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Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target a
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Jing Li , Sufang Han , Ziliang Qian , Xinying Su , Shuqiong Fan , Jiangang Fu , Yuanjie Liu , a
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Xiaolu Yin , Zeren Gao , Jingchuan Zhang , De-Hua Yu & Qunsheng Ji a
Innovation Center China; Asia & Emerging Market iMed; AstraZeneca Innovation Medicines and Early Development; Shanghai, PR China Published online: 19 Nov 2013.
Click for updates To cite this article: Jing Li, Sufang Han, Ziliang Qian, Xinying Su, Shuqiong Fan, Jiangang Fu, Yuanjie Liu, Xiaolu Yin, Zeren Gao, Jingchuan Zhang, De-Hua Yu & Qunsheng Ji (2014) Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target, Cancer Biology & Therapy, 15:1, 128-134, DOI: 10.4161/cbt.27146 To link to this article: http://dx.doi.org/10.4161/cbt.27146
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Research Paper Research Paper
Cancer Biology & Therapy 15:1, 128–134; January 2014; © 2014 Landes Bioscience
Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target Jing Li, Sufang Han, Ziliang Qian, Xinying Su, Shuqiong Fan, Jiangang Fu, Yuanjie Liu, Xiaolu Yin, Zeren Gao, Jingchuan Zhang, De-Hua Yu*, and Qunsheng Ji*
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Keywords: gastric cancer, lung cancer, PPME1 amplification, PP2A, shRNA-knockdown
Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.
Introduction Despite the significant progress that has been made regarding diagnosis and the clinical application of targeted therapies for subsets of patients with EGFR activation mutations and ALK gene rearrangements, lung cancer (LC), specifically non-small cell lung cancer (NSCLC), is still the leading cause of cancerrelated deaths worldwide.1 Surgical resection is the primary choice of treatment for LC, followed by radiation, chemotherapy, and/or targeted therapies.2 Metastatic and locally advanced disease, however, is not amenable to surgical resection, and the majority of patients who undergo surgery eventually relapse.3-5 The poor response and survival rates associated with the current treatment options clearly underscore the pressing need for the identification of new genetic lesions for the development of more effective therapies. Globally, gastric cancer (GC) is the fourth most common cancer diagnosed in men and the fifth most common cancer in women. In 2011, there were an estimated 640 000 cases in men and 350 000 cases in women, as well as 464 000 male deaths and 273 000 female deaths,6 accounting for approximately 8% and 10%, respectively, of annual cancer deaths worldwide. This translates into a high fatality-to-case ratio of 70%, which is
significantly higher than many other cancer indications, such as prostate and breast cancers, which have a fatality-to-case ratio of 30% and 33%, respectively. To date, only a combination regimen of trastuzumab, an HER2 neutralization antibody, and chemotherapy has successfully improved the survival of HER2-positive GC patients.7 Therefore, better management of this disease, particularly through the use of new targeted therapeutic agents, is sorely needed. Although the detailed molecular mechanisms for tumor development have not been fully characterized,8 genetic aberrations, including gene amplifications, mutations, or translocations, have been shown to be critical in carcinogenesis and tumor progression9 and are valid targets for cancer treatment.10-12 A key challenge that remains, however, is how to distinguish these so-called “driver” oncogenes from other co-existing “passenger” genes.13 In order to facilitate the identification and validation of “driver” genes, patient-derived, specimen-based molecular analysis and short-hairpin RNA (shRNA) knockdown techniques have been widely employed.14,15 PPME1 is a protein phosphatase 2A (PP2A)-specific methylesterase that mediates the demethylation and inactivation of PP2A. The reversible methylation of PP2A occurs at the carboxyl group of the carboxy terminal leucine 309 residue of PP2A (PP2Ac Leu
*Correspondence to: De-Hua Yu, Email: [email protected]; Qunsheng Ji, Email: [email protected] Submitted: 10/10/2013; Revised: 10/22/2013; Accepted: 11/10/2013 http://dx.doi.org/10.4161/cbt.27146 128
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309) and is catalyzed by an S-adenosylmethionine-dependent leucine carboxyl methytransferase and PPME1.16,17 Several lines of evidence show that this methylation is essential for the binding of the B55 subunit to the A/C heterodimer of PP2A.18 A predicted outcome of the B55 subunit association is the targeting of PP2A to different protein substrates in cells, such as AKT, AP-1/AP-2, HDAC4, KSR, P53, Src, Raf1, TGFBR1, TSC2, and Vimentin.19,20 Increased PPME1 expression has been found to be correlated with malignant progression and Erk pathway activity in human astrocytic glioma patient samples when compared with PPME1-negative tumors. Moreover, PPME1 immuno-positivity has been strongly correlated with an increased K i-67 proliferation index in tumor samples.21 In vitro, PPME1 depletion results in a significant reduction in the proliferation and reduced phosphorylation of Erk and its downstream target, Elk-1, which is a transcription factor that has been implicated in the proliferation of the U118MG and T98G glioblastoma cell lines.21 However, these data do not exclude the possibility that PPME1 partially contributes to malignant cell growth by regulating other PP2A target pathways in addition to its role in supporting Erk activity. In the current study, we have profiled the gene copy number changes in primary tumor samples from Chinese GC and LC patients and have identified PPME1 gene amplification in a subset of the tested samples. Further function studies have indicated that it is a potential therapeutic target.
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Results PPME1 is genetically amplified in GC and LC patient tumor samples and correlates with PPME1 protein expression Patient tumor samples for the array CGH analysis were provided by the Beijing Cancer Hospital and Guangdong General Hospital. Eligible samples (a tumor content of 70% or more) were collected from 131 Chinese GC patients and 124 Chinese LC patients. The median age of the GC patients was 62 y (ranging from 34 to 81 y) and the majority were male (n = 94; 71%). Twenty adjacent samples were used as control tissues. Amplification of the PPME1 gene was identified in 5 (3.8%) of the 131 GC samples analyzed (Fig. 1A). The median age of the LC patients was 61 y (ranging from 29 to 78 y) and the majority were male (n = 94; 72%). Twenty-three adjacent samples were used as control tissues. Amplification of the PPME1 gene was identified in 4 (3.1%) of the 124 LC samples analyzed. To confirm the observation of PPME1 amplification, 109 Chinese GC patient samples provided by the Shanghai Renji Hospital (median age 62 y [ranging from 18 to 86 y]; mainly males, n = 75 [69%]) and 41 Chinese LC patient samples provided by the Shanghai Chest Hospital (median age 60 y [ranging from 32 to 74 y]; mainly males, n = 33 [83%]) were screened using FISH analysis. PPME1 amplification was identified in 7 (6.4%) of the 109 samples from Chinese GC patients and 3 (7.3%) of the
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Figure 1. PPME1 gene amplification and protein expression levels in Chinese gastric cancer (GC) and lung cancer (LC) patient samples. (A) PPME1 gene amplification in a Chinese GC tumor detected by array comparative genomic hybridization. (B) PPME1 amplification and protein overexpression in GC and LC samples detected by FISH and immunohistochemistry (IHC) assays, respectively. PPME1 IHC score and PPME1 amplification status are embedded within the images (“AMP” denotes the gene amplified). For the fluorescence in situ hybridization analysis (FISH) images, the PPME1 gene probe signals appear red, CEP11 signals are green, and DAPI-counterstained nuclei appear blue. Scale bars represent 50 nm for H&E/IHC images and 30 nm for the FISH images. All images within each row are on the same scale.
41 samples from Chinese LC patients. Representative PPME1amplified FISH images are shown in Figure 1A. Preliminary results obtained using IHC analysis showed that PPME1 was overexpressed at the protein level in the FISH-amplified cases when compared with normal (Fig. 1B). shRNA knockdown of PPME1 expression in PPME1amplified GC and LC cells results in growth inhibition and apoptotic induction To understand the role of PPME1 amplification and expression in tumorigenesis, shRNA technology was used to deplete PPME1 expression in the cells. The PPME1-amplified human GC SNU668 and LC OKa-C-1 cells and nonamplified control cells were identified for use in a functional study. As shown in Figure 2, shRNA-mediated knockdown of PPME1 resulted in growth inhibition of 48.6% and 41.3% compared with scrambled shRNA in SNU668 and OKa-C-1 cells by Day 4 after transfection. In contrast, an equivalent knockdown of PPME1 showed no significant effects on the cell growth and survival of the PPME1
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nonamplified human MKN1 and HCC95 cancer cells (Fig. 2A), supporting an oncogenic role for PPME1 in GC and LC. We also explored the effect of the PPME1 knockdown on apoptotic induction in PPME1-amplified and nonamplified cancer cells. Cellular introduction of PPME1 shRNA for 96 h increased caspase 3/7 activity (a hallmark of apoptosis) in the amplified SNU668 and OKa-C-1 cells, but not in the nonamplified MKN1 and HCC95 cells (Fig. 2B). These results indicate that activation of caspase 3/7 following shRNA-induced PPME1 knockdown is a specific event in PPME1-amplified cells and that functional PPME1 is required for survival signaling in this cellular background. PPME1 regulates PP2Ac methylation and pAKT/pErK in PPME1-amplified cancer cells. Modulation of PP2A signaling by PPME1 knockdown To further ascertain the functional role of PPME1 in regulating PP2Ac Leu 309 methylation and the phosphorylation/activity of PP2A downstream targets, we analyzed the demethylation status of PP2Ac and the phosphorylation of two different PP2A
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Figure 2. Cell growth inhibition and apoptosis induction by a short-hair (sh)RNA-mediated knockdown of PPME1. (A) Characterization of two PPME1amplified cancer cell lines (SNU668 and Oka-C-1) and two PPME1 nonamplified cancer cell lines (MKN1 and HCC95) using western blot and fluorescence in situ hybridization analysis. (B) Cells were cultured for 72 h after PPME1 shRNA transfection, and cell viability was analyzed by Acumen assay. Graphs indicate the level of cell survival in the PPME1-amplified cancer cell lines and nonamplified cancer cell lines; PPME1 protein expression suppression by shRNA was analyzed by western blot analysis. (C) Cells were incubated with PPME1 shRNA at the indicated concentrations for 72 h and caspase 3/7 activity was then measured by ELISA assay. The results are representative from three independent experiments.
target proteins in both PPME1-amplified and nonamplified cells transfected with either scrambled or PPME1-specific shRNA. For this purpose, we used an antibody that specifically recognizes the demethylated form of PP2Ac Leu 309. We found that the PPME1 depletion resulted in a decrease of PP2Ac Leu 309 methylation in the PPME1-amplified cancer cell lines (data not shown). PP2Ac demethylation was not altered in the parental untreated cells and scrambled shRNA-transfected cells or in the PPME1 nonamplified cancer cells (MKN1 and HCC95). PPME1 depletion was also found to inhibit Erk and AKT phosphorylation in PPME1amplified cells without any notable effects on the nonamplified cells (MKN1 and HCC95) (Fig. 3). These results suggest that in the PPME1-amplified cells, PP2A methylation and MAPK/ AKT phosphorylation are relevant downstream signaling targets of PPME1 that appear to be necessary for maintenance of the oncogenic state.
Discussion PP2A is a human tumor suppressor that accounts for the majority of cellular serine/threonine phosphatase activity.22 Inactivating alterations of the PP2A subunits have been found in multiple human cancers.23 The PP2A core enzyme consists of a catalytic “C” subunit, a structural “A” subunit, and a “B” subunit that binds subunits “A” and “C” together as a heterodimer.24 The “B” subunits are subdivided into four distinct families that share no sequence similarities.25 The combinatorial assembly of these various “A”, “B”, and “C” subunits permits the formation of many distinct PP2A complexes, and various PP2A complexes have been implicated in the control of a diverse array of cellular processes, including cell proliferation, survival, adhesion, and cytoskeleton dynamics.26 The highly conserved “C” terminal tail of PP2Ac has been confirmed to play a key role in malignancy and can undergo two types of posttranslational modification: phosphorylation and methylation.27 Phosphorylation of Tyr307 in PP2Ac
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by several tyrosine-specific kinases or of an unidentified threonine residue by an auto-activated kinase results in inactivation of PP2A activity.28 PPME1 has been found to suppress PP2A activity, at least in part, by demethylating PP2Ac and modulating the binding interaction of the “C” subunit with regulatory “B” subunits. Recent structural studies have shed light on the physical interactions between PPME1 and the PP2A holo-enzyme. RNA-interference-mediated knockdown of PPME1 in PPME1overexpressing cancer cells leads to activation of PP2A and the corresponding suppression of protumorigenic phosphorylation cascades,21 suggesting a potential oncogenic role of PPME1 in glioma. In this study, through the use of array-CGH and IHC expression analysis, we have found for the first time that PPME1 amplification in Chinese GC and LC patients is correlated with protein overexpression. The role of this gene was explored by downregulating PPME1 expression in a cell culture. The evidence suggests that PPME1 represents a bona fide oncogene, as loss of PPME1 protein expression results in growth inhibition, and a significant induction of cell apoptosis in the PPME1-amplified GC and LC cell lines, but it has little effect on non-amplified cells. The Erk and AKT pathways represent key oncogenic mediators that are often dysregulated by genetic abnormalities in cancer as a consequence of their overarching control of many proteins critical for cell growth and metastasis. However, the mechanisms that sustain Erk and AKT pathway activity in malignant cells remain elusive. Our data show that a PPME1 depletion results in decreased demethylation of PP2Ac Leu309 and dephosphorylation of Erk and AKT in PPME1-amplified cells. The effect of PPME1 depletion on Erk activity in GC and LC cells is consistent with a previous report from Puustinen et al., who described a connection between Erk activation and PPME1 overexpression in astrocytic glioma.21 The involvement of the PP2A signaling pathway in AKT activation was initially reported by Chen et al. in SV40 small t antigen transformed cells.29 During the preparation
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Figure 3. Downregulation of AKT and Erk signaling by short-hairpin RNA-mediated knockdown of PPME1 expression. Cells treated with shRNA against PPME1 were subjected to western blot analysis for demethylation of PP2Ac and phosphorylation of AKT and Erk in PPME1. The results are representative from three independent experiments.
Materials and Methods Cell culture and reagents The following antibodies were purchased from Cell Signaling Technology (CST): total-PP2Ac, phospho-p44/p42 MAP kinase (Erk1/2), total-Erk, phospho-AKT (Ser473), total-AKT, cleaved caspase-3, poly-(ADP-ribose) polymerase (PARP), horseradish
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peroxidase (HRP)-linked anti-rabbit IgG, and HRP-linked antimouse IgG. Other antibodies used included PPME1 (GenWay Biothech) and demethylated-PP2A-C (Santa Cruz). An antibody that binds to GAPDH (CST) was used as a control. SNU-668 and HCC-95 cells were obtained from the Korea Cell Line Bank (KCLB), and OKa-C-1 and MKN1 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. SNU-668, MKN1, OKa-C-1, and HCC-95 cells were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). All cell lines were genetically tested and authenticated using the StemElite IDTM System Kit (Promega, G9530) and were not cultured for more than 3 mo prior to performing the work described here. All studies using human tissue were performed with the patients’ consent and the approval of the Local Research Ethics Committee. Array comparative genomic hybridization analysis of the GC and LC patient samples The PPME1 gene copy number was analyzed in frozen patient tissue samples using the Agilent 244K array comparative genomic hybridization (aCGH) platform. The quality of the raw data was checked with the Agilent CGH Analytics software, using the derivative of the log ratio spread (DLRSpread) as a surrogate for assay quality. Any sample with a DLRSpread of more than 0.3 was excluded from further analysis. Data for the samples that qualified for further assessment were subsequently analyzed using Nexus software (version 4) for the recovery of PPME1 segmental structure (segmentation) and the discrete copy number value at the single-sample level (calling). Segments with an array CGH logRatio (copy number of sample vs. the control) of more than 0.8 were classified as amplified. Fluorescence in situ hybridization analysis The PPME1 fluorescence in situ hybridization analysis (FISH) probe was generated internally by directly labeling the BAC (CTD-2128J17) DNA with Spectrum Red (Vysis, 30-803400). A CEP11 Spectrum Green probe (Vysis, 32-132011) for the centromeric region of chromosome 8 was used as an internal control. FISH assays were performed on four micron dewaxed and dehydrated formalin-fixed, paraffin-embedded (FFPE) sections. The SpotLight Tissue Pretreatment Kit (Invitrogen, 00-8401) was used for pretreatment (boiled in reagent 1 for approximately 15 min and then coated with reagent 2 for approximately 10 min; minor time adjustments were made for the individual samples). Sections and probes were codenaturated at 80 °C for 5 min and then hybridized at 37 °C for 48 h. After a quick washoff process (0.3% NP40; 1 × SSC at 75.5 °C for 5 min and twice in 2 × SSC at room temperature for 2 min), the sections were mounted with 0.3 μg/ml DAPI (Vector, H-1200) and stored at 4 °C away from light for at least 30 min prior to scoring. The target gene and CEP signals were observed using a fluorescence microscope equipped with the appropriate filters, allowing visualization of the intense red target gene signals, the intense green chromosome centromere signals, and the blue counterstained nuclei. Enumeration of the PPME1 gene and chromosome 11 was conducted by microscopic examination of 50 tumor nuclei,
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of this manuscript, Jackson et al. reported that exogenous overexpression of PPME1 leads to increased T308 phosphorylation on AKT in HEKTER-ASB56 g cells,30 which supports our observation that PPME1 plays a role in AKT activation in GC and LC cells. Although previous studies by Puustinen et al. have reported a promoting effect of PPME1 on the proliferation of glioma cells in vitro,21 no PPME1-based disease linkage in cancer has been reported to our knowledge. The differential phenotypes observed by shRNA-mediated PPME1 knockdown in PPME1-amplified vs. non-amplified cell lines support an oncogenic driving role of PPME1 amplification and overexpression in GC and LC. The amplification and overexpression of PPME1 observed in subsets of patients with GC and LC suggests its potential as a therapeutic drug target. This notion is further supported by the reduced PPME1 expression in normal tissues except for human adults’ brain, which may theoretically leave open a sufficient therapeutic window for targeting PPME1 in patients with a PPME1 amplification.31 Currently, two groups have demonstrated the feasibility of developing PPME1 selective small molecular inhibitors.32,33 The cell lines with PPME1 amplification described in this study will facilitate the development of PPME1 inhibitors. As the first targeted therapy for GC, trastuzumab has demonstrated efficacy in about 20% of GC patients with HER2 amplification or overexpression.34,35 Clinically, the FISH and IHC assays were approved the selection of patients with HER2 amplification and overexpression, which is based on HER2 FISH positivity or an IHC score of 3+ (a score of 2+ needs further FISH confirmation). Although we used the same criteria for HER2 to define PPME1 FISH positivity, it is still unclear whether they can be directly used for patient selection in the future. Further studies using the PPME1 selective inhibitor to determine a correlation between FISH and IHC scores and antitumor efficacy in preclinical models or in patients will help to define the use of PPME1 positivity for patient selection. Although the FISH assay is more clinically relevant than aCGH assay for patient selection, due to the non-availability of samples, the tumor samples used for FISH and aCGH analysis were collected from different cohorts, which might affect the higher amplification rates detected by FISH assay compared with aCGH (6.4% vs. 3.8% in GC and 7.3% vs. 3.1% in LC respectively). Further studies on the same cohort of samples will help to address this issue. In conclusion, we have identified PPME1 amplification, which is correlated with protein overexpression and has the potential to define new molecular segments in GC and LC. Our results suggest that the amplification of PPME1 is an oncogenic driver for the development of novel therapeutic approaches in GC and LC.
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blot assay. The viable cells were dyed with Hoechst (Invitrogen, H21486) and incubated at 37 °C for 1 h. Then the plates were determined using Acumen eX3 (TTP LabTech). In vitro pharmacodynamics study Cells were lysed in 1× sodium dodecyl sulfate (SDS) buffer 50 mM TRIS-HCl (pH 6.8; 100 mM dithiothreitol, 2% SDS [electrophoresis grade], and 10% glycerol) containing NaVo3 (Sigma), a complete protease inhibitor cocktail (Sigma), the complete phosphatase inhibitor cocktail 1 (Sigma), and the complete phosphatase inhibitor cocktail 2 (Sigma). Soluble proteins were quantified using a Pierce BCA™ Protein Assay Kit (Pierce, 23225) and separated on NuPage 4–12% Bis-tris gels (Invitrogen) in a 1× MES SDS running buffer (Invitrogen) for 2.5 to 3 h at 110 V. They were then electro-transferred to nitrocellulose polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc.) in a 1× NuPage transfer buffer (Invitrogen) containing 20% ethanol for 75 min at 340 mA. After blocking the membranes in a 5% fat-free milk solution (dissolved in the 1× TBST buffer, 1× Trisbuffered saline containing 0.1% [v/v] tween20), they were probed with the anti-PPME1 antibody (clone 4A12, GenWay Biothech, 20-614-460633), anti-demethylated-PP2A-C (sc-80990), antiPP2Ac (2259, Cell Signaling), anti-phospho-p44/42 MAPK (4370, Cell Signaling), phospho-Akt (Ser473) (M3628, Dako), and anti-GAPDH (2118, Cell Signaling). Antibodies were diluted 1:2000 for PPME1; 1:1000 for PP2Ac, MAPK, and P-AKT; 1:200 for anti-demethylated-PP2A-C; and 1:10 000 for GAPDH in TBST (50 mM Tris Base, 150 mM NaCl, 0.1% [v/v] Tween-20) containing 5% milk powder. After washing the membrane in TBST, it was incubated with the appropriate HRPconjugated secondary antibodies diluted 1:2000 in TBST. The immuno-reactive signals were detected using an ECL reagent Advance Western Blotting Detection Kit (GE Healthcare) and an ImageQuant LAS 4000 (GE Healthcare). ApoTox-Glo™ Triplex Assay The ability to induce Caspase 3/7 activation after exposure to shRNA was measured by ApoTox-Glo™ Triplex Assay (Promega, G6321) according to the manufacturer’s instruction. The cells were added to the shRNA/INTERFER in mixture, and at the end of incubation, 100 μl of the Caspase-Glo® 3/7 reagent was added to all wells and briefly mixed by orbital shaker (300 to 500 rpm for approximately 30 s). After incubation for at least 30 min at room temperature, the resultant luminescent light was measured in a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, LLC). Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We would like to thank Dr Paul Gavine for reviewing this manuscript.
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which yielded a ratio of PPME1 to CEP11. Tumors with a ratio of 2 or greater or a presence of at least 10% of a gene cluster were defined as amplification. Immunohistochemistry The primary antibody used for PPME1 immunohistochemical (ICH) staining was a commercially available mouse monoclonal antibody (clone 2C5, Genway Biotech). Sections (4-mm thick) were cut from FFPE blocks and mounted on silanized slides. For PPME1 staining, the sections were deparaffinized in xylene and dehydrated with graded ethanol. After washing them with distilled water, the sections were placed in the supplied buffer. For antigen retrieval, the slides were heated at 110 °C for 5 min and then cooled for at least 20 min at room temperature with running deionized water. After washing them a with Trisbuffered 0.9% NaCl solution containing Tween 20 (pH 7.6), the tissue sections were covered for 5 min with a peroxidaseblocking reagent (Dako) to block endogenous peroxidase, followed by an additional washing with the supplied buffer. After peroxidase blocking for 5 min, the sections were incubated with a primary antibody for 60 min at room temperature. They were then labeled with an HRP-labeled polymer (Envision kit, Dako) for 30 min at room temperature and reacted with a diaminobenzidene tetrahydrochloride solution. PPME1 immunopositivity was scored as follows: Reactivity was scored as zero when there were no interpretable signals within the tumor, and it was scored as positive when reactivity of the tumor cell was detected above the background level. The positive samples were classified further as 1+, 2+, and 3+ based on their reactivity intensity. The tissue replicate from each tumor with the highest intensity was used as the final IHC result for that tumor. Scores of 2+ and 3+ were classified as overexpression. Lentiviral-shRNA-mediated PPME1 gene knockdown in cell culture The pGCSIL-GFP (Genechem) lentiviral vector was used to express a short-hairpin RNA for PPME1 silencing. The targeted sequences for PPME1 were PSC5018: GATACATCTG AGTTCAAAT and PSC5020: GTACAGCTAT GGATGCACTT A; the scrambled control sequence was PSC3945: TTCTCCGAAC GTGTCACGT. Lentivirus stocks were produced by a transient transfection of HEK293T cells with the encapsidation plasmid, the pVSV-G plasmid, and the lentiviral recombinant vector as previously described.36 Forty-eight hours after the transfection, the lentiviral supernatants were harvested and centrifuged briefly (500 × g for 10 min) to remove cellular debris. The viruses were frozen at –80 °C until use. The titer of each virus stock was determined by real-time PCR using the SBI Ultra Rapid Lentiviral Titer Kit. The target cells were infected with lentivirus-expressing PPME1 shRNA and nontargeted shRNA at a suitable MOI supplemented with 5 ug/ml polybrene. After infection for 96 h, the PPME1 protein expression was measured in cells infected with the lentivirus by western
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24. Zhou J, Pham HT, Walter G. The formation and activity of PP2A holoenzymes do not depend on the isoform of the catalytic subunit. J Biol Chem 2003; 278:8617-22; PMID:12506124; http://dx.doi. org/10.1074/jbc.M211181200 25. Li X, Virshup DM. Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A. Eur J Biochem 2002; 269:546-52; PMID:11856313; http://dx.doi. org/10.1046/j.0014-2956.2001.02680.x 26. Bononi A, Agnoletto C, De Marchi E, Marchi S, Patergnani S, Bonora M, Giorgi C, Missiroli S, Poletti F, Rimessi A, et al. Protein kinases and phosphatases in the control of cell fate. Enzyme Res 2011; 2011:329098; PMID:21904669; http://dx.doi. org/10.4061/2011/329098 27. McCaddon A, Hudson PR. Methylation and phosphorylation: a tangled relationship? Clin Chem 2007; 53:999-1000; PMID:17517583; http://dx.doi. org/10.1373/clinchem.2007.086579 28. Chen J, Martin BL, Brautigan DL. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 1992; 257:12614; PMID:1325671; http://dx.doi.org/10.1126/ science.1325671 29. Chen W, Arroyo JD, Timmons JC, Possemato R, Hahn WC. Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity. Cancer Res 2005; 65:8183-92; PMID:16166293; http://dx.doi.org/10.1158/00085472.CAN-05-1103 30. Jackson JB, Pallas DC. Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner. Neoplasia 2012; 14:585-99; PMID:22904676 31. Ortega-Gutiérrez S, Leung D, Ficarro S, Peters EC, Cravatt BF. Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS One 2008; 3:e2486; PMID:18596935; http://dx.doi.org/10.1371/journal. pone.0002486 32. Xing Y, Li Z, Chen Y, Stock JB, Jeffrey PD, Shi Y. Structural mechanism of demethylation and inactivation of protein phosphatase 2A. Cell 2008; 133:15463; PMID:18394995; http://dx.doi.org/10.1016/j. cell.2008.02.041 33. Bachovchin DA, Mohr JT, Speers AE, Wang C, Berlin JM, Spicer TP, Fernandez-Vega V, Chase P, Hodder PS, Schürer SC, et al. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors. Proc Natl Acad Sci U S A 2011; 108:6811-6; PMID:21398589; http://dx.doi.org/10.1073/pnas.1015248108 34. Hechtman JF, Polydorides AD. HER2/neu gene amplification and protein overexpression in gastric and gastroesophageal junction adenocarcinoma: a review of histopathology, diagnostic testing, and clinical implications. Arch Pathol Lab Med 2012; 136:691-7; PMID:22646280; http://dx.doi. org/10.5858/arpa.2011-0168-RS 35. Gunturu KS, Woo Y, Beaubier N, Remotti HE, Saif MW. Gastric cancer and trastuzumab: first biologic therapy in gastric cancer. Ther Adv Med Oncol 2013; 5:143-51; PMID:23450234; http://dx.doi. org/10.1177/1758834012469429 36. Dropulić B. Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Hum Gene Ther 2011; 22:649-57; PMID:21486177; http://dx.doi.org/10.1089/hum.2011.058
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References
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Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target a
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Jing Li , Sufang Han , Ziliang Qian , Xinying Su , Shuqiong Fan , Jiangang Fu , Yuanjie Liu , a
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Xiaolu Yin , Zeren Gao , Jingchuan Zhang , De-Hua Yu & Qunsheng Ji a
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Click for updates To cite this article: Jing Li, Sufang Han, Ziliang Qian, Xinying Su, Shuqiong Fan, Jiangang Fu, Yuanjie Liu, Xiaolu Yin, Zeren Gao, Jingchuan Zhang, De-Hua Yu & Qunsheng Ji (2014) Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target, Cancer Biology & Therapy, 15:1, 128-134, DOI: 10.4161/cbt.27146 To link to this article: http://dx.doi.org/10.4161/cbt.27146
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Research Paper Research Paper
Cancer Biology & Therapy 15:1, 128–134; January 2014; © 2014 Landes Bioscience
Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target Jing Li, Sufang Han, Ziliang Qian, Xinying Su, Shuqiong Fan, Jiangang Fu, Yuanjie Liu, Xiaolu Yin, Zeren Gao, Jingchuan Zhang, De-Hua Yu*, and Qunsheng Ji*
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Keywords: gastric cancer, lung cancer, PPME1 amplification, PP2A, shRNA-knockdown
Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.
Introduction Despite the significant progress that has been made regarding diagnosis and the clinical application of targeted therapies for subsets of patients with EGFR activation mutations and ALK gene rearrangements, lung cancer (LC), specifically non-small cell lung cancer (NSCLC), is still the leading cause of cancerrelated deaths worldwide.1 Surgical resection is the primary choice of treatment for LC, followed by radiation, chemotherapy, and/or targeted therapies.2 Metastatic and locally advanced disease, however, is not amenable to surgical resection, and the majority of patients who undergo surgery eventually relapse.3-5 The poor response and survival rates associated with the current treatment options clearly underscore the pressing need for the identification of new genetic lesions for the development of more effective therapies. Globally, gastric cancer (GC) is the fourth most common cancer diagnosed in men and the fifth most common cancer in women. In 2011, there were an estimated 640 000 cases in men and 350 000 cases in women, as well as 464 000 male deaths and 273 000 female deaths,6 accounting for approximately 8% and 10%, respectively, of annual cancer deaths worldwide. This translates into a high fatality-to-case ratio of 70%, which is
significantly higher than many other cancer indications, such as prostate and breast cancers, which have a fatality-to-case ratio of 30% and 33%, respectively. To date, only a combination regimen of trastuzumab, an HER2 neutralization antibody, and chemotherapy has successfully improved the survival of HER2-positive GC patients.7 Therefore, better management of this disease, particularly through the use of new targeted therapeutic agents, is sorely needed. Although the detailed molecular mechanisms for tumor development have not been fully characterized,8 genetic aberrations, including gene amplifications, mutations, or translocations, have been shown to be critical in carcinogenesis and tumor progression9 and are valid targets for cancer treatment.10-12 A key challenge that remains, however, is how to distinguish these so-called “driver” oncogenes from other co-existing “passenger” genes.13 In order to facilitate the identification and validation of “driver” genes, patient-derived, specimen-based molecular analysis and short-hairpin RNA (shRNA) knockdown techniques have been widely employed.14,15 PPME1 is a protein phosphatase 2A (PP2A)-specific methylesterase that mediates the demethylation and inactivation of PP2A. The reversible methylation of PP2A occurs at the carboxyl group of the carboxy terminal leucine 309 residue of PP2A (PP2Ac Leu
*Correspondence to: De-Hua Yu, Email: [email protected]; Qunsheng Ji, Email: [email protected] Submitted: 10/10/2013; Revised: 10/22/2013; Accepted: 11/10/2013 http://dx.doi.org/10.4161/cbt.27146 128
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309) and is catalyzed by an S-adenosylmethionine-dependent leucine carboxyl methytransferase and PPME1.16,17 Several lines of evidence show that this methylation is essential for the binding of the B55 subunit to the A/C heterodimer of PP2A.18 A predicted outcome of the B55 subunit association is the targeting of PP2A to different protein substrates in cells, such as AKT, AP-1/AP-2, HDAC4, KSR, P53, Src, Raf1, TGFBR1, TSC2, and Vimentin.19,20 Increased PPME1 expression has been found to be correlated with malignant progression and Erk pathway activity in human astrocytic glioma patient samples when compared with PPME1-negative tumors. Moreover, PPME1 immuno-positivity has been strongly correlated with an increased K i-67 proliferation index in tumor samples.21 In vitro, PPME1 depletion results in a significant reduction in the proliferation and reduced phosphorylation of Erk and its downstream target, Elk-1, which is a transcription factor that has been implicated in the proliferation of the U118MG and T98G glioblastoma cell lines.21 However, these data do not exclude the possibility that PPME1 partially contributes to malignant cell growth by regulating other PP2A target pathways in addition to its role in supporting Erk activity. In the current study, we have profiled the gene copy number changes in primary tumor samples from Chinese GC and LC patients and have identified PPME1 gene amplification in a subset of the tested samples. Further function studies have indicated that it is a potential therapeutic target.
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Results PPME1 is genetically amplified in GC and LC patient tumor samples and correlates with PPME1 protein expression Patient tumor samples for the array CGH analysis were provided by the Beijing Cancer Hospital and Guangdong General Hospital. Eligible samples (a tumor content of 70% or more) were collected from 131 Chinese GC patients and 124 Chinese LC patients. The median age of the GC patients was 62 y (ranging from 34 to 81 y) and the majority were male (n = 94; 71%). Twenty adjacent samples were used as control tissues. Amplification of the PPME1 gene was identified in 5 (3.8%) of the 131 GC samples analyzed (Fig. 1A). The median age of the LC patients was 61 y (ranging from 29 to 78 y) and the majority were male (n = 94; 72%). Twenty-three adjacent samples were used as control tissues. Amplification of the PPME1 gene was identified in 4 (3.1%) of the 124 LC samples analyzed. To confirm the observation of PPME1 amplification, 109 Chinese GC patient samples provided by the Shanghai Renji Hospital (median age 62 y [ranging from 18 to 86 y]; mainly males, n = 75 [69%]) and 41 Chinese LC patient samples provided by the Shanghai Chest Hospital (median age 60 y [ranging from 32 to 74 y]; mainly males, n = 33 [83%]) were screened using FISH analysis. PPME1 amplification was identified in 7 (6.4%) of the 109 samples from Chinese GC patients and 3 (7.3%) of the
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Figure 1. PPME1 gene amplification and protein expression levels in Chinese gastric cancer (GC) and lung cancer (LC) patient samples. (A) PPME1 gene amplification in a Chinese GC tumor detected by array comparative genomic hybridization. (B) PPME1 amplification and protein overexpression in GC and LC samples detected by FISH and immunohistochemistry (IHC) assays, respectively. PPME1 IHC score and PPME1 amplification status are embedded within the images (“AMP” denotes the gene amplified). For the fluorescence in situ hybridization analysis (FISH) images, the PPME1 gene probe signals appear red, CEP11 signals are green, and DAPI-counterstained nuclei appear blue. Scale bars represent 50 nm for H&E/IHC images and 30 nm for the FISH images. All images within each row are on the same scale.
41 samples from Chinese LC patients. Representative PPME1amplified FISH images are shown in Figure 1A. Preliminary results obtained using IHC analysis showed that PPME1 was overexpressed at the protein level in the FISH-amplified cases when compared with normal (Fig. 1B). shRNA knockdown of PPME1 expression in PPME1amplified GC and LC cells results in growth inhibition and apoptotic induction To understand the role of PPME1 amplification and expression in tumorigenesis, shRNA technology was used to deplete PPME1 expression in the cells. The PPME1-amplified human GC SNU668 and LC OKa-C-1 cells and nonamplified control cells were identified for use in a functional study. As shown in Figure 2, shRNA-mediated knockdown of PPME1 resulted in growth inhibition of 48.6% and 41.3% compared with scrambled shRNA in SNU668 and OKa-C-1 cells by Day 4 after transfection. In contrast, an equivalent knockdown of PPME1 showed no significant effects on the cell growth and survival of the PPME1
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nonamplified human MKN1 and HCC95 cancer cells (Fig. 2A), supporting an oncogenic role for PPME1 in GC and LC. We also explored the effect of the PPME1 knockdown on apoptotic induction in PPME1-amplified and nonamplified cancer cells. Cellular introduction of PPME1 shRNA for 96 h increased caspase 3/7 activity (a hallmark of apoptosis) in the amplified SNU668 and OKa-C-1 cells, but not in the nonamplified MKN1 and HCC95 cells (Fig. 2B). These results indicate that activation of caspase 3/7 following shRNA-induced PPME1 knockdown is a specific event in PPME1-amplified cells and that functional PPME1 is required for survival signaling in this cellular background. PPME1 regulates PP2Ac methylation and pAKT/pErK in PPME1-amplified cancer cells. Modulation of PP2A signaling by PPME1 knockdown To further ascertain the functional role of PPME1 in regulating PP2Ac Leu 309 methylation and the phosphorylation/activity of PP2A downstream targets, we analyzed the demethylation status of PP2Ac and the phosphorylation of two different PP2A
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Figure 2. Cell growth inhibition and apoptosis induction by a short-hair (sh)RNA-mediated knockdown of PPME1. (A) Characterization of two PPME1amplified cancer cell lines (SNU668 and Oka-C-1) and two PPME1 nonamplified cancer cell lines (MKN1 and HCC95) using western blot and fluorescence in situ hybridization analysis. (B) Cells were cultured for 72 h after PPME1 shRNA transfection, and cell viability was analyzed by Acumen assay. Graphs indicate the level of cell survival in the PPME1-amplified cancer cell lines and nonamplified cancer cell lines; PPME1 protein expression suppression by shRNA was analyzed by western blot analysis. (C) Cells were incubated with PPME1 shRNA at the indicated concentrations for 72 h and caspase 3/7 activity was then measured by ELISA assay. The results are representative from three independent experiments.
target proteins in both PPME1-amplified and nonamplified cells transfected with either scrambled or PPME1-specific shRNA. For this purpose, we used an antibody that specifically recognizes the demethylated form of PP2Ac Leu 309. We found that the PPME1 depletion resulted in a decrease of PP2Ac Leu 309 methylation in the PPME1-amplified cancer cell lines (data not shown). PP2Ac demethylation was not altered in the parental untreated cells and scrambled shRNA-transfected cells or in the PPME1 nonamplified cancer cells (MKN1 and HCC95). PPME1 depletion was also found to inhibit Erk and AKT phosphorylation in PPME1amplified cells without any notable effects on the nonamplified cells (MKN1 and HCC95) (Fig. 3). These results suggest that in the PPME1-amplified cells, PP2A methylation and MAPK/ AKT phosphorylation are relevant downstream signaling targets of PPME1 that appear to be necessary for maintenance of the oncogenic state.
Discussion PP2A is a human tumor suppressor that accounts for the majority of cellular serine/threonine phosphatase activity.22 Inactivating alterations of the PP2A subunits have been found in multiple human cancers.23 The PP2A core enzyme consists of a catalytic “C” subunit, a structural “A” subunit, and a “B” subunit that binds subunits “A” and “C” together as a heterodimer.24 The “B” subunits are subdivided into four distinct families that share no sequence similarities.25 The combinatorial assembly of these various “A”, “B”, and “C” subunits permits the formation of many distinct PP2A complexes, and various PP2A complexes have been implicated in the control of a diverse array of cellular processes, including cell proliferation, survival, adhesion, and cytoskeleton dynamics.26 The highly conserved “C” terminal tail of PP2Ac has been confirmed to play a key role in malignancy and can undergo two types of posttranslational modification: phosphorylation and methylation.27 Phosphorylation of Tyr307 in PP2Ac
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by several tyrosine-specific kinases or of an unidentified threonine residue by an auto-activated kinase results in inactivation of PP2A activity.28 PPME1 has been found to suppress PP2A activity, at least in part, by demethylating PP2Ac and modulating the binding interaction of the “C” subunit with regulatory “B” subunits. Recent structural studies have shed light on the physical interactions between PPME1 and the PP2A holo-enzyme. RNA-interference-mediated knockdown of PPME1 in PPME1overexpressing cancer cells leads to activation of PP2A and the corresponding suppression of protumorigenic phosphorylation cascades,21 suggesting a potential oncogenic role of PPME1 in glioma. In this study, through the use of array-CGH and IHC expression analysis, we have found for the first time that PPME1 amplification in Chinese GC and LC patients is correlated with protein overexpression. The role of this gene was explored by downregulating PPME1 expression in a cell culture. The evidence suggests that PPME1 represents a bona fide oncogene, as loss of PPME1 protein expression results in growth inhibition, and a significant induction of cell apoptosis in the PPME1-amplified GC and LC cell lines, but it has little effect on non-amplified cells. The Erk and AKT pathways represent key oncogenic mediators that are often dysregulated by genetic abnormalities in cancer as a consequence of their overarching control of many proteins critical for cell growth and metastasis. However, the mechanisms that sustain Erk and AKT pathway activity in malignant cells remain elusive. Our data show that a PPME1 depletion results in decreased demethylation of PP2Ac Leu309 and dephosphorylation of Erk and AKT in PPME1-amplified cells. The effect of PPME1 depletion on Erk activity in GC and LC cells is consistent with a previous report from Puustinen et al., who described a connection between Erk activation and PPME1 overexpression in astrocytic glioma.21 The involvement of the PP2A signaling pathway in AKT activation was initially reported by Chen et al. in SV40 small t antigen transformed cells.29 During the preparation
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Figure 3. Downregulation of AKT and Erk signaling by short-hairpin RNA-mediated knockdown of PPME1 expression. Cells treated with shRNA against PPME1 were subjected to western blot analysis for demethylation of PP2Ac and phosphorylation of AKT and Erk in PPME1. The results are representative from three independent experiments.
Materials and Methods Cell culture and reagents The following antibodies were purchased from Cell Signaling Technology (CST): total-PP2Ac, phospho-p44/p42 MAP kinase (Erk1/2), total-Erk, phospho-AKT (Ser473), total-AKT, cleaved caspase-3, poly-(ADP-ribose) polymerase (PARP), horseradish
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peroxidase (HRP)-linked anti-rabbit IgG, and HRP-linked antimouse IgG. Other antibodies used included PPME1 (GenWay Biothech) and demethylated-PP2A-C (Santa Cruz). An antibody that binds to GAPDH (CST) was used as a control. SNU-668 and HCC-95 cells were obtained from the Korea Cell Line Bank (KCLB), and OKa-C-1 and MKN1 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. SNU-668, MKN1, OKa-C-1, and HCC-95 cells were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). All cell lines were genetically tested and authenticated using the StemElite IDTM System Kit (Promega, G9530) and were not cultured for more than 3 mo prior to performing the work described here. All studies using human tissue were performed with the patients’ consent and the approval of the Local Research Ethics Committee. Array comparative genomic hybridization analysis of the GC and LC patient samples The PPME1 gene copy number was analyzed in frozen patient tissue samples using the Agilent 244K array comparative genomic hybridization (aCGH) platform. The quality of the raw data was checked with the Agilent CGH Analytics software, using the derivative of the log ratio spread (DLRSpread) as a surrogate for assay quality. Any sample with a DLRSpread of more than 0.3 was excluded from further analysis. Data for the samples that qualified for further assessment were subsequently analyzed using Nexus software (version 4) for the recovery of PPME1 segmental structure (segmentation) and the discrete copy number value at the single-sample level (calling). Segments with an array CGH logRatio (copy number of sample vs. the control) of more than 0.8 were classified as amplified. Fluorescence in situ hybridization analysis The PPME1 fluorescence in situ hybridization analysis (FISH) probe was generated internally by directly labeling the BAC (CTD-2128J17) DNA with Spectrum Red (Vysis, 30-803400). A CEP11 Spectrum Green probe (Vysis, 32-132011) for the centromeric region of chromosome 8 was used as an internal control. FISH assays were performed on four micron dewaxed and dehydrated formalin-fixed, paraffin-embedded (FFPE) sections. The SpotLight Tissue Pretreatment Kit (Invitrogen, 00-8401) was used for pretreatment (boiled in reagent 1 for approximately 15 min and then coated with reagent 2 for approximately 10 min; minor time adjustments were made for the individual samples). Sections and probes were codenaturated at 80 °C for 5 min and then hybridized at 37 °C for 48 h. After a quick washoff process (0.3% NP40; 1 × SSC at 75.5 °C for 5 min and twice in 2 × SSC at room temperature for 2 min), the sections were mounted with 0.3 μg/ml DAPI (Vector, H-1200) and stored at 4 °C away from light for at least 30 min prior to scoring. The target gene and CEP signals were observed using a fluorescence microscope equipped with the appropriate filters, allowing visualization of the intense red target gene signals, the intense green chromosome centromere signals, and the blue counterstained nuclei. Enumeration of the PPME1 gene and chromosome 11 was conducted by microscopic examination of 50 tumor nuclei,
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of this manuscript, Jackson et al. reported that exogenous overexpression of PPME1 leads to increased T308 phosphorylation on AKT in HEKTER-ASB56 g cells,30 which supports our observation that PPME1 plays a role in AKT activation in GC and LC cells. Although previous studies by Puustinen et al. have reported a promoting effect of PPME1 on the proliferation of glioma cells in vitro,21 no PPME1-based disease linkage in cancer has been reported to our knowledge. The differential phenotypes observed by shRNA-mediated PPME1 knockdown in PPME1-amplified vs. non-amplified cell lines support an oncogenic driving role of PPME1 amplification and overexpression in GC and LC. The amplification and overexpression of PPME1 observed in subsets of patients with GC and LC suggests its potential as a therapeutic drug target. This notion is further supported by the reduced PPME1 expression in normal tissues except for human adults’ brain, which may theoretically leave open a sufficient therapeutic window for targeting PPME1 in patients with a PPME1 amplification.31 Currently, two groups have demonstrated the feasibility of developing PPME1 selective small molecular inhibitors.32,33 The cell lines with PPME1 amplification described in this study will facilitate the development of PPME1 inhibitors. As the first targeted therapy for GC, trastuzumab has demonstrated efficacy in about 20% of GC patients with HER2 amplification or overexpression.34,35 Clinically, the FISH and IHC assays were approved the selection of patients with HER2 amplification and overexpression, which is based on HER2 FISH positivity or an IHC score of 3+ (a score of 2+ needs further FISH confirmation). Although we used the same criteria for HER2 to define PPME1 FISH positivity, it is still unclear whether they can be directly used for patient selection in the future. Further studies using the PPME1 selective inhibitor to determine a correlation between FISH and IHC scores and antitumor efficacy in preclinical models or in patients will help to define the use of PPME1 positivity for patient selection. Although the FISH assay is more clinically relevant than aCGH assay for patient selection, due to the non-availability of samples, the tumor samples used for FISH and aCGH analysis were collected from different cohorts, which might affect the higher amplification rates detected by FISH assay compared with aCGH (6.4% vs. 3.8% in GC and 7.3% vs. 3.1% in LC respectively). Further studies on the same cohort of samples will help to address this issue. In conclusion, we have identified PPME1 amplification, which is correlated with protein overexpression and has the potential to define new molecular segments in GC and LC. Our results suggest that the amplification of PPME1 is an oncogenic driver for the development of novel therapeutic approaches in GC and LC.
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blot assay. The viable cells were dyed with Hoechst (Invitrogen, H21486) and incubated at 37 °C for 1 h. Then the plates were determined using Acumen eX3 (TTP LabTech). In vitro pharmacodynamics study Cells were lysed in 1× sodium dodecyl sulfate (SDS) buffer 50 mM TRIS-HCl (pH 6.8; 100 mM dithiothreitol, 2% SDS [electrophoresis grade], and 10% glycerol) containing NaVo3 (Sigma), a complete protease inhibitor cocktail (Sigma), the complete phosphatase inhibitor cocktail 1 (Sigma), and the complete phosphatase inhibitor cocktail 2 (Sigma). Soluble proteins were quantified using a Pierce BCA™ Protein Assay Kit (Pierce, 23225) and separated on NuPage 4–12% Bis-tris gels (Invitrogen) in a 1× MES SDS running buffer (Invitrogen) for 2.5 to 3 h at 110 V. They were then electro-transferred to nitrocellulose polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc.) in a 1× NuPage transfer buffer (Invitrogen) containing 20% ethanol for 75 min at 340 mA. After blocking the membranes in a 5% fat-free milk solution (dissolved in the 1× TBST buffer, 1× Trisbuffered saline containing 0.1% [v/v] tween20), they were probed with the anti-PPME1 antibody (clone 4A12, GenWay Biothech, 20-614-460633), anti-demethylated-PP2A-C (sc-80990), antiPP2Ac (2259, Cell Signaling), anti-phospho-p44/42 MAPK (4370, Cell Signaling), phospho-Akt (Ser473) (M3628, Dako), and anti-GAPDH (2118, Cell Signaling). Antibodies were diluted 1:2000 for PPME1; 1:1000 for PP2Ac, MAPK, and P-AKT; 1:200 for anti-demethylated-PP2A-C; and 1:10 000 for GAPDH in TBST (50 mM Tris Base, 150 mM NaCl, 0.1% [v/v] Tween-20) containing 5% milk powder. After washing the membrane in TBST, it was incubated with the appropriate HRPconjugated secondary antibodies diluted 1:2000 in TBST. The immuno-reactive signals were detected using an ECL reagent Advance Western Blotting Detection Kit (GE Healthcare) and an ImageQuant LAS 4000 (GE Healthcare). ApoTox-Glo™ Triplex Assay The ability to induce Caspase 3/7 activation after exposure to shRNA was measured by ApoTox-Glo™ Triplex Assay (Promega, G6321) according to the manufacturer’s instruction. The cells were added to the shRNA/INTERFER in mixture, and at the end of incubation, 100 μl of the Caspase-Glo® 3/7 reagent was added to all wells and briefly mixed by orbital shaker (300 to 500 rpm for approximately 30 s). After incubation for at least 30 min at room temperature, the resultant luminescent light was measured in a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, LLC). Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We would like to thank Dr Paul Gavine for reviewing this manuscript.
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which yielded a ratio of PPME1 to CEP11. Tumors with a ratio of 2 or greater or a presence of at least 10% of a gene cluster were defined as amplification. Immunohistochemistry The primary antibody used for PPME1 immunohistochemical (ICH) staining was a commercially available mouse monoclonal antibody (clone 2C5, Genway Biotech). Sections (4-mm thick) were cut from FFPE blocks and mounted on silanized slides. For PPME1 staining, the sections were deparaffinized in xylene and dehydrated with graded ethanol. After washing them with distilled water, the sections were placed in the supplied buffer. For antigen retrieval, the slides were heated at 110 °C for 5 min and then cooled for at least 20 min at room temperature with running deionized water. After washing them a with Trisbuffered 0.9% NaCl solution containing Tween 20 (pH 7.6), the tissue sections were covered for 5 min with a peroxidaseblocking reagent (Dako) to block endogenous peroxidase, followed by an additional washing with the supplied buffer. After peroxidase blocking for 5 min, the sections were incubated with a primary antibody for 60 min at room temperature. They were then labeled with an HRP-labeled polymer (Envision kit, Dako) for 30 min at room temperature and reacted with a diaminobenzidene tetrahydrochloride solution. PPME1 immunopositivity was scored as follows: Reactivity was scored as zero when there were no interpretable signals within the tumor, and it was scored as positive when reactivity of the tumor cell was detected above the background level. The positive samples were classified further as 1+, 2+, and 3+ based on their reactivity intensity. The tissue replicate from each tumor with the highest intensity was used as the final IHC result for that tumor. Scores of 2+ and 3+ were classified as overexpression. Lentiviral-shRNA-mediated PPME1 gene knockdown in cell culture The pGCSIL-GFP (Genechem) lentiviral vector was used to express a short-hairpin RNA for PPME1 silencing. The targeted sequences for PPME1 were PSC5018: GATACATCTG AGTTCAAAT and PSC5020: GTACAGCTAT GGATGCACTT A; the scrambled control sequence was PSC3945: TTCTCCGAAC GTGTCACGT. Lentivirus stocks were produced by a transient transfection of HEK293T cells with the encapsidation plasmid, the pVSV-G plasmid, and the lentiviral recombinant vector as previously described.36 Forty-eight hours after the transfection, the lentiviral supernatants were harvested and centrifuged briefly (500 × g for 10 min) to remove cellular debris. The viruses were frozen at –80 °C until use. The titer of each virus stock was determined by real-time PCR using the SBI Ultra Rapid Lentiviral Titer Kit. The target cells were infected with lentivirus-expressing PPME1 shRNA and nontargeted shRNA at a suitable MOI supplemented with 5 ug/ml polybrene. After infection for 96 h, the PPME1 protein expression was measured in cells infected with the lentivirus by western
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References
