Antitumor Impact of P14arf on Gefitinib-Resistant Non–Small Cell

Published OnlineFirst June 12, 2013; DOI: 10.1158/1535-7163.MCT-12-1239

Molecular Cancer Cancer Therapeutics Insights Therapeutics

Antitumor Impact of p14ARF on Geﬁtinib-Resistant Non–Small Cell Lung Cancers

Ken Saito1, Nagio Takigawa4, Naoko Ohtani6, Hidekazu Iioka1,7, Yuki Tomita1,2, Ryuzo Ueda8, Junya Fukuoka9, Kazuhiko Kuwahara10, Eiki Ichihara5, Katsuyuki Kiura5, and Eisaku Kondo1,3

Abstract Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates the proliferation of tumors. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as a molecular-targeting agent for the inactivation of EGFR signaling and show considerable therapeutic effect in non–small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib- resistant mutations in EGFR, which is a critical issue for current therapeutics. We aimed to characterize the distinct molecular response to gefitinib between the drug-resistant and drug-sensitive lung adenocarcinoma cells in order to learn about therapeutics based on the molecular information. From the quantitative ARF PCR analysis, we found a specific increase in p14 expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14ARF triggered the most augmented apoptosis in both clones. We identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14ARF (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of the gefitinib-resistant clones without affecting nonneoplastic cells. Notably, transduction of the minimized dose of the p14 38-65 peptide restored the response to gefitinib like that in the sensitive clones. These findings suggest that the region of p14ARF 38-65 a.a. is critical in the pharmacologic action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers. Mol Cancer Ther; 12(8); 1–13. 2013 AACR.

Introduction first-line therapy for EGFR mutation-positive cells of non– Molecular-targeting agents may exert a gene-specific small cell lung cancer (NSCLC; refs. 2–4). The sensitivity effect on key regulators of tumor growth in various to gefitinib is enhanced by the point mutation of exon 21 intractable malignancies (1). Epidermal growth factor (L858R) or the in-frame deletion of exon 19 in the EGFR receptor (EGFR)-targeting agents such as gefitinib are a tyrosine kinase domain (3, 5) and gefitinib inhibits signal transduction of the phosphoinositide 3-kinase-Akt pathway and the mitogen-activated protein kinase pathway, Authors' Affiliations: 1Division of Oncological Pathology, Aichi Cancer thereby inhibiting the growth of tumors (6–8). However, Center Research Institute, Chikusa-ku; 2Department of Medical Oncology and Immunology, Nagoya City University Graduate School of Medical the recurrence of these tumors with drug resistance in Science, Mizuhocho, Mizuho-ku; 3Department of Epidemiology, Nagoya patients treated with such agents can limit the overall 4 University Graduate School of Medicine, Showa-ku, Nagoya; Department utility of these agents (9, 10). The main cause of resistance of General Internal Medicine 4, Kawasaki Medical School; 5Department of Respiratory Medicine, Okayama University Hospital, Kita-ku, Okayama; to EGFR inhibitors in lung cancers is genetic alterations in 6Division of Cancer Biology, The Cancer Institute of Japanese Foundation the threonine residue at position 790 in exon 20 (T790M) of 7 for Cancer Research (JFCR), Tokyo; Advanced Medical Research Center; EGFR, a gatekeeper mutation (11, 12). This has led to an 8Department of Tumor Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi; 9Department of Surgical Pathology, Toyama effort to identify such genetic alterations and attempt to University Hospital, Toyama; and 10Department of Immunology, Graduate develop more powerful agents to overcome resistance School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan (13). However, identification of the effector molecules that Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). determine cellular fate within the EGFR signaling pathway is another important goal and may help facilitate the Corresponding Author: Eisaku Kondo, Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, development of novel strategies to overcome drug resis- Nagoya 464-8681, Japan. Phone: 81-52-764-9703; Fax: 81-52-764- tance. Previous studies reported that gefitinib induces 2972; E-mail: [email protected] cell-cycle arrest and triggers apoptosis in some cancer doi: 10.1158/1535-7163.MCT-12-1239 cells (14, 15), although the effector molecules that crucially 2013 American Association for Cancer Research. mediate the antiproliferation and apoptotic effect through

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gefitinib treatment remain unclear. Interestingly, it has Quantitative PCR been suggested that EGFR mutation correlates with low RNA was extracted using an RNeasy Plus mini kit CDKN2A expression (16). Therefore, identification of (Invitrogen Life Technologies Corp.) according to the gefitinib-driven cell-cycle regulators may contribute to manufacturer’s protocol. The RNA was quantified by a better understanding of NSCLCs harboring resistant absorbance at 260 nm and 500 ng of total RNA was reverse EGFR mutations. transcribed by 6-mer oligos and the Super Script III kit in The CDKN2A (INK4a/ARF) locus is encoded by alter- 20 mL of reaction solution. Reverse transcriptase (RT)-PCR INK4a ARF native splicing variants including p16 , p14 , and products were diluted in 1:20 and then subjected to p12 (17, 18). Recently, exon array analysis by PCR revealed quantitative real-time PCR (qPCR) using a TaqMan gene the existence of p16 variant 2 in lung cancer cell lines (19). expression assays and arrays kit (Invitrogen Life Tech- INK4a ARF Among these CDKN2A variants, p16 and p14 , nologies Corp.) in 20 mL of reaction solution. Data analysis which share exons 2 and 3 and use a unique exon 1, are was followed by DDCt methods using the step one plus well studied. They have distinct functions through instruction (Applied Biosystems, Ltd.). independent signaling pathways and the amino acids sequences between p16INK4a and p14ARF are produced Cell proliferation assay from different promoter regions (18). Moreover, methyl- A total of 15 mL of Cell Count Reagent SF (Nacalai ation of their promoter is also reported in many cancer Tesque, Inc.) was mixed with 500 mL of RPMI 1640 medi- cells (20). The p16INK4a inhibits the phosphorylation of Rb um. The mixture was added to cells, incubated for 30 through binding to CDK4/6 (21). The role of p14ARF minutes, and the optical density of the formazan products antagonizes the actions of MDM2 in the MDM2-p53 was measured at an absorbance at 490 nm with a micro- pathway, resulting in the stabilization of p53 (22, 23). plate reader (Tecan Group, Ltd.). Thus, p16INK4a and p14ARF function as modulators of cell cycle and apoptosis leading to tumor suppression. Cell fractionation Here we report the distinct molecular response to Cells were seeded at a density of 5 104 cells/0.5 mL in gefitinib of drug-resistant human lung adenocarcinoma 24-well dishes. The next day, the medium was replaced cells in comparison with drug-sensitive cells to highlight with fresh RPMI 1640 medium with or without gefitinib ARF the biological significance of p14 in the growth regu- (1 mmol/L). After 20-hour treatment with gefitinib, cells lation of these tumors. were lysed with the ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem Corp.) and the subcellular Materials and Methods fraction in each sample was concentrated with the Amicon Cell lines Ultra 3K device (Millipore Corp.) according to the man- Non–small cell lung adenocarcinoma cell lines PC-9 ufacturer’s instructions. The protein concentration in each and RPC-9 were used as previously described (24). PC-9 fraction was determined using a bicinchoninic acid pro- cells have a deletion in EGFR exon 19 (del E746–A750) tein assay kit (Pierce Corp.). as an activating EGFR mutation and are sensitive to gefitinib. Gefitinib-resistant RPC-9 cells were derived Detection of mitochondria membrane potential from parental PC-9 cells and have both an exon 19 PC-9 and RPC-9 cells were treated either with gefitinib deletion and an exon 20 mutation (T790M). EGFR muta- (1 mmol/L) for 24 hours or with the functional peptides tions were confirmed by a sequence analysis of the (20 mmol/L) for 14 hours. The cells were changed to genomic DNAs. HCC827 and H1975 (2 NSCLC cell medium containing 10 mg/mL JC-1 dye (Invitrogen Life lines), NuLi-1 (an immortalized normal human bron- Technologies Corp.) and cultured at 37C for 10 minutes. chus cell line), and NHDF (normal human dermal After washing with fresh medium, red or green fluores- fibroblast) cell lines were obtained from American Type cence was detected by inverted fluorescence microscopy Culture Collection. These cell lines were passaged for (Olympus IX-71; Olympus Japan Inc.). less than 6 months and then replaced with those of early For MitoTracker-based staining, cells were incubated passages. HCC827 (exon 19 deletion of EGFR) is sensi- with medium containing 0.2 mmol/L of MitoTracker Red tive to gefitinib and H1975 (EGFR T790M and L858R (Lonza Corp.) for 15 minutes at 37C. Then, they were mutations) is resistant to gefitinib (Supplementary Fig. placed in fresh medium for evaluation by fluorescence S1A; ref. 25). MMNK-1, an immortalized line from nor- microscopy. mal cholangiocytes, and TMNK-1 from normal endothelial cells were provided by Dr. N. Kobayashi (Okayama Immunofluorescence Saidaiji Hospital; refs. 26 and 27). Cell lines used in this Cells were fixed with PBS containing 4% paraformal- study were not authenticated. Cells were maintained in dehyde for 10 minutes, then incubated with 3% bovine RPMI 1640 medium (Invitrogen Life Technologies Corp.) serum albumin/PBS-Tween 20 blocking solution for 15 containing 10% FBS with 100 U/mL penicillin (Invitro- minutes at room temperature. After washing, cells were gen Life Technologies Corp.) and 100 mg/mL strepto- incubated with 0.1% Triton/PBS containing the primary mycin (Invitrogen Life Technologies Corp.) at 37Cwith antibodies for 1 hour. Cells were washed again with 0.1% 5% CO2. Triton/PBS, incubated for 1 hour with the secondary

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antibodies, and then washed 3 times with 0.1% Triton/ Results PBS. The nuclei were counterstained with Hoechst 33258 Specific induction and subcellular localization of dye (Sigma-Aldrich Corp.). Subcellular localization of p14ARF in gefitinib-sensitive NSCLC cells the endogenous p14ARF was visualized with fluorescein As a preliminary analysis for identifying gefitinib-spe- isothiocyanate-conjugated anti-mouse immunoglobulin cific molecular response, reactivity to gefitinib was first G (IgG) antibody as the secondary antibody. HSP60 assessed among 4 NSCLC cell lines, 2 pairs of drug- was visualized with Cy3-conjugated goat anti-rabbit sensitive and drug-resistant clones. The chemical struc- antibody. ture of gefitinib used in this study is shown in Fig. 1A. The For the fluorescent immunostaining of paraffin-embed- growth of PC-9 and HCC827 cells, both of which bear ded tumor tissues, primary antibodies were used for activating mutations in EGFR (exon 19 of EGFR), was p14ARF (1:500) and HSP60 (1:800), and Cy3-conjugated suppressed by gefitinib in a dose-dependent manner rabbit anti-goat IgG (1:500) or Alexa Fluor 488-conjugated (Supplementary Fig. S1A). For example, approximately rabbit anti-mouse IgG (1:250) were used as the secondary 60% of PC-9 cells were inhibited with 1 mmol/L gefitinib antibodies. The nuclei were stained with Hoechst 33258 and 90% of were inhibited with 10 mmol/L. However, the dye (1:1,000). The fluorescent images were detected by growth of RPC-9 and H1975 cells, both of which harbored confocal microscopy (Carl Zeiss, Inc.). the T790M mutation as the resistant EGFR mutation, was not affected by 1 mmol/L of gefitinib. However, 10 mmol/L Immunohistochemistry of gefitinib comparably suppressed their growth (50% Antigen retrieval of paraffin-embedded sections was inhibition in RPC-9), which suggested that the T790M conducted by Pascal S2800 systems (DakoCytomation, mutation does not completely abolish the response but Ltd.) in sodium citrate buffer and endogenous peroxide still inhibits it (11). When the effect of gefitinib on cell- activity was quenched by incubation in 3% hydrogen cycle progression was investigated in PC-9 and RPC-9 peroxide (Kanto Chemical, Inc.) for 10 minutes at room cells using the FUCCI (fluorescent ubiquitination-based temperature. Slides were then washed with 0.05% Tween- cell-cycle indicator) system (30) to monitor the cell-cycle 20 in PBS and incubated with the primary antibody progression, the number of G1-arrested living cells [p14ARF (1:500) or p53 (1:200)] diluted in CanGet signal markedly increased in response to gefitinib treatment solution (Toyobo, Ltd.) overnight at 4C. The signals were among PC-9 cells but not among RPC-9 cells. A similar developed with EnvisionþSystem-HRP (DAB) kit (Dako- effect was observed by fluorescence-activated cell sorting Cytomation, Ltd.). Nuclei were counterstained with analysis (Supplementary Fig. S1B and S1C). These results hematoxylin. suggest a distinct effect of gefitinib in cell-cycle regulation as a critical point of the drug action. Tissue samples procurement Based on this finding, the dynamics of CDKN1A and Formalin-fixed, paraffin-embedded tumor tissues CDKN2A gene expression were further examined by that were diagnosed by pathologists as a lung adeno- qPCR in PC-9 and RPC-9 cells with or without gefitinib carcinoma were obtained surgically or via core biopsies treatment. The mRNA level of CDKN2A but not that of from patients. All patients provided informed consent CDKN1A in PC-9 cells showed a time-dependent increase for the scientific use of their tissues. Tissues subjected to in response to gefitinib, whereas RPC-9 showed no the analysis in this study were selected according to the increase in both mRNA levels (Supplementary Fig. ARF mutational analysis of the EGFR gene, which was con- S1D). We found specific amplification of p14 expres- ducted by Okayama University Hospital and Toyama sion but not the expressions of other CDKN2A variants INK4a University Hospital. In total, 3 tumor tissues from (p16 , p16 variant 2, p12) upon gefitinib treatment by patients with an activating EGFR mutation and 3 tumor conventional RT-PCR and qPCR only in gefitinib-sensi- tissues from patients with a resistant EGFR mutation tive NSCLC lines (PC-9 and HCC827; Supplementary Fig. were examined. The peptide nucleic acid–locked S2A–S2C). This phenomenon was not observed in gefiti- nucleic acid PCR clamp-based detection test (Mitsubishi nib-resistant lines (RPC-9 and H1975). In PC-9 cells, ARF Chemical Medience Corp.) was used to detect EGFR p14 mRNA was augmented up to fourfold after 48 mutations (28, 29). hours of incubation with gefitinib, which was consistent The use of these tissues for this study was approved by with prominent induction of the p14ARF protein (Supple- INK4a the Institutional Review Boards (ethics committees) at mentary Fig. S2B and S2D). p16 and other CDKN2A Okayama University Graduate School of Medicine, Den- variants were not significantly amplified in response to tistry and Pharmaceutical Sciences, University of Toyama gefitinib (Supplementary Fig. S2C and S2D). Graduate School of Medicine and Pharmaceutical Science, Based on these data, we examined the subcellular and Aichi Cancer Center. localization of endogenously induced p14ARF in PC-9 cells in comparison with RPC-9 cells with or without treatment Statistical analysis of gefitinib. Immunofluorescence using anti-p14ARF anti- Statistical differences were analyzed by paired Student t body showed that endogenous p14ARF was distributed to test (MS Excel) and a value of P < 0.05 was regarded as the nucleoli, nucleus, and mitochondria in gefitinib- statistically significant. untreated PC-9 and HCC827 cells, whereas only

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A B p14ARF HSP60 Merge (+ Hoechst)

ON PC-9 N N O O HN Cl

F Gefitinib RPC-9

C WCL Cyt WCL Mito WCL Nuc M M M (kDa) : Gefitinib (kDa) : Gefitinib(kDa) : Gefitinib 15 p14ARF 15 p14ARF 15 p14ARF

α-Tubline HSP60 Histone 50 15 50 H3 PC-9 PC-9 PC-9

D WCL Cyt WCL Mito WCL Nuc M M M (kDa) : Gefitinib (kDa) : Gefitinib(kDa) : Gefitinib

ARF 15 p14 15 p14ARF 15 p14ARF

α Histone 50 -Tubline HSP60 50 15 H3 RPC-9 RPC-9 RPC-9

EFPC-9 1.2 si Control si p14ARF 1.0

ARF ARF 0.8 * 0.6 M si Control si p14 si Control si p14 0.4 (kDa) : Gefitinib 15 0.2 ARF p14 growth cell Relative 0.0

15 cleaved ARF ARF Ccaspase-3 si Control si p14 si Control si p14 50 Gefitinib (+) Gefitinib (–) β-Actin 40 Gefitinib :

Figure 1. Subcellular accumulation of p14ARF in the gefitinib-treated NSCLC clones. A, the chemical structure of gefitinib. B, the localization of endogenous p14ARF was examined by fluorescence microscopy. HSP60 was used as a mitochondrial marker. Nuclei were stained with Hoechst 33258 dye. Scale bar, 10 mm. C and D, after 24 hours of treatment with (þ) or without ()gefitinib, PC-9 (C) and RPC-9 (D) cells were fractionated into cytosol (Cyt), mitochondrial (Mito), and nuclear (Nuc) fractions. The expression of p14ARF, a-tubulin (cytosol marker), HSP60 (mitochondrial marker), and histone H3 (nuclear marker) in these fractions was assessed by immunoblotting. Whole cell lysate (WCL) is shown. E, endogenous p14ARF was knocked down by the specific siRNA to p14ARF mRNA (0.5 nmol/L). After 48 hours of gefitinib treatment, p14ARF and cleaved caspase-3 protein levels were detected by immunoblotting. F, the p14ARF siRNA was introduced into PC-9 cells. Growth inhibition was depicted by cellular morphology and measured by WST-8 reagent with or without gefitinib 48 hours after the siRNA introduction. , statistically significant (n ¼ 3, P < 0.05).

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mitochondrial localization of p14ARF was recognized in mutants showed lower expression than the wild-type the untreated RPC-9 and H1975 cells (Fig. 1B and Sup- (WT) and d1 expressors (Fig. 2A), the MTS-p14ARF d2 plementary Fig. S2E). Fraction analysis of cell lysates from most efficiently inhibited cell growth of both PC-9 and gefitinib-treated PC-9 cells revealed the prominent RPC-9 (Fig. 2B). For a detailed analysis, we further gen- increase of p14ARF in the mitochondria rather than in the erated the GFP-tagged MTS-p14ARF expressors to deter- nucleus (Fig. 1C). In contrast to PC-9 cells, no significant mine the essential region for growth inhibitory function accumulation of p14ARF was observed in the mitochon- within p14ARF (Fig. 2C). Among the p14ARF expressors drial or nuclear fraction in RPC-9 cells (Fig. 1D). When covering the amino acid residues 38-65 (MTS-GFP-p14ARF ARF knocking down p14 mRNA by the RNA interference, 38-65), MTS-GFP-p14ARF 65-132, and MTS-GFP-p14ARF activation of caspase-3 was affected (Fig. 1E). Moreover, WT, the protein products of which were localized in the partial rescue of the cells from apoptosis was observed in mitochondria in both PC-9 and RPC-9 cells (Supplemen- the gefitinib-treated PC-9 cells when they were treated tary Fig. S5), the MTS-GFP-p14ARF 38-65 showed the most ARF with the p14 -specific siRNA (Fig. 1F). These data sug- potent growth inhibition. The inhibitory ratio was 60% in ARF gested that p14 may play a crucial role in apoptotic the transfected PC-9 cells and 70% in the RPC-9 cells, induction by gefitinib. which was even more potent than in MTS-GFP-p14ARF ARF However, we examined p14 mRNA levels in PC-9 WT-transfected cells (Fig. 2D). In the MTS-GFP-p14ARF 38- and RPC-9 cells treated with conventional chemothera- 65-transfected cells, prominent mitochondrial aggrega- peutic agents such as cisplatin and etoposide to examine tion was observed, which was similar to the cells ARF whether the induction of p14 expression is a gefitinib- treated with gefitinib (Fig. 3A and B). Staining these specific response. Standard doses of these agents induced cells with JC-1 dye revealed that the aggregation trig- the same degree of growth suppression as was observed in gered mitochondrial dysfunction (Fig. 3B; ref. 32) and gefitinib treatment in PC-9 and RPC-9 cells (Supplemen- that eventually led to caspase-3 activation in MTS-GFP- tary Fig. S3A) and the qPCR showed no significant induc- p14ARF 38-65-expressing PC-9 and RPC-9 cells as well ARF tion of p14 mRNA by cisplatin or etoposide in contrast as in gefitinib-treated PC-9 cells (Fig. 3C). These find- to gefitinib (Supplementary Fig. S3B). Furthermore, ings suggested that the region encoding residues 38-65 knockdown of EGFR using specific siRNA increased within p14ARF plays an important role for gefitinib- ARF p14 mRNA both in PC-9 and RPC-9 cells suggested induced growth inhibition in NSCLC cells with EGFR that inactivation of EGFR signaling by gefitinib resulted in mutations. ARF specific induction of p14 among genes encoded by the CDKN2A locus on these lung adenocarcinoma cells (Sup- p14ARF functional peptide as an antitumor tool plementary Fig. S3C and S3D). Taking these results together, we designed a synthetic ARF Genomic sequence analysis of p14 in PC-9 and RPC- peptide with an antitumor property against the gefitinib- 9 revealed that p14ARF seemed functional because these sensitive and gefitinib-resistant NSCLC cells carrying cell lines are carrying only silent mutations (Supplemen- functional EGFR mutations. The peptide covering the tary Fig. S4A). However, both PC-9 and RPC-9 cells harbor amino acid residue from the 38 to 65 positions of the ARF a missense mutation (R248Q) in the DNA-binding domain p14 protein was generated by fusion of nona-D-argi- of p53 (31) and nuclear localization of p53 was not altered nine (r9) to create a cell-penetrating form. The p14-1C in response to gefitinib treatment (Supplementary Fig. S4B peptide encoded by the p14ARF MDM2 binding site, which and S4C). These findings suggest that p14ARF may func- suppresses the growth of p14ARF-negative glioblastoma tion in a p53-independent manner in these cells. H1975 cells (33), was used for a comparison to evaluate the had a point mutation of the glycine residue at position 83 antitumor efficacy (Fig. 4A). MTT cell-proliferation assays (G83V), but its biological role has not yet been elucidated revealed growth suppression in all 4 NSCLC lines after (Supplementary Fig. S4A). 48 hours of incubation with these peptides (Fig. 4B); however, the p14 38-65 peptide showed the maximum Functional core of p14ARF in the NSCLC cells bearing growth inhibition on all 4 lines in comparison with the EGFR mutations p14-1C peptide (90% inhibition vs. 60% inhibition; Fig. To identify the specific function of p14ARF and its 4B). The p14 38-65 peptide penetrated more than 99% of functional intracellular localization that efficiently med- the NSCLC cells and it successfully localized in the iates growth inhibition in the NSCLC cells with EGFR mitochondria after its incorporation into the cells as mutations, we generated several expressors of the trun- was shown by MitoTracker staining (Fig. 4C and Sup- cated p14ARF (Fig. 2A): the d1 expressor, which lacked the plementary Fig. S6B). The peptide functioned to trigger MDM2 binding site; the d2 expressor, which lacked the the reduction of mitochondrial membrane potential, nucleophosmin/B23 binding site; and the d3 expressor, corroborated by JC-1 dye (Fig. 4D), and it eventually which lacked the entire exon 1b region. These truncated caused apoptosis via the activation of caspase-9 in both ARF p14 expressors were fused with either the mitochon- PC-9 and RPC-9 cells (Fig. 4D and E). Moreover, the drial-targeting sequence (MTS) or nuclear localization antitumor effect (growth suppression) by the p14 38-65 signal (NLS) to regulate the subcellular localization of peptidewasthestrongestamongthe4differentfunc- each protein. Although the MTS-targeted d2 and d3 tional peptides targeting specific molecules other than

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A Exon1β Exon2 B PC-9 B23 1.2 * p14ARF MDM2 1.0 WT 0.8 0.6 164 132 0.4 d1 0.2 23 132 0.0 d2 growth cell Relative WTd1 d2 d3 WTd1 d2 d3 38 132 d3 Empty Empty ARF ARF 65 132 MTS-p14 p14 -NLS

MTS-p14ARF p14ARF-NLS RPC-9 1.2 * M 1.0 (kDa) WT d1 d2 d3 WT d1 d2 d3 0.8 Empty Empty 28 0.6 p14ARF 0.4 18 variants 0.2 0.0 Relative cell growth cell Relative 50 WTd1 d2 d3 WTd1 d2 d3 β-Actin 37 Empty Empty

MTS-p14ARF p14ARF-NLS

CDPC-9 * p14ARF 1.4 1.2 WT MTS GFP 1.0 0.8 1 132 0.6 0.4 38-65 MTS GFP 0.2 38 65 0.0 Relative cell growth cell Relative WT WT GFP GFP

65-132 MTS GFP 38-65 38-65 65 132 65-132 65-132 MTS (–) MTS (+) PC-9 RPC-9 MTS (–) MTS (+) MTS (–) MTS (+) RPC-9 1.4 * 1.2 1.0 M M 0.8 (kDa) GFP WT 38-65 65-132 GFP WT 38-65 65-132 (kDa) GFP WT 38-65 65-132 GFP WT 38-65 65-132 0.6 50 50 0.4 p14ARF p14ARF 0.2 40 40 0.0

variants variants growth cell Relative 30 30 WT WT

50 50 GFP GFP 38-65 38-65

β-Actin β-Actin 65-132 65-132 40 40 MTS (–) MTS (+)

Figure 2. Growth inhibition by mitochondrial and nuclear targeting of the p14ARF mutants. A, the construction of p14ARF wild-type (WT) and deletion mutants (d1, d2, and d3; top). The mitochondrial targeting signal (MTS; MSVLTPLLLRGLTGSARRLPVPRAKIHSL) was fused to the N-terminal of p14ARF and the nuclear localization signal [NLS; 3x(DPKKKRKV)] was fused to the C-terminal of p14ARF. The expression of p14ARF wild-type and deletion mutants in PC-9 cells was assessed by immunoblotting using anti-myc epitope-tag antibody (bottom). B, growth assays of PC-9 (top) and RPC-9 (bottom) cells expressing the MTS-p14ARF mutants (white bar) or p14ARF-NLS mutants (black bar). The cells were incubated for 48 hours and their growth was measured by WST-8 reagent. The ratio of viable cells to nontransfected cells is shown. "Empty" indicates an empty vector containing only the MTS or NLS sequence. Results are presented as means SD (n ¼ 3, P < 0.05). C, schematic representation of the GFP-p14ARF chimeric proteins (top). The GFP-p14ARF mutants without the N-terminal MTS (MTS) or the MTS-GFP-p14ARF mutants containing MTS (MTSþ) were transiently expressed into PC-9 and RPC-9 cells (bottom). Protein levels were assessed by immunoblotting probed with anti-GFP antibody. D, after 48 hours of transfection, cell growth of PC-9 (top) and RPC-9 (bottom) were measured by WST-8 reagent. The ratio of viable cells to nontransfected cell sample is shown. Results were presented as means SD (n ¼ 3, P < 0.05).

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MTS-GFP- MTS-GFP- MTS-GFP- A MTS-GFP vector p14ARF WT p14ARF 38-65 p14ARF 65-132 GFP MitoTracker Figure 3. Effect of the p14ARF mutants on the mitochondrial function of the NSCLC cells. A, RPC-9 cells were transfected with the MTS-GFP-p14ARF mutants for 24 hours. The cells were stained Merge with MitoTracker and visualized by inverted ﬂuorescence microscopy. Nuclei were stained with Hoechst m 33258 dye. Scale bar, 10 m. B, B Gefitinib No drug Gefitinib PC-9 and RPC-9 cells were treated with geﬁtinib for 24 hours. The cells were stained with both anti-HSP60 antibody and JC-1 dye. The dynamics of the mitochondrial

membrane potential were PC-9 PC-9 visualized by ﬂuorescence microscopy. Scale bar, 10 mm. HSP60 JC-1 JC-1 C, cleaved caspase-3 detected by immunoblotting using anticleaved caspase-3 antibody in the geﬁtinib-treated cells 36 hours posttransfection of the MTS-GFP- ARF

p14 mutants. RPC-9 RPC-9

HSP60 JC-1 JC-1

C PC-9 RPC-9 MTS (+) MTS (+)

M (kDa) No drug GFP WT No drug GFP WT 38-65 65-132 38-65 65-132 Gefitinib Gefitinib

15 cleaved Caspase-3

50 β-Actin 40

p14ARF, which can restore the impaired p16, p14, and Restoration of response to gefitinib in the resistant p21 functions (33) or inhibit Akt function (Supplemen- tumor cells using the p14 peptide tary Fig. S6A, S6C, and S6D). Thus, the peptide encoded Using the p14 38-65 peptide, we attempted to restore the by the amino acid position 38-65 of p14ARF was a gefitinib sensitivity in the resistant clones. As shown in the powerful antitumor tool against gefitinib-sensitive and graphs, pretreatment of the resistant cells with a small gefitinib-resistant NSCLC cells, irrespective of the type amount of p14 38-65 peptide (2–10 mmol/L conc.) sensi- of EGFR mutations. tized the response against the same dose of gefitinib

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A Peptide Sequence

r9 rrrrrrrrr p14-1C rrrrrrrrr-GPG-RRFLVTLRIRRACGPPRVRVFVVHIPR

p14 38-65 rrrrrrrrr-GPG-APAAVALVLMLLRSQRLGQQPLPRRPG ( r ; D-Arg)

B PC-9 RPC-9 HCC827 H1975 * * * * 1.2 * 1.2 * 1.2 * 1.2 * 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 Relative cell growth cellRelative Relative cell growth cellRelative growth cellRelative 0.0 growth cellRelative 0.0 0.0 0.0 r9 r9 r9 r9 pep (-) pep (-) pep (-) pep (-) p14-1C p14-1C p14-1C p14-1C p14 38-65 p14 38-65 p14 38-65 p14 38-65

C FITC-r9-p14 38-65 MitoTracker D pep (-) p14-1C p14 38-65 PC-9

JC-1 JC-1 JC-1 RPC-9

JC-1 JC-1 JC-1 FITC-r9- MitoTracker Merge p14 38-65

E PC-9 RPC-9

M M pep (-) r9 p14-1C p14 38-65 (kDa) (kDa) pep (-) r9 p14-1C p14 38-65 40 cleaved 40 cleaved Caspase-9 Caspase-9

30 30 50 50 β-Actin β-Actin 40 40

Figure 4. Growth inhibition by the p14ARF peptides. A, amino acid sequences of the synthesized peptides used for this study. The r9 sequence was fused to all peptides to generate a cell-permeable form. r, D-arginine. B, the indicated cells were treated with 20 mmol/L of each peptide for 48 hours at 37 C. Cell growth in each sample was measured by WST-8 reagent. The ratio of the viable cells to the peptide-untreated cells [pep()] is shown. Results are presented as means SD (n ¼ 3, , P < 0.05). C, the overlaid cellular images of RPC-9 with fluorescein-labeled r9-p14 38-65 peptide (green) and MitoTracker (red) in combination with bright-field viewing (top). Bottom panels show a hyperview of each fluorescent image from the squared region in the top panels. Scale bar, 20 mm. D, PC-9 and RPC-9 cells stained with JC-1 dye for 14-hour treatment with the indicated peptides. Scale bar, 10 mm. E, cleaved caspase-9 in PC-9 (left) and RPC-9 (right) detected by immunoblotting after 24-hour treatment with the indicated peptide (20 mmol/L).

OF8 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

p14ARF Regulation in Geﬁtinib-Treated NSCLC

(1 mmol/L conc.) as in the case of gefitinib-sensitive clones cause the development of secondary EGFR mutations that such as PC-9. For example, treatment with 1 mmol/L of lead to gefitinib resistance. Considering this situation, gefitinib enhanced growth suppression of RPC-9 cells characterization of the cellular and molecular pathways from 40% to 80% when the cells were pretreated with that underlie the development of the resistance is critical 5 mmol/L of the p14 peptide for 12 hours. However, for insight into novel therapeutic countermeasures. This 5 mmol/L of p14 peptide alone was not sufficient to study showed the altered expression of CDK inhibitors as eliminate the tumor cells. Similarly, a 20% increase in the key to characterize the mechanisms mediating gefiti- growth inhibition was obtained by 1 mmol/L of gefitinib nib sensitivity and its resistance in NSCLCs. The CDKN2A treatment in the peptide-pretreated H1975 cells in com- locus is encoded by the alternative splicing variants that parison with the peptide-untreated cells (Fig. 5A and B). serve as tumor suppressor genes, including p16INK4a, Unlike the case of the p14 peptide, pretreatment with the p14ARF, and p12 (17–19). Although little is known about same dose of the p21-S153A peptide did not augment the p12, both p16INK4a and p14ARF play crucial roles in reg- response to gefitinib in these resistant clones. In this case, ulating cell proliferation and their expression is frequently 5 mmol/L of the p14 peptide alone did not seriously impaired by epigenetic causes in biologically aggressive affect the mitochondrial membrane potential, whereas tumors (20). Here we show that the specific induction of ARF the potential was dramatically reduced in these pep- p14 in response to gefitinib was distinct between the tide-pretreated cells in the presence of 1 mmol/L of gefi- gefitinib-sensitive and resistant clones, although they tinib (Fig. 5C). We also found that the p14 38-65 peptide retained its basal expression. In the sensitive NSCLC cells (at 20 mmol/L conc.) has little cytotoxic effect on the represented by PC-9, approximately fourfold enhance- ARF cell growth of the human immortalized lines derived ment of p14 mRNA was observed in response to the ARF ARF from normal cell origins that retain expression of p14 drug, whereas there was no significant change in p14 (Fig. 5D and E). mRNA levels against gefitinib. Furthermore, induction of ARF p14 was gefitinib specific in these tumor cells, because Expression of p14ARF in NSCLC tissues harboring this phenomenon was not detected by treatment with any EGFR mutations other chemotherapeutic agents. Its induction was specif- As a next step, we examined the expression of p14ARF in ically mediated by the EGFR pathway, which was shown human NSCLC tissues to determine whether they by the siRNA-introduced EGFR-knockdown model. reflected the subcellular localization of p14ARF in the We show that mitochondrial accumulation of p14ARF corresponding cell lines in vitro. Double immunofluores- is an important response to gefitinib, although many cence studies using anti-p14ARF antibody and anti-HSP60 previous studies have reported that the inhibitory mech- antibody as mitochondrial markers revealed that lung anism of p14ARF is in the conventional MDM2-p53 path- adenocarcinoma tissues with the deletion of EGFR exon way, triggering cell-cycle arrest and apoptosis. Indeed, 19 (DEx19) or those with the secondary mutation in p14ARF function still remains controversial in various cells T790M in addition to the deletion of EGFR exon 19 (22, 23); however, we corroborated that mitochondrial (DEx19þT790M) expressed colocalized p14ARF with p14ARF efficiently inhibited the growth of NSCLC cells HSP60 (Fig. 6A). Immunohistochemistry with anti-p14ARF and triggered apoptosis through caspase-9 activation, antibody in these cases showed that abundant expression which was more effective than nuclear-translocated of p14ARF was observed in both the nuclei and mitochon- p14ARF. Because we detected a missense mutation in ARF dria of lung cancer cells in the cases carrying the activating p53 in these cells (31), p14 might trigger apoptosis in EGFR mutations (DEx19 or L858R in exon 21), whereas a p53-independent manner. To explain this, we tried to endogenous expression of p14ARF on tumor cells was find a proapoptotic molecule as a specific target of the comparably weaker in the cases with the resistant EGFR augmented p14ARF in the gefitinib-treated NSCLC cells, mutations (T790M and D761Y; Fig. 6B). Expression of the but p14ARF did not alter the expression of the mitochon- p14ARF seemed to be independent of p53 expression in drial proapoptotic proteins Bax, Bad, Bcl-2, and Bcl-xL. these cancerous tissues (Supplementary Fig. S7A and Although Bcl-2–interacting mediator of cell death induc- S7B). In normal bronchiolar epithelial cells and alveolar tion was detected by gefitinib treatment as reported in ARF epithelial cells, p14ARF was expressed in the nucleus with previous studies, it seemed to be p14 independent or without mitochondrial expression and not all of the (data not shown). Thus, the details of the apoptotic epithelial cells expressed it (Fig. 6B). Thus, the expression mechanism via mitochondrial p14ARF still remain to be pattern of endogenous p14ARF in cellular models repre- elucidated. sented by PC-9 and RPC-9 seemed to be coincident to the In this study, the region spanning the 38-65 amino acid genetically corresponding tumor tissues from patients. residues within p14ARF was found to be a functional core for mitochondrial targeting and apoptosis in the NSCLC Discussion cells. Based on this, we generated the novel antitumor Utility of the EGFR tyrosine kinase inhibitor "gefitinib" peptide p14 38-65 as a synthetic cell-permeable form, is restricted to NSCLCs carrying activating EGFR muta- which may enable in vivo application. This peptide con- tions at the present moment. Furthermore, the antagonist tains the amino acids positioned the 42-45 (AVAL) of actions are not durable, because the tumor cells inevitably p14ARF, which was reported to be a putative motif of

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Saito et al.

A RPC-9 RPC-9 1.2 1.2 1 : Gefitinib (-) 1 : Gefitinib (-) : Gefitinib (+) : Gefitinib (+) 0.8 0.8 * *P = 0.002 < 0.05 0.6 0.6 0.4 0.4 * * P = 0.27 > 0.05 Relative cell growth cellRelative 0.2 growth cellRelative 0.2 0 0 012345678910 012345678910 p14 38-65 (μmol/L) p21-S153A (μmol/L)

B H1975 H1975 1.2 1.2 1 : Gefitinib (-) 1 : Gefitinib (-) : Gefitinib (+) : Gefitinib (+) 0.8 0.8 *P = 0.004 < 0.05 0.6 0.6 * 0.4 0.4 * P = 0.57 > 0.05

Relative cell growth cellRelative 0.2 growth cellRelative 0.2 0 0 012345678910 012345678910 p14 38-65 (μmol/L) p21-S153A (μmol/L)

CERPC-9 H1975 D 1.4 * * 1.2 M (bp) MMNK-1 TMNK-1 NHDF 1 400 No treat ARF 300 p14 0.8 JC-1 JC-1 200 400 0.6 300 β-Actin 200 0.4 Relative cell growth cell Relative 0.2 p14 38-65 Gefitinib (-) M 0 JC-1 JC-1 (bp) NuLi-1 PC-9 RPC-9 400 ARF 300 p14 pep (-) pep (-) pep (-) pep (-) pep (-) 200 pep (-) p14 38-65 p14 38-65 p14 38-65 p14 38-65 p14 38-65 400 p14 38-65 300 β-Actin

p14 38-65 200 Gefitinib (+) C-9 P JC-1 JC-1 MNK-1 NHDF NuLi-1 RPC-9 M TMNK-1

Figure 5. Pretreatment of the p14 38-65 peptide restores gefitinib sensitivity in the resistant clones. A, growth inhibition of the RPC-9 cells in response to 1 mmol/L of gefitinib was in a dose-dependent manner of the p14 38-65 peptide used for the pretreatment (left). Growth inhibition of the RPC-9 cells pretreated with the p21-S153A peptide in the same condition (right). B, H1975 cells showed similar response to gefitinib as in the case of RPC-9. Pretreated with the p14 38-65 (left) and pretreated with the p21-S153A (right; 0, 2, 5, and 10 mmol/L conc.). A and B, 12 hours after the peptide introduction, each cell line was incubated with (þ) or without ()1mmol/L gefitinib for 48 hours. Cell viabilities were measured by the MTT assay. The ratio of viable cells to the untreated cells (without peptides and gefitinib) is shown. Shown as means SD (n ¼ 3, P < 0.05). C, RPC-9 and H1975 cells were treated with the same conditions as in A and B for 24 hours. Mitochondrial membrane potential was monitored by JC-1 dye. Scale bar, 10 mm. D, p14 mRNA expression in the immortalized normal human cells (MMNK-1, TMNK-1, NHDF, NuLi-1), PC-9, and RPC-9 cells by RT-PCR. E, MTT assays of the cells treated with 20 mmol/L of the peptide for 48 hours. The ratio of cell growth to nontreated cells [pep(-)] is shown. Shown as means SD (n ¼ 3, , P < 0.05).

OF10 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

p14ARF Regulation in Geﬁtinib-Treated NSCLC

A H&E p14ARF HSP60 + Hoechst p14ARF+ HSP60 Δ Ex19

Figure 6. Expression of p14ARF in NSCLC tissue. A, tumor tissue section analysis by immunohistochemistry. Histology of lung adenocarcinomas shown Δ Ex19 + T790M by hematoxylin and eosin staining m (H&E). Scale bar, 100 m. B ΔEx19 EGFR L858R EGFR L858R EGFR Subcellular localization of p14ARF in each case was assessed by ﬂuorescent coimmunostaining using anti-p14ARF and anti-HSP60 antibodies. NSCLC tissues have the activating mutation and mutation resistant mutation of EGFR as Activating indicated. Nuclear counterstain with Hoechst 33258 was applied. Scale bar, 20 mm. T790M EGFR T790M EGFR D761Y EGFR B, immunohistochemistry detecting p14ARF in the lung adenocarcinoma tissues with the activating mutations (top) and resistant mutations (middle) of EGFR are indicated. Three cases mutation of the NSCLC were used. Nuclei Resistant were stained with hematoxylin. Expression of p14ARF in normal bronchial epithelium and alveolar epithelium adjacent to the tumor lesions (bottom) is shown. Scale bar, 50 mm. C, summary by a schematic representation showing the intracellular dynamics of p14ARF in response to geﬁtinib in

the EGFR-mutated NSCLC cells. Alveolar epithelium Bronchial epithelium Unidentiﬁed steps are shown as "?." C EGF p14 38-65 peptide

EGFR ARF Cytosol p14 Δψ

Gefitinib ? p14ARF Mitochondria ? Caspase-9

ARF p14ARF p14 Caspase-3 Nucleolus CDKN2A locus

Nucleus Apoptosis

mitochondrial localization, although it lacks a mdm2/B23 peptide, and the Akt inhibitory peptide (36) showed binding site (34). This newly designed peptide is powerful growth inhibition to the NSCLC cells to some extent; for antitumor potency because its antiproliferative effect however, the p14 38-65 peptide was superior to them. was much stronger than that of the p14 38-50 peptide Although its precise molecular action is not fully clear, it (fourfold more; Supplementary Fig. S6). Other functional affects the mitochondrial membrane potential and proa- peptides including the p21 S153A peptide (35), the Smac poptotically functions on the NSCLC cells examined.

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Saito et al.

Previous studies have reported that nucleolar expression gefitinib action (Fig. 6C). We identified its functional core of p14ARF in cells lacking p53 affects cell growth by sequence and showed one of the utilities of the designed inhibiting ribosome biogenesis through association with antitumor peptide against gefitinib-resistant NSCLC cells nucleophosmin (37, 38). The p16-MIS peptide that restores in this study. Our study may contribute to the develop- the p16INK4a function comparably inhibited cell growth ment of therapeutics for these intractable cancers. because the tumor cell lines examined bore a missense mutation in p16, but p16 was not the target for gefitinib Disclosure of Potential Conflicts of Interest action as was shown in this study (Supplementary Fig. N. Takigawa and K. Kiura have honoraria from Speakers Bureau of AstraZeneca. No potential conflicts of interest were disclosed by the other S4A and S6; ref. 39). As shown in Fig. 5, one of the utilities authors. of the p14 38-65 peptide may be as a cooperative agent in the treatment of the resistant clones with gefitinib, to Authors' Contributions recover their drug sensitivity up to a level of the sensitive Conception and design: K. Saito, R. Ueda, E. Kondo Development of methodology: K. Saito, E. Kondo NSCLC cells. Acquisition of data (provided animals, acquired and managed patients, Histologic examination of lung adenocarcinoma spe- provided facilities, etc.): K. Saito, N. Takigawa, Y. Tomita, J. Fukuoka, E. cimens from patients with either DEx19 of EGFR or Ichihara, K. Kiura, E. Kondo Analysis and interpretation of data (e.g., statistical analysis, biostatis- L858R in Ex21 of EGFR (both are activating EGFR tics, computational analysis): K. Saito, N. Ohtani, J. Fukuoka, K. Kuwa- mutations) coincidently reflected the expression pattern hara, E. Kondo ARF Writing, review, and/or revision of the manuscript: K. Saito, N. Takigawa, of endogenous p14 and the subcellular localization N. Ohtani, R. Ueda, J. Fukuoka, E. Kondo that were observed in the in vitro studies. Expression of Administrative, technical, or material support (i.e., reporting or orga- p14ARF seemed weaker in the cases with either the nizing data, constructing databases): K. Saito, H. Iioka, K. Kiura Study supervision: K. Kiura, E. Kondo T790M or D761Y resistance mutation. Furthermore, the mutant p53 seemed to be independently expressed Acknowledgments ARF from p14 in all the cases examined (Supplementary The authors thank the members of the Kondo Lab for suggestions about Fig. S7). experimental design, for critical evaluation of this manuscript, and for technical assistance. In summary, here we show that p14ARF is induced by gefitinib through inactivation of the EGFR signaling path- Grant Support EGFR way in the -mutated NSCLC cells and it is one of the E. Kondo was supported by a Grant-in-Aid for Scientific Research essential molecules for the drug action. Moreover, mito- (Kiban-C; grant no. 23590442; E. Kondo) from the Japanese Ministry of ARF Education, Culture, Sports, Science, and Technology (MEXT). chondrial localization is important for p14 to exert The costs of publication of this article were defrayed in part by the efficient cell growth suppression, which seems to function payment of page charges. This article must therefore be hereby marked in a p53-independent manner. The mechanism of p14ARF advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. induction by gefitinib and the detailed interaction of ARF p14 at mitochondria still remain unclear; however, Received December 26, 2012; revised June 3, 2013; accepted June 3, 2013; mitochondrial p14ARF is one of the critical regulators of published OnlineFirst June 12, 2013.

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www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 OF13

Antitumor Impact of p14ARF on Gefitinib-Resistant Non− Small Cell Lung Cancers

Ken Saito, Nagio Takigawa, Naoko Ohtani, et al.

Mol Cancer Ther Published OnlineFirst June 12, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-12-1239

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