared by the log-rank test (n ¼ 47).

gefitinib-acquired resistance and high expression of DUSP1 was lished data). Because HDACi can inactivate EGFR signaling correlated with longer gefitinib efficacy duration. Our results through multiple mechanisms, its combination with EGFR TKI imply that HDAC1 inhibition to induce DUSP1 upregulation deserves further clinical development. could be a strategy to overcome gefitinib-acquired resistance. Protein kinase and phosphatase could maintain homeostasis DUSP1 might serve as a predictive biomarker for gefitinib treat- of cellular signaling such as the MAPK signaling pathway. ment and its decrease might be one of the mechanisms respon- Therefore, dysregulation of phosphatase activity or loss of its sible for gefitinib-acquired resistance. expression may contribute to cancer progression or drug resis- Overexpression of HDACs might mediate drug resistance like tance (33). Overexpression of DUSP1 has been reported to cisplatin, etoposide, and vincristine (28, 29). HDACi MS-275 has inhibit proliferation and metastasis of NSCLC cells in vitro and been reported to restore gefitinib sensitivity in lung cancer cells in vivo, and correlate with favorable outcome (34, 35). Its through upregulation of E-cadherin expression (30), and SAHA expression was inversely correlated with disease severity in could restore BIM function and gefitinib sensitivity in resistant colon, bladder, and prostate cancer (36). In our study, DUSP1 NSCLC cells harboring the BIM deletion polymorphism (31). In expression was induced by HDAC1 inhibition and enhanced by our study, HDAC1 was overexpressed in gefitinib-resistant the combination with gefitinib to inhibit gefitinib-resistant H1975, PC9/IR, and HCC827/IR cells, and its knockdown could tumor growth in preclinical animal models. Overexpression of overcome gefitinib resistance and induce DUSP1 expression in DUSP1 in NSCLC-resistant cells could overcome gefitinib resis- vitro and in vivo. Though SAHA has been approved clinically for tance, whereas its knockdown in sensitive cells conferred gefi- cutaneous T-cell lymphoma (12), its activity against solid tumor is tinib resistance. In patients with NSCLC, decreased DUSP1 limited. We developed a novel HDACi, WJ-26210-2, which was mRNA expression was associated with gefitinib-acquired resis- more potent than SAHA with less toxicity against normal cells in tance, and high DUSP1 expression correlated with longer PFS of clinical relevant dose. WJ-26210-2 in combination with gefitinib first-line gefitinib treatment. Therefore, DUSP1 expression might exerted promising activity to overcome gefitinib resistance in vitro be not only associated with reducing gefitinib resistance, but and in preclinical models through induction of protein phospha- also served as a predictive biomarker for gefitinib efficacy. tase DUSP1. The association between DUSP1 and EGFR was HDACi WJ-26210-2 could induce DUSP1 upregulation to sen- demonstrated (Fig. 4B). Therefore, we are the first to demonstrate sitize gefitinib-resistant cells to gefitinib. The mechanism of that DUSP1 induced by HDAC1 inhibition might restore gefitinib upregulation of DUSP1 involving SP1 was also demonstrated. sensitivity in TKI-resistant cells through inactivation of EGFR The relationship between DUSP family and drug resistance has signaling to induce apoptosis. HDACi entinostat in combination been reported. Deficiency of DUSP4 caused Ras–ERK pathway with erlotinib could improve the outcome of patients with NSCLC activation leading to chemotherapy resistance and poor out- with high E-cadherin expression in the clinical trial (32). We also come in breast cancer (37). In contrast, DUSP1 expression found that EGFR was downregulated by HDACi through tran- confers resistance to chemotherapy through dephosphorylation scription inhibition in colon cancer cells and through c-Cbl of JNK leading to antiapoptotic effect (38). Therefore, the role of induction to decrease protein stability in NSCLC cells (25; unpub- DUSP family in drug resistance needs to be further investigated,

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and cellular context as well as treatment agents might also be Analysis and interpretation of data (e.g., statistical analysis, biostatistics, considered. computational analysis): Yun-Chieh Lin, Yu-Chin Lin, Y.-L. Chang In conclusion, knockdown of HDAC1 could sensitize gefiti- Writing, review, and/or revision of the manuscript: Yun-Chieh Lin, Yu-Chin fi in Lin, J.-Y. Shih, C.-C. Chen nib-resistant cells to ge tinib and induce DUSP1 expression Administrative, technical, or material support (i.e., reporting or organizing vitro and in vivo. Combination of HDACi WJ-26210-2 with data, constructing databases): J.-Y. Shih, W.-J. Huang gefitinib upregulated DUSP1 mRNA and protein to inactivate Study supervision: C.-C. Chen EGFR signaling, and thus conferred its proapoptotic and in vivo Other (compound synthesis and in vitro HDAC activity assay): S.-W. Chao antitumor effects. Lower DUSP1 mRNA was found from NSCLC with gefitinib-acquired resistance, and DUSP1 overexpression Acknowledgments predicted prolonged PFS of gefitinib. These data suggested that The authors thank Dr. Hui-Kuan Lin (MD Anderson Cancer Center, TX) for fi giving us valuable suggestions; Dr. Chih-Hsin Yang (National Taiwan Univer- DUSP1 plays a crucial role in ge tinib sensitivity, and combi- sity, Taiwan) for providing PC9, PC9/IR, and HCC827 cell lines; and Dr. Win- fi nation of HDAC inhibition with ge tinib is a promising strategy Ping Deng (Taipei Medical University, Taiwan) for providing the pcDNA3.1/ to overcome gefitinib-acquired–resistant NSCLC. Our study not DUSP1 plasmid. only explored an additional mechanism for acquired resistance in NSCLC harboring EGFR-activating mutation, but also pro- Grant Support vided an effective combinatorial approach to overcome gefiti- This work was supported by a research grant from the National Science nib-acquired resistance. Council of National Research Program for Biopharmaceuticals (NSC-102-2325- B-002-007) and Excellent Translational Medicine Research Projects of National Disclosure of Potential Conflicts of Interest Taiwan University College of Medicine and National Taiwan University Hos- pital of Taiwan (102C101-B1). No potential conflicts of interest were disclosed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Authors' Contributions accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Conception and design: Yun-Chieh Lin, Yu-Chin Lin, C.-C. Chen Acquisition of data (provided animals, acquired and managed patients, Received May 8, 2014; revised October 13, 2014; accepted October 25, 2014; provided facilities, etc.): Yun-Chieh Lin, J.-Y. Shih, S.-W. Chao published online January 15, 2015.

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